RU2289585C2 - Novel aminoalkylglucosaminide phosphate compounds, immunostimulating pharmaceutical composition comprising thereof and method for inducing immune response - Google Patents

Abstract

FIELD: organic chemistry, medicine, immunology, pharmacy.

SUBSTANCE: invention relates to novel immune active aminoalkylglucosaminide phosphate compounds of the formula (V):

or its pharmaceutically acceptable salts wherein X is chosen from the group consisting of -O- and -NH-; R¹, R² and R³ are chosen independently of one another from the group consisting of (C₉-C₁₄)-acyl groups. Also, invention relates to the immunostimulating pharmaceutical composition comprising pharmaceutically acceptable carrier and the therapeutically effective dose of compound of the formula (V), and to a method for inducing the immune response in a patient.

EFFECT: improved inducing response method, valuable medicinal properties of compounds and pharmaceutical composition.

19 cl, 4 tbl, 9 ex

Description

FIELD OF THE INVENTION

The present invention, in General, relates to new immunoeffective compounds, their use in pharmaceutical compositions and methods for their preparation and use for prophylactic and / or therapeutic vaccination. In particular, the present invention relates to new compounds containing 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) linked by a glycosidic bond to a cyclic aminoalkyl group (aglycon), and their use in pharmaceutical adjuvant systems.

State of the art

Humoral immunity and cellular immunity are the two main branches of the immune response in mammals. Humoral immunity includes the formation of antibodies to foreign antigens. Antibodies are produced by B-lymphocytes. Cellular immunity involves the activation of T-lymphocytes, which either act on infected cells that carry foreign antigens, or stimulate other cells that act on infected cells. Both branches of the mammalian immune system are important in the fight against the disease. Humoral immunity is the main line of defense against bacterial pathogens. In the case of viral diseases, the induction of cytotoxic T-lymphocytes (CTLs) is crucial for protective immunity. Thus, an effective vaccine preferably stimulates both branches of the immune system to protect against disease.

Vaccines present foreign antigens to the host so that the host has a protective immune response. Often, vaccine antigens are represented by killed or attenuated forms of the microbes that cause the disease. The presence of essential components and antigens in such killed or attenuated vaccines has led to significant efforts to clean vaccine components, including the development of well-defined synthetic antigens using chemical and recombinant methods. The purification and simplification of microbial vaccines, however, led to a concomitant decrease in activity. Low molecular weight synthetic antigens, although devoid of potentially harmful impurities, are often not sufficiently immunogenic in themselves. Such observations have led researchers to add immune system stimulants, known as adjuvants, to vaccine compositions to enhance the activity of vaccine components.

Immune adjuvants are compounds that, when administered to an individual or when tested in vitro, enhance the immune response to an antigen in a subject to which a given antigen is administered, or enhance certain activities of cells of the immune system. A variety of compounds have been obtained and tested with varying degrees of adjuvant activity (see, for example, Shimizu et al. 1985, Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994, Miyajima et al. 1996 ) However, these and other previously developed adjuvant systems often exhibit toxic properties, are unstable and / or have an unacceptably low immunostimulating effect.

The only adjuvant currently approved for human use in the United States is alum, a group of aluminum salts (such as aluminum hydroxide, aluminum phosphate) that contain vaccine antigens. Alum-type particulate carriers have been reported to facilitate the capture, processing, and presentation of soluble antigens by macrophages. However, alum is not without side effects and, unfortunately, is limited only by humoral immunity (antibodies).

The discovery and development of effective adjuvant systems is necessary to improve the efficacy and safety of existing and future vaccines. Thus, there is a continuing need for new and improved adjuvant systems, especially those that enhance both effector branches of the immune system, which will facilitate the development of the next generation of synthetic vaccines. The present invention satisfies these and other needs.

Disclosure of invention

The compounds of the present invention are immunoeffective molecules that enhance the humoral and cellular immune responses to vaccine antigens. These compounds, in General, can be described as belonging to the class of cyclic compounds of AGF, where AGF means aminoalkylglucosaminide phosphates. The term “cyclic AGF” means an azacycloalkyl- or (azacycloalkyl) alkyl glucosaminide phosphate in which 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) is linked by a glycosidic bond with an azacycloalkyl or (azacycloalkyl) alkyl group) (

The compounds of this invention encompass 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) linked by a glycosidic bond to an azacycloalkyl or (azacycloalkyl) alkyl moiety (aglycon). These compounds are phosphorylated at position 4 or 6 of the glucosamine ring and acylated with alkanoyloxytetradecanoyl residues at the aglycon nitrogen and at positions 2 and 3 of the glucosamine ring. The compounds of this invention and their pharmaceutically acceptable salts are generally described by formula I:

where X is —O — or —NH—, Y is —O — or —S—; a R ¹ , R ² and R ³ independently from each other are (C ₉ -C ₁₄ ) acyl groups, including saturated, unsaturated and branched acyl groups; R ⁴ is —H or —PO ₃ R ⁷ R ⁸ where R ⁷ and R ^{8 are} independently represented by H or a (C ₁ -C ₄ ) aliphatic group; R ⁵ is —H, —CH ₃ or —PO ₃ R ⁹ R ¹⁰ , where R ⁹ and R ^{10 are} independently selected from —H or (C ₁ -C ₄ ) aliphatic groups; R ^{6 is} independently selected from H, OH, (C ₁ -C ₄ ) oxyaliphatic groups, —PO ₃ R ¹¹ R ¹² , —OPO ₃ R ¹¹ R ¹² , —SO ₃ R ¹¹ , —OSO ₃ R ¹¹ , —NR ¹¹ R ¹² , -SR ¹¹ , -CN, -NO ₂ , -CHO, -CO ₂ R ¹¹ and -CONR ¹¹ R ¹² , where R ¹¹ and R ^{12 are} independently selected from H or (C ₁ -C ₄ ) an aliphatic group; provided that one of the groups R ⁴ and R ⁵ contains phosphorus, and when R ⁴ is represented by -PO ₃ R ⁷ R ⁸ , then R ^{5 is} not -PO ₃ R ⁹ R ¹⁰ , with * ^1-3 and ** denote chiral centers; and the indices n, m, p and q independently from each other mean an integer from 0 to 6, provided that the sum of p and m is from 0 to 6.

In some embodiments of the compounds of the present invention, X and Y are oxygen, R ⁴ is PO ₃ R ⁷ R ⁸ , R ⁵ and R ⁶ are H, and indices n, m, p and q are integers from 0 to 3. In a more preferred embodiments R ⁷ and R ^{8 are} represented by H. In an even more preferred embodiment, n = 1, m = 2, and subscripts p and q = 0. In an even more preferred embodiment, R ₁ , R ₃ and R ₃ are (C ₉ -C ₁₃ ) acyl groups, more preferably (C ₁₀ -C ₁₂ ) vanilla groups. In an even more preferred embodiment, * ^1-3 are in the R-configuration, Y is in the equatorial position, and ** are in the s-configuration. Particularly preferred are N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-tetradecanoyloxytethecanoylamino] -3-O - [( R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside (formula II),

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

[N- (R) -3-decanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-decanoyloxytetradecanoylamino] -3-O - [(R) -3-decanoyloxytetradecanoyl] -β-D-glucopyranoside (formula IV)

and their pharmaceutically acceptable salts.

The present invention also provides pharmaceutical compositions containing compounds of the above general formula and specific formulas. Pharmaceutical compositions may be combined with various antigens and various drug formulations known in the art.

The compounds of the present invention are also useful in various methods of inducing an immune response in an individual. Such a method consists in administering to the individual a therapeutically effective amount of one or more compounds of the present invention, preferably in the form of a pharmaceutical composition also comprising a pharmaceutically acceptable carrier.

The present invention also encompasses methods of treating mammals suffering from or susceptible to infection by a pathogen, cancer, or autoimmune disease. Such a method consists in administering to the mammal a therapeutically effective amount of one or more compounds of the present invention, preferably in the form of a pharmaceutical composition also containing a pharmaceutically acceptable carrier.

In addition, the present invention also includes a method for treating diseases or conditions that improve with the formation of nitric oxide in a patient. The method comprises treating a patient with an effective amount of a compound or compounds of the present invention or an effective amount of a composition comprising one or more compounds of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the compounds of the present invention can be administered 48 hours before, immediately before or during ischemia.

The implementation of the invention

Definitions

The term “acyl” refers to those groups that come from an organic acid when its hydroxyl moiety is removed. Accordingly, acyl may mean, for example, acetyl, propionyl, butyryl, decanoyl and pivaloyl. For example, "(C ₉ -C ₁₄ ) acyl" means an acyl group containing from 9 to 14 carbon atoms.

The term "aliphatic" by itself or as part of another substituent means, unless otherwise indicated, a straight or branched chain or a cyclic hydrocarbon group, including groups containing both cyclic elements and chains that can be fully saturated or mono- or polyunsaturated containing the indicated number of carbon atoms (for example, C ₁ -C ₄ means from 1 to 4 carbon atoms). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, methylene, ethylene and n-butylene. An unsaturated alkyl group is a group containing one or more double bonds and / or triple bonds. Examples of unsaturated aliphatic groups include vinyl, 2-propenyl, crotyl, 2-butadienyl, 1-propynyl and 3-propynyl.

The term “hydroxyaliphatic” refers to those groups that contain an aliphatic group connected to the rest of the molecule through an oxygen atom.

Each of the above terms (for example, "alkyl", "acyl") may include both substituted and unsubstituted forms of these groups.

Substituents in aliphatic groups can serve a number of groups selected from the number -OR ', -O, = S, -NR', -N-OR ', -NR'R'',-SR', -halogen, -SiR ' R``R ''', -OC (O) R', -C (O) R ', -CO ₂ R', -CONR'R '', -OC (O) NR'R '', -NR '' C (O) R ', -NR'C (O) NR''R''', -NRC (O) 2R ', -NH-C (NH ₂ ) = NH, -NR'C (NH ₂ ) = NH, -NH-C (NH ₂ ) = NR ', -S (O) R', -S (O) ₂ R ', -S (O) ₂ NR'R'', -CN and NO ₂ , the number of which varies from 0 to (2m '+ 1), where m' = is the total number of carbon atoms in such a radical. R ′, R ″ and R ″ ″ independently of one another are hydrogen and unsubstituted (C ₁ -C ₄ ) aliphatic groups. From the foregoing discussion of substitutions, it should be understood that the term “alkyl” may include groups such as haloalkyl (for example, —CF ₃ and —CH ₂ CF ₃ ) and the like.

The term “halo” or “halogen” by itself or as part of another substituent means, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom. In compounds with several halogen substituents, the halogens may be the same or different.

The term “pharmaceutically acceptable salts” embraces those salts of the active compounds which are prepared with relatively non-toxic acids or bases, depending on the particular substituents found in the compounds described in the present invention. In the case where the compounds of the present invention contain relatively acidic functional groups, their salts with bases can be obtained by adding the desired base, both in the appropriate inert solvent and without it. Examples of pharmaceutically acceptable salts formed with bases include sodium, potassium, calcium, ammonium, organic amines, magnesium salts and the like. In the case where the compounds of the present invention contain relatively basic functional groups, their salts with acids can be obtained by adding the desired acid, either in an appropriate inert solvent, or without it. Examples of pharmaceutically acceptable salts formed with acids include salts of inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monosubstituted carbonic, phosphoric, monosubstituted phosphoric, disubstituted phosphoric, sulfuric, monosubstituted sulfuric, hydroiodic, phosphoric and the like. salts of relatively non-toxic organic acids, such as acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberinic, fumaric, almond, phthalic, benzenesulfonic, para-tolylsulfonic, citric, tartaric, methanesulfonic and the like. Also covered are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galacturonic acid and the like (see, for example, Berge SM et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977 66, 1-19). Certain compounds of the present invention contain both basic and acidic functional groups, which allows them to be converted into salts formed with either a base or an acid.

Neutral forms of the compounds can be regenerated by treating the salt with a base or acid and isolating the starting compound by a standard method. The starting form of the compound differs from the various salt forms in some physical properties, such as solubility in polar solvents, but otherwise these salts are equivalent to the starting form of the compound for the purposes of the present invention.

Along with salt forms, the present invention provides compounds that are in the form of prodrugs (precursors). Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to form the compounds of the present invention. In addition, Prodrugs can be converted into compounds of the present invention using chemical or biochemical methods under ex vivo conditions. For example, prodrugs can be slowly converted to the compounds of the present invention if they are placed in a transdermal patch-reservoir with an appropriate enzyme or chemical reagent.

Some compounds of the present invention may exist in unsolvated forms along with solvated forms, including hydrates. In general, solvated forms are equivalent to unsolvated forms and are intended to be encompassed by the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for use in accordance with the present invention and are intended to be encompassed by the scope of the present invention.

Some compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds. It is contemplated that racemates, diastereoisomers, geometric isomers, and individual isomers are encompassed by the scope of the present invention.

The compounds of the present invention may also contain atomic isotopes in unnatural proportions to one or more of the atoms that make up such compounds. For example, compounds can be radioactively labeled with radioactive isotopes, for example, such as tritium ( ³ H) iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C). It is intended that all isotopic variations of the compounds of the present invention, radioactive and non-radioactive, are encompassed by the scope of the present invention.

Introduction

In an effort to improve vaccine safety, their manufacturers avoid killed whole-cell vaccines and produce recombinant or subunit vaccines. Upon receipt of these safer vaccines, extraneous components of bacteria and viruses are eliminated, and minimal structures or epitopes remain that are considered necessary for protective immunity. The safety of these vaccines is improved by eliminating the foreign components of bacteria and viruses, which are often toxic and pyrogenic. However, it is these toxicity-causing components that provide non-specific immunostimulation that makes whole cell vaccines so effective. Without additional immunostimulation, minimal structures and epitopes that form recombinant and subunit vaccines often have weak immunogenicity.

The disaccharide molecule derived from LPS Salmonella minnesota R595 is an MPL immunostimulant (Corixa Corp.) with immunostimulating properties. The MPL immunostimulant, or monophosphoryl lipid A, is a structural derivative of lipid A (or LPS) and has a better therapeutic index than lipid A (see the structure of monophosphoryl lipid A in U.S. Patent 4,987,237 and the description of the preparation of monophosphoryl lipid A in U.S. Patent Nos. 4,436,727 and 4,436,728). Other useful immunostimulants include 3-de-O-acylated monophosphoryl lipid A (3D-MPL), which is described in U.S. Patent No.4,912,094. This compound can be safely administered to humans in doses of at least up to 20 mcg / kg, although some patients may have fever, flu symptoms, increased heart rate and a moderate increase in blood pressure at doses of ≥10 mcg / kg. Experiments on cell and animal cultures confirm that the MPL immunostimulant nevertheless retains some immunostimulatory activity of the initial LPS in that pyrogenicity and the ability to induce inflammatory cytokines such as TNF and IL-8, although at higher doses, are retained. Thus, the need for effective adjuvants for vaccines is well recognized. Ideally, such adjuvants should enhance the protective immune response, but not cause unwanted toxicity and pyrogenicity.

In an effort to obtain an immunostimulant with low pyrogenicity, synthetic molecules were obtained that have structural similarities with the MPL immunostimulant. These new molecules, collectively referred to as aminoalkyl glucosaminide phosphates (AGF), consist of an acylated glucose molecule coupled to an acylated aminoalkyl group (Johnson et al. (1999) Bioorg. Med. Chem. Lett. 9: 2273-2278; PCT / WO 98 / 50399 and the references cited therein). Each molecule has 6 fatty acid residues, which is considered the optimal number for the maximum adjuvant activity. The substitution of various chemical groups in the aminoalkyl structures of AGF was conceived in order to optimize stability and solubility. Thus, in general terms, AGF can be divided into several families based on the structure of aminoalkyl groups. After an initial biological assessment, it became apparent that aminoalkyl groups can strongly influence the pyrogenic properties of AGF (see US Patent Application Serial No.09 / 074,720 filed May 7 , 1998, and US Patent Nos. 6,113,918 and 6,303,347). During the initial screening process for synthetic adjuvant compounds, rabbit pyrogenicity data were obtained. It was noted that some of these compounds did not cause a temperature increase when administered intravenously at a dose of 10 μg / kg. In general, it was these compounds that did not induce the inflammatory cytokines TNF-α or IL-1β to a significant extent when analyzing ex vivo cytokine induction on human peripheral blood mononuclear cells. Next, we report on the study of the adjuvant properties of one of the classes of AGF that cause minimal activity when tested for pyrogenicity in rabbits and in the analysis of ex vivo cytokine induction.

Compounds and Compositions

The present invention provides compounds described by the general formula I:

and pharmaceutically acceptable salts thereof, wherein X is —O — or —NH—, Y is —O — or —S—; a R ¹ , R ² and R ³ independently from each other represent (C ₉ -C ₁₄ ) anilic groups, including saturated, unsaturated and branched acyl groups; R ⁴ is

- H or —RO ₃ R ⁷ R ⁸ , where R ⁷ and R ^{8 are} independently represented by H or a (C ₁ -C ₄ ) aliphatic group; R ⁵ is —H, —CH ₃ or —PO ₃ R ⁹ R ¹⁰ , where R ⁹ and R ^{10 are} independently selected from —H or (C ₁ -C ₄ ) aliphatic groups; R ^{6 is} independently selected from H, OH, (C ₁ -C ₄ ) oxyaliphatic groups, —PO ₃ R ¹¹ R ¹² , —OPO ₃ R ¹¹ R ¹² , —SO ₃ R ¹¹ , —OSO ₃ R ¹¹ , —NR ¹¹ R ¹² , -SR ¹¹ , -CN, -NO ₂ , -CHO, -CO ₂ R ¹¹ and -CONR ¹¹ R ¹² , where R ¹¹ and R ^{12 are} independently represented by H or (C ₁ -C ₄ ) an aliphatic group; provided that one of the groups R ⁴ and R ⁵ contains phosphorus, and when R ⁴ is represented by -PO ₃ R ⁹ R ⁸ , then R ^{5 is} not -PO ₃ R ⁹ R ¹⁰ , with * ^1-3 and ** denote chiral centers;

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

Although hexopyranoside in Formula I is presented in a gluco configuration, other glycosides are also within the scope of the invention. For example, glycopyranosides, including other hexopyranosides (allo-, altro-, manno-, gulo-, ido-, galacto-, thalo-), are included in the scope of the invention.

In the above general formula, the 3'-stereogenic centers at which the normal fatty acid residues are attached and which are denoted as * ¹ , * ² and * ³ are in the R or S configuration, preferably in the R configuration. The absolute stereochemistry of carbon atoms in a cyclic aglycone to which R ⁶ and glucosamine are attached directly or indirectly (denoted as **) can be represented by the R or S configuration. In the above general formula, Y may be in the equatorial or axial position, preferably equatorial. All stereoisomers, enantiomers, diastereomers and mixtures thereof are considered to be included in the scope of the present invention.

In preferred embodiments of the present invention, X and Y are —O—, R ⁴ is phosphono, R ⁵ and R ^{6 are} H, and n, m, p and q are integers from 0 to 3, more preferably from 0 to 2. Most preferably n = 1, m = 2, and p and q are 0. In this preferred embodiment, the compounds of this invention are 2-pyrrolidinylmethyl-β-D-glucosaminide-4-phosphates of the general formula V:

In a preferred embodiment of the present invention, the 3'-stereogenic centers (* ^1-3 ) at which they are attached are in the R configuration, Y is in the equatorial position, and the absolute stereochemistry of the stereogenic center of pyrrolidine (**) is represented by the S configuration.

Particularly preferred embodiments are N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamino] -3-O- [(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside and its pharmaceutically acceptable salts of formula II,

[N- (R) -3-dodecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-dodecanoyloxytethecanoylamino] -3-O - [(R) -3-dodecanoyloxytetradecanoyl] -β-D-glucopyranoside and its pharmaceutically acceptable salts of formula III,

[N (R) -3-decanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-decanoyloxytetradecanoylamino] -3-O - [(R) - 3-decanoyloxytetradecanoyl] -β-D-glucopyranoside and its pharmaceutically acceptable salts of formula IV.

Getting connections

Compounds of the present invention can be obtained by the methods described in Johnson et al., Bioorg. Med. Chem. Lett. 9: 2273-2278 (1999) and PCT / WO 98/50399 and the references therein. In general, the synthesis methods described in the above sources are broadly applicable to the preparation of compounds with various acyl groups and substituents. Specialists in this field should understand that the converging methods described in these sources can be modified using other acylating reagents or can come from commercially available materials that already have the corresponding acyl groups attached.

Compound Evaluation

Compounds of the present invention can be evaluated in various assay formats to select a compound with a suitable pharmacophore profile. For example, in U.S. Patent No.6,013,640 describes animal models suitable for evaluating the cardioprotective properties of the described compounds. The examples below also present methods for evaluating the pyrogenicity of the compounds in question and other methods for evaluating the pro-inflammatory properties of these compounds.

The present invention also provides pharmaceutical compositions containing the compounds provided therein in admixture with one or more pharmaceutically acceptable carriers. Suitable carriers depend on the disease to be treated together with the route of administration. Accordingly, a discussion of carriers is provided below in conjunction with methods of use.

Pharmaceutical compositions and their use

In one embodiment, the present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier. The compound is in a therapeutically effective amount necessary to achieve the desired effect in the sense of treating a disease or disease or achieving a biological manifestation. The pharmaceutical composition may act as an adjuvant when administered with an antigen.

The compositions of this invention include both compositions formulated for direct administration of the active compounds to patients without dilution, either together with a vaccine or other active agent, or alone, as well as more concentrated compositions of the compounds that can be formulated for subsequent dilution in order to avoid transport and / or storing large quantities of diluent (water, saline, aqueous materials). In general, pharmaceutical compositions of the invention intended for direct or immediate administration to a patient (i.e., without dilution) should contain one or more compounds in a therapeutically effective amount. Such an amount varies depending on the particular therapeutic compound or compounds and on the desired therapeutic effect. More concentrated compositions should contain such amounts of a compound or compounds of the invention that are suitable for such compositions.

In the preparation of pharmaceutical compositions, the pharmaceutically acceptable carriers may be in solid or liquid form. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that can also act as diluents, flavors, binders, preservatives, disintegrants, or encapsulating materials.

In powders, the carrier is a finely divided solid which is mixed with the finely divided active component. In tablets, the active component is mixed in an appropriate proportion with a carrier having the necessary binding properties and compressed to the desired shape and size.

Solid forms of the compositions can also be obtained by spray drying aqueous formulations of active adjuvants (eg, in the form of salt) or lyophilization and grinding together with fillers.

Suitable carriers for the solid compositions of this invention include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter, etc. The term " the preparation "includes formulations of the active compound with encapsulating material as a carrier providing capsules in which the active component, with or without other carriers, is surrounded by a carrier, which is thus in contact with it. Wafers and lozenges are likewise included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid dosage forms suitable for oral administration.

To obtain candles, a low-melting wax such as a mixture of glycerides of fatty acids or cocoa butter is first melted and the active component is dispersed homogeneously in it, for example, by stirring. The molten homogeneous mixture is then poured into molds of the appropriate size and cooled, while it solidifies.

Liquid form preparations include solutions, suspensions and emulsions, for example, aqueous solutions or solutions in a mixture of water / propylene glycol. For parenteral injections, liquid preparations can be prepared as a solution in an aqueous solution of polyethylene glycol. In certain embodiments, the pharmaceutical compositions are formulated as stable emulsions (eg, water-in-oil or oil-in-water emulsions) or aqueous formulations, preferably comprising one or more surfactants (detergents). In such emulsions, suitable detergents well known in the art can be used. In one embodiment, the composition is in the form of a micellar dispersion comprising at least one suitable detergent. Detergents suitable for such micellar dispersions include phospholipids. Examples of phospholipids include: diacylphosphatidylglycerols, such as dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG); diacylphosphatidylcholines such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids such as dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE). Other examples include, but are not limited to, ethanolamine derivatives (such as phosphatidylethanolamine above or cephalinum), serine (such as phosphatidylserine), and 3'-O-lysylglycerol (such as 3'-O-lysylphosphatidylglycerol).

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers and thickeners as desired. Aqueous suspensions suitable for oral administration can be prepared by dispersing the finely divided active component in water together with a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose and other well-known suspending agents.

Also included are solid preparations that need to be converted, shortly before use, to liquid forms for oral administration. Such preparations, along with the active component, may contain colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

The pharmaceutical preparations are preferably in dosage form. In this form, the drug is divided into unit doses containing an appropriate amount of the active ingredient. The dosage form may be a packaged preparation, the package containing a certain amount of the drug, for example, the packaging of tablets, capsules, and powders in vials or ampoules. Also, the capsule, tablet, cachet or lozenge itself, or an appropriate amount of them in packaged form, can serve as the dosage form.

Thus, the adjuvant systems of the invention are particularly advantageous in the manufacture and use of vaccines and other immunostimulating compositions for the treatment and prevention of diseases, inducing active immunity to antigens in mammals, preferably humans. Vaccine production is a well-developed field and general guidelines for the preparation and preparation of vaccines are readily available from numerous sources. One such example is New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., USA, 1978.

In one illustrative embodiment, the antigen in the vaccine composition of the invention is a peptide, polypeptide, or immunogenic portion thereof. The "immunogenic portion" as applied to this invention is a portion of a protein that is recognized (i.e., specifically binds to) an antigen receptor on the surface of B cells and / or T cells. Such immunogenic portions typically include at least 5 amino acid residues, more preferably at least 10, even more preferably at least 20 amino acid residues of an antigenic protein or variant thereof.

The immunogenic portions of antigenic polypeptides can generally be identified by well-known methods, such as those described in Paul, Fundamental Immunology, 3 rrd ed., 243-247 (Raven Press, 1993) and the references therein. Such methods include screening polypeptides for their ability to react with antigen-specific antibodies, antisera and / or T cell lines or clones. As applied to this invention, antisera and antibodies are "antigen-specific" if they specifically bind to the antigen (that is, they react with a protein in an ELISA or other method and do not noticeably react with unrelated proteins). Such antisera and antibodies can be obtained, as described in this invention, by well-known methods. The immunogenic part of a protein is the part that reacts with such antisera and / or T cells to no less extent than a full-sized polypeptide reacts (for example, when analyzed by ELISA and / or T cell reaction). Such immunogenic parts may respond to such an analysis to the same or greater extent than the full-sized polypeptide. Such screening can generally be carried out by methods well known in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide can be immobilized on a solid support and reacted with patient sera, which leads to the binding of antibodies in these sera to the immobilized polypeptide. You can then remove unbound serum and detect bound antibodies using, for example, ¹²⁵ I labeled protein A.

Peptide and polypeptide antigens are obtained by any of a number of well-known methods. Recombinant proteins encoded by DNA sequences can be easily obtained from isolated DNA sequences using a variety of expression vectors known in the art. Expression can be carried out in any suitable host cells transformed or transfected with an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast cells, and higher eukaryotes, such as mammalian cells and plant cells. Preferably, E. coli cells, yeast cells, or mammalian cell lines, such as COS or CHO, are used.

Parts and other variants of protein antigens containing less than 100 amino acids, usually less than 50 amino acids, can also be obtained by synthesis using methods well known in this field. For example, such polypeptides can be synthesized by any of the commercially available solid-phase methods, such as the Memfield solid-phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain, see Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Automated polypeptide synthesis equipment is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, CA) and can be operated according to the manufacturer's instructions.

In certain specific embodiments, the polypeptide antigen used in the vaccine compositions of the invention may be a fusion protein comprising two or more separate polypeptides. A fusion partner can, for example, facilitate the presentation of T helper epitopes (immunological fusion partner), preferably those that are recognized in humans, or it can promote protein expression (expression enhancer) in greater yield than that of a native recombinant protein. Some preferred fusion partners are both immunological and expression enhancing partners. Other fusion partners may be selected in order to increase the solubility of the protein or to ensure that it enters the desired intracellular compartments. Other fusion partners include affinity tags that facilitate protein purification.

Fusion proteins can generally be obtained by standard methods, including chemical conjugation. Preferably, the fusion protein is expressed as a recombinant protein, which allows to increase the level of production compared to the non-fused protein in the expression system. Briefly, DNA sequences encoding the polypeptide components can be assembled separately and ligated into the corresponding expression vector. The 3'-end of the DNA sequence encoding one polypeptide component is in the lead, with or without a peptide linker, with the 5'-end of the DNA sequence encoding the second polypeptide component so that the reading frames of these sequences are in the same phase. This makes it possible to translate a single fusion protein that preserves the biological activity of both polypeptides that make up its composition.

The sequence of the peptide linker can be used to separate the first and second polypeptide components at a distance that allows each of the polypeptides to fit into their secondary and tertiary structure. The sequence of such a peptide linker is inserted into the fusion protein by standard methods well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) the ability to accept a flexible expanded conformation, (2) the inability to accept such a secondary structure that could interact with functional epitopes on the first and second polypeptides, (3) the absence of hydrophobic or charged residues that could react with functional epitopes of polypeptides. Preferably, the peptide linkers contain residues of Gly, Asn and Ser. Other neutral amino acids in the linker sequence, such as Thr and Ala, can be used. Amino acid sequences that can advantageously be 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; U.S. Patent No.4,935,233 and U.S. Patent No.4,751,180. The linker sequence, in General, can have a length of from 1 to 50 amino acids. Linker sequences are not needed if I have the first and second polypeptides;

non-essential N-terminal amino acid sites that can be used to separate functional domains and prevent steric interference.

In preferred embodiments, the immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, the protein D derivative comprises about a third of the protein (e.g., the first 100-110 amino acids from the N-terminus) and may contain a lipid. In some preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included at the N-terminus to give the polypeptide additional exogenous T-cell epitopes and to increase expression in E. coli (so that they function as expression enhancers). The lipid tail provides optimal antigen presentation for antigen-presenting cells. Other fusion partners include the non-structural protein NS1 (hemagglutinin) of the influenza virus. Typically, the first 81 amino acids from the N-terminus are used, although other fragments containing T-helper epitopes can be used.

In another embodiment, the immunological fusion partner is a protein, known as LYTA, or a portion thereof (preferably a C-terminal region). LYTA originates from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alaninamidase, known as LYTA amidase (encoded by LytA gene, see Gene 43: 265-292, 1986). LYTA is an autolysin that specifically destroys certain bonds in the backbone of peptidoglycans. The C-terminal portion of the LYTA protein is responsible for affinity for choline or some choline analogues, such as DEAE. This property is used to develop plasmids that serve for the expression of C-LYTA in E. coli and are useful for the expression of fusion proteins. The purification of fusion proteins containing a C-LYTA fragment at the N-terminus is described (see Biotechnology 10: 795-798, 1992). In a preferred embodiment, a repeating portion of LYTA can be incorporated into the fusion protein. The repeating portion is located at the C-terminal portion starting at residue 178. A particularly preferred repeating portion extends to residues 188-305.

In a further embodiment of the invention, the adjuvant system described therein is used in the preparation of DNA-based vaccine compositions. Exemplary vaccines of this type contain DNA encoding one or more polypeptide antigens so that the antigen is formed in situ. DNA can be located in any of a number of delivery systems known in the art, including nucleic acid expression systems, bacterial and viral expression systems. Numerous gene delivery methods are known in the art, such as those described in Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998, and the references therein. Suitable nucleic acid expression systems contain the necessary DNA sequences for expression in a patient (such as a promoter and termination signal). Bacterial delivery systems include the introduction of bacteria (such as Calmette-Cheren bacilli) expressing the immunogenic portion of the polypeptide on the cell surface or secreting the epitope. In one preferred embodiment, the DNA is introduced using a viral expression system (e.g., vaccinia virus or another poxvirus, retrovirus or adenovirus), which usually involves the use of a non-pathogenic (defective) virus capable of replicating. Exemplary systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 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 No.4,777,127; GB 2,200,651; EP 0.345.242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502. 1993; Guzman et al., Circulation 88, 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993. Methods for incorporating DNA into such expression systems are well known in the art.

Alternatively, the DNA may be naked, as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993, and in Cohen, Science 259: 1691-1692, 1993. Absorption of naked DNA can be increased by applying DNA to biodegradable beads that are efficiently transported into cells. It should be borne in mind that the vaccine, if desired, can include both a polynucleotide and a polypeptide component.

Moreover, it is obvious that the vaccine may contain pharmaceutically acceptable salts of the desired polynucleotide, polypeptide and / or carbohydrate antigens. For example, such salts can be obtained 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 salts and magnesium).

The adjuvant system of the present invention exhibits strong adjuvant effects when administered in a wide range of doses and a wide range of proportions.

The amount of antigen in each dose of the vaccine is usually selected as the amount that causes protective immunity without significant adverse side effects in typical vaccines. This amount varies depending on which specific immunogen is used and how it is presented. In general, each dose should contain 1-1000 μg of protein, most often 2-100 μg, preferably 5-50 μg. Of course, the dose administered may depend on the age, weight, type of concomitant treatment, if any, and the nature of the antigen administered.

The immunogenic activity of a given amount of the vaccine composition of the present invention can be readily established, for example, by measuring the increase in antibody titer against the antigen used in the vaccine composition (Dalsgaard K. Acta Veterinia Scandinavica 69: 1-40, 1978). Another common method involves intradermally administering various doses of the vaccine composition to CD-1 mice, and then taking serum samples from these mice and testing for antibodies against the immunogen, for example, by ELISA. These and other similar approaches are known to those skilled in the art.

The antigen can be obtained and / or isolated from virtually any desired source depending on the infectious disease, autoimmune disease, condition, cancer, pathogen or disease to be treated with this vaccine composition. As an example: antigens can come from viral sources such as influenza virus, feline leukemia virus, feline immunodeficiency virus, human HIV-1, HIV-2, herpes simplex virus type 2, human cytomegalovirus, hepatitis A, B, C or E virus , respiratory syncytial virus, human papillomavirus, rabies, measles or foot and mouth disease viruses. Exemplary antigens can also come from bacterial sources such as anthrax, diphtheria, Lyme disease, malaria, tuberculosis, leishmaniasis, T.cruzi, Ehrlichia, Candida, etc., or protozoa such as Babesia bovis or Plasmodium. Typically, antigens consist of natural or synthetic amino acids, for example, in the form of peptides, polypeptides or proteins, and can consist of polysaccharides or be mixtures thereof. Exemplary antigens can be isolated from natural sources, synthesized by solid-phase synthesis, or obtained by recombinant DNA methods.

In another embodiment, tumor antigens are used in the vaccine compositions of the present invention for the prevention and / or treatment of cancer. Cancer cells often carry specific antigens on their surface, such as shortened epidermal growth factor, folate binding protein, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate specific membrane antigen, HER2-neu, which are candidates for use in therapeutic anti-cancer vaccines. Since tumor antigens are normal or related to normal components of the body, the immune system often cannot develop an effective immune response against such antigens and destroy cancer cells. To obtain such an answer, the adjuvant systems of the present invention can be used. In this case, exogenous proteins can enter the processing pathway of endogenous antigens, which leads to the production of cytolytic or cytotoxic T cells (CTK). This adjuvant effect promotes the formation of antigen-specific CTKs, which search for and destroy those cancer cells that carry the tumor antigen used for immunization on their surface. Representative types of cancer with which this approach can be applied include cancer of the prostate, colon, breast, ovary, pancreas, brain, head and neck, melanoma, leukemia, lymphoma, etc.

In a further embodiment of the invention, the adjuvant system of the present invention can be used alone, that is, without simultaneous administration of antigen, to stimulate the immune system in the treatment of chronic infectious diseases, especially in patients with immune disorders. Illustrative examples of infectious diseases in which this approach can be used for treatment or prophylaxis can be found in U.S. Patent No.5,508,310. Stimulating the immune system in this way can also be useful as a preventative measure to limit the risk of nosocomial and / or postoperative infections.

In another embodiment, the antigen in the vaccine composition is not a foreign antigen, but rather represents its own antigen, that is, the vaccine composition is directed against an autoimmune disease such as type I diabetes, common organ-specific autoimmune diseases, neurological diseases, rheumatic diseases, psoriasis, collagenosis , autoimmune cytopenia and other autoimmune diseases. Such common organ-specific autoimmune diseases include thyroiditis (Graves' disease and Hashimoto's disease), gastritis, adrenalitis (Addison's disease), oophoritis, primary biliary cirrhosis of the liver, myasthenia gravis, gonad failure, hypoparathyroidism, alopecia, malabsorption syndrome, malignant, malignant caused by the formation of antibodies to receptors, and vitiligo. Such neurological diseases include schizophrenia, Alzheimer's disease, depression, hypopituitarism, diabetes insipidus, sicca syndrome and multiple sclerosis. Such rheumatic diseases / collagenoses include rheumatoid arthritis, systemic lupus erythematosus or lupus erythematosus, scleroderma, polymyositis, chronic intestinal inflammation, dermatomyositis, ulcerative colitis, Crohn’s disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, common psoriatic dermatitis. Other autoimmune diseases include autoimmune uveoretinitis, glomerulonephritis, post-infarction postcardiotomy syndrome, pulmonary hemosiderosis, amyloidosis, sarcoidosis, aphthous stomatitis and other immunological diseases that we cited and are known in the relevant fields.

Although any suitable carriers known in the art may be used in the vaccine compositions of this invention, the type of carrier usually varies depending on the desired mode of use. The compositions of the present invention can be formulated for any suitable route of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, intracutaneous, subcutaneous or intramuscular administration. For parenteral administration such as subcutaneous injection, the carrier often includes water, saline, alcohol, fat, wax or a buffer. When administered orally, the above carriers are often used or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate are used. Biodegradable microspheres (e.g., from polylactate-polyglycolate) can also be used as carriers for the compositions of this 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; 5,820,883; 5,853,763; 5,814,344 and 5,942,252, the contents of which are included in the present invention by reference in its entirety. Carrier systems based on a modified hepatitis B virus core protein are also suitable, such as those described in WO 99/40934 and the references therein, all of which are incorporated herein by reference. A carrier containing particulate protein complexes of the type described in U.S. can also be used. Patent No.5,928,647, the contents of which are incorporated by reference in their entirety, which are capable of inducing a class I restriction response of cytotoxic T lymphocytes in the host.

In one illustrative embodiment, the vaccine composition is applied to the mucous membranes, in particular the oral cavity, preferably under the tongue, to generate an immune response. Application in the oral cavity may be preferred in many cases instead of traditional parenteral administration due to the ease and convenience of non-invasive methods of administration. Moreover, this approach also provides a method for generating mucosal immunity, which is difficult to achieve with traditional parenteral administration and which can provide protection against airborne pathogens and / or allergens. An additional advantage of the use in the oral cavity is that with sublingual administration of the vaccine, patient compliance with the prescription is improved, especially when used in pediatrics, or when traditionally require numerous injections over a long time, as in the treatment of allergies by desensitization.

Vaccine compositions may also contain buffers (e.g., saline with neutral buffer, saline with phosphate buffer or phosphate buffer without saline), carbohydrates (e.g. glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostatics, chelators such as EDTA, glutathione, adjuvants (e.g. aluminum hydroxide), soluble substances that support isotonicity, hypotension, or mild hypertonicity with respect to the blood of the recipient, suspending agents, thicken ate and / or preservatives. Alternatively, the compositions of the present invention can be prepared in lyophilized form. Compositions can also be enclosed in liposomes using well-known technology.

So, in one embodiment, the vaccine compositions are aqueous formulations containing 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 detergent, e.g., a phospholipid detergent. Representative examples of phospholipids include: diacylphosphatidylglycerols, such as dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG); diacylphosphatidylcholines such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids such as dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE).

Typically, the detergent: adjuvant molar ratio in the aqueous composition is from 10: 1 to 1:10, most often from 5: 1 to 1: 5, however, any effective amount of detergent in the aqueous composition that is best suited for specific applications can be used. goals.

In another embodiment, the composition is an emulsion, for example a water-in-oil or oil-in-water emulsion. Such emulsions are generally well known in the art.

The adjuvant system of the present invention can be used as a single adjuvant system or, alternatively, can be used in conjunction with other adjuvants or immunoeffectors. For example, such adjuvants may include oil adjuvants (e.g. Freund's complete and incomplete adjuvants), liposomes, mineral salts (e.g. AlK (SO ₄ ) ₂ , AlNa (SO ₄ ) ₂ , AlNH ₄ (SO ₄ ) ₂ , silica , alum, Al (OH) ₃ , Ca ₃ (PO ₄ ) ₂ , kaolin and carbon), polynucleotides (for example, polyIC and polyAU acids), polymers (for example, nonionic block copolymers, polyphosphazenes, cyanoacrylates, DL-lactide copolymers and glycoside), among other things, and some natural substances (for example, lipid A and its derivatives, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordete lla pertussis and in representatives of the genus Brucella), bovine serum albumin, diphtheria toxoid, tetanus toxoid, edetin, mollusk hemocyanin saucer, pseudomonas toxin A, choleragenoid, cholera toxin, pertussis toxin, viral proteins and such eukaryotic proteins as interferons or interferons tumor necrosis. Such proteins may be obtained from natural or recombinant sources in accordance with methods well known in the art. Upon receipt from recombinant sources, the adjuvant may contain a protein fragment, including at least the immunostimulating part of the molecule. Other known immunostimulatory macromolecules that can be used in the practice of the invention include, but are not limited to, polysaccharides, tRNAs, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (of relatively high molecular weight) 4,4'-diamino 3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (see Sela M., Science 166: 1365-1374, 1969) or glycolipids, lipids or carbohydrates.

In one embodiment, the adjuvant system is preferably intended to generate an immune response predominantly of the Th1 type. High levels of Th1 cytokines (e.g. IFN-γ, TNFα, IL-2, and IL-12) contribute to the development of cellular immune responses to the introduced antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) contribute to the development of humoral immune responses. After applying the vaccine in accordance with the invention, the patient has an immune response, including responses such as Th1 and Th2. In a preferred embodiment, in which the response is predominantly of the Th1 type, the level of Th1 type cytokines will increase more than the level of Th2 type cytokines. The levels of these cytokines can be easily determined by standard methods. See a review of cytokine families: Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

For example, additional adjuvants for generating a response primarily of the Th1 type include, for example, combinations of monophosphoryl lipid A of type 3-O-deacylated monophosphoryl lipid A (3D-MPL) and an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, WA; see U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is not methylated) also elicit predominantly Th1 responses. Such oligonucleotides are well known and described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352, 1996. Other representative adjuvants that can be included in vaccine compositions include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, USA) , ISCOMS (CSL), MF-59 (Chiron), Detox ™ Adjuvant (Corixa, Hamilton, MT).

The compositions described by us can be used in sustained release formulations (i.e., formulations such as capsules, sponges or gels, for example, consisting of polysaccharides), which provide a slow release of the substance after administration. Such formulations, in General, can be obtained using well-known technology (for example, see Coombes et al., Vaccine 14: 1429-1438, 1996) and apply, for example, by oral, rectal administration or implantation under the skin or another place. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and / or enclosed in a reservoir surrounded by a membrane that controls the rate of release. Carriers for such formulations are biocompatible and can also be biodegradable; preferably, the formulation provides a relatively constant level of release of the active ingredient. Such carriers include microparticles of copolymers of lactide and glycolide, polyacrylate, latex, starch, cellulose, dextran, etc. Other sustained-release carriers include supramolecular bio-vectors consisting of an internal non-liquid hydrophilic layer (e.g., cross-linked polysaccharide or oligos , an outer layer containing an amphiphilic compound, for example, a phospholipid (see US Patent No. 5,151,254 and PCT applications WO 94/20078, WO 94/23701 and WO 96/06638). The amount of active compound contained in a sustained release formulation varies depending on the implantation site, rate and expected duration of release, and the nature of the disease to be treated or prevented.

You can use any known means of delivery in pharmaceutical compositions and vaccines to facilitate the production of an antigen-specific immune response directed to cells. Delivery vehicles include antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and other cells that can serve as effective APCs. Such cells, although not necessarily, can undergo genetic modification to increase the ability to present an antigen, enhance activation and / or maintain a T-cell response, per se target action and / or immunological compatibility with the recipient (i.e., HLA haplotype match). In general, APC can be isolated from a number of biological fluid media and organs, including tumors and surrounding tissues, and they can be autologous, allogeneic, syngeneic, or xenogenic cells.

In some preferred embodiments of the present invention, dendritic cells or their precursors are used as antigen-presenting cells. Dendritic cells are very potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as physiological adjuvants for the development of prophylactic or therapeutic immunity against cancer (see Timmerman and Levy, Ann. Rev. Med. 50 : 507-529, 1999). In general, dendritic cells can be identified by their typical shape (stellate in situ, with noticeable cytoplasmic dendritic processes visible in vitro), their ability to capture, process and present antigens with high efficiency and their ability to activate “naive” T- cells. Of course, dendritic cells can be modified in such a way that they express specific surface receptors or ligands that are not normally found on dendritic cells in vivo or ex vivo, and such modified cells are provided by the present invention. As an alternative to dendritic cells in a vaccine, secreted vesicles (exosomes) from antigen-loaded dendritic cells can be used (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

Dendritic cells and their precursors can be obtained from peripheral blood, bone marrow, cells that infiltrate tumors or cells that infiltrate the surrounding tumor tissue, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or liquid medium. For example, dendritic cells can be obtained by ex vivo differentiation by adding a set of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to cultures of monocytes derived from peripheral blood. Alternatively, CD34-positive cells obtained from peripheral blood, cord blood or bone marrow can be differentiated into dendritic cells by adding various combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand to the culture medium and / or other compounds that cause differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are conveniently divided into "immature" and "mature" cells, which makes it easy to distinguish between two well-studied phenotypes. However, one should not assume that this nomenclature excludes all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with a high ability to capture and process antigens, which correlates with a high level of expression of the Fcγ receptor and mannose receptor. A mature phenotype is usually characterized by a decrease in the expression of these markers and a high level of expression of T-cells responsible for activation of surface molecules such as MHC class I and class II, adhesion molecules (e.g. CD54 and CD11) and costimulatory molecules (e.g. CD40, CD80 , CD86 and 4-1 BB).

Typically, the APC can be transfected with a polynucleotide encoding an antigen polypeptide (or part of it or another variant) so that the antigen polypeptide or its immunogenic part is expressed on the cell surface. Such transfection can be performed ex vivo, and then use a composition or vaccine containing transfected cells, and the adjuvants described by us for therapeutic purposes. Alternatively, a gene delivery vehicle directed to dendritic or other antigen-presenting cells can be introduced to the patient, resulting in in vivo transfection. Transfection of dendritic cells in vivo and ex vivo, for example, is usually carried out by any methods known in this field, such as those described in WO 97/24447, or by the "gene gun" method described in Mahvi et al., Immunology and Cell Biology 75: 456-460, 1997. Dendritic cell antigen loading can be accomplished by incubating the dendritic cells or their precursors with a polypeptide, DNA (naked or in plasmid vector) or RNA antigen, or with recombinant bacteria or viruses expressing antigen (eg. , vaccinia virus-based vectors, bird smallpox, adenovirus or lentivirus). Prior to loading, the polypeptide can be covalently conjugated to an immunological partner that facilitates the promotion of T cells (e.g., with a carrier molecule). Alternatively, dendritic cells can be incubated with an unconjugated immunological partner, alone or in the presence of a polypeptide.

Treatment for nitric oxide related diseases

One aspect of the present invention provides methods for treating diseases or conditions mediated by nitric oxide, in particular ischemia and reperfusion injury. These methods include administering to a patient in need of such treatment an effective amount of a compound of the present invention. Inductors of iNOS gene transcription and iNOS protein synthesis are believed to contribute to inflammation and therefore are somewhat “toxic” or poorly tolerated by animals and humans. Endotoxin (LPS) and inflammatory cytokines such as IL-1, TNF-α and IFN-γ are known as iNOS inducers. All of them are essentially toxic and can cause systemic inflammatory reactions, adult respiratory distress syndrome, multiple organ failure and cardiovascular failure when administered to animals.

Studies of the protective effect of the MPL immunostimulant on the heart have shown that the induction of nitric oxide synthases (iNOS) is important in the delayed cardioprotective effect of this compound. In addition, the nitric oxide (NO) signaling system, presumably via constitutive NOS pools, plays an important role in the acute cardioprotective effect of this compound. Due to the residual endotoxin-like activity of the MPL immunostimulant, it is not surprising that this compound may be capable of inducing a nitric oxide signaling system. Moreover, it is assumed that the nitric oxide signaling system is the way through which ischemic preconditioning has a protective effect on the heart. This observation, combined with the fact that nitric oxide donors have a protective effect on the heart, further confirms that the NOS / NO pathway mediates the cardioprotective effect of the MPL immunostimulant.

The compounds of the present invention are useful in methods for treating diseases or conditions modulated or ameliorated by nitric oxide, in particular ischemia and reperfusion injuries (see cardioprotective properties of aminoalkyl glucosaminide phosphates and methods for analyzing cardioprotective properties in US patent application No. 09/808669 of March 14, 2001) .

EXAMPLES

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

Example 1. Obtaining the triethylammonium salt of N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamino] -3- O - [(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside (triethylammonium salt of the compound of formula II)

(1a) Into a solution of 2-deoxy-4-O-diphenylphosphopo-3-O - [(R) -3-tetradecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2 - (2,2,2-trichloroethoxycarbonylamino) -β-D-glucopyranosyl bromide (1.05 g, 0.81 mmol) in anhydrous 1,2-dichloroethane (10 ml), a molecular sieve of 4 (0.5 g) was added, anhydrous CaSO ₄ (2.2 g, 16 mmol) and N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinemethanol (0.40 g, 0.75 mmol). This mixture was stirred for 1 hour at room temperature, treated with Hg (CN) ₂ (1.02 g, 4.05 mmol) and refluxed for 16 hours in the dark. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered. The filtrate was washed with a 1N aqueous KI solution, dried with Na ₂ SO ₄ and concentrated. Flash chromatography on silica gel (gradient elution, 15 → 20% EtOAc / Hexane) gave 0.605 g (43%) of N - [(R) -3-tetradecanoyloxytethecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4 -O-diphenylphosphono-3-O - [(R) -3-tetradecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2-trichloroethoxycarbonylamino) β-D-glucopyranoside as an amorphous substance.

(1b) A solution of the compound obtained according to the preceding paragraph 1a (0.50 g, 0.29 mmol) in AcOH (10 ml) at 60 ° C was treated with zinc dust (0.98 g, 15 mmol) in three equal portions over 1 hours. The reaction mixture was cooled, sonicated, filtered through a pad of celite and concentrated. The resulting precipitate was partitioned between CH ₂ Cl ₂ and saturated aqueous NaHCO ₃ and the layers were separated. The organic layer was dried with Na ₂ SO ₄ and concentrated. A solution of the resulting crude amino alcohol and (R) -3-tetradecanoyloxytetradecanoic acid (0.155 g, 0.34 mmol) in CH ₃ Cl _{2 was} stirred together with a molecular sieve powder of 4 A (0.25 g) for 0.5 h, and then treated with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0.11 g, 0.44 mmol). The resulting mixture was stirred at room temperature for 8 hours, filtered through celite and concentrated. Flash chromatography on silica gel using 50% EtOAc / Hexane afforded 0.355 g (68%) of N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphono 2 - [(R) -3-tetradecanoyloxytetradecanoylamino] -3-O - [(R) -3-tetradecanoyloxytetradecanoyl] -β-D-glucopyranoside as a colorless syrup.

(1 s) A solution of the compound (0.300 g, 0.166 mmol) obtained in the previous p. 1b in a mixture of AcOH (1 ml) and tetrahydrofuran (9 ml) was hydrogenated in the presence of PtO ₂ (0.15 g) at room temperature and pressure 70 pounds per square inch for 18 hours. The reaction mixture was diluted with 2: 1 CHCl ₃ -MeOH (50 ml) and briefly sonicated. The catalyst was collected, washed with a 2: 1 mixture of CHCl ₃ -MeOH, and the combined filtrate and washings were concentrated. Flash chromatography on silica gel using CHCl ₃ -MeOH-H ₂ O-Et ₃ N (90: 10: 0.5: 0.5) gave a partially purified product, which was dissolved in an ice-cold 2: 1 mixture of CHCl ₃ -MeOH (30 ml) and washed with ice-cold 0.1 N HCl (12 ml). The organic phase was filtered and lyophilized from a 2% solution of Et ₃ N (5 ml pyrogen-free) to give 0.228 g (79%) of the triethylammonium salt of N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl- 2-deoxy-4-O-phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamino] -3-O - [(R) -3-tetradecanoyloxytethecanoyl] -β-D-glucopyranoside as a colorless powder. Mp 67-70 ° C, IR spectrum (film): 3306, 2955, 2923, 2853, 1736, 1732, 1644, 1548, 1466, 1378, 1245, 1177, 1110, 1053, 844 cm ^-1 ; ¹ H-NMR (CDCl ₃ -CD ₃ OD): δ 0.88 (m, 18H), 1.0-1.205 (mH), 2.20-2.70 (m, 12H), 3.06 (q , 6H, J = 7.2 Hz), 3.3-3.25 (mH), 4.52 (d, 1H, J = 8 Hz), 5.05-5.28 (m, 4H), 7 44 (d, 1H, J = 9 Hz); ¹³ C-NMR (CDCl ₃ ): δ 173.3, 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.

Analysis: theoretically for C ₁₀₁ H ₁₉₄ N ₃ O ₁₇ P · H ₂ O: C = 68.47, H = 11.15, N = 2.37, P = 1.75; in fact: C = 68.79, H = 11.00, N = 2.24, P = 1.97.

Example 2. Obtaining the triethylammonium salt 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 of compound III)

(2a) Into a solution of 2-deoxy-4-O-diphenylphosphono-3-O - [(R) -3-dodecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2 - (2,2,2-trichloroethoxycarbonylamino) -α-D-glucopyranosyl bromide (1.60 g, 1.27 mmol) in anhydrous 1,2-dichloroethane (3.2 ml) was added a 4 A molecular sieve (0.6 d) anhydrous CaSO ₄ (1.0 g, 7.3 mmol) and N - [(R) -3-dodecanoyloxytetradecanoyl] - (S) -2-pyrrolidinemethanol (0.58 g, 1.14 mmol). This mixture was stirred for 1 hour at room temperature, treated with Hg (CN) ₂ (0.58 g, 2.3 mmol) and refluxed for 6 hours in the dark. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered through a pad of celite. The filtrate was washed with a 1 N suitable KI solution, dried with Na ₂ SO ₄ and concentrated. Flash chromatography on silica gel (gradient elution, 25 → 35% EtOAc / Hexane) gave 1.72 g (82%) of N - [(R) -3-dodecanoyloxytethecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy -4-O-diphenylphosphono-3-O - [(R) -3-dodecanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2- trichloroethoxycarbonylamino) -β-D-glucopyranoside as a colorless oil.

(2b) A solution of the compound obtained according to the previous item 2a (1.58 g, 0.806 mmol) in AcOH (40 ml) at 60 ° C was treated with zinc dust (2.6 g, 40 mmol) in three equal portions for 1 hour. The reaction mixture was cooled, sonicated, filtered through a pad of celite and concentrated. The resulting precipitate was partitioned between EtOAc and saturated aqueous NaHCO ₃ and the layers were separated. The organic layer was washed with brine, dried with Na ₂ SO ₄ and concentrated to give 1.3 g of a white substance. A solution of the resulting crude amino alcohol and (R) -3-dodecanoyloxytetradecanoic acid (0.45 g, 1.05 mmol) in CH ₂ Cl ₂ (20 ml) was treated with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0, 30 g, 1.21 mmol). The resulting mixture was stirred at room temperature for 18 hours and concentrated. Flash chromatography on silica gel using 40 → 50% EtOAc / hexane afforded 0.89 g (56%) of N - [(R) -3-dodecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4- O-diphenylphosphono-2 - [(R) -3-dodecanoyloxytetradecanoylamino] -3-O - [(R) -3-dodecanoyloxytethecanoyl] -β-D-glucopyranoside as a white foam.

(2 s) A solution of the compound obtained in the previous item 2b (0.75 g, 0.44 mmol) in a mixture of AcOH (4.5 ml) and tetrahydrofuran (45 ml) was hydrogenated in the presence of PtO ₂ (0.45 g ) at room temperature and a pressure of 70 psi for 18 hours. The reaction mixture was diluted with 2: 1 CHCl ₃ -MeOH (35 ml) and briefly sonicated. The catalyst was collected, washed with a 2: 1 mixture of CHCl ₃ -MeOH, and the combined filtrate and washings were concentrated. Flash chromatography on silica gel using CHCl _3- MeOH-H ₂ O-Et3N (gradient elution: 96: 4: 0.3: 0.3 → 90: 10: 0.5: 0.5) gave a partially purified product (0.51 g), which was dissolved in an ice-cold 2: 1 mixture of CHCl ₃ -MeOH (50 ml) and washed with an ice-cold solution of 0.1 N HCl (20 ml). The organic phase was filtered and concentrated. The resulting white wax was lyophilized from a 2% solution of Et ₃ N (70 ml free of plrogen) to obtain 0.54 g (78%) of the triethylammonium salt of N - [(R) -3-dodecanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl -2-deoxy-4-O-phosphono-2 - [(R) -3-dodecanoyloxy) adecanoylamino] -3-O - [(R) -3-dodecanoyloxytetracanoyl] -β-D-glucopyranoside as a white powder. Mp 146-151 ° C, 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, 722 cm ^-1 ; ¹ H-NMR (CDCl ₃ -CD ₃ OD): δ 0.88 (m, 18 N), 1.0-2.10 (mH), 2.20-2.75 (m, 12H), 3, 04 (q, 6H, J = 7.2 Hz), 3.3-4.3 (mH), 4.45 (d, 1H, J = 8.5 Hz), 5.0-5.28 (m , 4H); ¹³ C-NMR (CDCl ₃ ): δ 173.9, 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 mass spectroscopy: theoretically for [M + Na] ⁺ 1590, 1900, actually 1590, 1866.

Analysis: theoretically for C ₉₅ H ₁₈₂ O ₁₇ P · 3H ₂ O: C = 66.20, H = 10.99, N = 2.44; in fact: C = 66.36, H = 10.69, N = 2.15.

Example 3. Obtaining the triethylammonium salt 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 of compound IV)

(3a) Into a solution of 2-deoxy-4-O-diphenylphosphono-3-O - [(R) -3-decanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2 - (2,2,2-trichloroethoxycarbonylamino) -α-D-glucopyranosyl bromide (1.70 g, 1.38 mmol) in anhydrous 1,2-dichloroethane (3.5 ml) was added a molecular sieve of 4 (0.6 g ), anhydrous CaSO ₄ (1.2 g, 8.8 mmol) and N - [(R) -3-decanoyloxytetradecanoyl] - (8) -2-pyrrolidinemethanol (0.60 g, 1.24 mmol). This mixture was stirred for 1 hour at room temperature, treated with Hg (CN) ₂ (0.63 g, 2.5 mmol) and refluxed for 6 hours in the dark. The reaction mixture was cooled, diluted with CH ₂ Cl ₂ and filtered through a pad of celite. The filtrate was washed with a 1 N aqueous KI solution, dried with Na ₂ SO ₄ and concentrated. Flash chromatography on silica gel (gradient elution, 25 → 40% EtOAc / hexane) gave 1.82 g (80%) of N - [(R) -3-decanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy -4-O-diphenylphosphono-3-O - [(R) -3-decanoyloxytetradecanoyl] -6-O- (2,2,2-trichloro-1,1-dimethylethoxycarbonyl) -2- (2,2,2- trichloroethoxycarbonylamino) -β-D-glucopyranoside as a colorless oil.

(3b) A solution of the compound obtained in the preceding paragraph 3a (1.67 g, 1.02 mmol) in AcOH (50 ml) at 60 ° C was treated with zinc dust (3.33 g, 51 mmol) in three equal portions over 1 hours. The reaction mixture was cooled, sonicated, filtered through a pad of celite and concentrated. The resulting precipitate was partitioned between EtOAc and saturated aqueous NaHCO ₃ and the layers were separated. The organic layer was washed with brine, dried with Na ₂ SO ₄ and concentrated to give 1.25 g of a white substance. A solution of the resulting crude amino alcohol and (R) -3-decanoyloxytetradecanoic acid (0.53 g, 1.33 mmol) in CH ₂ Cl ₂ (20 ml) was treated with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0, 38 g, 1.53 mmol). The resulting mixture was stirred at room temperature for 18 hours and concentrated. Flash chromatography on silica gel using 40 → 50% EtOAc / hexane afforded 1.23 g (74%) of N - [(R) -3-decanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl-2-deoxy-4- O-diphenylphosphono-2 - [(R) -3-decanoyloxytetradecanoylamino] -3-O - [(R) -3-decanoyloxytethecanoyl] -β-D-glucopyranoside as a white foam.

(3 s) A solution of the compound obtained in the previous item 3b (1.07 g, 0.654 mmol) in a mixture of AcOH (6.5 ml) and tetrahydrofuran (65 ml) was hydrogenated in the presence of PtO ₂ (0.66 g) at room temperature and a pressure of 70 psi for 18 hours. The reaction mixture was diluted with a 2: 1 mixture of CHCl ₃ -MeOH (50 ml) and briefly sonicated. The catalyst was collected, washed with a 2: 1 mixture of CHCl ₃ -MeOH, and the combined filtrate and washings were concentrated. A waxy solid was obtained, which was lyophilized from a 2% solution of triethylamine to obtain ~ 1 g of crude triethylamine salt as a white powder. Flash chromatography on silica gel using CHCl _3- MeOH-H ₂ O-Et ₃ N (gradient elution: 96: 4: 0.3: 0.3 → 88: 12: 1: 0.6) gave a partially purified product (0.84 g), which was dissolved in an ice-cold 2: 1 mixture of CHCl ₃ -MeOH (168 ml) and washed with an ice-cold solution of 0.1 N HCl (67 ml). The organic phase was filtered and concentrated. The resulting white wax was lyophilized from a 2% aqueous solution of Et3N (70 ml pyrogen-free) to give 0.79 g (79%) of the triethylammonium salt of N - [(R) -3-decanoyloxytetradecanoyl] - (S) -2-pyrrolidinylmethyl- 2-deoxy-4-O-phosphono-2 - [(R) -3-decanoyloxytetradecanoylamino] -3-O - [(R) -3-decanoyloxytethecanoyl] -β-D-glucopyranoside in the form of a white powder. Mp 121-122 ° C, 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, 721 cm ^-1 ;

¹ H-NMR (CDCl ₃ -CD ₃ OD): δ 0.88 (m, 18H), 1.0-2.05 (mH), 2.20-2.75 (m, 12H), 3.04 (q, 6H, J = 7.2 Hz), 3.3-4.3 (mH), 4.45 (d, 1H, J = 8.5 Hz), 5.0-5.28 (m, 4H) ; ¹³ C-NMR (CDCl ₃ ): δ 173.7, 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 mass spectroscopy: theoretically for [M + Na] ⁺ 1506, 0961, in fact 1506, 1008.

Analysis: theoretically for C ₈₉ H ₁₇₀ N ₃ O ₁₇ P: C = 67.43, H = 10.81, N = 2.65; in fact: C = 67.26, H = 10.85, N = 2.47.

EXAMPLES 4-8

The main objective of Examples 4-8 is to determine whether the compound of formula II obtained in Example 1 (in the form of a triethylammonium salt) (hereinafter referred to as "compound II") will exhibit minimal pyrogenicity and have adjuvant activity when introduced into the vaccine together with antigens.

Example 4. Adjuvant activity against HBsAg (hepatitis B surface antigen)

Groups of BALB / c mice (Jackson Laboratories, Bar Harbor, Maine) aged 6-8 weeks were injected subcutaneously with 2 μg HBsAg (Laboratorio Pablo Cassara) ± 20 μg of adjuvant (MPL immunostimulant or compound II) on day 0 and day 21. Vaccines received by mixing adjuvant containing formulations in triethanolamine (TEOA) with recombinant HBsAg. Anti-HBsAg antibody titers were determined by ELISA in pooled sera (5 mice per group) obtained 21 days after secondary vaccination (Table 1). Immune controls were not vaccinated.

In the serum of mice treated with compound II, the titers of antibodies to HBsAg were significantly higher than in the serum of control mice treated with antigen alone (Table 1). Particularly noticeable was an increase in titers for IgG2a and IgG2b isotypes. These titers were equivalent to those in the control groups receiving the MPL immunostimulant.

Table 1.
Comparison of low pyrogenic adjuvants with HBsAg Group Pyrogenicity ^a Serum titers IgG IgG1 IgG2a IgG2b Non-immune - <100 <100 <100 <100 TheoA (r-rite) n / t 51200 102400 25600 1600 MPL to TEOA 2-3 409600 204800 204800 51200 Connection II in TEOA 0.3 409600 204800 204800 51200 ^a Pyrogenicity data represent the total temperature increase in ° C in 3 rabbits after an intravenous dose of 10 μg / kg. In the pyrogenicity assay, the compounds were dissolved in 10% EtOH / water for injection (USP, USP) at a concentration of 100 μg / ml, and then diluted with 5% glucose in water. N / m means that the compound was not tested.

Example 5. Adjuvant activity against hemagglutinin protein in FluZone vaccine against influenza

Groups of BALB / c mice (Jackson Laboratories, Bar Harbor, Maine) aged 6-8 weeks were subcutaneously injected with 0.2 μg hemagglutinin protein in FluZone influenza vaccine (Connaught Laboratories) ± 20 μg adjuvant (MPL immunostimulant or compound II) per day 0 and day 14. Anti-FluZone antibody titers were determined by FluZone ELISA in pooled sera from 5 mice obtained 14 days after secondary vaccination (Table 2). Immune controls were not vaccinated. Dilutions of sera from test groups were used, starting from 1: 1600.

The results were close to the results of the previous example. Again, in compound II-treated patients, antibody titers were significantly higher than in the serum of control mice treated with antigen alone (Table 2). The increase in titers was also expressed in an increase in IgG2a and IgG2b isotypes. These titers were equivalent to those in the control groups receiving the MPL immunostimulant.

Table 2.
Comparison of low-pyrogenic adjuvants with flu vaccine Group Pyrogenicity ^a Serum titers IgG IgGI IgG2a IgG2b Non-immune - <100 <100 <100 <100 TheoA (r-rite) n / t 12800 51200 1600 <1600 MPL to TEOA 2-3 102400 102400 25600 12800 Connection II in TEOA 0.3 51,200 102400 25600 6400 ^a Pyrogenicity data represent the total temperature increase in ° C in 3 rabbits after an intravenous dose of 10 μg / kg. In a pyrogenicity assay, the compounds were dissolved in 10% EtOH / water for injection (USP) at a concentration of 100 μg / ml, and then diluted with 5% glucose in water. N / t means that the compound was not tested.

Example 6. Adjuvant activity against HBsAg

Groups of BALB / c mice were injected subcutaneously with 2.0 μg HBsAg (Rhein Americana and Rhein Biotech) ± 25 μg adjuvant (MPL immunostimulant or compound II) on day O and day 21. Antibody titers of IgGI and IgG2a isotypes were determined by ELISA in pooled sera obtained 21 days after secondary vaccination (table 3). Immune controls were not vaccinated. In this experiment, compound II caused an increase in titers compared with the control group receiving antigen in phosphate buffered saline (PBS). RC-553 caused an increase in titers equally with the positive controls receiving the MPL immunostimulant (Table 3).

Table 3.
Comparison of low pyrogenic adjuvants with HBsAg Group Pyrogenicity ^a Serum titers IgGI IgG2a Non-immune - <100 <100 PBS (control) n / t 64000 4000 MPL to TEOA 2-3 128,000 1,024,000 Connection II in TEOA 0.3 32000 2048000 ^a Pyrogenicity data represent the total temperature increase in ° C in 3 rabbits after an intravenous dose of 10 μg / kg. In a pyrogenicity assay, the compounds were dissolved in 10% EtOH / water for injection (USP) at a concentration of 100 μg / ml, and then diluted with 5% glucose in water. N / t means that the compound was not tested.

Example 7. Compound II increases the activity of CTLs of immunized mice against HBsAg

Some mice from each group in Example 4 were also used as spleen cell donors in order to evaluate the activity of cytotoxic T lymphocytes (CTLs). Specific lysis induced by HBsAg was evaluated by the standard ^5l Cr release method over 4 hours (Moore et al. (1988) Cell 55: 777-785). Single cell suspensions were obtained from mouse spleens 9 days after vaccination. Spleen cells were treated with NH ₄ Cl in Tris buffer to remove red blood cells and resuspended to a concentration of 7.5 × 10 ⁶ cells / ml in RPMI medium with 10% calf serum with 5 mM HEPES, 4 mM L-glutamine, 0.05 mM 2-mercaptoethanol and antibiotics. A synthetic peptide representing the known epitope (IPQSLDSWWTSL) of CTLs undergoing L ^d- restriction by MHC class I at a final concentration of 75 nM was added to the cells. After incubation for 4 days, the cells were removed and CTL activity was determined. The specific death of ^5l Cr-labeled transfected P815S cells expressing an L-restricted epitope was measured. Target cells are transfected P815 (P815S) cells that express the CTL epitope undergoing L ^d- restriction. Nonspecific lysis was <10% with an effector: target ratio of 50: 1 relative to target cells of P815 (Table 4). In contrast to the antibody response, RC-553 caused a significant increase in the level of CTL activity compared to the control in the presence of antigen alone (Table 4).

Table 4.
Comparison of low pyrogenic adjuvants with HBsAg Group Pyrogenicity ^a Specific death in% (ratio effector: target) 50: 1 25: 1 12.5: 1 6.25: 1 Non-immune - 6 3 one 0 PBS (control) n / t 29th twenty eleven 7 MPL to TEOA 2-3 80 71 47 32 Connection II in TEOA 0.3 85 77 53 37 ^a Pyrogenicity data represent the total temperature increase in ° C in 3 rabbits after an intravenous dose of 10 μg / kg. In a pyrogenicity assay, the compounds were dissolved in 10% EtOH / water for injection (USP) at a concentration of 100 μg / ml, and then diluted with 5% glucose in water. N / t means that the compound was not tested.

Example 8. Induction of cytokines by compound II ex vivo

The effect of compound II on the production of TNF-α and IL-1β was measured under ex vivo conditions on human peripheral blood mononuclear cells. An MPL immunostimulant and compound II were prepared as aqueous solutions in 0.2% TEOA / water for injection.

Human whole blood was used to evaluate the ability of glycolipids (AGP) to induce pro-inflammatory cytokines. Human whole blood was collected in heparinized tubes, 0.45 ml were taken and mixed with 0.05 ml of saline with phosphate buffer (PBS, pH 7.4) containing glycolipid (i.e. test compounds). The tubes were incubated for 4 hours at 37 ° C on a shaker. Then the samples were diluted with 1.5 ml of sterile PBS and centrifuged. Supernatants were removed and analyzed for TNF-α and IL-1β in cells by ELISA sandwich using Quantikine kits from R&D Systems for immunoassay of human TNF-α and IL-1β.

Compound II at concentrations of 1, 5, and 10 μg / ml in the analysis did not cause the formation of TNF-α, which could be detected under the conditions of this method. On the contrary, a positive control - LPS effectively stimulated the secretion of TNF-a from cells at a concentration of 1 ng / ml. The MPL immunostimulant effectively induced TNF-α in a concentration range from 100 to 10,000 ng / ml.

Similarly, compound II (1, 5 and 10 μg / ml) did not cause a noticeable formation of IL-1β. To compare the effects of this compound, the level of IL-lp induced by the MPL immunostimulant was taken as 1, then the relative induction of cytokines by compound II was ≤0.05.

Discussion of Examples 4-8

The data from these experiments indicate that compound II is able to increase immunity to vaccine antigens. This compound increased serum titers to two different vaccine antigens - the flu antigen and the hepatitis surface antigen. Like the MPL immunostimulant, it caused a shift in the antibody profile from the response predominantly represented by the IgG1 isotype to the response with a high level of IgG2a antibodies. Along with an increase in antibody response, this compound was a good adjuvant in inducing CTL activity.

A remarkable feature of the results of this study is that compound II has an effect on the immune response without causing a noticeable formation of inflammatory cytokines TNF-a or IL-1β. These cytokines are produced by cells of the innate immunity system as a reaction to bacterial cell wall products, including lipid A. Since this compound has structural similarities with lipid A, it can be assumed that it will also stimulate the formation of TNF-α or IL-1β, as many AGF molecules do. As inflammatory cytokines, TNF-α and IL-1β trigger the cascade of other cytokines responsible for phagocyte activation and the mobilization of specific immunity. Initially, IL-1 was called endogenous pyrogen, since it causes an increase in temperature. Thus, the absence of a noticeable level of IL-1 after administration of compound II coincides with the apparent absence of an increase in temperature when tested for pyrogenicity in rabbits.

It remains possible that in these experiments, this compound actually stimulates the secretion of TNF-α and IL-1β at a level high enough to activate specific immunity, but too low to be detected by ex vivo analysis for cytokines. Another possibility is that this compound does not stimulate the formation of TNF-α and IL-1β, but other cytokines, which cause a specific immune response to co-administered vaccine antigens. Apparently, IFN-y is formed. This cytokine is believed to be responsible for the induction of isotype shift towards antibodies of the IgG2a subclass, and is also an activator of CTL responses mediated by TH-1. Thus, both an increase in IgG2a titer and the appearance of active CTL populations reflect the formation of IFN-γ.

Example 9. Stimulation of inducible nitric oxide synthase (iNOS) compound II

This example illustrates the effects of various glycolipids on iNOS induction in macrophages of J774 mice. The macrophage cell line of J774 mice is susceptible to in vitro priming of IFN-γ and is very sensitive to subsequent LPS stimulation of iNOS upregulation, which is measured by ELISA using standard Graiss reagent. In this method, J774 cells at a concentration of 1 × 10 ⁶ cells / ml are inoculated into a 30 ml flask and incubated for 16-24 hours with the addition of IFN-γ of 100 units / ml. The cells are then harvested, washed, resuspended and introduced at 2 × 10 ⁵ cells / well in a 96-well plate and left to attach to the walls. Serial dilutions of the glycolipid compounds of the test group are added to the wells, and these cultures are incubated for another 36-40 hours, after which the culture supernatants are selected for analysis of nitrite using the Greiss reagent (Green et al. (1982) Anal. Biochem. 126: 131- 138). The nitrite content is closely correlated with iNOS activity.

The effectiveness of the compounds was determined as the concentration (ng / ml) of glycolipid in the culture, capable of causing a half-maximum formation of nitrite (ED ₅₀ ). The lower the ED ₅₀ value, the stronger the ability to induce iNOS. ED ₅₀ values were calculated according to the methods set forth in Johnson et al. (1999) J. Med. Chem. 42: 4640-4649.

The MPL immunostimulant showed an ED ₅₀ value of about 2 ng / ml, which leads to a high level of nitrite formation, while compound II showed a nominal ED ₅₀ value of ≥3000 ng / ml.

The very low level of maximum iNOS activity observed in the presence of this compound indicates that it is practically inactive in this iNOS induction system.

It is intended that the examples and embodiments described herein be provided for illustrative purposes only and that various modifications and modifications will be proposed by those skilled in the art that should be included in accordance with the spirit and scope of this application and the scope of the appended claims. All cited publications, patents, and patent applications are included in the present invention by reference in their entirety for all purposes.

Claims (19)

1. The compound of the formula

or its pharmaceutically acceptable salts, where X is selected from the group consisting of —O— and —NH—; R ¹ , R ² and R ^{3 are} independently selected from the group consisting of (C ₉ -C ₁₄ ) acyl groups. 2. The compound of claim 1, wherein X is —O—. 3. The compound according to claim 1, in which R ¹ , R ² and R ³ each are (C ₁₀ -C ₁₂ ) acyl groups. 4. The compound according to claim 2, in which R ¹ , R ² and R ^{3 are} each represented by decanoyl residues. 5. The compound of claim 2, wherein R ¹ , R ^2, and R ^{3 are} each represented by dodecanoyl residues. 6. The compound according to claim 2, in which * ¹ , * ² and * ³ are in the R-configuration. 7. An immunostimulating pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound according to any one of claims 1 to 6. 8. The pharmaceutical composition according to claim 7, further comprising at least one antigen. 9. The pharmaceutical composition of claim 8, in which the antigen comes from the group consisting of herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, HIV, hepatitis A, B, C or E viruses, respiratory syncytial virus, human papillomavirus, influenza virus, tuberculosis, leishmaniasis, T.cruzi, Ehrlichia, Candida, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium and Toxoplasma. 10. The pharmaceutical composition of claim 8, in which the antigen is a human tumor antigen. 11. The pharmaceutical composition of claim 10, in which the tumor antigen is derived from cancer of the prostate, colon, breast, ovary, pancreas, head and neck, leukemia or lymphoma. 12. The pharmaceutical composition of claim 10, in which the antigen is an autoantigen. 13. The pharmaceutical composition of claim 12, wherein the autoantigen is associated with an autoimmune disease. 14. The pharmaceutical composition of claim 13, wherein the autoimmune disease is type I diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, or psoriasis. 15. The pharmaceutical composition according to claim 7 in the form of an aqueous composition. 16. The pharmaceutical composition according to clause 15, in which the aqueous composition includes one or more surfactants. 17. The pharmaceutical composition according to claim 7 in the form of an emulsion composition. 18. The pharmaceutical composition according to claim 7 in the form of a solid composition. 19. A method of inducing an immune response in a patient, comprising administering to the patient a therapeutically effective amount of a compound according to any one of claims 1 to 6 or a composition according to any one of claims 7 to 18.