RU2361577C2 - Sphingoid polyalkylamine conjugates for bacterination - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns application of the sphyngoid-polyalkylamine conjugate - N-palmitoile-D-erythrosphyngosilecarbamoilspermine (CCS) as a keeping agent for biologically active molecules, such as antigens. The invention also concerns the way of modulation of the immune response at the subject. In the specific version of realisation of the invention the pharmaceutical composition represents a vaccine against flu, including CCS and antigens of a virus of flu - hemagglutinin and a neuraminidase. Advantage of the invention consists in intensifying of the immune response.

EFFECT: intensifying of the immune response.

19 cl, 1 ex, 18 tbl, 3 dwg

Description

FIELD OF THE INVENTION

The present invention relates to vaccination using polyalkylamine conjugates of sphingolipids for the effective delivery of biologically active materials, in particular antigenic molecules.

BACKGROUND

The following is a list of all kinds of materials that can be considered essential for characterizing the achieved level in the technical field to which the present invention relates.

US 5334761: "Cationic lipids";

US 2001/048939: "Cationic reagents of transfection";

US 5,659,011: “Agents having high nitrogen content and high cationic charge based on dicyanimide dicyandiamide or guanidine and inorganic ammonium salts”;

US 5674908: "Highly packed polycationic ammonium, sulfonium and phosphonium lipids";

US 6281371: "Lipopolyamines, and the preparation and use thereof";

US 6075012: "Reagents for intracellular delivery of macromolecules";

US 5783565: "Cationic amphiphiles containing spermine or spermidine cationic group for intracellular delivery of therapeutic molecules";

Marc Antoniu Ilies & Alexandru T. Balaban, Expert Opin. Ther. Patents, 11 (11): 1729-1752 (2001);

Miller AD. Chem. Int. Ed. Eng. 37: 1768-1785 (1998);

Nakanichi, T. et al., J Control Release 61: 233-240 (1999);

Brunel F. et al., Vaccine 17: 2192-2193 (1999);

Guy B, et al. Vaccine 19: 1794-1805 (2001);

Lima KM et al. Vaccine 19: 3518-3525 (2001).

Many natural biological molecules and their analogues, including proteins and polynucleotides, foreign substances and drugs that can affect cellular function at a subcellular or molecular level, are mainly incorporated into the cell to manifest their effect. For such agents, the cell membrane presents a selective barrier that is impervious to them. The cell membrane has a complex composition, including phospholipids, glycolipids and cholesterol, as well as endogenous and exogenous proteins, whose function is mediated by the influence of cytoplasmic components, including Ca ⁺⁺ and other metal ions, anions, ATP, microfilaments, microtubules, enzymes and Ca ⁺⁺ - binding proteins, as well as extracellular glycocalyx (proteoglycans, glycosaminoglycans and glycoproteins). The interaction between the structural and cytoplasmic components of the cell and their response to external signals determine the importance of transport processes for the selectivity of the membrane, manifested both in the aspect of the intracellular environment and at the level of intercellular interactions.

Also the subject of the study was the successful delivery of funds that, in vivo, are not captured by the cell and do not penetrate into it. The membrane barrier can be overcome by complexes formed by binding agents to lipid compositions, which are very similar in lipid composition to natural cell membranes. Such compositions may fuse with the cell membranes with which they come in contact, or, more often, may be captured by the mechanism of pinocytosis, endocytosis and / or phagocytosis. In all these processes, the associated substances are delivered inside the cells.

Lipid complexes can also facilitate intracellular transport due to the fact that they overcome the charge-induced repulsion between cell surfaces, which are most often negatively charged. The lipids in such compositions include an amphipathic lipid, such as cell membrane phospholipids, and form various layers or aggregates, such as micelles or hollow lipid vesicles (liposomes) in aqueous systems. Liposomes can be used to capture substances introduced into liposomes; in other cases, the considered drug molecules can be incorporated into lipid vesicles as an internal component of the membrane, and not be captured in the space of an aqueous vesicle or electrostatically attached to the surface of the aggregate. However, most of the phospholipids used are either zwitterions (neutral molecules) or negatively charged molecules.

A major achievement in the study of intracellular drug delivery was the discovery that the positively charged synthetic cationic lipid chloride N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA) in the form of liposomes or small vesicles can spontaneously interact with DNA to form lipid-DNA complexes that are adsorbed on cell membranes, and, being captured by cells either by fusion, or, more likely, by the mechanism of adsorption endocytosis, lead to transg expression for [Felgner, P. L. et al., Prod. Natl. Acad. Sci. USA 84: 7413-7417 (1987); and U.S. Pat. No. 4897355 (Eppstein, D. et. Al.)]. In other cases, the DOTMA 1,2-bis (oleoyloxy) -3- (trimethylammonium) propane (DOTAP) analogue has been successfully used in combination with a phospholipid, which leads to the formation of DNA complexing vesicles. Lipofectin ™ Reagent (Bethesda Research Laboratories, Gaithersburg, MD) is an effective tool for delivering high anionic charge polynucleotides to living cells in tissue culture, which includes positively charged liposomes consisting of positively charged DOTMA lipid and neutral lipid dioleylphosphamide ethyl lipid (DOPE), which serves as the so-called helper lipid. These liposomes spontaneously interact with negatively charged nucleic acids to form complexes called lipoplexes. When using an excess of positively charged liposomes relative to negatively charged DNA molecules, the total charge of the complexes formed also becomes positive. The positively charged complexes obtained in this way spontaneously attach to negatively charged cell surfaces or enter the cells either by the mechanism of adsorption endocytosis or by the mechanism mediated by fusion with the plasma membrane, so that both processes lead to the delivery of a functional polynucleotide inside, for example, cells in tissue culture. DOTMA and DOTAP are good examples of monocationic lipids [Illis et al., 2001, ibid.].

Polyvalent cations (including polyamines, inorganic salts and complexes, as well as dehydrating solvents) have been shown to facilitate the delivery of macromolecules to cells. In particular, polyvalent cations provoke the breakdown of oligo- and polyanions (nucleic acid molecules, amino acids, etc.) into compact structures and facilitate the assembly of such polyanions into viruses, their incorporation into liposomes, transport into cells, etc. [Thomas T.J. et al., Biochemistry 38: 3821-3830 (1999)]. The smallest natural polycations that are capable of exerting such an effect through compact DNA conversion are the polyamines spermidine and spermine. By attaching a hydrophobic label via a linker to such molecules, a new class of transfection vectors, polycationic lipopolymers, can be obtained.

Cationic lipids and cationic polymers interact electrostatically with the anionic groups of DNA (or any other polyanionic macromolecule) to form DNA lipid complexes (lipoplexes) or polycationic DNA complexes (polyplexes). The formation of the complex is associated with the release of counterions of lipids or polymers, which is a thermodynamic driving force for the spontaneous formation of lipoplex and polyplex. Cationic lipids can be divided into four classes: (i) Quaternary ammonium salt of lipids (for example, DOTMA (Lipofectin ^TM ) and DOTAP) and phosphonium / arsonium pairs; (ii) lipopolyamines; (iii) cationic lipids containing both quaternary ammonium fragments and polyamine fragments; and (iv) amidinium, guanidinium and heterocyclic lipid salts.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to the use of a sphingoid-polyalkylamine conjugate for the preparation of a pharmaceutical composition for modulating an immune response in a subject.

According to a preferred embodiment of the present invention, the sphingoid-polyalkylamine conjugate comprises a sphingoid framework containing at least one polyalkylamine chain through a carbamoyl bond.

The term "sphingoid-polyalkylamine conjugate" in the context of the present description refers to chemical conjugation (binding) between a sphingoid base (which in the present context is also described by the term "sphingoid framework") and at least one polyalkylamine chain. The conjugation between the sphingoid base and at least one polyalkylamine chain is via a carbamoyl bond, which will be described in more detail below.

The term “sphingoid base / framework” as used herein includes long chain aliphatic amines containing two or three hydroxyl groups, where the aliphatic chain may be saturated or unsaturated. Another example of an unsaturated sphingoid base is a base containing a characteristic trans-double bond at position 4.

The term "modulation" in the context of the present description refers to any measurable regulatory or biochemical effect exerted by biologically active material delivered by conjugate on the immune response of a subject, including a cellular response and / or a humoral response. Modulation includes inhibiting or, conversely, stimulating or enhancing one or both types of responses when a sphingoid-polyalkylamine conjugate is administered to a specified subject in combination with biologically active substances. The term "modulation" is preferably used in relation to stimulation or amplification of two or more times relative to the level detected by the introduction of a biologically active molecule without conjugate. The present invention also relates to modulation of the immune response in cases where biologically active material administered without conjugate is substantially ineffective in generating such a response.

In addition, the term “modulation” refers to the inhibition or suppression of an immune response in a subject, for example, in the treatment of autoimmune diseases, as well as in the treatment of allergies.

Thus, the term "biologically active molecule" in the context of the present description refers to any substance that, when administered in combination with a sphingoid-polyalkylamine conjugate, affects the immune system of a subject. The biologically active material is preferably an antigenic protein, an antigenic peptide, an antigenic polypeptide or an antigenic carbohydrate.

In accordance with another aspect, the present invention relates to a method for modulating an immune response in a subject, comprising administering to the subject a sphingoid-polyalkylamine conjugate together with a biologically active molecule, wherein the sphingoid-polyalkylamine conjugate comprises a sphingoid framework containing at least one polyalkylamine linked via a carbamoyl bond chain.

In accordance with another aspect, the present invention relates to a pharmaceutical composition for modulating an immune response in a subject, wherein said composition comprises (i) at least one sphingoid-polyalkylamine conjugate and (ii) at least one biologically active molecule associated with given conjugate.

In accordance with another aspect, the present invention relates to a complex comprising (i) a sphingoid-polyalkylamine conjugate and (ii) a biologically active material capable of modulating an immune response in a subject.

And finally, the present invention relates to the use of a sphingoid-polyalkylamine conjugate, as defined above, as a means of retaining biologically active molecules (eg, antigenic molecules). In this context, the sphingoid-polyalkylamine conjugate may be part of a kit used to capture / retain biologically active molecules, preferably antigenic molecules and / or immunostimulants and / or immunosuppressants, where the kit includes, in addition to the conjugate indicated, instructions for using it for capture / retention of biologically active molecules. The conjugate in this kit may be in the form of a dry product, and in this case, the kit may also include a suitable liquid with which the conjugate is mixed before use to obtain a suspension or emulsion or solution, or the conjugate may already be in liquid form (suspensions, emulsions, solution etc.). This kit can have many uses. So, for example, the kit can be used to study the functions of various immunomodulating molecules in terms of modulating immune responses in the processes of isolation of biologically active molecules and their identification. Those skilled in the art know for what purpose such a retention aid can be used, in particular for research purposes.

The term "retention agent" in the context of the present description refers to a conjugate capable of association with biologically active molecules, where the latter bear a negative charge, represent a negative dipole or have a local negative charge (the area on the molecule carrying the total negative charge) due to the polycationic structure conjugate. The capture / retention process per se involves an electrostatic interaction between a captured / retained molecule that has such a negative charge, is a negative dipole or carries a local negative charge, and a positively charged conjugate of the present invention.

The conjugate of the present invention can also be used as a carrier for delivery by transfer of biologically active molecules, carried out by the capture mechanism, to the target site and to the target cell.

BRIEF DESCRIPTION OF THE DRAWINGS

To illustrate the present invention and to more clearly demonstrate the ways of its practical implementation, some variants of its implementation are described below using non-limiting examples and with reference to the accompanying drawings.

In FIG. 1A-1D show some possible chemical structures, “linear”, “branched” or “cyclic” lipid-like cationic compounds (LLC), which are encompassed by the general definition of sphingoid-polyalkylamine conjugate (I), where FIG. 1A is an illustration of a framework (ceramide), attached to one polyalkylamine chain, FIGS. 1B and 1C show the same frame attached to two polyalkylamine chains, and FIG. 1D again shows the same frame, however, in this case, the only polyalkylamine chain is attached by two hydroxyl moieties to form a cyclic polialkilaminovogo conjugate.

In FIG. 2A-2F show the biological distribution and pharmacokinetics of various fluorescently labeled lipid compositions in the gastrointestinal tract - ■ -, in the lungs - ♦ - or spleen ---, not detected - ♦ -; on figa shows the distribution of empty structures DMPC: DMPG (molar ratio 9: 1); 2B shows the distribution of DMPC: DMPG: HN; on figs shows the distribution of empty structures DOTAP: cholesterol; FIG. 2D shows the distribution of DOTAP structures: cholesterol: HN; FIG. 2E shows the distribution of empty CCS: cholesterol structures; and finally, FIG. 2F shows the distribution of CCS-cholesterol: HN structures.

In FIG. 3A-3D shows the biological distribution of various compositions of lipid structures containing ¹²⁵ I-HN in the gastrointestinal tract - ■ -, in the lungs - ♦ - or the spleen -◊-, not detectable -x-, and, in particular, in FIG. 3A illustrates the biological distribution of free ¹²⁵ I-HN; on figv shows containing ¹²⁵ I-HN lipid structure, which consists of DOTAP: cholesterol; on figs shows containing ¹²⁵ I-HN lipid structure, which consists of DMPC: DMPG, and fig.3D shows containing ¹²⁵ I-HN lipid structure, which consists of CCS: cholesterol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of sphingoid-polyalkylamine conjugates as retention agents for the transfer of biologically active molecules that are effective in modulating the immune response in a subject.

Sphingoid-polyalkylamine conjugates are lipid-like cationic compounds (LLC), which can be synthesized as follows. Long chains with N-substituted bases, in particular N-substituted sphingoids, or sphingoid bases bind to various polyalkylamines or their derivatives to form a polyalkylamine-sphingoid structure, which can be used as such or can be further alkylated.

Protonation at an appropriate pH or alkylation of the formed polyalkylamine-sphingoid structure gives the lipid-like compounds the desired positive charge needed to interact with the biologically active molecules delivered to the target cell and the target cells. Sphingoid-polyalkylamine conjugates are effectively associated with biologically active molecules due to electrostatic interactions between biologically active molecules of anionic character and polyalkylamine fragments of the conjugate to form complexes (lipoplexes).

Alternatively, sphingoid-polyalkylamine conjugates can form structures comprising biologically active molecules.

The sphingoid-polyalkylamine conjugate may take the form of individual lipid-like molecules or may be a combined structure. One example of a suitable combined structure includes the formation of vesicle micelles and, in particular, liposomes. Other examples of such combined structures include the formation of micelles, invert phases, phases from cubic forms, and the like. Obviously, the sphingoid-polyalkylamine conjugate can be in a combined vesicular / micellar form or as part of another kind of combined structures.

"Combined lipid structure" in the context of the present description refers to the organized formation of lipid molecules, forming, in particular, micelles and liposomes. The combined lipid structures are preferably stable combined lipid structures. "Stable combined lipid structure" in the context of the present description refers to such a combined structure that is chemically and physically stable under storage conditions (4 ° C, in physiological environment) for at least one month.

In the case when these combined structures have the form of vesicles (for example, liposomes), biologically active molecules can be encapsulated inside such vesicles, partly in a lipid bilayer, or they can be adsorbed on the surface of the vesicles (or any combination of these three options can take place) . When these combined structures are micelles, biologically active molecules can be incorporated into amphiphilic compounds that form micelles, and / or can be associated with them in a stable mode due to electrostatic interaction.

Thus, in the context of the present description, the terms “encapsulated in”, “contained in”, “included in” or “associated with” indicate a physical compound between the conjugate and the biologically active molecule. A physical compound may be the incorporation of a given molecule or its capture into combined structures (eg, vesicles, micelles or other structures) formed from a conjugate; non-covalent binding of a biological molecule to the surface of such combined structures or immersion of a biological molecule in the space between sphingoid-polyalkylamine conjugates forming such combined structures. It should be noted that due to the positive charge or positive dipole of the sphingoid-polyalkylamine conjugate under physiological conditions, the preferred association between the conjugate and biologically active material is due to electrostatic, dipole, or acid-base interactions.

Notwithstanding the foregoing, the present invention is not limited to the specific type of association that arises between a sphingoid-polyalkylamine conjugate and a biologically active molecule. Thus, association means any interaction between a conjugate or a combined structure formed on its basis and biologically active material, which can lead to the achievement of the desired therapeutic effect.

The association of biologically active molecules and conjugate can be achieved using any method known in the art. Such a method includes, but is not limited to, post- or co-lyophilizing a conjugate with a biologically active molecule or simply mixing a preformed sphingoid-polyalkylamine conjugate with a biological molecule. The method of co-lyophilization is described, in particular, in US patents Nos. 6156337 and 6066331, and post-encapsulation methods are described, for example, in WO 03/000227, and all of these documents are incorporated herein by reference.

Thus, the present invention in accordance with the first of its aspects relates to the use of a sphingoid-polyalkylamine conjugate for the preparation of a pharmaceutical composition for modulating an immune response in a subject, wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid framework containing at least via a carbamoyl linkage one, and preferably one or two polyalkylamine chains.

As indicated above, the sphingoid-polyalkylamine conjugate comprises linking the sphingoid backbone to at least one polyalkylamine chain, wherein said linkage is carried out via corresponding carbamoyl bonds. More preferably, the sphingoid-polyalkylamine conjugate has the general formula (I):

where R ₁ means hydrogen, branched or linear alkyl, aryl, alkylamine or a group-C (O) R ₅ ;

R ₂ and R ₅ independently from each other mean branched or linear C ₁₀ -C ₂₄ alkyl, alkenyl or polyenyl groups;

R ₃ and R ₄ independently from each other mean a group —C (O) —NR ₆ R ₇ , where R ₆ and R ₇ , which may be the same as or different from R ₃ and R ₄ , and independently mean hydrogen or a saturated or unsaturated, branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be quaternary ammonium; or

R ₃ means hydrogen; or

R ₃ and R ₄ form together with the oxygen atoms to which they are attached a heterocyclic ring comprising —C (O) —NR ₉ - [R ₈ —NR ₉ ] _m —C (O) -, R ₈ means saturated or unsaturated C ₁ -C ₄ alkyl and R ₉ means hydrogen or a polyalkylamine of the formula - [R ₈ -NR ₉ ] _n -, wherein said R ₉ or each alkyl amine unit of R ₈ NR ₉ may be the same or different in said polyalkylamine; and

n and m are independently an integer from 1 to 10, preferably from 3 to 6;

W is a group selected from —CH═CH—, CH ₂ —CH (OH) - or —CH ₂ —CH ₂ -.

Non-limiting examples of sphingoid or sphingoid bases that may be used in a more particular embodiment of the present invention include sphingosines, dihydrosphingosines, phytosphingosines, dehydrophytosphingosine and derivatives thereof. Non-limiting examples of such derivatives include acyl derivatives such as ceramide (N-acylsphingosine), dihydroceramides, phytoceramides and dihydrophytoceramides, respectively, as well as ceramins (N-alkylsphingosine) and corresponding derivatives (e.g. dihydroceramine, phytoceramine and the like). Accordingly, N-substituted sphingoids or sphingoid bases contain two hydroxyl groups that are activated and subsequently react with polyalkylamine to form a polyalkylamine sphingoid structure. Non-limiting examples of activating agents include N, N'-disuccinimidyl carbonate, di- or tri-phosgene or imidazole derivatives. The interaction of these activating agents with sphingoid or sphingoid bases leads to the formation of succinimidyloxycarbonyl, chloroformate or imidazole carbamate, respectively, on one or both hydroxyls. The reaction of activating sphingoids with polyalkylamines can lead to the formation of branched, linear (unbranched) or cyclic conjugates, as shown in figure 1.

In accordance with one preferred embodiment of the present invention, the sphingoid framework is a ceramide attached to one (FIG. 1A) or two (FIG. 1B or 1C) polyalkylamine chains, or this attachment involves the involvement of two hydroxyl moieties, resulting in the formation of a cyclic polyalkylamine fragment (fig.1D).

Formed sphingoid-polyalkylamine conjugates can further react with methylating agents to form quaternary amines. The resulting compounds carry a positive charge of various sizes, determined by the ratio between the quaternary, primary and / or secondary amines in the formed conjugates. As such, the sphingoid-polyalkylamine conjugate exists in the form of a quaternized nitrogen salt, including, but not limited to, quaternary ammonium chloride, quaternary ammonium iodide, quaternary ammonium fluoride, quaternary ammonium bromide, quaternary ammonium oxyanion, and a combination thereof.

The sphingoid-polyalkylamine conjugate is preferably used in combination with a biologically active molecule. A biologically active material is any molecule that, when administered together with a sphingoid-polyalkylamine conjugate, affects the immune system of a subject, and in accordance with one embodiment of the invention, it can be a stimulating or enhancing effect. Preferably, this effect is a two or more times enhanced effect relative to the effect of a biologically active molecule when administered to a subject without a given conjugate.

In accordance with one embodiments of the present invention, the biologically active material is a protein, polypeptide, peptide or carbohydrate. Specifically, the biologically active molecule may be an immunomodulator comprising an antigenic protein or antigenic peptide, immunostimulants and / or immunosuppressants. Antigenic proteins and peptides, immunostimulants and immunosuppressants are well known in the art. Preferably, the biologically active protein, peptide or carbohydrate at the physiological pH value has a total negative dipole moment or a total negative charge, or contains at least one region characterized by a total negative charge.

According to another embodiment of the present invention, the biologically active material is a nucleic acid molecule, such as oligodeoxynucleotides (ONE).

The preferred weight ratio between the sphingoid-polyalkylamine conjugate and the biologically active material is from 1000: 1 to 1: 1.

The sphingoid-polyalkylamine conjugate can also be combined with other active substances, for which it is known that they can be used in combination with antigenic molecules. Such substances include, for example, immunostimulating agents (also known as "immunostimulant" or "adjuvant"). Such agents include any substance that, when added to the vaccine, enhances the immune response, so that using a smaller amount of vaccine, a more pronounced response can be achieved. An immunostimulating agent may be administered along with the conjugate / biologically active material or for a certain period of time (for example, from several hours or days before or after administration of the conjugate / biologically active molecule).

Preferred immunostimulatory agents include, but are not limited to, cytokines such as interleukins (IL-2, IL-10, IL-12, IL-15, IL-18), interferons (IFN alpha, beta, gamma), oligodeoxynucleotides (ONE) , toxins (e.g., cholera toxin (ST), staphylococcal enterotoxin B (SEB)), thermolabile E. coli enterotoxin (HLT), as well as any other adjuvants known in the art for enhancing or stimulating an immune response to an antigenic molecule.

The combined structures may include a sphingoid-polyalkylamine conjugate (unmethylated or methylated) as the sole lipid-like ingredient, or may be combined with other helper lipid substances. Such helper lipid substances may include non-cationic lipids such as DOPE, DOPC, DMPC, cholesterol, oleic acid or other substances, in different molar ratios relative to the lipid-like compound. Cholesterol is one such preferred in vivo adjuvant, while DOPE may be the preferred helper lipid for in vitro use. In this particular embodiment of the present invention, the molar ratio of cholesterol to cationic lipid is in the range from 0.01 to 1.0, and preferably from 0.1 to 0.4.

Enhancers (known in the art, such as CaCl ₂ ) and soluble polyalkylamines may also be included in the combined structure.

Other components that may be included in the lipid structure and which are used in such structures include steric stabilizers. One example of such commonly used steric stabilizers includes members of the family of lipopolymers, for example, lipids derivatized with polyethylene glycol (PEG-lipid conjugate). It is known that this family of compounds, in particular, increases (increases) the circulation time of lipids in the blood.

In accordance with one embodiment of the present invention, the formed liposomes can be in the form of unsorted heterogeneous and heterolayer vesicles (UHV) with a diameter of from about 50 to 5000 nm. Further, the obtained UHV can be sorted and transformed by further processing into large (more homogeneous) single-layer vesicles (LUV) with a diameter of about 50-100 nm. The structure and size of the vesicles, such as their shape and size, can have a pronounced effect on their effectiveness as vesicles that deliver active biological structures to the target, that is, they are parameters that determine their ability to deliver.

A preferred group of polyalkylamine chains forming part of the sphingoid-polyalkylamine conjugate has been structurally defined above with reference to formula (I). According to this embodiment of the invention, the polyalkylamine chains, which may be the same or different in the conjugate of formula (I), are selected from spermine, spermidine, polyalkylamine analogue, or a combination thereof. The term "polyalkylamine analogue" is used to mean any polyalkylamine chain and, in accordance with one embodiment of the present invention, means a polyalkylamine comprising from 1 to 10 amino groups, preferably from 3 to 6 amino groups, and more preferably 3 or 4 amino groups. Alkylamines in the polyalkylamine chain may be the same or different and each of them may be a primary, secondary, tertiary or quaternary amine.

Alkyl moieties may be the same or different in the polyalkylamine chain and each of them is preferably a C ₁ -C ₆ aliphatic repeating unit. Some non-limiting examples of polyalkylamines include spermidine, N- (2-aminoethyl) -1,3-propanediamine, 3,3'-iminobispropylamine, spermine and bis (ethyl) spermine derivatives, polyethyleneimine.

The most preferred sphingoid-polyalkylamine conjugate of the present invention is N-palmitoyl-D-erythrosphingosylcarbamoyl spermine (CCS). This conjugate includes a ceramide attached via a carbamoyl bond to spermine.

The sphingoid-polyalkylamine conjugate of the present invention is preferably used to prepare a vaccine.

In accordance with one embodiment of the present invention, a sphingoid-polyalkylamine conjugate, and preferably CCS, is used to prepare an influenza vaccine. In this embodiment, the biologically active material is obtained from influenza virus or from a biologically active analogue of a molecule derived from influenza virus. Such analogs include any substance that is part of an antigenic fragment derived from an influenza virus and that elicits an immune response.

Specific antigenic material derived from influenza virus are hemagglutinin (HA) and neuraminidase (NA) molecules, the combination of which is referred to as NH.

The present invention also relates to a method for modulating an immune response in a subject, wherein the method comprises treating said subject with a sphingoid-polyalkylamine conjugate in combination with a biologically active material.

Combination treatment involves the administration of a sphingoid-polyalkylamine conjugate and biologically active material, either together or for a predetermined period of time, such as several hours or several days (optionally in combination with an immunostimulating agent). However, in accordance with a preferred embodiment of the present invention, the conjugate and biologically active material are mixed with each other before administration to the subject.

The administration of a sphingoid-polyalkylamine conjugate together with biologically active material relates to another aspect of the present invention. Accordingly, a pharmaceutical composition is provided that comprises a physiologically acceptable carrier and an effective amount of a sphingoid-polyalkylamine conjugate together with a biologically active material. The pharmaceutical composition optionally contains an immunostimulating agent.

With the introduction of a sphingoid-polyalkylamine conjugate in combination with biologically active material, its dose can be selected in accordance with the results of its practical use in medicine, taking into account the clinical condition of the individual, place and route of administration, mode of administration, patient age, gender, body weight and others significant factors known in medical practice.

The term "effective amount" in the context of the present description refers to the amount that is effective for modulating (enhancing or stimulating, in accordance with the above definition) the immune response in a subject with respect to the effect achieved when the biologically active material is administered to a subject without a sphingoid -polyalkylamine conjugate. Preferably, the amount is effective for immunizing a subject in relation to a particular disease or disorder.

Regardless of the foregoing, this amount may be effective in achieving suppression or inhibition of the immune response, for example, for the treatment of allergies or autoimmune reactions.

The composition of the present invention containing a sphingoid-polyalkylamine conjugate in combination with a biologically active material can be administered in various ways. Non-limiting examples of routes of administration include oral, subcutaneous (s / c), parenteral, including intravenous (iv), intraarterial (iv), intramuscular (iv), intraperitoneal (iv), intrarectal (and / r ) and the intranasal (and / n) route of administration, as well as infusion methods for administration to the eye region, intraocular administration. Preferred routes of administration include intranasal or intramuscular administration.

A physiologically acceptable carrier of the present invention generally relates to inert, non-toxic, solid or liquid substances, preferably not interacting with the biologically active material or the conjugate, and which is required for efficient delivery of the conjugate together with the biologically active molecule.

Non-limiting examples of a physiologically acceptable carrier include water, saline, 5% dextrose (glucose), 10% sucrose, and the like, either individually or with a small amount (up to 10%) of alcohol, such as ethanol.

Preferably, the composition of the present invention is a liquid composition, including suspensions, aqueous solutions, or is in the form of an aerosol, where all these forms are known to specialists in this field. Aerosol compositions can be formulated including suitable pressurized propellants such as propane, nitrogen, and the like. In addition, they can be incorporated into pharmaceutical preparations with non-pressurized carriers, such as carriers in nebulizers or atomizers.

DESCRIPTION OF SPECIFIC EXAMPLES

FLU

Characterization of cationic liposomes loaded with HN antigen

The efficacy of HN encapsulation (a commercial preparation of hemagglutinin and neuraminidase derived from influenza viruses), introduced into various cationic liposome compositions in different mass ratios of lipid / protein (3 / 1-300 / 1), with or without cholesterol (Chol) was investigated. . Table 1 shows the results of such an experiment using cationic lipids DOTAP and CCS.

Table 1 Effect of lipid ratio (DOTAP, CCS) / protein and cholesterol (Chol) on the efficiency of HN encapsulation Wt./wt. ratio
DOTAP / HN Molar ratio
DOTAP / Chol % encapsulation of HN Wt./wt. ratio
CCS / HN Molar ratio
CCS / Chol % encapsulation
Hn 300/1
100/1
50/1
30/1
10/1
3/1 1/1
1/1
1/1
1/1
1/1
1/1 93
90
90
88
79
35 300/1
100/1
30/1
10/1 1/0
1/0
1/0
1/0 73
64
38
one 100/1
100/1
100/1
100/1 1/0
1/1
2/1
4/1 90
92
89
80 300/1
100/1
30/1
10/1 3/2
3/2
3/2
3/2 71
64
41
0 A monovalent vaccine is used for DOTAP, and a trivalent vaccine is used for CCS.

The percent inclusion for DOTAP is 75-90% when using a lipid / protein mass ratio in the range of 50/1 to 300/1 both in the presence and absence of cholesterol (Chol). When using lipid / protein mass ratios in the range from 30/1 to 300/1, ~ 90% antigenic potential is achieved, decreasing to 79% and 35% in the case of the mass ratios 10/1 and 3/1, respectively. Adding Chol to the composition does not affect the antigenic potential when evaluating the DOTAP / Chol molar ratios of 1/1 and 2/1, however, there is a slight decrease in encapsulation (80%) in the ratio of 4/1. In the case of CCS, both in the presence and absence of Chol, the inclusion efficiency is lower (64-73% when the mass ratio is varied in the range 100 / 1-300 / 1).

The level of HN association with liposomes is also determined by simply mixing soluble antigen with preformed empty liposomes. In such cases, 40-60% of the antigen is associated with liposomes using a lipid / protein weight ratio of 100/1 to 300/1, regardless of the composition.

Together, these results indicate a very high inclusion efficiency (> 60%) in the case of all compositions using a simple and quick (5 minutes) procedure. In addition, even preformed liposomes in an aqueous suspension are capable of efficient association with surface antigens of the influenza virus.

The immunogenicity of the composition is also evaluated using different lipid / antigen weight ratios (in the presence or absence of cholesterol).

In the first experiment, serum levels of HI, IgG1 and IgG2a antibodies were determined after iv vaccination of young (two-month-old) BALB / mice with HN-containing neutral, anionic or cationic liposomes (Table 2A). HN antigen is a monovalent subunit vaccine derived from A / New Caledonia strain (H1N1). In the same experiment, pulmonary and nasal levels of HI, IgG1, IgG2a and IgA antibodies are also determined, as well as the amount of INFγ produced by spleen cells (Tables 2B and 2C).

Table 2A Serum levels of antibodies HI, IgG1 and IgG2a Group n = 5 Vaccine Serum HI (mean ± SD) IgG1 IgG2a one
2
3 FBI
F-hn
Lip (DMPC) -HN (Neutral) 0
0
3 ± 7 (0) 0
55
150 0
0
0 four Lip (DMPC / DMPG) -HN (anionic) 6 ± 13 (20) 500 0 5
6
7
8
9
10
eleven Lip (DC-Chol: DOPE) -HN
Lip (DSTAP: Chol) -HN
Lip (DDAB: Chol) -HN
Lip (DOTAP: Chol) -HN
Lip (DMTAP: Chol) -HN
Lip (CCS: Chol) -HN
F-HN + CT (1 mcg) 18 ± 7 (0)
28 ± 29 (40)
136 ± 32 (100)
576 ± 128 (100)
672 ± 212 (100)
2368 ± 1805 (100)
1664 ± 572 (100) 0
twenty
one hundred
15,000
30000
30000
55,000 0
0
0
730
470
9000
7000 F-HN - free antigen; Chol - cholesterol, CT - cholera toxin; groups 5-10: include the use of cationic liposomes. In parentheses are indicated:% seroconversion -% of mice with a HI titer ≥40.

In particular, a comparison is made between the administration of neutral (DMPC), anionic (DMPC / DMPG, molar ratio 9/1) and cationic (6 compositions) combined structures (Lip) with HN encapsulated antigens for the purpose of inducing local and systemic responses after two and / n introductions. In all compositions, the mass ratio of lipid / HN is 300/1, and the molar ratio of cationic lipid / Chol or cationic lipid / DOPE is 1/1. In parallel, free antigen (F-HN) and F-HN, administered together with cholera toxin (CT, 1 μg) as an adjuvant, are examined. This vaccine is administered on days 0 and 7 in the amount of 3 μg / dose (10 μl per nostril) and the responses are determined 4-6 weeks after the second dose of the vaccine.

Table 2B IgG1, IgG2a, and IgA antibody levels in lung and nasal washings Group
n = 5 Vaccine Lung Nose IgG1 IgG2a IgA IgG1 IgG2a IgA one
2
3 FBI
F-hn
Lip (DMPC) -HN (Neutral) 0
0
thirty 0
0
0 0
0
0 0
0
0 0
0
0 0
0
0 four Lip (DMPC / DMPG) -HN (anionic) 40 0 0 0 0 0 5
6
7
8
9
10
eleven Lip (DC-Chol: DOPE) -HN
Lip (DSTAP: Chol) -HN
Lip (DDAB: Chol) -HN
Lip (DOTAP: Chol) -HN
Lip (DMTAP: Chol) -HN
Lip (CCS: Chol) -HN
F-HN + CT (1 mcg) 0
0
0
730
470
9000
7000 0
twenty
80
1050
3000
30000
10,000 0
0
thirty
170
thirty
1900
1800 0
0
0
40
fifteen
15,000
40 0
80
thirty
180
80
thirty
120 0
0
0
thirty
70
300
thirty

Table 2C INFγ levels in the spleen (pg / ml) Group n = 5 Vaccine Spleen IFNγ (pg / ml) one
2
3
four
5
6
7
8
9
10
eleven FBI
F-hn
Lip (DMPC) -HN (Neutral)
Lip (DMPC / DMPG) -HN (anionic)
Lip (DC-Chol: DOPE) -HN
Lip (DSTAP: Chol) -HN
Lip (DDAB: Chol) -HN
Lip (DOTAP: Chol) -HN
Lip (DMTAP: Chol) -HN
Lip (CCS: Chol) -HN
F-HN + CT (1 mcg) 1800
1400
4200
4000
4900
2300
3100
8000
7800
10200
5200

As shown in tables 2A-2C, the free antigen, as well as neutral and anionic Lip-HN, were practically ineffective as a vaccine for the mucosa. Conversely, cationic Lip-HN, in particular vaccines such as DOTAP-HN, DMTAP-HN and CCS-HN, provoke a hard systemic and humoral response of the mucosa with the production of high levels of IgG1, IgG2a IgA antibodies, namely: they cause mixed Th1 -Th2 reaction. IgE antibodies are not detected. Cationic liposomal vaccines, including DOTAP-HN, DMTAP-HN and CCS-HN, also induce high levels of IFNγ (but not IL-4) in antigen-stimulated spleen cells. Responses elicited by CCS-HN are even more pronounced than responses induced by F-HN with CT added as an adjuvant. Based on the results obtained, only cationic liposome compositions are then used: DOTAP-HN, DMTAP-HN and CCS-HN.

In a second experiment, the effect of various lipid / HN weight ratios on the immunogenicity of HN-containing cationic liposomes and preformed liposomes is determined by simple mixing with a soluble antigen. The data shown in tables 3A-3C indicate that all three compositions induce potent responses of a systemic (serum) and local (mild) nature, while a decrease in the lipid / HN mass ratio below 100/1 affects a sharp decrease in the immune response.

Table 3A Serum levels of antibodies HI, IgG1, IgG2a and IgA No. Vaccine (n = 5) The mass ratio of lipid / HN Serum Hi IgG1 IgG2a IgA one F-hn 0 0 0 0 2
3
four
5
6 Lip (DOTAP) -HN 300/1
100/1
30/1
10/1
3/1 496 ± 295 (100)
196 ± 119 (100)
36 ± 50 (80)
28 ± 18 (60)
0 15,000
5000
1000
600
twenty 450
280
200
thirty
0 0
0
0
0
0 7
8
9
10
eleven Lip (DMTAP) -HN 300/1
100/1
50/1
30/1
10/1 388 ± 260 (100)
208 ± 107 (100)
130 ± 118 (80)
48 ± 71 (40)
24 ± 35 (40) 2500
2200
850
450
120 250
600
150
0
0 0
0
0
0
0 12
13
fourteen
fifteen
16 Lip (CCS) -HN 300/1
100/1
50/1
30/1
10/1 560 ± 480 (100)
752 ± 504 (100)
272 ± 156 (100)
112 ± 125 (80)
52 ± 68 (40) 2000
6500
1900
650
275 1800
6000
700
400
440 200
0
0
0
0 17
eighteen
19
twenty F-HN + CT (1 mcg)
F-HN + Lip (DOTAP)
F-HN + Lip (DMTAP)
F-HN + Lip (CCS) -
300/1
300/1
300/1 896 ± 350 (100)
864 ± 446 (100)
320 ± 226 (100)
704 ± 525 (100) 30000
5000
1900
30000 8000
1500
400
5000 120
0
0
500 For groups 18-20, preformed liposomes are mixed with a soluble antigen.

Table 3B HI, IgG1, IgG2a and IgA levels in the lungs No. Vaccine (n = 5) The mass ratio of lipid / HN Lung Hi IgG1 IgG2a IgA one F-hn 0 0 0 0 2
3
four
5
6 Lip (DOTAP) -HN 300/1
100/1
30/1
10/1
3/1 40
40
thirty
twenty
10 600
500
250
250
twenty 85
twenty
35
0
0 thirty
0
0
0
0 7
8
9
10
eleven Lip (DMTAP) -HN 300/1
100/1
50/1
30/1
10/1 0
0
0
0
0 5500
7000
4500
1500
500 200
350
250
110
0 1200
0
0
0
0 12
13
fourteen
fifteen
16 Lip (CCS) -HN 300/1
100/1
50/1
30/1
10/1 80
80
40
0
0 12500
7000
5500
1500
500 3000
5500
900
200
200 20000
65,000
20000
0
0 17
eighteen
19
twenty F-HN + CT (1mkg)
F-HN + Lip (DOTAP)
F-HN + Lip (DMTAP)
F-HN + Lip (CCS) -
300/1
300/1
300/1 80
0
0
80 45000
6000
3750
35,000 2250
500
225
3000 3000
1200
1500
80,000

Table 3C INFγ levels in the spleen (pg / ml) No. Vaccine (n = 5) Mass ratio
lipid / HN Spleen
IFNγ (pg / ml) one F-hn 7430 2
3
four
5
6 Lip (DOTAP) -HN 300/1
100/1
30/1
10/1
3/1 9780
42220
20440
20,400
27780 7
8
9
10
eleven Lip (DMTAP) -HN 300/1
100/1
50/1
30/1
10/1 Not investigated 12
13
fourteen
fifteen
16 Lip (CCS) -HN 300/1
100/1
50/1
30/1
10/1 17
eighteen
19
twenty F-HN + CT (1 mcg)
F-HN + Lip (DOTAP)
F-HN + Lip (DMTAP)
F-HN + Lip (CCS) -
300/1
300/1
300/1

In this case, the advantage of the Lip-CCS-HN vaccine in comparison with other vaccine compositions is also noted, determined by higher levels of IgG2a and IgA antibodies in the blood serum and in the lung (groups 12-16). It is interesting to note that simply mixing soluble antigen with preformed liposomes results in very effective vaccines (groups 18–20) that are equivalent to liposomes with encapsulated antigen. This suggests that the actual encapsulation of the antigen is not always necessary to achieve the adjuvant cationic combined structures / liposomes.

In another experiment, the effect of cholesterol on the immunogenicity of liposomes loaded with HN is investigated. Tables 4A-4C show the results of this experiment, which show that the addition of Chol slightly reduces the systemic HI response to DOTAP-HN in molar ratios of 2/1 and 4/1 (groups 4, 5), but not in a molar ratio of 1/1 (group 3), and also moderately increases the overall response to DMTAP-HN at all ratios (groups 7-9) and the local response (in the lung) to CCS-HN in the ratio 1/1 (group 11).

Table 4A Serum levels of antibodies HI, IgG1, IgG2a and IgA No. Vaccine (n = 5) The ratio of cationic lipid / cholesterol, wt./wt. Serum Hi IgG1 level IgG2a level IgA level one F-hn - 0 0 0 0 2
3 Lip (DOTAR) -HN
Lip (DOTAP: Chol) -HN 1/0
1/1 320 ± 0 (100)
496 ± 295 (100) 15,000
15,000 450
450 0
0 four
5 2/1
4/1 168 ± 216 (100)
195 ± 111 (100) 7000
15,000 800
250 0
0 6
7 Lip (DMTAP) -HN
Lip (DMTAP: Chol) -HN 1/0
1/1 320 ± 188 (100)
672 ± 419 (100) 20000
30000 290
300 0
0 8
9 2/1
4/1 576 ± 368 (100)
608 ± 382 (100) 25,000
30000 650
600 0
0 10
eleven Lip (CCS) -HN
Lip (CCS: Chol) -HN 1/0
1/1 2560 ± 1568 (100)
2368 ± 1805 (100) 30000
30000 7000
9000 one hundred
one hundred 12 F-HN + CT (1 mcg) - 1664 ± 572 (100) 55,000 7000 twenty

Table 4B Lung antibody levels HI, IgG1, IgG2a and IgA No. Vaccine (n = 5) The ratio of cationic lipid / cholesterol, wt./wt. Lung Hi IgG1 level IgG2a level IgA level one F-hn - 0 0 0 0 2
3
four
5 Lip (DOTAR) -HN
Lip (DOTAP: Chol) -HN 1/0
1/1
2/1
4/1 40
40
40
60 900
600
680
720 85
80
180
fifty 25
thirty
22
60 6
7
8
9 Lip (DMTAP) -HN
Lip (DMTAP: Chol) -HN 1/0
1/1
2/1
4/1 60
120
160
80 1000
3000
2500
4000 40
thirty
160
one hundred 0
fifteen
200
150 10
eleven
12 Lip (CCS) -HN
Lip (CCS: Chol) -HN
F-HN + CT (1 mcg) 1/0
3/2
- 640
1280
twenty 30000
30000
10,000 1500
1900
1800 9000
15,000
1000

Table 4C Spleen IFNγ levels (pg / ml) No. Vaccine (n = 5) The ratio of cationic lipid / cholesterol, wt./wt. The level of IFNγ in the spleen (PG / ml) one F-hn - 7430 2
3
four
5 Lip (DOTAR) -HN
Lip (DOTAP: Chol) -HN 1/0
1/1
2/1
4/1 7480
9780
12870
9330 6
7
8
9 Lip (DMTAP) -HN
Lip (DMTAP: Chol) -HN 1/0
1/1
2/1
4/1 8520
10900
8560
7490 10
eleven
12 Lip (CCS) -HN
Lip (CCS: Chol) -HN
F-HN + CT (1 mcg) 1/0
3/2
- 15550
13780
11110

The immunogenicity of CCS-HN vaccine is also evaluated in mature (18 months) C57BL / 6 mice after intramuscular (once, on day 0) or intranasal (twice, on days 0 and 7) administration of 1 and 2 μg, respectively, of subunit vaccine (HN) ( derived from A / Panama virus [H3N2]). The combined lipid structures are composed of CCS / cholesterol (3: 2 molar ratio) and the lipid / HN ratio is 200: 1 (w / w). In contrast to the zero activity of a commercial vaccine, the CCS-HN vaccine provokes the formation of high levels of HI and IgG2a antibodies (when tested 4 weeks after vaccination) and IgG2a and IgA antibodies in the lung (when tested 6 weeks after vaccination), as shown in tables 5A and 5B (the data presented are the average titers).

Table 5A Serum lung levels of HI, IgG1, IgG2a and IgA in mature mice No. Vaccine ^a (n = 5) Serum Hi IgG1 IgG2a one
2
3
four FBI, and / n × 2
F-HN, v / m × 1
F-HN, and / n × 2
Lip (CCS) -HN, and / n × 2 0
0
0
80 0
fifteen
0
130 0
0
0
350

Table 5B IgG1, IgG2a, and IgA antibody levels in the lung in mature mice No. Vaccine (n = 5) Lung IgG1 IgG2a IgA one
2
3
four FBI, and / n × 2
F-HN, v / m × 1
F-HN, and / n × 2
CCS-HN, and / n × 2 0
0
0
0 0
0
0
180 0
0
0
840

In addition, the induction of cellular responses by various vaccine compositions is also being investigated. In particular, young mice are immunized with i / n by administering various cationic liposome compositions and 6 weeks after vaccination, the splenocyte cell responses are evaluated: cytotoxicity, proliferation and IFNγ production. In the experiment, the results of which are shown in table 6, a comparison was made of the results of the use of liposomes containing HN (groups 3-10), and free antigen (F-HN), which is administered either individually (group 2), or in a mixture with previously formed empty liposomes (groups 11-13). The immunogenicity of Lip (DMTAP-HN) and Lip (CCS) -HN obtained by varying w / w is also determined. lipid / HN ratios (30 / 1-300 / 1).

Table 6 Induction of cellular responses with i / n administration of cationic liposomes No. Vaccine The ratio of lipid / HN, wt./wt. Cytotoxicity of P815 + P815 peptide,% Proliferation, Δimp / min (average value) IFNγ (pg / ml) one FBI - 6 four 7010 1900 2 F-hn - 8 5 7700 4500 3 Lip (DMTAP) -HN 300/1 16 13 10960 3500 four 100/1 9 9 12870 5850 5 50/1 3 2 17670 3400 6 30/1 3 2 17920 3050 7 Lip (CCS) -HN 300/1 four 2 20370 8000 8 100/1 21 7 24870 8250 9 50/1 6 3 20980 10650 10 30/1 8 5 11510 3500 eleven F-HN + Lip (DOTAP) 300/1 17 four 19390 3400 12 F-HN + Lip (DMTAP) 300/1 17 7 11850 5700 13 F-HN + Lip (CCS) 300/1 16 8 19270 4100

The most pronounced toxicity against the studied target cells (pulse exposure to P815 with a peptide from the influenza virus) is achieved only when using CCS-HN with a lipid / HN ratio (wt./wt.) 100/1 (group 8) and using all three previously formed liposomes (DOTAP, DMTAP and CCS), which are administered together with the free antigen. The maximum proliferative response is observed with the introduction of DMTAP-HN with wt./wt. lipid / HN ratios of 50/1 and 30/1, and when using CCS-HN in ratios of 300/1, 100/1 and 50/1. The proliferative and cytotoxic responses exhibited by the most effective liposome compositions are 2–3 times higher than the responses that the free antigen induces.

The above results suggest that, in comparison with the humoral response (table 3), where the highest levels of all types of studied antibodies are observed at ratios (wt./wt.) Lipid / HN in the range 100 / 1-300 / 1, in this case, achievement of cellular responses may be optimal reduced ratios (for example, in the range of 30 / 1-100 / 1). In addition, while DMTAP-HN elicits a pronounced humoral response, this composition is a weak inducer of cytotoxic activity compared to CCS-HN. It is interesting to note that vaccination using a mixture of free antigen with preformed cationic liposomes (all three compositions) in suspension causes suitable cellular responses that are similar in severity to the responses induced by the encapsulated antigen. Thus, simply mixing the free antigen with preformed cationic liposomes may be sufficient to induce both pronounced humoral (Tables 3A-3C) and cellular (Table 6) responses.

In another experiment, the results of which are shown in tables 7A-7C, a comparison was made between the introduction of 1 i / m dose, 1 or 2 i / n doses and 2 oral doses of monovalent cationic HN-containing liposomes, including DOTAP, DMTAP and CCS, by immunogenicity and ability to induce protective immunity when provoked by a live virus. In this experiment, the lipid / HN ratio (w / w) is 300/1, and the cationic lipid / Chol ratio is 1/1 in the case of DOTAP and DMTAP systems and 3/2 in the case of the CCS system. Of the three indicated methods, i / n administration induces a twice as strong humoral and cellular response and protective immunity. If we compare 3 of these compositions, then CCS induces the strongest response, especially with regard to the production of antibodies IgG2a and IgA.

Table 7A Serum levels of HI, IgG1, IgG2a and IgA No. Vaccine (n = 10) Method of administration Serum levels Hi IgGl IgG2a IgA one FBI 0 0 0 0 2 F-hn v / m × 1 60 ± 37 (70) 1000 40 0 3 Oral × 2 0 0 0 0 four and / n × 1 0 0 0 0 5 and / n × 2 0 55 0 0 6 Lip (DOTAP / Chol) -HN v / m × 1 424 + 141 (100) 21000 5500 0 7 Oral × 2 0 0 0 0 8 and / n × 1 40 ± 28 (50) 450 80 0 9 and / n × 2 409 + 172 (100) 25,000 1300 60 10 Lip (DMTAP / Chol) -HN v / m × 1 768 + 211 (100) 24000 8000 0 eleven Oral × 2 0 0 0 0 12 and / n × 1 10 + 10 (0) 300 60 0 13 and / n × 2 532 ± 763 (100) 10500 380 fifty fourteen Lip (CCS / Chol) -HN v / m × 1 864 ± 1100 (100) 25,000 10,000 0 fifteen Oral × 2 0 0 16 and / n × 1 34 ± 50 (20) 1000 thirty 0 17 and / n × 2 2289 ± 1576 25,000 20000 400 (one hundred) eighteen F-HN + CT (1 mcg) and / n × 2 756 ± 650 (100) 21000 15,000 twenty

Table 7B Lung antibody level No. Vaccine (n = 10) Method of administration Lung Hi IgGl IgG2a IgA one FBI 0 0 0 0 2 F-hn v / m × 1 0 80 0 0 3 Oral × 2 0 0 0 0 four and / n × 1 0 0 0 0 5 and / n × 2 0 70 twenty 0 6 Lip (DOTAP / Chol) -HN v / m × 1 40 900 500 0 7 Oral × 2 0 0 0 0 8 and / n × 1 0 fifty twenty 0 9 and / n × 2 120 10,000 1000 350 10 Lip (DMTAP / Chol) -HN v / m × 1 twenty 900 150 0 eleven Oral × 2 0 0 0 0 12 and / n × 1 0 35 twenty 0 13 and / n × 2 240 20000 700 2200 fourteen Lip (CCS / Chol) -HN v / m × 1 60 3500 900 0 fifteen Oral × 2 0 0 0 0 16 and / n × 1 0 120 0 35 17 and / n × 2 360 30000 5000 20000 eighteen F-HNH + CT (1 mcg) and / n × 2 240 22000 2500 1800

Table 7C Cellular response and protective immunity No. Vaccine (n = 5) Route of administration Spleen, Δimp / min (average value) IFNγ (pg / ml) Virus titer in the lung (log 10) one FBI 1641 0 7 2 F-hn v / m × 1 1909 0 four 3 orally × 2 2253 0 Nd four and / n × 1 669 0 Nd 5 and / n × 2 2813 0 5 6 Lip (DOTAP / Chol) -HN v / m × 1 3452 3300 0 7 orally × 2 0 1150 Nd 8 and / n × 1 482 1900 Nd 9 and / n × 2 8391 3200 0 10 Lip (DMTAP / Chol) -HN v / m × 1 5632 0 one eleven orally × 2 553 0 Nd 12 and / n × 1 1277 0 Nd 13 and / n × 2 7331 3150 0 fourteen Lip (CCS / Chol) -HN v / m × 1 6196 5750 0 fifteen orally × 2 476 550 Nd 16 and / n × 1 1705 6250 Nd 17 and / n × 2 4912 15500 0 eighteen F-HN + CT (1 mcg) and / n × 2 1933 5650 0

In the experiment described in tables 8-10, a commercial trivalent vaccine is examined and the results of administering one dose of CCS-based vaccine (using 2 or 4 μg of antigen [HN] of each of the viral strains) and two doses of the vaccine (2 μg / strain /) are compared. dose), administered at intervals of 3, 7 or 14 days between dosages. The combined lipid structures consist of CCS / Chol (cholesterol) in a molar ratio of 3/2, and in all compositions the ratio (w / w) lipid / HN of 100/1 is observed. As controls, a standard trivalent commercial vaccine (HN) is administered, either alone or in combination with 1 μg of cholera toxin (CT), used as an adjuvant for administration to the mucosa. 5-6 weeks after the introduction of the first dose of the vaccine in serum, lung homogenates, and nasal lavage fluids, HI antibody levels are determined (Table 8), as well as antigen-specific antibodies IgG1, IgG2a, IgA and IgE (table 9). In addition, in 5 mice in the selected groups, an / n challenge with live virus was performed (using the X-127 virus strain adapted for mice) and after 4 days the titer of the virus in the lung was quantified (Table 10).

Unlike the commercial influenza vaccine (HN), which shows little or no immunogenicity (groups 2-6), the CCS / Chol-flu vaccine induces high titers of all types of antibodies tested (with the exception of IgE, which was not detected), especially against two strains of virus A (groups 8-11; tables 8, 9). Moreover, it should be assumed that the regimen of administering two doses with an interval of one week is optimal (group 10). In the case of a single regimen of administration, the antigen in an amount of 4 μg, but not 2 μg (group 8 in comparison with group 7), induces high titers of serumal antibodies HI, IgG1 and IgG2a and IgG1 antibodies in the lung. However, in comparison with the option of administering two doses, a single regimen does not lead to the production of IgG2a and IgA antibodies in either the lung or nasal passages (Table 9).

In the test for the detection of a protective reaction (table 10), the CCS-flu influenza vaccine, given i / n in a single (4 μg) or double (2 μg / dose) regimen, provides complete protection against viral infection (decrease in lung titer by 6 log), while a standard vaccine reduces the titer of the virus by only 0.5-1 log. Thus, despite the fact that the influenza vaccine CCS-flu gives worse results in a single dose than in the case of two doses, for some isotypes of antibodies, both modes provide the same level of protection.

In this experiment, the authors also compared the options for the introduction of CCS separately and CCS / Chol as a vaccine carrier and did not reveal differences in immunogenicity between the two compositions (data not shown). Another modification of the composition included a reduction in the size of the lipid structures of CS / Chol (diameter 0.05-5 μm) by extrusion (diameter ≤0.02 μm). Antibody titers induced by the extruded vaccine were 50-80% lower than titers caused by the non-extruded vaccine (data not shown). Thus, untreated CCS lipid structures in the presence or absence of cholesterol are highly effective as vaccine carriers for their use in a trivalent influenza vaccine.

Table 8 Demonstration of antibody production for hemagglutination inhibition (HI) after intranasal administration of a trivalent influenza vaccine in free form and in the form of combined structures with CCS lipid, administered in single or double mode with different time intervals to young (two-month-old) BALB / C mice No. Vaccine ^a (n = 5) Dose Days The average value of the titer of HI (% seroconversion) ^b A / new caledonia A / panama B / yamanashi In serum In the lung In serum In the lung In serum In the lung one No vaccine (FBI) × 2 0.7 0 0 0 0 0 0 2 F-hn 2 mcg × 1 0 0 0 0 0 0 0 3 4 mcg × 1 0 0 0 0 0 0 0 four 2 mcg × 2 0, 3 0 0 0 0 0 0 5 2 mcg × 2 0, 7 0 0 0 0 0 0 6 2 mcg × 2 0, 14 0 0 0 0 0 0 7 Lip (CCS / Chol) -HN 2 mcg × 1 0 0 0 0 0 0 0 8 4 mcg × 1 0 336 (100) 40 328 (100) 40 52 (80) 0 9 2 mcg × 2 0, 3 544 (100) 80 408 (100) 40 52 (80) 0 10 2 mcg × 2 0, 7 544 (100) 80 544 (100) 120 88 (100) 0 eleven 2 mcg × 2 0, 14 480 (100) 60 368 (100) 40 80 (80) 0 12 F-HN + CT (1 mcg) 2 mcg × 2 0, 7 608 (100) 80 664 (100) 120 84 (80) 0 ^a - Mice are immunized with the trivalent subunit preparation of the Fluvirin® 2003/2004 vaccine consisting of A / New Caledonia / 20/99 (H1N1) -like, A / Moscow / 10/99 (H3N2) -like and B / Hong Kong / 330 / 2001-like, which is available either in free form (F-HN), or is included in the combined lipid complexes CCS / Chol (molar coefficient 3/2) (at a dose of 0.6 mg to animals in groups 7, 9, 10, 11 and 1.2 mg - in group 8).
^b - Serum HI titer is determined in each individual mouse 35 days after the first dose of the vaccine. HI titer in the lungs (when combined) is determined on day 42.
In parentheses is the percentage of mice in which the HI titer ≥40 and 0 denotes a titer HI <20.

Table 9 Demonstration of the production of antigen-specific antibodies IgG1, IgG2a and IgA in serum, lung and nasal passages after intranasal administration of a trivalent influenza vaccine in free form and in the form of combined structures with CCS lipid, administered in a single or double mode with different time intervals young (two-month) BALB / C mice No. Vaccine ^a (n = 5) Dose Days The average value of the titer of antibodies In serum In lung homogenate In the nasal passages IgGl IgG2a IgGl IgG2a IgA IgGl IgG2a IgA one No vaccine (FBI) × 2 0, 7 0 0 0 0 0 0 0 0 2 F-hn 2 mcg × 1 0 0 0 0 0 0 0 0 0 3 4 mcg × 1 0 320 90 1500 0 0 0 0 0 four 2 mcg × 2 0, 3 0 0 0 0 0 0 0 0 5 2 mcg × 2 0, 7 0 0 0 0 0 0 0 0 6 2 mcg × 2 0, 14 40 0 0 0 0 0 0 0 7 Lip (CCS / Chol) -HN 2 mcg × 1 0 300 0 600 0 0 0 0 0 8 4 mcg × 1 0 12000 4500 13000 0 0 0 0 0 9 2 mcg × 2 0, 3 15,000 10,000 15,000 2500 3500 0 10 0 10 2 mcg × 2 0, 7 15,000 12000 14000 2500 9000 200 thirty one hundred eleven 2 mcg × 2 0, 14 13000 5500 12000 1800 3000 fifty 0 0 12 F-HN + CT (1 mcg) 2 mcg × 2 0, 7 21000 15,000 20000 2500 2000 250 thirty 45 ^a - see table 8, which describes the experiment. Samples are pooled and tested by IFTP against three viral strains (with combined HN) 42 days after the first dose of the vaccine. 0 means caption <10.

Table 10 Immunity created in young BALB / c mice against viral challenge by intranasal administration of the trivalent influenza vaccine, in free form and when incorporated into combined structures with CCS lipid No. Vaccine ^a (n = 5) Dose Days Lung virus titer
(log 10) ^b one
2
3
four
5
6 No vaccine
F-HN, 4 mcg × 1
F-HN, 2 mcg × 2
Lip (CCS / Chol) -HN, 4 mcg × 1
Lip (CCS / Chol) -HN, 2 mcg × 2
F-HN, 2 μg + CT (1 μg) × 2 -
0
0, 7
0
0, 7
0, 7 6
5.5
5
0
0
0 ^a - See Table 8 for a description of the experiment. In groups 4, 5, the ratio (w / w) lipid / HN is 100/1.
^b - Mice are infected intranasally 42 days after the first dose of the vaccine using ~ 10 ⁶ 50% of the infectious dose for the chicken embryo (EID 50) adapted to the mice of the chimeric virus X-127 (A / Bejing / 262/95 [H1N1] × X-31 [A / Hong Kong / 1/68 × A / PR / 8/34]. The lungs are removed after 4 days, homogenized, diluted serially and injected into allantois of 10-day-old fertilized eggs. After incubation for 48 hours at a temperature of 37 ° C and for 16 hours at a temperature of 4 ° C, 0.1 ml of allantoin liquid is taken and the presence of the virus is checked by the hemag reaction glutination.

In the experiment described in tables 11 and 12, a trivalent influenza vaccine is obtained using CCS / Chol combined lipid structures using varying amounts of HN antigens and lipid. In this experiment, vaccines are prepared using (a) varying amounts of antigen (0.25–2 μg per strain of virus) and lipid (0.075–0.6 mg), keeping the ratio (w / w) lipid / HN constant 100/1; (b) increasing amounts of antigen (0.25-2 μg) and a constant amount of lipid (0.6 mg), which leads to a variation in the ratio (wt./wt.) lipid / HN from 100/1 to 800/1. As can be seen from the data shown in table 11 (titer HI) and table 12 (isotype titers), vaccines obtained at a ratio (wt./wt.) Lipid / HN equal to 100/1, using 2 or 1 μg of antigen each strain and 0.6 or 0.3 mg of lipid, respectively, create high and close in value levels of antibodies against 3 viral strains (groups 2, 3). When using lower doses of antigen (0.5, 0.25 μg / strain) and lipid (0.15, 0.075 mg), the response is markedly reduced (groups 4, 5), especially the response of the mucous membranes (lung / nose) (table 12 ) In the case of using a constant dose of lipid (0.6 mg), high levels of antibodies are achieved even when using two reduced doses of antigen (0.25, 0.5 μg / strain) (groups 6-8). Thus, the amount of CCS lipid is crucial, and when choosing the appropriate dose of lipid, the dose of antigen can be reduced by 4-8 times (from 1-2 μg to 0.25-0.5 μg).

Table 12 The effect of antigen dose and lipid dose on the induction in serum, lung and nasal passages of antigen-specific antibodies IgG1, IgG2a and IgA after intranasal administration of a trivalent influenza vaccine prepared using CCS lipid structures, which is administered twice (with an interval of 1 week) to young BALB / mice c Vaccine ^a (n = 5) HN (mcg) Lipid (mg) The ratio (wt./wt.) Lipid / HN The average value of the titer of antibodies In serum In lung homogenate In the flushing fluid from the nose IgGl IgG2a IgGl IgG2a IgA IgGl IgG2a IgA one F-hn 2 - - 0 0 0 0 0 0 0 0 2 Lip (CCS / Chol) -HN 2 0.6 100/1 15,000 12000 14000 2500 9000 200 thirty one hundred 3 one 0.3 100/1 14000 2500 10,000 1000 8000 one hundred 0 80 four 0.5 0.15 100/1 15,000 1300 8000 1500 4000 0 0 0 5 0.25 0,075 100/1 12000 400 3500 400 2500 0 0 0 6 one 0.6 200/1 20000 15,000 12000 2500 8000 200 fifteen 80 7 0.5 0.6 400/1 15,000 14000 15,000 5000 15,000 150 35 one hundred 8 0.25 0.6 800/1 15,000 9000 21000 2500 13000 250 25 90 ^a - See tables 8, 9, which describe the procedure for conducting the experiment.

In yet another experiment, subunit influenza vaccine, either in free form (HN) or in association with CCS / Chol combined lipid structures (LipHN), was tested for its ability to induce a cross-reaction of HI antibodies with various sub-strains of influenza A and B viruses that are not included in the vaccine. The data in table 13 show that intranasal (i / n) and intramuscular (i / m) vaccination with single or double administration using a monovalent or trivalent CCS-containing influenza vaccine leads to the manifestation of high serum titers of HI antibodies directed against strains used for immunization, as well as the cross-reaction of HI antibodies with several strains of A / H1N1, A / H3N2 and B, which circulated in 1986-1999 and were not included in the vaccine. Slightly lower HI titers were detected after a single and / or a dose of the vaccine (group 6 against group 7). HI titers in lung homogenate (group 4, 8) were lower than the corresponding titers in serum. Thus, parenteral or intranasal vaccination using a CCS-containing vaccine can lead to protection against a wide range of strains of virus A and B. Such antigenic variants can occur during an influenza epidemic / pandemic as a result of antigenic drift. And, on the contrary, the standard commercial vaccine administered by i / n (groups 1, 5) was completely ineffective in terms of inducing antibodies against both homologous and heterologous strains.

Biological distribution of anionic and cationic liposomes loaded with HN and introduced intranasally

In an experiment on the study of biological distribution, intranasally (200 μg of lipid, 2 μg of antigen per mouse) was administered to BALB / c mice 3 preparations of combined lipid structures: DMPC / DMPG (anionic), DOTAP / Chol (cationic) and CCS / Chol (cationic), which are either empty or containing HN influenza antigens. Further, fluorescently labeled lipid is monitored in homogenates of various tissues for 24 hours (at points 1, 5, and 24 hours after administration).

As shown in table 14 below and in FIG. 2A-2F, after 1 and 5 hours, 75-100% of the injected lipid is detected for all three studied compositions. However, the level of such reduction sharply decreases at 24 hours for all compositions except for the CCS-containing composition. This CCS composition containing HN antigens has the highest retention time (> 24 hours) in 3 target organs (nose, lungs, gastrointestinal tract), and lipid accumulation in the brain is not observed, as well as significant accumulation in other organs studied (liver) , kidneys, heart, spleen).

Table 14 Detection of lipid 1, 5, and 24 hours after intranasal administration of fluorescently labeled lipid pooled structures % detection (of total lipid injected) The composition of the lipid structure 1 hour 5 o'clock 24 hours DMPC / DMPG (empty) 100,2 99.3 26.9 DMPC / DMPG: HN 100,2 99.9 8.3 DOTAP / Chol (empty) 107.0 75.1 8.1 DOTAP / Chol: HN 99.9 106,4 6.7 CCS / Chol (empty) 99.6 96.9 74,2 CCS / Chol: HN 101.1 101.5 94.5

The biological distribution of ¹²⁵ I-labeled HN upon administration is similar to that shown for a fluorescent lipid (data not shown). The long-term retention of CCS vaccine components in the airways and gastrointestinal tract can be explained, at least in part, by its higher immunogenicity compared to other liposomal formulations. The same result was obtained in the following study, in which the antigenic component of the vaccine was monitored. HN proteins label ¹²⁵ I and are administered intranasally, either in free form or in association with one of the lipid compositions used in experiments to study the biological distribution of the fluorescent label. After 1, 5 and 24 hours after instillation, the radioactivity of different tissues is determined.

Table 15 shows that the level of antigen recovery in this experiment is also high. As shown in FIG. 3A-3D, the biological distribution pattern of ¹²⁵ I-labeled HN is similar to that for lipid (FIGS. 2A-2F), and it also follows that (a) there is indeed an in vivo association between the HN proteins and the combined lipid structures and (b) prolonged retention antigen in the nose when associated with cationic combined lipid structures can be associated specifically with cationic combined lipid structures and is not a property inherent in HN proteins, since such retention of HN is not observed if only one protein is introduced in soluble form.

This experiment also shows that the accumulation of HN protein in the brain is absent when it is administered either individually or in association with combined lipid structures (the main safety aspect concerns intranasal vaccination). Since the method of tracking a radioactive tag is more sensitive than the fluorescent method, this result is more reliable.

Table 15 Detection of ¹²⁵ I-labeled HN administered intranasally, either individually or in association with pooled lipid structures after 1, 5 and 24 hours Detected level (% of total injected amount) The preparation of combined lipid structures 1 hour 5 o'clock 24 hours Hn 77 48 17 Lip (DMPC: DVMPG) HN 88 fifty 26 Lip (DOTAP: Chol) HN 105 48 32 Lip (CCS: Chol) HN one hundred 74 41

In order to determine whether protein and lipid are retained and / or excreted by a mechanism characterized by the same or different kinetics in different tissues, another analysis of the obtained data was carried out, where the ratio between the% retention of antigen (of the total administered dose) and% retention was determined lipid in various tissues at different time points. If the indicated ratio does not change and is approximately equal to 1, this should mean that both components are kept at a close level in the same organ, whereas if this coefficient is greater or less than 1, this will indicate that the kinetics of the clearance of the components is different and one component can be output faster than another.

As can be seen from Table 16 below, the only coefficient that remains constant over time is the coefficient that determines the CCS / Chol-HN ratio in the nasal passages (ratio coefficient = ~ 0.45). This suggests that (a) the high level of antigen retention in the nose when using CCS and DOTAP correlates with the level of association and is determined by the binding of these compositions to the nasal mucosa, in contrast to DMPC / DMPG; and (b) unlike other compositions whose components disintegrate in the body and are excreted at different rates, the CCS-HN-based composition remains stable, especially in the nasal passages, which may contribute to the higher immunogenicity observed with CCS-containing vaccines.

Table 16 Retention of lipid and HN antigen after i / n administration Lip (DMPC: DMPG) HN Lip (DOTAP: Chol) HN Lip (GCS: Chol) HN 1 hour 5 h 24 h 1 hour 5 h 24 h 1 hour 5 h 24 h Hn Nose 9% four% 2% 38% 16% 2% 41% fourteen% 12% Lungs thirty% 3% four% 19% four% 12% 24% 21% eleven% Gastrointestinal tract 35% 32% eleven% 33% 27% 10% 22% 28% 8% Recovery 88% fifty% 26% 105% 58% 32% one hundred% 74% 41% Lipid Nose 0% 0% 0% 46% 56% 0% 88% thirty% 25% Lungs 67% 80% 5% 38% 3% 3% 12% 35% fourteen% Gastrointestinal tract 33% twenty% four% 16% 47% 3% one% 37% 55% Recovery one hundred% one hundred% 8% one hundred% 106% 7% 101% 102% 95% HN / lipid ratio Nose - - - 0.82 0.29 _ 0.47 0.45 0.47 Lungs 0.44 0,03 0.85 0.50 1.29 3.56 2.01 0.60 0.80 Gastrointestinal tract 1,07 1,56 2.96 2.12 0.57 2.86 16.08 0.76 0.15 Recovery 0.88 0.50 3.12 1.05 0.55 4.78 0.99 0.73 0.44 These data show% retention of the total amount of lipid or HN protein introduced.

Preliminary safety studies of intranasal influenza vaccine

Toxicity (local, systemic) is the main problem both in the case of v / m and / and the introduction of vaccines, in connection with which an appropriate pilot toxicity study was conducted. Cationic lipid compositions (based on DMTAP, DOTAP and CCS) containing hemagglutinin + neuraminidase (HN) influenza antigens are administered i / n (twice, with an interval of 1 week) to mice (n = 4 / group) and after 72 hours they are counted the number of blood cells (complete blood count, differential blood count), conduct a chemical blood test and histological examination (sections from the nose, lung). Mice showed no obvious signs of toxicity. The results of counting the number of blood cells and chemical analysis fall within the range of normal values and, as expected, mice injected with cationic compositions may exhibit an inflammatory reaction in the nasal mucosa and lungs, expressed in the degree from minimal to weak. Some mice that were injected with saline alone or with an unencapsulated antigen showed a similar in nature, but less pronounced inflammatory reaction.

Immunomodulatory activity of CCS-containing influenza vaccine in relation to mice

In the experiments described below, mice were injected with various liposome formulations (consisting of DMPC, DMPC / DMPG, DOTAP / Chol, CCS / Chol), 0.5-1 mg lipid, in the presence or absence of HN antigens. Mice are either left without further exposure, or are injected with thioglycolate (TG, to increase macrophage production) ip 2 days before the injection of liposomal formulations. Peritoneal cells were selected 24-48 hours after the injection of liposomes and used as such or after 4 hours of adsorption at 37 ° C on plastic plates followed by removal of non-adherent cells. In other experiments, peritoneal cells taken from TG-treated mice were incubated with liposome compositions for 24-48 hours. Cells are also examined by flow cytometry to evaluate the expression of the main tissue compatibility system of MHC II, as well as CD40 and B7 molecules that have a joint stimulating effect. Supernatants are examined for the presence of cytokines: interferon γ (IFNγ), tumor necrosis factor α (TNF α) and interleukin 12 (IL-12) and for the presence of nitric oxide (NO).

All cationic compositions (CCS / Chol, DOTAP / Chol, DMTAP / Chol) exert positive regulation of B7 and CD40 expression, to a greater extent than other compositions (DMPC [neutral composition], DMPC / DMPG [anionic composition]), and induce high levels of IFNγ and IL-12. In some cases, the CCS / Chol composition has a stronger effect than other cationic compositions. None of the compositions induce significant levels of TNFα and NO. The increased expression of molecules that have a joint stimulating effect on antigen-presenting cells, and the induction of IL-12 and IFNγ using cationic compositions, can be partially explained by the greater adjuvant activity of such compositions. These results, combined with the fact of prolonged retention of the CCS-influenza vaccine in the respiratory tract (FIGS. 2C and 2F, and FIGS. 3A-3D) after intranasal administration, may explain why CCS is such an effective carrier / adjuvant used in vaccines applied to mucous membrane.

Hepatitis A virus (HAV)

The ability of CCS-containing combined lipid structures to enhance the immune response was investigated using, in addition to the influenza vaccine, also a HAV vaccine administered intranasally (and / n) and intrarectally (and / p).

A HAV vaccine (Aventis Pasteur) in an amount of 10 IU (~ 1.5 μg protein) is administered twice at a two-week interval, and 3 weeks after the second dose is administered, the response is examined by the enzyme-linked immunosorbent assay (IFAS (ELISPOT)). CpG-ODN, used as an adjuvant for the mucosa, is administered in an amount of 10 μg / dose. HAV-CCS combined lipid structures are prepared according to the procedure described above for the influenza vaccine (table 1).

The data shown in table 17 show that while a commercial vaccine is not able to induce an IgA response in both test tissues (lamina propria and Peyer's plaques) and when using different modes of administration (and / n and / p ), the vaccine obtained using CCS or CpG-ONE, in most cases, causes a pronounced response. The combination of the combined lipid structures of HAV-CCS and CpG-ODN in all cases leads to a synergistic response. Thus, CCS-containing combined lipid structures, both individually and in particular in combination with CpG-ONE, are also effective as a carrier / adjuvant for mucosal HAV vaccine.

Table 17 Induction of IgA antibodies after intranasal (and / n) or intrarectal (and / r) administration to mice of BALB / c vaccine with hepatitis A virus (HAV), alone and in combination with CCS-containing combined lipid structures and / or CpG-ODN Vaccine The average number of IgA AFC / 10 ⁶ cells in: Own record (lamina propria) Peyer's Plaques and / n and / p and / n and / p HAV only
Hav-ccs
HAV + CpG-ONE
HAV-CCS + CpG-ONE 0
12
16
139 0
27
22
68 0
0
0
28 0
one
fourteen
23 AFC - antibody-forming cells

C. BOTULINUM

In another experiment, mice were immunized with a / n dose of 0.4 μg of the commercial toxoid C. botulinum (as a model for studying the bioterrorist agent, Uruguay, without aluminum) and the antibody titer was determined by IFTP 4 weeks after the second dose of the vaccine.

The results of the experiment using C. botulinum toxoid are summarized in table 18, which shows the improved properties of the CCS-toxoid composition compared to the standard vaccine in case of i / n instillation, in particular with regard to IgA levels in the small intestine and feces. Such antibodies are expected to neutralize the toxin when administered orally. In mice immunized with a / n administration of one such vaccine, IgA does not form.

Table 18 Induction of IgG1, IgG2a and IgA antibodies in BALB / c mice vaccinated intranasally (twice, 1 week apart) with free or CCS-associated toxoid Clostridium botulinum (CBT) Vaccine ^a n = 10 The average value of the titer of antibodies Serum Small intestine Feces IgG1 IgG2a IgG1 IgG2a IgA IgA CBT
CCS-CBT 0
400 0
24 1000
1600 180
0 0
1800 0
1800

MATERIALS

Chemical aspect

Synthesis of N-palmitoyl-D-erythrosphingosyl-1-carbamoyspermine (CCS)

(i) N-palmitoylsphingosine (1.61 g, 3 mmol) was dissolved in anhydrous THF (100 ml) with heating. The clear solution was brought to room temperature and N, N'-disuccinimidyl carbonate (1.92 g, 7.5 mmol) was added. DMAP (0.81 g, 7.5 mmol) was added with stirring and the reaction mixture was further stirred for 16 hours. The solvent was removed under reduced pressure and the residue was recrystallized from n-heptane to give 1.3 g (68%) of disuccinimidylceramidyl carbonate as a white powder, mp. 73-76 ° C.

(ii) Spermine (0.5 g, 25 mmol) and disuccinimidylceramidyl carbonate (0.39 g, 0.5 mmol) are dissolved in anhydrous dichloromethane with stirring and then treated with a catalytic amount of 4-dimethylaminopyridine (DMAP). The solution was stirred at room temperature for 16 hours, the solvent was evaporated and the residue was treated with water, filtered and dried in vacuo to give 0.4 g (82%) of the crude material, which was further purified by silica gel column chromatography using butanol as an eluent: AcOH: H ₂ O (60:20:20).

(iii) To obtain a compound based on a quaternary amine, the product in step (ii) can be methylated with DMS or CH ₃ I.

The CCS structure was confirmed by ¹ H and ¹³ C-NMR spectrometry (data not shown). A detailed description of the analysis is given in the jointly pending application for international patent No_____.

Other synthesis methods

The following procedures can also be used, as well as the procedure described above:

The synthesis of the linear monosubstituted ceramide-spermine conjugate shown in figa

The ceramide equivalent is reacted with 2.5 equivalents of disuccinimidyl carbonate in the presence of DMAP to give the corresponding 1,3-di-O-succinimidyl derivative.

The disuccinimidyl derivative thus obtained is reacted with spermine equivalent at room temperature using a catalytic amount of DMAP to give the 3-monosubstituted ceramin-spermine conjugate shown in FIG. 1B.

The synthesis of the linear disubstituted ceramide-spermine conjugate shown in figv

The equivalent of the 1,3-di-O-succinimidylsphingoid derivative obtained by the above procedure is reacted with 2.5 equivalents of spermine at a temperature of 80 ° C. in the presence of catalytic amounts of DMAP. In this case, a 1,3-disubstituted CCS is formed.

Synthesis of a linear disubstituted ceramide-branched spermine conjugate shown in FIG. 1C

The equivalent of the 1,3-di-O-succinimidyl ceramide derivative obtained by the above procedure is reacted with 2.5 equivalents of alpha-omega-disubstituted spermine at a temperature of 80 ° C. in the presence of catalytic amounts of DMAP.

The protecting group is removed to give a 1,3-branched disubstituted ceramide-spermine conjugate.

Synthesis of a linear disubstituted ceramide-cyclic spermine conjugate shown in Fig.1D

The equivalent of the 1,3-di-O-succinimidylceramide derivative obtained by the above procedure is reacted with 0.75 equivalents of spermine at a temperature of 80 ° C. in the presence of catalytic amounts of DMAP.

Flu antigens

A monovalent subunit antigen preparation obtained from the A / New Caledonia / 20/99-like influenza strain (H1N1) was kindly provided by Dr. Gluck and Zurbriggen, Berna Biotech, Bern, Switzerland. This drug (designated as HN) includes 80-90% hemagglutinin, 5-10 wt.% Neuraminidase and trace amounts of NP and M1 proteins. Commercial trivalent subunit vaccine (Fluvirin®) for use in the 2003/2004 season containing HN from A / New Caledоnia / 20/99 (H1N1), A / Panama / 2007/99 (H3N2) and B / Shangdong / 7/97 was obtained from Evans Vaccines Ltd. (Liverpool, UK). This vaccine is concentrated ~ x8 before encapsulation (Eppendorf Concentrator 5301 (Eppendorf Concentrator 5301, Eppendorf AG, Hamburg, Germany)). Complete inactivated virus is used in some experiments to stimulate in vitro.

Lipids

Phospholipids (FL) dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dioleylphosphatidylethanolamine (DOPE) are obtained from Lipoid GmbH (Lipoid GmbH, Ludwigshafen, Germany). In addition to liposomes, DMPC (neutral) and DMPC / DMPG (molar coefficient 9/1, anionic) prepare 6 compositions of cationic liposomes / combined lipid structures. Monocationic lipids dimethylaminoethanecarbamoylcholesterol (DC-Chol), 1,2-distearoyl-3-trimethylammonium-propane (chloride salt) (DSTAP), dioleyl-3-trimethylammonium-propane (chloride salt) (DOTAP) and dimyristoylmonium-3-tri (chloride salt) (DMTAP) is obtained from Avanti Polar Lipids (Avanti Polar Lipids (Alabaster, AL, USA)). Monocationic lipid dimethyldioctadecylammonium bromide (DDAB) and cholesterol (Chol) are obtained from Sigma. The new, patented polycationic sphingolipid N-palmitoyl-D-erythrosphingosylcarbamoyspermin (acetate salt) (ceramidecarbamoylspermin, CCS) is obtained from Biolab Ltd. (Biolab Ltd., Jerusalem, Israel). Where indicated, helper lipids (DOPE, Chol) are used in a molar lipid / helper ratio of 1 / 1-4 / 1.

Mice

In the experiment, special pathogen-free (SPF) females were used in 6-8-week-old BALB / c mice and 18-month-old C57BL / 6 mice (5-10 animals in the group). Animals are maintained under conditions conducive to maintaining SPF.

WAYS

Encapsulation of influenza antigen in liposomes / combined lipid structures

HN antigens (see above) encapsulate large (average diameter 0.1-5 μm) heterogeneous (not sorted) vesicles. The following procedure is usually used to prepare all vaccine compositions. Lipids (10-30 mg) were dissolved in 1 ml of tertiary butanol and then sterilized by filtration (GF92, Glasforser, Vorfilter no. 421051, Schleicher & Schuell, Dassel, Germany). The sterile lipid solution is frozen at a temperature of -70 ° C and then lyophilized for 24 hours until completely dried. Dried lipids can be stored at 4 ° C for> 2 years without significant (<5%) lipid degradation or loss of “encapsulation” ability. If necessary, the lipid powder is hydrated with a solution of antigen (in the FBI, pH 7.2) using the ratio (wt./wt.) Lipid: antigen (protein) from 3/1 to 800/1. The antigen solution is added stepwise, each time increasing the added amount by 20-50 μl and vigorously stirred in a vortex mixer after each addition, bringing the total final volume to 0.5-1 ml. In some experiments, dried lipids are hydrated using FBI, and such preformed "empty" pooled lipid structures are mixed with an antigen solution. The mixture is stirred in a vortex mixer for 1-2 minutes and used for 30-60 minutes.

To determine the effectiveness of “encapsulation”, depending on the type of composition, two procedures can be used that achieve ≥80% separation of the free antigen and the antigen associated with the lipid. For all vaccine compositions except CCS, the following separation procedure is used. The combined lipid structures (1-30 mg lipid) containing HN antigen (50-100 μg protein) are suspended in 0.5 ml PBS and carefully layered on 0.5 ml D ₂ O (99.9%, Aldrich Chemical Co. , Milwaukee, WI, USA). Then the specified sample is centrifuged for 1 hour at a temperature of 30 ° C at a speed of 45000 rpm Free unencapsulated HN is precipitated, while the HN and protein-free combined structures / liposomes that are part of the structures (liposomes) remain in the supernatant. All supernatant was collected and the combined structures / liposomes were dissolved by adding 0.2 ml of warm 10% Triton X-100 to the supernatant and sediment fractions. In both fractions, protein concentrations were determined using the modified Lowry technique. In the case of CCS, CCS-HN compositions are suspended in 0.5 ml of PBS-D ₂ O (1 volume of PBS X10 + 9 volumes of D ₂ O), and then mixed with 0.5 ml of PBS. The mixture is then centrifuged for 10 minutes at 20 ° C at a speed of 10,000 rpm. CCS +/- antigen is precipitated, while free HN remains in the supernatant. Dissolution of the lipid and determination of protein in both fractions is carried out according to the method described above. Using both separation techniques, the overall recovery of HN antigens reaches> 95%.

Immunization

Vaccines with free HN (F-HN) and with HN included in the combined structures / liposomes (Lip-HN) containing 0.25-4 μg of antigen / strain / dose and 0.075-1.2 mg of lipid / dose are administered either once intramuscularly (intramuscularly, in an amount of 30 μl), or once or twice intranasally (i / n, 5-50 μl in one nostril) with an interval of 3, 7 and 14 days, or two times orally (in an amount 50 μl) with an interval of 1 week. In all cases, mice were lightly anesthetized using 0.15 ml of 4% chloral hydrate in the FBI by intraperitoneal administration. During oral vaccination, mice are given 0.5 ml of an antacid solution (8 parts of a balanced Henk solution + 2 parts of a 7.5% sodium bicarbonate solution) in their mouth 30 minutes before vaccination. Cholera toxin (CT, Sigma, USA) in an amount of 1 μg / dose was used in all experiments as a standard mucosal adjuvant for comparative analysis. In two experiments, CpG-ODN (ODN 1018, kindly provided by Dr. E. Raz, University of California, San Diego, California, USA), in free form and in the form of liposomes, was used in an amount of 10 μg / dose as an adjuvant.

Evaluation of humoral responses

4-6 weeks after vaccination, serum, lung homogenates and lavage fluids from the nasal passages are examined, analyzing the samples individually or in a combined form, starting with a sample dilution of 1/10 or 1/20. Hemagglutination inhibiting antibodies are determined by a standard hemagglutination inhibition (HI) assay starting with a 1/10 sample dilution. Mice with an HI titer ≥40 (which is considered a protective titer for humans) are considered capable of seroconversion. The amounts of antigen-specific antibodies IgG1, IgG2a, IgA and IgE are determined by the method of IFTP. The highest dilution of the sample, giving an absorption of 0.2 OD higher than the control (antigen + serum from a normal mouse, OD <0.1), is considered as the antibody titer for IFTP.

Assessment of cellular responses

Splenocytes are obtained 5-6 weeks after vaccination and examined for proliferative activity, production of IFNγ and IL-4, as well as for cytotoxic activity after antigenic stimulation in vitro. The cultures were grown at 37 ° C in an enriched RPMI 1640 or DMEM medium supplemented with 5% (for evaluating proliferation, cytokine production) or 10% (for assessing cytotoxicity) of fetal calf serum (FST) with a content of 5 × 10 ^-5 M 2- mercaptoethanol (to assess cytotoxicity) or without it. Cell cultures are treated as follows: (i) to evaluate proliferation, cells are incubated in U-shaped 96-cell plates in an amount of 0.5 × 10 ⁶ cells / cell, in triplicate, with or without antigen (0.5-5 μg per well) in a final volume of 0.2 ml. After 72-96 hours, the cultures are pulsed.

1 μCi ³ H-thymidine for 16 hours. The results are expressed as Δimp / min = (average number of pulses per minute in the case of cells cultured with antigen) - (average number of pulses per minute in the case of cells cultured without antigen). (ii) To determine the level of cytokines, cells are incubated in 24-cell plates in an amount of 2.5 × 10 ⁶ - 5 × 10 ⁶ cells / cell, in duplicate with or without antigen (5-10 μg per cell) in the final volume of 1 ml. Supernatants were harvested after 48-72 hours and tested by IFTFA for the presence of murine IFNγ and IL-4 using the OptEIA kit (Pharmingen, USA). (iii) To assess cytotoxicity: reactive splenocytes (2.5 × 10 ⁶ ) are incubated as described in (ii) for 7 days together with an equal number of BALB / c stimulating splenocytes that were infected with the influenza virus X / 127 ( H1N1) (see description below). The splenocytes used for infection are incubated at 37 ° C, occasionally mixing for 3 hours in RPMI 1640 medium (without FST) with splenocytes having an activity of 150 hemagglutination units per virus / 1 × 10 ⁶ splenocytes, followed by washing. Next, the primed effector cells are re-stimulated for 5 days using infected irradiated (3000 rad) splenocytes with an effector / stimulator cell ratio ј in the presence of 10 IU / ml rhIL-2. Cytotoxicity is determined using a standard 4-hour ⁵¹ Cr release test at an effector / target cell ratio of 100/1. As labeled target cells, unmodified P815 and P815 are used, subjected to pulse treatment for 90 minutes at a temperature of 37 ° C 189-199 with the HA2 peptide (IYSTVASSLVL, 20 μg / 1 × 10 ⁶ cells).

Assessment of protective immunity

Mice under anesthesia are injected with a suspension of live viruses of 25 μl per nostril, ~ 10 ⁷ EID 50 (50% infectious dose for chicken embryo) using the chimeric virus X-127 (A / Beijing / 262/95 (H1N1) × X-31 ( A / Hong Kong / 1/68 × A / PR / 8/34), which is infectious in mice and cross-reacts with A / New Caledonia. The lungs are removed on day 4, washed three times with cold PBS and homogenized in PBS ( 1.5 ml per lung of one mouse, which is considered to be 1/10 dilution.) Lung homogenates from each animal group are combined, centrifuged at 2000 rpm for 30 minutes supernatants are collected at 4 ° C. Serial 10-fold dilutions are performed and 0.2 ml of each dilution is injected in duplicate in allantois chicken eggs with 11-day embryos. After 48 hours at 37 ° C and For 16 hours at 4 ° C, 0.1 ml of allantoin liquid was taken and checked for the presence of the virus in a hemagglutination test (for 30 minutes at room temperature) using chicken red blood cells (0.5 wt.%, 0.1 ml). Virus titer in the lung is defined as the largest dilution of the homogenate of the lung producing the virus in allantoin fluid (positive hemagglutination).

Biological distribution and pharmacokinetics of various fluorescently labeled lipid compositions and radioactively labeled HN antigen

Mice are subjected to a single vaccination using compositions of lissamine-rhodamine-labeled lipid structures, either empty or associated with a trivalent subunit influenza vaccine (HN), in a volume of 20 μl. After 1, 5 or 24 hours, the mice are sacrificed and different organs are selected. The organs are stored at −20 ° C. overnight and the next morning they are homogenized in lysis buffer. Next, 0.2 ml of the homogenate is transferred into Eppendorf tubes, 0.8 ml of isopropanol is added and turned off for 15 minutes to release the fluorescent sample into the supernatant. 50 μl of the supernatant was applied to 384 black plates and the fluorescence level was determined (emission wavelength: 545, excitation wavelength: 596).

In another test, 450 μg of the trivalent HN-containing vaccine (5 ml) is dialyzed against DDW (for desalination) and then concentrated 1000 times to 5 μl. Then the protein is diluted with 0.1 M borate buffer (pH 8.5) to obtain a stock solution containing 450 μg in 15 μl. Next, the protein is labeled at ¹²⁵ I using the Bolton Hunter reagent in accordance with the manufacturer's instructions. Mice were injected with ¹²⁵ I-labeled HN (2 μg) and after 1, 5 and 24 hours the mice were sacrificed, different organs were selected into vials (see FIG. 3) and the level of radioactivity was determined in a γ-counter calibrated at ¹²⁵ I.

The present invention is defined by the following claims, the contents of which should be considered as part of the present description.

Claims (21)

1. A pharmaceutical composition for modulating an immune response in a subject, comprising (1) N-palmitoyl-D-erythrosphingosylcarbamoyl spermine (CCS) and (ii) at least one biologically active molecule, wherein said biologically active molecule is an immunomodulator selected from amino acid molecules, nucleic acid molecules, or low molecular weight molecules. 2. The composition according to claim 1, containing at least one physiologically acceptable carrier. 3. The composition according to claim 1, where the specified modulation includes stimulation or enhancement of the immune response. 4. The composition according to claim 1, where the specified biologically active molecule at a physiological pH value has a total negative dipole moment or total negative charge, or contains at least one region having a total negative charge. 5. The composition according to claim 1, wherein said biologically active molecule is selected from an antigenic protein, antigenic peptide, antigenic polypeptide or carbohydrate. 6. The composition according to claim 1, in which the specified nucleic acid molecule is an oligodeoxynucleotide (ONE). 7. The composition according to claim 1, containing an immunostimulating agent. 8. The composition according to claim 1, in which N-palmitoyl-D-erythrosphingosylcarbamoyl spermine (CCS) forms lipid structures. 9. The composition of claim 8, in which N-palmitoyl-D-erythrosphingosylcarbamoyspermin (CCS) forms vesicles or micelles, or combinations thereof. 10. The composition according to claim 1 for vaccinating a subject against influenza virus. 11. The composition of claim 10, wherein said biologically active molecule is derived from an influenza virus, or it is an analogue of a molecule derived from an influenza virus. 12. The composition of claim 11, wherein said biologically active molecule is a combination of hemagglutinin and neuraminidase (HN). 13. The composition according to any one of claims 1 to 12 in a dosage form suitable for intranasal or intramuscular administration. 14. A method for modulating an immune response in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 13. 15. The method according to 14, comprising intranasal or intramuscular administration of the specified pharmaceutical composition. 16. The method according to 14, including the introduction of an immunostimulating agent at the same time or during a certain period of time before or after the introduction of the specified pharmaceutical composition. 17. The method according to clause 16, including intranasal or intramuscular administration of these immunostimulating agents and pharmaceutical compositions. 18. An influenza vaccine containing N-palmitoyl-D-erythrosphingosylcarbamoylspermin (CCS) in combination with hemagglutinin and neuraminidase. 19. A method of modulating a subject's immune response to an influenza virus, comprising administering to said subject N-palmitoyl-D-erythrosphingosylcarbamoyl spermine (CCS) together with an influenza antigen.
Priority on points: 06/17/2004 according to claims 7, 16 and 17; 06/18/2003, 09/25/2003, 01/21/2004, 02/19/2004 according to claims 1-6, 8-15, 18 and 19.

Images