CZ407492A3 - pharmaceutical composition - Google Patents

Abstract

The solution lies in a pharmaceutical composition capable of dissolving or dispersing in an aqueous medium; the composition consists of a lipophilic active ingredient s pharmacological action in water insoluble and osidic a derivative which is itself water-soluble and acceptable from the pharmaceutical point of view it helps dissolve or dispersing the lipophilic active agent. This osidic effect derivative is based on hydrocarbon interactions the chains of the osid derivative and the lipophilic active substance for non-covalent binding.

Description

Pharmaceutical composition

Field of the invention

The invention relates to pharmaceutical compositions comprising, as an active ingredient, an active ingredient of a hydrophobic nature which is nevertheless soluble in an aqueous solvent. The invention also relates to a process for the manufacture of such pharmaceutical compositions and their use.

BACKGROUND OF THE INVENTION

Many of the compounds that are or could be used in pharmacy are not soluble in aqueous solvents.

This insolubility in aqueous solvents is particularly disadvantageous for substances intended for administration in a physiological aqueous solution of the conventional type, as commonly used in the manufacture of injectables. However, this property is also disadvantageous when administered orally, since it is usually a factor that reduces the efficacy and bioavailability of the active ingredient.

Among the water-insoluble pharmacologically active substances, mention may be made in particular of the lipophilic derivatives of muramyl peptides which have been described in European Patent Specification No. 165,123 of May 10, 1985 and also in European Patent Specification No. 4,512 of March 20, 1979 .

Said products are immunomodulators capable of increasing a specific immunological response, for example, during vaccination, or increasing the non-specific resistance of an organism, for example, to infection.

Because they are hydrophobic in nature, they provide aqueous suspensions that are difficult to disperse to a completely satisfactory degree, especially when the dispersion should be administered by injection. A number of possibilities have been exploited in a known manner to bring these substances into solution or dispersion in an aqueous medium, in particular mechanical means or the use of organic co-solvents or wetting agents.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for dissolving or forming a homogeneous dispersion of these hydrophobic substances in an aqueous solvent, particularly in the case of lipophilic muramyl peptides, but also other active lipophilic substances containing hydrocarbon chains. Indeed, these chains themselves contribute to the low water solubility of these substances and to the instability of their aqueous dispersions.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition capable of forming a solution or homogeneous and stable dispersion in a pharmaceutically acceptable aqueous medium, comprising a lipophilic pharmacologically active substance having at least one hydrocarbon chain of at least 3 carbon atoms and a water soluble osidic derivative. which is pharmaceutically acceptable and capable of ensuring the solubility of the active ingredient, wherein said osidic derivative comprises a hydrocarbon group of at least 3 carbon atoms and one or more hydroxyl groups, at least one of which is substituted with a phosphate or sulfate group, to a lipophilic active ingredient in a ratio sufficient to form a solution or dispersion of the entire composition in an aqueous medium.

Obviously, when a solution or dispersion of the two components of the pharmaceutical composition as defined above is formed, Van der Waals bonds are formed between the hydrocarbon chains of these substances. Often the bonds of this type are the more efficient the greater their amount and thus the longer the hydrocarbon chains. On the other hand, the chain length, especially with respect to the water-soluble osidic derivative, must not exceed the length at which the lipophilicity of the compound would already increase.

By lipophilic is meant that in a two-phase system consisting of an aqueous solution and an organic solution, said substance has a partition coefficient whose value is more favorable to the organic phase. In other words, lipophilic substances are more soluble in an organic solvent than in an aqueous solvent. Lipophilic substances may even be practically insoluble in water.

On the other hand, the term water-soluble has the opposite meaning. The partition coefficient of a water-soluble substance in the two-phase system is more favorable for the aqueous phase.

It is believed that the solubilizing ability of an oside derivative is due to the simultaneous presence of hydrocarbon groups and sulfate or phosphate groups in the derivative. The hydrocarbon chains allow the formation of a Van der Waals-like base with the hydrocarbon chains of the active substance, and the sulfate and phosphate groups allow dissolution of the non-covalent complex thus formed.

In each individual case, the length of the hydrocarbon chain is chosen in the lipophilic substance on the one hand and in the solubilizing water-soluble substance on the other hand so that the ratios are favorable for the formation of the above-mentioned Van der Waals bonds.

The pharmacologically active ingredient may consist of any substance that meets the above criteria.

In general, the lipophilicity of the pharmacological component, either based on the length of the hydrocarbon chain carried by the compound, or, if these chains are short, may be due to the number of chains or a combination of the length of the hydrocarbon chains and their number.

As already mentioned, the active ingredient may be any muramyl peptide derivative bearing a hydrocarbon chain which reduces the water solubility of muramyl peptide. A preferred category of these lipophilic muramyl peptides are the compounds that will be discussed in more detail below.

Another group of preferred compounds are carbohydrate derivatives or analogs, in particular monosaccharide or disaccharide derivatives of lipid A. Some preferred examples of these will also be given below.

Other pharmaceutically active substances can also be made more soluble by the process.

In particular, cyclic peptides from the cyclosporin family, for example, cyclosporin A described by JF Borel et al., Agents Actions 6, 1976, + 68, Immunology 32, 1977, 1017, PJ Tudchka et al., Blood 61, 1983, 318. They may also be cyclic pseudopeptides such as valinomycin or polyene macrolides such as lucensomycin and fat-soluble vitamins, in particular vitamins A, D, Ξ and K ·

Generally, it can be any pharmacologically active substance of the polyene type, mono- or polysaccharide type, polypeptide type, cyclic or non-cyclic, or also a glycopeptide compound carrying at least one hydrocarbon chain of the above type. In particular, the invention relates to the solubilization of pharmacologically active substances whose solubility is not sufficient for medical use on the basis of the intended route of administration, in particular compounds having a hydrocarbon chain which can be linked by Van der Waals-type linkages to the corresponding hydrocarbon chain bound to the above-mentioned type.

As regards the water-soluble derivative of the osidic type, it is to be understood that the term osidic, as used throughout the application, generally refers to any substance that contains one or more carbohydrate moieties in its structure.

In general, any osidic derivative in which the lipophilic-type active substance is desirable to be solubilized, unless the pharmacological properties of the active substance are modified, in particular in terms of loss of efficacy or toxicity, is useful for the purposes of the invention.

The hydrocarbon chains which are bound to the pharmacologically active substance or to the osidic derivative are preferably also lipophilic groups. These are preferably strings of the following groups:

at least one linear hydrocarbon chain of 3 to 20, preferably 4 to 20 carbon atoms, optionally containing one or more ether, ester, thioether, thioester or amide linkages, this linear structure will hereinafter be referred to as a type A lipophilic group, at least one hydrocarbon a branched chain of 5 to 50, preferably 10 to 50, carbon atoms, optionally containing one or more ether, ester, thioether, thioester or amide linkages, this branched structure will hereinafter be referred to as a type 3 lipophilic group.

A preferred category of osidic derivatives for the pharmaceutical compositions of the invention are compounds of Formula I

where

R represents a hydrocarbon group of at least 3 carbon atoms, in particular a type A or B lipophilic group,

R 2 is hydrogen, hydroxy, acetamido, sulfate, sulfamido, phosphate or phosphamido, and R 6, the same or different, is hydrogen, hydroxy, methoxy, sulfate or phosphate, is hydrogen, hydroxy, methoxy, sugar residue ( -ozy) or with osamines the anomeric carbon in the 1-position (C), linked by a glycosidic linkage, the positions C2, C3 and C4 are substituted with groups ^R 2 ^{from R} 3 ^{and R} 4 ^'in said ^{Y 2narnu'} which was previously given for R 1 and R _d and the C 6 position is substituted with R 1 as defined for R, provided that at least one of R ₂ , R 1, R 8,

R 8, or R 8 is a phosphate or sulfate moiety.

Examples of osido derivatives corresponding to formula I include the following compounds:

alpha- or beta-methylglycoside-N-acetyl-4-sulfate-6-OR-2-desoxyglucosamine,

N-acetyl-4-sulfate-6-OR-2-desoxyglucosamine, alpha- or beta-methylglycoside-4-sulfate-6-OR-glucopyranose,

4-sulphate-6-OR-glucopyranose,

6,6'-di-OR-4,4'-disulfate-alpha, alpha * -D-trehalose, wherein R is as defined above.

Obviously, lipophilic groups cannot be of such length or cannot be present in such a total number. that the resulting osidic derivative is itself already insoluble

Preferred osidic derivatives may be selected from the following group of compounds:

alpha- or beta-methylglycoside-N-acetyl-4-sulfate-6-octanoyl-2-desoxyglucosamine,

-. -alpha- or beta-methylglycoside-N-acetyl-4-sulfate-6- (2,3-dipalmitoyl) glyceroyl-2-desoxyglucosamine,

- N-acetyl-4-sulphate-6-octanoyl-2-desoxyglucosamine,

N-acetyl-4-sulfate-6- (2,3-dipalmitoyl) glyceroyl-2-desoxy-D-glucosamine, alpha, or beta-methyltlycoside-4-sulfate-6-octanoylglucopyranoside, alpha- or beta-methylglycoside-4 -Sulfate-6- (2,3-diplalmitoyl) glyceroyl-D-glucopyranoside

4-Sulfate-6-octanoyl-D-glucopyranose,

4-Sulfate-6- (2,3-dipalmitoyl) glyceroylglucopyranose

4,4'-disulphate-6,6'-dioctanoyl-alpha, alpha'-D-trehalose,

4,4'-disulfate-6,6 '- (2,3-dipalmitoyl) glyceroyl-alpha, alpha'-D-trehalose.

Some other classes of compounds that can be used in the practice of the invention will be set forth below without limiting the invention thereto.

The above-mentioned dual type of hydrocarbon groups, which may themselves be lipophilic, can provide the solubility of the complex upon non-covalent association of the pharmacologically active agent and the oside derivative. According to a preferred embodiment, one of the compounds comprises a linear hydrocarbon chain, while the other comprises a branched hydrocarbon chain.

The branched hydrocarbon chain is preferably a chain branched on two adjacent carbon atoms or on two carbon atoms separated by another carbon atom.

The linear hydrocarbon chain should be of sufficient length to link with the branched hydrocarbon chain to allow the formation of non-covalent bonds, in particular Van der

Of the Waals type between at least one branching of a hydrocarbon branched structure and a linear chain.

£

Thus, the association of the pharmacologically active substance and the osidic derivative takes place via their hydrocarbon chains, which in themselves may be lipophilic and which produce non-covalent bonds that are sufficiently strong to maintain the linear hydrocarbon chain between two branches of a branched hydrocarbon chain in a certain position, which is often called a sandwich position.

Preferably, the linear hydrocarbon chain has at least the same number of hydrocarbon atoms as the branched chain hydrocarbon branched residue.

The combination of the osidic derivative as described above confers amphiphilic properties on a pharmacologically active substance which is otherwise lipophilic and water insoluble, thereby permitting the formation of a homogeneous dispersion thereof in a solvent of an aqueous nature.

In particular, the present invention provides any composition which can be dissolved or homogeneously dispersed in an aqueous solvent and which comprises:

1) a pharmacologically active substance, water-insoluble, substituted

lipophilic group A, or

- lipophilic group B,

(2) an osidic derivative bound in a non-covalent manner to the compound of (1), wherein said osidic derivative is itself substituted:

- a lipophilic group of type A when the pharmacologically active substance is substituted with a lipophilic group of type B, or

AND

at least one lipophilic group B in the case where the pharmacologically active substance is substituted by a lipophilic group A, wherein at least one hydroxyl group of the osidic derivative is substituted by at least one phosphate or sulfate group.

In general, any pharmacologically active substance, in particular insoluble in aqueous solvent, capable of binding at least one of the aforementioned type A or type lipophilic group can be used as the pharmacologically active substance in the composition according to the invention.

The process for linking lipophilic groups or hydrocarbon chains to osidic derivatives can be applied in the same way to the binding of like chains to insoluble pharmaceutically active substances.

The pharmacologically active compounds useful as a component of the composition of the present invention include lipophilic derivatives of muramyl peptide of formula II

OH

CO-X-NH-CH-CO-R

HO I i I k

CH

NHCOCH

CH

CH-CO- (A) -Y 2 where n

R @ - is hydrogen or methyl,

R 8 is amino, hydroxy or -OW 4, where it is a hydrocarbon radical having 1 to 10 carbon atoms,

X is an aminoazyl group selected from alanyl, valyl, isoleucyl, norleucyl, leucyl, threonyl, prolyl, glutaminyl, asparaginyl, methionyl, tryptopanyl, phenylanynyl, tyrosyl, or glycyl,

Y is hydroxy, amino or OW ₂ , wherein W ₂ is a C 1 -C 4 hydrocarbon radical, or is a type A or B lipophilic group,

Z represents a hydrogen atom or a type A or B lipophilic group, wherein at least one of Y and Z is in each case a type A or type B lipophilic group, n and n and the same or different are 0 or 1,

A 1 and B 2, the same or different, are groups containing one to three aminoacyl residues, also the same or different, or -NH- (CH ₂ ) -CO-, where p is an integer from 2 to 10.

Particularly preferred compounds are those compounds of formula II wherein n 1 and n ₂ are 0,

Rg is methyl,

R 8 is hydroxy, amino or

On (CH 2) H, where x. is an integer from 1 to 6, with an X throw of 4,

- X is L- or D-alanyl, L-threonyl, L-valyl, when Y is -O-CH ₂ -CHO (R ₇ ) -CH ₂ O (R g) Z is hydrogen, and when Z is

II

-C-CHO (R 6) -CH ₉ O (R g), Y represents a hydroxy group, an amino group or a group as defined above, and R 1, and R 1 are oalmitoyl groups.

A further preferred embodiment of the invention is wherein the compound for use in the compound of formula (II), i.e.

- n-j. and n, 0,

- R- represents methyl,

R 8 represents a hydroxy group, an amino group or a group

On (CH ~) H, where x. denotes an integer from 1 to 6, ^x 1 ' ¹ preferably 4, denotes L- or D-alanyl, L-threonyl, L-valyl, when Y is -O- (CH-),

A is Z is hydrogen and in the case of? -CO (CH ₂ ) _x H, where = 8, Y represents an amino or OW ₂ group, as defined above.

H, where x ₂ = 3, that Z represents the hydroxy group W ₂ has the above

The biologically active agent which can be attached to the above-mentioned osido derivatives to form the soluble or dispersible compositions of the present invention may belong to other groups. For example, it may be an immunomodulator selected from the group consisting of components of bacterial origin, for example bacterial wall components, trehalose derivatives, in particular TDM (trehalose dimycolate products) and L? S liposaccharides having lipid chains on one side and on the other hand, they contain at least one sulfate or phosphate group, of the lipid fractions obtained from said LPS-type substances, in particular it may be the so-called lipid A described in Ernst Th. Rietschel et al., Molecular structure of bacterial endotoxin in relation to bioactivity in Csllular and molecular asoacts of endotoxin reactions, under the control of A. Ncwothy, J. J. Spitzsr and Ξ. J. Ziegler, 1990, Elsevier Science Publishers, Ε. V., Division biomedicale, pp. 15-32, the following is a formula of lipids A, III

CH 3 CH 3 (CH ₂ ) y (CH 2) _X

WHAT

CH?

CH—

I (CH ₂ ) x

CHj

OCC r

_(CH2) y

CHj

CH3

CH χ = 10 y = 12 (III)

- analogues of previous components of bacterial origin or natural, semi-synthetic or synthetic products corresponding to fragments of components of bacterial origin, in particular detoxified derivatives. Particular mention should be made of lipid A derivatives or analogs, whether mono- or disaccharidated, mono- or diphosphorylated or bearing branched or linear hydrocarbon chains, which may still be further substituted by other hydrocarbon linear chains via an ester, thioester, ether or thioether bond, in in the case of synthetic or semi-synthetic products, these substances usually have reduced toxicity compared to that of natural lipid A.

The first group of lipid A analogs or disaccharide derivatives is made up, for example, of the MPL derivative, which is monophosphorylated lipid A (Ribi Immunochem, Research Inc).

They are very hydrophobic substances dispersible in water on a homogeneous dispersion in the presence of the above-mentioned osido derivatives.

These lipid A analogs can be represented by Formula IV

wherein and R * is hydrogen or -PO4, at least one of which is PO4,

R 1 and R 1 and R 2 are CO-CH (OR) - (CH ₂ ) _x -CH ₃ , wherein x is an integer of 5 to 15, preferably 10, and

R represents a CO- (CH 2) -CH 3 group, wherein z y J y is an integer of 5 to 15, preferably 10 or 12.

A second group of compounds are lipid A analogs or monosaccharide derivatives, in particular the MRL 953 derivative described in PL Stuetz et al., Cellular and Molecular Aspects of Endotoxin Reaction, A. Nowotny, JJ Spitzec, EJ Ziegler eds., Elsevier Science Publishers, 1990, pp. 129 to 144.

These analogs and monosaccharide derivatives of lipid A can be represented by the general formula V

HIM-

^R 4 °

NHR ₂ where

R 6 represents a hydrogen atom or a PO 4 group,

R ₂ and R ₃ represent a CO-CH (R) - _{(CH2) χ -ΟΗ 3,} wherein X denotes an integer of 5-15, preferably 10, and R represents a hydrogen atom, a hydroxyl group or a CO- (CH ₂ ) y-CH ₃ , wherein Y is an integer of 5 to 15, preferably 10 to 12, represents a group PO ^ when R ^ has a meaning different from PO ^ or a hydrogen atom or has the meaning given for R ₂ and R ^.

Due to the niophophobicity of some muramyl peptides, it is often difficult to formulate these compounds into galenic pharmaceutical compositions. However, these lipophilic muramyl peptides can also be converted to homogeneous aqueous solutions or dispersions in the presence of the above-described osidics.

The invention also relates to pharmaceutical compositions which can be used to increase the general defense capability of an organism or to increase its defense against a specific antigen.

The weight ratio of the oside derivative to the pharmacologically active substance is preferably in the range of 0.25 to 5, in particular 0.5 to 1.5.

The invention also relates to the oside derivatives of the general formula I as such, if they carry at least one sulfate group and also to a process for their preparation by analogous methods to those commonly used in the synthesis of mono- and oligosaccharides.

By way of example, the process is carried out in several stages, starting with -oza or oligosaccharide, which is then chemically modified to obtain the desired derivative, in particular substituting it at the desired positions. The functional groups at positions 1 to 4 and 6 of the starting materials are usually temporarily protected by the attachment of protective groups such as benzyl, benzelidene or acetyl, these protective groups being introduced specifically and gradually into positions which are not to be chemically modified, consider the different reactivity of these groups.

The introduction of the type A or type B lipophilic groups as mentioned above or even shorter hydrocarbon chains can be advantageously carried out by reacting the unsubstituted hydroxyl groups in a form substituted with a tosyl group with a saturated aliphatic acid salt with %, or reacted in free form with a saturated aliphatic acid chloride or reacted with a carboxylic function of a saturated aliphatic acid in the presence of a coupling aid.

Upon introduction of the chemical modifications at the designated positions, all of the protecting groups are removed from the derivative thus obtained to yield the desired mono- or oligosaccharide derivative.

The introduction of one or more phosphate groups may be accomplished prior to deprotection, for example, by a procedure commonly referred to as the phosphidtriester procedure described in Perich J.W. et al., Tetrahedron Lett., 1988, 29, 2369.

The introduction of one or more sulfate groups may be accomplished by deprotection as described above, in particular using trimethylamine-sulfuric anhydride as described by Ichikawa Y. et al., Carbohydrate Research, 172 (1988) 37 to 64.

The invention also relates to pharmaceutical compositions comprising the above complexes or a mixture thereof dispersed in a physiologically acceptable aqueous solvent.

Preferably, the compositions of the present invention are lyophilized prior to being introduced into an aqueous solution or aqueous dispersion and are therefore available in powder form.

These dry powder formulations are readily reconstituted or dispersed in an appropriate volume of aqueous solvent.

The invention also relates to a method of modifying the solubility of a pharmacologically active compound of the type I described above, the method comprising contacting a type I compound with a type II osidic derivative as described above, provided that, when the compound of formula I is described above. they are brought into contact with water to form a solution or dispersion which can be used as such for the preparation of injectables when sterile water has been used.

The solubilization of the type I pharmacologically active substance is such that a liquid is characterized by only a slight opalescence which can be measured nephelometrically, but this opalescence is often absent.

The liquid thus obtained behaves as a solution and can be administered to the living organism as an injectable solution. As to the dispersion of a substance in an aqueous medium, it is a constant and homogeneous distribution of the molecules of the substance in the medium to form complete or partial solvation of these molecules with water molecules, and is not a dispersion from which the dispersed substance settles over time. In particular, no change in the physical state of the dispersion thus prepared can be observed within a period of 24 hours from the time of dispersion preparation.

The invention also relates to a process for the production of the aforementioned pharmaceutical compositions, the process comprising solubilizing one or more osidic derivatives in a certain amount of an aqueous solvent, adding a predetermined amount of one or more pharmacologically active substances to the solution thus obtained. a suspension in an aqueous solvent or a solution in an organic solvent, if necessary removing the co-organic solvent used, if necessary, lyophilizing the aqueous solution thus obtained.

The process may also be modified by adding one or more osido derivatives to a predetermined amount of one or more pharmaceutically active derivatives under the above conditions.

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Process for the preparation of 6,6'-dioctanoyl-4,4'-disulfonyl-alpha, alpha'-D-trehalose sodium (compounds pp

6,6-octanoyl-2,3,2 ', 3'-tetra-O-benzyl-alpha, alpha'-D-trehalose mmols 2,3,2', 3'-tetra-O-benzyl-alpha, alpha D-trehalose (AF Hadfield, L. Hough, and AC Richardson, Carbohyd. Res., 63, 1978, p. 51), 7.5 mmol of octanoic acid and 7.37 mmol of triphenylphosphine are dissolved in 50 mL of anhydrous tetrahydrofuran THF in an argon atmosphere in the absence of light. The solution was cooled to 0 ° C and a solution of 8.02 mmol of diisopropyl azodicarboxylate in 25 mL of anhydrous THF was added over 15 minutes. The material was purified on silica 60 H under pressure using toluene: methylene chloride: ethyl acetate 30: 10: 5. 2.58 g of an oil were obtained in 90% yield.

- Sodium salt of 6,6'-dioctanoyl-2,3,2 ', 3'-tetra-O-benzyl-4,4'-disulfonyl-alpha, alpha'-D-trehalose

To 1.5 mmol of the product from the previous step in solution in 15 ml of anhydrous DMF was added 15 mmol, 22.1 g of trimethylamine-sulfuric anhydride complex, and the mixture was incubated at 50 ° C for two days in the absence of water. Then 10 ml of methanol and 10 ml of chloroform were added and the mixture was passed through a Sephadex LH _2Q column using a 1: 1 mixture of chloroform and methanol. After evaporation, the material was purified under pressure on a 60H silica column using chloroform / methanol / water 15: 5: 0.5 to give 1.7 g of an oil. This oil is dissolved in 80 ml of a 1: 1 mixture of DMF and water and the solution is passed through a column of 85 ml of Dowex 50 WX4 H ⁺ resin, eluting with the above solvent mixture and neutralizing the material with 0.1N sodium hydroxide and lyophilizing. yield 1.51 g (87%).

Sodium salt of 6,6'-dioctanovl-4,4'-di-O-sulfonyl-alpha, alpha'-D-trehalose ^C 28 ^H 48 ^Na 2 ° 19 ^S 2 ' ^{m e} sp = 798.79.

Dissolve 1.5 g of the derivative from the preceding step in

150 ml of a 6.5: 10 mixture of water and ethanol and then hydrogenated in the presence of 750 mg of 10% palladium on charcoal. The mixture was filtered, evaporated and the material was lyophilized from an aqueous solution. The material was then purified on 60-G silica using a 40: 20: 8: 2 mixture of chloroform, methanol, acetic acid and water. After evaporation, further evaporation from the toluene solution was freeze-dried from the aqueous solution. The product was passed through a column of Sephadex L12 in methanol. The appropriate fractions were evaporated, lyophilized from aqueous solution to give 525 mg of the product in 51% yield.

The sulfate content is 2.6 eq / g (conductometry).

The octanoic acid content was 1.97 equiv / mol (CPV).

Example 1

1) Preparation of an aqueous dispersion of MDP-GDP, i.e., alpha- (N-acetylmuramyl-L-alanyl-D-isoglutamine) -beta-gamma-dipalmitoyl-sn-glycerol by treatment with compound P 1

A first embodiment of mg P4 _is dissolved in 10 ml of distilled water. 10 mg of MDP-GDP was added with vigorous stirring. The obtained aqueous dispersion has an opalescent appearance and does not settle or settles only slightly, then a further homogeneous dispersion is obtained by further mixing. After lyophilization, a dispersion with the same properties can be recovered by adding water or saline.

- Second version

Dissolve 2.20 mmol, 57 mg of MDP-GDP in 2 mL of chloroform to give solution A. 20 mg of compound P4 is dissolved in 1 mL of chloroform to give solution B.

The chloroform solutions A and B are combined. Distilled water (5-10 ml) was added and a stream of nitrogen was bubbled through the solution until the chloroform had completely evaporated.

In this way, a homogeneous, stable, slightly opalescent aqueous dispersion is obtained.

The aqueous dispersion is lyophilized and the obtained powder is dissolved in an aqueous solvent at the desired concentration. In this way, a dispersion having the same appearance and properties as the above-mentioned dispersion is obtained.

This procedure allows to obtain galenic formulations of lipophilic pharmaceutically active substances corresponding to the general formula II. Pharmaceutical compositions are obtained in which the pharmacological activity of the compounds is maintained, in particular the ability to control the immunological response as described in European Patent Specification No. 165 123.

2) Effect of the use of P ^ on the efficacy of MDP-GDP in guinea pig

a) Preparation of an emulsion mg of Compound P4 is dissolved in 0.5 ml of saline and this solution is added dropwise to 1 mg of MDP-GDP powder.

The dispersion obtained is mixed with 0.5 ml of an isotonic solution of ovalbumin containing 20 mg / ml of ovalbumin. Add 1 ml of mineral oil (AIF) incomplete Freund's adjuvant in incomplete form to form an emulsion containing 0.5 mg of incomplete adjuvant in 1 ml,

0.5 mg P 5 and 5 mg ovalbumin.

(b) Pharmacological tests

Control animals are given: (a) only water and mineral oil emulsions containing the antigen; and (b) the same emulsion containing only MDP-GDP. The test animals are administered an emulsion containing a further osid derivative P 1. Injection is carried out subcutaneously in the plantar area of the hind paw of male Hartley guinea pigs weighing 350 g. After 3 weeks, the animals are treated intradermally with either 0.1 ml of saline or 0.1 ml of saline with ovalbumin. After 48 hours, the diameter of the swelling, which is a sign of late hypersensibility, is measured. After 24 hours, the animals are sacrificed by cardiac puncture and blood serum is collected. Blood titration of antibodies is performed by the classical Elisa procedure of F. Audibert, L. Chedid, P. Lefrancier and J. CHOAY, Immunol., 1976, 21, 143 and M. Jolivet, F. Audibert, H. Gras-Masse A. Tartar, D. Schlessinger, R. Witz, and L. Chedid, Infect. Immunol., 1987, 55, 1498.

(c) Results

The results are summarized in the following table:

Table

immunisace ^x retarded hypersensitivity Elisa antibody titer AIF 0.0.0.0 22.000, 37.000 65,000, 95,000 AIF-P 100 yUg 0.0.0.0 7,000, 8,000, 21.000 AIF + MDP-GDP 100 µg 8,14,15,17 241.000, 295.000, 325,000, 641,000 AIF + MDP-GDP 100 / Ug + Unit 100 / Ug 5,16,20,25 157.000, 275.000, 275,000, 318,000.

All animals received 1 mg of ovalbumin. The diameter of the swelling is given in mm.

The data represent the maximum dilution of sera at which an optical density corresponding to the optical density of normal serum diluted 50-fold can be obtained.

The results show that MDP-GDP is a very effective adjuvant for enhancing cellular response and humoral response and that adding compound P1 does not alter the efficacy of the compound.

3) Effect P ^ osidového derivative on immunostimulatory properties of muramyl peptides and - Infection efficiency Swiss mice were administered Compound P-MDP-GDP or MDP-GDP + P intravenously 24 hours before infection of ¹ x 10 4 cells of Klebsiella pneumoniae. Solution P4 is added to MDP-GDP to give a composition with a mass equivalent amount of both.

The results shown in Table I show that compound Ρ-j- by itself does not protect against infection. Comparison of the efficacy of different doses of MDP-GDP alone or mixtures of MDP-GDP with P4 shows no difference in efficacy when using the compound as such or a mixture thereof with P4.

From the results, it can be concluded that Compound P 1 does not inhibit the effects of MDP-GDP in increasing resistance to infectious diseases.

b - Effect of MDP-GDP on TNF production according to its content in circulating blood

TNF, i.e., tumor necrosis factor, is a protein that is synthesized by cells from the monocyte / macrophage family when stimulated. LPS, i.e. bacterial lipopolysaccharide, is a substance that very effectively stimulates TNF production. Muramyl peptides stimulate TNF production in vitro, but are somewhat less effective than LPS. In vivo, the amount of TNF in the serum after administration of muramyl peptides is below the detection limit.

MDP-GDP and other derivatives with a pro-infective effect may increase the response to LPS given after administration, thus significantly increasing the amount of TNF in the blood serum. The results of the corresponding experiments are shown in Table II below. Obviously, administration is most effective hours prior to LPS administration, particularly in the case of co-administration of MDP-GDP + P ^, but even 16 hours prior to LPS administration, MDP-GDP injection has a demonstrable effect on potentiation of the response.

c) - In vitro action of MDP-GDP on monocytes in human blood

Human blood monocytes are isolated and incubated for 24 hours in the presence of various doses of product. Supernatants were assayed for TNF and IL-6, cytokines produced by activated monocytes.

In Table III, it is apparent from the results shown that significant amounts of TNF are produced only at 10 µg / ml MDP-GDP and that the product solubilized by compound P ₁ has somewhat higher efficacy. The values for IL-6 are somewhat more obvious and it is also possible to demonstrate comparable monocyte stimulation for MDP-GDP per se and for solubilized material by P 1.

d) - Influencing polynuclear cells in humans

Muramyl peptides have a direct effect on polynuclear cells, as evidenced by phagocytosis and Candida albicans destruction.

Determination of this effect can be demonstrated based on the capacity to incorporate glucose by the remaining yeast cells.

In the two assays whose results are summarized in Table IV, control cells were not stimulated with somewhat increased activity. However, the addition of muramyl peptide alone or together with P 1 increases the inhibitory activity of cells on yeast growth.

Thus, it can be concluded that in vivo experiments in mice or in vitro on cells of human origin do not result in a decrease in efficacy in the solubilization of MDP-GDP with compound P P. Compound P4 alone is always ineffective.

Table I

Protective effect of MDP-GDP solubilized by compound P ^ in mice in Klebsiella pneumoniae infection

i.v. den 1 Day 15 survival % protection * control 1/16 Pj. 100 / Ug 0/8 MDP-GDP 3 / Ug 3/8 37.5 ns 10 / Ug .3 / 8 37.5 ns 30 / Ug 12/16 68.7, P <0.01 100 / Ug 4/8 37.5 ns 14DP-GDP / P ₁ 3/3 / Ug 3/8 37.5 ns 10/10 / Ug 3/8 37.5 ns 30/30 / Ug 8/16 43.7, P <0.02 100/100 / Ug 7/8 75, P < 0.02

In each case, statistical calculations were performed separately compared to the respective control.

ns = statistically insignificant result.

Table II

The effect of pretreatment with MDP-GDP, solubilized with compound P ^, on the increase in TNF levels in blood serum after induction by the addition of LPS

treatment with 100 µg i.v. mice, before administration LPS LPS i. 25 / Ug v. serum TNF efficacy U / ml / Ug / ml h-6 _{χ χ} + 189 .> 3,4 h-6 MDP-GDP / P 1 - / 10 / 0,18 h-6 MDP-GDP + 11 475 206.55 h-6 MDP-GDP / P ₁ + 19 038 342.68 h-16 MDP-GDP + 1 899 34.18 H-16 MDP-GDP / P ₁ 4 296 77.33

hours after injection of the LPS-containing sample.

Efficacy is expressed in units / ml (U / ml) compared to an equivalent amount of recombinant TNF mice in a cytotoxicity assay using cell line L292.

Table III

Treatment of MDP-GDP in vitro on human-derived monocytes active substance µg / ml activity in TNF U / ml IL-6 U / ml supernatant

^P 1 0 <10 MDP-GDP 0.1 0 <10 0.1 0 410 1 0.3 <10 10 6.9 \ 94,9 MDP-GDP / P ₁ 0.01 0 <10 0.1 0 <10 1 5.9 83 10 10.5 594

* Μ £

Cells were used at a density of 2 x 10 ml, incubated with the individual products for 24 hours.

The potency of the supernatant is given in units / ml (U / ml).

Table IV

Effect of NDP-GDP on polynuclear cells of human origin active substance / µg / ml inhibition of Candida albicans growth in%

1.pokus 2.pokus

control 35.3 41.5 ^? 1 10 34.6 38.7 MDP-GDP 0.1 44.1 47.2 1 75.1 78.1 10 86.2 86.1 MDP-GDP / P ₁ 0.1 32.2 77.2 1 41.4 93.6 10 65.9 95.1

Polynuclei cells at a density of 1 x 10 ⁵ ml were incubated with the active ingredients for 30 minutes, and then were divided into wells of a microtiter plate with suspension 4.

C. albicans with a cell density of 1 x 10 / ml. After 18 hours 3, the culture medium was removed, H-D-glucose uptake by viable yeast cells was then measured after an incubation time of 3 hours.

4) Effect of osidic derivative P ^ on pyrogenic effect

MDP-GDP

Pyrogenicity tests were conducted in New Zealand rabbits weighing 3-4 kg, kept in cages. Animals were dosed with 0.5 ml / kg intravenously at zero time and the temperature of the animal was recorded continuously for 5 hours.

a) Evaluation of pyrogenicity P ^

Two groups of 3 rabbits were used:

- Group I: 10 µg / kg P ^,

Group II: 100 µg / kg P 1.

The following temperature differences were noted:

- Group I: 0,15, 0,25 and 0,25 ° C,

The average value for Group I is 0.22 + 0.06 ° C.

- Group II: 0.40, 0.15 and 0.30 ° C.

The average value for Group II is 0.28 + 0.12 ° C.

Accordingly, Compound P 1 had essentially no pyrogenic effect in the rabbit, even with continuous observation over 5 hours.

b) Increased pyrogenicity of MDP-GDP in the presence of P 1

The minimum pyrogenic dose for MDP-GDP that has been predetermined is 2 µg / kg.

For administration, a dose of 10 µg / kg was chosen at which the temperature was increased by approximately 1 ° C.

Two groups of 3 rabbits were created:

- Group I: 10 µg / kg MDP-GDP

Group II: 10 µg / kg MDP-GDP + 10 / µg / kg P 4

The following temperature differences were noted:

- Group I: 0,70, 0,50, 0,85 ° C,

The average value for Group I is 0,68 + 0,18 ° C,

Group II: 1.15, 0.90, 0.65 ° C.

The average value for Group II is 0.90 + 0.25 ° C.

By unpaired Student's t at p = 0.2866, it can be shown that the difference is not statistically significant and that, therefore, compound P1 does not exhibit a synergistic pyrogenic effect when administered with other products, such as MDP-GDP.

The dispersed pharmaceutical compositions obtained as described above have the same pharmacological properties as those described for the dispersions of the NDP-GDP-P1 compounds. Further data will be given in Example 5, which is particularly interesting in pharmacological test results using MDP (Thr) ¹ -GDP and P4 dispersible compositions.

Example 2

A process for the preparation of a pharmaceutical composition dispersible in an aqueous solution based on alpha- (acetylmuramyl-L-threonyl-D-isoglutamine) -beta, gamma-dipalmitoyl-sn-glycerol (MDP (Thr) ¹ -GDP).

This dispersion in the presence of compound P _{1 e} j can be obtained under exactly the same conditions as in the case of MDP-GDP.

Example 3

A process for the preparation of a pharmaceutical composition dispersible in aqueous solution containing a monophosphorylated lipid A analogue.

2.1 mg of monophosphorylated lipid A derivative (MPL, Ribi Immunochem. Research, Inc) was dissolved in 0.5 ml of chloroform to give solution A. 3.8 mg of P4 was dissolved in ml of chloroform to give solution B.

The chloroform solutions A and B are combined. Up to 3 ml of distilled water was added and a stream of nitrogen was bubbled through the solution until the chloroform was completely evaporated.

In this way a homogeneous, stable aqueous dispersion is obtained which is slightly opalescent.

The aqueous dispersion is lyophilized and the powder obtained is dissolved in an aqueous solvent at the desired concentration. In this way, a dispersion of the same appearance and quality as the original dispersion was obtained.

Biological properties of aqueous dispersion of MPL and compound P ^ Antibacterial activity

MPL was dissolved according to the manufacturer's recommendations (Ribi Immunochem. Research, Inc). A 4 mg / ml solution of MPL is then prepared from a solution of 20 mg of MPL in a 4: 1 mixture of chloroform and methanol, and then a solution containing 2 mg / ml of MPL is added by adding a calcium-free saline solution.

The bioassay was carried out as described in Example 1 (3a).

Compound P1 does not alter the protective effect of MPL against K. pneumoniae infection. The results obtained are shown in Table V below

Table V

Protective effect of MPL (Ribi Immunochem. Research Inc) individually or in the presence of compound P. against infection, i

induced by a large dose of 4 x 10 K. pneumoniae in Swiss mice active substance survival dose / number of animals after 15 days

control 0/8 MPL 0.1 mg 0/8 MPL 1 mg 4/8 MPL 10 mg 7/8 MPL / P ^ 0.1 mg 1/8 MPL / P-1 1 mg 4/8 MPL / Pj 10 mg 8/8

Example 4

Aqueous Dispersion of Monosaccharide Analog Lipid A, MRL-953 (Monosaccharide Monophosphorylated Analog) of Formula VI with Compound P 1

(VI)

a) Preparation of the dispersion

Dissolve 10.65 mg of MRL 953 in 0.5 ml of chloroform to form solution A. 16.91 mg of PRL are dissolved in 0.5 ml of chloroform to form solution B.

The chloroform solutions A and B are combined, 2-3 ml of water are added and nitrogen is bubbled from the bottom of the vessel until the chloroform is completely evaporated to give a homogeneous, stable, slightly opalescent aqueous dispersion.

The aqueous dispersion is lyophilized and the powder obtained is dissolved in an aqueous solvent at the desired concentration. In this way, a dispersion having the same appearance and properties as the original dispersion is obtained.

b) Biological properties of the aqueous dispersion of MRL 953 and P 1

Antibacterial efficacy

The protective effect against K. pneumoniae infection was evaluated in Swiss female mice aged 5 to 6 weeks.

Mice were injected intravenously in the evening prior to infection:

- 0,2 ml saline,

- LPS (lipopolysaccharide from Salmonella enteritidis) in suspension in 0,2 ml saline,

- 0.2 ml (1) of an aqueous dispersion of P + MRL 953 containing 30, 10 or 1 µg of MRL 953,

2) dispersion of MRL 953 in glucose solution containing 30, 10, 3 and 1 mg of MRL 953.

K. pneumoniae infection is accomplished by intravenous administration of 0.2 ml of a suspension of bacteria containing 4.5 x 10 bacteria. The death of the animals is recorded 2 weeks after infection.

The results obtained are shown in Figures 1a and 1b and demonstrate that the protective effect of MRL 953 is substantially the same when using any dispersion. In the drawings, account is taken of the variation in the number of surviving mice as a percentage of the ordinate axis as a function of the time T applied to the abscissa axis in days. The test substances LPS and MPL 953 were administered at the doses indicated on the right side of the drawing for

- the glucose solution of Fig. 1a,

a solution in the presence of the oside derivative P 1 in Fig. 1b.

It is apparent from the drawing that the protective effect of the test substances, in particular MRL 953, against K. pneumoniae infection in mice is approximately the same in both cases. When MRL 953 is administered in the form of a dispersion together with compound P 1, the effect is somewhat higher.

Example 5

Influence of dispersion according to the invention on biological properties of the third active substance

The association of some muramyl peptides with an osido derivative may have interesting effects on the third active ingredient, namely

- enhancement of the biological effect of the third substance,

- reduction of toxicity of the third substance,

- both effects mentioned.

For example,

- a significant increase in the antiviral effect of interferon is observed when the active substance is administered after administration of the MDP (Thr) -GDP complex with compound P1 under conditions in which an aqueous dispersion may form,

a decrease in the synergistic toxic effect of LPS with muramyl peptide, inducing pyrogenic effects, for example MDP-GDP and Compound P1, can be observed under conditions that correspond to those in the previous examples. For example, doses of LPS, which are lethal doses to the mouse when administered with LPS alone, are no longer lethal when co-administered with the MDP-GDP complex with the Pβ compound.

In other words, the invention also relates to the administration of the pharmaceutical compositions of the invention in order to alter the biological properties of the third substances which are administered simultaneously or sequentially.

This effect can be further elucidated by the following examples, which illustrate individual cases of action on third substances, but are not intended to limit the invention.

1) Effect of compound P 1 on the immunostimulatory properties of lipophilic muramyl peptides

a) Increasing the antiviral effect of interferon by action

MDP (Thr) ¹ -GDP

The experimental model for this test is encephalomyocarditis in mice. Male Swiss mice weighing 17 g were used as experimental animals. Animals were administered intraperitoneally with either an aqueous suspension of MDP (Thr) - GDP, or an aqueous dispersion of the MDP (Thr) - GDP complex with P 1.

After 24 hours, encephalomyocarditis virus was administered intraperitoneally at a dose of 100 x DL 50. 1 hour after administration, some animals received 25,000 U.I intraperiitoneally. (International Units) interferon alpha, beta-mouse origin.

Table VI summarizes the results obtained. These results indicate that the use of compound P does not decrease, but in turn, increases the antiviral effect of MDP (Thr) -GDP as well as potentiating the antiviral effect of interferon by this lipophilic muramyl peptide.

í ž

Table VI

Enhancement of the antiviral effect of interferon alpha, beta in encephalomyocarditis virus infection in mice active substance i.p. day 1 interferon% alpha survival, beta control

P ^ (600 / Gg)

MDP (Thr) ^{* 1} -GDP (600 / µg) | P ₁ (600 / Ug) + | MDP (Thr) ¹ -GDP (600 / µg) +

P ^ (600 / ug) +

MDP (Thr) ¹ -GDP (600 / Ug) +

IMDP (Thr) ¹ -GDP (600 / ug) +

O

b) Effect of lipophilic muramylpeptide pretreatment on the rise in circulating TNF after injection of LPS or synthetic lipid A analogue

Swiss mice were injected intravenously:

0.2 ml saline,

MDP-GDP in suspension in 0.2 ml saline,

MDP (Thr) ^ - GDP aqueous suspension without additives or in an aqueous dispersion together with a compound Ρ. ¹ and

- Murabutide in solution in 0.2 ml saline. 'í

i i

and

Pre-treatment is performed by intravenous injection 6 hours prior to injection of S. enteritidis LPS in saline or MRL 953, in an aqueous dispersion in saline in the presence of compound P 1.

Animals are bled from the abdominal aorta for 1.5 hours to 2 hours of the last injection at which the highest effect occurs. TNF activity is evaluated by a cytotoxicity assay that measures the number of viable cells. The unit of TNF represents the concentration that produces 50% cytotoxicity under the given conditions.

Aqueous suspensions of lipophilic MDP stimulate TNF response to stimulation by LPS or aqueous dispersion of MRL 953 in the presence of P 1. This effect is somewhat more pronounced when using an aqueous dispersion of lipophilic MDP in the presence of compound P 1.

The results obtained are summarized in Table VII below.

Table VII

Effect of lipophilic MDP pretreatment on serum TNF levels after injection of LPS or synthetic lipid A analogue (MRL 953)

active ingredient i.v. 6 h. Before the experiment substance used to elicit a response LPS 2 / Ug 53 / P ^ 300 / Ug TNF efficacy U.I./ml 1.5 h. 2 h. MRL 9 - LPS 879 - MDP-GDP 100 / Ug LPS 2,947 - MDP (Thr) ¹ -GDP 100 / Ug LPS 4,235 - - LPS 900 - MDP (Thr) ¹ -GDP 100 / Ug LPS 3,311 - MDP (Thr) ¹ -GDP 100 / Ug l ^{+ P} 1 LPS 7,347 - - MRL953 / P ₁ 4 10 4 10 MDP (Thr) ¹ -GDP 100 µg MRL953 / P ₁ 1,089 488 IMDP (Thr) ¹ -GDP 100 / Ug l ^{+ P} 1 MRL953 / P ^ - 1,838 Murabutid 100 µg MRL953 / P ₁ - 1,150 - MRL953 / P- ₁ 76 - Murabutid MRL / P ₁ 820 -

*

c) Reduction of the synergistic toxic effect of LPS and MDP-GDP in the presence of compound P 1

MDP-GDP in suspension in 0.2 ml saline without any additives or in glucose solution in the presence of pluric acid (F 58 SERVA), or in dispersion in 0.2 ml saline in the presence of compound P ^ is administered intravenously with LPS from S. enteritidis to adrenalectomized Swiss mice.

Death is recorded after 3 days, but death occurs within 24 to 48 hours.

Suspension of MDP-GDP in saline or glucose solution together with pluric acid increases the toxicity of co-injected LPS. Dispersion of MDP-GDP in the presence of P 1 reduces this effect.

This reduction in toxic effect is not a direct effect of compound P ^ on LPS, whose toxicity in a normal mouse in the presence of P ^ remains (results not shown).

It should be noted that the results of interest are that the osidic derivative could be used in vivo for a large number of lipophilic substances, for example lipophilic muramyl peptides such as MDP-GDP, which may have some pyrogenic effect without producing a synergistic effect. toxic effect in vivo with LPS of infectious microorganisms that may be present in the host.

The results are summarized in the following table

VIII.

Tables 1 to VIII

Reduction of the synergistic toxic effect of LPS and MDP-GDP in the presence of P ^ medium MDP-GDP LPS death / g number 25 lg animals

physiological saline solution + + + 0/18 100 ALIGN! 300 300 7/18 17/18 0/18 p <0.0 2 p <0.01 glucose solution acid Pluronic F68 - + 0/18 100 ALIGN! + 8/18 p <0.01 300 + 17/18 p <0.01 300 - 1/18 aqueous dispersion, in the presence of P ₁ - + 0/18 100 ALIGN! + 0/18 300 + 6/18 p <0.05 300 - 0/18

Represented by:

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Claims (5)

PATENT CLAIMS A pharmaceutical composition capable of solubilizing or dispersing a homogeneous and stable solution or dispersion in a pharmaceutically acceptable aqueous medium, comprising a lipophilic, pharmacologically active substance having at least one hydrocarbon chain of at least 3 carbon atoms and a solubilising osidic derivative , water-soluble and pharmaceutically acceptable, comprising a hydrocarbon group of at least 3 carbon atoms and one or more hydroxyl groups, at least one of which is substituted with a phosphate or sulphate group, the pharmaceutical composition comprising the osido derivative relative to the lipophilic substance with pharmacological an effect in a ratio sufficient to form a solution or dispersion of the entire composition in an aqueous medium. 2. A pharmaceutical composition according to claim 1, wherein the lipophilic pharmacologically active substance is a compound of the saccharide, peptide or polypeptide type, cyclic or non-cyclic, glycopeptide, cyclic or non-cyclic with at least one hydrocarbon chain of at least 3 carbon atoms. Pharmaceutical composition according to claim 1 or 2, characterized in that the hydrocarbon structure comprises at least one linear hydrocarbon chain of 3 to 20, preferably 4 to 20, carbon atoms, optionally containing one or more ether, ester, thioether, thioester or aitic linkages, this linear structure will hereinafter be referred to as a linear lipophilic group of type A or amide linkages; referred to as lipophilic group type 3. Pharmaceutical composition according to claim 1 or 3, characterized in that it comprises, as the lipophilic component, a pharmacologically active lipophilic muramyl peptide. Pharmaceutical composition according to claim 4, characterized in that it contains a compound of the formula II as a pharmacologically active ingredient.