CA2087536A1 - Compositions based on active ingredients of hydrophobic drugs that can be sulubilized in an aqueous solvent - Google Patents

Abstract

The invention relates to any composition caracterized in that it is comprised of: on the one hand, an active principle of a medicament or a pharmacologically active substance, water insoluble, said substance being substituted by at least one lipophylic group, and on the other hand, an osidic derivative of which at least one of the hydroxyl functions is substituted by one or a plurality of phosphate or sulphate group or groups, said compound being also substituted by a lipophylic group, the composition being capable of homogeneous solubilization or dispersion in an aqueous solvent. The invention also relates to the use of such compositions, particularly as pharmaceutical compositions dissolved in a physiologically acceptable aqueous solvent.

Description

WO 92/0147 ~ PC ~ r / FR91 / 00595 ~ 7 ~ 3 ~

COMPOSITIONS BASED ON ACTIVE INGREDIENTS OF MEDICINES '-HYDROPHOBES SOLUBILIZABLE IN AN AQUEOUS SOLVENT

The invention relates to compositions comprising active ingredients in hydrophobic drugs, nevertheless soluble in an aqueous solvent.
The invention also relates to a method obtaining such compositions, and their uses in particular as compositions pharmaceutical.
Many products used in the field pharmaceutical, or likely to be, cannot be dissolved in an aqueous solvent.
This insolubility in an aqueous solvent is particularly disadvantageous for products -intended to be administered in a solution physiological aguous of the type of those usually used for injections. She also for products intended to be taken orally and mistletoe have efficacy and reduced bioavailability.
Among these pharmacologically active products and insoluble in an aqueous solvent, there may be mentioned in particular, the lipophilic derivatives of mura ~ yl-peptides, such as those described in European patent n-0 165 123 of 05/10/1985, or in the European patent 4512 dated 20/03/1979.
In fact, these products are immunomodulators able to increase specific immune response (vaccine adjuvant), or to increase resistance non-specific (anti-infective agent).

:,. . ~: ~,, Very hydrophobic in nature, they give aqueous suspensions difficult to disperse so completely satisfactory (especially with regard to level of dispersion compatible with the possibility inject them into a patient) l using the various means for dissolution or aqueous dispersion, bases on known techniques, in particular mechanical treatments, or the use of organic solvents or surfactants.
The object of the present invention is precisely to provide means for solubilization or homogeneous dispersion in an aqueous solvent of products hydrophobic, such as lipophilic muramyl-peptides, or other active drug ingredients lipophilic, carriers of hydrocarbon chains.
These sometimes themselves contribute to the low solubility of these products in water, or even the instability of their aqueous dispersions.
The invention relates generally to a composition capable of being solubilized or homogeneously and stably dispersed in a pharmaceutically acceptable aqueous solution, characterized by the association:
- on the one hand, a lipophilic active principle of drug or "pharmacologically active substance", comprising at least one hydrocarbon group comprising itself at least 3 carbon atoms, and - on the other hand, of a solubilizing osidic drift, itself water-soluble, pharmaceutically acceptable, itself carrying a hydrocarbon group comprising at minus 3 carbon atoms and comprising one or several hydroxyl functions, of which at least one is substituted by a phosphate or sulfate group, this osidic derivative being in a sufficient proportion compared to the lipophilic active ingredient for ,,. .-'. ~ .:, '. ''.: '~''''',.''-~ 087 ~ 3 ~
promote the solubilization or dispersion of the whole composition in a solution aqueous.
Dissolution or dispersion of the two above-defined constituents of the composition made apparently intervene the constitution of bonds of Van der Walls type between their hydrocarbon chains respective. Often the connections of Van der Walls will be all the more effective as they are numerous and therefore, that the hydrocarbon chains will be longer. It goes without saying that these lengths of chains should not, especially when it comes to the water-soluble sugar derivative, exceed those which would increase his lipophilicity too much.
It is understood that in the foregoing and what follows, "lipophilic" should be understood as meaning that the substance concerned is present in a system two-phase (aqueous solution - organic solution) a partition coefficient favorable to the phase organic, in other words the lipophilic substance is more soluble in organic solution than in the aqueous solution. The lipophilic substance can even be practically insoluble in water.
Conversely, and in the present description, the adjective "water-soluble" has the opposite meaning.
The partition coefficient of the water-soluble substance in the above two-phase system is in favor of aqueous phase.
We can assume that power the drift solubilizer is due to simultaneous presence in the structure of this derivative osidicity of said group (s) hypocarbon (s) and sulfate group (s) or phosphate. Hydrocarbon groups allow the establishment of Van der Waals-type connections with ''":

WO ~ 2/01475 pCT / FR91 / 00595 the (the ~ hydrocarbon chain (s) of the active ingredient and the sulfate or phosphate group (s) allows (solves) the solubilization of the complex not covalent thus forms.
If applicable, the lengths of the chains respectively carried by the substance active lipophilic on the one hand, the solubilizing derivative water soluble, on the other hand, are chosen so that favor the establishment of the above-mentioned links Van der Waals type.
The pharmacologically active substance may be made up of any substance meeting the criteria indicated above. Generally, we notice that the lipophilicity of the substance pharmacologically active can result either from the length of the chains hydrocarbons it carries, either - and in particular when these hydrocarbon chains are short - from many of these channels, or again, of course, the combination of the lengths and the number of these hydrocarbon chains.
As we have already seen, the active substance can be consisting of any muramyl peptide derivative comprising a hydrocarbon chain tending to reduce the hydrosolubility of the corresponding muramyl-peptide, since it would be devoid of said chain hydrocarbon. A preferred category of such muramyl-peptides called "lipophilic" is still illustrated below.
Another category of preferred compounds is consisting of daggerboards or the like saccharides, especially mono- or di-saccharides lipid A. Some preferred examples of these analogues are discussed below.

. . .,., ... : ~. . :, ..... .: ~. . .

2087 ~ 3 ~
Other pharmaceutical substances may also be solubilized by implementing the ~ ues techni of the invention. ~ -Among these, particular mention should be made of cyclic peptides of the cyclosporine family, for example cyclosporin A (JF Borel et al, Agents Actions - 6 (1976) l + 68; is, Immumnology 32 (1977) 1017; PJ Tudchka et al., Blood 61 (1983) 318) but also cyclic pseudopeptides, for example valinomycin or polyene macrolides, for example lucensomycin, and vitamins fat-soluble, in particular vitamins A, D, E, K, Generally any substance pharmacologically active of the polyenic, mono or polysaccharide, polypeptide, with character cyclic or not, or glycopeptide, which carries -at least one hydrocarbon chain as it was defined above, may be the constituent lipophilic compositions according to the invention.
The invention applies in particular to the solubilization of pharmacologically active substances active, whose solubility character would prove insufficient for medical application, whatever either the route of administration envisaged, that it contains a hydrocarbon chain which can to associate, via type links Van de Waals, with a hydrocarbon chain corresponding carried by an osidic derivative, such that it has been defined above.
As for the water-soluble sugar derivative, it it should be noted that the term "oily" as used above, and in what follows, generally relates to any product including in its structure one (or more) residue (s) saccharide (s).

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2 ~ 8r ~ 3 ~ 6 ~.
In general, any sugar derivative such as:
that defined above can be used in compositions of the invention, since this derivative does not change the properties pharmacological of the active substance above mentioned, such that the latter would become inactive or toxic.
The hydrocarbon chains carried, either by the.
pharmacologically active substance, either by derived from oside, are advantageously themselves lipophilic groups. They are notably constituted:
. either by at least one hydrocarbon structure linear from 3 to 20, in particular from 4 to 20 atoms of carbon, including any, one or more:
ether, ester, thioether, thioester, or amide, this linear structure being designated below after by "type A" lipophilic group, . either by at least one hydrocarbon structure branched from 5 to 50, in particular from 10 to 50 atoms of carbon, including if applicable, one or more ether, ester, thioether, thioester, or.
amide, this branched structure being designated below:
afterwards by "type B" lipophilic group.
A category of preferred sugar derivatives for the production of compositions in accordance with the invention are those which answer the following formula (I)::

7 ~ 87 ~ 3 ~ -~ ' in which :
- R represents a hydrocarbon group comprising at least 3 carbon atoms, in particular a group lipophilic "type A" or "type B", - R2 represents H, or OH, or an acetamido group, sulfate, sulfamido, phosphate, phosphamido, - R3 and R4, identical or different, represent either H, OH, or OCH3, or a group sulfate or phosphate, - R1 represents either H, or OH, or OCH3, or else a ose or osamine residue of which the anomeric carbon in position l (Cll is engaged in a link glycoside, positions C2, C3 and C4 being respectively substituted by groups R '2, R' 3, and R'4 having respectively the meanings given above for R2, R3, and R4, and position C6 by a group R 'having the meaning given above for R, at least one of the groups R2, R'2, R3 ~ R'3 ~ R4 or R'4 representing a sulfate or phosphate residue.
As an example of the sugar derivatives corresponding in the above-mentioned formula (I), mention may be made of following compounds:
- a or ~ -methyl-glycoside N-acetyl-4-sulfate-6-OR-2-deoxy-glucosamine, ,: ':''','' ,, 75 ~

- N-acetyl 4-sulfate-6-OR-2-desoxy-glucosamine, - ~ or ~ -methyl-glycoside 4-sulfate-6-OR-glucopyrano-is, - 4-sulfate-6-OR-glucopyranose, - 6,6'-di-OR-4,4'-di-sulfate-a, ~ '-D-trehalose, R having the meaning indicated above.
It goes without saying that the lipophilic groups do not must not be of such length or number such that the whole osidic derivative would tend to become insoluble.
Preferred sugar derivatives are chosen from the following compounds:
- ~ or ~ -methyl-glycoside N-acetyl-4-sulfate-6-octanoyl-2-deoxy -glucosamine, - ~ or ~ -methyl-glycoside-N-acetyl-4-sulfate-6- (2,3-dipalmitoyl) glyceroyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6-octanoyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6- (2,3-dipalmitoyl) -glyceroyl-2-deoxy-D-glucosamine, - a or ~ -methyl-glycoside 4-sulfate-6-octanoyl-glu-copyranoside, - ~ or ~ -methyl-glycoside 4-sulfate-6- (2,3-dipalmi-toyl) -glyceroyl-D-glucopyranoside, - 4-sulfate-6-octanoyl-D-glucopyranose, - 4-sulfate-6 (2,3-di-palmitoyl) -glyceroyl-glucopyranose - 4.4 'di-sulfate-6.6' di-octanoyl ~ '-D-trehalose, - 4,4'di-sulfate-6,6 '(2,3-dipalmitoyl ~ -glyceroyl-~
D-trehalose.
Certain categories of compositions conforming to the invention will be even more - and not so limiting - illustrated in the following.
The two hydrocarbon groups, the case due, lipophilic by themselves, ensuring respectively a non-covalent association between the 2n ~ 7 ~ 3 ~
9,.
pharmacologically active substance and the derivative of oside, are linear hydrocarbon structures.
Advantageously, however, these groups are such that one of them is made up of a structure linear hydrocarbon, while the other is consisting of a branched hydrocarbon structure.
The branched hydrocarbon structure is then preference chosen from those consisting of one branched hydrocarbon chain, the ramifications of which originate on two carbon atoms adjacent, or at most separated by an atom of carbon.
Preferably the linear hydrocarbon structure must then, to associate with the hydrocarbon chain branched, have a sufficient length allowing the establishment of non-covalent bonds, in particular Van der Walls type, with at least one of ramifications of the branched hydrocarbon structure.
So the association of the substance pharmacologically active and of the drift in sugar is it done through their hydrocarbon or even lipophilic groups respective, as soon as links are established noncovalent strong enough to allow the linear hydrocarbon structure, to be maintained in a so-called "sandwich" position between the two ramifications of the branched hydrocarbon structure.
Preferably, the hydrocarbon structure linear has a maximum number of carbon atoms equal to the number of branched carbon atoms of the branched hydrocarbon structure.
The association of the sugar derivative as described above, of amphiphilic and water-soluble nature, ~ the hydrophobic pharmacologically active substance and -. . .
. ~. ... . . .
. ,:,., :::: ... :.

2 ~ 3 ~

insoluble in water, allows the homogeneous dispersion of the latter in an aqueous solvent.
- The subject of the invention is more particularly, any composition likely to be solubilized or homogeneously dispersed in an aqueous solvent, characterized in that it comprises:
(1) a pharmacologically active, insoluble substance ble in water, this substance being substituted:
. either by a "type A" lipophilic group, . either by a "type B" lipophilic group, (2) an osidic drift associated noncovalently has substance (1), this osidic derivative being itself substituted:
. either by at least one lipophilic group "of type A "when said substance pharmacologically active is substituted by a lipophilic group "of type B ", . either by at least one lipophilic group "of type B "when the said substance pharmacologically active is substituted by a lipophilic group "of type A ", at least one of the hydroxyl functions of the derivative the above mentioned sugar being substituted by one, or several, phosphate or sulfate group (s).
Generally, any substance pharmacologically active, especially of a nature insoluble in an aqueous solvent, and on which is - likely to be grafted at least one group lipophilic "type A" or "type B" mentioned above or more generally a hydrocarbon chain more short, can be used as a substance pharmacologically active entering into compositions of the invention.
The techniques described below as examples ~, ..,. ,, ''. ''''.

~ 8 ~ 53l ~

for the grafting of these lipophilic groups or hydrocarbon chains on oside derivatives are also applicable to the grafting of chains of the same nature on pharmacologically active substances insoluble, as soon as they are themselves lacking.
Among the pharmacologically active substances (or lipophilic active ingredients) used in compositions of the invention, there may be mentioned the derivatives muramyl peptide lipophiles with the formula (II) following:

L- (31) nl ~ 0 ~

~ _, OH
HO /

/ \
R5 Co-x-NH-clH-co-R6 CH2-cO- ~ Al) n2 Y

in which ~ Rs represents H or CH3;
- R6 represents a group -NH2, -OH or a group -OW1, W1 being a hydrocarbon group comprising from l to l0 carbon atoms;
- X represents an aminoacyl residue of the group including alanyl, valyle, isoleucyle, norleucyle, leucyle, threonyle, prolyle, glutaminyle, asparaginyl, methionyl, tryptophanyl, phenylalanyl, tyrosyl, glycyl;

: '.-': '":' :: ' WO92 / 01475 PCT ~ FR91 / 00595 - 2 Q ~ ~ ~ J ~ 12 - Y represents either OH or NH2, or a group -OW2, W2 being a hydrocarbon group of 1 to 4 atoms of carbon, or a type A lipophilic group or type B, -- Z represents either H or a lipophilic group "type A" or "type B", it being understood, however, that at least one of the two groups Y and Z is always made up of a "type A" or "type B" lipophilic group;
- nl and n2, identical or different, represent. .
zero or l, - A ~ and B1 are either groups respectively identical or different comprising from 1 to 3 residues aminoacyles, themselves identical or different between them, or a group -NH- (CH2) p-CO- with p values between 2 and 10.
A particularly preferred composition of the invention comprises compounds of formula (II) - ~
mentioned above, in which -- n1 and n2 represent zero, -- R5 represents CH3, - R ~ represents -OH, -NH2 or -On (CH2) H with x1 included between l and 6, with a preference for x1 = 4, --X is L or D-alanyl, L-threonyle, L-valyle,::
- when Y represents -O-CH2-CHO (R7) -CH2O (RB), Z represents H, and when Z represents O
He . ,.
-C-CHO (R7) -CH2O (R8), Y represents -OH, -NH2 or a group -OW2, W2 having the meaning given above above, and R7 and R8 being palmitoyl groups.
Another preferred composition of the invention comprises compounds of formula (II) mentioned above, in which :

,.,. ,,,,., ~,. :. , ~. , - 20 ~ 53 ', - nl and n2 represent zero, - ~ represents CH3 - ~ represents -OH, or -NH2 or On (CH2) 1H with x ~
between l and 6, with a preference for x1 = 4, -X is k or D-alanyl, L-threonyl, L-valyle, - when Y represents -O- (CH2) X2H with x2 8, Z
represents H, and when Z represents -CO (CH2) ~ 2H, with x2 = 8, Y represents -OH, -NH2, or a group -OW2, W2 having the meaning indicated above.
The biologically active substance susceptible to be associated with the derivatives of osides, defined above, to form dissolvable compositions or dispersible according to the invention, can also belong to other product categories. ~ It these are, for example, immunomodulatory substances, selected from:
- constituents of bacterial origin, among others constituents of the bacterial wall: derivatives of trehalose, in particular of the type of TDM (products of class of trehalose dimycolates), and lipolysaccharides (LPS) which on the one hand contain lipid chains and, on the other hand, at least one phosphate or sulfate group:
- lipid fractions obtained from these LPS, in particular "Lipid A" (see for example article by Ernst Th. Rietschel et al entitled "Molecular structure of bacterial endotoxin in relation to bioactivity "(Molecular structure of bacterial endotoxins in relation to their bioactivity) in the work itself entitled "Cellular and molecular aspects of endotoxin reactions "(Cellular and molecular aspects of reactions using endotoxins), under the 7 ~ 3 ~

direction of A. Nowotny, JJ Spitzer and EJ
Ziegler; 1990, Elsevier Science Publishers BV
(Biomedical Division), pages 15-32); we call back for remember the formula for "Lipid A" (III):
HO

COO - C ~ CH
(~ H2) y (CH2 ~ x CONH HO CH - OOC
CH ~ CH ~ CH2 CONH (CH2) X (CH2) y COO ~ CH CH2 CH ~ 3 ~ CH2) y (cH2) x COO CH
CH3 CH ~ (CH2) y (cH2) x CH ~ CH ~ x - ~ o - analogues of the original constituents bacterial or products (natural, semi-synthetic or synthetic) corresponding to fragments of these constituents of bacterial origin, in particular detoxified derivatives. We will mention everything in particular derivatives or analogs of lipid A, mono- or di-saccharide, mono- or diphosphorylated, carriers of branched chains or chains lin hydrocarbons ~ areas, which can be themselves substituted or not, by hydrocarbon chains linear by means of ester, thioester, ether bonds or thioether, products which, obtained by synthesis or hemisynthesis, have a toxicity ~ attenuated compared to Natural Lipid A.

~ 7 ~

A first family is that of analogues or disaccharide derivatives of Lipid A, such as MPL
(monophophoryl Lipid A, produced by Ribi immunochem, Research Inc.). These are very structures hydrophobic homogeneously dispersible in water, in the presence of the above derivatives of osides.
These Lipid A analogs have the formula general (IV), HO -R $ 40 NHR2 OR t in which:
R4 and R'1 represent H or P04, with at least R4 or R ' being a group P04 R2, R3 and R'2, R'3 represent CO-CH (OR) - (CH2) X-CH3, x being between 5 and 15, with a preference for x = 10, and R being CO- (CH2) y-CH3 y being included between 5 and 15 with a preference for y = 10 or 12.
A second family is that of analogues or monosaccharide derivatives of Lipid A, in particular the MRL 953 (product described in PL Stuetz et al, in Cellular and molecular aspects of endotoxin reactian A. Nowotny, JJ Spitzec, EJ Zlegler editors.
(Elsevier Science Publishers) 1990, pages 129-144).
These analogs and monosaccharide derivatives of Lipid A respond to the general formula (V):

WO 92/01475 PCI '/ FR91 / 00595 r ~ ~) 3 b HO--~ ORI

in which : -R ~ represents H or PO3 R2 and R3 represent CO-CH (R) - (C ~ 2) x-CH3, x being between 5 and 15, with a pre ~ erence for x = 10, and R being H or OH or also CO- (CH2) y ~ CH3, y being between S and 15 with a preference for y = 10 or 12, R4 represents PO3 if R1 is not PO3, or H or even one of the meanings given to R2 and R3.
For the same hydrophobicity reasons as already mentioned about certain Muramylpeptides, their Galenic formulation is often difficult.
However, like the lipophilic Muramylpeptides they can lead to solutions or dispersions homogeneous aqueous in the presence of the above derivative of oside.
The present invention therefore relates to the use compositions according to the invention either alone for stimulate the body's general defenses, i.e.
with an antigen.
The oside derivative and the substance pharmacologically active are, in the combination, advantageously in a weight ratio of 0.25 to 5, especially from 0.5 to 1.5.

2og7r ~, 3 ~ d The invention also relates to derivatives of formula (I) themselves, as soon as they contain at least one sulfate group, as well as their process of obtaining according to methods which are conventionally used in synthetic chemistry of mono and oligosaccharides.
As an illustration, this methodology proceeds in successive stages from the dare or from the oligosaccharide to be chemically modified, to achieve research drift, specifically substituted to the desired position. To do this, the functions in positions C1, C2, C3, C4 and C6 of a dare and those corresponding to an oligosaccharide, are protected temporarily by protective groups (such as that benzyl, benzylidene, acetyl ...) introduced specifically and sequentially on the positions which will not ultimately be the subject of the chemical modification envisaged, taking advantage of differences in reactivity of said functions.
Then the introduction of lipophilic groups "type A" or "type ~" above or moreover of shorter hydrocarbon chains, is advantageously carried out in the following manner:
(or the) primary hydroxyl functions not protected, can react either in substituted form by a tosyle group with a fatty acid salt, in presence of ether crown, either in free form with a fatty acid chloride, or with the function carboxylic of a fatty acid in the presence of a reagent of coupling.
After introduction of (or the) modifications chemicals considered in the (or the) positions left - ~
free, the derivative obtained is released (step of deprotection) of all its protective groups for lead to the desired mono or oligosaccharide derivative.

~ 2 ~ ~ r` ~ r ~ 18 If applicable, the introduction of (or) groups phosphates is carried out prior to the stage of above-mentioned protection, by the so-called method phosphite triester described in Perich, JW et al, Tetrahedron Lett., (1988), 29, 2369.
The introduction of the sulfate group (s) is carried out after the above-mentioned deprotection step, in particular by using the trimethylamine- complex -sulfuric anhydride according to the method in particular described in Ichikawa Y. et al, Carbohydrate Research, 172, ('988), 37-64.
The invention also relates to compositions pharmaceuticals comprising a composition of the type of those described above, or a mixture of these compositions, dispersed (s) in a solvent physiologically acceptable.
Advantageously, the compositions of the invention can be freeze-dried from their aqueous solution or dispersion, and then present themselves in powder form.
These compositions in the form of dry powder, are easily solubilized or dispersed again in a appropriate volume of aqueous solvent.
The invention also relates to a method of modification of the solubility properties of a pharmacologically active substance of type (l) described above, this process consisting of - `-association of substance (l) with a derivative osidic type (2) as described above, in conditions such as when this association of (l) and (2) is put in water, we obtain a solution (or a dispersion) usable in particular in as an injection, when the water above mentioned is sterile.

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The solubilization of the substance phar-macologically active (1) is such that one obtains a fluid characterized by a weak opalescence (measurable by nephelometry), this one being even often absent.
This fluid behaves like a solution and is likely to be administered into the body as than solution for injection. Dispersion of a substance in an aqueous medium corresponds to a distribution stable and homogeneous molecules of this substance in the mass of this medium, resulting from a total or partial solvation by molecules water, not a dispersion of this substance sedimentable over time. In particular we does not observe any change in physical state in the dispersed within 24 hours of preparation.
The invention also relates to a method for obtaining the compositions described above including: -- the solubilization of the osidic derivative (s) in a determined quantity of an aqueous solvent, - addition with stirring to the solution previously obtained, of a determined quantity of one (or several) pharmacologically active substance, i.e.
suspended in an aqueous solvent, either in solution in an organic solvent, `
- if necessary, the elimination of the organic solvent, - the case still falling, the lyophilization of the previous solution.
As a variant, the process for obtaining compositions of the invention is carried out in adding, under the conditions prec ~ demment described, the (or) derivative (s) osidique (s) to a :

,. .

W092 / Ot475 PCTtFR9t / 00595 ~ 0 ~

specified quantity of one (or more) substance (s) pharmacologically active.
The invention is further illustrated in the detailed description which follows, without this the latter is not limiting.
Description of the ~ ynthese of the sodium salt of 6,6 ' di-octanoyl- ~, 4'-disulfonyl- ~, ~ -D-trehalose ~ or com-posed P1) .6,6'octanoyl-2,3-2 ~, 3 ', tetra-0-benzyl- ~, ~' -D-trehalo-get Dissolution in dry THF (50 ml), under argon, protected from light by 3 mmoles of 2.3-2 ', 3'-tetra-0-benzyl ~ -D-trehalose (AF Hadfield, L. Hough, and AC Richardson - Carbohyd. Res. 63, (1978), 51) 7.5 mmol of octanoic acid, and 7.87 mmol of triphenylphosphine. To the solution cooled to OC, addition in 15 minutes of a solution of diisopropylazodicarboxylate (8.02 mmol) in the Dry THF (25 ml). Purification on silica 60 H under -pressure in the toluene / CH2Cl2 / AcoET mixture 10/30/5. Oil: m = 2.58 g (90%).
. Sodium salt ~ u 6,6'-dioctanoyl-2,3-2 ~, 3'-tetra-0-benzyl-4.4 ~ -di- ~ ulfonyl-, a ~ -D-trehalose.
1.5 mmol of the above product in solution in dry DMF (15 ml), addition of 15 oles of complex trimethylamine / sulfuric anhydride (2.1 g ~ and incubation at 50 C in anhydrous conditions for 2 days. Addition of C ~ 30H (10 ml), then, extension by chloroform (10 ml) and percolation on Sephadex LH20 in the mixture CHCl3-CH30H: I / I. After concentration, purification on a 60H silica column under pressure, in the mixture CHCl3-CH30 ~ -H20 5/15/05 (1.7 g: oil). Dissolution in the mixture DMF-H20: I / I (80 ml) and percolation on column of ~ '~. '. . '''.''. `. ''''' '~ `";' ................ 1 1 ';'. ... '', ``,, '~,'. '' .

WO92 / 01475 pCT / FR9l / 00595 ~ 0875 '~
Dowex 50 WX4 ~ resin (85 ml) by elution with same mixture of solvents: neutralization with NaOH
0.1N: lyophilization m = 1.51 g (87%).
. Sodium salt of 6.6 ~ -dioctanoyl-4.4 ~ -di-O-sulfonyl-a ~ ts ~ -D-treh ~ lOse (C28H ~ 8Na2019B2 798.79) 1.5 g of the previous derivative, dissolved in 150 ml of a water / alcohol mixture (6.5 / 10), is hydrogenated presence of 750 mg of Pd / C at 10 ~. After filtration, concentration, lyophilization in aqueous solution.
Purification on silica 60-G in CHCl3-CH30H-CH3COOH-H2O: 40/20/8/2. Concentration, coevaporation with toluene, lyophilization in aqueous solution.
Percolation on Sephadex LH20 in methanol.
Drying concentration, lyophilization in solution in water m = 525 mg (51%). -Sulphate content: 2.7 Eq / g (conductimetry).
octanoic acid content: 1.97 Eq / mole (CPV).
EXE ~ PLES OF IMPLEMENTATION ~ OF THE OSIDE P DERIVATIVE ~ DAN8 EXEMP ~ E - I:
1): Description of implementation of the diseersion aqueuise de MDP-GDP, ¢ 'est- ~ -dire le ~ N- ~ cetyl-mura-myl ~ lanyl- D-iisoglutamine) - ~ B, ~ -dip ~ lmitoyl-sn-Gly-cerol, with the composite Pl - First mode of implementation mg of P1 are dissolved in 10 ml of water distilled. To the solution are added 10 mg of MDP-GDP, under strong agitation. The aqueous dispersion obtained is opalescent in appearance and does not sediment or very little, (a new agitation makes it possible to obtain a homogeneous dispersion again). After lyophilization, we can by addition of water or physiological solute, reconstitute a dispersion of identical appearance.
- Second mode of implementation WO92 / 01475 PCTtFR91 / 00C ~ 5 r ~ ~ 3 ~ d 22 Method of use 2.20.57 mg of MDP-GDP are dissolved in 2 ml of chloroform (solution A). 20 mg of P1 are dissolved in l ml of chloroform (solution B).
The chloroform solutions (A) and (B) are reunited. 5 to 10 ml of distilled water are added and bubbled a stream of nitrogen from the bottom of the tube containing the two phases, until elimination complete with chloroform.
~ n obtains a homogeneous aqueous dispersion and stable, slightly opalescent.
The aqueous dispersion is lyophilized and the powder obtained is taken up in an aqueous solvent with desired concentration. We obtain a dispersion of same appearance and quality as before.
The process allows an easy dosage formulation active ingredients of lipophilic drugs corresponding to the general formula (II). It results from preparations which keep the activities pharmacology of these products, especially as that regulate the immune response, such as have been claimed in European patent no. 0 165 123.
2 ~: Influence of the use of Pl on wtivity adjuvant to MDP-GDP studied in guinea pigs.
a) Preparation of the emulsion l mg of P1 is dissolved in 0.5 ml of solute physiological, this solution is added drop by drop drop to 1 mg of MDP-GDP powder.
The dispersion obtained is mixed with 0.5 ml of a isotonic solution of ovalbumin at 20 mg / ml. She is to which 1 ml of mineral oil has been added (Adjuvant Incomplete from Freund, AIF) and we proceed to the emulsion.
This preparation therefore contains per ml, 0.5 mg adjuvant, 0.5 mg of Pl and 5 mg of ovalbumin . ~

~ 87 ~ t '~ u b) Methodology Control lots receive a) emulsion water / mineral oil containing the antigen alone, b) same emulsion containing adjuvant molecules only (MDP-GDP). The experimental lots receive the emulsions containing the P1 sugar derivative. The injections are done subcutaneously in rear guinea pig pads Hartley weighing 350 g. After 3 weeks the animals receive 0.1 ml of solute intradermally physiological alone or containing 0.1 ml of ovalbumin.
After 48 hours, measure the induration diameter which is the sign of the establishment of a state delayed hypersensitivity. After 24 hours, animals are sacrificed by cardiac puncture and will be collected. Titration of circulating antibodies is done by the Elisa method according to the modalities classics, especially those described in:
F.AUDIBERT, L. CHEDID, P. LEFR ~ NCIER and J. CHOAY, Immunol. t1976), (21), 143; and M. JOLIVET, F.
AUDIBERT, H. GRAS-MASSE, A. TARTAR, D. SC ~ hESSINGER, R. WITZ and ~. CHEDID, Infect. Immunol. (1987), (55), 1498.

',',, ' WO92 / 01475 PCT / FR91 / 00 ~ a5 i ~ a '~,' l 'à3 ~ -c) Results The results obtained3 are con ~ ignes n ~ ns following table:
IMMUNIZATION ~ ANTI- TITLE HYPERSENSITIVITY
DELAYED - ELISA BODY
diameter of indu-ration mm AIF oØ0.0 22,000, 37,000, 65,000, 95,000 AIF + P1 oOo0 7,000, 8,000, 100 ~ g 21,000.
AIF + MDP-GDP 100 ~ g 8,14,15,17 241,000,295,000 325,000,641,000.

AIF + MDP-GDP 5,16,20,25 157,000,275,000, 100 ~ g + P1 275,000,318.0000 100 ~ g : All animals received 1 mg ovalbumin.
The figures represent the maximum dilution of the serum to obtain an optical density equal to that obtained using a normal serum diluted 50 times.
These results show that MDP-GDP is a strong adjuvant cell-mediated response and humoral response and that the addition of P1 did not changed their activity.

3): Influence of the osidic derivative Pl 8US
immunostimulatory properties ~ of muramyl-peptides.
a - Anti-infectious activity Swiss mice received Pl, MDP-GDP, or MDP-GDP + P1 by vein 24 hours before ~ -. , ''' :. . . .
'. : '''.

'~ a 87 ~ 3lj 1 x 104 Klebsiella pneumoniae infection. The solution of P ~ is added to the MDP-GDP to obtain a preparation containing an equivalent amount of 2 products (weight / weight).
The results of Table I show that P1 alone -has no protective action against infection. The comparison of the activity of the different doses of MDP-GDP, alone or with P1, does not show any differences significant.
We can conclude from these experiments that the compound P1 does not inhibit the anti-infective activity of MDP-GDP.
b - Action of the MDP-GDP on the production of TNF
flowing TNF (tumor necrosis factor), is a protein - synthesized by cells c- the line monocytes / macrophages when stimulated. The LPS (bacterial lipopolysaccharide) is an inducer very powerful TNF production. The muramyl-peptides stimulate the production of TNF in vitro but they are significantly less effective than LPS. In vivo, serum TNF levels produced after administration of muramyl-peptides are below or at the limit of detection possibilities.
MDP-GDP, and other derivatives, which have a anti-infective action, have the property of increasing response to LPS injected after, causing considerable increase in serum TNF levels. The table II represents the results of an experiment of this type. We see that the pretreatment is more effective 6 hours before the LPS, especially in the case of MDP-GPD + P1 association, but only 16 hours before, the injection of MDP-GDP still has a potentiation of the response.

"'.'':~''~''' ~ '''''".

WO 92tO1475 PCr / FR91 / 00.

~ 8 ~ rj3 ~ 26 c - Action of MDP-GDP in vitro on monocYtes human sana.
~ blood monocytes are isolated and incubated for 24 hours with different doses of product. ~ -Supernatants are taken to assay TNF and IL-6, cytokines that are produced by monocytes active.
Table III shows that the production of TNF
is significant only with 10 ~ g / ml of MDP-GDP and that the product solubilized with compound P1 is of slightly higher activity. The dosage of IL-6 gives sharper results and also shows Comparative stimulation of monocytes between MDP-GDP
and the solubilized MDP-GDP in association with P1.
d - Action on human nuclear Pol ~
Muramyl peptides have a direct action on Polynucleaires which is highlighted in particular by phagocytosis and destruction of Candida ~:
albicans.
The estimate of this action is made by the ability to incorporate glucose treated with fungus cells remained viable.
In the two experiments reported on the Table IV, unstimulated control cells have already a relatively high basic activity.
However, the addition of the muramyl peptide, with or without Pl, produces an increase in inhibitory activity cells on the growth of the fungus.
In conclusion, whether in the experiments done in vivo in mice or in vitro with human cells, the solubilization of MDP-GDP by the compound P ~ never decreases its activity. Compose P1seul is still inactive.

-; . - ~

,:,:, '' -,. ~ "':
- ~. ..

~, a87 ~ 3, ~

~ ABI, WATER I
Protective Action of MDP-GDP dissolves By com ~ dared P, in mice infected with Klebsiella pneumoniae :
IV treatment, Survival at D + 15% Protection ~
at D-1 .
Witnesses 1/16 P ~ 100 ~ g 0/8 MDP-GDP 3 ~ g 3/8 37.5 ns .-3/8 37.5 ns 12/16 68.7 P <0.01 - -100 4/8 37.5 ns MDP-GDP / Pl 3/3 ~ g 3/8 37.5 ns 10/10 3/8 37.5 ns 30/30 8/16 43.7 P <0.02 100/100 7/8 75 P <0.02 *: Statistical calculations are made taking account for each batch of their witnesses respective.

:. . ~,. ,. ,., ~, WO92 / 0147s PCT / FR91 / 00 ~ 5 ~ ~ ~ 7 d 3 d 28 TABLEA ~ II
Influence of Pretreatment By MDP-GDP solubilizes by P, on the LPS-induced TNF response Treatment with 100 ~ g LPS iv TNF activity in the iv / mouse before LPS 25 ~ g serum U / ml ~ g / ml : - '' h-6 P1 189 3.4 h - 6 MDP-GDP / P, - <10 <O, 18 h-6 MDP-GDP +11.475 206.55 h-6 MDP-GDP / Pl +19.038 342.68 h-16 MDP-GDP +1.899 34.18 h-16 MDP-GDP / Pt 4,296 77.33 : 2 hours after the test injection with the LPS
Activity expressed in U / ml and in TNF equivalent murine recombinant, in the cytotoxicity test for cell line L292.

,, ... , ..,. , ~, : `` ~; ~
':,. : ''.,.,. ~ '"'..,.:.

~ 7 ~
~ A ~ LEAU III
Action of MDP-GDP in vitro on human monocvts .
Stimulant ~ g / ml Activity in the supernatant TNF U / ml IL-6 U / ml ... .
Pl O <10 MDP-GDP 0.1 0 <10 0.1 0 <10:. , 1 0.3 <10 ~.
6.9 94.9 MDP-GDP / Pl 0.01 0 ~ 10 0.1 0 <10 -.-1 5.9 83 10.5 594 ..
: '' Cells at 2 x 106 / ml, incubated for 24 hours with products.

WO92 / 01475 PCT / FR91 / 00C ~

`2 ~ 3 ~ 30 $ ABLEA ~ IV
Action of MDP-GDP on human PolYnuclear Stimulant ~ g / ml Growth inhibition Candida (%) 1st exp. 2nd exp.

Witnesses 35.3 41.5 Pl 10 34.6 38.7 MDP-GDP 0.1 44.1 47.2 1 7S, 1 78.1 86.2 86.1 MDP-GDP / P1 0.1 32.2 77.2 1 41.4 93.6 65.9 95.1 .
Polynucleares (1 x 106 / ml) are incubated with stimulants for 30 minutes and distributed in microplate wells with a suspension of C.
albicans at 1 x 104 / ml. After 6 p.m. the middle of culture is eliminated and incorporation of 3 ~ -D-glucose by Candida cells is measured after a 3 hour incubation.

..,,, l . ~ -,. : -. ,: - ~
,:. . ., j. .

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. ::: ..

31 ~ 7 ~ 3i3

4): Influence of the osidic derivative Pl on the effect MDP-GDP pyrogen.
The pyrogenicity tests were carried out on New Zealand rabbits weighing 3 to 4 kilograms in a contention box. The animals received a dose of product in a volume of OS ml / kg per route venous at time zero and their temperature recorded for 5 hours continuously.
(at). Assessment of the ~ vroqenicity of P1 Two batches of three rabbits were made up:
. group I: 10 ~ g / kg of P1;
. group II: 100 ~ g / kg of P1.
The recorded temperature differences were:
. for group I: 0.15, 0.25; 0.25 (C), either an average of: 0.22 C + 0.06;
. for group II: 0.40, 0.15, 0.30 (-C), or an average of: 0.28-C + 0.12.
This experiment indicates that P1 is totally reduced pyrogenic effect in rabbits and drastic conditions for a registration made for 5 hours.
(b). Evaluation of the P ~ roqicity of MDP-GDP in ~ P1 resence The minimum pyrogenic dose of MDP-GDP beforehand determined, is equal to: 2 ~ g / kg.
A dose of 10 ~ g / kg was chosen which gives -approximately an increase in temperature of lC.
Two batches of three rabbits were made up:
. group I: 10 ~ g / kg of MDP-GDP;
. group II: 10 ~ g / kg of MDP-GDP ~ 10 ~ g / kg of Pl. -The recorded temperature differences are:
. for group I: 0.70, 0.50, 0.85 (-C), or an average of: 0.68-C + 0.18;
. for group II: 1.15, o.so, 0.65 (oC), . : -:. . : ~:. ~:
,.

W092 / 01 ~ 7 ~ PCT / FR91 / OOC45 ~ a; ~ i 32 i.e. an average of: o.9o ~ C + 0.25.
A Student statistical test (unpaired two-tailed t test, p = 0.2866) indicates that this difference is not significant and therefore that P1 does not induce a synergy of the pyrogenic effect of a other product like MDP-GDP.
The dispersed compositions obtained have pharmacological properties which are similar nature than those reported above for dispersions of the composition MDP-GDP-P ~. We do again back in example V on results particularly interesting pharmacological which also use dispersible compositions based on MDP (Thr) 1-GDP and P1.
EXAMPLE II Production of a dispersible composition in an aqueous solution based on ~ -acetyl-muramyl- ~ -threonyl-D-isoglutamine) - ~, ~ -dipalmitoyl- ~ n -glycerol (M9P ~ hr) l-GDP) This dispersion in the presence of the compound Pl, is obtained in exactly the same conditions as for MDP-GDP
EXAMPLE III Production of a dispersible composition in an aqueous solution of an a ~ alogue ~ ipide A monophosphoryl 2.1 mg of the monophosphoryl derivative of lipid A (MPL
produced by Ribi lmmunochem. Research, Inc.) is dissolved in 0.5 ml of chloroform (solution A).
3.8 mg of P1 are dissolved in l ml of chloroform (solution B).
The chloroform solutions (A) and (~) are reunited. Add 2 to 3 ml of distilled water and make bubbling a stream of nitrogen from the bottom of the tube containing the two phases, until elimination complete with chloroform.

, ~ ''. :!
. '' `:` :: ':. . :

WO92t01475 PCT1FR91 / 00595 2n87 ~ 3 ~

A homogeneous aqueous dispersion is obtained and stable, slightly opalescent.
The aqueous dispersion is lyophilized and the powder obtained is taken up in an aqueous solvent at the desired concentration. We obtain a dispersion of same appearance and quality as before. `:
Biological properties of the sharpness of MPL
~ monophosphoryl Lipid A, Ribi immunochem. Research, Inc.) ~ with compound P1 Antibacterial activity.
The MPL is dissolved according to the procedure recommended by Ribi immunochem. Research, Inc .. A
from a solution in the chloroform mixture:
methanol (4: l) 20 mg MPL, a stock solution containing 4 mg / ml of MPL is thus prepared, then by addition of physiological serum devoid of calcium, 2 mg / ml solution.
The test is carried out as described in example l, paragraph 3a.
The compound P1 does not alter the protective effect of MPL vis-à-vis infection by ~ .pneumoniae. ``

'' ~ ', "''~,`' WO92 / 0147

5 PCT / FR91 / 00545 7 ~

~ ABLEA ~ V
Protective effect of MPL (Ribi immunochem. Research Inc.) alone or in the presence of compound P1, opposite infection with a high dose (4 x 10 5) of K. pneumoniae, in Swiss mice.

TRAITBMBNT 8URVIVANT8 / ~ OTAL
A ~ DAY + lS
-MPL 0.1 mg 0/8 MPL l mg 4/8 MPL 10 mg 7/8 MPL / P10, l mg l / 8 MPL / P1 l mg 4/8 MPL / P1 10 mg 8/8 WO92tO1475 PCT / FR91 / 00595 ; ~ Q ~ 753 ~

EXAMPLE I ~ Agous dispersion of an analog mono ~ accharidic of Lipid A, MRL 953 (analog monosaccharidigue ~ onophosphoryl), of formula VI, with compound P1 0 ~

~ HO ~ OH

a) Production of the dispersion 10.65 mg of MRL 953 are dissolved in 0.5 ml of chloroform (solution A). 16.91 mg P1 is dissolved in 0.5 ml of chloroform (solution B).
The chloroform solutions (A) and (B) are reunited. Add 2 to 3 ml of water and bubbled a stream of nitrogen from the bottom of the tube containing the two phases, until complete elimination of chloroform. An aqueous dispersion is obtained homogeneous and stable, very slightly opalescent.
The aqueous dispersion is lyophilized and the powder obtained is taken up in an aqueous solvent at WO92 / 01475 PCT / FR9l / 00 ~ 5 3 ~

desired concentration. We obtain a dispersion of same appearance and quality as before.
b) Biological properties of the aqueous dispersion of the MRL 953 with comPose P1 Antibacterial activity (Figures l (a) and l (b) Protective effect against K infection pneumoniae has been evaluated in female Swiss mice (ages ~ e 5 to 6 weeks). -The mice received the day before infection with intravenous route:
- 0.2 ml of physiological saline, - LPS (lipopolysaccharide of Salmonella enteritidis) suspended in 0.2 ml of physiological saline, - 0.2 ml of ~ 1) the aqueous dispersion of P1 + MRL 953, containing 30, 10, 3 and 1 ~ g of MRL 953 ~ 2) the dispersion in a solution of ~ RL 953 glucose containing 30, 10, 3, and 1 mg of MRL 953.
Infection with ~ .pneumoniae is carried out by intravenous injection of 0.2 ml suspension a suspension of bacteria (containing 4.5 x 103 bacteria). Mortality is noted during the two weeks after bacteria are inoculated.
The comparison of Figures l (a) and l (b) shows that the protective effect of MRL 953 is comparable, let ~ be the dispersion method used. The figures l (a) and l ~ b) indeed include curves representing variations in the percentages of surviving mice (% on the ordinate axes) in function of time (T in days on the axes of abscissa) when the substances studied (LPS and MPL953) were administered at the doses indicated at the right of the figures, in dispersed form in:
- a glucose solution (figure l (a)) WO92 / 0147s PCT / FR91 / 00595 ~ 7 ~ 3 ~

- a solution of the P1 oside derivative (figure l (b)) It follows from examining the figures that the effect protector of the substances studied, plus especially MRL9S3, vis-à-vis an infection by K. Pneumoniae in mice is of the same order of greatness in both cases. He would even more effective when MLR953 is administered under form of dispersion, in association with drift of oside P ~.
EXENP ~ EV - I ~ fluence of dispersions according to the invention on the biological properties of an active ingredient third The association of certain muramylpeptides with a oside derivative can very interestingly lead to a third active ingredient ~
. either an increase in biological activity the third active ingredient, . either a decrease in the toxicity of this third active ingredient, . either both.
So, for example, we notice:
. a significant increase in activity antiviral of interferon, when the latter principle active is administered after administration of the combination of MDP (Thr) 1-GDP and compound P1 in the conditions allowing the formation of their aqueous dispersion;
. a decrease in the toxic synergy of- LPS
with a muramylpeptide with some effect pyrogen, for example MDP-GDP of compound P1, in conditions corresponding to those already described in the previous examples. Especially doses of LPS, lethal to mice when they are administered alone, are no longer administered when the same -. .:,:.
.: ,, '''..,: -,. ~ ''' 3 ~ 38 doses are administered in combination with the mixture of MDP-GDP and the compound P1.
In other words, the invention also relates to applying the compositions according to the invention to the modulation of biological properties of third-party compounds, administered concomitantly or not.
These modulation effects are further illustrated by non-limiting way by the examples whose results are reported below.
~ l) - Influence of the use of the P1 component on the immunostimulatory properties of muramylpeptides lipophilic a) - Increase in antiviral activity interferon by MDP (Thr ~ 1-GDP
The experimental model is encephalomyocarditis of the mouse. The animals are male mice (Swiss, 17 g). They receive intraperitoneally either an aqueous suspension of the only MDP (Thr) 1-GDP, or an aqueous dispersion of MDP (Thr) 1-GDP and of compound After 24 hours, the virus is injected into them.
encephalomyocarditis intraperitoneally 10 times the LD 50. One hour after this injection, some animals receive intraperitoneal 25,000 IU of interferon ~, ~ of mouse.
Table VI presents the results obtained. he as a result, the use of compound P1 does not decrease not, but on the contrary increases antiviral activity of MDP (Thr) 1-GDP, and the effect of potentiation of the antiviral activity of interferon by this lipophilic muramylpeptide.

1, ... , ... ,,. . ,,. I '~., ",,:' ~ ', ..'-",.",'..........

. ~

w ~ 75 '~

T ~ BLEAU VI

. ~. .
Increased antiviral activity of interferon ~, ~ vis-à-vis an infection by the virus of.: -encephalomyocarditis, in mice .:
T ~ HOLD IP INTERFERON ~, ~% OF S ~ RIE
AJ - l .
Witness -%
P1 ~ 600 ~ g) _ 0%
MDP (Thr) 1-GDP (600 ~ g) `- 20%
1P1 (600 ~ g) +
¦MDP (Thr) 1-GDP (600 ~ g) - 35%
_ + 30%
P1 (600 ~ g) + 30%
MDP (Thr) 1-GDP (600 ~ g) + 46%
¦P1 (600 ~ g) +
¦MDP (Thr) 1-GDP (600 ~ g3 + 70%

b) - Influence of a Pre-treatment by MuramvlpePtides liPophiles on the Production of TNF circulating following LPS injection or a LiPide A sport analog (Table VII ~
Swiss mice receive an injection intravenous from:
- 0.2 ml of physiological saline, - - MDP-GDP suspended in 0.2 ml of serum physiological, - MDP (Thr) 1-GDP in aqueous suspension, alone, or in aqueous dispersion, in the presence of the compound Pl, - Murabutide in solution in 0.2 ml of serum physiological.

WO 92/01475 PCI` / FR91 / 00!; 95 r ~

The above intravenous injection is given 6 hours before injecting LPS (S. enteritidis) into physiological saline or MRL 953 dispersed in physiological saline in the presence of P1.
The animals are bled from the abdominal aorta 1.5 to 2 hours (optimum response time) after this last injection. TNF activity is assessed by the -cytotoxicity test being measured by the number of viable cells. One unit of TNF represents the concentration which produces 50% cytotoxicity in given conditions.
Aqueous suspensions of lipophilic MDP
stimulate the TNF response to LPS stimulation or by the aqueous dispersion of MRL 953 in the presence of P1.
This effect is slightly accentuated with the dispersions aqueous of these lipophilic MDP in the presence of COMPOSITION P1 ~

WO92 / ~ 1475 PCT / FR91 / 00595 41 ~ 7 ~ 3i3 TAB ~ EA ~ VIS; ~:

Influence of pre-treatment with lipophilic CDM on serum TNF production following injection LPS or a synthetic analogue of Lipid A
(MRL 953).

TREATMENT iv CHALLENGE ACTSVS ~ B TN ~
H - 6 LPS l ~ g (in IU / ml) ~ RL953 / P1 300 ~ g 1,5h2h no LPS 879 MDP-GDP 100 ~ g LPS 2.947 MDP (Thr) t-GDP 100 ~ g LPS 4.235 none LPS 900 - ~ -MDP (Thr) 1-GDP 100 ~ g LPS 3.311 ¦MDP (Thr) 1-GDP 100 ~ g, ~
I + P1 LPS 7,347 none MRL953 / P1 <10 ~ 10 MDP (Thr) l-GDP 100 mg MRL953tP11.089 488 ¦MDP (Thr) 1-GDP 100 mg ¦ + P1 MRL953 / P1 ~ 1,838 Murabutide 100 mg MRL953 / P1 ~ 1,150 none MRL953 / P1 76 Murabutide MRL953 / P1 820 WO 92/01475 PCr / FRgl / 00595 r ~

c) - Reduction of syner ~ ie toxioue LPS and MDP-GDP in the Presence of compound P1 (TABLE ~ III) MDP-GDP, ~ is suspended in 0.2 ml of serum physiological, alone, or in a glucose solution in presence of pluronic acid (F 68, SERVA), OR
dispersion in 0.2 ml of physiological serum in presence of compound P1, is administered by the intravenously with LPS (S. enteritidis) to mice Swiss adrenalectomisees.
Mortality is noted in 3 days, but it is most often produced in 24 to 48 hours.
MDP-GDP suspensions in serum physiological or in a glucose solution of acid pluronic increases the toxicity of LPS in ~ ecte simultaneously. The dispersion of MDP-GDP in the presence of P1 reduces this effect.
This reduction in the toxic effect is not due to a direct effect of the compound Pt on the LPS ion the toxicity remains in normal mice in the presence of -compound P1 (results not shown). ~ -We note that the interest of these results -lies in the fact that the association with the drift - ~ -of oside makes it possible to envisage the in vivo use of a greater number of lipophilic products, for example lipophilic muramylpeptides which, like MDP-GDP, may have some residual pyroginicity, without having to fear a synergy:
significant toxic, in vivo, of these products with LPS of possible infectious organisms with the host.

,; .. '', ':. ',,', 'i,. ~ ~.

43 `~ '7 ~ ~ ~
TABLEA ~ VIII

Reduction of toxic synergy of LPS and MDP-GDP
in the presence of P1 .
VE ~ IC ~ LE CDM - GDP LPSMORTS / TOTAL
~ lg) ~ 25 lg) physiological serum - + 0/18 ~.
100 + 7/18 p <O, 02 300 + 17/18 p <O, 01 glucose solution pluronic acid (F68) - + 0/18 100 + 8/18 p <0.01 300 + 17/18 p <0.01 ~ -aqueous dispersion, in the presence of P1 ~ + 0/18 100 + 0/18 300 + 6/18 p <O, 05 ~
300 - 0/18::

,:. . :. :,

Claims (29)

1. Composition capable of being dissolved or homogeneously and stably dispersed in a pharmaceutically acceptable aqueous solution, characterized by the association:
- on the one hand, a lipophilic active principle of medicinal product or "pharmacologically active substance", comprising at least one hydrocarbon group comprising itself at least 3 carbon atoms, and - on the other hand, a solubilizing osidic derivative, itself water-soluble, pharmaceutically acceptable, itself bearing a hydrocarbon group comprising at least least 3 carbon atoms and comprising one or several hydroxyl functions, at least one of which is substituted by a phosphate or sulphate group, this saccharide derivative being in a sufficient proportion compared to the lipophilic active ingredient to promote the solubilization or dispersion of the whole of the composition in an aqueous solution. 2. Composition according to claim 1, characterized in that the substance pharmacologically active lipophilic is a saccharide-like substance, peptide or polypeptide, cyclic or not, cyclic or non-cyclic glycopeptides, carrying least one hydrocarbon chain comprising at least three carbon atoms. 3. Composition according to claim 1 or claim 2, characterized in that the group . hydrocarbon consists of:
. either by at least one hydrocarbon structure linear from 3 to 20, in particular from 4 to 20 atoms of carbon, including, where appropriate, one or more bond(s) ether, ester, thioether, thioester, or amide, this linear structure being referred to below by "type A" lipophilic group, . either by at least one hydrocarbon structure branched from 5 to 50, in particular from 10 to 50 atoms of carbon, including, where appropriate, one or more bond(s) ether, ester, thioether, thioester, or amide, this branched structure being referred to below after by "type B" lipophilic group. 4. Composition according to claim 1 or claim 3, characterized in that the substance pharmacologically active is a lipophilic derivative of muramyl-peptide. 5. Composition according to claim 4, characterized in that the substance pharmacologically active is chosen from the compounds corresponding to the following formula (II):
in which - R5 represents H or CH3;
- R6 represents a group - NH2, -OH or a group -OW1, W1 being a hydrocarbon group comprising from 1 to 10 carbon atoms;

- X represents an aminoacyl residue of the group including alanyl, valyl, isoleucyl, norleucyl, leucyl, threonyl, prolyl, glutaminyl, asparaginyl, methionyl, tryptophanyl, phenylalanyl, tyrosyl, glycyl;
- Y represents either OH or NH2, or a group -OW2, W2 being a hydrocarbon group of 1 to 4 carbon atoms carbon, or even a lipophilic group "of type A" or "type B", - Z represents either H or a lipophilic group "of type A" or "type B", it being understood, however, that at least one of the two groups Y and z is always constituted by a group type A or type B lipophilic;
- n1 and n2, identical or different, represent zero or 1, - A1 and B1 are either groups respectively identical or different comprising from 1 to 3 residues aminoacyls, themselves identical or different between them, or an -NH-(CH2)p-CO- group with p values between 2 and 10. 6. Composition according to claim 5, characterized in that the substance pharmacologically active is chosen from the compounds of formula (II) in which :
- n1 and n2 represent zero, - R5 represents CH3, - R6 represents -OH, -NH2 or with x1 included between 1 and 6, with a preference for X1=4, - X is L or D-alanyl, L-threonyl, L-valyl, - when Y represents -O-CH2-CHO(R7)-CH2O(R8), Z represents H, and when Z represents ?

-C-CHO(R7)-CH2O(R8), Y represents -OH, -NH2 or a group -OW2, W2 having the meaning indicated in the claim 6, and R7 and R8 being groups palmitoyls. 7. Composition according to claim 5, characterized in that the substance pharmacologically active is chosen from the compounds of formula (II) in which :
- n1 and n2 represent zero, - R5 represents CH3, - R6 represents -OH, or -NH2 or -On (CH2)x1H with x1 between 1 and 6, with a preference for x1=4, - X is L or D-alanyl, L-threonyl, L-valyl, - when Y represents -O-(CH2)x2H with x2 = 8, Z
represents H, and when Z represents -CO(CH2)x2H, with x2 = 8, Y represents -OH, -NH2, or a group -OW2, W2 having the meaning indicated in the claim 6. 8. Composition according to claim 1, characterized in that the substance pharmacologically active is formed from parent constituents bacterial, among other cell wall constituents bacterial: derivatives of trehalose, in particular of the type TDM (products of the trehalose class dimycolates), lipolysaccharides (LPS), including on the one hand lipid chains and, on the other hand, least one phosphate or sulfate group. 9. Composition according to claim 1, characterized in that the substance pharmacologically active is a lipid fraction obtained from these LPS, in particular "Lipid A", this fraction comprising at least one phosphate or sulphate group. 10. Composition according to claim 9, characterized in that the substance pharmacologically active is made up of derivatives or analogs of lipid A, mono- or di-saccharides, mono- or diphosphorylated, carriers of branched chains or chains linear hydrocarbons, which may themselves be substituted or not, by hydrocarbon chains linear by means of ester, thioester, ether bonds or thioether. 11. Composition according to any of the claims 8 to 10, characterized in that the pharmacologically active substance consists of a monophosphorylated lipid A, in particular MPL. 12. Composition according to any of the claims 8 to 10, characterized in that the pharmacologically active substance consists of an analogue of lipid A corresponding to the general formula (IV), in which:
R4 and R'1 represent H or PO4, with at least R4 or R'1 being a PO4 group R2, R3 and R'2, R'3 represent CO-CH(OR)-(CH2)x-CH3, x being between 5 and 15, with a preference for x = 10, and R being CO-(CH2)y-CH3 y being between 5 and 15 with a preference for y = 10 or 12. 13. Composition according to any of the claims 8 to 10, characterized in that the pharmacologically active substance consists of a monosaccharide derivative of lipid A meeting the general formula (V):
in which :
R1 represents H or PO3 R2 and R3 represent CO-CH(R)-(CH2)x-CH3, x being between 5 and 15, with a preference for x = 10, and R being H or OH or alternatively CO-(CH2)y-CH3, y being between 5 and 15 with a preference for y = 10 or 12, R4 represents PO3 if R1 is not PO3, OR H or even one of the meanings given to R2 and R3. 14. Composition according to claim 13, characterized in that the monosaccharide derivative of lipid A is the compound of formula VI:
15. Composition according to any of the claims 1 or 2, characterized in that the lipophilic pharmacologically active substance is a cyclic peptide substance, such as a cyclosporin. 16. Composition according to claim 1 or claim 2, characterized in that the substance pharmacologically active is a substance of the type peptide, such as gramicidin, or a substance of macrolide type, such as leucensomycin, or a pseudopeptide-like substance, such as valinomycin, or a fat-soluble vitamin, including type vitamin A, D, E, K, 17. Composition according to any of the claims 1 to 16, characterized in that the derivative saccharide is represented by the following formula (I):
in which :
- R represents a hydrocarbon group comprising at least least 3 carbon atoms, in particular a group "type A" or "type B" lipophilic, - R2 represents H, or OH, or an acetamido group, sulfate, sulfamido, phosphate, phosphamido, - R3 and R4, identical or different, represent either H, either OH, or OCH3, or a sulfate group or phosphate, - R1 represents either H, or OH, or OCH3, or a ose or osamine residue whose anomeric carbon in position 1 (C1) is engaged in a bond glycosidic, the positions C2, C3 and C4 being respectively substituted by groups R'2, R'3, and R'4 having respectively the meanings given above for R2, R3, and R4, and position C6 by a group R' having the meaning given above for R, at least one of the groups R2, R'2, R3, R'3, R4 or R'4 representing a sulfate or phosphate residue. 18. Composition according to claim 17, characterized in that the group R or the group R' is consisting of:

. either by at least one hydrocarbon structure linear from 3 to 20, in particular from 4 to 20 atoms of carbon, including, where appropriate, an ether bond, ester, thioether, thioester, or amide, this structure linear being designated hereinafter by grouping "type A" lipophilic, . either by at least one hydrocarbon structure branched from 5 to 50, in particular from 10 to 50 atoms of carbon, including, where appropriate, one or more bond(s) ether, ester, thioether, thioester, or amide, this branched structure being referred to below after by "type B" lipophilic group. 19. Composition according to claim 17 or claim 18, characterized in that the drift saccharide is chosen from the following compounds:
- .alpha. or .beta.-methyl-glycoside N-acetyl-4-sulfate-6-OR-2-deoxy-glucosamine, - N-acetyl 4-sulfate-6-OR-2-desoxy-glucosamine, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-OR-glucopyrano-himself, - 4-sulfate-6-OR-glucopyranose, - 6,6'-di-OR-4,4'-di-sulfate-.alpha.,.alpha.'-D-trehalose, R having the meaning indicated in the claim 17 or 18. 20. Composition according to claim 17, characterized in that the saccharide derivative is chosen among the following compounds:
- .alpha. or .beta.-methyl-glycoside N-acetyl-4-sulfate-6-octanoyl-2-deoxy-glucosamine, - .alpha. or .beta.-methyl-glycoside-N-acetyl-4-sulfate-6-(2,3-dipalmitoyl) glyceroyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6-octanoyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6-(2,3-dipalmitoyl)-glyceroyl-2-deoxy-D-glucosamine, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-octanoyl-glu-copyranoside, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-(2,3-dipalmi-toyl)-glyceroyl-D-glucopyranoside, - 4-sulfate-6-octanoyl-D-glucopyranose, - 4-suyl-D-glucopyranoside, - 4-sulfate-6-octanoyl-D-glucopyranose, - 4-sulfate-6(2,3-di-palmitoyl)-glyceroyl-glucopyranose - 4,4'di-sulfate-6,6'di-octanoyl-.alpha.,.alpha.'-D-trehalose, - 4,4'di-sulfate-6,6'(2,3-dipalmitoyl)-glyceroyl-.alpha.-.alpha.'-D-trehalose. 21. Composition according to any of the claims 18 to 20 in combination with one any one of claims 3 to 8, characterized in that that it includes:
(1) a pharmacologically active substance, insoluble wheat in water, this substance being substituted:
. either by a "type A" lipophilic group, . either by a "type B" lipophilic group, (2) an saccharide derivative non-covalently associated with the substance (1), this saccharide derivative being itself substituted:
. either by at least one lipophilic group "of type A" when said pharmacologically active is substituted by a lipophilic group "of type B", . either by at least one lipophilic group "of type B" when said pharmacologically active is substituted by a lipophilic group "of type A", at least one of the hydroxyl functions of the derivative oside mentioned being substituted by one, or several, phosphate or sulphate group(s). 22. Composition according to any of the claims 18 to 20 in combination with one any one of claims 9 to 13, characterized in what it includes:
(1) a pharmacologically active substance, insoluble wheat in water, this substance being substituted:
. either by a "type A" lipophilic group, . either by a "type B" lipophilic group, (2) an saccharide derivative non-covalently associates with the substance (1), this saccharide derivative being itself substituted:
. either by at least one lipophilic group "of type A" when said pharmacologically active is substituted by a lipophilic group "of type B", . either by at least one lipophilic group "of type B" when said pharmacologically active is substituted by a lipophilic group "of type A", at least one of the hydroxyl functions of the derivative oside mentioned being substituted by one, or several, phosphate or sulphate group(s). 23. Composition according to any of the claims 1 to 22, characterized in that the derivative of oside and the pharmacologically active substance are advantageously in a weight ratio of 0.25 to 4, in particular from 0.5 to 1.5. 24. Composition according to any of the claims 1 to 23, characterized in that it is at the anhydrous state, in particular in the form of a lyophilisate. 25. Pharmaceutical composition characterized in that that it comprises a composition according to any of claims 1 to 24, where appropriate in combination with a pharmaceutical vehicle acceptable either in the anhydrous state or in the state dispersed in an aqueous solvent physiologically acceptable. 26. Oside derivative corresponding to the general formula (I) following:
in which - R represents a hydrocarbon group comprising at least minus 3 carbon atoms, - R2 represents H, or OH, or an acetamido group, sulfate, sulfamido, phosphate, phosphamido, - R3 and R4, identical or different, represent either H, either OH, or OCH3, or a sulfate group or phosphate, - R1 represents either H, or OH, or OCH3, or a ose or osamine residue whose anomeric carbon in position 1 (C1) is engaged in a bond glycosidic, the positions C2, C3 and C4 being respectively substituted by groups R'2, R'3, and R'4 having respectively the meanings given above for R2, R3, and R4, and position C6 by a group R' having the meaning given above for R, at least one of the groups R2, R'2. R3, R'3, R4 or R'4 representing a sulphate or phosphate residue, it being understood that at least one of the preceding groups is a sulfate group. 27. Oside derivative according to claim 26, characterized in that the group R or the group R' is consisting of:

. either by at least one hydrocarbon structure linear from 3 to 20, in particular from 4 to 20 atoms of carbon, including, where appropriate, an ether bond, ester, thioether, thioester, or amide, this structure linear being designated hereinafter by grouping "type A" lipophilic, . either by at least one hydrocarbon structure branched from 5 to 50, in particular from 10 to 50 atoms of carbon, including, where appropriate, one or more bond(s) ether, ester, thioether, thioester, or amide, this branched structure being referred to below after by "type B" lipophilic group. 28. Oside derivative according to claim 26 or claim 27, responding to one of the formulas following:
- .alpha. or .beta.-methyl-glycoside N-acetyl-4-sulfate-6-OR-2-deoxy-glucosamine, - N-acetyl 4-sulfate-6-OR-2-desoxy-glucosamine, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-OR-glucopyrano-himself, - 4-sulfate-6-OR-glucopyranose, - 6,6'-di-OR-4,4'-di-sulfate-.alpha.,.alpha.'-D-trehalose, R having the meaning indicated in the claim 26 or claim 27. 29. Oside derivative according to claim 26, corresponding to one of the following formulas:
- .alpha. or .beta.-methyl-glycoside N-acetyl-4-sulfate-6-octanoyl-2-deoxy-glucosamine, - .alpha. or .beta.-methyl-glycoside-N-acetyl-4-sulfate-6-(2,3-dipalmitoyl) glyceroyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6-octanoyl-2-deoxy-glucosamine, - N-acetyl-4-sulfate-6-(2,3-dipalmitoyl)-glyceroyl-2-deoxy-D-glucosamine, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-octanoyl-glu-copyranoside, - .alpha. or .beta.-methyl-glycoside 4-sulfate-6-(2,3-dipalmi-toyl)-glyceroyl-D-glucopyranoside, - 4-sulfate-6-octanoyl-D-glucopyranose, - 4-sulfate-6(2,3-di-palmitoyl)-glyceroyl-glucopyranose - 4,4'di-sulfate-6,6'di-octanoyl-.alpha.,.alpha.'-D-trehalose, - 4,4'di-sulfate-6,6'(2,3-dipalmitoyl)-glyceroyl-.alpha.-.alpha.'-D-trehalose.