CN101374551A - Novel glycolipid adjuvant compositions - Google Patents

Abstract

This invention relates to compositions and methods of preparing stable adjuvant diluent stock solutions and final adjuvant solutions comprising glycolipids, weak acids, alcohols, nonionic surfactants and buffers.

Description

New glycolipid adjuvant compositions

Invention field

The present invention relates to new glycolipid adjuvant compositions and using method thereof and preparation method.These new compositionss are for a long time stable and do not have a flocculation among the present invention.They especially help to comprise the release of the multiple medicine of vaccine.

Background of invention

Vaccine is generally used for protecting human and veterinary animal avoids infecting the infectious disease that is caused by antibacterial, virus and parasite body.Can be any medicament although be used for the antigen of vaccine, though it is made of the survival of pathogenic organism body pathogenic organism body, albumen, recombiant protein or its fragment modified or attenuation of having killed usually.Regardless of antigenic source, be necessary to add adjuvant usually to strengthen the host to antigenic immunne response.

Adjuvant is used to realize two purposes: it makes antigen slow down and stimulating immune system from the release of injection site.

First adjuvant of being reported in the document is Freund's complete adjuvant (FCA).FCA contains water-in-oil emulsion and Mycobacterium extract.Mycobacterium extract provides the immunostimulating molecule with native form.Water-in-oil emulsion produces the storage effect, and antigen slowly discharges therein.Unfavorable is that FCA toleration difference and its can cause property inflammation out of control.Found that before more than 80 years people reduce the adverse side effect of adjuvant hardy since the FCA.

Known glycolipid analogs contains the class noval chemical compound with adjuvant character.United States Patent (USP) 4; 855; 283; (hereinafter being ' 283) discloses the synthetic of glycolipid analogs; this glycolipid analogs comprises N-glycosyl amide, N-glycosyl urea, and N-glycosyl carbamate and specific chemical compound: N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate (is called Bay R1005; O Lockhoff, Angew.Chem.Int.Ed.Engl. (1991) 30:1611-1620).Chemical compound described in ' 283 patents is particularly suited for as adjuvant.

Glycolipid adjuvant preparation (formulation) need be easy to make and be stable and do not have lipid composition and flocculate when long term storage.The non-acetate form of glycolipid amide or glycosyl amide is for highly insoluble, and flocculates from solution when storing under room temperature or lower temperature usually.

The few flocculation of solution that comprises the glycosyl amide that this paper provided and adjuvant and extremely stable.They are easy to make and can the commercial size preparations.Liquid glycolipid adjuvant formulation can be used as the diluent of rehydrated lyophilization antigen preparation.Instant test is provided and tests the method for these stability of formulation by the accelerated stability method of testing.

Summary of the invention

The present invention comprises compositions and the manufacturing or the production method of glycosyl amide stock solution and glycolipid adjuvant solution.Glycosyl amide stock solution is to be dissolved in the alcohol and in conjunction with an amount of weak acid by the glycolipid with formula 1 to add the preparation of " nonionic " surfactant.Weak acid added in the glycolipid alcoholic solution, and it is a molar excess with respect to glycolipid.In one embodiment, glycolipid is N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl dodecanoyl amide hydration acetate (hydroacetate).In one embodiment, alcohol is ethanol.In one embodiment, weak acid is acetic acid.In one embodiment, non-ionic surface active agent is various sorbitans

Or polyoxyethylene sorbitan

Especially be sorbitan monolaurate (Span

) and polyoxyethylene sorbitan monolaurate (Tween

).Glycolipid adjuvant solution is by introducing an amount of glycosyl amide stock solution preparation in " suitable buffer ".The final pH value of stable glycolipid adjuvant solution as herein described should be between about 6 and about 8.Preferable final pH value is between about 6 and about 7.Final pH value between about 6.3 and about 6.4 has been described.Should avoid glycolipid adjuvant above the NaCI high salt concentration of 30mM.

Below illustrate this two kinds of solution in more detail:

Glycosyl amide stock solution is the compositions that comprises following each material:

A) glycolipid of formula I,

Its Chinese style I is

Wherein:

R ¹For hydrogen or have the saturated alkyl of 20 carbon atoms at the most;

X is-CH ₂-,-O-or-NH-;

R ²For hydrogen or have the saturated alkyl of 20 carbon atoms at the most;

R ³, R ⁴And R ⁵Be independently hydrogen ,-SO ₄ ²-,-PO ₄ ²-,-COC _1-10Alkyl;

R ⁶For L-alanyl, L-alpha-amido butyl, L-arginyl-, altheine acyl group, L-aspartyl, L-cysteinyl-, L-glutamyl, L-glycyl, L-histidyl-, L-hydroxypropyl, L-isoleucyl-, L-leucyl-, L-lysyl-, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-seryl-, L-Threonyl, L-tyrosyl-, L-tryptophanyl with L-is valyl or their D-isomer;

This glycolipid is the form of salt, and wherein the form of this salt is derived from weak acid;

B) alcohol should alcohol be HO-C wherein _1-3Alkyl;

C) weak acid, wherein 1) this weak acid is molar excess with respect to glycolipid content; And 2) for using standard scale or any acid of standard value pKa value between about 1.0 and about 9.5; With

D) non-ionic surface active agent, wherein this non-ionic surface active agent makes the capillary reagent of its dissolved material and has a kind of hydrophobic components and another kind of hydrophilic component for reducing.

Glycolipid adjuvant solution is the compositions that contains following each material:

A) glycosyl amide stock solution; With

B) suitable buffer, wherein this buffer is suitable for veterinary or medical application and can keeps relative constant pH value between about 6.0 and about 8.0 in aqueous solution.

Detailed Description Of The Invention

Except that illustrating in addition, employed following term has following implication in this description and the claim:

Term " alcohol " refers to following formula: compound: HO-C _1-3Alkyl.It can be methanol, ethanol or any type of propanol, as normal propyl alcohol or isopropyl alcohol.Preferred alcohol.

Term " alkyl " refers to the saturated hydrocarbons part of straight chain and side chain.

Term " glycolipid " refers to the chemical compound of following formula I.These chemical compounds are described in United States Patent (USP) 6,290, in the United States Patent (USP) 4,855,283 of promulgation on August 8th, 971 and 1989.United States Patent (USP) 6,290,971 all quote in full at this as a reference with United States Patent (USP) 4,855,283.The trade mark that the glycolipid of the specific description of this paper has when being the acetate form is called Bay

With chemistry " N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate " by name.The trade mark that the amide form of this chemical compound has is called Bay15-

With chemistry " N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide " by name.

The glycolipid of formula I is:

Formula I

Wherein

R ¹For hydrogen or have the saturated alkyl of 20 carbon atoms at the most;

X is-CH ₂-,-O-or-NH-;

R ²For hydrogen or have the saturated alkyl of 20 carbon atoms at the most;

R ³, R ⁴And R ⁵Be independently hydrogen ,-SO ₄ ²-,-PO ₄ ^2-,-COC _1-10Alkyl;

R ⁶For L-alanyl, L-alpha-amido butyl, L-arginyl-, altheine acyl group, L-aspartyl, L-cysteinyl-, L-glutamyl, L-glycyl, L-histidyl-, L-hydroxypropyl, L-isoleucyl-, L-leucyl-, L-lysyl-, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-seryl-, L-Threonyl, L-tyrosyl-, L-tryptophanyl with L-is valyl or their D-isomer;

Or its pharmaceutically acceptable salt.

Another particular has been described the glycolipid of formula 1, wherein:

R ¹Be hydrogen or saturated C _12-18Alkyl;

R ²Be hydrogen or saturated C _7-11Alkyl;

X is-CH ₂

R ⁴And R ⁵Be hydrogen independently;

R ⁶Be selected from the L-leucyl-;

The variable of formula I be separately and independently, and the combination of all variablees is all described and claimed in this.

In another embodiment, glycolipid is the described glycolipid of formula II (a):

Formula II (a)

In another embodiment, glycolipid is the described glycolipid of formula II (b):

Formula II (b)

In another embodiment, glycolipid has the structure of formula III:

Formula III

The formula III chemical compound can amide form or the existence of acetate form.The amide form of this chemical compound has trade (brand) name Bay 15-

The acetate form has trade (brand) name Bay

The glycolipid of formula I can use the following United States Patent (USP) 4,855 that is selected from, 283 step preparation.

As seeing from formula 1, chemical compound of the present invention is based on the 2-amino-2-deoxyhexamethylose that is substituted.These sugar always combine the N-glycosidic bond via anomeric carbon atom C-1 with amide groups, urea groups or alkoxy carbonyl group amido

R wherein ¹, R ²Has above-mentioned implication with X.

In formula I chemical compound of the present invention, the 2-amino of amino sugar combines with a-amino acid or alpha-amino acid derivatives through amido link.

Aminoacid is natural L-aminoacid, for example glycine, sarcosine, hippuric acid, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, ornithine, citrulline, arginine, aspartic acid, N acid, glutamic acid, glutamine, phenylalanine, tyrosine, proline, tryptophan and histidine.For example D-alanine of D-aminoacid has also been described, or amino carboxylic acid such as butyrine, norvaline, Amicar or alpha-amido enanthic acid, it is D-and L-form, as the substituent group of amino sugar.

Also be provided for the method for preparation I compound.The method need be from the shielded 2-amino of amino-2-deoxidation glucopyranose derivatives (formula IV),

R wherein ¹⁰Expression is used to protect amino protecting group, and it is synthetic as can be known by peptide, and selectivity is eliminated in due course.

The example of suitable protecting group is acyl group (for example trifluoroacetyl group or tribromo-acetyl base, an adjacent nitre benzene sulfenyl, 2; 4-dinitrobenzene sulfenyl) or the lower alkoxycarbonyl that arbitrarily replaces (for example methoxycarbonyl group, tertbutyloxycarbonyl, benzyloxycarbonyl group, to methoxyl group benzyloxy carbonyl or 2; 2, the 2-trichloro-ethoxycarbonyl).Suitable N-protected aminohexose derivant is known.For example, M.Bergmann and L.Zervas, Ber.64,975 (1931); D.Horton, J.Org.Chem.29,1776 (1964); P.H.Gross and R.W.Jeanloz, J.Org.Chem.32,2759 (1967); M.L.Wolfrom and H.B.Bhat, J.Org.Chem.32,1821 (1967); Summary: J.F.W.McOmie (editor).Prot.Groups.Org.Chem., PlenumPress (1973); Geiger, ＂ The Peptides ＂, the 3rd volume, 1-99 page or leaf, (1981) Academic Press; And the document of wherein being quoted.The preferred amino protecting group that is used for preparation I compound is BOC base (tertbutyloxycarbonyl) or Z base (benzyloxycarbonyl group).

In first reactions steps, make by end-blocking amino sugar derivative (IV) and amine (formula V) reaction

H ₂N-R ¹ (V)

R wherein ¹Has above-mentioned implication, to generate glycosyl amine (formula VI)

The preparation of this type glycosyl amine is known (ELLIS, Advances inCarbohydrate Chemistry 10,95 (1955)) in principle, and is described in clearly among No. the 3rd, 213,650, the DE-OS (German prospectus).

In second reactions steps, make glycosyl amine (VI) and arbitrary suitable carboxylic acid derivates (formula VII), for example carboxyl halogenide or carboxylic acid anhydrides reaction,

R ¹¹-CO-CH ₂-R ⁴ (VII)

R ²Have above-mentioned implication, and R ¹¹Expression halogen (for example chlorine) or expression-O-CO-R ²(R ²Have above-mentioned implication) or expression-O-CO-O-low alkyl group.Obtain glycosyl amide (formula VIII) in this way

R wherein ¹And R ²Have above-mentioned implication, and R ¹⁰With R ⁶Identical, and X represents-CH ₂-.The condition of this type of N-acidylate is pointed out in No. the 3rd, 213,650, DE-OS (German prospectus).

In preferred embodiments, in the presence of organic additive alkali,, make the reaction of formula VI glycosyl amine and 1 to 2 equivalent phosgene (formula VII) or with 1 to 2 normal by relevant carboxylic acid R by the known method of document ²-CH ₂-CO ₂The mixed anhydride reaction that H and ethyl chloroformate or isobutyl chlorocarbonate obtain generates tool X=-CH ₂-the glycosyl amide of formula VIII.

This reaction is between 0 ℃ and 50 ℃, to carry out under the suitable existence of organic base or inorganic base in organic or water-organic solvent.The diluent that is fit to is for example methanol, ethanol, 1-propanol or a 2-propanol of alcohol, or ether for example ether, oxolane or 1,4-dioxane, or halogenated hydrocarbon for example dichloromethane, chloroform or 1,2-dichloroethanes or N, dinethylformamide.

When the glycosyl amine (VI) that obtains in the first step reacts with haloformate (IX),

R ¹²-CO-O-R ² (IX)

R ¹²Expression halogen (for example chlorine or bromine), and R ²Have above-mentioned implication, obtain glycosyl carbamate (VIII) then, the X among the formula VIII represents oxygen.

In one embodiment, formula VIII glycosyl amine and 1 to 2 equivalent chlorinated carbonates IX are reacted to generate the glycosyl carbamate.Preferably in organic or aqueous-organic solvent, temperature but is more preferably at room temperature carried out this reaction between 0 ℃ and 50 ℃.Alcohol, ether, halogenated hydrocarbons or dimethyl formamide that suitable solvent is as indicated above.

When the glycosyl amine (VI) that obtains in the first step reacts with 1 to 2 equivalent organic isocyanate (formula X),

R ²-NCO (X)

R wherein ²Have above-mentioned implication, acquisition formula VIII glycosyl urea and X are-NH-.With above-mentioned response class seemingly, this acylation reaction is preferably carried out in organic solvent, reaction temperature is between-20 ℃ and 60 ℃, preferably between 0 ℃ and 25 ℃.The suitable solvent is above-mentioned alcohol, ether, halogenated hydrocarbons or dimethyl formamide.

Glycosyl amide (formula VIII, X=-CH by the method acquisition ₂-), the glycosyl carbamate (formula VIII, X=-O-) or the glycosyl urea (formula VIII X=-NH-) is separated into crystalline or unbodied form by known procedures separately, if necessary can for example purification is carried out in recrystallization, chromatograph, extraction etc. by standardization program.

In many examples, it also is favourable carrying out chemical derivatization with the similar or alternative above-mentioned steps of above-mentioned purification step, the glycosyl amide with well-crystallized character, glycosyl carbamate and the glycosyl urea derivative of its production VIII.In the example of glycosyl amide of the present invention, glycosyl carbamate and glycosyl urea, this type chemically derived turns to the esterification on the saccharide residue hydroxyl for example.The example of suitable ester group is acetyl group, benzoyl or p-nitrophenyl formoxyl.

Be three-O-acyl derivative of preparation glycosyl amide, glycosyl urea or glycosyl carbamate, make corresponding triol (formula VIII) in the presence of inorganic or organic additive alkali with acylation reaction.Suitable acylating agent is sour chloride such as chloroacetic chloride, Benzenecarbonyl chloride. or p-nitrophenyl methyl chloride, or anhydride is such as acetic anhydride.The ester of this reaction result production XI,

R wherein ¹, R ², R ¹⁰Have above-mentioned implication with X, and

R ¹³Expression acetyl group, benzoyl or p-nitrophenyl formoxyl.

The O-acylation reaction is preferably carried out in inert organic solvents.Spendable solvent is halogenated hydrocarbons (as dichloromethane, chloroform or 1,2-dichloroethanes), ether (as oxolane or 1, the 4-dioxane), ester (as ethyl acetate) and amide (as dimethyl formamide).

Also may for independent organic base (such as triethylamine or pyridine) as suitable solvent.Spendable alkali is all alkali that are used for O-acidylate organic chemistry.The preferred mixture that uses triethylamine, pyridine or pyridine/4-dimethylaminopyridine.Easily crystallization from organic solvent of three esters (formula XI).The preferred polar solvent of crystallization, as short chain alcohol, just methanol, ethanol, normal propyl alcohol or isopropyl alcohol.Other is applicable to that the crystalline solvent of three esters (formula XI) is the mixture of organic solvent and polar inorganic or organic solvent, for example oxolane-methanol, oxolane-water, alcohol-water and isopropyl alcohol-water.Three esters (formula XI) by independent or suitable repeatedly recrystallization purifying are reduced to triol (formula VIII) by three O-acetyl group hydrolysis or transesterification.Polytype ester cracking in the known organic chemistry.Relevantly can in the presence of the Feldalat NM of methanol and catalytic amount, carry out transesterification by known ZEMPLEN hydrolysis the organic chemistry from three esters (formula XI) preparations triols (formula VIII).

The 3rd reactions steps of relevant formula I compound of the present invention comprises the sugared protecting group that goes up 2-amino in the selective splitting formula VIII chemical compound.In this reaction, need pay special attention to the 1-acylamino-on the sugar or 1-amine formamido group or elimination simultaneously of 1-(alkoxy carbonyl group acylamino-) in the formula VIII chemical compound.

Under the hydrogenolysis condition, the preferred benzyloxycarbonyl group that uses can be quantitatively on aminohexane C-2 and selective splitting, keeps 1-acylamino-, 1-amine formamido group or 1-alkoxy carbonyl group acylamino-.This hydrogenolysis provides the glycosyl amide that has free 2-amino on sugar, glycosyl urea or the glycosyl carbamate with following structural formula (XII),

R wherein ¹, R ²Has above-mentioned implication with X.

The example of hydrogenolysis appropriate catalyst is the noble metal that is adsorbed on the active carbon, such as platinum or palladium.Preferred palladium/the charcoal (5% or 10%) that uses.Hydrogenolysis can be carried out in the suitable pressure vessel under atmospheric pressure or high pressure.Be suitable for the hydrogenant atent solvent that is, such as alcohol (methanol, ethanol or propanol), ether (such as oxolane or 1, the 4-dioxane) or carboxylic acid (such as acetic acid) or its mixture.Solvent mixes with water or diluted acid (such as hydrochloric acid or sulphuric acid) in the time of suitably.Certainly, when adding these acid, the 2-amino of formula XII-2-deoxidation-glycosyl amide ,-carbamate and-urea obtains as these sour ammonium salts.The tertbutyloxycarbonyl protecting group equally is applicable to formula VIII chemical compound, and it can use mineral acid (as hydrochloric acid or sulphuric acid) cracking by the known method of document.Wherein same, the 2-amino of formula XII-2-deoxidation-glycosyl amide ,-carbamate and-urea is as being used for the ammonium salt of cracked acid and optionally being obtained.

The 4th reactions steps of relevant formula I chemical compound of the present invention, it comprise the amino glycosyl amide that makes formula XII, amide ,-carbamate or-urea or its salt and suitable amino acid derivativges bonding.Suitable amino acid derivativges is a N-end-blocking aminoacid (formula XIII)

R wherein ⁷Have above-mentioned implication,

R ⁸Expression hydrogen or methyl, and

R ¹⁴Expression is generally used for the synthetic and selective removal and keep the protecting group of peptide bond once more of peptide.

Amino protecting group is above-mentioned protecting group among the preferred formula XIII that uses, and benzyloxycarbonyl group or tertbutyloxycarbonyl are especially preferred.The 2-amino of formula XII-2-deoxidation-glycosyl amide ,-carbamate or-bonding of urea and formula XIII amino acid derivativges can undertake that (E.Wunsch etc.: Synthese von Peptiden (Synthesis ofpeptides): Methoden der Org.Chemie (Methods of org.chemistry) is (E.Muller (Houben-Weyl) by the synthetic conventional method of peptide, Editor), XV/I volume and XV/2 volume, the 4th edition, by Thieme, Stuttgart publishes (1974)).

The example of conventional method is the amino and the condensation in the presence of dehydrant (for example dicyclohexylcarbodiimide or DIC) of formula XIII amino acid derivativges of formula XII chemical compound.

The condensation of formula XII chemical compound and formula XIII chemical compound also can be carried out when activated carboxylic.Can activable carboxyl for for example anhydride, be preferably mixed acid anhydride, such as the acetate of acid or the amide of acid, such as imidazoles; Or Acibenzolar.The example of Acibenzolar is cyano methyl ester, five chlorophenyl ester and N-hydroxyphthalimide ester.Acibenzolar also can be from acid (formula XIII) and N-maloyl imines or the acquisition of 1-hydroxybenzothiazole in the presence of dehydrant such as carbodiimide.Amino acid whose derivant is known and can prepares in a known way.The peptidoglycolipid of the formula XIII carboxyl compound condensation prepared formula XIV that the amino-compound of formula XII and selectivity are activated.

R wherein ¹, R ², R ⁷, R ⁸, R ¹⁴Has above-mentioned implication with X.

In the step of the final process for preparing relevant formula I chemical compound, the protecting group R in the formula XIV chemical compound ¹⁴Be eliminated.Be noted that in this step other acylamino-, carbamate groups or the urea groups that are present in the formula XIV chemical compound are not cleaved.Be preferred for the protecting group R in the XIV chemical compound ¹⁴, N-benzyloxycarbonyl group and N-tertbutyloxycarbonyl can be eliminated, and keeps acylamino-, carbamate groups or urea groups.Benzyloxycarbonyl group is in the presence of noble metal such as palladium charcoal, and (as ethanol, methanol, glacial acetic acid or oxolane) is by hydrogenolysis selective removal benzyloxycarbonyl group in suitable solvent.This solvent can neat solvent or is mixed or mix with water use mutually.This reaction can be carried out under atmospheric pressure or high pressure.Tertbutyloxycarbonyl R in the formula XIV chemical compound ¹⁴Can eliminate by the acidolysis process.The example of appropraite condition at room temperature in suitable solvent (such as glacial acetic acid, ether, dioxane or ethyl acetate) use sodium chloride.Be known on the methodological principle of this type of cracking t-butyl carbamate.In this way the peptide glycosyl amide of the formula I of Huo Deing ,-carbamate and-urea is separated into the form of crystallization or amorphous solid with original known method, and if necessary, carries out purification by standard method (as recrystallization, chromatograph, extraction etc.).

The chemical compound of formula I of the present invention can also be by obtaining same good result's second route of synthesis preparation.This second route of synthesis is different with above-mentioned first route of synthesis, wherein synthon amino sugar aminoacid, amine R ¹-NH ₂With carboxylic acid R ²-CH ₂-CO ₂-H or carbonic acid derivative R ²-O-CO-halogen or R ²-NCO (R wherein ¹And R ²Have above-mentioned implication) bonding order difference.In this second approach, the 2-N-of suitable formula XV (aminoacyl) amino sugar is used as starting ingredient,

R wherein ⁷And R ⁸Have above-mentioned implication, and R wherein ¹⁴Known amino protecting group in the expression chemistry of peptides is preferably benzyloxycarbonyl group or tertbutyloxycarbonyl.Then, so the amino-compound condensation of the formula XV chemical compound that obtains and formula III is with the glycosyl amine of generation general formula X VI,

R wherein ¹, R ⁷, R ⁸And R ¹⁴Have and formula I and R ⁶The implication of definition unanimity.

Above-mentionedly be useful on the chemical compound that the method for preparing general formula VI chemical compound all can be used for preparing general formula X VI.Then, the reaction of formula XVI chemical compound and above-mentioned carboxylic acid derivates (formula VII) or haloformate (formula IX) or organic isocyanate (formula X) is with 2-(aminoacyl)-amino glycosyl amide (X=-CH of production XIV ₂-) or formula XIV-carbamate (X=-O-) or formula XIV-urea (X=-NH-).These acylation reactions can be undertaken by the reactions steps of glycosyl amine mentioned above and carboxylic acid or carbonic acid derivative usually.

The intermediate formula XIV of Huo Deing can come purification by above-mentioned physical purification method by this method.Yet, preferably by formula XIV chemical compound being changed into three-O-acetas or the three-O-benzoate of general formula X VII by above-mentioned O-process for acylating,

Wherein the implication of variable is consistent with formula 1.

Therefore the easy crystallization of these chemical compounds especially from organic solvent for example methanol or the ethanol, and is able to purification.By being widely used in the above-mentioned ester method for hydrolysis of carbohydrate chemistry, the purified crystals derivant of formula XVII is transformed the triol of accepted way of doing sth XIV then.The final removal of amino acid whose protecting group is addressed hereinbefore with preparation I compound in the formula XIV chemical compound.The invention still further relates to the salt of formula I chemical compound.These salt are mainly the nontoxic salts that can be used for pharmaceutics usually, for example the chloride of formula I chemical compound, acetate and lactate or indifferent salt.

Term " weak acid " be meant the pKa value of using standard scale or standard value (Ka-log) any acid between about 1.0 and about 9.5.Describe following weak acid example with title, chemical formula and approximate pKa value, and be not desire restriction the present invention.Acetic acid, H (C ₂H ₃O ₂) (pKa 4.76); Ascorbic acid (1), H ₂(C ₆H ₆O ₆) (pKa 4.10); Aspirin, H ₈(C ₉O ₄), (pKa3.5); Butanoic acid H (C ₄H ₇O ₂) (pKa 4.83); Carbonic acid, H ₂CO ₃, (pKa 4.83 forms 1); Chromic acid, HCrO ₄ ^-, (pKa 6.49 forms 2); Citric acid, H ₃(C ₆H ₅O ₇), (pKa3.14 form 1); Citric acid, H ₂C ₆H ₅O ₇ ^-, (pKa 4.77 forms 2); Citric acid, (HC ₆H ₅O ₇) ⁼, (pKa 6.39 forms 3); Formic acid, H (CHO ₂), (pKa 3.75); Fumaric acid, H ₄(C ₄O ₄) (pKa 3.03); Enanthic acid, H (C ₇H ₁₃O ₂), (pKa 4.89); Caproic acid, H (C ₆H ₁₁O ₂), (pKa 4.84); Fluohydric acid. (hyrofluoric acid), HF, (pKa3.20); 1-Hydroxy-1,2,3-propanetricarboxylic acid. (isocitrate), H ₈(C ₆O ₇), (pKa 3.29); Lactic acid, H (C ₃H ₅O ₃), (pKa 3.08); Maleic acid, H ₄(C ₄O ₄) (pKa 1.83); Nicotinic acid, H ₅(C ₆NO ₂) (pK3.39); Oxalic acid, H ₂(C ₂O ₄), (pKa 1.23 forms 1); Oxalic acid, (HC ₂O ₄) ^-, (pKa4.19 form 2); Valeric acid, H (C ₅H ₉O ₂), (pKa 4.84); Phosphoric acid, H ₃PO ₄, (pKa 2.16 forms 1); Propanoic acid, H (C ₃H ₅O ₂), (pKa 4.86); Acetone acid, H4 (C ₃O ₃), (pKa 2.39); Succinic acid H ₆(C ₄O ₄) (pKa 4.19) and trichloroacetic acid, H (C ₂C ₁₃O ₂), (pKa 0.70).Any combination of these acid is also enumerated.

Preferred acetic acid.Aspirin, citric acid, formic acid, fumaric acid, Fluohydric acid., 1-Hydroxy-1,2,3-propanetricarboxylic acid., maleic acid, nicotinic acid, phosphoric acid, acetone acid, succinic acid and trichloroacetic acid are weak acid more commonly used, these weak acid respectively, combination and include as a set.

Term " non-ionic surface active agent " is meant a kind of surfactant, and this material reduces the surface tension that makes its dissolved material, and nonionic is meant to have uncharged polar group.The term amphiphilic surfactant be meant a surfactant molecule part for hydrophobic and a part of for hydrophilic a kind of surfactant.Suitable surfactant will for nonionic with amphipathic, and be suitable for veterinary or medical application.Specific non-ionic surface active agent whether is suitable for medical treatment or veterinary purpose can be easy to determine by those of ordinary skills.Many suitable non-ionic surface active agents can be used for the present invention and hereinafter provide a plurality of examples.

This paper includes the non-ionic surface active agent of two kinds of well-known types.They be called as sorbitan (usually with

Trade mark is sold) and polyoxyethylene sorbitan (usually with

Trade mark is sold), this paper includes following each thing especially:

Sorbitan monolaurate (Span

), sorbitan monopalmitate (Span

), sorbitan monostearate (Span

), sorbitan tristearate (Span

), dehydrated sorbitol mono-fatty acid ester (Span

), sorbitan trioleate (Span

), polyoxyethylene sorbitan monolaurate (Tween

), polyoxyethylene sorbitan monopalmitate (Tween

), polyoxyethylene sorbitan monostearate (Tween

), polyoxyethylene sorbitan monoleate (Tween 80) and polyoxyethylene sorbitan trioleate (Tween 85).These descriptions mean comprise trade (brand) name composition that these surfactant Supply Catalogs are listed or etc. effective constituent.Surfactant can be individually or combination in any use.

Sorbitan monolaurate (Span is described especially

), polyoxyethylene sorbitan monolaurate (Tween

Dehydrated sorbitol mono-fatty acid ester (Span ), sorbitan trioleate (Span

), polyoxyethylene sorbitan monoleate (Tween 80), polyoxyethylene sorbitan trioleate (Tween 85).

Term " suitable buffer " is meant a kind of buffer, and it is applicable to veterinary or medical application and can keeps relative constant pH value between about 6 and about 8 in aqueous solution.Phosphate buffer is an embodiment as herein described.Can be by means of the dihydric salt of blended sodium phosphate and/or potassium phosphate in varing proportions and monohydric salt and make the phosphate buffer with specific pH value of wide region.Those skilled in the art know the preparation and the purposes of various sodium buffer and potassium buffer.

Other example of buffer is as follows:

2-(N-morpholino) ethane sulfonic acid (being also referred to as MES);

3-(N-morpholino) propane sulfonic acid (being also referred to as MOPS);

N-[three (methylol)]-2-aminoethane sulphonic acid (being also referred to as TES);

4-(2-ethoxy) piperazine-1-ethane sulfonic acid (being also referred to as HEPES);

[three (methylol) methyl] glycine (being also referred to as TRIS).

Part i: the preparation of solution

New preparation disclosed herein is 1) glycosyl amide stock solution and 2) glycolipid adjuvant solution.

1) glycosyl amide stock solution is by being dissolved in glycolipid in the alcohol and mixing an amount of weak acid preparation.Weak acid added in the glycolipid alcoholic solution, and weak acid is molar excess with respect to glycolipid.Non-ionic surface active agent is added in the glycolipid alcohol acid blend makes glycosyl amide stock solution.The glycolipid of example is N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate.Example alcohol is ethanol.Example weak acid is acetic acid.Non-ionic surface active agent is aforesaid.

The preparation of glycosyl amide stock solution.The weak acid adding is contained in the alcoholic solution of glycolipid.The weak acid that adds is molar excess with respect to glycolipid content.The weak acid component of adding should be 1.25 to 5 times with the glycolipid molar equivalent.In specific embodiment, recommend the following relative quantity of acid.Weak acid should be 2.0 times, 2.5 times, 2.7 times, 3.0 times and 5.0 times of glycolipid molal quantity, and the best is 2.7 times.

Before or after adding weak acid, non-ionic surface active agent is added in the above-mentioned pure glycolipid mixture to obtain final glycosyl amide stock solution.

In the presence of weak acid, the glycosyl amide is changed into the acetate form of glycolipid.When only directly introducing in the aqueous buffer solution, the glycolipid of formula I can not fully dissolve.The solution that obtains from the buffer solution of dissolution type I glycolipid typically is milky mixt.Thereby early stage researcher has been attempted to make these mixture solutions even by means of this milky solution of supersound process.Yet ultrasonic Treatment can not guarantee that but solution keeps even between the storage life.The chemical method that these chemical compounds are suspended can make aqueous buffering glycolipid solution dissolve fully in proper pH value, near optically clear.When the weak acid that adds during, guarantee that all glycolipids all change into soluble form, and prevent that it from replying and be soluble form with respect to the glycolipid molar excess.

Weak acid makes glycolipid change into pharmaceutically acceptable salt.Preferred salt is nontoxic salts, and it is generally used for medicine and biological preparation.For example, chloride, acetate, lactate and the indifferent salt of the formula I chemical compound that obtains of described herein and weak acid.

The alcohol that is used to dissolve glycolipid can be propanol or its any combination of methanol, ethanol, any isomeric forms.Gained glycolipid alcoholic solution will be for clarifying on the optics.Any chemical reaction that the acetate form of glycolipid can be gone back to non-acetate form all will cause glycolipid to flocculate in aqueous solution.When the glycolipid flocculation occurred, the glycolipid molecule is laminar separated out from solution, is deposited in container bottom.Faintly acid initial concentration has determined whether will exist the flocculation of any glycolipid in the glycosyl amide stock solution of glycolipid and alcohol.Weak acid answers molar excess to avoid flocculation with respect to glycolipid.

2) glycolipid adjuvant solution is to prepare by an amount of glycosyl amide stock solution is introduced in " suitable buffer ".It is as herein described that finally the pH value of stable glycolipid adjuvant solution should be between about 6 and about 8.Preferred final pH value is between about 6 and about 7.Final pH value between about 6.3 and about 6.4 has been described.

Because glycosyl amide stock solution contains excess acid, so it has resiliency, can be used as adjuvant.For example, can mix the phosphate buffer that makes specific pH value by dihydric salt and monohydric salt with different proportion sodium phosphate or potassium phosphate with wide region.If use phosphate buffer, then can it be made, and its pH value be about 7.8 with about 20mM.When being added into glycosyl amide stock solution in the buffer, the pH value of buffer reduces.The phosphate buffered solution of pH 7.8 produces pH value and is about 6.4 final glycolipid adjuvant solution.Final pH value can be regulated, but it is also unnecessary usually.

The pH value of glycosyl amide stock solution that contains weak acid and glycolipid is extremely low.Can be necessary pH value is increased to acceptable level.Should avoid highly basic for this purpose,, cause that salt-independent shape precipitates (flocculation) in aqueous environments because alkaline adding can transform back salt-independent shape with the salt form of glycolipid.Yet,, should only use a small amount of if need highly basic.For example, recommend to use the NaOH of maximum 100mM, and the best is 4.0mM or littler.

Buffer solution optionally comprises some NaCl, but unessential.NaCl concentration can between about 1 and about 50mM between.More a spot of NaCl is better than more amount.The example of this paper does not have NaCl or 15mM NaCl.Owing to flocculation can occur, so the NaCl of 100mM and improper.15mM or lower NaCl concentration expection can not produce flocculation.30mM or lower NaCl concentration expection can not produce flocculation.50mM or lower NaCl concentration expection can not produce flocculation.

Part ii: the evaluation of glycolipid adjuvant solution

The stability of glycolipid adjuvant solution can be by simple range estimation or by using suitable analytical tool to monitor between the storage life.When in aqueous solution, the glycolipid molecule forms micelle and can use laser-diffractometer accurately to measure micellar size.This measurement can be used for determining whether to exist the flocculation of glycolipid molecule.

The alternative method of real-time stabilization measurement is for carrying out accelerated stability test.Accelerated stability test makes assist agent solution stand about 37 ℃ temperature lasting about 7 days, cultivated about 2 days down at about 4 ℃ then, and constantly jolting.Cultivate the about 1 year period that stores under about 4 ℃ that was illustrated in about 7 days down at about 37 ℃.Under continuous jolting, cultivate the stress condition of representing that glycolipid adjuvant solution during transportation can be faced in about 2 days at about 4 ℃.

For determining that whether glycolipid adjuvant solution open with Cytoplasm etc., can measure osmotic pressure.Can add different concentrations of sodium chloride and use permeability manometer to measure the osmotic pressure of gained solution.Except that increasing osmotic pressure, the concentration that increases sodium chloride also tends to make solution muddy more.Turbidity is considered to be gathered into than macroparticle by micelle and causes.Use 0.2 μ m filter to be difficult to or can't be unsuitable for commercialization usually by filtering solution, be generally used for guaranteeing that the assist agent solution of commercial size preparation is aseptic because filter latter stage.Electronic Micro-Analysis can be used for determining whether assembling owing to too much salt causes micelle.

Non-glycolipid adjuvant in addition also can be used for glycolipid adjuvant solution with the above-mentioned substance combination.In another embodiment of the present invention, other immunostimulating molecule can be added in the glycolipid adjuvant solution.The immunostimulating molecule is known in this technology, and they comprise Saponin, Quil A, GERBU Adjuvant 100 (DDA) and carbopol (Carbopol).

Quil A obtains for the bark from South America alkalium wood (Quillaja saponaria) extracts purification.Reaction of Quil A inductor fluidity and cell-mediated reaction.Quil A usually uses jointly with cholesterol, because can eliminate comparatively bad side effect during cholesterol adding proper proportion.Cholesterol and Quil A form insoluble complex, and when cholesterol combined with Quil A, these complex formed similar helicoidal structure, therefore expose the sugar unit of molecule, thereby help immune response stimulating.

GERBU Adjuvant 100 DDA is the cationic surfactant with 18 carbon alkyl chains.It is amphipathic quaternary amine.Since DDA on oil/water termination by directly combine with antigen as antigenic carrier, therefore need make the direct and AI of DDA with the acquisition optimal immune response.It stimulates body fluid immunoreation and cell-mediated immunoreation.

Carbopol is to can be used for another useful immunostimulating molecule of the present invention.It is and the crosslinked acrylate homopolymer of polyalkenyl ether.

III part: the purposes of glycolipid adjuvant solution

The glycolipid adjuvant solution of pharmaceutically acceptable salt form can be mixed with antigen.The antigen that is fit to comprises: microbial pathogens albumen, glycoprotein, lipoprotein, peptide, glycopeptide, lipopeptid, toxoid, carbohydrate and tumor specific antigen.Antigen can be derived from multiple source.Antigen from microbial pathogens comprises malignant bacteria, virus and parasite body.Can use two or more antigenic mixture.Antigen can be through kill, the active antigen of natural attenuation, modification, or the albumen, chemical synthesising peptide or the immune stimulatory that produce for protein extract, reorganization any other material of replying.Peptide antigen can be used as free peptide and exist or with the glycolipid conjugation or with other known B cell or T cellular antigens determinant conjugation.

Stable glycolipid adjuvant solution can with other adjuvant or known combination of components with adjuvant character.Can comprise the derivant of polymer, naturally occurring terpenoid rough or partially purified form, amphiphilic quaternary amine, bacterial cell wall material and the synthetic analogues of bacteria cell wall or DNA component with the other adjuvant of glycolipid adjuvant solution combination.Glycolipid adjuvant solution can use or combination jointly with one or more reagent (such as antibiotic or synantigen not).Antibacterial or virus antigen can be the active antigen through killing or modify.By making virus growth and make inactivation of virus prepare virus antigen in tissue culture through killing via chemical treatment.Some virus can be grown in fertilized ovum.Killed virus antigen can add in the solution that contains glycolipid adjuvant solution, and gained solution can be used for the animal inoculation vaccine to realize avoiding the protection of viral infection.

In one embodiment of the invention, glycolipid adjuvant solution can be used as the antigenic diluent of live virus of modification.Can make the viral pathogen perform toxic attenuation through tissue culture or by virus genomic special handling from generation to generation by several that make viral pathogen.These attenuated viral strains can become in tissue culture very high tire and can be used as vaccine antigen.Attenuated viral strains is called as the live virus antigen of modification.Although these Strain toxicity are less, when as the antigen in the vaccine, its still for hyperimmunization originality and provide and avoid the protection that the virulent virus strain is infected.If glycolipid adjuvant solution is as the antigenic diluent of modifying of live virus, then glycolipid adjuvant solution should be after tested to guarantee that it does not have any effect of killing the virus to relevant specific virus.

Can in experiment in vitro, measure live virus antigenic the kill the virus character of glycolipid adjuvant solution to modifying.Make through cryodesiccated virus antigen rehydrated with glycolipid adjuvant solution or water.Gained virus solution is applied on the monolayer that allows cell.Tiring of virus antigen by the bacterial plaque counting that forms on the monolayer is determined.Among the rehydrated sample of the water sample rehydrated with using glycolipid adjuvant solution, the difference of the virus titer that is obtained can be used for determining whether to exist any glycolipid adjuvant solution that any live virus is had the effect of killing the virus.

The live virus antigen of modifying can be cryodesiccated, and in the commercial vaccine preparation as providing through cryodesiccated cake.Usually, the live virus of these modifications is antigenic rehydrated through cryodesiccated cake and diluent solution, and is used for non-vaccine through enteral administration.The example of diluent comprises the aqueous solution that contains phosphate buffer salt.If diluent solution contains known immunostimulating molecule, can improve the effect of the antigenic vaccine of live virus that has modification.In one embodiment of the invention, glycolipid adjuvant solution is used as diluent solution.

Embodiment

Embodiment 1. is with isocyatic Bay

Prepare insoluble glycosyl amide compositions with acetic acid.

Table 1. is not suitable for the compositions of commercial use

Bay

By Bayer register of company, its commodity are called N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide.When this chemical compound was used for assist agent solution that the described chemical compound of manufacturing table 1 uses, wherein acetic acid was to use with molar concentration such as glycolipid and glycolipid is the form of its free alkali, the soluble and flocculation of this glycolipid.

Embodiment 2. solubility glycosyl amide stock solutions

Use the component identical but acetic acid concentration increases generation solubility glycosyl amide stock solution to some extent with respect to glycolipid concentration with embodiment 1.

The composition of table 2. glycosyl amide stock solution

Use 60% ethanol (v/v) herein, and the mol ratio of acetic acid and glycolipid is 2.0.200-standard (proof) ethanol that replaces embodiment 1 with 60% ethanol water.Gained glycosyl amide stock solution is clarifying and do not have sedimentation at container bottom on the optics.This glycosyl amide stock solution is added in the various buffer with the glycolipid adjuvant solution in preparation following examples 3.

Embodiment 3. preparation glycolipid adjuvant solution

The phosphate buffered solution for preparing different pH value.By restraining NaH with 138 ₂PO ₄H ₂O salt is dissolved in the 250mL deionized water in the beaker and makes final volume is 500mL, makes 2M sodium dihydrogen phosphate stock solution.Similarly, by restraining NaH with 142 ₂PO ₄Being dissolved in the 300mL deionized water in the beaker and making final volume is 500mL, makes 2M sodium hydrogen phosphate stock solution.Two kinds of stock solutions all use 0.2 micron filter aseptic filtration.

The composition of the 1M stock solution of the buffer solution of sodium phosphate of the different pH value of table 3.

The pH value of calculation	Na 2HPO 4Solution (ml)	NaH 2PO 4·H 2O solution (ml)	The cumulative volume of 2M stock solution (ml)	The sterile deionized water that is added (ml)	The cumulative volume of 1M stock solution (ml)
						6.0	87.7	12.3	100	100	200
6.5	68.5	31.5	100	100	200
						7.0	39.0	61.0	100	100	200
7.5	16.0	84.0	100	100	200
						7.8	8.5	91.5	100	100	200

The 2M sodium dihydrogen phosphate stock solution and the sodium hydrogen phosphate stock solution of the different volumes of preparation shown in the table 3 obtain the 1M stock solution of the buffer solution of sodium phosphate of different pH value then.Then, with 50 * dilution 1M phosphate buffered solution to be to obtain the 20mM phosphate buffer.

Glycolipid adjuvant solution is to use these deposit buffer and from the preparation of the glycosyl amide stock solution of embodiment 2.

Prepared 5mL glycosyl amide stock solution in every 96mL of these 20mM phosphate solutions, adding as embodiment 2.Gained glycolipid adjuvant solution contains 12.5mM acetic acid and 6.33mM glycolipid.Glycolipid is the acetate form at this moment.

The importance of the final pH value of embodiment 4. glycolipid adjuvant solution.

In another group experiment, how the importance of testing the final pH value of various solution influences flocculation to estimate pH value.Preparation 20mM phosphate buffer, original ph is 7.8.Glycolipid adjuvant shown in the table 4 is to use as the preparation of glycosyl amide prepared among the embodiment 1, wherein with etc. molar concentration use glycolipid and acetic acid.Notice that final pH value that descend and few (table 4) demonstrates the effect of buffer.NaCl concentration changes.The optical density (OD) reading (O.D.) at 600nm place in the table 4 is compared with the similar reading in the table 5, and the glycolipid adjuvant solution of table 5 is to prepare with the glycosyl amide stock solution as the acetic acid that contains the glycolipid twice as high molar ratio prepared among the embodiment 2.Use higher concentration or relatively large acetic acid can produce minimum flocculation.The flocculation of filtered sample is more than filtered sample.In addition, along with NaCI concentration increases, flocculation increases even occurs precipitating.With original ph is that 8.0 phosphate buffer prepares the glycolipid adjuvant solution described in the table 5, and the final pH value of glycolipid adjuvant solution is between 6.8 and 7.0.The glycolipid adjuvant solution that the further reduction of the final pH value of glycolipid adjuvant solution can cause having low turbidity and not have flocculation.

Table 4. preparation contains the acetic acid of equimolar amounts and the glycosyl amide compositions of glycolipid (referring to embodiment 1)

NaCl concentration (mM)	Buffer volume (ml)	The volume (ml) of glycolipid deposit compositions	Final pH value	The O.D. at 600nm place
					0	480	25	7.42	1.693
15	480	25	7.39	1.873
					100	480	25	7.33	2.742

Table 5. uses the glycosyl amide stock solution of the acetic acid that contains glycolipid mole twice to prepare glycolipid adjuvant solution (referring to embodiment 2)

NaCl concentration (mM)	Buffer volume (ml)	The volume (ml) of glycosyl amide stock solution	Final pH value	The O.D. at 600nm place
					0	480	25	6.93	0.146
15	480	25	6.88	0.487
					100	480	25	6.84	2.826

Optical density (OD) is represented translucent solution less than 0.1 (O.D.).For evenly and slightly muddy, optical density (OD) 0.5 to 1.0 has a little muddiness to optical density (OD) between 0.1 and 0.5, and optical density (OD) 1.0 to 1.5 is assert muddy.Optical density (OD) be higher than 1.5 for muddy and can not use 0.2 micron filter to filter.It is generally acknowledged that the latter is not suitable for commerce.

Embodiment 5. demonstrates flocculation with acetic acid titration glycolipid adjuvant and can reverse.

For determining whether the acetic acid that adds recruitment in the glycolipid adjuvant that shows flocculation will reverse flocculation, preparation glycolipid adjuvant as described in example 1 above.Even in the presence of no any NaCl, flocculation also appears in this glycolipid adjuvant.In this flocculation glycolipid adjuvant mixture, add the acetic acid that increases concentration.With 16.6 times of water dilution acetic acid to obtain the working solution concentration of 1 molar concentration.Then, this 1M solution of 15 μ l is added in the 15ml glycolipid adjuvant mixture so that acetic acid concentration increases to 1mM.Along with acetic acid concentration increases, the pH value of glycolipid adjuvant reduces and the flocculation dissolving.But glycolipid adjuvant is still muddy in a measure.This observed result confirms that the acetic acid concentration that increases makes the free alkali of Bay 15-5381 change into the acetate form, and this salt form more can dissolve in aqueous solution.

Table 7. is with acetic acid titration glycolipid adjuvant

The volume of glycolipid adjuvant	The volume of the 1N acetic acid that is added	The pH value of solution
			15mL	0	7.25
15mL	15μl(1mM)	7.21
			15mL	30μl(2mM)	7.10
15mL	60μl(4mM)	6.97
			15mL	150μl(10mM)	6.44
15mL	750μl(50mM)	4.57

Embodiment 6. preparations contain NaCl and do not contain second of NaCl stablizes the glycolipid assist agent solution

Establishing being increased in after the importance of keeping in the glycolipid stability of solution of acetic acid amount, compositions shown in the decision use table 8 at first prepares glycosyl amide stock solution, and uses this formulations prepared from solutions another kind to contain NaCl then and do not contain the glycolipid adjuvant solution of NaCl.This glycosyl amide stock solution is similar to the solution among the embodiment 2, and cumulative volume is its 4 times and acetic acid and polysorbas20 with relatively large amount.

The composition of table 8. glycosyl amide stock solution

Reagent	Amount (200ml)
		60% ethanol (volume/volume)	179ml
Polysorbas20	4.0ml
		Acetic acid	3.0ml
Bay 15-5381	13.96 gram

The phosphate buffer that uses embodiment 3 with as the glycosyl amide stock solution of preparation in the table 8 prepare 3 kinds of different glycolipid assist agent solutions with different N aCl concentration.

As the preparation in embodiment 4, the table 5, make the glycolipid adjuvant solution of the NaCl that contains 0mM, 15mM and 100mM.Can filter 0mM and 15mMNaCl solution via 0.2 micron filter.The glycolipid adjuvant solution that contains the NaCl of 100mM can not filter by 0.2 micron filter.

Table 9. contains NaCl and does not contain the preparation of the stable glycolipid assist agent solution of NaCl

Sodium chloride concentration (mM)	Buffer volume (ml)	The volume (ml) of glycosyl amide stock solution	Final pH value	The O.D. of 600nm
					0	465	35	6.39	0.039
15	465	35	6.37	0.073
					100	465	35	6.29	0.439

Each glycolipid adjuvant solution of 20mL is placed the 30ml vial and cultivate down room temperature and 4 ℃.Estimate at predetermined distance.At first, the glycolipid adjuvant solution with NaCl of 0mM is clarifying on the optics.It is muddy slightly and be 0.073 at the O.D. of 600nm to contain the glycolipid adjuvant solution of NaCl of 15mM.It is muddy and be 0.439 (table 9) at the O.D. of 600nm to contain the glycolipid adjuvant solution of NaCl of 100mM.Room temperature and 4 ℃, these glycolipid adjuvant solution all do not show the sign of any flocculation.These glycolipid adjuvant solution are observed a term, its outward appearance no change.

Embodiment 7: stablize the glycolipid assist agent solution with the NaOH titration.

Initially, obtain not contain clarification and stable glycolipid adjuvant solution on the optics of NaOH.Removing or use minimum NaOH for establishment is necessary to preventing to flocculate, and is necessary to show that adding NaOH gradually can induce flocculation in the stabilizing sugar lipoprotein mixture that does not add NaOH.The NaOH of the 1N of proper volume is added in 15mL such as the following table 10 in the prepared glycolipid adjuvant solution that does not add any NaCl.Gradually NaOH is increased to 12mM (table 10) from 1mM.Use the glycosyl amide stock solution preparation described in the embodiment 6 to be used for the glycolipid adjuvant solution of this experiment.Along with the NaOH concentration in the glycolipid adjuvant solution increases gradually, the pH of preparation increases gradually, occurs flocculation simultaneously.

Table 10. is with the stable glycolipid adjuvant solution of NaOH titration

The volume of glycolipid adjuvant	The NaOH volume (mM) of the 1N that is added	The pH value of solution
			15ml	0	6.21
15ml	15μl(1mM)	6.38
			15ml	30μl(2mM)	6.48
15ml	60μl(4mM)	6.68
			15ml	150μl(10mM)	7.11
15ml	750μl(50mM)	12.17

Embodiment 8. uses HPLC quantitative to the glycolipid component.

Following methodology is used for Bay

HPLC analyze.Use the HPLC parameter described in the table 11.

Table 11. is used for quantizing the summary of the used parameter of HPLC method of Bay 15-5381

Parameter	Details
		Chromatographic column	Hamilton PRP-1,7 microns, 250 * 4.6mm
Flow velocity	1.5ml/min
		Sample size	10μl
Detect wavelength	210nm
		Mobile phase A	0.4% perchloric acid, v:v
Mobile phase B	Acetonitrile
		Gradient	0min，40％ A/60％ B 15min，30％ A/70％ B 20min，30％ A/70％ B 35min，10％ A/90％ B 50min，10％ A/90％ B 51min，40％ A
Running time	65min
		Bay 15-5381 retention time	About 25 minutes

Table 12.Bay

Reference substance

Reference substance	Concentration
		1	0.103mM
2	0.206mM
		3	0.412mM
4	0.618mM
		5	0.824mM
6	1.03mM

Reference substance in preparation 0.10 to the 1.03mM scope also injects HPLC.The summary of these reference substances is as shown in table 12.The sample temperature is inverted 5 times to room temperature and before using.In the 10ml volumetric flask, the 1ml sample is added in the 6ml methanol.Then the sample ultrasonic ripple was handled 10 minutes, it is diluted to scale and mixing.With peak area concentration is mapped, reference substance is carried out linear regression analysis.According to standard curve calculation sample amount.

Embodiment rose scale preparation in 9: three ten (30).

Prepare a collection of 30 liters of glycolipid adjuvant solution forming as described in example 6 above that have.This batch solution contains the NaCl of 15mM.

Use this 30L preparation, to increase by 5 kinds of different sub-solution of NaOH preparation of concentration.NaOH concentration increases to 1mM, 2mM, 4mM, 8mM and 12mM from 0mM.The sample aliquot of each NaOH concentration is used for pH value measurement and range estimation.Along with the increase of NaOH amount, the pH value of glycolipid adjuvant also increases, and follows flocculation to increase.At room temperature flocculating begins to occur in the NaOH of 2mM concentration, and begins to occur among the NaOH of flocculation at 4mM down at 4 ℃.

Table 13. is along with the characteristic of the increase 30L batch glycolipid adjuvant of NaOH concentration

Sample number	Describe	Measure concentration (mM)	The O.D. at 600nM place	The pH value
					30-L sample 1	15mMNaCl， 0mM NaOH	6.21	0.218	6.42
30-L sample 2	15mMNaCl， 1mM NaOH	6.3	0.137	6.52
					30-L sample 3	15mMNaCl， 2mM NaOH	6.24	0.137	6.59
30-L sample 4	15mMNaCl， 4mM NaOH	6.17	0.15	6.80
					30-L sample 5	15mMNaCl， 8mM NaOH	6.26	0.129	7.06
30-L sample 6	15mMNaCl， 12mM NaOH	6.15	0.062	7.54

The amount of using the HPLC method described in the embodiment 8 to come Bay 15-5381 in 6 samples of all shown in the quantization table 13.Sample with different pH value shows the Bay 15-5381 of same concentrations, and this explanation adjuvant component is along with NaOH adding pH value increase and with not degraded during the flocculation.

Embodiment 10: use the acceleration stress test to carry out estimation of stability.

This embodiment describes the method and the result of the acceleration stress test of glycolipid adjuvant solution.With 3 batches of glycolipid adjuvant solution as described in example 6 above of 500L scale preparation.All 3 batches all have the NaCl of 15mM and do not contain NaOH.To use accelerated stability test from this 500L of 3 batch glycolipid adjuvant solution, be used to study the stability of glycolipid.

For quickening stress test, this glycolipid adjuvant solution is 37 ℃ of following joltings 7 days, then 4 ℃ of following joltings 2 days.Being illustrated in 4 ℃ in 7 days 37 ℃ of following joltings placed 1 year down.Stress between 2 days expression delivery periods of 4 ℃ of following joltings.

One group of glycolipid adjuvant solution keeps static down at 37 ℃ and continues 7 days, then in jolting 2 days in addition under 100rpm under 4 ℃.At 4 time points, i.e. T=0,3,7 and 9 days, record observed result and photo.Then at 2 time points, that is T=0 and 9 days, carry out refractive index and grain size analysis.

Make second group of glycolipid adjuvant solution in jolting 7 days under 100rpm under 37 ℃; Then in addition jolting 2 days under 100rpm under 4 ℃.At 4 time points, that is T=0,3,7 and 9 days, record observed result and photo.Then at 2 time points, that is T=0 and 9 days, carry out refractive index and grain size analysis.

Make the 3rd group of glycolipid adjuvant solution keep down static and continue 9 days, with in contrast at 4 ℃.At 4 time points, that is T=0,3,7 and 9 days, record observed result and photo.Then at 2 time points, that is T=0 and 9 days, carry out refractive index and grain size analysis.

Not owing to stress test causes change of granularity.Observation sample immediately after sample preparation, all samples is all kept submicron particle size.In addition, remaining in 4 ℃ of Bay down or in 37 ℃ of samples that stand stress

The HPLC of component measures and does not show Bay

Any variation of amount.

Table 15. is at the back Bay of stress test

Quantification.

The lot number of glycolipid adjuvant solution and processing	Measure concentration (mM)
		1 batch-4 ℃	6.23
1 batch of-37 ℃ of jolting	6.29
		2 batches-4 ℃	6.32
2 batches of-37 ℃ of joltings	6.30
		3 batches-4 ℃	6.28
3 batches of-37 ℃ of joltings	6.29

In table 15, reference substance was kept 7 days down at 4 ℃, and with specimen 37 ℃ of following joltings 7 days.Has similar concentration at 7 days sample of 37 ℃ of following joltings to the sample that stores down at 4 ℃.

Embodiment 11: the test of killing the virus of glycolipid adjuvant solution.

To the test of killing the virus of above embodiment 9 described glycolipid adjuvant solution with the 30L scale preparation.This glycolipid adjuvant solution contains the NaCl of 15mM and does not contain NaOH.

The test glycolipid adjuvant is as the adaptability of the diluent of the live virus of modifying.With the live virus antigen preparation of modifying is through cryodesiccated material.When making these materials rehydrated, confirm that used glycolipid adjuvant solution does not kill the live virus of modification with suitable glycolipid adjuvant solution.3 kinds of bovine viral antigens of test glycolipid adjuvant solution antagonism: bovine respiratory syncytial virus (BRSV), parainfluenza virus 3 (PI3) and infectious bovine rhinotrachetis (IBR) virus.

Use glycolipid adjuvant solution, make viral material rehydrated.After room temperature (RT) is cultivated 1 hour down, sample is applied in the monolayer permissive cell strain of serial dilution.By obtaining every milliliter 50% TCID (TCID50/ml) value with each rehydrated virus antigen of sterilized water or glycolipid adjuvant solution to appearing at viral filler counting on the monolayer.In this measured, the reduction by 0.7 of tiring after the glycolipid adjuvant solution of test is rehydrated was considered as killing the virus.

The results are shown in Table 16.Glycolipid adjuvant solution does not show any effect of killing the virus to these 3 kinds of bovine virals.

The mensuration of killing the virus of table 16. glycolipid adjuvant solution

Virus	Tire at first	Finally tiring of glycolipid adjuvant	Finally tiring of sterilized water	The loss of tiring
					tsIBR 051404	7.3±0.5	7.08	7.49	0.42
tsPI3 052604	7.6±0.5	7.74	7.49	-0.25
					BRSV 081103	6.4±0.5	6.57	6.82	0.25

This embodiment shows that glycolipid adjuvant solution can be used in the commercialization preparation of animal health vaccine. Contain the antigenic three kinds of different cattle disease viral diseases of the live virus that utilizes 3 kinds of modifications.These bovine viral antigens are cattle on the hoof herpesvirus, the cattle on the hoof respiratory syncytial virus of modification and the parainfluenza viruses alive 3 of modification of modifying.These virus antigens are made through cryodesiccated cake, and the glycolipid adjuvant solution of the present invention's preparation can be used as these antigenic diluent solutions.Used glycolipid is N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate.Provide these embodiment with explanation the present invention.These embodiment should be considered as limitation of the scope of the invention.To those skilled in the art, many changes of the present invention, variation, improvement and other purposes and application will be conspicuous.

Claims (15)

1. compositions, it comprises:

A) glycolipid of formula I,

Its Chinese style I is

Wherein

R ¹And R ²Be hydrogen or have the saturated alkyl of 20 carbon atoms at the most independently;

X is-CH ₂-,-O-or-NH-;

R ²For hydrogen or have the saturated or unsaturated alkyl of 20 carbon atoms at the most;

R ³, R ⁴And R ⁵Be independently hydrogen ,-SO ₄ ^2-,-PO ₄ ^2-,-COC _1-10Alkyl;

R ⁶For L-alanyl, L-alpha-amido butyl, L-arginyl-, altheine acyl group, L-aspartyl, L-cysteinyl-, L-glutamyl, L-glycyl, L-histidyl-, L-hydroxypropyl, L-isoleucyl-, L-leucyl-, L-lysyl-, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-seryl-, L-Threonyl, L-tyrosyl-, L-tryptophanyl with L-is valyl or their D-isomer;

This glycolipid is the form of salt, and wherein the form of this salt is derived from weak acid;

B) alcohol should alcohol be HO-C wherein _1-3Alkyl;

C) weak acid, wherein this weak acid is 1) be molar excess with respect to glycolipid content, and 2) use standard scale or standard value pKa (Ka-log) any acid of value between 1.0 and 9.5;

D) non-ionic surface active agent, wherein this non-ionic surface active agent is that a kind of reduction makes the capillary reagent of its dissolved material and has a kind of hydrophobic components and another kind of hydrophilic component.

2. the compositions of claim 1, wherein this glycolipid is the chemical compound of formula II (a),

Formula II (a)

And this weak acid is a kind of or combination in any that is selected from the following weak acid: acetic acid, H (C ₂H ₃O ₂) (pKa 4.76); Ascorbic acid (1), H ₂(C ₆H ₆O ₆) (pKa 4.10); Aspirin, H ₈(C ₉O ₄), (pKa 3.5); Butanoic acid, H (C ₄H ₇O ₂) (pKa 4.83); Carbonic acid, form 1, H ₂CO ₃, (pKa 4.83); Chromic acid, form 2, HCrO ₄ ^-, (pKa 6.49); Citric acid form 1, H ₃(C ₆H ₅O ₇), (pKa 3.14); Citric acid form 2, H ₂C ₆H ₅O ₇) ^-, (pKa 4.77); Citric acid form 3, (HC ₆H ₅O ₇) ⁼, (pKa 6.39); Formic acid, H (CHO ₂), (pKa 3.75); Fumaric acid, H ₄(C ₄O ₄) (pKa 3.03); Enanthic acid, H (C ₇H ₁₃O ₂), (pKa 4.89); Caproic acid, H (C ₆H ₁₁O ₂), (pKa 4.84); Fluohydric acid., HF, (pKa 3.20); 1-Hydroxy-1,2,3-propanetricarboxylic acid., H ₈(C ₆O ₇) (pKa 3.29); Lactic acid, H (C ₃H ₅O ₃), (pKa 3.08); Maleic acid, H ₄(C ₄O ₄) (pKa 1.83); Nicotinic acid, H ₅(C ₆NO ₂) (pK 3.39); Oxalic acid form 1, H ₂(C ₂O ₄), (pKa 1.23); Oxalic acid form 2, (HC ₂O ₄) ^-, (pKa 4.19); Valeric acid, H (C ₅H ₉O ₂), (pKa 4.84); Phosphoric acid 1, H ₃PO ₄, (pKa 2.16); Propanoic acid, H (C ₃H ₅O ₂), (pKa 4.86); Acetone acid, H4 (C ₃O ₃) (pKa 2.39); Succinic acid H ₆(C ₄O ₄) (pKa 4.19) and trichloroacetic acid, H (C ₂C ₁₃O ₂), (pKa 0.70).

3. the compositions of claim 2, wherein this glycolipid is the chemical compound of formula II (b),

Formula II (b)

And this weak acid is selected from following faintly acid a kind of or combination in any: acetic acid, aspirin, citric acid, formic acid, fumaric acid, Fluohydric acid., 1-Hydroxy-1,2,3-propanetricarboxylic acid., maleic acid, nicotinic acid, phosphoric acid, acetone acid, succinic acid and trichloroacetic acid.

4. the compositions of claim 3, wherein this glycolipid is N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate with formula III structure,

Formula III

And this weak acid is acetic acid.

5. the compositions of claim 2, wherein this weak acid is selected from the group that following each material is formed: acetic acid, aspirin, citric acid form 1, citric acid form 2, citric acid form 3, formic acid, fumaric acid, Fluohydric acid., 1-Hydroxy-1,2,3-propanetricarboxylic acid., maleic acid, nicotinic acid, phosphoric acid 1, acetone acid, succinic acid and trichloroacetic acid.

6. each compositions among the claim 1-5, wherein this faintly acid amount is greater than the molar equivalent of this glycolipid, or this faintly acid amount is with the molar equivalent of following multiple greater than this glycolipid:

A) 1.25 times,

B) 2.0 times,

C) 2.5 times,

D) 2.7 times,

E) 3.0 times,

F) 5.0 times,

Molar equivalent greater than this glycolipid.

7. each compositions among the claim 1-6 should alcohol be an ethanol wherein.

8. each compositions among the claim 1-7, wherein this non-ionic surface active agent is selected from any or combination in the group that following each material forms:

Sorbitan monolaurate, the sorbitan monopalmitate, sorbitan monostearate, the sorbitan tristearate, dehydrated sorbitol mono-fatty acid ester, sorbitan trioleate, the polyoxyethylene sorbitan monolaurate, the polyoxyethylene sorbitan monopalmitate, the polyoxyethylene sorbitan monostearate, the polyoxyethylene sorbitan monoleate, polyoxyethylene sorbitan trioleate and other are generally used for sorbitan and the polyoxyethylene sorbitan in the vaccine.

9. compositions, it comprises:

A) glycolipid of formula I;

Its Chinese style I is

Wherein

R ¹And R ²Be hydrogen or have the saturated alkyl of 20 carbon atoms at the most independently;

X is-CH ₂-,-O-or-NH-;

R ³, R ⁴And R ⁵Be independently hydrogen ,-SO ₄ ^2-,-PO ₄ ^2-,-COC _1-10Alkyl;

R ⁶For L-alanyl, L-alpha-amido butyl, L-arginyl-, altheine acyl group, L-aspartyl, L-cysteinyl-, L-glutamyl, L-glycyl, L-histidyl-, L-hydroxypropyl, L-isoleucyl-, L-leucyl-, L-lysyl-, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-seryl-, L-Threonyl, L-tyrosyl-, L-tryptophanyl with L-is valyl or their D-isomer;

This glycolipid is the form of salt, and wherein the form of this salt is derived from weak acid;

B) alcohol should alcohol be HO-C wherein _1-3Alkyl;

C) weak acid, wherein this weak acid is 1) be molar excess with respect to glycolipid content, and 2) use standard scale or standard value pKa (Ka-log) any acid of value between about 1.0 and about 9.5;

D) non-ionic surface active agent, wherein this non-ionic surface active agent is that a kind of reduction makes the capillary reagent of its dissolved material and has a kind of hydrophobic components and another kind of hydrophilic component; With

E) aqueous buffer solution, wherein this suitable buffer pH value of being suitable for vaccine use and can keeping other composition between pH value about 6 to about 8,

The NaCl that condition is to use is no more than 50mM at most.

10. the compositions of claim 9, wherein the pH of this solution is adjusted to the relative constant pH between about 6 and about 7 in the aqueous solution, and this buffer is selected from the group that the two phosphate buffer of one of the dihydric salt of sodium phosphate with identical or different ratio and/or potassium phosphate and monohydric salt or dihydric salt and monohydric salt is formed.

11. the compositions of claim 9, wherein this buffer is selected from the group that following each material is formed:

A) 2-(N-morpholino) ethane sulfonic acid (being also referred to as MES);

B) propane sulfonic acid (being also referred to as MOPS) 3-N-morpholino);

C) n-[three (methylol)]-2-aminoethane sulphonic acid (being also referred to as TES);

D) 4-(2-ethoxy) piperazine-1-ethane sulfonic acid (being also referred to as HEPES); With

E) [three (methylol) methyl] glycine (being also referred to as TRIS);

Or its any combination.

12. each compositions among the claim 1-11, it also comprises antigen or its any combination in the group that is selected from following each material composition: the parainfluenza virus alive 3 of the cattle on the hoof herpesvirus of modification, the cattle on the hoof respiratory syncytial virus of modification and modification.

13. a compositions, it comprises:

A) have N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate of formula III structure:

Formula III

B) ethanol;

C) acetic acid;

D) non-ionic surface active agent, this non-ionic surface active agent is selected from: sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, dehydrated sorbitol mono-fatty acid ester, sorbitan trioleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monoleate, polyoxyethylene sorbitan trioleate;

E) aqueous buffer solution, wherein the pH value of this solution is adjusted to the constant relatively pH value between about 6 and about 7 in the aqueous buffer solution, and this buffer is selected from the group that following each material is formed:

(a) 2-(N-morpholino) ethane sulfonic acid (also being called MES);

(b) propane sulfonic acid (also being called MOPS) 3-N-morpholino);

(c) n-[three (methylol)]-2-aminoethane sulphonic acid (also being called TES);

(d) 4-(2-ethoxy) piperazine-1-ethane sulfonic acid (also being called HEPES); With

(e) [three (methylol) methyl] glycine (also being called TRIS);

Or its any combination,

The NaCl that condition is to use is no more than 15mM at most; With

F) antigen of forming by the parainfluenza virus alive 3 of the cattle on the hoof respiratory syncytial virus of the cattle on the hoof herpesvirus of modifying, modification and modification basically.

14. one kind prepares method for compositions, it comprises following each material is mixed:

A) glycolipid of formula I;

B) alcohol should alcohol be HO-C wherein _1-3Alkyl;

C) weak acid, wherein this faintly acid amount is a molar excess with respect to glycolipid content; With

D) non-ionic surface active agent.

15. one kind prepares method for compositions, it comprises following each material is mixed:

A) glycolipid of formula I;

B) alcohol should alcohol be HO-C wherein _1-3Alkyl;

C) weak acid, wherein this faintly acid amount is a molar excess with respect to glycolipid content;

D) non-ionic surface active agent; And add

E) suitable buffer.