CN101374551B

CN101374551B - Glycolipid adjuvant compositions

Info

Publication number: CN101374551B
Application number: CN200780003499.XA
Authority: CN
Inventors: P·J·多米诺夫斯基; R·M·曼南; S·麦迪拉塔
Original assignee: Pfizer Products Inc
Current assignee: Zoetis LLC
Priority date: 2006-01-26
Filing date: 2007-01-15
Publication date: 2013-06-12
Anticipated expiration: 2027-01-15
Also published as: UA90569C2; ZA200806149B; CN101374551A; SI1991266T1

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.In the present invention, these new compositionss are for a long time stable and without flocculation.They especially help to comprise the release of the multi-medicament of vaccine.

Background of invention

Vaccine is generally used for to protect mankind 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 or pathogenic organism body, albumen, recombiant protein or its fragment attenuation modified by the pathogenic organism body survival of having killed consist of usually for they.Regardless of the source of antigen, usually be necessary to add adjuvant to strengthen the host to the immunne response of antigen.

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

First adjuvant of reporting in 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.Undesirable is, the FCA toleration poor and its can cause property inflammation out of control.Found FCA before more than 80 years since, people reduce the adverse side effect of adjuvant hardy.

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 the N-glycosyl amide, N-glycosyl urea, and N-glycosyl carbamate and specific compound: N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide acetate (are called Bay R1005; O Lockhoff, Angew.Chem.Int.Ed.Engl. (1991) 30:1611-1620).Compound described in ' 283 patents is particularly suited for as adjuvant.

Glycolipid adjuvant preparation (formulation) need be easy to make and stablize when long term storage and flocculate without lipid composition.The non-acetate form of glycolipid amide or glycosyl amide is for highly insoluble, and flocculates from solution when usually storing under room temperature or lower temperature.

The few flocculation of the solution that comprises glycosyl amide that this paper provides 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 the stability of these preparations by the accelerated stability method of testing.

Summary of the invention

The present invention comprises compositions and manufacturing or the production method of glycosyl amide stock solution and glycolipid adjuvant solution.The glycosyl amide stock solution is to be dissolved in alcohol and in conjunction with appropriate 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 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 sorbitan (Span

) or polyoxyethylene sorbitan (Tween

), be especially that (Span 20 for sorbitan monolaurate

) and Tween 20 (Tween 20

).Glycolipid adjuvant solution is by introducing appropriate glycosyl amide stock solution preparation in " suitable buffer ".The final pH value of stable glycolipid adjuvant solution as herein described should be between approximately 6 and approximately between 8.Better final pH value is between approximately 6 and approximately between 7.Described between approximately 6.3 and the about final pH value between 6.4.Should avoid the glycolipid adjuvant over the NaCl high salt concentration of 30mM.

Below illustrate in more detail this two kinds of solution:

The 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, 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 wherein HO-C _1-3Alkyl;

C) weak acid, wherein 1) this weak acid is molar excess with respect to glycolipid content; And be 2) Application standard table or standard value pKa value between approximately 1.0 and the approximately any acid between 9.5; With

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

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

A) glycosyl amide stock solution; With

B) suitable buffer, wherein this buffer be suitable for veterinary or medical application and can keep in aqueous solution approximately 6.0 with the about relative constant pH value between 8.0.

Detailed Description Of The Invention

Except illustrating in addition, the following term of using in this description and claim has following implication:

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 compound of following formula I.These compounds are described in United States Patent (USP) 6,290, in the United States Patent (USP) 4,855,283 that on August 8th, 971 and 1989 promulgated.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 by name

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 compound has Bay by name and chemistry " N-(2-deoxidation-2-L-leucyl-amino-beta--D-Glucopyranose. by name

Base)-N-octadecyl lauramide ".

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 compound can amide form or the existence of acetate form.The amide form of this compound has trade (brand) name Bay 15-1583

The acetate form has trade (brand) name Bay R1005

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, compound of the present invention is based on the 2-amino that is substituted-2-deoxyhexamethylose.These sugar always are combined 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 compound of the present invention, the 2-amino of amino sugar is combined 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, ASPARTIC 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 to be from the shielded 2-amino of amino-2-deoxidation glucopyranose derivatives (formula IV),

R wherein ¹⁰Expression is for the protection of the protecting group of amino, 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, 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; 2,2-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, (1981) Academic Press; And the document of wherein quoting.Preferred amino protecting group for the preparation of formula I compound is BOC base (tertbutyloxycarbonyl) or Z base (benzyloxycarbonyl group).

In the 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 in No. the 3rd, 213,650, DE-OS (German Prospectus).

In the 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 in this way glycosyl amide (formula VIII)

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, under the existence of organic additive alkali, by the known method of document, make formula VI glycosyl amine and 1 to 2 equivalent phosgene (formula VII) reaction or with the relevant carboxylic acid R that passes through of 1 to 2 equivalent 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 2-propanol of alcohol, or ether for example ether, oxolane or Isosorbide-5-Nitrae-dioxane, or halogenated hydrocarbon for example dichloromethane, chloroform or 1,2-dichloroethanes or DMF.

When the glycosyl amine (VI) that obtains in 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, then obtain glycosyl carbamate (VIII), the X in 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 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 ℃.Suitable solvent is above-mentioned alcohol, ether, halogenated hydrocarbons or dimethyl formamide.

The glycosyl amide (formula VIII, the X=-CH that obtain by the method ₂-), glycosyl carbamate (formula VIII, X=-O-) or glycosyl urea (formula VIII, X=-NH-) be separated into crystalline or unbodied form by known separately process, can carry out purification by standardization program such as recrystallization, chromatograph, extraction etc. if necessary.

In many examples, it is also 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 of its production VIII, glycosyl carbamate and glycosyl urea derivative.In the example of glycosyl amide of the present invention, glycosyl carbamate and glycosyl urea, this type chemically derived turns to the esterification on 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) under inorganic or organic additive alkali exist 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 Isosorbide-5-Nitrae-dioxane), ester (as ethyl acetate) and amide (as dimethyl formamide).

Also may be for independent organic base (such as triethylamine or pyridine) as suitable solvent.Spendable alkali is for being used for all alkali of 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, namely methanol, ethanol, normal propyl alcohol or isopropyl alcohol.Other solvent that is applicable to three esters (formula XI) crystallization is the mixture of organic solvent and polar inorganic or organic solvent, for example oxolane-methanol, oxolane-water, alcohol-water and isopropanol-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 known organic chemistry.Relevantly can by ZEMPLEN hydrolysis known organic chemistry, under existing, the Feldalat NM of methanol and catalytic amount carry out transesterification from three esters (formula XI) preparations triols (formula VIII).

The 3rd reactions steps of relevant formula I compound preparation of the present invention comprises the protecting group of the upper 2-amino of sugar in selective splitting formula VIII compound.In this reaction, need pay special attention to the 1-acylamino-on sugar or 1-amine formamido group or elimination simultaneously of 1-(alkoxy carbonyl group acylamino-) in formula VIII 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 the catalyst that hydrogenolysis is suitable is the noble metal that is adsorbed on active carbon, such as platinum or palladium.Preferred palladium/the charcoal (5% or 10%) that uses.Hydrogenolysis can under atmospheric pressure or high pressure, be carried out in suitable pressure vessel.Be suitable for the atent solvent that is of hydrogenation, such as alcohol (methanol, ethanol or propanol), ether (such as oxolane or Isosorbide-5-Nitrae-dioxane) or carboxylic acid (such as acetic acid) or its mixture.In the time of suitably, solvent mixes with water or diluted acid (such as hydrochloric acid or sulphuric acid).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 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 optionally obtained as the ammonium salt of the acid that is used for cracking.

The 4th reactions steps of relevant formula I 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 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 again of peptide.

In the preferred formula XIII that uses, amino protecting group is above-mentioned protecting group, 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 by the conventional method that peptide synthesizes that (E.Wunsch etc.: Synthese von Peptiden (Synthesis ofpeptides): Methoden der Org.Chemie (Methods of org.chemistry) is (E.Muller (Houben-Weyl), Editor), XV/I volume and XV/2 volume, the 4th edition, by Thieme, Stuttgart publishes (1974)).

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

The condensation of formula XII compound and formula XIII 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 HP ester.Acibenzolar also can be from acid (formula XIII) and N-maloyl imines or the acquisition of 1-hydroxybenzothiazole under the existence of dehydrant such as carbodiimide.Amino acid derivatives is known and can prepares in a known way.The peptidoglycolipid of the formula XIII carboxyl compound condensation preparation 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 compound, the protecting group R in formula XIV compound ¹⁴Be eliminated.Should be noted in this step, other acylamino-, carbamate groups or the urea groups that are present in formula XIV compound are not cleaved.Be preferred for the protecting group R in the XIV compound ¹⁴, N-benzyloxycarbonyl group and N-tertbutyloxycarbonyl can be eliminated, and keeps acylamino-, carbamate groups or urea groups.Benzyloxycarbonyl group is under the existence 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 mutually mixed or mix with water use.This reaction can be carried out under atmospheric pressure or high pressure.Tertbutyloxycarbonyl R in formula XIV 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.Known on the methodological principle of this type of cracking t-butyl carbamate.The peptide glycosyl amide of the formula I that obtains in this way ,-carbamate and-urea is separated into the form of crystallization or amorphous solid, and if necessary, carries out purification by standard method (as recrystallization, chromatograph, extraction etc.) with original known method.

The compound of formula I of the present invention can also be by obtaining the second route of synthesis preparation of same good result.This second route of synthesis is different from above-mentioned the 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) the bonding order different.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, the formula XV compound that so obtains and the glycosyl amine of the amino-compound condensation of formula III with generation general formula X VI,

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

Above-mentioned all methods for the preparation of general formula VI compound all can be used for preparing the compound of general formula X VI.Then, the reaction of formula XVI compound and above-mentioned carboxylic acid derivates (formula VII) or haloformate (formula IX) or organic isocyanate (formula X) is with the 2-(aminoacyl) of production XIV-amino glycosyl amide (X=-CH 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 that obtains by this method can come purification by above-mentioned physical purification method.Yet, preferably by by above-mentioned O-process for acylating, formula XIV compound being changed into three-O-acetas or the three-O-benzoate of general formula X VII,

Wherein the implication of variable is consistent with formula 1.

Therefore the easy crystallization of these compounds especially from organic solvent for example methanol or ethanol, and is able to purification.Then 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.In formula XIV compound, the final removal of amino acid whose protecting group is addressed hereinbefore with preparation I compound.The invention still further relates to the salt of formula I compound.These salt are mainly the nontoxic salts that usually can be used for pharmaceutics, for example the chloride of formula I compound, acetate and lactate or indifferent salt.

Term " weak acid " refer to Application standard table or standard value the pKa value (Ka-log) between approximately 1.0 and the approximately any acid between 9.5.Describe following weak acid example with title, chemical formula and approximate pKa value, and be not wish 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, be combined as a set and include.

Term " non-ionic surface active agent " refers to a kind of surfactant, and this material reduces the surface tension of the material that makes its dissolving, and nonionic refers to have uncharged polar group.The term amphiphilic surfactant refer to 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 Multi-instance.

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

Trade mark is sold) and polyoxyethylene sorbitan (usually with Tween

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

(Span 20 for sorbitan monolaurate

), (Span 40 for span 40

), (Span 60 for sorbitan monostearate ), (Span 65 for the sorbitan tristearate

), (Span 80 for dehydrated sorbitol mono-fatty acid ester ), (Span 85 for sorbitan trioleate

), Tween 20 (Tween20

), (Tween 40 for the polyoxyethylene sorbitan monopalmitate

), (Tween 60 for the polyoxyethylene sorbitan monostearate

), Polysorbate 80 (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.

(Span 20 to describe especially sorbitan monolaurate

), (Tween 20 for Tween 20

), dehydrated sorbitol mono-fatty acid ester (Span80 ), (Span 85 for sorbitan trioleate

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

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

Other example of buffer is as follows:

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

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

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

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

[three (methylol) methyl] glycine (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) the glycosyl amide stock solution is by being dissolved in glycolipid in alcohol and mixing appropriate 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 glycolipid alcohol acid blend makes the 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 as above.

The preparation of glycosyl amide stock solution.Weak acid is added in the alcoholic solution that contains 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, the following relative quantity of recommending 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 above-mentioned pure glycolipid mixture to obtain final glycosyl amide stock solution.

Under weak acid exists, glycosyl amide is changed into the acetate form of glycolipid.When only directly introducing in 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 compounds are suspended can make aqueous buffered glycolipid solution dissolve fully in proper pH value, near optically clear.During with respect to the glycolipid molar excess, guarantee that all glycolipids all change into soluble form when the weak acid that adds, and prevent that it from replying and be soluble form.

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 compound that obtains of described herein and weak acid.

The alcohol that is used for the dissolving glycolipid can be propanol or its any combination of methanol, ethanol, any isomeric forms.Gained glycolipid alcoholic solution will be for clarifying on 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.In the glycosyl amide stock solution of glycolipid and alcohol, the initial concentration of weak acid has determined whether will exist the flocculation of any glycolipid.Weak acid answers molar excess to avoid flocculation with respect to glycolipid.

2) glycolipid adjuvant solution is to prepare by appropriate glycosyl amide stock solution is introduced in " suitable buffer ".The pH value of final stable glycolipid adjuvant solution as herein described should be between approximately 6 and approximately between 8.Preferred final pH value is between approximately 6 and approximately between 7.Described between approximately 6.3 and the about final pH value between 6.4.

Because the 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 the specific pH value with wide region by dihydric salt and the monohydric salt with different proportion sodium phosphate or potassium phosphate.If the use phosphate buffer can it be made with about 20mM, and its pH value is about 7.8.When being added into the glycosyl amide stock solution in buffer, the pH value of buffer reduces.The phosphate buffered solution of pH7.8 produces pH value and is about 6.4 final glycolipid adjuvant solution.Final pH value can be regulated, but it is usually also unnecessary.

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, because adding of highly basic can transform back salt-independent shape with the salt form of glycolipid, cause that salt-independent shape precipitates (flocculation) in aqueous environments.Yet, if need highly basic, should only use a small amount of.For example, the NaOH of the maximum 100mM of recommendation, and the best is 4.0mM or less.

Buffer solution optionally comprises some NaCl, but unessential.NaCl concentration can be between approximately 1 and approximately between 50mM.The NaCl of small amount is better than more amount.The example of this paper does not have NaCl or 15mM NaCl.Because flocculation appears in meeting, 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

Between the storage life, the stability of glycolipid adjuvant solution can be by simple range estimation or by using suitable analytical tool to monitor.When in aqueous solution, the glycolipid molecule forms micelle and can use laser-diffractometer accurately to measure the size of micelle.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.The temperature that accelerated stability test makes assist agent solution stand approximately 37 ℃ continues approximately 7 days, then approximately cultivating approximately 2 days under 4 ℃, and constantly jolting.Store the approximately period of 1 year under approximately being illustrated in approximately 4 ℃ in 7 days approximately cultivating under 37 ℃.Approximately 4 ℃ cultivate under continuous jolting and approximately be illustrated in the stress condition that In transit glycolipid adjuvant solution can be faced in 2 days.

For determine glycolipid adjuvant solution whether with Cytoplasm etc., can measure osmotic pressure.Can add the sodium chloride of variable concentrations and use permeability manometer to measure the osmotic pressure of gained solution.Except increasing osmotic pressure, the concentration that increases sodium chloride is also tended to make solution more muddy.Turbidity is considered to be gathered into than macroparticle by micelle and causes.The solution that uses 0.2 μ m filter to be difficult to maybe can't filter is unsuitable for commercialization usually, is 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 because 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 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 (Quillaj a saponaria) extracts purification.Quil A inductor fluidity is reacted and cell-mediated reaction.Quil A usually and cholesterol jointly use because cholesterol can be eliminated comparatively bad side effect when adding proper proportion.Cholesterol and Quil A form insoluble complex, and when Quil A was combined, these complex formed similar helicoidal structure when cholesterol, 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 the quaternary amine of amphiphilic.Due to DDA on oil/water termination by with antigen directly in conjunction with and as the carrier of antigen, therefore need to make DDA directly with AI 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 the acrylate homopolymer crosslinked with 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 derive from multiple source.Antigen from microbial pathogens comprises malignant bacteria, virus and parasite body.Can use the mixture of two or more antigens.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, restructuring 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 through killing via chemical treatment in tissue culture.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 diluent of the live virus antigen of modification.Can by make viral pathogen several from generation to generation through tissue culture or make the viral pathogen perform toxic attenuation by virus genomic special handling.These attenuated viral strains can become very high tiring and can be used as vaccine antigen in tissue culture.Attenuated viral strains is called as the live virus antigen of modification.Although these Strain toxicity are less, when as the antigen in 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 diluent of the live virus antigen of modifying, 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 measure glycolipid adjuvant solution to the character of killing the virus of the live virus antigen of modification in experiment in vitro.Make through cryodesiccated virus antigen rehydrated with glycolipid adjuvant solution or water.Gained virus solution is applied on the monolayer that allows cell.Tiring by the bacterial plaque counting that forms on monolayer is determined of virus antigen.Among the rehydrated sample of the water sample rehydrated with using glycolipid adjuvant solution, the difference of the virus titer that obtains can be used for determining whether to exist any glycolipid adjuvant solution to have to any live virus 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 antigen of these modifications rehydrated through cryodesiccated cake and diluent solution, and be 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 with the vaccine of the live virus antigen of modifying.In one embodiment of the invention, glycolipid adjuvant solution is used as diluent solution.

Embodiment

Embodiment 1. is with isocyatic Bay 15-5831

Prepare insoluble glycosyl amide compositions with acetic acid.

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

Bay 15-5831

By Bayer register of company, its commodity are called N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl)-N-octadecyl lauramide.During the assist agent solution that uses for the manufacture of the described compound of table 1 when this compound, wherein acetic acid to be using with the 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 with embodiment 1 but acetic acid concentration increases to some extent with respect to glycolipid concentration, generate solubility glycosyl amide stock solution.

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 be clarify on optics and at container bottom without sedimentation.This glycosyl amide stock solution is added in 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 with 138 gram NaH ₂PO ₄H ₂O salt is dissolved in 250 mL deionized waters in beaker and makes final volume is 500 mL, makes 2 M sodium dihydrogen phosphate stock solutions.Similarly, by with 142 gram NaH ₂PO ₄Being dissolved in 300 mL deionized waters in beaker and making final volume is 500 mL, makes 2 M sodium hydrogen phosphate stock solutions.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 adds (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

Then the 2M sodium dihydrogen phosphate stock solution of the different volumes shown in preparation table 3 and sodium hydrogen phosphate stock solution obtain the 1M stock solution of the buffer solution of sodium phosphate of different pH value.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 glycosyl amide stock solution preparation of embodiment 2.

Add as 5mL glycosyl amide stock solution prepared in embodiment 2 in every 96mL of these 20mM phosphate solutions.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 the final pH value of the various solution of test affects flocculation to estimate pH value.Preparation 20mM phosphate buffer, original ph is 7.8.Glycolipid adjuvant shown in table 4 is to use as glycosyl amide preparation prepared in embodiment 1, wherein with etc. molar concentration use glycolipid and acetic acid.Note, final pH value is 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 table 4 is compared with the similar reading in table 5, the glycolipid adjuvant solution of table 5 be with as the glycosyl amide stock solution of the acetic acid that contains the glycolipid twice as high molar ratio prepared in embodiment 2 prepare.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 NaCl concentration increases, flocculation increases even appearance precipitation.Take original ph as the glycolipid adjuvant solution described in 8.0 phosphate buffer preparation table 5, the final pH value of glycolipid adjuvant solution is between 6.8 and 7.0.The further reduction of the final pH value of glycolipid adjuvant solution can cause having low turbidity and without the glycolipid adjuvant solution of flocculation.

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

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 of glycosyl amide stock solution (ml)	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) represents 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 it is muddy that optical density (OD) 1.0 to 1.5 is identified.Optical density (OD) is higher than 1.5 be muddy and can not use 0.2 micron filter to filter.It is generally acknowledged that the latter is not suitable for business.

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

For determining to add the acetic acid of recruitment whether will reverse flocculation in the glycolipid adjuvant that shows flocculation, preparation glycolipid adjuvant as described in example 1 above.Even under existing without any NaCl, flocculation also appears in this glycolipid adjuvant.Add the acetic acid that increases concentration in this flocculation glycolipid adjuvant mixture.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 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 adds	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

Establish the acetic acid amount be increased in importance in keeping the glycolipid stability of solution after, determine that at first the compositions shown in use table 8 prepares the glycosyl amide stock solution, and then use the another kind of glycolipid adjuvant solution that contains NaCl and do not contain NaCl of this solution preparation.Solution phase in this glycosyl amide stock solution and embodiment 2 seemingly, cumulative volume is its 4 times and have relatively a large amount of acetic acid and polysorbas20.

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

Use the phosphate buffer of embodiment 3 and prepare 3 kinds of different glycolipid assist agent solutions with different N aCl concentration as the glycosyl amide stock solution for preparing in table 8.

As the preparation in embodiment 4, table 5, make the glycolipid adjuvant solution of the NaCl that contains 0mM, 15mM and 100mM.Can filter 0mM and 15mM NaCl 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 of glycosyl amide stock solution (ml)	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 in the 30ml vial and cultivates under room temperature and 4 ℃.Estimate at predetermined distance.At first, the glycolipid adjuvant solution that has the NaCl of 0mM is to clarify on optics.Contain the glycolipid adjuvant solution of NaCl of 15mM slightly muddy and be 0.073 at the O.D. of 600nm.Contain the glycolipid adjuvant solution of NaCl of 100mM muddy and be 0.439 (table 9) at the O.D. of 600nm.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, and its outward appearance is unchanged.

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 from flocculating, and is necessary to show to add gradually NaOH meeting induction of 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 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 described in embodiment 6 for the preparation of the glycolipid adjuvant solution in this experiment.Along with the NaOH concentration in glycolipid adjuvant solution increases gradually, the pH of preparation increases gradually, flocculation occurs 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 adds	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 15-5831

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

Table 11. is used for the summary of the HPLC method parameter used of quantification 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	Approximately 25 minutes

[0204]Table 12.Bay 15-5831

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 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 6ml methanol.Then the sample ultrasonic ripple was processed 10 minutes, it is diluted to scale and mixes.With peak area, concentration is mapped, reference substance is carried out linear regression analysis.According to standard curve calculation sample amount.

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

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

Use this 30L preparation, prepare 5 kinds of different sub-solution with the NaOH that increases 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 ， Ban Sui flocculation and increases.At room temperature flocculating begins to occur in the NaOH of 2mM concentration, and begins to occur in the NaOH of flocculation at 4mM under 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

Come the amount of Bay 15-5381 in 6 samples of all shown in quantization table 13 with the HPLC method described in embodiment 8.Sample with different pH value shows the Bay 15-5381 of same concentrations, and this explanation adjuvant component adds pH value to increase along with NaOH and there is no degraded with during flocculating.

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

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

For accelerating stress test, this glycolipid adjuvant solution is 37 ℃ of lower joltings 7 days, then 4 ℃ of lower joltings 2 days.Be illustrated in 7 days under 4 ℃ 37 ℃ of lower joltings and placed 1 year.Stress at 2 days expression In transits of 4 ℃ of lower joltings.

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

Not because stress test causes change of granularity.Observe immediately sample after sample preparation, all samples is all kept submicron particle size.In addition, remaining under 4 ℃ or Bay 15-5831 in 37 ℃ of samples that stand stress

The HPLC of component measures and does not show Bay15-5831

Any variation of amount.

Table 15. is at the rear Bay 15-5831 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 under 4 ℃, and with specimen 37 ℃ of lower joltings 7 days.Has similar concentration at 37 ℃ of lower joltings sample of 7 days to the sample that stores under 4 ℃.

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

To the test of killing the virus of the described glycolipid adjuvant solution with 30L scale preparation of above embodiment 9.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.The live virus antigen of modifying is prepared as through cryodesiccated material.When making these materials rehydrated with suitable glycolipid adjuvant solution, confirm that glycolipid adjuvant solution used does not kill the live virus of modification.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 the lower cultivation of room temperature (RT) 1 hour, sample is applied in the monolayer permissive cell strain of serial dilution.By obtaining every milliliter of 50% TCID (TCID50/ml) value with sterilized water or rehydrated each virus antigen of glycolipid adjuvant solution to appearing at viral filler counting on 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.Rispoval

Three kinds of different cattle disease viral diseases that contain the live virus antigen 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 the diluent solution of these antigens.Glycolipid used 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 apparent.

Claims

1. compositions, it comprises:

A) glycolipid of formula IIa, its Chinese style IIa is

Formula II (a)

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

C) weak acid, wherein this weak acid is 1) for the molar equivalent of glycolipid, be the 1.25-5 amount doubly of glycolipid amount, and 2) Application standard table or any acid of standard value pKa value between 1.0 and 9.5;

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

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

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

Formula II (b)

And this weak acid is selected from a kind of or combination in any of following weak acid: 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 or succinic acid.

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

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 forms: 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 and succinic acid.

6. the compositions of any one in claim 1-5, wherein the amount of this weak acid is the following multiple of the molar equivalent of this glycolipid:

A) 1.25 times,

B) 2.0 times,

C) 2.5 times,

D) 2.7 times,

E) 3.0 times, or

F) 5.0 times.

7. the compositions of any one in claim 1-5, should alcohol be wherein ethanol.

8. the compositions of any one in claim 1-5, wherein this non-ionic surface active agent is selected from any or combination in the group that following each material forms: sorbitan monolaurate, span 40, sorbitan monostearate, the sorbitan tristearate, dehydrated sorbitol mono-fatty acid ester, sorbitan trioleate, Tween 20, the polyoxyethylene sorbitan monopalmitate, the polyoxyethylene sorbitan monostearate, Polysorbate 80, polyoxyethylene sorbitan trioleate and other are generally used for sorbitan and the polyoxyethylene sorbitan in vaccine.

9. compositions, it comprises:

A) glycolipid of formula IIa;

Its Chinese style IIa is

Formula II (a)

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

D) non-ionic surface active agent, wherein this non-ionic surface active agent is the capillary reagent of a kind of reduction material of making its dissolving 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 6 to 8,

Condition is that the NaCl that uses is no more than at most 50mM.

10. the compositions of claim 9, wherein the pH of this solution is adjusted to the relative constant pH between 6 and 7 in aqueous solution, and this buffer is phosphate buffer, it has one of the dihydric salt of sodium phosphate of identical or different ratio and monohydric salt or dihydric salt and monohydric salt both, perhaps have one of the dihydric salt of potassium phosphate of identical or different ratio and monohydric salt or dihydric salt and monohydric salt both, perhaps its combination.

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

A) 2-(N-morpholino) ethane sulfonic acid;

B) 3-(N-morpholino) propane sulfonic acid;

C) n-[three (methylol)]-the 2-aminoethane sulphonic acid;

D) 4-(2-ethoxy) piperazine-1-ethane sulfonic acid; With

E) [three (methylol) methyl] glycine;

Or its any combination.

12. the compositions of any one in claim 1-5 and 9-11, 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 the N-(2-deoxidation-2-L-leucyl-amino-beta--D-glucopyranosyl) of formula III structure-N-octadecyl lauramide acetate:

Formula III

B) ethanol;

C) acetic acid, its amount for the molar equivalent of glycolipid for the 1.25-5 of glycolipid amount doubly;

D) non-ionic surface active agent, this non-ionic surface active agent is selected from: sorbitan monolaurate, span 40, sorbitan monostearate, sorbitan tristearate, dehydrated sorbitol mono-fatty acid ester, sorbitan trioleate, Tween 20, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, Polysorbate 80 and polyoxyethylene sorbitan trioleate;

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

(a) 2-(N-morpholino) ethane sulfonic acid;

(b) 3-(N-morpholino) propane sulfonic acid;

(c) n-[three (methylol)]-the 2-aminoethane sulphonic acid;

(d) 4-(2-ethoxy) piperazine-1-ethane sulfonic acid; With

(e) [three (methylol) methyl] glycine;

Or its any combination,

Condition is that the NaCl that uses is no more than at most 15mM; With

F) antigen that is formed 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.

14. the compositions of any one in claim 1-5,9-11 and 13, the amount of wherein said weak acid be described glycolipid molar equivalent 2.0-5.0 doubly.

15. a method for preparing compositions, it comprises following each material is mixed:

A) glycolipid of formula IIa, IIb or III;

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

C) weak acid, wherein the amount of this weak acid for the molar equivalent of glycolipid for the 1.25-5 of glycolipid amount doubly; With

D) non-ionic surface active agent.

16. a method for preparing compositions, it comprises following each material is mixed:

A) glycolipid of formula IIa, IIb or III;

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

C) weak acid, wherein the amount of this weak acid for the molar equivalent of glycolipid for the 1.25-5 of glycolipid amount doubly;

D) non-ionic surface active agent; And add

E) suitable buffer, wherein said suitable buffer are suitable for vaccine use and can keep in the pH scope of pH value between 6.0 and 8.0 of other composition, and its condition is that the NaCl that adds in said composition is no more than 50mM.

17. the method for claim 15 or 16, the amount of wherein said weak acid be described glycolipid molar equivalent 2.0-5.0 doubly.