CN1353615A - Compositions of A-beta peptide and processes for producing same - Google Patents

Abstract

The invention is directed to compositions comprising solubilized A beta peptide or suspension of A beta peptide and to processes for producing the same by adjusting the pH sufficient to effect the solubilization, and sterile filtration thereof, to producing method of the compositions and methods of treating and preventing Alzheimer's disease with the obtained compositions.

Description

The composition and method of making the same of A β peptide

The technical field of the invention

The present invention relates generally to and comprises the proteinic pharmaceutical composition that causes that mammal antibody response aspect is very useful.More particularly, the present invention relates to comprise the amyloid beta that can cause the mammalian immune reaction of effective dose and the Pharmaceutical composition that medicinal diluent is formed.This diluent is preferably aseptic injectable water.

Background technology related to the present invention

Amyloid beta is also referred to as A-β or A β peptide, is the shearing product of amyloid precursor protein (APP).It is the main constituent of amyloid plaque in the mammal brain, and amyloid plaque is common in Alzheimer disease, and is big characteristics of this disease.Because protease is to the multiformity of APP effect, A β peptide be one adjustable length, contain the amino acid whose peptide chain of 39-43.

The more proteic sudden changes of APP are relevant with the appearance of Alzheimer.For example, (Nature) 349,704 (1991) (valines of Goate etc. " nature " ⁷¹⁷Be mutated into isoleucine (valine ⁷¹⁷To isoleucine)); " natures " such as Chartier Harlan be 353,844 (1991) (valines (Nature) ⁷¹⁷Be mutated into glycine (valine ⁷¹⁷To glycine)); Murrell etc. " science " are 254,97 (1991) (valines (Science) ⁷¹⁷Be mutated into phenylalanine (valine ⁷¹⁷To phenylalanine)); Mullan etc., (two sudden changes make lysine to " natural genetics " (Nature Genet.) 1,345 (1992) ⁵⁹⁵, aspartic acid ⁵⁹⁶Be mutated into methionine ⁵⁹⁵, leucine ⁵⁹⁶(a double mutation changinglyisne ⁵⁹⁵-methionine ⁵⁹⁶To asparagines ⁵⁹⁵-leucine ⁵⁹⁶)).It has been generally acknowledged that these sudden changes cause Alzheimer by the course of processing that strengthens or change from APP to A β peptide, particularly the processing of APP causes A β 42 and A β 43 quantitative increases.The sudden change of other genes as presenilin genes PS1 and PS2, is considered to the course of processing of remote-effects APP and increases amount (Hardy, TINS20,154 (1997) of A β 42 and A β 43.Observed result shows that A β peptide, particularly A β 42 are paathogenic factors of Alzheimer.In brain, A β peptide condenses and forms amyloid deposition, comprises that polypeptide passes through the βZhe Die structure and forms fibril.

About treating or prevent the motive force of the therapeutic studies of Alzheimer, concentrate on the generation that stops or slow down the interior A β peptide of brain recently, perhaps stop its back dispose procedure, perhaps stop the amyloidogenic speckle of precipitation form.A treatment approach of the particular importance that the present invention uses is to use A β peptide to cause that the immunoreation of body offsets its effect.For example, the open text No.WO99/27944 of PCT is incorporated herein by reference.

The present invention relates to a kind of novelty and unexpected method, be used for realizing the open disclosed invention of text No.WO99/27944 of PCT.Especially, the present invention includes and give patient's medication, to cause immunoreation the particular formulations of the longer chain forms of A β peptide.Yet in the prior art, the longer chain forms of A β peptide is difficult to be dissolved in conventional formulation system.

Especially, Hilbich etc. " molecular biology magazine " (J. Mol.Biol.), 218 (1), pP.149-64, report: although A β 1-43 peptide is soluble to a certain extent in pure water, but the adding ion component as buffer or salt or organic solvent, can cause that peptide is precipitated out from solution with the form of noncrystal aggregation.For example, Hilbich finds that (" PBS " is KCl, the Na of 8mM that contains NaCl, the 3mM of 135mM to phosphate buffer in the present example ₂HPO ₄H ₂The KH of O and 2mM ₂PO ₄, pH is 7.5), it makes in the said composition peptide of 90-94% soluble.PBS is the carrier of the composition for injection used always, and it is similar with the pH level with the ion concentration in the biosystem.5mM NaCl can cause the precipitation of 42-50% peptide class.(the citation document is the same, p.153, and Table2).Peptide solution in the pure water is hypotonic, and the pH value of these solution is defined as 5.5 by Hilbich (same author).Usually, the pH of human blood is about 7.4.Report A β peptides such as Dyrks are insoluble under physiological condition.Dyrks, T., Weidemann, A., Multhaup, G etc., " European molecular biology magazine " (EMBOJ) 7, p.949-57 (1988).

The conformation of A β peptide in solution can be measured with circular dichroism method (C.D.).Hilbich etc. (the same author) have reported with the research of circular dichroism method to A β peptide and segmental conformation.In addition, the treatise of M.Manning " measuring proteic structure and solubility by the circular dichroism method " (Protein Structure and Stability Assessment by Circular DichrosimSpectroscopy) has been told about the stability and the The specificity of enzyme.Himmel, M.E. and Georgiou, G., ACS Symposium Series 516 (1993) is P.36.Here quoted from, and the document that Manning quoted from is classified list of references as at this.

Kline etc., U.S. Patent No. 5,851,996 (' 996 patent) and No.5,753,624 (' 624 patents) have described very a small amount of (10 ^-2Mg or still less) A β peptide or segmental medication are with liquid or solid carrier sublingual administration, as making carrier with the phenyl saline solution.' 996 patents point out that there are various versions (col.2, line 13) in A β peptide, can be used for treating Alzheimer.This patent is not specifically defined the implication of various versions, the used 28 seed amino acid fragments, does not also specify its characteristics in embodiment.The scope of the A β peptide dosage that ' 996 patents are pointed out is that 10-10 is to 10-2mg (the 8th volume, 442-43 page or leaf).

From above-mentioned viewpoint, the difficulty aspect dissolving and the dissolving of maintenance A β peptide that prior art is verified.And the low-solubility of the A β peptide of longer chain forms causes and is difficult to realize it is carried out the standardization sterilization.Most standard sterilizing methods is incompatible with peptide class medicament, and as radiation, autoclaving and chemical sterilization techniques (as using oxirane and glutaraldehyde), said method can cause the degraded of peptide.Therefore, can select the filtration of peptide to carry out the sterilization of A β peptide.But the insoluble meeting of A β peptide causes that filter membrane stops up, and stops the recovery and the commodity production of capacity A β peptide.

The object of the invention

The present invention relates to surprising and a discovery beyond expectation, promptly can reach the pH value scope that is suitable for dissolving A β peptide, with the A β peptide aqueous solution of preparation high concentration by regulating aqueous solution acid/basicity.The pH value scope is about 8.5-12 preferably, is preferably about 9-10.

The present invention finds that further the solubility solution of A β peptide can reclaim 50% A β peptide at least by a suitable micropore filter paper sterilization behind filtration sterilization.Under the good conditions, the A β peptide of at least 70% filtration sterilization obtains reclaiming, and is preferably 90%.This sterile solution can be made the Pharmaceutical composition that contains capacity A β peptide, can cause immunoreation to the mammal administration.Preferably, it is non-through enteral administration to adopt suspension composition to carry out.

Therefore, aspect of said composition invention, behind sterilising filtration, the pH value of said composition is adjusted to the physiology suitable condition, to form a kind of suspension that contains 0.1mg/mlA β peptide at least.Said composition can be used to carry out non-through enteral administration.The pH of this suspension compositions is preferably 5.5-6.5 between 5-7.A kind of preferred compositions comprises that the QS-21 of capacity and A β peptide are common forms a kind of sterile suspension of looking limpid.

The present invention further finds, can handle solvable aseptic A β peptide solution with lyophilization, and the lyophilized formulations that comprises A β peptide is provided.Can rebuild these compositionss in the suitable time, so that the water preparation that comprises A β peptide to be provided.

Said composition the present invention relates to contain the aqueous solution of 0.01mg/ml A β peptide at least on the other hand, and wherein this aqueous solution keeps the suitable dissolved pH value of this A β peptide.Preferably, after using the medicinal buffer of effective dose, this solution can keep the pH value that suits.

The one side again of said composition the present invention relates to comprise the aseptic aqueous solution of 0.01mg/ml A β peptide at least, and wherein this aqueous solution keeps the suitable dissolved pH value of A β peptide.Preferably, after using the medicinal buffer of effective dose, this solution can keep the pH value that suits.

On the other hand, the present invention relates to composition freeze-drying is handled, this freeze-dried composition comprises A β peptide, prepares with the following step:

A) it is freezing to comprise the aseptic aqueous solution of 0.01mg/ml A β peptide at least, and wherein this aqueous solution keeps the suitable dissolved pH value of A β peptide; And

B) the above-mentioned a) frozen composition of preparation being carried out lyophilizing handles.

Preferably, compositions of the present invention comprises the longer chain forms (as mentioned below) of A β peptide.More preferably, said composition comprises medicinal buffer, is selected from aminoacid, salt and derivant thereof, medicinal basifier, alkali metal hydroxide and aqua ammonia, organic and inorganic acids and salt; And the mixture of above-mentioned substance.

In said composition on the other hand, the present invention relates to comprise the compositions of the aqueous solution of 0.01mg/ml A β peptide at least, wherein this aqueous solution keeps the suitable dissolved pH value of A β peptide, and A β peptide wherein is the conformation of random coil basically.

Compositions of the present invention can be made medicinal forms, is fit to give the mammal medication of suffering from Alzheimer, or to there being this sick risk person of suffering to carry out the administration prevention.This inventive compositions aspect relates to Pharmaceutical composition, and A β peptide wherein is soluble random coil, or the stable aqueous suspensions of 0.01mg/ml A β peptide at least, or freeze dried form, and above-mentioned form can be degerming, and can be used for non-through enteral administration.

Aspect of this technology, this invention relates to the technology of the aseptic composite for preparing a kind of longer chain forms A β peptide, comprising:

Regulate pH value of aqueous solution, make it to dissolve A β peptide wherein;

A certain amount of A β peptide is dissolved in solution, makes it to reach the concentration that can obtain mammalian immune;

By the resulting solution of membrane filtration in a unified aperture, antibacterial can be got rid of in this aperture, makes nearly all A β peptide pass through film; And

For the solution that contains 0.01mg/ml or more A β peptide, selectively the pH value with the solution of gained is adjusted to about 5-7, obtains the suspension of peptide.

In this technology on the other hand, the present invention relates to the method for a kind of prevention and treatment mammal Alzheimer, comprise the aseptic aqueous solution compositions of using capacity to mammal, said composition comprises the A β peptide of 0.05mg/ml at least, to cause mammiferous immunoreation.

More preferably, filtration treatment of the present invention is employed is the A β peptide of random-coil conformation form basically.

Brief Description Of Drawings

Fig. 1 is the function of the mean residue ellipticity measured value of circular dichroism spectral representation to two kinds of different A β 42 peptide solution wavelength.Absorption value when dotted line is represented pH6 is owing to the βZhe Die structure of molecule.Solid line is the absorption values of A β 42 peptide solutions at pH9, expression peptide random-coil conformation.

Fig. 2 be A β 42 peptides in solution corresponding to the dissolving peptide spirogram of calculated by peak area, determine by reversed phase high-performance liquid chromatography, prove the solubility property of A β peptide 42.

The present invention describes in detail

The present invention relates to use composition and the application process thereof of the aqueous solution of the A β peptide that contains effective treatment concentration. Before the present invention is discussed in detail, at first used term is carried out as giving a definition.

Definition:

The sequence of two peptides of term " essential homogeny " expression, when in ideal alignment, when in program GAP or BESTFIT, using default weight differential (gap weight), there is at least 65% sequence identical, the sequence that better is at least 80%-90% is identical, be preferably at least 95% or more sequence identical (as 99% or higher sequence identical). Preferably, the residue position is not both because the replacement of conserved amino acid forms.

For the comparison of sequence, usually adopt a sequence as the reference sequence, cycle tests is compared with it. Suitable reference sequences is people's A β peptide sequence, particularly hereinafter described 42 amino acid sequences. Other suitable types are forms of brachymemma, such as A β 39; The form of perhaps extending is such as A β 43 (at the last disconnected unnecessary threonine that has of C). When using sequence comparison algorithm, with cycle tests and reference sequences input computer, if necessary, specified order coordinate, and specified sequence algorithm routine parameter. According to the program parameter of appointment, utilize sequence comparison algorithm to calculate the same percentage of cycle tests and reference sequences.

Optimum comparative sequences is arranged, local homology algorithm such as " senior applied mathematics " (adv.Appl.Math.2:482 (1981)) of Smith﹠Waterman, Needleman and Wunsch " molecular biology magazine ", the homology permutation algorithm of (J.Mol.Biol.48:443 (1970)), Pearson and Lipman, the similitude algorithm research of " institute of NAS periodical " (Pro.Nat ' l. AcadSci.USA) 85:2444 (1988); But the appliance computer of these algorithms is processed (GAP, BESTFIT, FASTA and the TFASTA of state of Wisconsin heredity software kit (Gemics Software Package), science of heredity calculates unit (Genetics Computer Group), 575 Science Dr., Madison, WI), perhaps by visual check (with reference to generally Ausubel etc., supra). Algorithm examples that be fit to measure sequence homogeny and sequence similarity percentage is the BLAST algorithm, by Altschul etc., in " molecular biology magazine " (J.Mol.Biol.) elaboration among the 215:403-410 (1990). The BLAST analysis software can be obtained by NCBI (http: ∥ www.ncbi.nlm.nih.gov/). Although can use concrete parameter, generally also can adopt default program parameter to carry out sequence relatively. For amino acid sequence, BLASTP can be with default 3W (wordlength), 10E (expectation), and BLOSUM62 scoring matrix (referring to Henicoff and Henikoff " institute of NAS periodical " (Pro.Nat ' l.Acad Sci.USA) 89:10915 (1989)).

Conservative and nonconservative for amino acid whose replacement is divided into, amino acid is divided into following several groups: first group (hydrophobic side chain): nor-leucine, methionine, alanine, valine, leucine, isoleucine; Second group (neutral hydrophilic side chain): cysteine, serine, threonine; The 3rd group (acid side-chain): aspartic acid, glutamic acid; The 4th group (basic side chain): asparagine, glutamine, histidine, lysine, arginine; The 5th group (affecting the residue of side chain direction): glycine, proline; With the 6th group (aromatic side chain): tryptophan, tyrosine and phenylalanine. Conservative replacement occurs between the phase amino acid on the same group. Nonconservative replacement occurs between a group membership and another group membership.

APP ⁶⁹⁵、APP ⁷⁵¹、APP ⁷⁷⁰Refer to respectively the polypeptide chain of 695,751,770 amino acid residues of people's app gene coding. Referring to Kang etc., " nature " (Nature) 325,773 (1987); Ponte etc. " nature " (Nature) 331,525 (1988), and Kitaguchi etc. " nature " (Nature) 331,530 (1988). The amino acid of people's amyloid precusor protein (APP) is by the sequence numbering of APP770 isomers.

In the present invention and document, the A β peptide of the about 70%-80% of the weight of A β peptide representative and salt and the moisture of about 15%-30%. This is drawn by amino acid analysis and/or nitrogen elementary analysis. For example, A β 42 peptides of 0.1mg are calculated wherein peptide components, represent A β 42 peptides that it contains 0.075mg and moisture and the salt of 0.025mg; 0.6mg A β 40 expression contain A β 40 peptides of 0.45mg and moisture and the salt of 0.15mg; 2.0mg A β 42 expression contain A β 42 peptides of 1.5mg and moisture and the salt of 0.5mg.

A β peptide involved in the present invention is that those can form the β-pleated sheet conformation, and can cause immunoreactive A β fragments of peptides when being combined the administration mammal separately or with assistant agent. Those skilled in the art know the method for measuring the β-pleated sheet conformation, for example ring two color methods mentioned in this article. Can measure immunogenicity with the method that following embodiment biologically-active moiety is described.

" the A β peptide of longer chain forms " comprises A β 38, A β 39, A β 40, A β 41, A β 42 and the A β 43 of any nature, and essentially identical peptide sequence, is preferably the form that exists in the human body. A β 39, A β 40, A β 41, A β 42 and A β 43 refer to that those comprise respectively the A β peptide of 1-39,1-40,1-41,1-42,1-43 amino acid residue, excise respectively the amino acid of the C end of peptide chain. Therefore, A β 41, A β 40, A β 39 are different from A β 42, are that its C end lacks respectively alanine, alanine-isoleucine and alanine-Ile-Val, and these can be found out from following A β peptide sequence. A β 43 is different from A β 42, is that there is threonine residues in its C end. The sequence of these peptides and with the relation of APP referring to the TINS such as Hardy 20,155 Fig. 1 (1997).

The sequence of A β 42: H₂N-Asp-Ala-glutamine-phenylalanine-arginine-histidine-aspartic acid-serine-Gly-Tyr-glutamic acid-valine-histidine-histidine-Gln-Lys-Leu-Val-Phe-Phe-alanine-glutamic acid-aspartic acid-valine-glycine-Ser-Asn-LYS-GLY-alanine-Ile-Ile-Gly-Leu-methionine-valine-Gly-Gly-valine-valine-isoleucine-alanine-OH.

" the A β peptide of longer chain forms " also comprises its analog. Analog comprises the gene of equipotential, species and induced mutation. Usually, analog is different in one or several site from the peptide that naturally exists, and often is because conservative replacement. Usually analog has 80%～90% sequence homogeny at least with the peptide that naturally exists. Some analogs also comprise non-natural amino acid or the modification of N end or C end. Non-naturally amino acid whose example has alpha amino acid, α-two substituted amino acids, N to hold alkylating amino acid, lactic acid, 4-hydroxy-proline and γ-carboxyl glutaric acid, ε-N, N, N-trimethyl lysine, ε-N-acetyllysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methyl-histidine, 5-oxylysine, ω-N-methylarginine.

A β peptide can be from about 0.05mg/ml to the about 2.0mg/ml of upper limit solubility (seeing Fig. 2) in composition and the application in the technique of this invention. The scope of suitable peptide is from about 0.1 to about 0.8mg/ml, and the scope of being preferably is to about 0.6mg/ml from about 0.3.

It is reported, find that in amyloid plaque the A β peptide of insoluble form is the conformation of β-pleated sheet. Soto, C. etc., " Neuscience communication " (Neuroscience Leters), 186 (2-3), pp. 115-118, (1995). Other has Simmons, " molecule pharmacy " (the Molec. Pharmacol.) such as L.K., 45 (3) pp.372-379 (1994) reported that the neurotoxicity of the conformation of β-pleated sheet and peptide is relevant, and the random coil form only have very low toxicity or do not have toxicity. From as can be known above, method and composition of the present invention can use the random coil form of A β peptide. The applicant has proved the random coil form can cause immune response in the experiment mammal. The random coil form is suitable for micro porous filtration most and processes.

" random coil " refers to the open chain conformation of A β peptide. Random coil is the secondary conformation of peptide backbone, is not the conformation that forms orderly rule, such as the conformation of alpha-helix or β-pleated sheet. Random coil is the unordered effect of the unordered folding and hydrogen bond of hydrophobic group, rather than the spread pattern of rule. Random coil still can form the folding of some peptide backbones or part in order, yet these features be at random and dynamic, be not typical in all random coil therefore. In the conformation of random coil, peptide presents good dissolubility and filterability. The conformation of alpha-helix or β-pleated sheet is the known peptide conformation of prior art. For example, in Lehninger " biochemistry " (Biochemistry) (2^ndEd., Worth Publishers, 1975)) alpha-helix and the β-pleated sheet described in the PP.128-9, at this as a reference.

Can use circular dichroism method (" CD ") to measure the random-coil conformation of A β peptide, easy and β-pleated sheet conformation difference. The circular dichroism method shows, random coil 190 and 200nm place an individual very strong negative band is arranged, almost can't see signal at wavelength greater than the place of 215nm. If at longer wavelength place very weak positive signal is arranged, its center is at the 220-225nm place. This can find out in Fig. 1. On the other hand, the β-pleated sheet conformation presents a point and strong just band in the place of 200nm. Illustrate the secondary structure of peptide if need to look back the CD spectroscopic methodology, please refer to the list of references of the Manning that quotes previously.

When having in the A β peptide when surpassing 50% random coil, its conformation just is in random-coil conformation. Preferably greater than 70% or 80% random coil, be random coil greater than 85% or 90% in the preferred A β peptide conformation.

" non-composition through enteral administration " refers to that those are sterile; and the composition of suitable direct administration human body; can adopt the form of injection or transfusion, that is to say by some can directly enter blood and not be subjected to barrier or the administering mode of immune system protection. Other modes are for entering human body through skin, mucous membrane or digestion, respiratory system. So, non-sterilization through the enteral administration composition is very important.

" transfusion ", as a kind of administering mode, with basically continue, drug solution fluid at a slow speed, the mode that within the relatively long time, enters blood. " injection " then is the quick delivering medicament mode of an agent solution or suspension.

The mode of quiet notes (IV), intramuscular injection (IM), lumbar injection (IP), subcutaneous injection (SC) and breastbone inner injection all right and wrong through the dosing way of enteral administration composition.The human body of injection or transfusion administration has been described with anatomical term in these methods.This composition that just requires this invention has the pH value under the physiological condition by above-mentioned approach effect on human body the time, concrete numerical value depends on that concrete patient and doctor judge.Higher pH solution (pH＞8) is suitable for venous transfusion or drop slowly most.

In order to understand " buffer " and " buffer agent " among the present invention, recall acid-base titration curve earlier, this curve has a relative flat zone, extends to the pH value unit of titration mid point both sides 1.0.Proton donor and proton acceptor (bronsted lowry acids and bases bronsted lowry just) that equivalent is arranged at the titration mid point.In this zone, when adding small amount of H ⁺Or OH ^-The time, pH value only can produce minor variations.Conjugate Acid-Base Pairs is as buffer system in this zone, and it can add H ⁺Or OH ^-The time stop the variation of pH value.If pH value has exceeded this zone, the buffer capacity of buffer will weaken.The buffer capacity of buffer is a titration curve midpoint maximum at its pH value, that is to say, contain the proton donor and the proton acceptor of equivalent concentration this moment, and the pH value of this moment equals its pK ' (dissociation constant).Preparation about buffer has a detailed description in " Biochemical Research data " (Data for Biochemical Reserch) (Rex, M.C., OxfordScience Publications, 1995).

Take place pH value that a lot of in vivo physiological mechanisms can keep blood 7.35-7.45 very among a small circle in.The some of them buffering is based on carbonic acid one phosphoric acid balance sysmte, and many other bufferings relate to aminoacid and protein.For example, the pH value of tear remains on 7.4 by protein as buffer.Single amino acid also is a buffer system of great use, and its titration curve is more complicated, because at the existing proton acceptor of same intramolecularly proton donor is arranged again.This molecule is amphoteric, that is to say, at the existing positive charge of same intramolecularly position the negative charge position is arranged again.As follows, adding H ⁺Or OH ^-The time aminoacid can play cushioning effect.

In order to make the present invention that one " medicinal buffer system " be arranged, used buffer extensively comprises Conjugate Acid-Base Pairs and number acid alkali cpd, can regulate and keep A β peptide solution on desired pH value level.The pH value of the present invention in dissolving/filter process is 8.5 or higher, and pH value will be lower than 4 on the other hand.

The composition of buffer can be selected from aminoacid, salt and derivant thereof; Pharmaceutically useful basifier, alkali-metal hydroxide, ammonium hydroxide, organic and inorganic acid, salt; And some mixture of the above material.The concentration of each composition will be enough to reach and keep desired pH value in the buffer, and its concentration is got due to its Acidity of Aikalinity.Can utilize the method for prior art to determine valid density, as using pH meter and titration technique.

Employed composition has the material of hydroxide class in the embodiment of the invention, comprises alkali-metal hydroxide and ammonium hydroxide, and the basifier of pharmaceutics includes but not limited to trihydroxy methyl aminomethane (tris), sodium borate (Na ₂B ₄O ₇) and disodium citrate, aminoacid, amino acid whose salt, ester or amide and some simple derivants (as the acetylizad derivant of amino acid whose N-).And be particularly suited for doing buffer glycine (as Glycine sodium), arginine and lysine and sodium hydroxide and ammonium hydroxide arranged.

Implement the employed pharmaceutically useful acid of the inventive method, comprise to indefiniteness hydrochloric acid, phosphoric acid, citric acid, acetic acid, maleic acid, malic acid and succinic acid etc.In addition, these acid can also be used to the pH value of titration aqueous slkali, make it reach a level lower, that be more suitable for physiological requirement, to obtain the suspension of peptide.

On the contrary, above-mentioned various alkali compoundss can be used for the filtering solution of the low pH value of titration, make it more meet physiological requirement, also can obtain the suspension of peptide.

The mixture of pH value regulator also can be put into limit of consideration.The Conjugate Acid-Base Pairs of above-mentioned salt form (as Ammonium Acetate) also can be used for buffer of the present invention.With the alkaline solution of acetimetry ammonium hydroxide,, prepare such conjugation buffer system until forming nearly neutral liquor ammonii acetatis.

Noun used herein---" tension regulator " is meant the factor of various formation osmolarities.The tension regulator that uses in the embodiment of the invention includes, but is not limited to sugar, as mannitol, sucrose, glucose, and salt, as NaCl, KCl or the like.The consumption of tension regulator should make the osmotic pressure of solution be lower than 350 the milli ooze/kilogram, better can arrive 250-350 oozes in the least/kilogram (mOsm/kg), and preferably remain on 280-320 ooze in the least/kilogram scope in.What deserves to be mentioned is that some electrically charged chemical compounds as buffer composition also can exert an influence to tension force.So, before adding the tension force of tension regulator, determine the tension force of A β peptide buffer solution earlier with further regulator solution.

Can select some chelating agen for use in composition of the present invention and the manufacturing process.The preferred embodiment of used chelating agen comprises ethylenediaminetetraacetic acid (EDTA) and salt (as sodium salt) thereof, and these salt are in the concentration range of 0.05-50mM, and concentration is better effects if when 0.05-10mM, and preferred concentration is 0.1-5mM.Also can use other known chelating agen (or interleaving agent) as polyvinyl alcohol.

Optionally, can also comprise some surfactants or cleaning agent in the said composition, as polysorbate (as tween (Tween )) or 4-(1,1,4, the 4-tetramethyl butyl) (4-(1 for the phenoxy group polyethoxy ethanol, 1,4,4-tetramethylbutyl) phenyoxypolyethoxyehthanols) (Triton ), or many propyl group of polyethylene glycol (polyethylenepolypropylene glycols) (Pluronics ).The content of surfactant can be 0.005-1%, is preferably 0.02-0.75%.A kind of polysorbate that is more suitable for is PS-80, and its trade name is Tween  80.

Wetting agent also is one of adjuvant used in the present invention.Polyethylene Glycol as PEG3350, can combine with A β peptide at aqueous phase, stablizes its hydrophilic segment, thereby regulates the association and the dissolubility thereof of A β peptide particles.The content of wetting agent is 0.5-5% (w/v (the gram number of expression active component in 100 ml solns, percentage by weight in the unit volume)) preferably.

Pharmaceutically useful sugar (as sucrose, glucose, maltose or lactose) or sugar alcohol (as mannitol, xylitol, sorbitol) are to the not influence of drug effect of active component.In one aspect of the invention, the used sugar or the molecular weight of sugar alcohol are easy to be dispersed or dissolved in water less than 500, to be applicable to the present invention.Sugar that is adopted and sugar alcohol comprise xylitol, mannitol, sorbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose, lactose in an embodiment of the present invention, or the like.They can use separately, also can mix in twos or multiple mixing.Preferred sugar is mannitol, is particularly useful in the freeze-dried composition; Sucrose is applicable to liquid composite.

Also find to use a kind of QS-21 adjuvant, can with the suspendible protein interaction of the present composition, form transparent clarifying suspension.In this required interaction process, the peptide section exists with the conformation of beta sheet, and is suspended in the solution with minimum graininess, can improve the stability of suspension and promote the immunogenic effectiveness of said composition.Similar to QS-21 to the model of action of A β peptide, can utilize DPPC of the prior art (dipalmitoyl phosphatidyl choline), produce the suspension that contains molecule with the effect of A β peptide, therefore also can be used as adjuvant, to form transparent clarification suspension.Can also select other adjuvant to mix use with QS-21.

Lyophilization is a technology commonly used in the pharmacy procedure, and it can keep the freezing dry process stability of peptide section thereafter that neutralizes.Lyophilization can make protein or peptide section stabilisation in aminoacid, also is well-known to those skilled in the art with sugar as substrate.With reference to Lueckel B., Deng " prescription-its concentration ratio of sugar and aminoacid or mannitol is to freeze concentration and cryodesiccated influence " (Formulations of sugars with amino acids ormannitol-Influence of concentration ratio on the properties of thefreeze-concentrate and the lyophilzate), " drug technique progress " be 3 (3) pp.325-336 (1998) (PHARM.DEV.TECHNOL.).Medicine seminar association (The Royal Pharmacutical Society of GB Symp) of Britain imperial family: ' describing the new analytical method of biological technology products ' (New Analytical Approaches to theCharacterization of Biotechnology Procucts).In June, 1996 Lueckel B. etc., " the method optimizations of lyophilization biotechnology goods " (A strategy foroptimizing the lyophilization of biotechnolofical products), " pharmaceutical science " (Britain) 3 (PHARM.SCI. (UK) 3) (1) pp.3-8 (1997).With above two pieces of documents at this hereby incorporated by reference document.

Without any harmful or harmful effect, this component is used to administration to any component of term " medicinal " modification in context for the main body that is acted on." medicinal diluent " and " medicinal adjuvant " is meant any activity that keeps or do not change reactive compound, and the main body that is applied is not poisoned or dysgenic component, this component is used to administration in context, for example avirulent pH value regulator, buffer agent, tension regulator or chelating agen or the like.

" comprise " composition that one or more are listed and the composition of not listing especially in compositions or the method.For example, in the compositions that contains A β peptide, both comprised the A β peptide in the compositions of listing, comprised also that with A β peptide be other components that are not listed in the compositions of composition.

Other are described as follows: abbreviation explanation ℃ degree centigrade cc cubic centimetre C.D. circular dichroism method pH log[H ⁺], hydrogen ion concentration and Acidity of Aikalinity amount in a kind of solution

The dissociation constant of degree PK ' acid is relevant with pH:

PH=PK '+log[(H+ receptor) ÷ (H+ donor)] PS80 polysorbate80 or Tween80  (polysorbate

And ethylene oxide copolymer); " Merck index " (Merck

Index) the molar concentration M molar concentration of Min minute ml milliliter of monograph No.7559 (the 11st edition) μ m micron N equivalent concentration-expression solution, with mol metering mM mM DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid, normally its sodium salt Tris Tris " Merck index " (Merck

Index) the non-complete adjuvant of thematic No.9684 (the 11st edition) complete adjuvant/Fu Shi of the anti-phase high pressure liquid chromatography RPM revolutions per minute of RP HPLC CFA/IFA Fu Shi (Chang etc.,

" senior delivering drugs systematic review " (Acvance Drug

Delivery Reviews) 32,173-186 (1998)) MPL 3-oxygen-deacylated tRNA base monophosphoryl lipid A (MPL ^TM) (referring to

GB2220211)。QS21 extracts from the bark of the Quillaia Mo Nila tree in South America

The triterpenoid saponin that comes (with reference to Kensil etc., " vaccine is established

Meter " (Vaccine Design): adjuvant and adjuvant research

(Subunit?and?Adjuvant?Approach)

(Powell﹠amp; Newman, Plenum publishing house, New York,

1995; U.S. Patent No. 5,057,540).

(Stimulon ^TMQS-21) FIO information only for reference

Filtration is a flow of liquid through having the filter membrane in unified aperture, according to the size of impurity and with the process of its eliminating.Although micron particles can be removed by non-membrane material or fiber medium material, and has only filter membrane to have accurate aperture, it can get rid of littler granule quantitatively.So just can by miniature displacer the time, utilize filter membrane quantitatively antibacterial to be removed, to reach the effect of degerming.In the past when purification A β peptide, the peptide section is often insoluble or assemble agglomeratingly, can make granule stop up fenestra like this, or/and the peptide section is very low in the filtering response rate, thereby can't carry out commercial-scale filtration.

What be suitable for is to get rid of particulate filtration device more than 0.2 micron.

Filter membrane is unified making on substrate, and according to film surface apertures magnitude classification.The filter membrane of this surface type both can be stayed granule in the film, also can stay on the film.The classification in different apertures allows to pass through than its little granule, and the granule bigger than the aperture is stayed on the film.These filters that definite aperture is arranged are that (that is to say of certain load limit arranged, when filter is full of the granule that is excluded, can make along with the rising of filtrate pressure the material that is excluded on a small quantity by), they all are the devices that can select controlling microbial for use, and the surface type membrane filter of " sterilization level " is known by pharmacy industry.

In all filtration application, the permeability of filter medium can be subjected to the influence of aspect character such as filtrate chemistry, molecule, static.Yet, hydrophilic miniature displacer used in the present invention, all very stable under desired high pH value of experimental technique or low ph environment, can also accurately get rid of unwanted particulate matter and not by its obstruction.Hydrophilic miniature displacer can be selected and use to persons skilled in the art just.Utilize some commercially available descriptions of product or network address can help us to select required filter, as following network address: http: ∥ millispider.millipore.com/corporate/sitemap.nsf/catalogs (from May, 1999).

Filter selected in the example of the present invention has: Millipore Durapore , also claim Millex GV (Millipore company, general headquarters are at Massachusetts Bedford), it is the fluoride (polyvinylidene fouoride) of polyvinylidene, a kind of hydrophilic polymers, good stability and lower albumen affinity are arranged, and its aperture is 0.22 μ m; MillexGN ^TM, a kind of hydrophilic nylon material, the aperture is 0.2 μ m; Millex Gp ^TM, a kind of polyether sulfone (polyethersulfone) polymer of hydrophilic finishing, the aperture is 0.22 μ m.It is stable that Durapore still can keep when pH value 9-9.5, thereby more suitable.

A β peptide after the filtration will comprehensively reclaim, and the response rate just can be thought to reclaim more completely greater than 50%, and is better greater than 80% as the response rate of the A β peptide after filtering, and is preferably more than 90%.

Method

Method of the present invention comprises that the preparation least concentration is the A β peptide aqueous solution of 0.01mg/ml.Prepare such compositions and will regulate pH value of aqueous solution, so that A β peptide dissolves and reach desired concentration (0.1-2.0mg/ml).

What use is that traditional method is regulated pH value of aqueous solution, by adding acid or alkali meets the requirements of pH value.Preferably the amount of acid or alkali is an acceptable on pharmaceutics.For in storage process, keeping the pH value of solution, allow to contain pharmaceutically useful buffer in the compositions.It is techniques well known that pH value is as requested selected suitable buffer.

Preferably pH value of aqueous solution is adjusted to about 2-4 or 8.5-12, under this pH value, be surprised to find that A β peptide is easy to dissolving very much.

The low pH value that needs in the operating process of the present invention (approximately pH 2-4) can be realized by selecting halogen acids (example hydrochloric acid, hydrobromic acid), phosphoric acid, citric acid, acetic acid and other pharmaceutically useful acid for use.Select suitable acid to be well known to those skilled in the art.

The high pH value that needs in the operating process of the present invention (approximately 8.5-12) can be by selecting some pharmaceutically useful alkali for use, and for example, alkali metal, ammonium hydroxide (being NaOH, NH4OH) wait the pH value that improves solution to reach requirement.It is relatively good to adopt buffer that pH value is stabilized between the 8.5-10 under high pH value condition, preferably controls pH value between 9-10.

Be to adjust earlier or the back is adjusted to pH value no matter, the A β peptide that adds ormal weight is essential.Certainly, dissolve immediately, add A β peptide again after preferably adjusting pH value in order to make the peptide section.Additionally, can gentle agitation, heating is with accelerate dissolution.

Any available supplementary element as pharmaceutically useful buffer, tonicity agents, adjuvant or the like, can be selected to add when an opportunity arises, all can before or after adding A β peptide.

In case the aqueous solution that requires preparation is finished, just can carry out sterilising filtration or/and lyophilization according to the operating process of knowing in this area, its idiographic flow is seen following embodiment.

Following solution is preferred buffer system, is used to make the dissolving of A β peptide and meets the requirements of concentration range (0.6-2.0mg/ml):

The preferred amino acids compositions:

The 10mM Glycine sodium, pH value 9.0,9.5 or 10.0 and/or 0.02-1.0% (w/v)

Polysorbate80 (PS-80)

Non-essential one or more tension regulators that contains are beneficial to non-through enteral administration;

10mM L-arginine-HCl, or 10mM L-lysine, pH value is 9.0

Or 10.0 and/or the polysorbate80 (PS-80) of 0.02-1.0% (w/v) arranged

Non-essential one or more tension regulators that contains are beneficial to non-through enteral administration;

Composition of the present invention is suitable for cryopreservation, with degraded or the gathering that prevents the peptide section.Preferred storage temperature is 2 to 8 ℃.

Below with embodiment enforcement of the present invention is illustrated, these embodiment only in order to reach the purpose of being convenient to understand, can not limit scope of the present invention.

Detailed description of preferred embodiment

The dissolubility of embodiment 1 peptide

Peptide and reagent

The trifluoroacetate of A β 42 is from U.S. peptide company, and lot number is M05503T1, M10028T1, and do not carry out any modification before using.Other salt all can obtain and successful Application in the present invention.The example of alternative A β peptide equilibrium ion salt sees Table 3.

All acid, alkali and buffering solution all are to prepare with the reagent that meets requirement of experiment, for convenience, and all through preserving after the aseptic filtration.Polysorbate80 (polysorbate 80) (has another name called Tween 80, PS80) preparation: according to w/v (bulk density calculating), with standard compliant low peroxide PS80 solution (the single oleate of the polysorbate of 10% low peroxide of Aldrich-Sigma company), dilute that to be mixed with concentration be 4% mother solution.

The dissolving of peptide:

The A β peptide of weighing 500-700 μ g is dissolved in an amount of buffer, and it is 0.6,0.8 or A β 42 solution of 1.0mg/ml that preparation obtains ultimate density.Peptide is placed directly in the Wheaton vial of 4ml band screw cap and weighs.Added after an amount of buffer gentle agitation about 15 to 30 minutes.Visual observation solution is also evaluated dissolubility in order to following standard: (+)=poorly soluble, muddy suspension; (++)=have clear and bright suspension of insoluble particles; (+++)=clear and bright solution.

The dissolubility of table 1. range estimation solution

Buffer character	A β 42 aimed concns (mg/ml)	The range estimation dissolubility
			0.01 equivalent NaOH	??1.0，0.8	??+++
0.01 equivalent NH 4OH	??1.0，0.8	??+++
			The 50mM Glycine sodium, pH9.0	??1.0	??+++
The 10mM Glycine sodium, pH9.0	??1.0，0.8， ??0.6	??+++
			The 10mM Glycine sodium, pH9.5	??0.6	??+++
The 10mM Glycine sodium, pH10.0	??1.0，0.8	??+++
			The 10mM Glycine sodium, pH9.0,0.02%PS80	??1.0	??+++
The 10mM Glycine sodium, pH9.5,0.1%PS80,5% sucrose	??0.6	??+++
			The 10mM Glycine sodium, pH9.5,4% mannitol, 1% sucrose	??0.6	??+++
The 10mM Glycine sodium, pH9.5,4% mannitol	??0.6	??+++
			The 10mM Glycine sodium, pH10.0,0.02%PS80	??1.0	??+++
10mM L-arginine hydrochlorate, pH9.0	??0.6	??+++
			10mM L-arginine hydrochlorate, pH10.0	??0.8	??+++
10mM aminutrin sodium, pH9.0	??0.8，0.6	??+++
			10mM aminutrin sodium, pH10.0	??0.8	??+++
The 10mM L-Lysine sodium salt, pH9.5,4% mannitol	??0.6	??+++
			The 10mM ammonium acetate, pH9.0	??1.0	??+++
The 10mM ammonium acetate, pH9.0,0.02%PS80	??1.0	??+++
			The 50mMTris-hydrochloride buffer, pH10.0, EDTA (0.5mM), 0.02%PS80	??1.0	??++
The 10mM sodium borate, pH9.0	??1.0	??+++
			10mMN-acetyl group dextrorotation sodium glutamate, pH9.0	??0.8	??+++
The 10mM glycine hydrochloride, pH3.0	??1.0	??++
			0.01 equivalent HCl	??1.0	??+++
0.01 mol phosphoric acid	??1.0	??+++

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Buffer character	A β 42 aimed concns (mg/ml)	The range estimation dissolubility
			Dimethyl sulfoxine (DMSO) (pure)	??0.6	??+++
The 10mM Glycine sodium, pH8.0	??1.0	??++
			10mM L-arginine hydrochlorate, pH9.0	??0.8，1.0	??++
10mM ammonium bicarbonate sodium, pH9.0	??0.8	??++
			10mM ammonium carbonate sodium, pH9.0	??0.8	??++
The 50mMTris-hydrochloride buffer, pH10.0, EDTA (0.5mM)	??1.0	??++
			The 10mM sodium borate, pH10.0	??1.0	??++
10mM L-arginine hydrochlorate, pH8.0	??1.0	??+
			The 10mM ammonium acetate, pH8.0	??1.0	??+
The 10mM ammonium acetate, pH8.0,0.02%PS80	??1.0	??+
			The 50mMTris-hydrochloride buffer, pH8.0, pH9.0, or pH10.0	??1.0	??+
The 50mMTris-hydrochloride buffer, pH8.0 or pH9.0, EDTA (0.5mM)	??1.0	??+
			The 50mMTris-hydrochloride buffer, pH8.0 or pH9.0, EDTA (0.5mM), 0.02%PS80	??1.0	??+
50mM or 100mMNaCl, pH 9.0	??1.0	??+
			The 50mM sodium phosphate, pH8.0, pH9.0, or pH10.0	??1.0	??+
The 50mM Glycine sodium, pH8.0 or pH10.0	??1.0	??+
			10mMN-acetyl group dextrorotation aminoglucose sodium, pH9.0 or pH10.0	??0.8	??+
10mMN-acetyl group dextrorotation sodium glutamate, pH10.0	??0.8	??+
			The 10mM sodium citrate, pH3.0 or pH4.0	??1.0	??+
The 10mM sodium acetate, pH4.0	??1.0	??+

The solution that is labeled as " +++" shows that solution is clear and bright when aimed concn.The pH value of buffer changes in the scope of 9-10.Inorganic solute is NaOH and NH for example ₄The solution of OH is alkaline, and HCl and phosphoric acid are highly acid.PH greater than 9 condition under, the dissolubility of this peptide is 0.6-1.0mg/ml.Polysorbate80, sucrose, mannitol and ethylenediaminetetraacetic acid additives such as (EDTA) does not influence the dissolubility of peptide, and improve to filter the back peptide tension force and the response rate aspect assosting effect is arranged, perhaps as the chela mediating recipe.Acid solution (pH 4 or lower) also can dissolve the A β 42 in the 0.6-1.0mg/ml concentration range.Be labeled as the solution of " ++ ", show that peptide is partly dissolved when dissolving with aimed concn.The dissolubility of this peptide may be lower than the concentration of detection.Be labeled as the solution of "+", show not fully dissolving of peptide under aimed concn.The dissolubility of embodiment 2 A β peptides in buffer

A β 42 (0.6,1.0,1.5,2.0,3.0 and 3.5mg/ml) is dissolved in 10mM, in the Glycine sodium buffer of pH9.0.Every kind of solution is centrifugal in bench top centrifuge (greater than 10,000 rev/mins, about 10 minutes), and it is muddy suspension (concentration is that the solution of 0.6-1.5mg/ml is clear and bright) that the observation peptide concentration reaches 2.0-3.5mg/ml.

Get the supernatant of sample aliquot in each solution, detect (RP HPLC) by reversed-phase high-performance liquid chromatography and analyze.

Table 2 is a peptide peak area table, shows the solubility curve of A β 42 in the Glycine sodium buffer of pH 9.0 with the data mapping.

Table 2 solubility limit, A β 42 peptides, trifluoroacetate (TFA)

A β 42 mg/ml (the 10mM glycine, pH9.0)	The HPLC peak area
		????0.6	????3613553
????1	????5921792
		????1.5	????8850393
????2	????9213446

In addition, trifluoroacetate, ammonium salt, hydrochlorate and the sodium-salt form of A β 42 peptides can purification.These salt are dissolved in the different buffer, and after revising according to the counter ion contribution of various salt, the concentration that makes A β 42 peptides is 0.45mg/ml.After the filtration, the peak area of the reversed-phase high-performance liquid chromatography by described peptide before and after relatively filtering is determined its response rate.All salt is all solvable and can filter in the concentration of 0.45mg/ml, the results are shown in table 3.

The dissolubility of the various salt of table 3.A β 42 peptides

???????0.45mg/ml?Aβ42	The response rate (%)
		Contain 1mM NH 4The ammonium salt solution of OH	????70
Contain 2mM NH 4The ammonium salt solution of OH	????106
		The ammonium salt solution that contains the 10mM Glycine sodium, pH9.0	????115
The ammonium salt solution that contains the 10mM Glycine sodium, 5% sucrose, pH9.0	????96
		The ammonium salt solution that contains the 10mM Glycine sodium, pH9.5	????101
The ammonium salt solution that contains the 10mM Glycine sodium, pH10.0	????106
		The trifluoroacetic acid saline solution that contains the 10mM Glycine sodium, pH9.0	????101
The hydrochlorate solution that contains the 10mM Glycine sodium, pH9.0	????101
		The sodium salt solution that contains the 10mM Glycine sodium, pH9.0	????100

Embodiment 3 reclaims dissolved peptide by typical hydrophilic filter

Experiment (is filtered diameter 25mm, filter area 3.9cm with the injector type filter ²) as follows: Millex GV, 0.22uM: a kind of hydrophilic polyvinylidene fluoride (PVDF, Durapore ) film with low protein binding characteristic; Millex GN, 0.20uM: a kind of hydrophilic nylon membrane with low protein binding characteristic; And Millex GP, 0.22uM: a kind of hydrophilic surface reworked polyethylene sulfone (PES) with low protein binding characteristic.

Above-mentioned filter is as the hydrophilic microstrainer of the representational various models that can be obtained by the commercial channel.Carry out filtration studies by following dissolution system:

1. the 0.01 equivalent NH that contains 0.6mg/ml A β 42 ₄OH solution

2. the solution that contains the 10mM Glycine sodium of 0.6mg/ml A β 42, pH9.0

3. the solution that contains the 10mM L-Lysine sodium salt of 0.6mg/ml A β 42, pH9.0

4. the 10mM arginine monohydrochloride solution that contains 0.6mg/ml A β 42, pH9.0

Each filter filters every kind of A β 42 solution of about 2ml.The concentration of peptide detects with reversed-phase high-performance liquid chromatography (RP HPLC).Measure its response rate by the peak area that compares micro-filtration front and back peptide, the results are shown in following table 3 and table 4.

Table 3-filtered and recycled rate

Solution component 0.6mg/ml A β is dissolved in:	Filter model (response rate %)
				MillexGV	MillexGN	??MillexGP
	The 10mM Glycine sodium	????99.1	????97.2				????98.0
The 10mM arginine monohydrochloride				????91.9	????86.6	????92.5
	The 10mM L-Lysine sodium salt	????95.0	????94.7				????97.4
0.01 equivalent NH 4OH				????102.2	????104.8	????105.1

Embodiment 4 adds excipient and does not add under the excipient condition being dissolved in the filtration of the A β 42 in Glycine sodium and the L-Lysine sodium salt buffer

Concentration is the Glycine sodium solution that 0.6mg/ml A β 42 is dissolved in 10mM in these researchs, this solution contains 0.1% polysorbate80 (PS80), 0.9% NaCl, perhaps the mixture of 0.1%PS80,0.9% NaCl, 5% sucrose, 1% sucrose and/or 4% mannitol.Getting every kind of about 2-5ml of solution filters with Millex GV.The sample aliquot supernatant in the bench top micro centrifuge with 〉=10, under 000 rev/min of condition centrifugal about 3 minutes.Measure its response rate by the peak area of peptide in reversed-phase high-performance liquid chromatography before and after comparing micro-filtration.

The filtered and recycled rate of table 4 peptide in various buffer

Solution component 0.6mg/ml A β 42 is dissolved in:	Recovery percent
		The 10mM Glycine sodium, pH9.0	104.5％
The 10mM Glycine sodium, pH9.0,0.1%PS80	106.2％
		The 10mM Glycine sodium, pH9.0,5% sucrose	115.4％
The 10mM Glycine sodium, 4% mannitol, pH9.5	103.0％
		The 10mM Glycine sodium, pH9.5,4% mannitol, 1% sucrose	98.0％
The 10mM Glycine sodium, pH9.0,0.9% NaCl	76.7％
		The 10mM Glycine sodium, pH9.0,0.1%PS80,0.9% NaCl	65.0％
10mM lysine/citrate, pH9.5	93.2％
		10mM lysine/citrate, 4% mannitol, pH9.5	80.0％

Millex GV filter, as a kind of defecator of preferred filtration A β peptide solution, its main advantage is the response rate of peptide higher, and can use in commercialization process.The peptide solution that contains 0.9%NaCl, its visual observation result is insufficient for dissolving, causes by the detected response rate of reversed-phase high-performance liquid chromatography lower.In order to increase the dissolubility of the peptide under the existence conditions such as inorganic salt such as NaCl, preferably after the peptide micro-filtration in buffer, add aseptic tension adjustment agent solution.

On the other hand, as the different lytic activity of saccharide embodiment of tension regulator, and has the state maintenance that is beneficial to the peptide suspension.This specific character is called hydration again and increases preface, thereby makes the peptide chain hydration become α beta sheet conformation more stable on the thermodynamics.

But the pH value excursion of A β peptide filter operation approximately is 8.5-12, and pH value has the good response rate in the scope of 9-10.It is the MillexGV film of 0.2 μ m that manufacturing industry is filtered normal employing aperture.

In order to obtain being suitable for clinical practice, meeting physiology's requirement, and stablize, have the A β peptide formulations of biologic activity, the key step in the formulation preparation comprises preferably carries out sterilising filtration to peptide in the scope of pH8.5-10.Embodiment 5 dissolubility liquid formulations

Assay method

Three kinds of A β 42 peptides are produced by U.S. peptide company (APC) and are provided.Carry out the production of duplicating of the correction of preparation conclusive evidence, chemistry and biology property description of this APC peptide and A β 42 by california peptide institute (Canifornia Peptide Research).

Obtain describing in detail in preparation explanation the 1st, the 2nd and the 3rd part below.

Stability analysis:

The detailed description that pair preparation is hereinafter arranged.The peptide of different preparations is sub-packed in the different vials, stores some months at 2-8 ℃, regularly analyzes.The concentration and the purity of periodic monitoring preparation outward appearance, pH value, peptide in the Study on Stability process.And measure the concentration and the regional percent purity of A β peptide with reversed-phase high-performance liquid chromatography.A poly reversed-phase column is arranged in the reversed-phase high-performance liquid chromatography, carry out gradient elution, and detect at the 220nm place with ammonium bicarbonate (or Tris (tris))/acetonitrile.Measure the concentration of peptide by standard control; Calculate to such an extent that the percentage rate of A β peptide is measured purity by whole chromatogram being carried out the area-method integration.

Characteristic description

By the architectural characteristic under circular dichroism method mensuration A β 42 dissolved states, as shown in Figure 1.

Biologic activity

Give A β 42 and adjuvant such as CFA/IFA, MPL (Corixa immunochemistry company) or the QS 21 (AquilaPharmaceuticals) of the different preparations of Switzerland Webster mice (every group of 4-8 mice) injection, according to the antigen that is indicated in the specialty research/adjuvant concentration injection.Unless otherwise specified, injection generally is whenever biweekly, promptly in the injection of the 0th, the 2nd and the 4th week.Mice blood drawing after the injection of the 2nd and the 4th week.Serum is done standard and is intersected enzyme-linked immunosorbent assay (ELISA), makes the anti-rat immune globulin of goat that antigen is connected with Radix Cochleariae officinalis peroxide (change) the thing enzyme antibody that serves as a mark with A β 42.Tiring of each animal internal antibody represented with loose point or geometric mean of some data in every group, with the result with the A β 42/CFA/IFA of coherent condition in contrast tiring of antibody of immunogen generation compare.Tire and the results are shown in Table 8 and 10.

1. mixed solution (preparation)

A β peptide is dissolved in the concentration of 0.6mg/ml and contains the 10mM Glycine sodium, in the buffer of pH9-9.5, and filters with the Millex GV in 0.2 μ m aperture.Every 0.5ml mixed solution is packed in the little glass medicine bottle (Gensia P/N X34-113-002) of a 2cc volume, and covers sealing with gray butyl synthetic rubber stopper (Gensia P/N X66-113-030).Medicine bottle is in 2-8 ℃ of preservation.

2. stability test

Concentration and purity with A β 42 in the reversed-phase high-performance liquid chromatography monitoring mixed solution.Sample solution direct analysis or analyze again after centrifugal through little.Following form (table 5) is listed the analytical data of consoluet preparation.Before analyzing centrifugal or not the centrifuged sample analysis result do not have notable difference, show that peptide has lasting dissolubility under current concentration.Area percentage rate purity to peptide is compared with standard control: do not find tangible peptide signs of degradation.The dissolving preparation of preserving under 2-8 ℃ of condition keeps stable three middle of the month.

Table 5. is preserved the result after three months

0.6mg/ml A β 42 is dissolved in:	Outward appearance	??pH	Area percentage rate purity	A β concentration (mg/ml)
					Reference standard	Do not have	Do not have	????68％	Do not have
The 10mM Glycine sodium, pH9.0	Clear and bright solution	????8.6	????69％	?0.65

3. characteristic description

By circular dichroism method Characteristics Detection to A β peptide formulations, show that peptide is in the state of random coil in solution, there is distinctive negative ellipticity to absorb at 189-205 millimicron place.

Biologic activity

The dissolving preparation of the A β 42 of pH9 is injected into Swiss Webster mice with adjuvant improves antibody titer.Matched group is compared, the injection of per fortnight once (0 week, 2 weeks, 4 weeks) 50 μ gMPL or 25 μ gQS21 adjuvant and 33 μ gA β 42 are injected together, cause that tangible immunizing potency replys.

Embodiment 6 liquid mixed suspension preparations

Glycine/acetate preparation

A β peptide is dissolved in the Glycine sodium of 10mM with 0.6mg/ml or also contains in the buffer of pH9.0-9.5 of other excipient.The solution of peptide is regulated pH value with the acetic acid of 0.1M and is reached about 6 after filtering with Millex GV, obtains the suspension of peptide.In the little glass medicine bottle of a 2cc 0.5ml mixed solution is packed into (Gensia P/N X34-113-002), and cover sealing with gray butyl synthetic rubber stopper (Gensia P/N X66-113-030).Phial is 2-8 ℃ of preservation.

Glycine/citrate preparation

0.6mg/ml A β peptide formulations is to prepare containing 0.1%PS-80 or also contain in the Glycine sodium solution of 10mM of 5% sucrose (25ml).After the solution of peptide filters with Millex GV, with the about pH6.0 of 0.1M citric acid titration.Join in glycine/citrate/PS 80 mixed systems being titrated to the NaCl that can select after pH value is 6 0.9%.0.5ml mixed solution is packed in the little glass medicine bottle of a 2cc (Gensia P/NX34-113-002), and cover sealing with gray butyl synthetic rubber stopper (Gensia P/NX66-113-030).Phial is 2-8 ℃ of preservation.

Glycine/citrate preparation is mixed with the solution that has buffer capacity under the condition of pH6.Prepare the 0.6mg/ml A β peptide of several 100ml with Glycine sodium and 5% sucrose of 10mM or the preparation that also contains the pH9 of 0.1%PS 80.Also prepared in addition with containing 5% sucrose or also containing 0.1mg/ml A β 42 preparations of Glycine sodium preparation of the 10mM of 0.1%PS 80.Before regulating pH, filter this peptide solution with Millex GV.Regulate pH value to 6 with the sodium citrate of 1M pH5.5, and make that sodium citrate concentration is 10mM and 20mM in the buffer.In the little glass medicine bottle of a 2cc 0.5ml mixed solution is packed into (Gensia P/N X34-113-002), cover gray butyl synthetic rubber stopper, and use alumiseal.Phial is 2-8 ℃ of preservation.

Stability test

Concentration and purity with A β 42 in the reversed-phase high-performance liquid chromatography monitoring preparation.Earlier the preparation of peptide is used the 0.01 equivalent NaOH solution equal-volume dilution that contains 2% dodecyl sodium sulfate (SDS) before analysis after, heating is 1 minute under 100 ℃ of conditions, makes the peptide resolubilization, and the total concentration of peptide in A β 42 suspensions is measured in the back.The method of dissolved A β 42 concentration of a kind of alternative measurement is that specimen is placed microcentrifuge, with more than or equal to 10,000 rev/mins of speed centrifugal treating 3-5 minute.This resolubilization peptide solution or supernatant with rp-hplc analysis equity volume.In research process, constantly grope the condition of reversed-phase high-performance liquid chromatography post, with resolution that improve to measure and more accurate quantitative.The accepted standard contrast was compared when therefore the area percentage rate purity of this peptide was with analysis, and compared the chromatogram of each time point, measured the palliating degradation degree of peptide with this.In the process of stability study, monitor outward appearance and pH value simultaneously.

Table 6 has been listed the data of several A β 42 suspension formulations.All preparation visual observation are suspension.These preparations are with the various sour adjust pH to 6 listed in the table.According to the not same-action of different excipient, the suspension of peptide form at once or could form through fortnight or longer time.In the preparation of this peptide, add NaCl or citrate is replaced to acetate, can accelerate the formation of suspension.

The concentration of peptide is aimed concn 0.6mg/ml in all preparations.The accepted standard contrast when analyzing of the area integral percentage rate purity of peptide equates.Under 2-8 ℃ of condition, store degraded and the loss of not finding peptide in 3 months.The data of listing in the outward appearance of said preparation and pH value and the table 1 are identical.

9%NaCl and 5% sucrose provide physiological tonicity for non-through enteral administration.Same described suspension pH value is also in the desired pH value scope of injection.

The liquid mixed suspension preparation of table 6

Preparation is formed	Outward appearance	??pH	Percentage purity	???Aβ42(mg/ml)
							0.6mg/ml A β is dissolved in:	The total concentration of peptide	Supernatant
T＝0				T=2 week
					10mM Glycine sodium/sodium acetate, pH6	Suspension			????5.9	?70％	0.54	?0.23	.04
10mM Glycine sodium/sodium acetate, pH6,5% sucrose	Suspension	????5.9	?68％	0.65			?0.21	.04
					10mM Glycine sodium/sodium acetate, pH6,0.1%PS80,5% sucrose	Suspension			????6	?67％	0.62	?0.43	.08
10mM Glycine sodium/sodium acetate, pH6,0.1%PS80,0.9%NaCl	Suspension	????5.9	?68％	0.55			?0.04	.04
					10mM Glycine sodium/sodium acetate, pH6,0.9%NaCl	Suspension			????6	?70％	0.53	?0.26	.05
10mM Glycine sodium/sodium citrate, pH6,0.1%PS80,5% sucrose	Suspension	Do not survey	?77％	0.63			Less than 0.01	Do not survey
					10mM Glycine sodium/sodium citrate, pH6,0.1%PS80,0.9%NaCl	Suspension			Do not survey	?71％	0.63	?0.02	Do not survey

The 10mM that provided by citrate or the buffer capacity of 20mM are provided preparation in the table 7, and making solution keep pH in process for preparation is 6.The very fast formation suspension of these preparations.According to the self-characteristic of A β peptide, the peptide structural change mainly occurs in the sterilization core of its inside in the process that forms the suspendible system.Compare with titration, can make the operation in the sterilizing room be more prone to handle and improve the repeatability of experiment with the buffer of concentration known.

Table 7. buffer-type suspension

0.6mg/ml A β 42 is dissolved in:	Outward appearance	pH	Percentage purity	The total concentration mg/ml of peptide
					The 10mM sodium citrate, 10mM glycine, pH6.0,0.1%PS80,5% sucrose	Suspension	6.0	?81％	????0.66
The 20mM sodium citrate, 10mM glycine, pH6.0,0.1%PS80,5% sucrose	Suspension	5.9	?83％	????0.69
					The 20mM sodium citrate, 10mM glycine, pH6.0,5% sucrose	Suspension	6.0	?81％	????0.67
0.1mg/ml A β 42 is dissolved in:
					The 20mM sodium citrate, 10mM glycine, pH6.0,0.1%PS80,5% sucrose	Suspension	5.9	?82％	????0.2
The 20mM sodium citrate, 10mM glycine, pH6.0,5% sucrose	Suspension	6.0	?81％	????0.09

The composition of these preparations further increases.With the Glycine sodium solution that the trifluoroacetate or the acetate of A β 42 peptides is dissolved in 10mM, pH9-9.5.Regulate salinity and make that the concentration of A β 42 is 0.45mg/ml.The PS80 that before filtering with the concentration change scope is 0.02%-0.5% is dissolved in the peptide solution.Similarly, after the adding of sucrose and NaCl can or add acid before adding acid.Regulate the peptide solution pH value with citric acid or hydrochloric acid, be listed in the table below.All preparations form suspension, and the concentration of A β 42 peptides reaches desired value.

Table 7b mixed suspension preparation 0.45mg/ml A β 42 is dissolved in:
	The 10mM Glycine sodium, 20mM citrate, 0.1%PS80, pH6.0
The 10mM Glycine sodium, 20mM citrate, 0.5%PS80, pH6.0
	The 10mM Glycine sodium, 20mM citrate, 154mM NaCl, pH6.0
The 10mM Glycine sodium, 20mM citrate, 154mM NaCl, 0.1%PS80, pH6.0
	The 10mM Glycine sodium, 154mMNaCl, pH6.0 (hydrochloric acid)
The 10mM Glycine sodium, 154mMNaCl, 0.1%PS80, pH6.0 (hydrochloric acid)

Feature description

The circular dichroism and the fourier-transform infrared testing result of A β 42 suspensions of pH6 show that the peptide configuration that is in suspension is a βZhe Die.The peptide configuration of the suspension in this configuration and the concentration 0.6mg/ml solution is identical, with whether to add buffer agent (citrate, acetate, phosphate) and excipient (sucrose, NaCl) in the preparation irrelevant.Add PS-80 in the said preparation and can make suspendible system homodisperse more.

Biologic activity

After A β peptide suspension and accessory drugs are injected in the Swiss Webster mice body jointly antibody titer is increased.As shown in table 8, compare the titre reaction that injection (0,2,4 week) every two weeks will cause q.s with matched group.

The antibody response of table 8. pair suspension formulation

Peptide formulations 0.6mg/ml A β peptide exists:	Peptide (μ g)	Accessory drugs (μ g)	Antibody titer geometric mean (blood drawing for the second time) **
				10mM Glycine sodium/sodium acetate, pH6,5% sucrose	33μg	?50μg ?MPL *	≈7000
10mM Glycine sodium/sodium acetate, pH6,5% sucrose	33μg	?25μg ?QS21	≈10,000
				10mM Glycine sodium/sodium acetate, pH6,0.1%PS-80,5% sucrose	33μg	?50μg ?MPL *	≈10,000
10mM Glycine sodium/sodium acetate, pH6,0.1%PS-80,5% sucrose	33μg	?25μg ?QS21	≈7,000
				10mM sodium citrate or sodium acetate, pH6,0.1%PS-80,5% sucrose	33μg	?50μg ?MPL *	≈10,000
10mM sodium citrate/sodium acetate, pH6,0.1%PS-80,5% sucrose	33μg	?25μg ?QS21	≈8,000
				10mM sodium citrate/sodium acetate, pH6,0.9%NaCl	33μg	?50μg ?MPL *	≈12,000
10mM sodium citrate or sodium acetate, pH6,0.9%NaCl	33μg	?25μg ?QS21	≈14,000
				Contrast: Calif. peptide lot number MF0639	33μg	?CFA/IFA	≈1,200

^*The MPL preparation contains triethanolamine

^*Sample titre value is calculated embodiment 7 lyophilized formulations with 50% maximum absorbance

Preparation

0.6mg/ml A β 42 peptides, glycinate or the lysine buffer of 10mM, and add four kinds of components of mannitol or mannitol and sucrose, make through the filtration sterilization of Millex GV filter.A kind of preparation, A β 42 peptides of 0.6mg/ml and pH are 9.5 10mM glycine sodium salt component, add 4% mannitol, titration just is not loaded in the bottle of jumping a queue to the physiology pH value.Remaining three kinds of solution (lysine/citrate/4% mannitol; Glycinate/citrate/4% mannitol; Glycinate/HCl/4% mannitol/1% sucrose) subsequently respectively with citric acid or HCl titration to pH7.5.Get three kinds of preparation 0.5ml then respectively and pack in the vial (Gensia P/N X34-113-002) of 2cc, and clog (Gensia P/N X66-113-030) gently with the elastomeric lyophilizing plug of gray butyl.

Lyophilizing

With said preparation lyophilizing in programmable Virtis freeze dryer (Gensia Sicor provides).Selecting mannitol for use is main matrix components.For making the mannitol crystal, said preparation is carried out freezing in the mode of thermal cycle (annealing), carry out once subsequently, the secondary product drying.

Stability analysis

Concentration and purity with reverse high performance liquid chromatography monitoring lyophilized formulations.Rebuild this every kind preparation with 1.0ml water for injection (WFI).Rebuild the back (through or without the bench top micro centrifuge with 〉=10, the centrifugal 3-5 of the speed of 000rpm minute), perhaps analyze immediately after the dissolving again as mentioned above.

Listed the concentration of four kinds of A β 42 peptide formulations after lyophilizing and reconstruction in the following form (table 9).Shown in data, these preparations are all solvable.Sample is all analyzed with two kinds of forms: reconstituted formulation and the reconstituted formulation of the peptide suspension being carried out the resolubilization processing.Do not see significant difference between reconstituted formulation and total peptide solution (resolubilization sample).In to the stability test of embodiment 5, determine, under 2-8 degree centigrade, do not occur the degraded and the loss of tangible peptide in the trimestral storage process with reverse high performance liquid chromatography.

Table 9. lyophilized formulations

Preparation 0.6mg/ml A β 42 peptides are dissolved in	Outward appearance	pH	Purity %	????mg/ml?Aβ42
						Rebuild peptide solution	Peptide is handled in resolubilization
				The 10mM Glycine sodium, pH9.5,4% mannitol	Settled solution			8.2	?82％	??0.59	??0.61
10mM Glycine sodium/sodium citrate, pH7.5,4% mannitol	Settled solution	7.4	?82％			??0.57	??0.60
				10mM L-Lysine sodium salt/sodium citrate, pH7.5,4% mannitol	Settled solution			7.6	?81％	??0.57	??0.55
10mM Glycine sodium/HCl, pH7.5,4% mannitol, 1% sucrose	Settled solution	7.4	?81％			??0.56	??0.59

Characteristic description

The circular dichroism method and the Fourier transformation infrared spectroscopy that to pH are 7.5 A β 42 peptide lyophilized formulations the analysis showed that in the process of lyophilizing and reconstruction, peptide chain can both keep the conformation of random coil.Glycine sodium solution A β 42 peptide formulations for pH9 have also obtained same observed result, and it also is soluble, and are filtrable and present the conformation of random coil in solution.

Biological activity

Contain 0.6mg/ml A β 42 peptides, the A β 42 peptide lyophilized formulations of 10mM Glycine sodium/HCl/4% mannitol/1% sucrose pH7.5 elect it as representational lyophilized formulations.When with after MPL and any one accessory drugs of QS21 mix, this product can cause the antibody titer of certain level in Swiss Webster mice.Listed in the table 10 with the geometric mean of matched group comparison and tired.

The antibody response of table 10 pair lyophilized preparation

Peptide formulations 0.6mg/ml A β 42 peptides are dissolved in:	μ g peptide	μ g accessory drugs	Antibody titer geometric mean (blood drawing for the second time) **
				10mM Glycine sodium/HCl, pH7.5,4% mannitol, 1% sucrose	33μg	?50μg ?MPL *	??≈14,000
10mM Glycine sodium/HCl, pH7.5,4% mannitol, 1% sucrose	33μg	?25μg ?QS21	??≈3,000
				Contrast: Calif. peptide lot number: MF0639	33μg	?CFA/IFA	??≈1,200

^*The MPL preparation contains triethanolamine

^*Sample titre value is calculated embodiment 8 with 50% maximum absorbance and is scaled up use GMP production standard

The suspension of peptide scales up to 1.5 liters scale, and is produced and encapsulation by signatory GMP pharmaceutical manufacturer.Encapsulate with two kinds of concentration: 0.6mg/ml and 0.1mg/ml.The product of two kinds of concentration all successfully scales up, encapsulates, and keeps stable in 2-8 degree centigrade and 25 degrees centigrade of following bimestrial times.

A β peptide is dissolved in pH9, contains in the 10mM Glycine sodium buffer of 5% sucrose with the concentration of any 0.6mg/ml or 0.1mg/ml.Peptide solution enters aseptic center through 0.2 micron Millipak20 micropore germ tight filter filtration sterilization.The reversed phase high efficiency liquid phase is presented at the loss of no tangible peptide in the filter process.Preparation 1M pH is 5.5 sodium citrate buffer solution, also allows it equally through the filtration sterilization of 0.2 micron Millipak20 micropore germ tight filter, enters aseptic center.Peptide solution is fixed through claiming, the sodium citrate buffer solution that adds appropriate amount is mixed with the 20mM citric acid, and pH is 6 preparation.

Under the concentration of 0.6mg/ml, peptide forms suspension immediately after adding citrate buffer solution; PH value in this process is measured as 6.4.The peptide suspension is packed into the amount of every bottle of 1.2ml under not stopping to stir in the 2cc pyrex bottle, and seals with West 4416 stoppers and seal.0.1mg/ml the solution of concentration had kept several hours clarification (in that to add fashionable also be clarifying) before forming suspension in second day in vial

Table 11 has been listed the A β 42 peptide suspension data of relevant 0.6mg/ml:

The suspension of table 11 A β peptide 6mg/ml

Add 10mM glycine, 20mM citrate, 5% sucrose, pH is 6

Test	Specification	The result
			Outward appearance	No color blurring suspension, impurity particle is up to standard	By
pH	????5.5-6.5	????6.3
			Concentration	????0.5-0.7mg/ml	????0.64mg/ml
Area purity percentage rate (high performance liquid chromatogram)	????FIO	????84.4％
			Volume, ml	????FIO	????1.13ml
Overall sterilizing state	????FIO	There is not growth
			Container final sterilization state	There is not growth	By
The endotoxin of antibacterial	????FIO	Less than 2 units/ml

Embodiment 9

The A β 1-42 peptide buffering suspension that adds the QS-21 accessory drugs

The single vial formulation that A β 1-42 peptide and accessory drugs merge is studied with QS-21, and this is a kind ofly to have the active triterpene soap of immune activation Saponin, and surprisingly finds to have formed the clear and bright peptide suspension of vision.

1.A the single vial formulation of β 1-42 peptide/QS-21

Freeze dried QS-21 is dissolved in 10mM Glycine sodium and the A β 1-42 peptide solution of pH9.Among each embodiment below, freeze dried QS-21 uses A β 1-42 peptide solution (trifluoroacetic acid (TSF) salt, 0.45mg/ml) dissolving of the 10mM glycine (Gly) of pH9.The pH value that adding citrate buffer (Cit) will contain the component of dissolved QS-21 transfers to 6.0 rapidly, and forms the clear and bright suspension of vision.The clarity of the suspension that measures at the spectrophotometer of 405nm with wavelength set.The result hints that particulate size is littler than catoptrical wavelength in the peptide suspension.These preparations have kept trimestral stability under 2-8 degree centigrade condition of storage under the STABILITY MONITORING of whole process.Find that the A β 1-42 peptide that contains in all preparations mainly exists with the beta sheet conformation.When measuring under 405nm with spectrophotometer, turbid phenomenon does not all appear in all preparations.With 0.45mg/ml A β 1-42 peptide, 0.2mg/ml QS-21,10mM glycine, 20mM citrate, 5% sucrose, pH is that 6 preparation has carried out the titre test in mice, and the result has caused high titre reaction.

Table 12A

Pharmaceutical formulation	Outward appearance
		0.45mg/ml A β 1-42 peptide, 0.2mg/ml QS-21, the 10mM glycine, the 20mM citrate, 5% sucrose, pH are 6.01	Clear and bright
0.45mg/ml A β 1-42 peptide, 0.1mg/mlQS-21, the 10mM glycine, the 20mM citrate, 5% sucrose, pH are 6.0	Clear and bright
		0.225mg/ml A β 1-42 peptide, 0.3mg/ml QS-21, the 10mM glycine, the 20mM citrate, 5% sucrose, pH are 6.0	Clear and bright
0.225mg/ml A β 1-42 peptide, 0.15mg/mlQS-21, the 10mM glycine, the 20mM citrate, 5% sucrose, pH are 6.0	Clear and bright

1 finds to have high titre reaction in mice

2.A the titration of β 42 peptides and QS-21

It is in 9.5 the 10mM glycine solution that A β 42 peptides (2mg/ml) are dissolved in pH.With 1 normal sodium hydroxide pH value is transferred to 9.5.A β 42 peptide solutions pass through the filtration of Millex GV filter more then.QS-21 (5mg/ml) is dissolved in pH and in 6.0 the 10mM sodium citrate solution, passes through the filtration of Millex GV filter again.Etc. A β 42 peptides and the partially mixed ratio that meets the requirements of of QS-21 solution of duplicate samples, mixing is the glycine solution of 9.5 10mM again with pH, and pH is 5.2 1M citrate solution, is diluted to the listed ultimate density of following table.

The initial AN1792/QS-21 of table 12B. is group type preparation altogether

Pharmaceutical formulation is in the glycine of 10mM, the citrate of 20mM, pH6.0 solution	Outward appearance	Absorbance 405nm
			1.0mg/ml A β 42 peptides, 1.0mg/ml QS-21	Clear and bright	??0.018
1.0mg/ml A β 42 peptides, 0.5mg/ml QS-21	Opaque (+)	??0.013
			1.0mg/ml A β 42 peptides, 0.25mg/ml QS-21	Opaque (++)	??0.030
1.0mg/ml A β 42 peptides, 0.125mg/ml QS-21	Opaque (+++)	??0.175
			1.0mg/ml A β 42 peptides, 0.063mg/ml QS-21	Opaque (+++)	??0.308
1.0mg/ml A β 42 peptides, 0.03 1mg/ml QS-21	Opaque (+++)	??0.315
			1.0mg/ml A β 42 peptides, 0.016mg/ml QS-21	Opaque (+++)	??0.268
1.0mg/ml A β 42 peptides, 0.0mg/ml QS-21	Opaque (+++)	??0.213
0.5mg/ml A β 42 peptides, 1.0mg/ml QS-21	Clear and bright	??0.005
			0.5mg/ml A β 42 peptides, 0.5mg/ml QS-21	Clear and bright	??0.006
0.5mg/ml A β 42 peptides, 0.25mg/ml QS-21	Clear and bright	??0.005
			0.5mg/ml A β 42 peptides, 0.125mg/ml QS-21	Opaque (+)	??0.021
0.5mg/ml A β 42 peptides, 0.063mg/ml QS-21	Opaque (++)	??0.148
			0.5mg/ml A β 42 peptides, 0.031mg/ml QS-21	Opaque (+++)	??0.172
0.5mg/ml A β 42 peptides, 0.016mg/ml QS-21	Opaque (+++)	??0.162
			0.5mg/ml A β 42 peptides, 0.0mg/ml QS-21	Do not have	Do not have
			0.3mg/ml A β 42 peptides, 1.0mg/ml QS-21	Clear and bright	??0.004
0.3mg/ml A β 42 peptides, 0.5mg/ml QS-21	Clear and bright	??0.004
			0.3mg/ml A β 42 peptides, 0.25mg/ml QS-21	Clear and bright	??0.006
0.3mg/ml A β 42 peptides, 0.125mg/ml QS-21	Clear and bright	??0.005
			0.3mg/ml A β 42 peptides, 0.063mg/ml QS-21	Opaque (+)	??0.013
0.3mg/ml A β 42 peptides, 0.031mg/ml QS-21	Opaque (+)	??0.077
			0.3mg/ml A β 42 peptides, 0.016mg/ml QS-21	Opaque (+)	??0.068
0.3mg/ml A β 42 peptides, 0.0mg/ml QS-21	Opaque (++)	??0.046
0.1mg/ml A β 42 peptides, 1.0mg/ml QS-21	Clear and bright	??0.004
			0.1mg/ml A β 42 peptides, 0.5mg/ml QS-21	Clear and bright	??0.002
0.1mg/ml A β 42 peptides, 0.25mg/ml QS-21	Clear and bright	??0.002
			0.1mg/ml A β 42 peptides, 0.125mg/ml QS-21	Clear and bright	??0.001
0.1mg/ml A β 42 peptides, 0.063mg/ml QS-21	Clear and bright	??0.002
			0.1mg/ml A β 42 peptides, 0.031mg/ml QS-21	Clear and bright	??0.003
0.1mg/ml A β 42 peptides, 0.016mg/ml QS-21	Clear and bright	??0.004
			0.1mg/ml A β 42 peptides, 0.0mg/ml QS-21	Clear and bright	??0.008

3.A other preparations of β 42 peptides and QS-21

The villaumite of A β 42 peptides with the concentration dissolving of 1mg/ml, is with or without 5% sucrose, 0.1%PS-80 or 0.4%PS-80 in the solution in the solution of the 10mM of pH9.0-9.5 glycinate.Peptide solution is through the filtration sterilization of Millex GV injection filter.Freeze dried QS-21 concentration with 5mg/ml in the 10mM of pH6.0 citrate solution is dissolved, then through the filtration sterilization of Millex GV injection filter.

A β 42 peptides of proper volume with make A β 42 peptides that table 12C lists and each final concentration solution of QS-21 after QS-21 solution mixes.At last, with pH be 5.4 1M citrate buffer the most at last pH transfer to 6, at this moment the concentration of citrate buffer is 20mM.The visual appearance of preparation is estimated to muddy (+extremely +++) with clear and bright.Adjust the concentration of A β 42 peptides and QS-21 and the visual appearance of the whole preparation of adjustable ratio.In addition, sucrose and surfactant can be used as the additional excipient of this preparation.The supplementing preparation of table 12C.A β 42 peptides and QS-21

Pharmaceutical formulation			Outward appearance
							QS-21 (mg/ml)	Aβ42 (mg/ml)	The ratio of A β 42/QS-21	No excipient	5% sucrose	0.1％PS- 80	0.4％PS- 80
??0.05	??0.05	?1.0	Clear and bright	Clear and bright	Clear and bright	Clear and bright
							??0.1	??0.1	?1.0	Clear and bright	Clear and bright	Clear and bright	Clear and bright
??0.2	??0.2	?1.0	Clear and bright	Clear and bright	Clear and bright	Muddy (+)
??0.1	??0.23	??2.3	Muddy (+)	Muddy (+)	Muddy (+)	Muddy (+)
							??0.2	??0.23	??1.1	Clear and bright	Clear and bright	Clear and bright	Muddy (+)
??0.3	??0.23	??0.8	Clear and bright	Clear and bright	Clear and bright	Muddy (+)
??0.1	??0.45	??4.5	Muddy (++)	Muddy (++)	Clear and bright	Muddy (+)
							??0.2	??0.45	??2.3	Muddy (++)	Muddy (++)	Muddy (+)	Muddy (+)
??0.3	??0.45	??1.5	Clear and bright	Clear and bright	Clear and bright	Muddy (+)

The C.D. spectroscopic assay and the explanation of embodiment 10 A β 42 peptide formulations

Circular dichroism method data are drawn by an Aviv 62-DS type optically-active spectrophotometer (New Jersey Lakewood).Suitably the preparation sample and pack into one do not have tension force, optical path length is in 1 millimeter the quartzy cell, carries out the mensuration of near ultraviolet and far-ultraviolet spectrum respectively.Sample carrier is under stable 25 degrees centigrade.With average 4 seconds was that unit is by 0.5nm interval value.In the far-ultraviolet region (wavelength is 180-250nm), the signal of peptide backbone is occupied an leading position in the spectrum, thereby can draw the estimation of secondary structure.What the analysis of near ultraviolet band (wavelength is 1250-350nm) drew then is distinct information: this regional main signal is from aromatic series side chain (phenylalanine, tyrosine and tryptophan).The feature of signal and intensity show that the extensibility of side chain of each position is all relevant with the peptide chain backbone with directivity.Because the number of chromophore lacks than the number of Ammonia group, and these chromophores are dispersed throughout whole molecule, so information of some relevant partial structurtes can be provided.

As the form of above embodiment to a large amount of modifications and variations that the present invention did, same those skilled in the art also can make various versions.Thereby the present invention is only limited by claim.The various variations that do not break away from the present invention's spirit and essence are all in claim scope of the present invention.

Claims (47)

1. one kind contains the compositions that concentration is at least the A β peptide aqueous solution of 0.01mg/ml, and wherein said aqueous solution is kept and made the abundant dissolved pH value of described A β peptide.

2. compositions according to claim 1 wherein is by adding the medicinal buffer of effective dose to keep suitable pH value in described solution.

3. one kind contains the compositions of aqueous solution of degerming that concentration is at least the A β peptide of 0.01mg/ml, and wherein said aqueous solution is kept certain pH value with the described A β of enough dissolvings peptide.

4. compositions according to claim 3 wherein is to keep described pH value by the medicinal buffer that adds effective dose in described solution with it.

5. according to claim 1 or 3 described compositionss, wherein said A β peptide is the longer chain forms of A β peptide.

6. according to claim 1 or 3 described compositionss, wherein said A β peptide is A β 42.

7. according to claim 1 or 3 described compositionss, wherein said pH value scope is approximately 8.5 to 12.

8. compositions according to claim 7, wherein said pH value scope is approximately 9 to 10.

9. according to claim 2 or 4 described compositionss, wherein said medicinal buffer is selected from aminoacid, salt and derivant thereof, pharmaceutically useful basifier, alkali metal hydroxide and ammonium hydroxide, organic or inorganic acid and its esters, and the mixture of the above material.

10. compositions according to claim 9, wherein said medicinal buffer are glycine (Glycine sodium) or arginine (arginine hydrochloride).

11. freeze-dried composition with the A β peptide of following step preparation:

A) freezing a kind of concentration is at least the degerming aqueous solution of the A β peptide of 0.01mg/ml, and wherein said aqueous solution keeps certain pH value so that the dissolving of described A β peptide; And

B) the described frozen composition with above-mentioned a) step preparation carries out lyophilizing.

12. compositions according to claim 11, wherein said A β peptide is the longer chain forms of A β peptide.

13. compositions according to claim 11, wherein said A β peptide is A β 42.

14. compositions according to claim 11 wherein adds the medicinal buffer of effective dose, to keep described pH value in described solution.

15. compositions according to claim 14, wherein said medicinal buffer is selected from aminoacid, salt and derivant thereof; Medicinal basifier, alkali metal hydroxide, and ammonium hydroxide, organic or inorganic acid and salt thereof; And the mixture of the above material.

16. according to claim 1,3 or 11 described compositionss, wherein said A β peptide is in the random coil state basically.

17. according to claim 1,3 or 11 described compositionss, the concentration of wherein said A β peptide is approximately 0.05mg/ml to 2.0mg/ml.

18., further contain medicinal adjuvant in the wherein said compositions according to claim 1,3 or 11 described compositionss.

19. compositions according to claim 18, wherein said adjuvant are selected from the non-complete adjuvant of Fu Shi, MPL, QS-21 and Alumen.

20. one kind comprises pH value and is approximately 5 to 7, concentration is at least the compositions of degerming suspension of the A β peptide of 0.1mg/ml.

21. compositions according to claim 20 also contains the medicinal buffer of effective dose in the suspension of wherein said peptide.

22. according to claim 20 or 21 described compositionss, wherein said A β peptide is the longer chain forms of A β peptide.

23. according to claim 20 or 21 described compositionss, wherein said A β peptide is A β 42.

24. compositions according to claim 21, wherein said medicinal buffer is selected from aminoacid, salt and derivant thereof; Medicinal basifier, alkali metal hydroxide, and ammonium hydroxide, organic or inorganic acid and salt thereof; And the mixture of the above material.

25. compositions according to claim 20 contains concentration and is approximately 0.1 to 0.8mg/ml A β peptide, glycine and a kind of acid that enough pH value is transferred to about 5.5-6.5 of 10mM.

26. according to claim 24 or 25 described compositionss, wherein said compositions further comprises one or more excipient, described excipient is selected from tension regulator, surfactant and wetting agent.

27. compositions according to claim 24, wherein said compositions further comprises medicinal adjuvant.

28. compositions according to claim 26, wherein said compositions further comprises medicinal adjuvant.

29. compositions according to claim 28, wherein said adjuvant are selected from the non-complete adjuvant of Fu Shi, MPL, QS-21 and Alumen.

30. compositions according to claim 28 contains concentration and is approximately 0.1 to the A β peptide of 1.0mg/ml, is dissolved in pH value and is approximately the glycine of 6 10mM and the QS-21 that concentration is at least 0.1mg/ml, its amount can effectively form the clear and bright suspension of vision.

31. a degerming method for compositions that is used to prepare the longer chain forms of A β peptide comprises:

Adjust pH value of aqueous solution so that the dissolving of wherein said A β peptide;

In aqueous solution, add the A β peptide that can cause immunoreactive q.s in the mammalian body; And

The filter membrane of gained solution through an even filter opening filtered, described pore size should the filtering antibacterial again can be by q.s the described A β peptide of all kinds basically.

32. method according to claim 31, wherein said filtration adopt a kind of homogeneous aperture to be approximately 0.22 micron hydrophilic polymer film.

33. method according to claim 31, the yield that wherein filters back A β peptide surpasses 50%.

34. contain at least a diluent in the solution before the method according to claim 31, wherein said filter, be selected from concentration and be approximately the medicinal buffer of 5mM to 45mM.

35. contain the tension regulator of a kind of 0.9%-6.0% of being approximately (w/v) before the method according to claim 34, wherein said filter in the solution.

36. contain the surfactant of a kind of about 0.02%-1.0% (w/v) before the method according to claim 34, wherein said filter in the solution.

Be approximately the chelating agen of 0.1mM 37. contain a kind of concentration in the solution before the method according to claim 34, wherein said filter to 1.0mM.

38. according to claim 34,35,36 or 37 described methods, the pH value of the degerming solution after wherein will filtering is adjusted to about 5-7 to form the peptide suspension.

39. method that is used in mammalian body, preventing or treating Alzheimer, comprise the degerming aqueous solution composition that concentration is at least the A β peptide of 0.05mg/ml that contains that described mammal is given effective dose, to bring out immunoreation in described animal body, wherein said aqueous solution keeps certain pH value so that the dissolving of described A β peptide capacity.

40. one kind excites the method to the antibody response of A β peptide, comprising in needing the mammalian body of medication:

Non-ly remove bacteria composition through the longer chain forms A of enteral administration immunizing dose β peptide.

41. according to claim 39 or 40 described methods, wherein said method further is included in the described independent or mixing adding medicinal adjuvant in the bacteria composition that removes.

42. according to claim 39 or 40 described methods, the wherein said bacteria composition that removes is a compositions according to claim 30.

43. a compositions includes concentration and is at least the A β peptide of 0.1mg/ml and the QS-21 of certain effective dose, with form vision clear and bright, the pH value scope is 5 to 7 suspension.

44. a compositions includes concentration and is at least the A β peptide of 0.1mg/ml and the DPPC of certain effective dose, the pH value scope is 5 to 7, to form the clear and bright suspension of vision.

45. the bacteria composition that removes that contains longer chain forms A β peptide brings out the application in the medicine of the antibody response of A β peptide in preparation.

46. the application of degerming waterborne compositions in the medicine of preparation prevention or treatment Alzheimer that contains A β peptide.

47., further comprise medicinal adjuvant in the wherein said medicament according to claim 45 or 46 described application.

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