HRP20010901A2 - Compositions of a-beta peptide and processes for producing same - Google Patents

Description

PREVIOUS KNOWLEDGE

Field of invention

This invention relates generally to pharmaceutical compositions containing proteins useful for enhancing antibody responses in mammals. More specifically, the present invention relates to pharmaceutically acceptable compositions comprising an amount of amyloid beta peptide effective to elicit an immunogenic response in a mammal, and a pharmaceutically acceptable diluent. Preferably, the diluent is a sterile parenterally acceptable aqueous phase.

Previous knowledge

Amyloid beta peptide, also known as A-beta or Aβ peptide, is a cleavage product of the amyloid precursor protein (APP). It is the basic component of the amyloid plaque in the brain of mammals, which is the basic characteristic of Alzheimer's disease. Aβ peptide is a 39-43 amino acid chain, the length of which is variable due to the different process of obtaining it from APP, and from several proteases.

Several mutations within the APP protein have been correlated with the existence of Alzheimer's disease. See eg Goate et al. Nature, 349, 704 (1991) (valine717 to isoeucine); Chartier Haran et al. Nature 353, 844 (1991) (valine717 to glycine); Murrell et al. Science 254, 97 (1991) (valine717 to phenylalanine); Mullan et al. Nature Genet. I, 345 (1992) (double mutation changing lysine595-methionine596 to asparagine595-leucine596). It has been shown that such mutations cause Alzheimer's disease, and by increased or changed processing of APP into Aβ peptide, especially by processing APP into an increased amount of Aβ42 and Aβ43. Mutations in other genes, such as the presenilin genes, PSI and PS2, are thought to act indirectly to process APP and generate increased amounts of Aβ42 and Aβ43 (see Hardy, TINS 20, 154 (1997)). The observation shows that Aβ peptide, especially Aβ42, is the causative element of Alzheimer's disease. In the brain, aggregation of Aβ peptides occurs and amyloid deposits are formed, which contain peptides organized into the structure of fibrils or β-pleated plates.

Efforts in recent research into the therapeutic treatment or prevention of Alzheimer's disease are focused on stopping or slowing down the production of Aβ peptides in the brain, by blocking processing or deposition into amyloidin plaques after personalization. One therapeutic approach of particular importance is the application of the present invention to the use of Aβ peptides to induce the body's immune response against it. See, for example, PCT publication no. WO99/27944, which is incorporated herein by reference in its entirety for all purposes.

This invention is directed to new unexpected methods for using the invention described in PCT publication WO99/27944. More particularly, it involves administering some formulation of the long form of Aβ peptide to a patient to induce an immune response. However, as indicated in the art, the longer forms of the Aβ peptide are poorly soluble in conventional formulation systems.

Hilbich et al. J. Mol. Biol., 218 (1), p. 149-64 (1991) have shown that the Aβ-43 peptide is soluble to some degree in pure water, the addition of ionic components such as buffers or salts, or organic solvents cause the peptide to precipitate in solution in the form of amorphous aggregates. For example, Hibich found that physiological phosphate buffer solution ("PBS" here containing 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 · 2H2O and 2 mM KH2PO4 at pH 7.5) made 90-94% of peptides insoluble in the preparation. PBS is a common carrier of parenteral preparations, approximating tonicity and pH levels to the living system. Five (5) mM NaCl causes 42-50% of the peptide to precipitate, (ibid, p. 153, Table 2). A peptide solution in pure water would be hypotonic, pH 5.5 of such a solution was determined by Hibich (idem.). Typically, the pH of human blood is 7.4. Dyrks et al. have also shown that Aβ42 is insoluble under physiological conditions. Dyrks, T., Weidemann, A., Multhaup, G., et al. EMBO J.. 7, p. 949-57 (1988).

The conformation of the Aβ peptide in solution can be measured using circular dichroism (C.D.) spectroscopy. Conformational studies of Aβ peptides and fragments using C. D. are presented by Hilbich et al. I am going to. See also the monograph by M. Manning entitled: Protein Structure and Stability Assessment by Circular Dichroism Spectroscopy, by Bioctalyst design for Stability and Specificity; Himmel M. E. and Georiou, G., editors, ACS Symposium Series 516 (1993) at p. 36. That reference hereinafter refers to the Manning reference, which is incorporated herein by quotation in its entirety for all purposes.

Kline et al., U.S. Patent No. 5,851,996 ('996 patent) and 5,753,624 ('624 patent) describe the administration of a very small amount (10-2 mg or less) of Aβ peptide or fragment thereof given sublinally in a liquid or solid carrier, such as phenylated saline. The '996 patent claims that amyloid beta protein "exists in various structural forms" (column 2, paper 31) which can be compared for the treatment of Alzheimer's disease. Elsewhere, what was meant by the various structural forms was not defined, nor was any characterized other than the 28 amino acid fragment used in the examples. The '996 patent claims doses of Aβ peptide from 10-10 to 10-12 mg (column 8, lines 442-32).

From the above point of view, previous findings have shown the difficulty in dissolving Aβ peptides and keeping them dissolved. Furthermore, the insolubility of long forms of Aβ peptides presents difficulties from the point of view of sterilization and standardization. Most standard sterilization methods are incompatible with peptide formulations, including radiation, sterilization by autoclave or chemical techniques, such as ethylene oxide gas or glutaraldehyde, all of which cause peptide degradation. Therefore, peptide filtration would be the method of choice for sterilization of Aβ peptide formulation. Unfortunately, the insolubility of Aβ peptides causes clogging of filtration membranes and prevents the isolation of a sufficient amount of Aβ peptides in a commercial-scale process.

Summary of the invention

This invention is directed to the anticipated discovery that aqueous solutions containing a high concentration of Aβ peptide can be prepared by adjusting the acidity/basicity of such aqueous solutions to a pH effective to dissolve the Aβ peptide. Preferably the pH is adjusted to a pH range of from about 8.5 to about 12, more preferably from about 9 to about 10.

This invention is further directed to the discovery that dissolved Aβ peptide in solution can be sterile filtered through suitable micropore filters, with isolation of at least 50% of the Aβ peptide after sterile filtration. Preferably, 70% of the Aβ peptide is isolated after sterile filtration, and even more preferably, 90% of the Aβ peptide is isolated after sterile filtration. Such sterile solutions can be formulated as pharmaceutical compositions containing a sufficient amount of A p peptide to elicit an immunogenic response when administered to a mammal. Preferably, such administration is parenteral administration, in the form of a suspension of the preparation.

Therefore, in one aspect of the composition of the invention, after sterile filtration, the pH of the composition is adjusted to a physiologically acceptable pH to create a peptide suspension containing at least 0.1 mg/mL of Aβ peptide. The preparation is useful for parenteral administration. The pH of the preparation suspension is between pH 5 and about pH 7, preferably between pH 5.5 and 6.5. Most preferably, the composition contains a sufficient amount of QS-21 together with the Aβ peptide to form a visually clear, sterile suspension.

This invention is further directed to the discovery that thawed and sterile solutions of Aβ peptide can be lyophilized to obtain lyophilized formulations containing Aβ peptide. These preparations can be prepared at appropriate times to obtain an aqueous preparation containing the A peptide.

In another aspect of the preparation, the invention is directed to aqueous solutions containing at least 0.01 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide. Preferably, the solution is maintained at such a suitable pH using an effective amount of a pharmaceutically acceptable buffer.

In another aspect of the preparation, the invention is directed to sterile aqueous solutions containing at least 0.1 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide. Preferably, the solution is maintained at such a suitable pH using an effective amount of a pharmaceutically acceptable buffer.

In another aspect of the preparation, the invention is directed to lyophilized preparations containing a lyophilized preparation with Aβ peptide, and the preparation is prepared by the following procedure:

a) freezing a sterile aqueous solution containing at least 0.01 mg/mL Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve the Aβ peptide, and

b) lyophilization of the frozen preparation prepared above in a).

Preferably, the composition of the invention contains the long form (defined below) of the Aβ peptide. More preferably, the preparation contains a pharmaceutically acceptable buffer which is preferably selected from the group consisting of: amino acids, salts or derivatives thereof; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides and ammonium hydroxides, organic and inorganic acids and their salts; and their mixtures.

In another aspect of the preparation, the invention is directed to a preparation containing an aqueous solution containing at least 0.01 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide, and further wherein Aβ peptide is mainly in a random coil conformation.

The compositions of the invention can be formulated into pharmaceutical compositions suitable for administration to a mammal having Alzheimer's disease or at risk of developing Alzheimer's disease. Aspects of the preparation of the invention are directed to pharmaceutical preparations that are in the conformation of a random ball of stable Aβ peptide, and in a stable aqueous suspension of at least 0.1 mg/mL of Aβ peptide suspended in said preparation, or a lyophilized preparation, and any of them can be sterile and given parenterally .

In one of the aspects of the process, this invention is directed to the process of preparing sterile preparations of long-form Aβ peptides containing:

adjusting the pH of the aqueous solution so that the Aβ peptide dissolves in it, dissolving in the solution such an amount of Aβ peptide as is sufficient to achieve an immunogenic concentration for mammals,

filtering the resulting solution through a membrane of the same pore size, and the size of said pores is in the range that allows the exclusion of bacteria, and the passage of almost the entire amount of Aβ peptide through the membrane, and

for solutions containing 0.1 mg/mL or more of Aβ peptide, it is possible to adjust the pH of the resulting solution between about pH 5 and about pH 7, to obtain a peptide suspension.

In another aspect of the method, the invention is directed to a method of preventing or treating Azheimer's disease in a mammal, and the method comprises administering to said mammal a sufficient amount of a sterile aqueous preparation containing at least 0.05 mg/mL Aβ peptide, to induce an immunogenic response of said mammal.

Most preferably, the filtration process of the present invention utilizes an Aβ peptide that is predominantly in a random coil conformation.

Short description of the pictures

Figure 1 is C. D. spekar showing the mean value of the residual ellipticity measurement plotted as a function of wavelength for two different Aβ42 solutions. The dashed line shows that the absorbance at pH 6 is attributed to a β-pleated sheet from the molecule. The solid line is the absorbance curve of the Aβ42 solution at pH 9 and shows that the peptide is in a random coil conformation.

Figure 2 is a curve of Aβ42 in solution versus the calculated signal area for the amount of dissolved peptide, as determined by reverse phase high pressure liquid chromatography, showing the solubility of Aβ42.

Detailed description of the invention

This invention is directed to compositions and methods using aqueous compositions containing a therapeutically effective concentration of Aβ peptide. However, before discussing the invention in further detail, the following terms will be defined.

Definitions

The term "sufficient identity" means that two peptide sequences, when optimally compared, such as by the Aβ or BEDFIT programs using a given mass gap, share at least 65 percent sequence identity, preferably at least 80-90 percent sequence identity, more preferably at least 95 percent sequence identity or higher (eg, 99 percent sequence identity or higher). Preferably, non-identical residue positions differ by conservative amino acid substitution.

For sequence comparison, typically one sequence acts as a reference sequence, and the tested sequence, specifically, the 42 amino acid sequence shown below, is compared to it. Other suitable forms would be truncated forms, such as Aβ39 or the extended form, Aβ43 (with an additional threonine group at the C-terminal end). When the sequence comparison algorithm is used, the tested and reference sequences are input data to the computer, the coordinates of the subsequences are indicated if necessary, and the program parameters for the sequence algorithm are indicated. The sequence comparison algorithm program then calculates the sequence identity percentage for the tested sequence(s), relative to the reference sequence, based on the specified program parameters.

Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needeman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity test method of Pearson & Lipman, Proc. Nat'll. Acad. Sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection (see generally Ausubel et al supra ). One example of an algorithm that has been added to determine percent sequence identity is the BLAST algorithm described by Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (http://www.ncbi.nm.nih.gov/). Default program parameters can typically be used to perform a sequence comparison, although parameter tuning can also be used. For the amino acid sequence, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and a BOSUM62 matrix as default parameters (see Henikoff&Henikoff, Proc. Natl, Acad. Sci. USA 89, 10915(1989) ).

For purposes of classifying amino acid substitution as conservative or non-conservative, amino acids are grouped into the following groups: Group I (hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group I (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): ly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitution includes amino acids from the same group. A non-conserved substitution replaces a member of one group with a member of another group.

APP695, APP751, and APP770 refer to amino acid residues 695, 751, and 770 of long polypeptides encoded by human APP genes. See Kan et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525 (1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids within the human amyloid precursor protein (APP) are assigned numbers according to the sequence of the APP770 isoform.

In this invention and in the literature, the mass of Aβ peptide represents about 70% to about 85% Aβ peptide and about 15% to about 30% salt and water. This is determined by amino acid analysis and/or elemental nitrogen analysis. For example, when 0.1 mg of Aβ42 peptide is corrected for peptide content, it represents 0.075 mg of Aβ42 peptide and 0.25 mg of water and salt; 0.6 mg of Aβ40 peptide represents 0.45 mg of Aβ40 peptide and 0.15 mg of water and salt; 2.0 mg of Aβ42 peptide represents 1.5 mg of Aβ42 peptide and 0.5 mg of water and salt.

Aβ peptide, as used in the present invention, refers to a segment of the Aβ peptide that can form a β-pleated sheet conformation and enhance an immunogenic response when administered to a mammal alone or together with an adjuvant. Determination of β-pleated sheet conformation by, for example, measurements of circular of dichroism is within the skill of the profession. Immunogenicity can be determined as described in the Biological Activity section of the Examples below.

The term "long forms of Aβ" peptides include naturally occurring forms of Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43 and peptide sequences sufficiently identical to these, and preferably in human form. Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43 refers to a peptide containing amino acid residues 1-39, 1-40, 1-41, 1-42 and 1-43, amino acids truncated at the C-terminal end of the peptide. Therefore, Aβ41, Aβ40, and Aβ39 differ from Aβ42 by omitting Ala, Ala-Ile, and Ala-Ile-Val from the C-terminal end, as can be seen from the Aβ peptide sequence below. Aβ43 is distinguished by the presence of a threonine residue at the C-terminal end. The sequences of these peptides and their relationship to APP is illustrated in Figure 1 of Hardy et al., TINS 20, 155 (1997).

Aβ42 has the sequence: H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-ln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val -Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-ly-Leu-Met-Val-Gy-Gly-Val-Val-Ile-Ala-OH.

The term "long forms of Aβ" peptides includes their analogs. Analogues include allergic species and induced variants. Analogues typically differ from natural peptides at one or several positions, often due to conservative substitution. Analogues typically have at least 89% sequence identity to native peptides. Some analogs also include synthetic amino acids or modifications of the N or C terminal amino acids. Examples of synthetic amino acids are α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetiizine, O-phosphoserine, N- acetylserine, N-formylmethylnine, 3-methyl-histidine, 5-hydroxyisine, ω-N-methylarginine.

Aβ can be used in the compositions and methods of the invention from about 0.05 mg/mL to an upper solubility limit of about 2.0 mg/mL (refer to Figure 2). The preferred range of the peptide is from about 0.1 to about 0.8 mg/mL, and the most preferred range is from about 0.3 to about 0.6 mg/mL.

It has been shown that the insoluble form of the Aβ peptide is found in the amyloid plaque in a P-pleated sheet conformation, Soto, C., et al. Neuroscience Letters, 186 (2-3), pp. 115-118 (1995). Also Simmons, L.K. et al. Molec, PharmacoL, 45 (3) p. 373-379 (1994) showed that the β-pleated sheet conformation is associated with the neurotoxic effects of the peptide, while the random coil form is only weakly toxic or inactive. As indicated above, the methods and compositions of the invention can utilize the random coil conformation of the Aβ peptide. Applicants herein demonstrate that the random coil conformation is capable of enhancing the immunogenic response in tested mammals. The random coil conformation is the most preferred in the microfiltration process.

The term "random coil" refers to the open chain conformation of the Aβ peptide. A random coil is a secondary conformation of the peptide backbone and is not ordered into a regular conformation such as the α-coil or β-pleated sheet forms. The regularity of the random coil structure is disturbed due to hydrophobic structuring and hydrogen bond interactions, which are characterized by other more irregularly ordered forms. A random coil still has some form of peptide skeleton or partial ordering, however, such characteristics are random and dynamic, and thus not typical of all random coil populations. In the random coil conformation, peptides have good solubility and filterability. α-Helix and β-pleated sheet are well-known peptide conformations. They are described, for example, in Lehniner, Biochemistry (2nd edition, Worth Publishers, 1975) p. 128-9, for α-coil and β-corrugated plate, on pp. 133-4, are incorporated here by quotation.

The random coil conformation of the Aβ peptide is characterized by the corresponding circular dichroism ("CD") and is easily distinguished from the β-pleated sheet conformation. As represented by the CD spectrum, a random coil is characterized by a strong negative band between 190 and 200 nm, and a small CD signal observed at wavelengths greater than 215 nm. This is shown in Figure 1 in the Drawings. In contrast, the β-pleated sheet conformation shows a strong positive signal centered around 200 nm. For a review of the secondary structure determined by CD spectra, reference should be made to Mannin's reference cited above.

The Aβ peptide is sufficiently in the random coil conformation when more than 50% of the Aβ peptide is in the random coil conformation. More than 70% or 80%, and most preferably more than 85% or 90% of the Aβ peptide is in a random coil conformation is preferred.

"Parenteral preparations" are those preparations that are sterilized and suitable for administration directly into the body, for example by injection or infusion, i.e. by the routes by which they immediately come into contact with the blood, without barrier or protection by the immune system, and by giving forms that enter the body through skin, mucus, through the gut or respiratory system. For these reasons, sterility of the parenteral preparation is required.

"Infusion" refers to the administration of a drug, and includes a sufficiently continuous slow flow of the drug solution and blood flow over a relatively long period of time. An "injection," in contrast, is the rapid administration of a unit dose of a solution or suspension.

Intravascular (or intravenous IV), intramuscular (IM), intraperitoneal (IP), subcutaneous (SC), and intrasternal refer to routes of administration of parenteral preparations. They describe in anatomical terms the parts of the body where the injection or infusion from the invention should be introduced. It is contemplated that compositions of the present invention having a physiologically acceptable pH may be administered by any of the above routes, depending on the individual patient and the judgment of the physician. Solutions with a higher pH (pH>8) are best administered by slow IV infusion or IV drip.

It is understood that with the terms "buffer" and "buffering agent" used herein, it should be remembered that the titration curve of an acid or base has a relatively flat zone extending about 1.0 pH units on either side of the titration inflection point. At the inflection point, an equivalent amount of proton-donor and proton-acceptor particles of the acid or base is present. In this zone, the pH of the system changes relatively little when a little H* or OH is added". This is the zone where the conjugate acid-base pair acts as a buffer, a system that resists a change in pH when a little H+ or OH- is added. At at a pH level outside this zone, the capacity of the buffer to resist the change in pH is lower. The power of the buffer is maximal when the pH is exactly equal to the pH at the inflection point of the titration curve, i.e. when the concentration of proton acceptor and proton donor and pH is equal to pK+ (acid dissociation constant ).Buffer preparations are described in detail in Data for Biochemical Research, Rex, M.C., Oxford Science, Publications, 1995.

Many physiological mechanisms work within the body to maintain blood pH within a narrow range of 7.35 to 7.45. While some major buffering mechanisms are based on carboxylic acid or phosphoric acid balance, many other mechanisms involve amino acids and proteins. For example, the pH of tears is maintained at 7.4 with a protein buffer. Certain amino acids are also useful buffers, although they show more complex titration curves due to the fact that they have atoms that are proton donors and proton acceptors within the same molecule. Molecules of this type are called dipolar ions, i.e. they exist in a form that has a positive or negative charge within the same molecule. Amino acids are capable of being buffered by the addition of H+ and OH- ions, as shown below.

[image]

For the purposes of this invention, a "pharmaceutically acceptable buffer" is broadly defined and includes conjugated acid-base pairs as well as acidic or basic compounds that have the ability to adjust or maintain the H solution of the Aβ peptide at a desired level. The dissolution/filtration processes of this invention are carried out at pH 8.5 or higher or in another aspect below pH 4.

Such powders are preferably selected from the group consisting of: amino acids, salts or their derivatives, pharmaceutically acceptable alkalizing substances, alkali metal hydroxides, organic and inorganic salts and their salts, and their mixtures. Buffers are used in concentrations sufficient to reach and maintain the desired pH and therefore the concentrations depend on the acidity/basicity of the individual buffer or on the combination of the selected ones. The selection of an effective concentration is within the skill of the profession, and for example a pH meter and titration techniques are used.

Exemplary classes of compounds useful in this invention are hydroxides, including alkali metal hydroxides, basifying agents known in the pharmaceutical art including, but not limited to, tris, borate (Na2B4O7) and disodium citrate, amino acids, amino acid salts or esters or amides, and their simple derivatives, for example N-acetyl derivatives of amino acids. Particularly preferred buffers are glycine (eg sodium glycinate), arginine and lysine, sodium hydroxide and ammonium hydroxide.

Examples of pharmaceutically acceptable salts for practicing the methods of this invention are, without limitation, hydrochloric acid, phosphoric acid, citric acid, malic acid and succinic acid and the like. In addition, these acids can be used to titrate the base solution to a lower, more pharmaceutically acceptable pH level, resulting in a suspension of the preparation.

In contrast, base compounds such as those enumerated above can be used to titrate a low pH filtered solution to a more pharmaceutically acceptable pH, thereby also forming a peptide suspension.

Combinations of pH adjusting agents were also considered. Conjugate base and acid pairs exemplified by salts such as ammonium acetate are also within the scope of the buffering agents of this invention. Such a conjugated pair can be formed, for example, by trituration of a basic solution of ammonium hydroxide with acetic acid to a nearly neutral solution of ammonium acetate.

As used herein "tonicity modifier" includes agents that contribute to the osmotic pressure of a solution. Examples of tonicity modifiers useful in the present invention include, but are not limited to, saccharides (sugars) such as mannitol, sucrose, and glucose, and salts such as sodium chloride, potassium chloride, and the like. Preferably, the tonicity modifier will be used in such an amount as to give a final osmotic value of less than about 350 mOs/kg, more preferably between about 250 to about 350 mOs/kg. It should be noted that charged compounds that serve as formulation buffers can also affect tonicity. Therefore, the tonicity of the Aβ peptide buffer solutions was first determined before further adjustment of the tonicity modifying agent.

A chelating agent can be used in the methods and compositions of the invention. Examples of preferred chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts (such as sodium) which are normally used at concentrations of 0.05 to 50 mM, more preferably at concentrations of 0.05 to 10 mM or from about 0.1 to 5 mM, as most preferred. Other known chelating agents can be used, such as polyvinyl alcohol.

The preparations may contain surfactants or detergents such as polysorbate (eg Tween®) or 4-(1,1,4,4-tetramethylbutyl)phenyloxypolyethoxyethanol (Triton®) or polyethylenepropylpropylene glycol polymers (Pluronics®). Surfactants range from about 0.005 to 1%, and about 0.02 to 0.75% of the agent is preferred. A preferred polysorbate is PS-80 which is commercially available as Tween® 80.

Gliding agents are also contemplated as excipients useful in the invention. Polyethylene glycols, eg PE3350, are useful for modifying the association of Aβ particles and therefore solubility is related to the polymer surface and the orientation of hydrophilic residues towards the aqueous phase. Lubricants can be present from 0.5 to 5% (w/v).

Pharmaceutically acceptable sugars (for example sucrose, dextrose, maltose or lactose) or pharmaceutically acceptable sugar alcohols (for example mannitol, xylitol or sorbitol) have no influence on the medicinal effect of the active substance. In one aspect, sugars or sugar alcohols that have a molecular weight of less than 500 and are capable of easily dispersing and dissolving in water can be used. Examples of sugars and sugar alcohols that can be used in this invention are: xylitol, mannitol, sorbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose, lactose and the like. They can be used alone or as a mixture of two or more compounds. The most preferred sugar is mannitol, especially in the lyophilized preparation, and sucrose in the solution of the preparation is also preferred.

The use of QS-21 as an adjuvant in the repairs of the invention was also found to interact with the suspended protein to form a visually clear suspension. This interaction is desirable because the peptide is in a β-pleated sheet conformation, but is suspended in a phase with very small particles and can lead to favorable properties of the preparation by increasing the stability of the suspension or improving the immunogenic properties. DPPC (dipalmitoyl phosphoatiphil choline) is known in the art to be expected to interact with the Aβ peptide in a similar manner to QS-21 to produce a suspension of small particles, and the use of other adjuvants of the invention is permitted, resulting in a clear suspension. . Alternatively, other adjuvants can be used in admixture with QS-21.

Lyophilization is a technique well known in the pharmaceutical art, as are techniques for stabilizing peptides during the lyophilization process. Stabilization of protein or peptide lyophilizate in amino acids and saccharide matrix is known to experts. See, for example: Lueckel B., et al., Formulations of sugars with amino acids or mannitol - Influence of concentration ratio on the properties of the freeze-concentrate and the lyophilizate, PHARM. DEV. TECHNOLOGY. 3(3) p. 325-336 (1998). The Royal Pharmaceutical Society in GB Symp: fNew Anaytical Approaches to the Characterization of Biotechnology Products' June 19%, presented by Luckel B., et al: A strategyfor optimizingthelyophilizationofbiotechnoloicalproduts, PHARM. SCI. (UK) 3(1) p. 3-8 (1997). Both of these references are reproduced here in full for all purposes.

The term "pharmaceutically acceptable" modifies any preparation to indicate that it has no harmful or adverse effect on the person to whom it is administered and under the conditions under which it is administered. The term "pharmaceutically acceptable diluent" refers to a compound that preserves or does not alter the activity of the active compound(s) and has no harmful or adverse effect on the subject under the conditions in which it is administered, such as non-toxic pH adjusting agents or buffers or agents to modify tonicity or chelators and the like.

Preparations or processes that contain "one or more of the listed elements may include other elements that are not specifically listed. For example, a preparation containing Aβ peptide includes Aβ peptide in the preparation as specified and Aβ peptide as a component of the preparation that has unspecified components.

Further definitions are as follows

Abbreviation Definition

°C degree of Cesius

cc cubic centimeter

C. D. circular dichroism

pH log[H], a measure of the hydrogen content and therefore the acidity or basicity of a solution

p K' acid dissociation constant

refers to pH according to pH = pK' + [H + acceptor]

[H + donor]

PS80 polysorbate 80 or Tween 80® copolymer of polysorbate and ethylene oxide;

Merck Idex monograph no. 7559 (11th ed.)

μm micron

min minutes

mL milliliter

N normality - an indicator of the molarity of the solution

M molarity, value expressed in mol/liter

mM millimole

DMSO dimethylsulfoxide

EDTA ethylenediamine tetraacetate, usually the disodium salt

Tris trimethamine, tris(hydroxymethyl)aminomethane; Merck Idex monograph no. 9684 (11th ed.)

RP HPLC high precision reverse phase liquid chromatography

RPM revolutions per minute

CFA/IFA complete Freund's adjuvant/incomplete Freund's adjuvant

(Chang et al. Advanced Drug Delivery Reviews 32,173-186 (1998)

MPL 3-O-deacylated monophosphoroyl lipid A (MPL™) (see for example B 2220211).

QS 21 triterpene glycoside isolated from the bark of Quillaja Saponaria Molina trees from South America (see Kensil et al. in Vaccine Design: The Subunit and Adjuvant Approach (ed. Powell & Newman, Plenum Press, NY, 1995); US Pat. No. 5,057,540 ), (Stimulon™QS-21)

FIO for information only

Filtration is the process of removing contaminants by excluding particles from the liquid by size, and passing them through a membrane filter that has the same pore size. Although micron-sized particles can be removed without a membrane, and by using materials found in a fibril medium, only membrane filters that have precisely defined pore sizes can ensure the quantitative removal of particles that have a smaller size. Therefore, with membrane filtration, it is possible to quantitatively remove bacteria from the solution, and when it passes through microfilters, which results in a sterilization effect. By previous purification, the Aβ peptide was so insoluble or remained in aggregates to the extent that the solution could not be microfiltered on a commercial scale due to clogging of the membrane pores and/or poor isolation of the peptide in the microfilter solution.

Preferred filters are generally by definition those that have the ability to remove particles over 0.2 microns.

Membrane filters are uniformly prepared according to the substrate, and generally can separate based on the mass on the membrane surface. However, membrane filters of the surface type receive the particle in the depth of the structure and on the surface of the membrane. In mass exclusion separation, particles smaller than the pores of the membrane filter pass, while larger particles are retained on the membrane surface. Since these defined filter pore sizes do not "saturate" (ie, when the filter begins to fill with retained particles, the increase in flow pressure allows the passage of small amounts of retained solids), they are the devices of choice for microbiological control. The "degree of sterilization" of surface-type membrane filters is well known in the pharmaceutical industry.

In all filtration applications, the permeability of the filter media can be changed by chemicals, molecular or electrostatic properties of the filtrate, however, the hydrophilic microfilters used in this invention were found to be stable at high or low pH environments, depending on the method used and the reliability of removing unwanted particles substances without clogging. Usually the skilled person will be able to select and use hydrophilic microfilters. Commercially available product specifications or websites such as http://millispider.miipore.com/corporate/ditemap.nsf/catalogs (May 1999) allow selection of filters that have the desired characteristics.

Preferred examples of hydrophilic filters used in this invention are Millipore Durapore®, also called Millex V, (Millipore Corporation, headquartered in Bedford, MA), a hydrophilic polyvinylidene fluoride polymer is stable and has weak protein binding characteristics, and a pore diameter of 0.2 μm ; and Milex GP™, a modified polyethersulfone polymer with a hydrophilic surface, with a pore size of 0.22 μm. Durapore is preferred because of its stability at pH 9-9.5.

It is preferred that almost all of the Aβ peptide is isolated after filtration. Almost all isolated Aβ peptides are defined so that the average isolated protein after sterile filtration is greater than about 50% of the peptides. More than 80% of the Aβ peptide isolated after sterile filtration is preferred, and most preferred more than 90% of the Aβ peptide is isolated after sterile filtration.

Methods

The methods in this invention include the preparation of aqueous preparations with a concentration of at least 0.01 mg/mL of Aβ peptide. Such preparations are prepared by adjusting the pH of the aqueous solution so that the Aβ peptide dissolves at the desired concentration (eg, at a concentration of about 0.1 mg/mL to about 2.0 mg/mL).

Adjusting the pH of an aqueous solution is achieved by common methods, a typical example of which is the addition of acid or base to achieve the desired pH. A pharmaceutically acceptable acid and base are preferably used. In order to maintain the pH of the solution during long-term storage, it is preferred to use pharmaceutically acceptable buffers in the preparation. The selection of suitable buffers depends on the desired pH and is within the skill of the art.

Preferably, the pH of the aqueous preparation is adjusted between about pH 2 to 4 or about pH 8.5 to 12. At such a pH, the Aβ peptide surprisingly becomes well soluble.

When the process of the invention is carried out at a low pH (around pH 2 to 4), acids such as hydrohalic acids (e.g. HCl, HBr), phosphoric acid, citric acid and acetic acid and others can be used to lower the pH of the solution to the desired pH pharmaceutically acceptable acids. Selection of suitable acids is within the skill of the art.

When the process of the invention is carried out at a high pH (about pH 8.5 to 12), bases such as alkali metals, ammonium hydroxides (eg NaOH, NH4OH) and the like can be used to increase the pH of the solution to the desired pH. At high pH, the pH of the solution in the given buffer is preferably adjusted between 8.5 and 11. A more preferred pH level is between pH 9 and 10.

Before or immediately after pH adjustment, Aβ peptide at the required concentration was added to the solution. It is preferred, of course, to add the Aβ peptide after adjusting the pH so that it dissolves immediately. After addition, gentle stirring and heating may be necessary to aid the dissolution process.

Addition to the composition of any additives, such as pharmaceutically acceptable buffers, tonicity adjusting agents, adjuvants, etc., can occur at any convenient time before or after the addition of the Aβ peptide.

When the aqueous solution described above is prepared, sterile filtration and/or lyophilization can be performed according to procedures well known in the art, examples of which are given below.

The following buffer solutions were prepared for dissolving Aβ peptide in the desired concentration ranging from 0.6 to 2.0 mg/mL peptide:

Preferred Amino Acid Preparations

10 mM sodium glycinate, pH 9.0, 9.5 or 10.0

and/or with 0.02 to 1.0% (w/v) polysorbate 80 (PS-80)

may contain one or more tonicity modifiers suitable for parenteral administration

10 mM L-arginine-HCl and 10 mM L-lysinate, both at pH 9.0, 10.0

and/or with 0.02 to 1.0% (w/v) polysorbate 80 (PS-80)

may contain one or more tonicity modifiers suitable for parenteral administration

The compositions of this invention are preferably stored at low temperatures to prevent degradation of the peptide chain. The preferred temperature range for storage of the Aβ peptide preparation is 2 to 8 °C.

The following examples illustrate the practice of the invention. These examples are for illustrative purposes and are not intended to limit the scope of the claimed invention.

Examples

Example 1 - Peptide solubility

Peptide and reagents

Aβ42 and its trifluoroacetate salt were obtained from American Peptide Co., well numbers M05503T1, M10028T1 were used without further modification. Other salts are available and have been used successfully in the processes of the invention. See Table 2 for examples of alternative Aβ peptide salt counterions.

All acid, base and buffer solutions were prepared from laboratory grade reagents that were stored as sterile filtered solutions. Polysorbate 80 (Tween 80, PS 80); A 4% solution was prepared by dissolving a w/v solution of PS 80 with a low peroxide content (for example, a 10% solution of low peroxide polysorbate monooleate is available from Adrich-Sima Chemicals).

Peptide dissolution:

Approximately 500-700 μg of Aβ peptide was weighed and dissolved in an appropriate amount of buffer solution to obtain a theoretical final Aβ42 concentration of 0.6, 0.8, or 1.0 mg/mL. Peptide was weighed directly into a 4 mL Weaton glass screw cap. A suitable amount of buffer solution was added and the peptide was gently mixed for 15 to 30 minutes. The solution was evaluated visually for solubility as follows: (+)=poor solubility, cloudy suspension; (++)= clear suspension with insoluble particles; (+++)=clear solution.

Table 1. Apparent solubility in solution

[image] [image]

Solutions marked +++ were visually clear at target concentrations. The pH range is from pH 9 to pH 10 for buffer solutions. The pH of inorganic solutions such as NaOH and NH4OH is basic, while HCl and phosphoric acid were strongly acidic. Peptide solubility was achieved at 0.6 mg/mL peptide at pH values approximately >pH 9. Additives such as polysorbate 80, sucrose, mannitol, and EDTA did not affect peptide solubility but helped increase tonicity and isolation after filtration, or may act as chelators. Acidic solutions (approximately pH 4 or less) also dissolve Aβ42 peptides at 0.6 to 1 mg/mL. Solutions marked ++ appear partially dissolved after dissolving the peptide at the target concentration. The peptide may be soluble at lower concentrations than tested here. Solutions marked + did not visually achieve complete dissolution at the target concentration.

Example 2 - Solubility of Aβ peptide in a buffered solution

Aβ42 (0.6, 1.0, 1.5, 2.0, 3.0 and 3.5 mg/mL) was dissolved in 10 mM sodium-lycinate buffer, pH 9.0. Each solution was centrifuged in a benchtop centrifuge (> 10000 RPM, ~10 min) and a cloudy suspension was observed for concentrations of 2.0-3.5 mg/mL (0.6-1.5 mg/mL solutions were visually clear).

An aliquot of the supernatant of each solution was analyzed by RP HPLC.

Table 2 contains tabulated peptide signal sizes and a graph, indicating the solubility of Aβ42 in sodium glycinate buffer at pH 9.

Table 2. Solubility limits of Aβ42 peptide, TFA salt

[image]

Furthermore, the Aβ42 peptide was purified as the trifluoroacetate, ammonium, chloride and sodium salts. These salts were dissolved in several buffers at an Aβ42 peptide concentration of 45 mg/mL, corrected for the contribution of counterions from different salts. Peptide solutions were filtered and peptide isolation was determined by comparing the magnitude of the RP-HPC signal before or after filtration. All salts are soluble and easily filtered at 0.45 mg/mL, as shown in Table 3.

Table 3. Solubility limits of Aβ42 peptide, TFA salt

[image]

Example 3 - Isolation of a dissolved peptide from a representative of hydrophobic filters

Syringe filters were tested (filter diameter 25 mm, 3.9 cm2 filter area): Millex GV, 0.22 μM: hydrophilic polyvinylidene difluoride (PVDF, Durapore®) membrane with low protein binding capacity, Millex GN, 0.20 μM: hydrophilic nylon membrane with low protein binding capacity protein binding, and Millex GP, 0.22 μM: a hydrophilic polyethylene-sulfone membrane (PES) with a modified surface with a low protein binding capacity.

The above filters were used as representatives of the types of commercially available hydrophilic microfilters. Filtration tests were performed using the following dissolution systems:

1. 0.6 mg/mL Aβ42 in 0.01 M NH4OH

2. 0.6 mg/mL Aβ42 in 10 mM sodium glycinate, pH 9.0

3. 0.6 mg/mL Aβ42 in 10 mM sodium lysinate, pH 9.0

4. 0.6 mg/mL Aβ42 in 10 mM arinine-HCl, pH 9.0

A volume of approximately 2 mL of each Aβ42 solution was filtered over each filter. Peptide concentrations were determined by reverse phase chromatography (RP HPLC). Filter regeneration was determined by comparing the magnitude of the peptide signal before and after microfiltration, and the results are shown in Tables 3 and 4 below.

Table 3 - Filter regeneration

[image]

Example 4 - Filtration of Aβ42 in sodium glycinate and sodium lysinate buffers with and without the addition of excipients

In these studies, 0.6 mg/mL Aβ42 was dissolved in 10 mM sodium glycinate containing 0.1% polysorbate 80 (PS80), 0.9% sodium chloride, and a combination of 0.1% PD80, 0.9% NaCl, 5% sucrose, 1% sucrose, and/or 4 % mannitol. Approximately 2-5 mL of each formulation was filtered through a Millex GV filter. An aliquot of the filtrate was centrifuged in a benchtop centrifuge set at >10,000 RPM for ~3 minutes. Filter regeneration was determined by comparing the RP-HPLC signal magnitude of the peptides before and after filtration.

Table 4 - Isolation by filtration for different buffer formulations

[image]

The Millex V filter has been shown to be the preferred filter for Aβ formulation due to good peptide isolation and acceptability for use in commercial processes. Peptide formulations containing 0.9% NaCl are visibly less soluble and there is less filter regeneration, as established by RP HPLC. To increase the solubility of the peptide in the presence of inorganic salts, such as sodium chloride, it may be preferred to add a sterilized solution of the tonicity modifier to the buffer solution after microfiltration of the peptide.

On the other hand, tonicity modifiers that are saccharides (sugars) show different types of activity in solution, and can help maintain the peptide suspension. This property, known as "water-ordering", tends to "hydrate" the peptide chain in solution, so that it assumes a thermodynamically more stable β-pleated sheet conformation.

Aβ peptide can be filtered in the range of about 8.5 to about 12, preferably pH 9 to 10 with good isolation. Filtration can be performed through a 0.2 μm Millex V (Millipore Durapore) membrane that is acceptable for the manufacturing process.

For a stable biologically active formulation of the Aβ peptide under physiologically acceptable conditions suitable for clinical use, the initial step of the formulation process will preferably involve pH 8.5 to 10 sterile filtration of the peptide.

Example 5 - Soluble liquid formulations

Method testing

Three groups of Aβ peptides are produced by American Peptide Co (APC). Confirmatory formulations, chemical and biological characterization of APC peptide results were repeated with Aβ42 prepared at California Peptide Research.

A description of the formulation is described in Parts 1, 2, and 3 below.

Solubility testing

Descriptions characteristic of the formulations are presented in individual lower parts. Individual dishes were analyzed periodically during several months of storage at 2-8 °C. Appearance, pH, peptide concentration and purity were routinely monitored throughout the research. Reverse phase HPLC (RP HPLC) was used to quantitatively determine the concentration and purity range of Aβ peptides. RP HPLC uses a polymeric reverse ammonium bicarbonate (or tris)/acetonitrile gradient elution column for peptides and detection at 220 nm. Peptide concentration was measured according to the reference standard, purity was calculated as the percentage of the Aβ peptide signal area detected in the resulting integrated chromatograph.

Characterization

Characterization of the AJ342 structure solution was obtained by circular dichroism research, as shown in Figure 1.

Biological activity

Swiss Webster mice (4-8 per group) were injected with different Aβ42 in different formulations and adjuvants with CFA/IFA, MPL (Corixa Immunochemicals) or QS 21 (Aquila Pharmaceuticals) at the indicated antigen/adjuvant concentrations for the specific study. Injections were routinely given biweekly at weeks 0, 2, and 4, unless otherwise indicated. Blood was taken from the mice on the second and fourth week after the injection. Sera were analyzed by a standard "sandwich" ELISA, using Aβ42 as antigen and HRP-conjugated goat anti-mouse IgG as the antibody shown. Antibody titers are plotted or as mean data for each animal in each group, and are generally compared to titers obtained using aggregated Aβ42/CFA/IFA as control immunogens. See Tables 8 and 10 for titer results.

1. Formulations

Aβ peptide was dissolved at 0.6 mg/mL in 10 mM sodium glycinate, pH 9-9.5 buffer and filtered through a Millex GV 0.2 μm filter. Formulations were made up to 0.5 mL in 2 cc glass vials (Genisa P/N X34-113-002) and sealed with gray butyl caps (Genisa P/N X66-1 13-030) and sealed. The dishes were stored at 2-8 °C.

2. Stability test

The concentration and purity of Aβ42 in the formulations was monitored by RP HPLC. Soluble samples were analyzed alone or after microcentrifugation of the samples.

The following table (Table 5) presents the analytical data for soluble liquid formulations. No difference was seen between samples that were and were not centrifuged prior to analysis, indicating sustained peptide solubility at the desired concentration. % purity by signal area was compared to a reference standard: no significant peptide degradation was observed. The soluble formulation remained stable for a period of 3 months, and when stored at 2 to 8 °C

Table 5. Results after three months of storage

[image]

3. Characterization

Characterization of the Aβ formulation by circular dichroism shows that the peptide occupies a random coil conformation in solution with a characteristic negative elliptical absorbance between 189-205 mn.

Biological activity

A soluble Aβ42 formulation having a pH of 9 increased antibody titers in Swiss Webster mice when injected with adjuvant. Injections at biweekly intervals (0, 2, 4 weeks) of 33 μg Aβ42 with 50 μg MPL or 25 μg QS21 elicited a corresponding titer response compared to control.

Example 6 - Soluble suspension formulations Glycine/acetate formulations

Aβ peptide was dissolved at 0.6 mg/mL in 10 mM sodium glycinate, pH 9-9.5 buffer alone or with various excipients. Peptide solutions were filtered through a Millex G V filter and titrated with 0.1 M acetic acid to pH-6 to form a peptide suspension in buffers. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed. The formulations were stored at 2-8 °C.

Glycine/citrate formulations

0.6 mg/mL Aβ42 formulations were prepared in 10 mM sodium glycinate containing 0.1% PS-80 alone or in combination with 5% sucrose (25 mL), pH 9-9.5 buffer alone or with various excipients. Peptide solutions were filtered through a Millex G V filter and titrated with 0.1 M citric acid to pH~6.0. Glycine/citrate/PS 80 can be added after titration with 0.9% sodium chloride. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed. The formulations were stored at 2-8 °C.

Glycine/citrate formulations were further developed to include buffer capacity at pH 6. Several 100 mL formulations of Aβ peptide were prepared in 10 mM sodium glycinate pH 9 containing 5% sucrose with or without 0.1% PS 80. Additional formulations were prepared at 0.1 mg/mL Aβ42 in 10 mM sodium glycinate containing 5% sucrose with or without 0.1% PS 80. Peptide solutions were filtered using Millex filters before pH adjustment. The pH was then adjusted to pH 6.0, and 10 mM and 20 mM citrate buffer solution using 1 M sodium citrate solution, pH 5.5. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed.

Stability test

The concentration and purity of Aβ42 in the formulations was monitored by RP HPLC. The total concentration of the v:v peptide Aβ42 suspension was measured by redissolving the peptide with a v dilution with 2% sodium dodecyl sulfate (SDS) in 0.01 M NaOH and 1 min. heating at 100 °C before analysis. Alternatively, the concentration of soluble Aβ42 in the suspension formulation was determined by centrifugation of the test sample in a microcentrifuge at > 10,000 rpm for 3-5 minutes. Aliquots of redissolved peptide or supernatant were analyzed by RP HPLC. It should be noted that during these investigations, the improved chromatographic RP HPLC method was determined relative to the signal area of the reference standard at the time of analysis and the chromatograms were compared at each time point to determine the degradation. pH and appearance were also monitored during stability testing.

Table 6 presents data for several liquid suspension formulations. All suspensions look like suspensions. These formulations were titrated to pH 6 with the specified acid listed in the table. Depending on the excipient, the peptide suspension can be formed immediately or over a period of 2 weeks or more. A faster formation of the suspension formulation can be achieved by adding sodium chloride or by substituting citrate for acetate in the peptide formulation.

All formulations reached the desired concentration of 0.6 mg/mL peptide. The % purity by signal area for all formulations is comparable to the purity of the reference standard. No loss or degradation of the peptide was observed over a period of 3 months, when stored at 2 to 8 °C. pH and appearance remain comparable to the data shown in Table 1.

0.9% NaCl and 5% sucrose lead to physiological tonicity for parenteral administration. As with suspensions, a pH of 6 is within the acceptable pH range for injections.

Table 6 Liquid formulations of suspensions

[image] nd: not performed

The formulations of Table 7 were prepared with a 10 mM or 20 mM buffer capacity of citrate buffer to ensure that a pH of 6 was achieved during the preparation process. These formulations immediately form a suspension. Due to the properties of the Aβ peptide, the conformational change of the peptide in suspension occurs within the sterile core. The use of known buffer concentrations instead of titration allows easy handling and reproducibility of operations within a sterile unit.

Table 7. Buffered suspensions

[image]

These formulations were further expanded. Aβ42 peptide trifluoroacetates or chlorides were dissolved in 10 mM sodium glycinate, pH 9-9.5. The concentrations were adjusted according to the salt content to obtain an Aβ42 concentration of 0.45 mg/mL. Polysorbate 80, at a concentration range of 0.02% to 0.5% can be added to the peptide solution before filtration. Similarly, sucrose and sodium chloride can be added to the formulations, either before or after the addition of the acid. The pH of the peptide solution was adjusted with citrate or HCl, as indicated below. All formulations form suspensions and the age of veins is the expected concentration of Aβ42 peptide.

Table 7b

[image]

Characterization

Characterization of Aβ42, pH 6 suspension by circular dichroism and FT-IR shows that the peptide adopts a beta-pleated sheet conformation and forms a suspension. Beta-pleated plate structures were comparable in 0.6 mg/mL suspensions, regardless of the buffering acid (citrate, acetate, phosphate) or added excipient (sucrose, NaCl) added to the formulations. The addition of PS-80 appears to generate a more uniformly dispersed suspension.

Biological activity

Aβ peptide suspension, when injected with adjuvant, increases the antibody titer of Swiss Webster mice. Injections at two-week intervals (0, 2, 4 weeks) as shown in Table 8 elicit an adequate titer response compared to control.

Table 8. Antibody response to suspension formulation

[image] * MPL formulation containing triethanolamine

** titers calculated as units at 50% max. FROM

Example 7 - Lyophilized formulations

Formulations

Four combinations of 0.6 mg/mL Aβ42 in 10 mM glycinate or lysine buffer with mannitol or mannitol and sucrose were prepared and sterile filtered through a Millex V filter. One formulation, 0.6 mg/mL Aβ42 in 10 mM sodium glycinate pH 9.5, 4% mannitol was placed in a vial and capped without titration to physiological pH. The remaining three solutions (lysine/citrate/4% mannitol; glycinate/citrate/4% mannitol; glycinate/HCl/4% mannitol/1% sucrose) were then titrated to pH 7.5 with Immune acid or HCl as indicated.

Formulations were made up to 0.5 mL in 2 cc glass vials (Genisa P/N X34-113-002) and loosely capped with gray butyl caps for lyophilization (Genisa P/NX66-113-030).

Lyophilization

The formulations were lyophilized in a programmable Virtis lyophilizer (from Genisa Sicor). Mannitol was chosen as the primary matrix component. To achieve the crystallization of mannitol, the formations are frozen by thermal cyclization (annealing) followed by primary and secondary cake drying.

Stability analysis

The concentration and purity of the lyophilized formulations was monitored by RP HPLC. 1.0 mL of water for injection (WFT) was added to each formulation. Formulations were analyzed immediately after reconstitution (with or without centrifugation on a benchtop microcentrifuge at >10,000 rpm for 3-5 minutes) or redissolved as described earlier.

The following table (Table 9) shows the concentrations of the four A(342 formulations after lyophilization and redissolution. As can be seen from the data, the formulations were soluble. The samples were analyzed as reconstituted formulations and as "redissolved" reconstituted formulations, as described for peptide suspensions. No difference was observed in the reconstituted formulation in terms of total peptide concentration (redissolved sample). No degradation or loss of peptide was observed over a period of 3 months, when stored at 2 to 8 °C, as determined by RP HPLC as in the stability test in Example 5.

Table 9. Lyophilized formulations

[image]

Characterization

Characterization of Aβ42, pH 7.5 lyophilized formulations by circular dichroism and FT-IR shows that the peptide adopts a random coil conformation during lyophilization and reconstitution. This observation is consistent with the sodium glycinate pH 9 formulation of Aβ42 which is also soluble, filterable, and assumes a random coil conformation in solution.

Biological activity

A lyophilized formulation of Aβ42, 0.6 mg/mL Aβ42 in 10 mM sodium glycinate/HCl/4% mannitol/1% sucrose, pH 7.5 was chosen as a representative lyophilized formulation. This product, when mixed with MPL or QS21 adjuvant, increases the antibody titer of Swiss Webster mice. The geometric means of the titers are shown in Table 10 compared to the control.

Table 10. Antibody response to the lyophilized formulation

[image] * MPL formulation containing tnetanolamm

** titers calculated as units at 50% max. FROM

Example 8 - Preparation on an increased scale using GMP preparation standards

The peptide suspension was prepared on an enlarged scale of up to 1.5 liters and was filled by the drug manufacturer. Two concentrations were filled: 0.6 mg/mL and 0.1 mg/mL. Both concentrations were successfully scaled up, filled and remained stable at 2-8 °C and at 25 °C after two months.

Aβ peptide was dissolved at concentrations of 0.6 mg/mL and 0.1 mg/mL in a 10 mM solution of glycinate buffer pH 9 containing 5% sucrose. The peptide solution was sterile filtered through a Molopak 20 Mollopore Durapore 0.2 fim sterilizing filter in the septic core. RP HPLC analysis showed no significant loss of peptide during the filtration process. 1M sodium citrate pH 5.5 was added to the compound and it was sterile filtered through a 0.2 μm Durapor filter into an aseptic core. The peptide solution was weighed and an appropriate amount of sodium citrate buffer was added to make a 20 mM citrate formulation, pH 6.

At a concentration of 0.6 mg/mL, the peptide immediately formed a suspension after the addition of citrate buffer, and during the measurement the pH was 6.4. The peptide suspension was constantly mixed, filled 1.2 mL into 2 cc borosilicate glass vials, which were closed with West 4416 stoppers and sealed. At a concentration of 0.1 mg/mL, the peptide remained dissolved in the vials (and the filling dissolved) for several hours, before forming a suspension the next day.

Table 11 presents the data obtained for this suspension of 0.6 mg/mL Aβ42:

Table 11.

Aβ peptide suspension 0.6 mg/mL in 10 mM glycine, 20 mM citrate 5% sucrose, pH 6

[image]

Example 9 - Buffered suspensions of Aβ 1-42 with QS1 adjuvant

Single-vial formulations in which Aβ1-42 and adjuvant were tested using QS-21 and a triterpene glucoside having immune-stimulating activity were surprisingly found to result in the formation of a visually clear peptide suspension formulation.

1. Aβ1-42/QS-21 formulation in one vial

QS-21 was dissolved and lyophilized with a solution of Aβ1-42 in 10 mM sodium glycinate at pH 9.0. In each subsequent example, lyophilized QS-21 was dissolved with Aβ1-42 (TFA salt 0.45 mg/mL) solution in 10 mM glycine (Gly), pH 9.0. The preparation contains dissolved QS-21, and the pH is adjusted to 6.0 with the addition of citrate buffer (Cit) to create a visually clear suspension. Turbidity measurements were taken with a spectrophotometer set at a wavelength of 405 nm to measure the clarity of the resulting suspension. The results show that the particle sizes of the suspended peptide are smaller than the wavelength of the reflected light. These formulations showed stability when stored at 2-8 °C, and when monitoring stability, all formulations were found to contain Aβ1-42 predominantly in conformation (3-pleated plates; none of the formulations showed turbidity when spectrophotometrically analyzed at 405 nm. A formulation of 0.45 mg/mL Aβ1-42, 0.2 mg/mL QS-21, 10 mM Cit, 5% sucrose, pH 6.0 was tested in a mouse titer assay, producing a higher titer response.

Table 12A

[image] 1 found to have a high titer in mass 2. Aβ42titrationsQS-21

Aβ42 (2 mg/mL) was dissolved in 10 mM glycine, pH 9.5. The pH was adjusted to 9.5 by adding 1 M NaOH. The Aβ42 solution was then filtered through a Millex GV filter. QS-21 (5 mg/mL) was dissolved in 10 mM citrate, pH 6.0, and filtered through a Millex GV filter. An aliquot of Aβ42 was mixed with an aliquot of QS-21 to form the desired ratio, mixed and diluted with 10 mM glycine, pH 9.5, and 1 M citrate pH 5.2 to obtain the final concentration indicated in Table 12 below.

Table 12B. Initial AN1792/QS21 Co-formulation

[image] [image]

3. Additional formulations of Aβ42 and QS-21

Aβ42 and corrid were dissolved at 1 mg/mL in 20 sodium glycinate, pH 9.0-9.5 with or without sucrose, 0.1% PS-80, or 0.4% PS-80. The peptide solution was sterile filtered through a Millex GV syringe filter. Lyophilisate QS-21 was dissolved at 5 mg/mL in 10 mM citrate, pH 6.0 and sterile filtered through a Millex GV syringe filter.

Appropriate volumes of Aβ42 and QS-21 solutions were combined to obtain the final concentration of Aβ42 and QS-21 indicated in the formulations in Table 12C below. Finally, the pH was adjusted with 1 M citrate buffer, pH 5.4 to give a final pH of 6 in 20 mM citrate buffer. The visual appearance of the formulation was evaluated as clear to cloudy (+ to +++). By changing the concentration ratios of Aβ42 and QS-21, the appearance of the formulations can be adjusted. Furthermore, sugars and surfactants are acceptable formulation excipients.

Table 12C. Additional formulations A(342 and QS-21

[image]

Example 10 - Measurement and interpretation of C.D. spectra of Aβ42 formulations

Surgical dichroism data were collected using an Aviv model 62-DS spectropolarimeter (Lakewood, NJ). Appropriate samples were prepared to collect the near UV and far UV spectra, and were loaded into a 1 mm quartz cell. The sample holder was maintained at a temperature of exactly 25 °C. Data were collected at 0.5 nm intervals using an averaging time of 4 seconds. In the far UV region (λ, ~1250-350 nm) completely different information is obtained, in that region the primary signals increase due to the aromatic side chain (Phe, Tyr and Trp). The label and magnitude of the signal indicate the degree of flexibility at each site as well as the orientation of the side chain relative to the peptide backbone. As the number of these chromophores is smaller than the number of amide groups, and therefore the chromophores are distributed throughout the entire molecule, they provide information only about the local structure.

It is expected that numerous modifications and variations in the invention, such as those described above in the illustrative examples, will be made by those skilled in the art, and therefore the limitation is only that which results from the appended claims. Accordingly, the claims are intended to cover such equivalent variations as fall within the scope of the invention as claimed.

Claims (47)

1. A preparation containing an aqueous solution of Aβ peptide with a concentration of at least 0.1 mg/mL, indicated by the fact that said solution is maintained at a pH sufficient to dissolve said Aβ peptide. 2. The composition of claim 1, characterized in that the solution is maintained at such a suitable pH using a pharmaceutically acceptable buffer. 3. A preparation containing an aqueous solution containing Aβ peptide with a concentration of at least 0.1 mg/mL, indicated by the fact that said solution is maintained at a pH sufficient to dissolve said Aβ peptide. 4. The composition of claim 3, characterized in that the solution is maintained at such a suitable pH using a pharmaceutically acceptable buffer. 5. The preparation according to claim 1 or 3, characterized in that said Aβ peptide is a long chain of Aβ peptide. 6. The preparation from claim 1 or 3, characterized in that said Aβ peptide is Aβ42. 7. The composition of claim 1 or 3, characterized in that the pH is from about 8.5 to about 12. 8. The composition of claim 7, characterized in that the pH is from about 9 to about 10. 9. The preparation from patent claims 2 or 4, characterized in that the pharmaceutically acceptable buffer is selected from the group consisting of: amino acids, salts and their derivatives; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides and ammonium hydroxides; organic and inorganic acids and their salts; and their mixtures. 10. The preparation of claim 9, characterized in that the pharmaceutically acceptable buffer is glycine (sodium glycinate) or arginine (arginine hydrochloride). 11. Lyophilized preparation of Aβ peptide, indicated by the fact that the preparation is prepared by the process: a) freezing a sterile aqueous solution that has at least 0.1 mg/mL Aβ peptide, whereby the aqueous solution is maintained at a pH sufficient to dissolve the Aβ peptide, and b) lyophilization of the frozen preparation prepared in the above a). 12. The preparation according to claim 11, characterized in that the Aβ peptide is a long-chain Aβ peptide. 13. The preparation according to claim 11, characterized in that the Aβ peptide is the long chain of Aβ42. 14. The composition of claim 11, characterized in that the solution is maintained at such pH using an effective amount of a pharmaceutically acceptable buffer. 15. The preparation from claim 14, characterized in that the pharmaceutically acceptable buffer is selected from the group consisting of: amino acids, salts and their derivatives; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides and ammonium hydroxides; organic and inorganic acids and their salts; and their mixtures. 16. The preparation according to claim 1, 3 or 11, characterized in that the Aβ peptide is mainly in a random coil conformation. 17. The composition of claim 1, 3 or 11, characterized in that the Aβ peptide has a conformation of about 0.05 mg/mL to about 2.0 mg/mL. 18. The preparation from patent claims 1, 3 or 11, characterized in that the preparation further contains a pharmaceutically acceptable adjuvant. 19. The preparation from patent claim 18, characterized in that the adjuvant is selected from the group consisting of: Freund's incomplete adjuvant, MPL, QS-21, and alum. 20. A composition, characterized in that it contains a sterile aqueous peptide suspension with at least 0.1 mg/mL of Aβ peptide at a pH of about 5 to about 7. 21. The composition of claim 20, characterized in that the aqueous peptide suspension also contains an effective amount of a pharmaceutically acceptable buffer. 22. The preparation according to claim 20 or 21, characterized in that the Aβ peptide is a long-chain Aβ peptide. 23. The preparation according to claim 22, characterized in that the Aβ peptide is the long form of Aβ42. 24. The preparation from patent claim 21, characterized in that the pharmaceutically acceptable buffer is selected from the group consisting of: amino acids, salts and their derivatives; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides and ammonium hydroxides; organic and inorganic acids and their salts; and their mixtures. 25. The composition of claim 20, comprising 0.1 to 0.8 mg/mL Aβ42 peptide, 10 mM glycine, and acid sufficient to adjust the pH to from about 5.5 to about 6.5. 26. The preparation from patent claims 24 or 25, characterized in that it further contains one or more excipients selected from the group consisting of: agents for tonicity modification, surface active agents and sliding agents. 27. The preparation from claim 24, characterized in that the preparation further contains a pharmaceutically acceptable adjuvant. 28. The preparation from claim 26, characterized in that the preparation further contains a pharmaceutically acceptable adjuvant. 29. The preparation from claim 28, characterized in that the adjuvant is selected from the group consisting of: Freund's incomplete adjuvant, MPL, QS-21, and alum. 30. The composition of claim 28, comprising from about 0.1 to about 1.0 mg/mL AΒ42 in 10 mM glycine and at least 0.1 mg/mL QS-21 in an amount effective to form a visually clear suspension having a pH of about 6. 31. Process for the preparation of a sterile preparation of the long form of Aβ peptide, indicated by the fact that it contains adjustments to the pH of an aqueous solution sufficient to dissolve the Aβ peptide in it, dissolving in the solution an amount of Aβ peptide that is sufficient to achieve an immunogenic concentration in a mammal, and filtering the resulting solution to bacteria would be excluded, and almost all Aβ peptide would pass through the membrane. 32. The method from patent claim 31, characterized in that the filtering is performed over a hydrophilic polymer membrane that has the same pore size of about 0.2 microns. 33. The method of patent claim 31, characterized in that the amount of Aβ peptide isolated after filtering is greater than 50%. 34. The method of claim 31, characterized in that the solution before filtration contains at least one diluent selected from the group consisting of pharmaceutically acceptable buffers having a concentration of about 5 mM to about 45 mM. 35. The method of claim 34, characterized in that the solution before filtering contains a tonicity modifier from about 0.9% to about 6.0% (w/v). 36. The method of claim 34, characterized in that the solution before filtering contains a surface-active substance of about 0.02% to about 1.0% (w/v). 37. The method of claim 34, characterized in that the solution before filtering contains a chelating agent from about 0.1 mM to about 1.0 mM. 38. The method of patent claims 34, 35, 36 or 37, characterized in that the pH of the sterile solution obtained after filtration is adjusted to a pH of about 5 to about 7, in order to obtain a peptide suspension. 39. A method of preventing Alzheimer's disease in mammals, characterized in that it comprises administering to said mammal a sufficient amount of a sterile aqueous preparation containing at least 0.05 mg/mL Aβ peptide, and to induce an immunogenic response in said mammal, and said aqueous solution is maintained at a sufficient pH to dissolve the Aβ peptide. 40. A method of inducing an antibody response to an Aβ peptide in a mammal in need of such an antigenic response, characterized in that it contains: parenteral administration of an immunogenic amount of a sterile preparation of the long form of Aβ peptide. 41. The method from patent claims 39 or 40, characterized in that the method further comprises administering a pharmaceutically acceptable adjuvant separately or mixed into said sterile preparation. 42. The method of claim 39 or 40, characterized in that the sterile solution is in accordance with claim 30. 43. Composition, characterized in that it contains a suspension of at least 0.1 mg/mL of Aβ peptide and QS-21 in an amount effective to form a visually clear suspension in the pH range of 5 to 7. 44. The preparation, characterized in that it contains a suspension of at least 0.1 mg/mL of Aβ peptide and DPPC (dipalmitoylphosphatidyl chloride) in an amount effective to form a visually clear suspension in the pH range of 5 to 7. 45. Use of a sterile aqueous preparation of the long form of the Aβ peptide, characterized in that for the production of a medicament for inducing an antibody response to the Aβ peptide. 46. Use of a sterile aqueous preparation of Aβ peptide, characterized in that it is for the production of a drug useful in the prevention of Alzheimer's disease. 47. The use of claim 45 or 46, characterized in that said drug further contains a pharmaceutically acceptable adjuvant.