EP1181040A1 - Zusammensetzungen mit a-beta peptide und verfahren zu ihrer herstellung - Google Patents

Zusammensetzungen mit a-beta peptide und verfahren zu ihrer herstellung

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Publication number
EP1181040A1
EP1181040A1 EP00942668A EP00942668A EP1181040A1 EP 1181040 A1 EP1181040 A1 EP 1181040A1 EP 00942668 A EP00942668 A EP 00942668A EP 00942668 A EP00942668 A EP 00942668A EP 1181040 A1 EP1181040 A1 EP 1181040A1
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EP
European Patent Office
Prior art keywords
peptide
composition
pharmaceutically acceptable
solution
suspension
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00942668A
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English (en)
French (fr)
Inventor
Pamela Hirtzer
Naina Patel
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Neuralab Ltd
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Neuralab Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • compositions of A-Beta Peptide and Processes for Producing Same are Compositions of A-Beta Peptide and Processes for Producing Same
  • This invention relates generally to pharmaceutical compositions comprising proteins, which are useful in raising a mammalian antibody response. More specifically, this 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.
  • the diluent is a sterile parenterally acceptable aqueous phase.
  • Amyloid-beta peptide also known as A-beta or A ⁇ peptide, is a cleavage product of the amyloid precursor protein (APP). It is the principal component of amyloid plaques in the mammalian brain that are fundamentally involved in and characteristic of Alzheimer's disease.
  • a ⁇ peptide is a 39-43 amino acid chain varying in length owing to the variability of its processing from APP by several proteases.
  • Such mutations are thought to cause Alzheimer's disease by increased or altered processing of APP to A ⁇ peptide, particularly processing of APP to increased amounts of A ⁇ 42 and A ⁇ 43. Mutations in other genes, such as the presenilin genes, PSl and PS2, are thought indirectly to affect processing of APP to generate increased amounts of A ⁇ 42 and A ⁇ 43 (see Hardy, TINS 20, 154 (1997)).
  • the observations indicate that A ⁇ peptide, and particularly A ⁇ 42, is a causative element in Alzheimer's disease. In the brain, the A ⁇ peptide aggregates and forms the amyloid deposits comprising the peptide organized into fibrils of ⁇ -pleated sheet structures.
  • a ⁇ peptide in inducing an immune response of the body against it. See, for example, PCT Publication No. WO99/27944, which is incorporated by reference in its entirety for all purposes.
  • the present invention is directed to novel and unexpected methods for practicing the invention described in PCT Publication No. WO99/27944.
  • it involves administering certain formulations of the long forms of A ⁇ peptide to the patient to induce an immune response.
  • the longer forms of A ⁇ peptide are difficult to solubilize in conventional formulation systems.
  • Hilbich et al., J. Mol. Biol, 218 (1), pp. 149-64, (1991) report that while A ⁇ 1 -43 peptide is soluble to a degree in pure water, the addition of ionic components, such as buffers or salts, or organic solvents cause the peptide to precipitate out of solution in the form of an amorphous aggregate.
  • ionic components such as buffers or salts, or organic solvents
  • Hilbich found phosphate-buffered saline (“PBS,” which in this instance contained 137mM NaCl, 3 mM KC1, 8 mM Na 2 HPO 4 -2H 2 O and 2 mM KH 2 PO 4 at pH 7.5) rendered 90-94% of the peptide in the composition insoluble.
  • PBS phosphate-buffered saline
  • PBS is a conventional carrier for parenteral compositions, approximating the tonicity and pH level of the living system.
  • Five (5) mM NaCl caused 42-50% of the peptide to precipitate. (Ibid., p.153, Table 2).
  • the solution of peptide in pure water would be hypotonic and the pH of such a solution was determined to be 5.5 by Hilbich (idem.).
  • the pH of the blood supply in man is about 7.4.
  • Dyrks, et al. have also reported A ⁇ 42 is insoluble at physiological conditions. Dyrks, T., Weidemann, A., Multhaup, G., et al. EMBO J.l, p. 949-57 (1988).
  • the conformation of A ⁇ peptide in the solution can be measured using circular dichroism (CD.) spectroscopy.
  • CD. circular dichroism
  • Conformational studies of A ⁇ peptide and fragments using CD. are reported by Hilbich, et al, idem. See also, monograph by M. Manning entitled: Protein Structure and Stability Assessment by Circular Dichrosim Spectroscopy, from Biocatalyst design for Stability and Specificity; Himmel, M. E. and Georgiou, G., eds., ACS Symposium Series 516 (1993) at p. 36.
  • This reference, hereinafter referred to as the Manning reference is hereby incorporated by reference in its entirety for all purposes.
  • Kline, et al, U.S. Patent Nos. 5,851,996 ('996 patent) and 5,753,624 ('624 patent) describe administration of very minute amounts (10 " mg or less) of A ⁇ peptide or a fragment thereof administered sublingually in a liquid or solid carrier, such as a phenylated saline solution.
  • the '996 patent states that the amyloid beta protein "exists in various structural forms" (col. 2, line 31) that can be used to treat Alzheimer's disease. It is not elsewhere defined what is meant by various structural forms, neither are any characterized beyond the 28 amino acid fragment used in the examples.
  • the doses of A ⁇ peptide are stated in the '996 patent to be from 10 " '°to 10 "2 mg (col. 8, lines 442-43).
  • aqueous solutions comprising high concentrations of A ⁇ peptide can be prepared by adjusting the acidity /basicity of such an aqueous solution to a pH effective to solubilize A ⁇ peptide.
  • the pH is adjusted to a pH range of from about 8.5 to about 12, and more prefereably from about pH 9 to 10.
  • This invention is further directed to the discovery that the solubilized solutions of A ⁇ peptide lend themselves to sterile filtration through a suitable micropore filter with at least 50% recovery of the A ⁇ peptide after the sterile filtration.
  • at least about 10% of A ⁇ peptide is recovered after sterile filtration and, more preferably at least 90%.
  • Such sterile solutions can be formulated as pharmaceutical compositions comprising a sufficient amount of A ⁇ peptide to effect an immunogenic response when administered to a mammal.
  • such administration is by parenteral administration, in the form of a suspension composition.
  • the pH of the composition is adjusted to a physiologically acceptable pH to form a peptide suspension containing at least 0.1 mg/ml of A ⁇ peptide.
  • the composition being useful for parenteral administration.
  • the pH of a suspension composition is between about pH 5 and about 7, preferably between 5.5 and 6.5.
  • a more preferred composition comprises a sufficient amount of QS-21 in conjunction with A ⁇ peptide to form a visually clear, sterile suspension.
  • This invention is still further directed to the discovery that the solubilized and sterile solutions of A ⁇ peptide can be lyophilized to provide for lyophilized formulations comprising A ⁇ peptide. These compositions can be reconstituted at the appropriate time to provide for an aqueous composition comprising A ⁇ peptide.
  • this invention is directed to an aqueous solution comprising at least 0.01 mg/ml of A ⁇ peptide wherein said aqueous solution is maintained at a pH sufficient to solubilize said A ⁇ peptide.
  • the solution is maintained at such a suitable pH by use of an effective amount of a pharmaceutically acceptable buffer.
  • this invention is directed to a sterile aqueous solution comprising at least 0.01 mg/ml of A ⁇ peptide wherein said aqueous solution is maintained at a pH sufficient to solubilize said A ⁇ peptide.
  • the solution is maintained at such a pH by use of an effective amount of a pharmaceutically acceptable buffer.
  • this invention is directed to lyophilized compositions comprising a lyophilized composition comprising A ⁇ peptide which composition is prepared by the process of: a) freezing a sterile aqueous solution comprising at least 0.01 mg/ml of A ⁇ peptide wherein said aqueous solution is maintained at a pH sufficient to solubilize said A ⁇ peptide; and b) lyophilizing the frozen composition prepared in a) above.
  • compositions of this invention comprise a long form (as defined below) of A ⁇ peptide.
  • composition comprises a pharmaceutically acceptable buffer which is preferably selected from the group consisting of amino acids, salts and derivatives thereof; pharmaceutically acceptable alkalizers, alkali metal hydroxides and ammonium hydroxides, organic and inorganic acids and salts thereof; and mixtures thereof.
  • this invention is directed to a composition
  • a composition comprising an aqueous solution comprising at least 0.01 mg/ml of A ⁇ peptide wherein said aqueous solution is maintained at a pH sufficient to solubilize said A ⁇ peptide and further wherein said A ⁇ peptide is substantially in a random coil conformation.
  • compositions of this invention can be formulated into pharmaceutical compositions suitable for delivery to a mammal having Alzheimer's disease or at risk of developing Alzheimer's disease.
  • composition aspects of the invention are directed to pharmaceutical compositions which are in a soluble random coil conformation of A ⁇ peptide or a stable, aqueous suspension of at least 0.1 mg/ml of A ⁇ peptide suspended in said composition, or a lyophilized composition, any or all of which may be sterile and pareterally administerable.
  • this invention is directed to a process for preparing a sterile composition of a long form of A ⁇ peptide comprising: adjusting the pH of an aqueous solution sufficient to solubilize the A ⁇ peptide therein; dissolving into the solution an amount of the A ⁇ peptide sufficient to achieve an immunogenic concentration for a mammal; filtering the resulting solution through a uniform pore size membrane, said pore size being in a range capable of excluding bacteria and passing substantially all of the A ⁇ peptide through the membrane; and optionally, for solutions containing 0.1 mg/mL or more of A ⁇ peptide, adjusting the pH of the resulting solution to between about pH 5 to about pH 7 to obtain a peptide suspension.
  • this invention is directed to a process for preventing or treating Alzheimer's disease in a mammal which method comprises administering to said mammal a sufficient amount of a sterile aqueous composition comprising at least 0.05 mg/ml of A ⁇ peptide to induce an immunogenic response in said mammal.
  • the filtration processes of this invention employ A ⁇ peptide which is substantially in the random coil conformation.
  • Figure 1 is a CD. spectrum showing the mean residue ellipticity measurement plotted as a function of wavelength for two different solutions of A ⁇ 42.
  • the dotted line shows the absorbance at pH 6 and is attributable to the ⁇ -pleated sheet form of the molecule.
  • the solid line is a plot of the absorbance of a solution of A ⁇ 42 at pH 9 and is indicative of the random coil conformation of the peptide.
  • Figure 2 is a plot of A ⁇ 42 placed into a solution against the peak area calculation for the amount of dissolved peptide as determined by reverse phase high performance liquid chromatography, demonstrating the solubility of A ⁇ 42.
  • compositions and methods employing aqueous compositions comprising therapeutically effective concentrations of A ⁇ peptide.
  • a ⁇ peptide A ⁇ peptide
  • substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65 percent sequence identity, preferably at least 80 or 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity or higher). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
  • a suitable reference sequence would be the human A ⁇ peptide sequence, specifically the 42 amino acid sequence as reported below.
  • Other suitable forms would be the truncated forms: such as A ⁇ 39, or the extended form, A ⁇ 43 (with an additional threonine group at the C-terminal end).
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, 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).
  • BLAST Altschul et al, J. Mol. Biol. 215:403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • default program parameters can be used to perform the sequence comparison, although customized parameters can also be used.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989)).
  • amino acids are grouped as follows: Group I (hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
  • APP 695 , APP 751 , and APP 770 refer, respectively, to the 695, 751, and 770 amino acid residue long polypeptides encoded by the human APP gene. See Kang 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.
  • the weight of A ⁇ peptide represents about 10% to about 85% A ⁇ 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 A ⁇ 42 peptide is corrected for the peptide content, this represents 0.075 mg of A ⁇ 42 peptide and 0.025 mg water and salt; 0.6 mg A ⁇ 40 peptide represents 0.45 mg of A ⁇ 40 peptide and 0.15 mg of water and salt; and 2.0 mg A ⁇ 42 peptide represents 1.5 mg of A ⁇ 42 peptide and 0.5 mg of water and salt.
  • a ⁇ peptide refers to those segments of the A ⁇ peptide capable of forming a ⁇ -pleated sheet conformation and raising an immunogenic response when administered either alone or in conjunction with an adjuvant to a mammal. It is within the ordinary skill of the art to determine ⁇ -pleated sheet conformation as described herein by use of, for example, circular dichroism measurements. Immuongenicity may be determined as described in the Biological Activity sections of the Examples given below.
  • a ⁇ long forms of A ⁇ peptide includes any of the naturally occurring forms of A ⁇ 38, A ⁇ 39, A ⁇ 40, A ⁇ 41, A ⁇ 42, and A ⁇ 43 and peptide sequences substantially identical thereto, and preferably the human forms.
  • a ⁇ 39, A ⁇ 40, A ⁇ 41, A ⁇ 42 and A ⁇ 43 refer to A ⁇ peptide containing amino acid residues 1-39, 1-40, 1-41, 1- 42 and 1-43, respectively, amino acids being truncated from the C-terminal end of the peptide.
  • a ⁇ 41, A ⁇ 40 and A ⁇ 39 differ from A ⁇ 42 by the omission of Ala, Ala-Ile, and Ala-Ile-Val respectively from the C-terminus, as can be seen by reference to the A ⁇ peptide sequence below.
  • a ⁇ 43 differs from A ⁇ 42 by the presence of a threonine residue at the C-terminus. The sequences of these peptides and their relationship to APP are illustrated in Figure 1 of Hardy et al., TINS 20, 155 (1997).
  • a ⁇ 42 has the sequence: H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu- Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala- Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-IIe-Ala-OH.
  • a ⁇ long forms of A ⁇ peptide also includes analogs thereof.
  • Analogs include allelic, species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with natural peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids.
  • unnatural amino acids are ⁇ , ⁇ -disubstituted amino acids, N- alkyl amino acids, lactic acid, 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N- trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methyl-histidine, 5-hydroxylysine, ⁇ -N-methylarginine.
  • a ⁇ may be utilized in the compositions and processes of the invention from about
  • 0.05 mg/ml to its upper solubility limit of about 2.0 mg/ml (referring to Fig.2).
  • Preferred ranges of the peptide are from about 0.1 to about 0.8 mg/ml and more preferred is a range of from about 0.3 to about 0.6 mg/ml.
  • the methods and compositions of this invention may employ a random coil conformation of A ⁇ peptide. Applicants demonstrate herein that the random coil conformation is capable of raising an immunogenic response in test mammals. The random coil conformation is most preferred in the microfiltration of the processes.
  • random coil refers to an open chain conformation of the A ⁇ peptide.
  • the random coil is a secondary conformation of the peptide backbone and is not ordered into a regular conformation such as the ⁇ -helix or ⁇ -pleated sheet forms. Rather the random coil is disordered from the hydrophobic folding and hydrogen bonding interactions that characterize the other, more regularly arranged forms.
  • a random coil may still possess some turns of the peptide backbone or partial ordering, however, such features are random and dynamic, and thus are not typical of all the random coil population.
  • the peptide exhibits good solubility and filterability.
  • the ⁇ -helix and ⁇ -pleated sheet conformations are well-known peptide conformations in the art.
  • the random coil conformation of A ⁇ peptide can be characterized by its circular dichroism ("CD") spectrum and is easily distinguished from the ⁇ -pleated conformation.
  • CD circular dichroism
  • a random coil is typified by a strong negative band between 190 and 200 nm, and little CD signal is seen at wavelengths longer than 215 nm. If there is a CD signal at longer wavelengths is very weakly positive, centered at 220-225 nm. This is shown in Figure 1 of the Drawings.
  • the ⁇ -pleated sheet conformation on the other hand displays a sha ⁇ and strong positive band centered at about 200 nm.
  • a ⁇ peptide is substantially in a random coil conformation when greater than 50% of the A ⁇ peptide is the random coil conformation.
  • Parenteral compositions are those compositions which are sterilized and suitable for administration directly into the body by, for example, injection or infusion, i.e. by routes which have immediate contact with the blood and without the barrier or immune system protections afforded by administration forms that enter the body via the dermal, mucosal, digestive or respiratory systems. For these reasons, sterility is a necessity for a parenteral composition.
  • Intravascular or intravenous or IV
  • intramuscular IM
  • intraperitoneal IP
  • subcutaneous SC
  • intrasternal all refer to different modes of adminstration of parenteral compositions. They describe in anatomical terms the area of the body in which the parenteral composition is introduced by injection or infusion. It is contemplated that the compositions of the present invention having a physiologically acceptable pH can be administered by any of the above modes, depending on the individual patient and the judgment of the administering physician.
  • the higher pH solutions pH > 8 may most advantageously be administered by a slow IV infusion or IV drip.
  • the titration curve of an acid or base has a relatively flat zone extending to about 1.0 pH unit on either side of the titration midpoint. At the midpoint, an equivalent amount of the proton-donor and proton-acceptor species of the acid or base are present. In this zone, the pH of the system changes relatively little when small increments of H + or OH " are added. This is the zone in which a conjugate acid- base pair acts as a buffer, a system which tends to resist change in pH when a given increment of H + or OH " is added. At pH levels outside this zone there is less capacity of the buffer to resist changes to the pH.
  • a buffer's power is maximum at the pH of the exact midpoint of its titration curve, i.e., where there are equal concentrations of the proton acceptor and proton donor and the pH is equal to the pK' (the acid dissociation constant). Buffer preparations are described in detail in Data for Biochemical Research, Rex, M.C, Oxford Science Publications, 1995.
  • a “pharmaceutically acceptable buffer” is defined broadly to include conjugate acid - base pairs as well as acidic or basic compounds that have the ability to adjust or to maintain the pH of a solution of A ⁇ peptide at a desired level.
  • a solubilization/filtration processes of this invention that is pH 8.5 or above or in another aspect below pH 4.
  • buffers are selected from the group consisting of amino acids, salts and derivatives thereof; pharmaceutically acceptable alkalizers, alkali metal hydroxides and ammonium hydroxides, organic and inorganic acids and salts thereof; and mixtures thereof.
  • Buffering agents are employed in concentrations sufficient to reach and maintain the desired pH and thus the concentrations are dependent on the acidity /basicity of the individual buffer or combination chosen. Selection of an effective concentration is within the ordinary skill of the art, using for example, pH meters and titration techniques.
  • hydroxides including alkali metal hydroxides and ammonium hydroxides
  • alkalizers known in the pharmaceutical arts, including but not limited to tris, sodium borate (Na B 4 O 7 ) and disodium citrate, amino acids, salts or esters or amides of amino acids and simple derivatives thereof, for example, N-acetyl derivatives of amino acids.
  • Particularly preferred buffers are glycine (e.g., sodium glycinate), arginine and lysine, sodium hydroxide and ammonium hydroxide.
  • Examples of pharmaceutically acceptable acids for practice of the methods of the present invention are, without limitation, hydrochloric acid, phosphoric acid, citric acid, acetic acid, maleic acid, malic acid and succinic acid, and the like. Additionally these acids may be used to titrate the pH value of a basic solution to a lower, more physiologically acceptable level, resulting in a peptide suspension composition.
  • Combinations of the pH adjusting agents are also contemplated.
  • Conjugate acid base pairs as discussed above and exemplified by salts such as ammonium acetate are also within the scope of buffering agents for the invention.
  • Such a conjugate pair could be formed, for example, by the titration of a basic solution of ammonium hydroxide with acetic acid to a more nearly neutral solution of ammonium acetate.
  • the term "tonicity modifier" includes agents that contribute to the osmolality of the solution.
  • tonicity modifiers suitable 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.
  • the tonicity agent will be employed in an amount to provide a final osmotic value of less than about 350 mOsm/kg and more preferably between about 250 to about 350 mOsm/kg, and most preferably between about 280 to about 320 mOsm/kg.
  • the charged compounds that serve as buffers in the formulations can also affect tonicity.
  • the tonicity of a buffered solution of A ⁇ peptide is first determined before being adjusted further by addition of tonicity modifying agents.
  • a chelating agent may optionally be used in the processes and compositions of the invention.
  • preferred chelating agents include ethylenediaminetetraacetic acid (EDTA), and its salts (such as sodium) which are normally employed at a concentration of 0.05 to 50 mM, more preferred at concentrations of 0.05 to 10 mM, or about 0.1 to 5 mM being most preferred.
  • EDTA ethylenediaminetetraacetic acid
  • Other known chelating (or sequestering agents) such as certain polyvinyl alcohols can also be used.
  • compositions may contain a surfactant or detergent such as polysorbate (e.g. Tween®) or 4-(l,l,4,4-tetramethylbutyl) phenyoxypolyethoxyehthanols (Triton®), or polymers of polyethylenepolypropylene glycols (Pluronics®).
  • a surfactant or detergent such as polysorbate (e.g. Tween®) or 4-(l,l,4,4-tetramethylbutyl) phenyoxypolyethoxyehthanols (Triton®), or polymers of polyethylenepolypropylene glycols (Pluronics®).
  • the surfactant ranges from about 0.005 to 1%, with about 0.02 to 0.75% preferred.
  • a preferred polysorbate is PS-80, which is commercially available as Tween® 80.
  • Wetting agents are also contemplated as excipeints useful in the invention.
  • the polyethylene glycols e.g. PEG 3350, are useful in modifying the association of A ⁇ particles and the solubility thereof by associating with the polymer suface and ordering its hydrophilic moieties in the aqueous phase.
  • Wetting agents may be present form 0.5 to
  • sugars for example sucrose, dextrose, maltose or lactose
  • pharmaceutically acceptable sugar alcohols for example mannitol, xylitol or sorbitol
  • sugars or sugar alcohols having a molecular weight of less than 500, and capable of easily dispersing and dissolving in water
  • sugars and sugar alcohols usable in the present invention include xylitol, mannitol, solbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose, lactose, and the like. They can be used alone, or as a mixture of two or more of these compounds.
  • the most preferred sugar is mannitol, especially in the lyophilized compositions; sucrose is also preferred in the solution compositions.
  • QS-21 an adjuvant
  • the use of QS-21 in the compositions of the invention interacts with the suspended protein in such a way that a visually clear suspension can be formed.
  • DPPC dipalmitoyl phosphatidyl choline
  • a ⁇ peptide is known in the art and is anticipated to interact with A ⁇ peptide in a similar fashion to QS-21 to provide the same small particle suspension and allow for use of other of the adjuvants in the invention, while providing a visually clear suspension.
  • other adjuvants may be used in admixture with QS-21.
  • Lyophilization is a technique well known in the pharmaceutical arts, as are techniques for stablizing peptides during and after the lyophilization process. Stabilization of a protein or peptide lyophilisate in an amino acid, saccharide matrix is also known to those of skill in the art. See, for example: Lueckel B., et al., Formulations of sugars with amino acids or mannitol - Influence of concentration ratio on the properties ofthefreeze-concentrate and the lyophilizate, PHARM. DEV. TECHNOL. 3(3) pp. 325-336 (1998).
  • pharmaceutically acceptable modifies any composition to mean that the composition does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered.
  • pharmaceutically acceptable diluent and “pharmaceutical acceptable excipient” refer to any compound which preserves or does not alter the activity of the active compound(s) and does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered, such as a non-toxic pH adjusting or buffering agent or a tonicity modifying agent or chelating agent, and the like.
  • compositions or processes "comprising" one or more recited elements may include other elements not specifically recited.
  • a composition that comprises A ⁇ peptide encompasses both A ⁇ peptide in the composition as recited and A ⁇ peptide as a component of a composition having other non-recited components.
  • MPL 3-O-deacylated monophosphoryl lipid A (MPLTM) (see for example GB 2220211).
  • QS 21 triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree of South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Pat. No. 5,057,540). (StimulonTM QS-21)
  • FIO for information only Filtration is the process of removing contaminants by size exclusion from fluids passing through a membrane filter having a uniform pore size.
  • micron-sized particles can be removed by use of non-membrane or depth materials such as those found in fibrous media, only a membrane filter, having a precisely defined pore size, can ensure quantitative exclusion of particles having a smaller size.
  • membrane filtration it is possible to quantitatively remove bacteria from a solution when passed through a microfiiter and thus effect sterilization.
  • Preferred filters are generally defined as those filters having the capability to remove particles from 0.2 microns up.
  • the membrane filter is uniformly cast on a substrate, and generally performs separations based on size exclusion at the membrane surface.
  • a surface type membrane filter may trap particles both in the depth of its structure and at the surface of the membrane. In size exclusion separations, particles smaller than the pores of the membrane filter pass through while larger particles are retained at the membrane surface. Because these defined pore size filters do not "unload,” (i.e. as the filter begins to fill up with trapped particles, increasing the flow pressure can allow passage of small amounts of trapped solids) they are the devices of choice for microbiological control. "Sterilizing grade" surface type membrane filters are well known in the pharmaceutical industry.
  • the permeability of a filter medium can be affected by the chemical, molecular or electrostatic properties of the filtrate, however it has been found that the hydrophilic microfilters used in the present invention are stable to the high or, conversely, low pH environment depending on the method employed and reliably remove undesired parti culate matter without clogging. It is within the skill level of the ordinary skilled artisan to be able to select and use hydrophilic microfilters.
  • Commercially available product specifications or websites such as: http://millispider.millipore.com/corporate/sitemap.nsf/catalogs (as of May 1999) enable the selection of filters having the desired characteristics.
  • Examples of preferred hydrophilic filters operative in the present invention are Millipore Durapore®, also called Millex GV, (Millipore Corporation, headquartered in Bedford, MA), a polyvinylidene fluoride hydrophilic polymer having good stability and low protein binding characteristics and a 0.22 ⁇ m pore diameter; Millex GNTM, a hydrophilic nylon material having a 0.2 ⁇ m pore size; and Millex GPTM, a hydrophilic surface modified polyethersulfone polymer of pore size 0.22 ⁇ m. More preferred is the Durapore filter owing to its stability at pH 9-9.5.
  • substantially all of the A ⁇ peptide is recovered after filtration.
  • Recovering substantially all the A ⁇ peptide is defined to mean greater than about 50% of the peptide is recovered after sterile filtration. Preferably, greater than 80% of the A ⁇ peptide is recovered after sterile filtration, and most preferably, greater than 90% is recovered after filtration.
  • compositions comprising concentrations of at least O.Olmg/ml of the A ⁇ peptide.
  • Such compositions are prepared by adjusting the pH of the aqueous solution such that the A ⁇ peptide will dissolve therein in requisite concentrations (e.g., at a concentration of from about 0.1 mg/ml to about 2.0 mg/ml).
  • Adjusting the pH of the aqueous solution is accomplished via conventional methods typically exemplified by the addition of either an acid or a base to arrive at the desired pH.
  • the acid or base employed is pharmaceutically acceptable at the amounts employed.
  • a pharmaceutically acceptable buffer in the composition. The selection of a suitable buffer relative to the desired pH is within the skill of the art.
  • the pH of the aqueous composition is adjusted to between about pH 2 to 4 or about pH 8.5 to 12.
  • the A ⁇ peptide is surprisingly quite soluble.
  • acids such as hydrohalide acids (e.g., HCl, HBr), phosphoric acid, citric acid and acetic acid and other pharmaceutically acceptable acids may be employed to lower the pH of the solution to the desired pH. Selection of suitable acids in within the skill of the art.
  • the process of the invention is practiced at high pH (about pH 8.5 to 12), then pharmaceutically acceptable bases such as alkali metals, ammomium hydroxides (e.g., NaOH, NH4OH) and the like may be employed to raise the pH of the solution to the desired pH.
  • pharmaceutically acceptable bases such as alkali metals, ammomium hydroxides (e.g., NaOH, NH4OH) and the like may be employed to raise the pH of the solution to the desired pH.
  • the pH of the solution in a given buffer is preferably adjusted to between 8.5 to 11. More preferred is a pH level of between pH 9 and 10.
  • requisite concentrations of the A ⁇ peptide are added to the solution. It is preferred, of course, to add the A ⁇ peptide after pH adjustment in order to immediately solubilize the peptide. Upon addition, gentle stirring and heating may be necessary to assist in the solubilization process.
  • any optional additives such as pharmaceutically acceptable buffers, tonicity agents, adjuvants, etc. to the composition can occur at any convenient time prior to or subsequent the addition of the A ⁇ peptide.
  • sterile filtration and/or lyophilization can proceed according to procedures well known in the art which procedures are exemplified by the examples below.
  • Preferred Amino Acid Compositions 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) optionally containing one or more tonicity modifiers sufficient for parenteral administration; 10 mM L-Arginine-HCl, or 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) optionally containing one or more tonicity modifiers sufficient for parenteral administration
  • the compositions of the present invention are preferably stored at low temperature to discourage degradation or aggregation of the peptide chains.
  • the preferred temperature range for storage of the A ⁇ peptide compositions is 2 to 8° C
  • a ⁇ 42 as its trifluoroacetate salt was obtained from American Peptide Co., Lot Numbers M05503T1, M10028T1 and used without further modification. Other salts are available and have been successfully employed in the processes of the invention. For examples of alternative counter-ion salts of the A ⁇ peptide see Table 2.
  • the solutions ranked as +++ were visually clear at the target concentration.
  • the pH range was from pH 9 to pH 10 for the buffered solutions.
  • the pH of the inorganic solutions such as NaOH and NH 4 OH were alkaline while HCl and phosphoric acid were strongly acidic. Solubility of the peptide was achieved from 0.6 to 1 mg/ml peptide at pH values approximately > pH 9.
  • Additives such as polysorbate 80, sucrose, mannitol and EDTA did not affect the solubility of the peptide and assist in increasing tonicity and recovery after filtration, or may act as chelating agents.
  • Acidic solutions (approximately pH 4 or below) also solubilize the A ⁇ 42 peptide at 0.6 to 1 mg/ml. Solutions ranked as
  • the peptide may be soluble at a lower concentration than tested herein.
  • a ⁇ 42 (0.6, 1.0, 1.5, 2.0, 3.0 and 3.5 mg/ml) was solubilized in 10 mM sodium glycinate buffer, pH 9.0. Each solution was centrifuged in a bench top centrifuge (>10,000 RPM, ⁇ 10 mins) as cloudy suspensions were observed for concentrations 2.0 - 3.5 mg/ml (solutions of 0.6 - 1.5 mg/ml were visually clear).
  • the A ⁇ 42 peptide was purified as the trifluoroacetate, ammonium, chloride and sodium salts. These salts were dissolved in several buffers, at a 0.45 mg/mL A ⁇ 42 peptide concentration, correcting for the counterion contribution of the various salts. Solutions of the peptide were filtered and the recoveries of the peptides were determined by comparing the RP-HPLC peak areas of the peptide before and after filtration. All salts were soluble and readily filtered at 0.45 mg/mL, as shown in Table 3. Table 3. Solubility of Alternate Salts of the A ⁇ 42 peptide
  • Syringe filters tested (25mm filter diameters, 3.9 cm filter area): Millex GV, 0.22 uM: a hydrophilic polyvinylidene difluoride (PVDF, Durapore®) membrane with low protein binding properties, Millex GN, 0.20 uM: a hydrophilic nylon membrane with low protein binding properties, and Millex GP, 0.22 uM: a hydrophilic surface- modified polyethenesulfone (PES) membrane with low protein binding properties.
  • PVDF polyvinylidene difluoride
  • Millex GN a hydrophilic polyvinylidene difluoride
  • Millex GP 0.22 uM: a hydrophilic surface- modified polyethenesulfone (PES) membrane with low protein binding properties.
  • Example 4 Filtration of A ⁇ 42 in Sodium Glycine and Sodium Lysine Buffers With and Without Additional Excipients.
  • the Millex GV filter was established as a preferred filter for A ⁇ formulations owing to good recoveries of peptide and acceptability for use in commercial processes. Peptide formulations containing 0.9% NaCl were visually less soluble, leading to lower filter recoveries by RP HPLC.
  • the tonicity modifying agents which are saccharides (sugars) display a different type of solution activity, and may favor the maintenance of peptide suspensions. This property, known as water-ordering, tends to "hydrate" the peptide chain in solution so that it adopts its thermodynamically more stable conformation, a ⁇ - pleated sheet.
  • the A ⁇ peptide can be filtered at pH ranges from about 8.5 to about 12, preferably from pH 9 to pH 10 with good recoveries. Filtration can be accomplished with a 0.2 um Millex GV (Millipore Durapore) membrane, which is acceptable for a manufacturing process.
  • Millex GV Millipore Durapore
  • the initial step in the formulation process will preferably include a pH 8.5 to 10 sterile filtration of the peptide.
  • a ⁇ 42 peptide Three lots of A ⁇ 42 peptide were manufactured by American Peptide Co (APC). Confirmatory formulations, chemical and biological characterization of the APC peptide results were reproduced with A ⁇ 42 manufactured by California Peptide Research.
  • mice (4 -8 mice per group) were injected with A ⁇ 42 in various formulations and adjuvanted with CFA/IFA, MPL (Corixa Immunochemicals) or QS 21 (Aquila Pharmaceuticals) at antigen/adjuvant concentrations noted in the particular study. Injections were routinely administered biweekly at 0, 2, and 4 weeks unless otherwise noted. Mice were bled following the 2-week and 4-week injections. Sera were analyzed in a standard sandwich ELISA assay, utilizing A ⁇ 42 as the antigen and HRP-conjugated goat anti-mouse IgG as the reporter antibody.
  • Antibody titers were reported in scatter plots or as geometric means of the data for each of the animals in each group, and generally compared to titers obtained using aggregated A ⁇ 42/CFA/IFA as the control immunogen. See Table 8 and 10 titer results. 1. Formulations.
  • a ⁇ peptide was solubilized at 0.6 mg/ml in 10 mM sodium glycinate, pH 9 - 9.5 buffer and filtered through a Millex GV 0.2 um filter.
  • the formulation was filled at a 0.5 ml volume in 2 cc glass vials (Gensia P N X34-113-002) and capped with gray butyl stoppers (Gensia P/N X66-113-030) and seals. Vials were stored at 2-8°C.
  • HPLC Soluble samples were analyzed either neat or after microcentrifugation of the samples.
  • Table 5 presents the analytical data for the Soluble Liquid Formulation. No difference was seen between samples which were or were not centrifuged prior to analysis, suggesting continuing solubility of the peptide at the intended concentration. The area % purity is comparable to the reference standard: no significant degradation of the peptide has been seen. The soluble formulation has remained stable over a period of 3 months when stored at 2°C to 8°C
  • Characterization of the A ⁇ formulation by circular dichroism demonstrates that the peptide assumes a random coil conformation in solution, with the characteristic negative ellipticity absorbance between 189-205 nm.
  • the pH 9 soluble A ⁇ 42 formulation when injected with adjuvant, raised an antibody titer in Swiss Webster mice. Injections at biweekly intervals, (0, 2, 4 weeks) of 33 ug A ⁇ 42 with either 50 ug MPL or 25 ug QS21 adjuvants elicited an adequate titer response in comparison to controls.
  • a ⁇ peptide was solubilized at 0.6 mg/ml in 10 mM sodium glycinate, pH 9 - 9.5 buffer alone or with various excipients.
  • the peptide solutions were filtered through a Millex GV filter and then titrated with 0.1M acetic acid to pH ⁇ 6 to create a suspension of the peptide in the buffers.
  • the formulations were filled at a 0.5 ml volume in 2 cc glass vials (Gensia P/N X34-1 13-002) and capped with gray butyl stoppers (Gensia P/N X66-113-030) and seals. Formulations were stored at 2-8°C
  • a ⁇ 42 formulations were prepared in 10 mM sodium glycinate containing 0.1 % PS-80 alone or in combination with 5 % sucrose (25 mis).
  • the peptide solutions were were filtered through a Millex GV filter and then titrated to approximately pH 6.0 with 0.1M citric acid.
  • 0.9% sodium chloride was added to the gly cine/citrate/PS 80 after titration to pH 6.
  • the formulations were then vialed at 0.5 ml fills in 2 cc glass vials (Gensia P/N X34-1 13-002) and capped with gray butyl stoppers (Gensia P/N X66-113-030). Formulations were stored at 2-8°C.
  • the Glycine/Citrate formulations were further developed to include buffering capacity at pH 6.
  • Several 0.6 mg/ml A ⁇ peptide formulations at 100 ml were prepared in 10 mM sodium glycinate pH 9, containing 5% sucrose with or without 0.1% PS 80. Additional formulations at 0.1 mg/ml A ⁇ 42 in 10 mM sodium glycinate containing 5% sucrose with or without 0.1% PS 80 were also prepared.
  • the peptide solutions were filtered through Millex GV filters before pH adjustment. The pH was then adjusted to pH 6.0 with 10 mM and 20 mM sodium citrate buffer using a 1 M sodium citrate pH 5.5 stock solution.
  • the concentration and purity of A ⁇ 42 in the formulations were monitored by RP HPLC.
  • Total peptide concentrations of the A ⁇ 42 suspensions were measured by resolubilization of the peptide with a v:v dilution with 2 % sodium dodecyl sulfate (SDS) in 0.01 N NaOH and 1 min heating at 100°C before analysis.
  • SDS sodium dodecyl sulfate
  • concentration of soluble A ⁇ 42 in the suspension formulation was determined by centrifuging the test sample in micro-centrifuge at > 10,000 rpm for 3-5 minutes. Aliquots of either the resolubilized peptide or the supernatant were then analyzed by RP HPLC.
  • Table 6 presents the data for several liquid suspension formulations of A ⁇ 42. All formulations are visually a suspension. These formulations are titrated to pH 6 with the respective acid listed in the table. Depending upon the excipient, a peptide suspension may occur either immediately or over a period of up to 2 weeks or more. More rapid suspension formation can be achieved by addition of sodium chloride or substitution of citrate for acetate in the peptide formulation.
  • Table 7 formulations were prepared with either 1 OmM or 20mM buffering capacity provided with the citrate buffer to assure a pH of 6 is obtained during the manufacturing process. These formulations formed a suspension immediately. Due to the properties of the A ⁇ peptide, the change of conformation of the peptide giving a suspension occurs inside the sterile core. The use of a known buffer concentration, as opposed to titration, allows for ease of handling and reproducibility during operations within a sterile filling suite.
  • Characterization of the A ⁇ 42, pH 6 suspensions by circular dichroism and FT-IR demonstrates that the peptide assumes a beta sheet conformation as the peptide forms a suspension.
  • the beta sheet structures are comparable in the 0.6 mg/ml suspensions, regardless of the buffering acid (citrate, acetate, phosophate) or additional excipients (sucrose, NaCl) added to the formulations.
  • the addition of PS-80 appears to generate a more uniformly dispersed suspension.
  • the A ⁇ peptide suspension when injected with adjuvant, raises an antibody titer in Swiss Webster mice. Injections at biweekly intervals, (0, 2, 4 weeks) as shown in Table 8 elicited an adequate titer response in comparison to the control.
  • the formulations were lyophilized in a programmable Virtis lyophilizer (provided by Gensia Sicor). Mannitol was chosen as the primary matrix component. To achieve crystallization of the mannitol, the formulations were frozen with thermal cycling (annealing), followed by primary and secondary drying of the cakes.
  • the concentration and purity of the lyophilized formulations was monitored by RP HPLC. Each formulation was reconstituted with 1.0 ml water for injection (WFI). Formulations were analyzed immediately after reconstitution (with or without centrifugation in a bench top micro-centrifuge at > 10, 000 rpm for 3-5 minutes) or resolubilized as described previously.
  • Table 9 tabulates the concentration of the four A ⁇ 42 formulations following lyophilization and reconstitution. As can be seen from the data, these formulations were soluble. Samples were analyzed both as the reconstituted formulation, and as the reconstituted formulation "resolubilized" as described for the peptide suspensions. No significant difference was observed in the reconstituted formulation versus the total peptide concentration (resolubilized sample). No significant degradation or loss in peptide was seen over the course of 3 months storage at 2 to 8 °C as determined by RP HPLC as in the stability testing for Example 5.
  • the A ⁇ 42 lyophilized formulation 0.6 mg/ml A ⁇ 42 in lOmM sodium glycinate/HCl/4% mannitol/1% sucrose, pH 7.5, was chosen as a representative lyophilized formulation.
  • This product when mixed with either MPL or QS21 adjuvant, raises an antibody titer in Swiss Webster mice. Geometric mean titers are shown in
  • the peptide suspension was scaled up to a 1.5-liter scale, manufactured and filled at a contract GMP drug product manufacturer. Two concentrations were filled: 0.6 mg/ml and 0.1 mg/ml. Both concentrations were successfully scaled up, filled, and remain stable at 2 - 8°C and at 25 °C after two months.
  • a ⁇ peptide was dissolved at either 0.6 mg/ml or 0.1 mg/ml concentrations in a 10 mM sodium glycinate pH 9 buffer containing 5% sucrose.
  • the peptide solution was sterile filtered through a Millipak 20 Millipore Durapore 0.2 um sterilizing filter, into the aseptic core.
  • RP HPLC analysis demonstrated no significant loss of peptide throughout the filtration process.
  • a I M sodium citrate pH 5.5 buffer was compounded, and likewise sterile filtered through a 0.2 um Durapore filter into the aseptic core.
  • the peptide solution was weighed, and an appropriate amount of sodium citrate buffer added to yield a 20 mM citrate, pH 6 formulation.
  • the peptide immediately formed a suspension upon addition of the citrate buffer; the in-process pH measurement was 6.4.
  • the peptide suspension was constantly stirred, filled at 1.2 ml per vial into 2cc borosilicate glass vials, and sealed with West 4416 stoppers and seals.
  • the 0.1 mg/ml concentration remained soluble (and was filled soluble) for several hours before forming a suspension in the vials by the next day.
  • Table 11 presents the data generated for this 0.6 mg/ml A ⁇ 42 suspension:
  • Solubilization of lyophilized QS-21 with A ⁇ l-42 solution in 10 mM sodium glycinate at pH 9.0 was solubilized with AD 1-42 (TFA salt, 0.45 mg/mL) solution in 10 mM Glycine (Gly), pH 9.0.
  • the pH of the composition containing the solubilized QS-21 was then rapidly adjusted to 6.0 by the addition of citrate buffer (Cit) to form a visually clear suspension.
  • Turbidity measurements were determined with a spectrophotometer set at a wavelength of 405 nm to measure the clarity of the resulting suspensions. The results indicate that the particle size of the suspended peptide is smaller than the wavelength of the reflected light.
  • a ⁇ 42 (2 mg/mL) was solubilized in 10 mM Glycine, pH 9.5. The pH was readjusted to 9.5 with 1 N NaOH. The A ⁇ 42 solution was then filtered through a Millex GV filter. QS-21 (5 mg/mL) was solubilized in 10 mM Citrate, pH 6.0 and was then filtered through Millex GV filter. Aliquots of A ⁇ 42 were mixed with aliquots of QS-21 to give the desired ratio, mixed, and diluted with 10 mM Glycine, pH 9.5 and 1 M Citrate, pH 5.2, to yield the final concentrations noted in Table 12 below.
  • a ⁇ 42, chloride salt was solubilized atl mg/mLin 10 sodium glycinate, , pH 9.0 - 9.5 with or without 5 % sucrose, 0.1 % PS-80, or 0.4 % PS-80.
  • Peptide solutions were sterile filtered through Millex GV syringe filters.
  • Lyophilized QS-21 was solubilized at 5 mg/mL in 10 mM citrate, pH 6.0, and sterile filtered through a Millex GV syringe filter. Appropriate volumes of the A ⁇ 42 solutions and the QS-21 solutions were combined to yield the final concentrations of A ⁇ 42 and QS-21 noted in the formulations in Table XX below.
  • the circular dichroism data were collected using an Aviv model 62-DS spectropolarimeter (Lakewood, NJ). Samples were prepared appropiately for collection of near UV and far UV spectra, respectively, and loaded into a strain-free 1 mm pathlength quartz cell. The sample holder was held at a steady temperature of 25° C. Data were collected at 0.5 nm intervals using a 4 second averaging time. In the far UV region ( ⁇ ⁇ 180-250 nm), signals from the peptide backbone dominate the spectrum and estimates of the secondary structure composition can be obtained. Examination of the near UV ( ⁇ ⁇ 1250-350 nm) region provides quite different information: in this region the primary signals arise from aromatic side chains (Phe, Tyr, and Tip).
  • the sign and magnitude of the signal indicates 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 less than the number of amide groups, and because the chromophores are distributed throughout the molecule, they provide some indication of local structure.

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