US20180200325A1 - Stabilized peptide composition - Google Patents

Stabilized peptide composition Download PDF

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Publication number
US20180200325A1
US20180200325A1 US15/527,938 US201515527938A US2018200325A1 US 20180200325 A1 US20180200325 A1 US 20180200325A1 US 201515527938 A US201515527938 A US 201515527938A US 2018200325 A1 US2018200325 A1 US 2018200325A1
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Prior art keywords
amino acid
seq
acid sequence
peptide
composition according
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Inventor
Takashi Murakami
Kazuyuki TAKATA
Yoshie NIWA
Shuichi HATANO
Hidenori KAWASAKI
Satoko FUJITA
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Shionogi and Co Ltd
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Shionogi and Co Ltd
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Assigned to SHIONOGI & CO., LTD. reassignment SHIONOGI & CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUJITA, Satoko, HATANO, Shuichi, NIWA, Yoshie, TAKATA, Kazuyuki, KAWASAKI, Hidenori, MURAKAMI, TAKASHI
Publication of US20180200325A1 publication Critical patent/US20180200325A1/en
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/04—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is a composition comprising peptides.
  • the present invention is also a composition comprising multiple types of peptides which is stabilized by containing a saccharide and an inorganic salt, especially sucrose and sodium chloride.
  • peptides having cytotoxic T-lymphocyte inducibility are known, which can be used as a vaccine for the treatment or prevention of cancer (Patent Documents 1 to 3). Some peptides generate analogues, which decrease their residual rate in a long term stability test.
  • the effect of the vaccine preparation comprising multiple types of peptides increases depending on the number of the kinds of the peptides. However, it is difficult to dissolve all peptides entirely if an effective amount of multiple types of peptides is included in the same preparation, because the amount of peptides per unit volume increases. Furthermore, it is difficult to keep all peptides stable, because multiple types of peptides having different characteristics are combined.
  • a peptide formulation containing sodium carbonate and/or arginine is disclosed as a peptide formulation used as cancer vaccines (Patent Document 4).
  • a saccharide such as sucrose and inorganic salts such as sodium chloride
  • Patent Document 5 the formulation including inactivated dengue virus, poloxamer detergent, sodium chloride, sucrose and the like
  • Patent Document 7 the formulation including the VIII factors, detergent, calcium chloride, sucrose, sodium chloride, trisodium citrate, buffer containing no amino acid and the like
  • Patent Document 8 the formulation including high concentrated protein, sodium chloride, sucrose and the like
  • Patent Document 8 the formulation including sodium carboxymethylcellulose, sodium chloride, polysorbate 20, Tween 20, 50:50 DL PLG 4A polymer, exendin-4, sucrose and the like
  • the stability of the peptides is greatly different, if the amino acid sequences of the peptides are different. Therefore, even if a peptide formulation is prepared in accordance with the above-disclosed formulation, it is not predictable whether the stability of the peptide in the formulation is improved.
  • the present invention provides a composition comprising one or more kinds of peptides, which is stable and can be preserved for a long time, and which can be dissolved in water.
  • the inventors of the present invention intensively studied and found that multiple types of peptides in the formulation can be dissolved in water by adding a saccharide and an inorganic salt in the formulation. They also provide a stabilized composition.
  • the present invention relates to the following items:
  • a composition comprising one or more kinds of peptides, a saccharide and an inorganic salt, wherein said peptide consists of an amino acid sequence selected from SEQ ID NOs: 1 to 5, or an amino acid sequence that one or two amino acid(s) may be each independently substituted, deleted or added in the amino acid sequence selected from SEQ ID NOs: 1 to 5;
  • the composition of item [1], wherein the second amino acid from the N-terminus of the amino acid sequence of the peptide is phenylalanine, tyrosine, methionine, tryptophan or threonine;
  • step b) a step of adjusting the solution prepared by said step a) to pH 10.0 or more and less than pH 12.0,
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0, and
  • a method of manufacturing the lyophilized formulation which comprises at least the following steps:
  • step b) a step of adjusting the solution prepared by said step a) to pH 10.0 or more and less than pH 12.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0 with an acid substance
  • a method of manufacturing the lyophilized formulation which comprises at least the following steps:
  • step b) a step of adjusting the solution prepared by said step a) to pH 11.0 or more and less than pH 12.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 9.0 or more and less than pH 11.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0 with an acid substance
  • a stable composition which can be preserved a long time, comprising one or more kinds of peptides which can be dissolved in water. Moreover, increasing stability of the peptides is realized by controlling pH of the solution. In other embodiments, increasing stability of the peptides is realized by controlling the concentration of oxygen in the vial.
  • FIG. 1 shows pH dependence of solubility of each peptide.
  • FIG. 2-1 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 1 compared to just after the preparation after the solution of the each composition which was pH 8 to 11 was incubated for 6 hours at 25° C.
  • FIG. 2-2 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 2 compared to just after the preparation after the solution of the each composition which was pH 8 to 11 was incubated for 6 hours at 25° C.
  • FIG. 2-3 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 compared to just after the preparation after the solution of the each composition which was pH 8 to 11 was incubated for 6 hours at 25° C.
  • FIG. 2-4 shows the survival rate of the peptide having an amino acid sequence of SEQ ID NO: 4 compared to just after the preparation after the solution of the each composition which was pH 8 to 11 was incubated for 6 hours at 25° C.
  • FIG. 2-5 shows the survival rate of the peptide having an amino acid sequence of SEQ ID NO: 5 compared to just after the preparation after the solution of the each composition which was pH 8 to 11 was incubated for 6 hours at 25° C.
  • FIG. 3 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 compared to just after the preparation after the lyophilized formulation containing any one of sucrose, trehalose, glucose, dextran or glycerol was incubated for 2 weeks at 40° C. under 75% humidity.
  • FIG. 4 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 compared to just after the preparation after the lyophilized formulation containing 30 mg, 60 mg or 100 mg of sucrose was incubated for 3 months at 40° C. under 75% humidity.
  • FIG. 5-1 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 compared to just after the preparation after the lyophilized formulation containing 30 mg, 60 mg or 100 mg of sodium chloride was incubated for 3 months at 40° C. under 75% humidity.
  • FIG. 5-2 shows the maintained shape of lyophilized formulation containing 2 mg of sodium chloride.
  • FIG. 5-3 shows the complete shrunken shape of lyophilized formulation containing 4 mg of sodium chloride.
  • FIG. 6 shows the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 compared to just after the preparation after the lyophilized formulation whose pH is 8.0, 8.5, 9.0 or 9.5 was incubated for 3 months at 25° C. under 60% humidity.
  • peptide refers to a polymer of 2 to 100 amino acids. This polymer may be linear, branched, or cyclic. The amino acid may be naturally-occurring, non-naturally-occurring, or may be an altered amino acid. This term can also include an assembly of a plurality of peptide chains into a complex. This term also includes natural or artificially altered amino acid polymers. Such alteration includes disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or alteration (e.g. conversion into a bound body with a labeling component). This definition also includes, for example, peptides including one or two or more analogs of amino acids (e.g. including non-naturally-occurring amino acids), peptide-like compounds (e.g. peptoids) and other alterations known in the art.
  • amino acids e.g. including non-naturally-occurring amino acids
  • peptide-like compounds e.g. peptoids
  • peptide cocktail means a composition containing two or more kinds of peptides.
  • amino acid may be naturally-occurring or non-naturally-occurring, as far as the object of the present invention is satisfied.
  • “Substituted” amino acid results from the substitution of one or more amino acids with different amino acids respectively when compared to the reference amino acid sequence (amino acid sequence of the wild type protein).
  • Deleted amino acid is defined as a change in any of the amino acid sequences in any of the amino acid sequences in which one or more amino acid residues are not present respectively when compared to the reference amino acid sequence (amino acid sequence of the wild type protein).
  • Added amino acid is defined as the addition of one or more amino acid residues in the middle, N-terminal or C-terminal amino acid sequence, when compared to the reference amino acid sequence (amino acid sequence of the wild type protein).
  • “Stable peptides in the solution condition” means the survival rate of the peptides is 95% or more, preferably 98% or more after the solution of composition normally which is pH 12 is incubated for 6 hours or more at 25° C. under 60% humidity and the level of increase of analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 is 0.9% or lesser, preferably 0.8% or lesser, more preferably 0.5% or lesser compared to just after the preparation after the solution of composition normally which is pH 11 is incubated for 6 hours or more at 25° C. under 60% humidity
  • “Stable lyophilized peptide formulation” means lyophilized peptide formulation consisting of the peptide having an amino acid sequence of SEQ ID NO: 3 and the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 is 2.0% or lesser, preferably 1.5% or lesser, more preferably 1.0% or lesser compared to just after the preparation after the lyophilized formulation is incubated for 3 months at 25° C.
  • “Stable peptide cocktail” means the peptide cocktail in which the level of increase of the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 is 1.0% or lesser, preferably 0.5% or lesser, more preferably 0.3% or lesser compared to just after the preparation after the lyophilized formulation is incubated for 3 months at 25° C. under 60% humidity or 1.0% or lesser, preferably 0.5% or lesser, more preferably 0.3% or lesser after the lyophilized formulation is incubated for 2 weeks at 40° C. under 75% humidity or 1.5% or lesser, preferably 1.0% or lesser, more preferably 0.5% or lesser after the lyophilized formulation is incubated for 3 months at 40° C. under 75% humidity.
  • “Analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3” includes oxidant and/or dimer of the peptide having an amino acid sequence of SEQ ID NO: 3. Preferably, it means oxidant in this description.
  • Oxidant means the peptide having the sulfinic acid structure by oxidizing the thiol of cysteine constituting the peptide.
  • W/O emulsion or “water-in-oil emulsion” means dispersion of aqueous phase in oil phase.
  • Weight ratio indicates ratio of the target composition against the peptide in the composition. Especially, the ratio of the weight of the target composition when the weight of the peptide is to be 1.
  • Adjuvant enhances the level of immune response in body fluid or cell and lengthens response time. Therefore, frequency of injections and volume of the antigen containing the vaccine can be reduced, and the cost of vaccination can be reduced.
  • Oxygen concentration is the ml volume of oxygen contained in 100 mL of medium such as air. Its value is expressed in %.
  • One dose vial is a vial containing the composition for using for one administration. This means not only in the case of the whole quantity of composition in the vial, but also in the case of the partial quantity of composition in the vial.
  • composition containing the peptide in the present invention can be used as a solution (example: injection) in which the peptide and the following additives are dissolved.
  • the liquid medium of the solution is dried to give as a solid preparation (example: lyophilized preparation). If it is a solid preparation, it is also possible to store the composition for a long time.
  • Examples of peptides included in the present invention include one or more peptides selected from peptides derived from DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1 proteins.
  • the peptide derived from DEPDC1 protein the peptide having an amino acid sequence EYYELFVNI (SEQ ID NOs: 1) is exemplified (Patent Document 1 (WO2008/047473)).
  • the peptide derived from MPHOSPH1 protein the peptide having an amino acid sequence IYNEYIYDL (SEQ ID NOs: 2) is exemplified (Patent Document 1 (WO2008/047473)).
  • the peptide having an amino acid sequence RYCNLEGPPI As the peptide derived from URLC10 protein, the peptide having an amino acid sequence RYCNLEGPPI (SEQ ID NOs: 3) is exemplified (Patent Document 2 (WO2006/090810)).
  • the peptide derived from CDCA1 protein As the peptide having an amino acid sequence VYGIRLEHF (SEQ ID NOs: 4) is exemplified (Patent Document 3 (WO2009/153992)).
  • the peptide derived from KOC1 protein As the peptide derived from KOC1 protein, the peptide having an amino acid sequence KTVNELQNL (SEQ ID NOs: 5) is exemplified (Patent Document 2 (WO2006/090810)).
  • one or more amino acids in the amino acids of these peptides can be substituted, deleted or added, or those one or two amino acids can be substituted.
  • the second amino acid from the N-terminus in the peptides of the present invention is preferably phenylalanine, tyrosine, methionine, tryptophan or threonine, and/or the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methionine.
  • the peptide in the present invention is especially preferably five kinds of peptides consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5
  • a content of peptides in the present formulation for one dose vial is preferably 1.0 to 20 mg, more preferably 2.0 to 15 mg, further preferably 5.0 to 14 mg, especially preferably 10 to 13 mg, more preferably 12 mg.
  • a content of peptides is preferably 9 to 13 mg, especially preferably 10 to 12 mg.
  • the concentration of the peptides is preferably 0.05 to 1.0% by weight, further preferably 0.1 to 1.0% by weight, more preferably 0.3 to 1.0% by weight, further preferably 0.4 to 0.8% by weight, especially preferably 0.6% by weight.
  • the concentration of peptides is preferably 0.45 to 0.65% by weight, especially preferably 0.5 to 0.6% by weight.
  • a saccharide those described in Japanese Pharmacopoeia, Japanese Pharmaceutical Codex, Japanese Pharmaceutical Excipients or the like, may be used.
  • monosaccharides glucose, fructose, ribose, xylose, mannose, maltotriose
  • disaccharides lactose, cellobiose, purified sucrose, maltose, trehalose and the like
  • trisaccharides raffinose and the like
  • sugar alcohols D-sorbitol, inositol, D-mannitol, and the like
  • polysaccharides disaccharides (dextrin, dextran, chondroitin sulfate, hyaluronic acid, dextrin sulfate and the like) and salts thereof (sodium chondroitin sulfate, sodium hyaluronate and the like), cyclic sugars (cyclodextrin, branched cyclodextr
  • the saccharide is one or more selected from the group consisting of monosaccharide, disaccharides and polysaccharide. More preferably, the saccharide is one or more selected from the group consisting of glucose, galactose, fructose, sucrose, lactose, maltose, trehalose, dextran and glycerol. Especially preferably, the saccharide is sucrose.
  • a content of saccharide in the present formulation for one dose vial is preferably 20 to 70 mg, more preferably 40 to 70 mg, further preferably 50 to 70 mg, more preferably 55 to 65 mg, especially preferably 60 mg.
  • the weight ratio of the peptide and the saccharide in the lyophilized composition is preferably 1:1.7 to 1:29.2, further preferably 1:3.3 to 1:5.8, more preferably 1:4.2 to 1:5.8, further preferably 1:4.6 to 1:5.4, especially preferably 1:5.
  • the concentration of the saccharide is preferably 1.0 to 3.5% by weight, further preferably 2.5 to 3.5% by weight, more preferably 2.8 to 3.2% by weight, further preferably 2.9 to 3.1% by weight, especially preferably 3.0% by weight.
  • the content of saccharide in the present formulation is preferably in the above range.
  • the inorganic salt in the present invention is preferably one or more selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium sulfite and sodium metabisulfite, more preferably sodium chloride.
  • a content of inorganic salt in the present formulation for one dose vial is preferably 0.1 to 3.0 mg, more preferably 1.0 to 3.0 mg, further preferably 1.5 to 2.5 mg, especially preferably 2.0 mg.
  • the weight ratio of the peptide and the inorganic salt in the lyophilized composition is preferably 1:0.008 to 1:1.25, further preferably 1:0.083 to 1:0.25, more preferably 1:0.125 to 1:0.208, especially preferably 1:0.17.
  • the concentration of the inorganic salt is preferably 0.01 to 0.3% by weight, further preferably 0.05 to 0.2% by weight, more preferably 0.05 to 0.15% by weight, especially preferably 0.1% by weight.
  • the amount of the inorganic salt contained in the composition of the present invention is lower than the above range, the stability of the peptide is reduced and the amount of impurities derived from the peptide may be increased, especially the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 may be increased.
  • the amount of the inorganic salt contained in the composition of the present invention exceeds the above range, poor freeze-drying occurs and the shape of the lyophilized product (cake) becomes bad when the composition of the present invention is freeze-dried. Therefore, the amount of the inorganic salt in the composition is preferably in the above range.
  • pH control agents those described in Japanese Pharmacopoeia, Japanese Pharmaceutical Codex, Japanese Pharmaceutical Excipients or the like, may be used.
  • carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid, sodium hydroxide, arginine, lysine and salts thereof, meglumine and the like can be used.
  • hydrochloric acid and sodium hydroxide can be used.
  • the amount of these pH adjusting agents in the composition may be any amount as long as it can be adjusted to a predetermined pH value.
  • the pH is measured according to the pH measurement method described in the general test method of the Japanese Pharmacopoeia.
  • the pH value is measured with a pH meter (HORIBA pH METER F-52) manufactured by HORIBA Ltd. with the range of 20° C. to 25° C.
  • pH of the solution is preferably 7.0 to 9.5, more preferably 8.0 to 9.5, further preferably 8.2 to 9.0, especially preferably 8.4 to 8.6.
  • the stability of the peptide is reduced and the amount of impurities derived from the peptide, especially the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 may be increased.
  • the solubility of the peptide contained in the composition of the present invention decreases and deposited peptide may appear.
  • the peptides having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 may have low solubility in lower pH below the above range and they may become deposited.
  • composition of the present invention is a composition wherein the saccharide is sucrose and the inorganic salt is sodium chloride.
  • composition of the present invention comprises a peptide having the amino acid sequence represented by SEQ ID NO: 1, a peptide having the amino acid sequence represented by SEQ ID NO: 2, a peptide having the amino acid sequence represented by SEQ ID NO: 3, a peptide having the amino acid sequence represented by SEQ ID NO: 4, a peptide having the amino acid sequence represented by SEQ ID NO: 5, sucrose, and sodium chloride.
  • a further preferable composition of the present invention comprises 1.0 to 2.4 mg of the peptide having the amino acid sequence represented by SEQ ID NO: 1, 1.0 to 2.4 mg of the peptide having the amino acid sequence represented by SEQ ID NO: 2, 1.0 to 2.4 mg of the peptide having the amino acid sequence represented by SEQ ID NO: 3, 1.0 to 2.4 mg of the peptide having the amino acid sequence represented by SEQ ID NO: 4, 1.0 to 2.4 mg of a peptide having an amino acid sequence represented by SEQ ID NO: 5, 30 to 60 mg of sucrose and 1 to 2 mg of sodium chloride.
  • the composition is a solution formulation, a lyophilized formulation, an emulsion formulation or the like.
  • it is a lyophilized preparation.
  • the lyophilization is carried out according to a known method by a freeze dryer (manufactured by Kyowa Vacuum Engineering Co., Ltd.).
  • the peptide(s) is(are) dissolved in water such as distilled water, purified water, preferable purified water for injection, physiological saline or the like to be the concentration of one type of peptide is 2.0 mg/ml (10 mg/ml when 5 kinds of peptides are contained), and the resulting mixture is mixed with an oil phase such as an adjuvant to prepare a water-in-oil (W/O) emulsion.
  • W/O water-in-oil
  • the present composition is oral drug or injectable drug but preferably injectable drug.
  • the concentration of oxygen in a vial in the case of the present composition is encapsulated into the vial is preferably 0.01% or more and less than 3.0%, more preferably 0.01% or more and less than 2.0%, further preferably 0.01% or more and less than 1.5%, especially preferably 0.01% or more and less than 1.0%.
  • the stability of the peptides deteriorate and the amount of impurities derived from peptides may increase, especially a peptide consisting of an amino acid sequence of SEQ ID NO: 3). Therefore, the stability of a peptide consisting of an amino acid sequence of SEQ ID NO: 3) deteriorates under the presence of oxygen.
  • the concentration of oxygen in a vial can be maintained in the above range over a long duration when the vial for freeze-dried formulation of the present invention is preserved hermetically with bag such as aluminum pouch.
  • the concentration of oxygen can be measured non-destructively by irradiation of laser to the gas in the vial and by monitoring of the amount of absorption using non-destructive headspace-analyzer (manufactured by Lighthouse Instruments and the like).
  • the present invention provides a kit including any one of (a) freeze-dried formulation of composition of the present invention, (b) reconstructed solution consisting of freeze-dried formulation of composition of the present invention or (c) water-in-oil emulsion formulation mixing reconstructed solution consisting of freeze-dried formulation of composition of the present invention and an adjuvant.
  • composition of the present invention included as freeze-dried formulation in a kit of the present invention may be any one of the composition above.
  • the present invention provides a method of manufacturing the lyophilized formulation which comprises at least the following steps:
  • step b) a step of adjusting the solution prepared by said step a) to pH 10.0 or more and less than pH 12.0,
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0, and
  • the present invention provides a method of manufacturing the lyophilized formulation which comprises at least the following steps:
  • step b) a step of adjusting the solution prepared by said step a) to pH 10.0 or more and less than pH 12.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0 with an acid substance
  • the present invention provides a method of manufacturing the lyophilized formulation which comprises at least the following steps:
  • step b) a step of adjusting the solution prepared by said step a) to pH 11.0 or more and less than pH 12.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 9.0 or more and less than pH 11.0 with an acid substance
  • step c) a step of adjusting the solution prepared by said step c) to pH 7.0 or more and less than pH 9.0 with an acid substance
  • the solution of pH 12.0 or more and less than pH 13.0 of above step a), the solution of pH 10.0 or more and less than pH 12.0 of above step b), and the solution of pH 7.0 or more and less than pH 9.0 of above step d) are able to be adjusted by pH adjustment agents.
  • the present invention also provides a method of preparing the lyophilized formulation including a step of controlling to 0.01% or more and less than 3.0% of the oxygen concentration in a vial of the lyophilized formulation obtained from above final step. This step can increase stability of the peptide (especially, the peptide represented by the amino acid sequence of SEQ ID NO: 3).
  • the present invention also provides a lyophilized formulation prepared by above preparation method of the lyophilized formulation.
  • the present invention also provides a composition comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or two amino acid(s) may be substituted, deleted or added in the amino acid sequence of SEQ ID NO: 3, and the oxygen concentration is 0.01% or more and less than 3.0% in the vial.
  • the analogues derived from the peptide having an amino acid sequence of SEQ ID NO: 3 may be increased when the oxygen concentration is 3.0% or more. Therefore, the peptide having an amino acid sequence of SEQ ID NO: 3 is preferably conserved under low oxygen concentration conditions since the peptide is affected by oxygen.
  • composition of the present invention obtained under the above condition is excellent in stability in long term storage. Therefore, the composition of the present invention is useful for a formulation that exerts a stable medicinal effect with excellent storage stability.
  • pH of the prepared solution was adjusted to 8.4-8.6 with hydrochloride solution, and the total solution weight was adjusted to 12500 g by adding water for injection.
  • Sterile filtration was performed for the prepared solution described above, 2.0 g of the solution was dispensed into 3 mL glass vial in each, and the dispensed vials were lyophilized.
  • the lyophilized sample described above was sealed with the glass vial and stored at 25° C. 60% RH or 40° C. 75% RH for the specified period. Then, appearance, contents, and related substances of the sample were evaluated.
  • Standard solution for measuring content of the peptide described as SEQ ID NO: 1 or SEQ ID NO: 2 was prepared in each by dissolving the peptide described above into 1.6 mg/mL phosphate solution and by adjusting each peptide concentration into 0.15 mg/mL.
  • Standard solution for measuring content of the peptide described as SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 was prepared in each by dissolving the peptide described above into 12.1 mg/mL of 2-amino-2-hydroxymethyl-1,3-propanediol aqueous solution and by adjusting each peptide concentration to 0.15 mg/mL.
  • column with octadecyl group was used, and the measurement was performed by gradient elution method. Flow rate in the measurement was set to 1.0 mL/min.
  • Solubility of the peptides described as SEQ ID NOs: 1-5 was evaluated with the condition of pH 2-12. Hydrochloride solution or sodium hydroxide solution was added to water for injection to adjust pH, and each peptide was mixed with the adjusted solution, where the peptide concentration was set to 3 mg/mL. The mixed solution was filtered, the filtered solution was diluted with 10-20 parts of water for injection, and peak area of each peptide was obtained by liquid chromatography (same test procedure described above). From peak area of each peptide, dilution ratio of the filtered solution, peak area and concentration of each standard solution described above, peptide concentration was obtained. Solubility of each peptide against pH was shown in FIG. 1 . It was observed that solubility of the peptides described as SEQ ID NO: 1 and SEQ ID NO: 2 decreased with decreasing pH of the solution. Solubility of the peptides described as SEQ ID NO: 4 was low when solution pH was around neutral.
  • the peptide described as SEQ ID NO: 1 or SEQ ID NO: 2 was dissolved into the solution with pH10 and solubility was examined by decreasing the solution pH in each. 15 mg of the peptide described as SEQ ID NO: 1 or SEQ ID NO: 2 was mixed with water, 0.1 mol/L sodium hydroxide solution was added for dissolution, and pH was adjusted to 10. Peptide concentration was adjusted to 1.5 mg/g. In each sample solution, hydrochloride solution was added to decrease pH gradually, and the appearance of the solution was checked. In the peptide described as SEQ ID NO: 1, when pH of the solution was 5.6 or below, white turbidity was observed in the solution (Table 1-1). In the peptide described as SEQ ID NO: 2, when pH of the solution was 5.5 or below, white turbidity was observed in the solution (Table 1-2).
  • Each sample was prepared with the concentration in Table 3, pH was adjusted to 8 with hydrochloride solution, and 2.5 g of each solution shown in Table 3 was dispensed into 3 mL vial. The dispensed vials were lyophilized.
  • the lyophilized sample was stored for 2 weeks at 40° C.75% RH, and the level of increase of the related substances derived from the peptide described as SEQ ID NO: 3 after storage was measured against the ones when the lyophilized sample was prepared. The results were shown in FIG. 3 . From the results, it was found that sucrose was the most effective for stabilizing formulations of the peptide mixtures, as the level of increase of the related substances was the lowest.
  • Each sample was prepared with the concentration in Table 4, and pH was adjusted to 8.5 with hydrochloride solution. 2.0 g of each solution shown in Table 3 was dispensed into 3 mL vial, and the dispensed vials were lyophilized. The lyophilized sample was stored for 3 months at 40° C.75% RH, and the level of increase of the related substances derived from the peptide described as SEQ ID NO: 3 after storage was measured against the ones when the lyophilized sample was prepared. The results were shown in FIG. 4 .
  • sucrose amount was 60 mg for a vial (30 mg/g as concentration of the solution), the level of increase of the related substances was lower than any others, and that the amount of sucrose impacted the stabilization of the formulation of the peptide mixtures.
  • compositions were mixed according to the Table 5-1, each solution was adjusted to pH 8.5 with hydrochloric acid aqueous solution, and 2.0 g of the resultant solution was dispensed into a 3 mL glass vial and lyophilized.
  • the prepared lyophilized products were stored at 40° C.75% RH for 3 months, and the level of increase of the related substances derived from the peptide described as SEQ ID NO: 3 was measured. The results are shown in FIG. 5 . From the results, it was found that the composition that contains 4.0 mg of sodium chloride per vial (2.0 mg/g as concentration of the solution) contributes to stabilization of the peptide mixtures better because the level of increase of the related substances was smaller.
  • FIG. 5-3 is the photo of the product which contains 2 mg/g of sodium chloride and was completely shrunken. From the results, it was found that the composition that contains 2 mg of sodium chloride per vial (1.0 mg/g as concentration of the solution) is more appropriate for lyophilization in terms of appearance of the products because the number of the completely shrunken product was less.
  • the above composition was mixed according to the Table 6 and adjusted to pH 8.0, 8.5, 9.0 or 9.5 with hydrochloric acid aqueous solution, and 2.0 g of the resultant solution was dispensed into a 3 mL glass vial and lyophilized.
  • the prepared lyophilized products were stored at 25° C.60% RH for 3 months, and the level of increase of the related substances derived from the peptide described as SEQ ID NO: 3 was measured. The results are shown in FIG. 6 . From the results, it was found that the formulation of which solution pH is 8.0 contributes to stabilization of the peptide mixtures because the level of increase of the related substances is smaller.
  • pH 8.5 is the best as pH of the solution of the peptides. It was decided that peptides are to be dissolved in the following order; peptides which has good stability in the solution state (the peptides described as SEQ ID NO: 1 and SEQ ID NO: 4) first, then peptides which have poor stability in the solution state (the peptides described as SEQ ID NOs: 2, 5 and 3). The peptides which have good stability in the solution state should be dissolved preferably in the order of the peptide described as SEQ ID NO: 1 and the peptide described as SEQ ID NO: 4. And it was concluded that the peptides which have poor stability in the solution state should be dissolved preferably in the order of peptides described as SEQ ID NOs: 2, 5 and 3.
  • the lyophilized products were stored at 25° C. for 3 months, oxygen concentration in the vials was measured with a non-destructive headspace analyzer and the related substances derived from the peptide described as SEQ ID NO: 3 were measured. The results are shown in Table 7-2. From the results, it was found that the lower the oxygen concentration, the less the level of increase of related substances derived from the peptide described as SEQ ID NO: 3. Therefore it was concluded that oxygen concentration in the vials should preferably be less than 3.0% in terms of stability.
  • the present invention is useful for a composition including peptides, especially a peptide vaccine formulation including peptides for treatment of cancer.

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