JP6671141B2 - Suspension - Google Patents

Description

  The present invention belongs to the field of cancer immunotherapy, and relates to a suspension and a lyophilized preparation containing a WT1 protein-derived cancer antigen peptide having cytotoxic T cell-inducing activity. Regarding stabilization.

Generally, the WT1 protein-derived cancer antigen peptide is a partial peptide derived from human WT1 protein (SEQ ID NO: 2) consisting of 449 amino acids, specifically, a peptide having 8 to 12 amino acids or a dimer thereof, Antigen recognition by major histocompatibility complex (MHC) class I antigen and presented by cytotoxic T cells (cytotoxic T lymphocytes, Cytotoxic T-lymphocyte, Cytotoxic T-cell; hereinafter CTL). Containing the peptide to be prepared. MHC is called human leukocyte antigen (HLA) in humans.

From the amino acid sequence represented by the WT1 _235-243 peptide Cys-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 3) consisting of 9 amino acids in the partial peptide derived from the WT1 protein It has been reported that a partial peptide, a variant in which some amino acids of the peptide are modified (modified peptide), and a dimer of the peptide are useful as a peptide that binds to HLA and induces CTL ( Patent Documents 1 to 4). Examples of the modified peptide and its dimeric peptide include a peptide having an amino acid sequence represented by Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 1); A peptide represented by the formula (a cystine form of the peptide having the amino acid sequence represented by SEQ ID NO: 1) and a peptide represented by the formula (2) (a dimer of the peptide having the amino acid sequence represented by SEQ ID NO: 1) Peptide).

  In general, cancer antigen proteins and cancer antigen peptides used for cancer vaccines are often administered together with an adjuvant (immune enhancer) for the purpose of more efficiently inducing CTL. Therefore, it is significant to develop emulsions, solutions or suspensions containing peptide-containing compounds that can easily combine cancer antigen proteins and cancer antigen peptides with various adjuvants according to the purpose.

  Patent Documents 3 to 5 disclose emulsion preparations containing the above-mentioned modified peptide and its dimer peptide as an active ingredient, but all documents disclose the dispersion state and stability of the peptide in the preparation. There is no.

  The solubility of the peptide in water or aqueous solution at physiological pH (2-7.5) is less than 0.5 mg / mL, and the peptide is considered substantially insoluble in water or aqueous solution.

In general, techniques used to formulate a drug that is substantially insoluble in water or an aqueous solution (poorly soluble in water or poorly water-soluble) include organic solvents, surfactants, strongly acidic or strongly basic components, and the like. And a solubilization technique using However, when this technique was applied to the above-mentioned peptide, it was found that when a solution was formed using the solubilization technique, impurities such as an oxidized form and a deamino form were generated immediately after dissolution, and the purity was reduced.
Further, an emulsification technique in which a drug is dispersed or dissolved in an oily raw material and then dispersed in an aqueous phase using a component such as an emulsifier may be used. However, the chemical stability of the peptide is not good, and it is known that oxidized forms and dimers are formed under refrigerated storage even in the state of the drug substance alone, and the purity is reduced. It has also been difficult to apply emulsification techniques involving oily raw materials and emulsifiers that are likely to occur.

  Finally, application of a suspension technique in which a drug is dispersed in a water-soluble solvent in a suspended state can be mentioned. In this case, although chemical stability is ensured, there is a problem in terms of physicochemical stability, such as a phenomenon in which particles precipitated during storage adhere to the bottom of the container and a phenomenon in which a large aggregate is generated. It is known. It is considered that such a physicochemical change makes it difficult to administer an appropriate amount or makes it impossible to obtain a desired medicinal effect.

  Patent Document 6 discloses a sterile suspension of a basic peptide complex and a freeze-dried preparation that hardly dissolve, but when applied to the above-mentioned peptide, the precipitated peptide rapidly aggregates, precipitates, and becomes uniform. No suspension could be obtained.

International Publication No. 00/06602 International Publication No. 02/079253 International Publication No. 2004/063217 International Publication No. 2007/063903 International Publication No. 2004/024175 JP 2008-524284 A

An object of the present invention is to provide a partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a cystine form of the peptide (a peptide represented by the formula (1)) or a dimer peptide of the peptide (a formula (2)) A peptide having improved suspension stability of the peptide which can be used for the preparation of a cancer vaccine preparation containing the peptide.
Another object of the present invention is to provide a freeze-dried preparation obtained by freeze-drying the suspension, to ensure the stability of the peptide, and to provide a freeze-dried preparation having good redispersibility when the suspension is prepared.

  As a result of intensive studies to solve the above problems, the present inventors have found that a partial peptide having the sequence represented by SEQ ID NO: 1, a cystine form of the peptide (a peptide represented by the formula (1)) or a A suspension containing a dimeric peptide (a peptide represented by the formula (2)) (hereinafter, sometimes simply referred to as the peptide of the present invention) contains an anionic polymer, so that it can be treated for 1 day. It has been found that a good suspension state is maintained even after placing. In addition, they have found that by containing a water-soluble anionic polymer, a particle size that can be sterilized or sterilized using a filter is secured. Further, they have found that a suspension and / or a lyophilized preparation having excellent preparation stability can be obtained by containing a sugar or a sugar alcohol, thereby completing the present invention.

  That is, the present invention relates to the following.

Item 1. An injectable pharmaceutical composition comprising the following components:
(A) a peptide having an amino acid sequence represented by Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 1);

(In the formula, Leu-OH indicates that the C-terminal of Leu is a free carboxyl group.)
A peptide represented by the formula (2):

(In the formula, the bond between Cys and Cys represents a disulfide bond, Leu-OH has the same meaning as described above, and the other bonds represent peptide bonds.)
Or one or more peptides selected from salts thereof, and (b) an anionic polymer.

Item 2. Item 2. The composition according to Item 1, wherein the component (a) is at least one peptide selected from the peptide represented by the formula (1), the peptide represented by the formula (2), or a salt thereof.

Item 3. Item 2. The composition according to Item 1, wherein the component (a) is a peptide represented by the formula (1).

Item 4. Item 4. The composition according to any one of Items 1 to 3, wherein component (b) is a water-soluble anionic polymer.

Item 5. Item 5. The composition according to any one of Items 1 to 4, wherein component (b) is an anionic polymer having a carboxylic acid.

Item 6. Item 6. The composition according to any one of Items 1 to 5, wherein component (b) is one or more selected from the group consisting of carmellose sodium, sodium alginate, xanthan gum and gum arabic.

Item 7. Item 7. The composition according to item 6, wherein component (b) is one or more selected from the group consisting of carmellose sodium, sodium alginate and xanthan gum.

Item 8. Item 8. The composition according to Item 7, wherein component (b) is carmellose sodium.

Item 9. Item 9. The composition according to any one of Items 1 to 8, further comprising one or more components (c) selected from the group consisting of sugars and sugar alcohols.

Item 10. Item 10. The composition according to item 9, wherein component (c) is one or more selected from the group consisting of mannitol, xylitol, trehalose, and sucrose.

Item 11. Item 10. The composition according to Item 9, wherein the component (c) is mannitol or xylitol, and trehalose.

Item 12. Item 10. The composition according to Item 9, wherein component (c) is mannitol and trehalose.

Item 13. Item 11. The composition according to any one of Items 1 to 10, wherein the composition is a suspension or a lyophilized preparation.

Item 14. Item 13. A suspension containing the components according to any one of Items 1 to 12, wherein the component (b) is 0.1 to 20 mg in 1 mL of the suspension.

Item 15. Item 13. A suspension comprising the components according to any one of Items 9 to 12, wherein the component (c) is 1 to 200 mg in 1 mL of the suspension.

Item 16. Item 16. The suspension according to Item 15, wherein the component (b) is 0.1 to 20 mg in 1 mL of the suspension.

Item 17. Item 17. The suspension according to any one of Items 14 to 16, wherein the component (a) is 0.5 mg to 200 mg in 1 mL of the suspension.

Item 18. Item 18. A freeze-dried preparation produced by freeze-drying the suspension according to any one of Items 14 to 17.

Item 19. Item 18. The composition according to any one of Items 1 to 12 or 14 to 17, which is a suspension produced by a step comprising the following steps using the components:
(1) a step of dissolving the component (a) in an alkaline solvent,
(2) a step of adding an acidic solvent to a solution of the component (a) obtained in the step (1) to precipitate the component (a);
(3) a step of separating and recovering the component (a) precipitated in the steps (1) and (2) from the solvent, and a step (4) of the component (a) recovered in the steps (1) to (3). Is redispersed in a solution containing the component (b) and, if necessary, the component (c), and then crushed and sized.

Item 20. Item 20. The composition according to Item 19, further comprising the following step (5):
(5) A step of reducing the number of microorganisms by filtering the suspension obtained in steps (1) to (4) through a filter having a pore size of 0.1 to 0.5 μm.

Item 21. Item 21. A freeze-dried preparation obtained by freeze-drying the composition according to item 19 or 20.

Item 22. 18. The method for producing a composition according to any one of Items 1 to 12 and 14 to 17, which is a suspension, comprising the following steps (1) to (4).
(1) a step of dissolving the component (a) in an alkaline solvent,
(2) a step of adding an acidic solvent to a solution of the component (a) obtained in the step (1) to precipitate the component (a);
(3) a step of separating and recovering the component (a) precipitated in the steps (1) and (2) from the solvent, and a step (4) of the component (a) recovered in the steps (1) to (3). Redispersing in a solution containing component (b) and, if necessary, component (c), and then pulverizing and sizing.

Item 23. Item 23. The production method according to Item 22, further comprising the following step (5):
(5) A step of reducing the number of microorganisms by filtering the suspension obtained in steps (1) to (4) through a filter having a pore size of 0.1 to 0.5 μm.

Item 24. Item 24. A method for producing a freeze-dried preparation by freeze-drying a composition obtained by the method according to Item 22 or 23.

Item 25. Item 13. A cancer vaccine composition comprising the composition according to any one of Items 1 to 12.

Item 26. Item 18. A cancer vaccine composition comprising the suspension according to any one of Items 14 to 17.

Item 27. Item 19. A cancer vaccine composition comprising the freeze-dried preparation according to item 18.

  By using the suspension of the present invention, a suspension containing the peptide of the present invention having cytotoxic T cell-inducing activity stably can be produced, and a cancer vaccine excellent in formulation stability is prepared. be able to. Further, according to the present invention, when the suspension is lyophilized into a lyophilized preparation, a lyophilized preparation with stable peptide retention and good redispersibility can be prepared.

FIG. 1 is a diagram showing the test results obtained for the CTL inducing activity in Test Example 6.

  Hereinafter, embodiments of the present invention will be described in detail.

  In this specification, each exemplified preferred embodiment may be combined with other exemplified preferred embodiments, and may be incorporated into corresponding examples described in the above items 1 to 27.

  The `` suspension agent '' in the present invention is a liquid agent for preparing a cancer vaccine, wherein the main solvent is water, the peptide of the present invention is in suspension, and can be mixed with various adjuvants. . The `` solvent '' in the present invention generally includes water, but a pharmaceutically acceptable solvent such as propylene glycol and polyethylene glycol can be partially mixed with water as long as the effect of the present invention is not affected. . Preferably only water.

  The “lyophilized preparation” in the present invention is a freeze-dried “suspension” in the present invention. It is usually performed to improve the chemical and physical stability of the peptide in the “suspension”. When used as a cancer vaccine, an appropriate amount of water is added and stirred to form a suspension, which is then mixed with various adjuvants to prepare a cancer vaccine.

The “peptide” in the present invention is a cancer antigen peptide for preparing a cancer vaccine, and is an amino acid represented by Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 1) A peptide consisting of the sequence, formula (1):

(Wherein Leu-OH indicates that the C-terminus of Leu is a free carboxyl group), formula (2):

Wherein the bond between Cys and Cys represents a disulfide bond, Leu-OH has the same meaning as described above, and the other bonds represent peptide bonds. It is a peptide that is more selected. Preferably, it is a peptide represented by the formula (1).

“A peptide consisting of an amino acid sequence represented by Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 1)” is a protein that is a gene product of the Wilms tumor tumor suppressor gene WT1. In particular, of the partial peptide derived from the human WT1 protein (SEQ ID NO: 2) consisting of 449 amino acids, Cys-Met-Thr-Trp-Asn-Gln-Met- consisting of 9 amino acids Asn-Leu (SEQ ID NO: 3) consisting of the amino acid sequence represented by the sequence of the peptide (WT1 _2
35-243 peptide) in which the second amino acid from the N-terminus is changed from methionine to tyrosine. The peptide is a peptide presented to an MHC class I antigen and recognized by CTL.

The “peptide represented by the formula (1)” includes a cystine form in which a sulfur atom of cysteine is cystine-bonded to a sulfur atom of a cysteine residue of the peptide having the amino acid sequence represented by SEQ ID NO: 1. The peptide is a peptide presented to an MHC class I antigen and recognized by CTL.

The “peptide represented by the formula (2)” includes a dimer in which sulfur atoms of cysteine residues of a peptide having two amino acid sequences represented by SEQ ID NO: 1 are cystine-bonded. The peptide is a peptide presented to an MHC class I antigen and recognized by CTL.

  The salt comprising the amino acid sequence represented by SEQ ID NO: 1, the peptide represented by the formula (1), or the peptide represented by the formula (2) is not particularly limited as long as it is a pharmaceutically acceptable salt. . The “salt” in the present invention includes an acid addition salt and a base addition salt. For example, acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, citrate, oxalate, acetate, and formate. , Propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, and other organic acid salts. , Sodium salts, potassium salts, calcium salts, magnesium salts, inorganic base salts such as ammonium salts, triethylammonium salts, triethanolammonium salts, pyridinium salts, organic base salts such as diisopropylammonium salts, and the like. Amino acid salts such as basic or acidic amino acids such as aspartic acid and glutamic acid are exemplified.

  Peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, peptide represented by the formula (1) or a salt thereof, hydrate of the peptide represented by the formula (2) or a salt thereof, ethanol solvent Solvates such as solvates are also included in the peptides of the present invention. Furthermore, the peptide of the present invention includes all possible stereoisomers, such as all diastereomers, enantiomers, and the like, of the peptide, and crystal forms of all aspects.

  The peptide of the present invention or a salt thereof can be produced by a known method (see Patent Documents 2 to 4 and the like). Further, the peptide of the present invention or a salt thereof can be produced by a method usually used in the art. See, for example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc. , New York, 1976; Peptide synthesis, Maruzen Co., Ltd., 1975; Basics and experiments of peptide synthesis, Maruzen Co., Ltd. 1985; Development of pharmaceuticals Continued, Vol. 14, Peptide synthesis, Hirokawa Shoten, 1991, etc. It can be synthesized by a peptide synthesis method.

  In the suspension of the present invention, the content of “peptide” per volume is not particularly limited, and may be any content per volume that is pharmacologically or physically acceptable. Among the “peptides”, for example, a preferable content of the peptide represented by the formula (1) is 0.5 mg to 200 mg / mL, more preferably 0.5 mg to 100 mg / mL. May be selected according to

  In the freeze-dried preparation of the present invention, the content of “peptide” per volume is not particularly limited. The freeze-dried preparation of the present invention is obtained by freeze-drying the suspension of the present invention, and the content of the peptide may be any as long as it satisfies the content of the suspension.

In the present specification, the left side of the above peptide is the N-terminus, and each amino acid symbol indicates that the following amino acid residue.
Ala or A: alanine residue
Arg or R: arginine residue
Asn or N: asparagine residue
Asp or D: Aspartic acid residue
Cys or C: cysteine residue
Gln or Q: glutamine residue
Glu or E: glutamic acid residue
Gly or G: glycine residue
His or H: histidine residue
Ile or I: isoleucine residue
Leu or L: Leucine residue
Lys or K: lysine residue
Met or M: methionine residue
Phe or F: phenylalanine residue
Pro or P: Proline residue
Ser or S: Serine residue
Thr or T: Threonine residue
Trp or W: tryptophan residue
Tyr or Y: tyrosine residue
Val or V: Valine residue

  Generally, "water-soluble" refers to a component that is soluble in water. “Water-soluble” in the present invention means having a solubility of 1 mg / mL or more.

  Generally, “anionic polymer” refers to a polymer containing a cationic metal ion. As the `` anionic polymer '' in the present invention, for example, carmellose sodium, carmellose potassium, carmellose calcium, sodium alginate, potassium alginate, calcium alginate, xanthan gum, gum arabic, sodium hyaluronate, pectin, carboxyvinyl polymer, Tragacanth, carrageenan, chondroitin sodium sulfate and the like. Carmellose sodium, carmellose potassium, sodium alginate, potassium alginate, xanthan gum, gum arabic, sodium hyaluronate, pectin, carrageenan, or sodium chondroitin sulfate, which are preferably water-soluble. More preferably, it is carmellose sodium, sodium alginate, xanthan gum, or gum arabic, even more preferably, it is sodium carmellose, sodium alginate, or xanthan gum, and most preferably, it is sodium carmellose.

The expression "having a carboxylic acid" in the present invention means that the anionic polymer contains a free carboxyl group which becomes a carboxylate ion in an aqueous solution. Examples of the anionic polymer containing a carboxylic acid include carmellose sodium, carmellose potassium, carmellose calcium, sodium alginate, potassium alginate, calcium alginate, xanthan gum, gum arabic, sodium hyaluronate, pectin, carboxyvinyl polymer, and tragacanth. And sodium chondroitin sulfate. Carmellose sodium, carmellose potassium, sodium alginate, potassium alginate, xanthan gum, gum arabic, sodium hyaluronate, pectin, or sodium chondroitin sulfate, which are preferably water-soluble. More preferably, it is carmellose sodium, sodium alginate, xanthan gum, or gum arabic, even more preferably, it is sodium carmellose, sodium alginate, or xanthan gum, and most preferably, it is sodium carmellose.

  In the suspension of the present invention, the content of “anionic polymer” per volume is not particularly limited, but is preferably 0.1 to 20 mg / mL, more preferably 0.5 to 10 mg / mL, Most preferably, it is 1 to 5 mg / mL.

  In general, “sugar” refers to a monosaccharide having a polyhydroxylated aldehyde or ketone and a compound in which two or more monosaccharides are linked by glycosidic bonds, and “sugar alcohol” refers to a ketone group or an aldehyde of a monosaccharide. Represents a compound in which the group has been reduced to an alcohol. Examples of the “sugar or sugar alcohol” in the present invention include mannitol, xylitol, trehalose, sucrose (sucrose), lactose, fructose, erythritol and the like. Preferably, it is mannitol, xylitol, trehalose, and / or sucrose.

  In the suspension of the present invention, the content of “sugar or sugar alcohol” per volume is not particularly limited, but is preferably 1 to 200 mg / mL, more preferably 10 to 150 mg / mL, and most preferably 50 to 150 mg / mL. １００100 mg / mL.

  The sugar or sugar alcohol can be contained alone, but can also be contained in combination of two or more sugars or sugar alcohols. Although there is no particular limitation on the combination, preferred combinations are mannitol and trehalose, and xylitol and trehalose, and more preferred combinations are mannitol and trehalose.

  The ratio of the content of two or more sugars or sugar alcohols is not particularly limited, but the ratio of mannitol and trehalose is preferably 1: 2 to 100: 1, and more preferably 1: 1 to 10: 1. The ratio of xylitol and trehalose is preferably from 1: 2 to 100: 1, more preferably from 1: 1 to 10: 1.

  The pH of the suspension of the present invention is from 3 to 7.5, but if it is not the desired pH at the time of production, it can be adjusted usually by using a pH adjuster. From the viewpoint of stability, a preferred pH is 4 to 7, and a more preferred pH is 5 to 7.

  The pH adjuster is appropriately selected from pH adjusters generally used in pharmaceutical preparations. Specifically, hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, tartaric acid, lactic acid, maleic acid, phosphoric acid, sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium acetate hydrate, anhydrous sodium acetate, sodium citrate Hydrates, sodium dihydrogen citrate, sodium tartrate, disodium phosphate, dipotassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, trisodium phosphate and the like can be mentioned. Preferred are hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide and potassium hydroxide, and more preferred are hydrochloric acid and / or sodium hydroxide.

  In addition, the suspension of the present invention, in addition to the above components, as appropriate within a range that does not affect the effects of the present invention, stabilizers, solubilizers, buffers, isotonic agents, solubilizing agents, etc. Additives commonly used in pharmaceutical formulations can be used. In particular, it is necessary to keep the amount of the additive that dissolves to generate metal ions in an amount that does not cause salting out of the anionic polymer.

The suspension of the present invention can be produced by a method generally used in pharmaceutical production and the like. Specifically, for example, it can be manufactured by a process including the following processes.
(1) A step of dissolving the component (a) in an alkaline solvent.
(2) A step of adding an acidic solvent to the solution of step (1) to precipitate the component (a).
(3) A step of separating and recovering the precipitate of the step (2) from the solvent.
(4) A step of redispersing the precipitate of the step (3) in a solution containing the component (b) and, if necessary, the component (c), and pulverizing and sizing.
(5) a step of reducing microorganisms by filtering the suspension of step (4) with a filter having a pore size of 0.1 to 0.5 μm, if necessary.

  The step (1) specifically includes, for example, suspending the peptide as the component (a) in water for injection, and adding an alkaline pH adjuster to dissolve the peptide. Further, the peptide may be dissolved in an aqueous solution which has been made alkaline in advance. If necessary, the solution may be sterilized using a filter having a pore size of 0.22 μm. The pH adjuster used is not particularly limited as long as it is alkaline, but sodium hydroxide or potassium hydroxide is preferred, and sodium hydroxide is more preferred.

  The step (2) specifically includes, for example, adding an acidic pH adjuster to the solution produced in the step (1) and stirring the solution to precipitate the solution. The pH adjuster used is not particularly limited as long as it is acidic, but hydrochloric acid, sulfuric acid or nitric acid is preferred, and hydrochloric acid is more preferred.

  In step (3), specifically, for example, the peptide is precipitated by centrifuging an aqueous solution containing the peptide which is the component (a) precipitated in step (2), and then the supernatant is removed. Separation and recovery methods are mentioned.

  In the step (4), specifically, for example, the precipitate collected in the step (3) is generally used for the component (b), optionally the component (c), and further optionally, the pharmaceutical preparation. Add to the aqueous solution containing the additives, stir and redisperse. The aqueous solution may be sterilized in advance using a filter having a pore size of 0.22 μm, if necessary. If the redispersed suspension does not have the desired pH, the pH is adjusted using a pH adjuster, and the dispersion treatment is performed once or more than once using a high-pressure homogenizer to perform pulverization and sizing. Thus, a production method for obtaining a suspension can be mentioned.

  In step (5), for example, the microorganisms are reduced by, for example, filtering the suspension obtained in step (4) using a filter having a pore size of 0.45 μm or 0.22 μm. And a method of removing.

  In the production of the suspension of the present invention, in addition to the above-mentioned steps, sterilization, filtration, pH adjustment, liquid volume adjustment, washing, etc., in the course of pharmaceutical production and the like, as long as they do not affect the next step The steps used may be added as appropriate.

  The freeze-dried preparation of the present invention can be produced by a usual method using the suspension of the present invention. Specifically, for example, a vial is filled with the suspension, and lyophilization is performed using a lyophilizer under ordinary production conditions. The production conditions are not particularly limited, but specifically, for example, after freezing at around -40 ° C, the inside of the refrigerator is depressurized to a vacuum, and at the same time, the temperature in the refrigerator is raised to -20 ° C, and After drying for about 30 hours, the temperature in the refrigerator is raised to 30 ° C. and drying is performed for about 10 to 30 hours.

By preparing a cancer vaccine composition using the suspension of the present invention, it can be used as a cancer vaccine. When the freeze-dried preparation of the present invention is used, it is prepared into a suspension in advance by a known method and used. The method for preparing a cancer vaccine composition using the suspension of the present invention is not particularly limited. For example, a method for preparing a cancer vaccine by mixing with an appropriate adjuvant; A method for preparing a cancer vaccine composition (cancer vaccine preparation) by freeze-drying or the like; a method for preparing the cancer vaccine composition by mixing the suspension of the present invention with various adjuvants by preparing it before use. Examples of adjuvants include Freund's adjuvant; mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol; and BCG (Calmet- Guerlain bacillus) and human adjuvants such as Corynebacterium parvum.

  As the adjuvant, another classification includes a sedimentation adjuvant and an oily adjuvant. Precipitating adjuvant refers to a mineral suspending agent to which the peptide adsorbs. Specific examples of the sedimentation adjuvant include sodium hydroxide, aluminum hydroxide (Alum, Alum), calcium phosphate, aluminum phosphate, alum, PEPES, carboxyvinyl polymer and the like. Oily adjuvants represent oil emulsions in which an aqueous solution containing the peptide is wrapped with mineral oil to form micelles and emulsify. Specific examples of the oil adjuvant include liquid paraffin, lanolin, Freund's adjuvant (complete Freund's adjuvant, incomplete Freund's adjuvant), Montanide, W / O emulsion (see WO 2006/078059), and the like.

  The cancer vaccine composition prepared using the suspension of the present invention can be used as a cancer vaccine for cancers with an increased expression level of the WT1 gene, such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, and the like. Used for the prevention or treatment of solid cancers such as blood cancer, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, etc. be able to.

  The suspension of the present invention can be lyophilized, if necessary, to stably retain the cancer antigen peptide as an active ingredient. Therefore, various administration forms can be selected. Specific examples include oral, nasal, pulmonary, transdermal, intradermal, subcutaneous, intramuscular, intravenous, intraperitoneal, and the like, and the above method is used to prepare a cancer vaccine composition according to purpose. I just need. In general, parenteral administration is known as a preferred administration route for immunostimulation as a cancer vaccine. For example, intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, nasal administration, Administration and transdermal administration. Among them, injection administration such as subcutaneous administration, intradermal administration, intraperitoneal administration, and intramuscular administration is preferred.

  Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, Test Examples, and the like, but the present invention is not limited thereto.

As a peptide represented by the formula (1) (also referred to as a peptide (cystine body)), a peptide produced by a method described in Patent Document (WO 2007/063903) is used, and a peptide represented by the formula (2) is used. As a peptide represented (also referred to as peptide (dimer)), a peptide produced by a method described in Patent Document (WO 2004/063217) was used, and as sucrose, "sucrose (Nacalai Tesque, Inc.) ")," Xylitol (manufactured by Nacalai Tesque) "as xylitol," sodium alginate (manufactured by Nacalai Tesque) "as sodium alginate, and" mannitol (manufactured by Mitsubishi Foodtech) "as mannitol. And "trehalose" (manufactured by Hayashibara Biochemical Laboratory) as the trehalose. As carmellose sodium, "CMC-Na (Daiichi Kogyo Seiyaku Co., Ltd.)" was used, and as xanthan gum, "Xanthan gum (CP Kelco)
"Pluronic F-68 (MP)" as Pluronic (polyoxyethylene (160) polyoxypropylene (30) glycol) using Macrogol 4000 (manufactured by NOF CORPORATION) as macrogol. Biochemical Co., Ltd.) as a polyoxyethylene hydrogenated castor oil 60 (POE hydrogenated castor oil 60) as "NIKKOL"
HCO-60 (Nikko Chemicals) "was used. "1 mol / l-hydrochloric acid (manufactured by Nacalai Tesque)" is used as hydrochloric acid as a pH adjuster, and "1 mol / l-sodium hydroxide solution (manufactured by Nacalai Tesque)" is used as sodium hydroxide as a pH adjuster. Was used.

  "NOFABLE EO-85S (manufactured by NOF CORPORATION)" is used as ethyl oleate, "NIKKOL ODM-100 (manufactured by Nikko Chemicals)" is used as octyldodecyl myristate, and "NOFABLE SOL is used as sorbitan monooleate. -991 (manufactured by NOF CORPORATION) "and glycerin monooleate used was" NOFABLE GO-991 (manufactured by NOF CORPORATION) ". As the concentrated glycerin, using "Japan Pharmacopoeia concentrated glycerin (manufactured by NOF CORPORATION)", as polyoxyethylene hydrogenated castor oil 20, using "NIKKOL HCO-20 (manufactured by Nikko Chemicals)" as sodium dihydrogen phosphate And "sodium dihydrogen phosphate dihydrate (manufactured by Nacalai Tesque)".

(Preparation of suspension)
[Example 1]
The peptide (cystine form) was added to an appropriate amount of water for injection and mixed, and then sodium hydroxide was added to adjust the pH to 10 or more, and mixed until the peptide was dissolved. Next, hydrochloric acid was added to adjust the pH to 6 or less, and the precipitated peptide was isolated by centrifugation. Each component was added to the isolated peptide so as to have the composition shown in Table 1, adjusted for pH, dispersed by a stirrer, and subjected to a dispersion treatment using a high-pressure homogenizer under a treatment pressure of 210 MPa and a number of passes of 30. By doing so, a suspension was obtained.

[Examples 2 to 6] [Comparative Examples 1 and 2]
After dissolving and isolating in the same manner as in Example 1 using the peptide (cystine form), it was produced in the same manner as in Example 1 using the amounts shown in Tables 1 and 2 to obtain a suspension.

(Preparation of freeze-dried preparation)
[Examples 4A to 6A]
After filling the suspensions produced in Examples 4 to 6 into glass vials, they were put into a freeze dryer and freeze-dried to produce freeze-dried preparations. In addition, freeze-drying, after freezing the suspension at around −40 ° C., depressurizing the inside of the refrigerator to a vacuum, and simultaneously raising the temperature in the refrigerator to around −20 ° C. and drying for about 20 hours. The temperature was raised to around 30 ° C. and drying was performed for about 12 hours.

(Preparation of suspension)
[Examples 7 to 20]
After dissolving and isolating in the same manner as in Example 1 using the peptide (cystine form), it was produced in the same manner as in Example 1 using the amounts shown in Tables 3 to 5 to obtain a suspension.

(Preparation of freeze-dried preparation)
[Examples 7A to 19A]
The suspensions prepared in Examples 7 to 19 were filtered using a filter having a pore size of 0.22 μm, and then filled into glass vials. The glass vial filled with the suspension was placed in a freeze dryer and freeze-dried under the same conditions as in Example 4A to produce a freeze-dried preparation.

(Preparation of suspension)
[Examples 21 to 24]
After dissolving and isolating in the same manner as in Example 1 using the peptide (dimer), it was produced in the same manner as in Example 1 using the amounts shown in Table 6 to obtain a suspension.

[Test Example 1] Suspension evaluation (1)
For the suspensions produced in Examples 1 to 6 and Comparative Examples 1 and 2, the particle size distribution and properties were evaluated. The particle size distribution was measured by a dynamic light scattering method after diluting a sample immediately after preparation with water for injection. The properties were evaluated visually for the samples immediately after preparation and after standing for one day. Table 7 shows the evaluation results.

  All of the preparations were in a suspended state immediately after production, but it was confirmed that the suspension was maintained even after standing for one day by containing an anionic polymer, and the usability as a suspension was good. It was suggested that The suspension in a suspended state has a 50% D of 1 μm or less and a 90% D of 2 μm or less, and contains suspended particles in a liquid as fine particles. It was confirmed that. In particular, by containing sodium alginate and xanthan gum, sterilization using a filter having a pore size of 0.45 μm is possible. By containing carmellose sodium, sterilization using a filter having a pore size of 0.22 μm becomes possible. It has been suggested. Further, in Examples 4 and 5, in which 90% D was less than 0.20 μm, filtration was performed using a filter having a pore size of 0.22 μm. It was confirmed that sterilization was possible.

[Test Example 2] Suspension evaluation (2)
The particle size distribution and properties of the suspensions produced in Examples 7 to 20 were evaluated in the same manner as in Test Example 1. Table 8 shows the evaluation results.

  Each of the suspensions containing sugar or sugar alcohol in the sample containing carmese sodium was able to maintain a suspended state even after standing for one day, and 90% D was 0.20 μm or less. Further, in Examples 7 to 20, when filtration was performed using a filter having a pore size of 0.22 μm, no blockage of the filter by the suspended matter and an increase in filtration resistance were not recognized, and it was confirmed that sterilization was possible.

[Test Example 3] Suspension evaluation (3)
The particle size distribution and properties of the suspensions produced in Examples 21 to 24 were evaluated in the same manner as in Test Example 1. Table 9 shows the evaluation results.

  It was also confirmed that a suspension containing a peptide (dimer) having a high molecular weight was able to maintain a suspension even after standing for one day. Further, in Example 23, when filtration was performed using a filter having a pore size of 0.22 μm, no blockage of the filter by the suspended matter and an increase in filtration resistance were not recognized, and it was confirmed that sterilization was possible.

[Test Example 4] Stability evaluation (1)
After storing the suspensions prepared in Examples 4 to 6 at 5 ° C. for 3 months and the lyophilized preparations prepared in Examples 4A to 6A at 5 ° C., 25 ° C. and 40 ° C. for 3 months, the lyophilized preparation was injected. After adding water to make a suspension, the particle size distribution, properties, and purity were evaluated. The particle size distribution was measured in the same manner as in Test Example 1. The properties are as follows: at the time immediately after the suspension was removed from the storage, and at the time both immediately after the lyophilized preparation was removed from the storage and resuspended by adding water for injection and after standing at room temperature for one day, The samples were evaluated visually. Purity was determined by reversed-phase high-performance liquid chromatography using a C18 reverse-phase column (4.6 mm × 150 mm, 5 μm) using pure water, acetonitrile, 2-propanol, and trifluoroacetic acid as the mobile phase (detection wavelength: 220 nm). Was measured by Using the peak area measured by this method, the relative purity (vs. the initial value) when the peptide purity and the purity at the start were set to 100%, was calculated by the following formula.
Peptide purity (%) = Peak area of peptide / Total peak area including analogs × 100
Relative purity (vs. initial value) (%) = Peptide purity at storage / Peptide purity at start × 100
Tables 10 to 13 show the evaluation results.

[Test Example 5] Stability evaluation (2)
After the lyophilized preparations prepared in Examples 7A to 19A were stored at 5 ° C and 25 ° C for 6 months, and stored at 40 ° C for 3 months, water for injection was added to make a suspension, and the same as in Test Example 4 The particle size distribution, properties and purity were evaluated. Tables 14 to 17 show the evaluation results.

  As shown in Test Examples 4 and 5, although it was stable under storage at 5 ° C., it was confirmed that the addition of trehalose suppressed the increase in particle size. In addition, it was confirmed that by adding trehalose and / or mannitol, the purity of the peptide was maintained even under storage at 40 ° C.

  In summary, the addition of an anionic polymer makes it possible to make the peptide of the present invention finely divided and stabilize the dispersion, and furthermore, by adding a specific component (c), improves the formulation stability. It was suggested that an excellent lyophilized preparation could be obtained.

Test Example 6 Confirmation of Specific CTL Inducing Activity Using the suspension of Example 11, a cancer vaccine composition was prepared by mixing with the adjuvant.

1) Preparation of cancer vaccine composition In advance, ethyl oleate (595 g), octyldodecyl myristate (595 g), sorbitan monooleate (85 g), glycerin monooleate (119 g), and polyoxyethylene cured castor Oil 20 (17 g), concentrated glycerin (17 g), and an aqueous solution (272 g) containing 25 mM sodium dihydrogen phosphate hydrate are placed in a container, and the mixture is treated with CLEAMIX CLM-1.5 (M Technique). An adjuvant was obtained by stirring at 10,000 rpm for 60 minutes.
Next, a cancer vaccine composition was obtained by mixing 300 μL of the suspension (Example 11) and 700 μL of the adjuvant using a test tube mixer.

2) CTL induction activity rating above 1) specific CTL inducibility cancer vaccine composition prepared by the ^{HLA-A * 2402 / K b} 1 transgenic mice (WO2002 / 47474 Patent reference, hereinafter, HLA-
A24 mice).
200 μL of the cancer vaccine composition (peptide dosage 300 μg) was administered intradermally to the ridge of HLA-A24 mice, and 7 days after administration, the spleen was excised to prepare splenocytes. Thereafter, the MABTECH ELISpot PLUS for Mouse I by the Elispot method (see J. Immunological Methods, 1995, 181, 45-54).
CTL-inducing activity of the cancer vaccine composition was evaluated using interferon-γ (manufactured by Mabtech). The method was performed according to the attached instructions, and 1 × 10 ⁶ splenocytes were poured per well, and a cell culture medium containing the peptide of SEQ ID NO: 1 (peptide concentration: 10 μg / mL) was further poured. Thereafter, the cells were cultured in a 37 ° C. CO ₂ incubator for about 18 hours. The plate was washed according to the attached document, and the number of spots was detected with a dedicated analyzer (KS Elispot Research system).
The results are shown in FIG. The number of spots is represented by the average value of three mice. As shown in FIG. 1, the cancer vaccine composition prepared using the suspension of the present invention was found to be capable of inducing HLA-A24-specific cellular immunity, and was found to be useful as a cancer vaccine. .

  According to the present invention, it is possible to provide a suspension containing WT1 protein-derived cancer antigen peptide, which has good suspendability, and a highly stable lyophilized preparation obtained by freeze-drying the suspension, Can be used as a cancer vaccine.

Claims (11)

An injectable pharmaceutical composition comprising the following components:
(A) a peptide having an amino acid sequence represented by Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 1);

(In the formula, Leu-OH indicates that the C-terminal of Leu is a free carboxyl group.)
A peptide represented by the formula (2):

(In the formula, the bond between Cys and Cys represents a disulfide bond, Leu-OH has the same meaning as described above, and the other bonds represent peptide bonds.)
In represented by peptides, and one or more peptides selected from a salt thereof and (b) carmellose sodium, sodium alginate, one or more anionic polymers selected from the group consisting of xanthan gum and arabic gum . The composition of claim 1, wherein component (b) is one or more selected from the group consisting of sodium carmellose, sodium alginate, and xanthan gum. The composition according to claim 1 , wherein component (b) is carmellose sodium. The composition according to any one of claims 1 to 3 , further comprising one or more components (c) selected from the group consisting of sugars and sugar alcohols. The composition according to claim 4 , wherein component (c) is one or more selected from the group consisting of mannitol, xylitol, trehalose, and sucrose. The composition according to claim 4 , wherein component (c) is mannitol or xylitol, and trehalose. The composition according to claim 4 , wherein component (c) is mannitol and trehalose. The composition according to any one of claims 1 to 7 , wherein the composition is a suspension or a lyophilized formulation. A method for producing a composition according to any one of claims 1 to 7 , which is a suspension, comprising the following steps (1) to (4) :
(1) a step of dissolving the component (a) in an alkaline solvent,
(2) a step of adding an acidic solvent to a solution of the component (a) obtained in the step (1) to precipitate the component (a);
(3) a step of separating and recovering the component (a) precipitated in the steps (1) and (2) from the solvent, and a step (4) of the component (a) recovered in the steps (1) to (3). Is redispersed in a solution containing the component (b) and, if necessary, the component (c), and then crushed and sized. The production method according to claim 9 , further comprising the following step (5):
(5) A step of reducing the number of microorganisms by filtering the suspension obtained in steps (1) to (4) through a filter having a pore size of 0.1 to 0.5 μm. Method for producing a lyophilized preparation by freeze-drying the composition obtained by the method according to claim 9 or 1 0.