WO2021020446A1 - 単純ヘルペスウイルスを含む組成物 - Google Patents

単純ヘルペスウイルスを含む組成物 Download PDF

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Publication number
WO2021020446A1
WO2021020446A1 PCT/JP2020/029068 JP2020029068W WO2021020446A1 WO 2021020446 A1 WO2021020446 A1 WO 2021020446A1 JP 2020029068 W JP2020029068 W JP 2020029068W WO 2021020446 A1 WO2021020446 A1 WO 2021020446A1
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Prior art keywords
composition
hsv
present
herpes simplex
simplex virus
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English (en)
French (fr)
Japanese (ja)
Inventor
大嗣 津田
朱純 河合
京子 上萩
多佳久 月原
蝶野　英人
舞紀 田中
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Takara Bio Inc
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Takara Bio Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K47/16—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
    • 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
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to a composition containing a herpes simplex virus.
  • Herpes simplex virus is a type of virus belonging to the order Herpesvirus, Herpesvirus family, Alphaherpesvirus subfamily, and Simplex virus genus, and has a double-stranded DNA of 150 kbp as a genome.
  • HSV includes herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).
  • HSV-1 presents gingival aphthous ulcer and facial herpes as primary infection symptoms, and herpes labialis, corneal conjunctivitis herpes, and herpes encephalitis as recurrent infection symptoms.
  • the latent infection site of HSV-1 is mainly the trigeminal ganglion.
  • HSV-2 presents genital herpes as a primary infection symptom, and genital herpes and neonatal herpes as recurrent infection symptoms.
  • the latent infection site of HSV-2 is mainly the sacral ganglion.
  • herpes simplex virus is pathogenic to humans, vaccines to prevent primary infection and recurrent onset are being developed.
  • herpes simplex virus since herpes simplex virus has strong neurogenicity and high tumor-killing ability, medical use is being attempted not only as a vaccine but also in the fields of gene therapy and virus therapy.
  • Oncolytic virus is a general term for viruses that selectively proliferate and spread in tumor cells and destroy tumor cells without causing excessive damage to normal cells.
  • Many viruses such as HSV, adenovirus, leovirus, measles virus, Newcastle disease virus, and bovine hemolytic virus have been studied as oncolytic viruses, but the most researched and clinically applied as oncolytic viruses. Is the HSV.
  • Non-Patent Document 1 the US Food and Drug Administration
  • T-VEC trade name IMLYGIC
  • HF10 the oncolytic virus preparation talimogene laherparepvec
  • HSV composition The composition containing herpes simplex virus (hereinafter referred to as HSV composition) used for these uses must be retained in a state of retaining desired biological activity (antigenicity, infectivity, cytotoxic activity, etc.). ..
  • desired biological activity antigenicity, infectivity, cytotoxic activity, etc.
  • cryopreservation or lyophilization is common.
  • a technique for storing the HSV composition in a liquid state is desired.
  • Patent Document 2 discloses a method for stabilizing an HSV vaccine with freeze-drying or liquid by using "inactivated or attenuated virus, cell culture medium, hydrolyzed gelatin, and sugar". ing.
  • the HSV vaccine is stabilized in liquid by using "inactivated or attenuated virus, cell culture medium, hydrolyzed gelatin, sugar, L-glutamic acid, and L-arginine". Discloses how to do this.
  • Patent Document 4 discloses stabilization of herpes simplex virus in a frozen state by a composition containing "hydrolyzed gelatin, sugar, sodium chloride, and sodium phosphate having a pH of 7.4".
  • the present invention is intended to solve the problems of the conventional HSV storage method, and is a composition for maintaining the activity of HSV for a long time, and a method for storing HSV using the composition.
  • the purpose is to provide.
  • the present invention provides a composition comprising herpes simplex virus, hydrolyzed gelatin, and at least a low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton.
  • the composition of the present invention can store HSV for a long period of time while maintaining the infectivity of HSV to cells even under freezing conditions or dry conditions.
  • the compositions of the present invention also prevent viral inactivation due to freeze-thaw damage. That is, even if freeze-thaw is repeated, HSV can be preserved while maintaining the infectivity of HSV to cells.
  • the present invention [1] A composition containing herpes simplex virus, hydrolyzed gelatin, and a low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain. [2] The composition according to [1], wherein the low molecular weight compound is a basic amino acid having an amino group, a guanidyl group or an imidazole group in the side chain. [3] The composition according to [2], wherein the basic amino acid is selected from the group consisting of lysine, ornithine, arginine, homoarginine and histidine.
  • a method for storing herpes simplex virus (A) A step of preparing a composition containing herpes simplex virus, hydrolyzed gelatin, and a low molecular weight compound having an amino group and a carboxyl group, and further having a nitrogen atom capable of accepting a proton, and (b) the prepared composition.
  • a kit for storing herpes simplex virus which comprises hydrolyzed gelatin and a low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain.
  • the low molecular weight compound is a basic amino acid having an amino group, a guanidyl group or an imidazole group in the side chain.
  • the basic amino acid is selected from the group consisting of lysine, ornithine, arginine, homoarginine and histidine.
  • the kit according to any one of [14] to [16] further comprising glycerol.
  • the kit according to any one of [14] to [17] which does not contain sugar.
  • [20] Use of a composition containing hydrolyzed gelatin and a low molecular weight compound having an amino group and a carboxyl group and a nitrogen atom capable of accepting a proton in the side chain for storing herpes simplex virus.
  • the low molecular weight compound is a basic amino acid having an amino group, a guanidyl group or an imidazole group in the side chain.
  • the basic amino acid is selected from the group consisting of lysine, ornithine, arginine, homoarginine and histidine.
  • the composition further comprises glycerol.
  • the present invention provides a composition containing a herpes simplex virus and a method for preserving the herpes simplex virus using the composition.
  • the composition can be stored for a long period of time while maintaining the infectivity of herpes simplex virus to cells under any of non-frozen (for example, liquid), frozen or dry conditions. ..
  • the composition obtained by the present invention is suitably used for medical treatment for, for example, cancer treatment. Therefore, the present invention is expected to make a great contribution to the medical field, such as reducing the burden on the medical staff who handles the herpes simplex virus and improving the convenience of the herpes simplex virus.
  • composition of the present invention is characterized by containing herpes simplex virus, hydrolyzed gelatin, and a low molecular weight compound having an amino group and a carboxyl group, and further having another nitrogen atom capable of accepting a proton.
  • Herpesvirus refers to any virus belonging to the herpesvirus family, which is a family of viruses.
  • Herpesviruses double chain linear DNA molecular weight 80 ⁇ 150 ⁇ 10 6 daltons around the core protein comprises 162 capsomeres, characterized by latent infection.
  • the virus particles are spherical with a diameter of about 100 to 200 nm, and there are icosahedron-shaped capsids with a diameter of about 100 nm in the envelope.
  • the genome is double-stranded DNA, the size of which is, for example, 152260 base pairs for herpes simplex virus type 1, 172,282 base pairs for EB virus, and 229,400 base pairs for cytomegalovirus.
  • Alpha herpesvirus subfamily Human herpesvirus-1 (herpes simplex virus type 1; HSV-1), human herpesvirus-2 (herpes simplex virus type 2; HSV) -2), Alerton virus (Allerton virus), B virus (B virus), bovine mammilitis virus (BMV), feline rhinotracheitis virus (feline herpesvirus virus), infectious throat.
  • HSV-1 human herpesvirus-1
  • HSV-2 human herpesvirus-2
  • Alerton virus Allerton virus
  • B virus B virus
  • BMV bovine mammilitis virus
  • feline rhinotracheitis virus feline herpesvirus virus
  • Beta herpesvirus subfamily Cytomegalovirus, human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), etc.
  • Gamma Herpesvirus subfamily Human herpesvirus-4 (HHV-4), human herpesvirus-8 (HHV-8), EB virus (EBV), Marek's disease virus, etc.
  • the viral construct of the present invention is derived from the subfamily Alphaherpesvirinae.
  • herpes simplex virus or "HSV” means any virus belonging to the genus Simplex virus of the family Alphaherpesvirinae of the herpesvirus family.
  • Herpes simplex virus is 100-200 mm in diameter and has an envelope containing viral glycoprotein in the outermost layer, which contains an icosahedral capsid. What is a capsid? There is a structure called a segment between the envelope and the envelope, which contains a large number of viral proteins. Double-stranded linear DNA (about 150,000 base pairs) exists in the center (core) of the capsid.
  • HSV there are at least 74 types of genes in genomic DNA, and half of them are replication non-essential genes (accessory genes) that are not essential for proliferation in cultured cells.
  • the replication of genomic DNA and the formation of capsids are carried out in the nucleus, and the capsids containing DNA are transported from the nucleus to the cytoplasm by budding from the inner membrane.
  • HSV there are typically two closely related viral species named type 1 (HSV-1) and type 2 (HSV-2).
  • HSV includes wild-type HSV and its derivatives, unless otherwise specified.
  • Mutant HSVs that are functionally different from the wild type are exemplified as the above-mentioned derivatives, and the mutant HSVs are HSVs that are artificially produced (for example, by mutagen treatment or recombinant DNA technology) or that appear in nature. Include. Functional differences include, for example, strains in which the gene function of wild-type HSV is enhanced, attenuated or deficient, or strains into which a foreign gene has been introduced. For strains lacking gene function, “attenuated HSV” with reduced toxicity, “restricted proliferative HSV” that can proliferate in specific cells, and "cell affinity modified HSV” with modified affinity with cells. Is included.
  • the HSV that can be used in the present invention is preferably HSV-1.
  • the present invention can preferably use therapeutic HSV that requires infectivity.
  • therapeutic HSV include HSV that can be used as a vaccine and oncolytic HSV that is useful as a cancer therapeutic agent.
  • the oncolytic HSV may be any one having the property of selectively proliferating in the tumor tissue and destroying the tumor, and the HSV spontaneously acquired selective oncolytic virus and artificially selectively oncolytic virus. Any of the sex-imparted HSVs can be used in the present invention. Known oncolytic HSV can be used in the present invention, and examples thereof include, but are not limited to, canerpaturev, talimogene laherparepvec, and G47 ⁇ .
  • C-REV cancerpathurev
  • T-VEC total herpes simplex virus
  • G47 ⁇ refers to Proc Natl Acad Sci U S A. 2001, 98 (11): An oncolytic virus produced by modifying the HSV-1 gene reported in 6396-401.
  • the composition of the present invention is for maintaining the infectious activity of HSV contained in the composition on cells for a long period of time.
  • the present invention provides a composition capable of preserving the desired HSV while preserving the activity and properties it had prior to storage.
  • the composition of the present invention may be in a non-frozen, frozen or dried state, but is preferably in a non-frozen state, and is not particularly limited to the present invention, but for example, a liquid composition is suitable.
  • “liquid” is synonymous with “solution” and can be paraphrased as “liquid state”, “liquid shape”, “solution state”, and “solution shape”.
  • the amount of HSV is not particularly limited as long as it can exert the effect of the present invention, has a type and concentration of hydrolyzed gelatin, an amino group and a carboxyl group, and further accepts protons. It can be appropriately determined in consideration of the type and concentration of the low molecular weight compound having a nitrogen atom, the type and concentration of other components, the storage temperature and the like.
  • the concentration of HSV is not particularly limited, but is at least 1 ⁇ 10 5 TCID 50 / mL, preferably at least 2 ⁇ 10 5 TCID 50 / mL, at least 5 ⁇ 10 5 TCID 50 / mL, and at least 1 ⁇ 10 6 TCID 50.
  • ⁇ 10 6 TCID 50 / mL at least 5 ⁇ 10 6 TCID 50 / mL, at least 1 ⁇ 10 7 TCID 50 / mL, at least 2 ⁇ 10 7 TCID 50 / mL, at least 5 ⁇ 10 7 TCID 50 It is present in excess of / mL, at least 1 ⁇ 10 8 TCID 50 / mL, at least 2 ⁇ 10 8 TCID 50 / mL, or at least 5 ⁇ 10 8 TCID 50 / mL.
  • the appropriate amount varies depending on the situation in which it is used, and can be appropriately determined by those skilled in the art depending on the state of the active ingredient present in the composition and the like.
  • the HSV manufacturing method is not particularly limited.
  • the method for producing HSV may be any method. In general, after culturing infected cells obtained by infecting an appropriate host cell with HSV, a culture solution supernatant and / or a disrupted product of infected cells can be obtained as a virus-containing sample. Purified HSV can be obtained by subjecting these virus-containing samples to a known virus purification operation.
  • Hydrolyzed gelatin is obtained by extracting collagen contained in the skin, bones, scales, etc. of living organisms and further reducing the molecular weight by hydrolysis. Hydrolysis can be carried out by heat treatment, enzymatic digestion treatment, acid treatment, alkali treatment, or a combination thereof.
  • the hydrolyzed gelatin contained in the composition of the present invention is preferably derived from porcine.
  • the composition of the present invention may contain one or more hydrolyzed gelatins. Further, as a means of hydrolysis, treatment by enzymatic digestion is preferable, and from the viewpoint of immunogenicity, the average molecular weight of the hydrolysis product is preferably 10,000 or less.
  • hydrolyzed gelatin manufactured by Nippi can be preferably used.
  • the concentration of hydrolyzed gelatin contained in the composition of the present invention is not particularly limited as long as it can exert the effect of the present invention, and the type and concentration of HSV, the type and concentration of other components, the storage temperature, etc. Can be appropriately determined in consideration of.
  • the concentration of hydrolyzed gelatin is not particularly limited, but is 0.0001 to 10% (w / v), 0.0005 to 5% (w / v), 0.001 to 2.5% (w / v), and so on. 0.005-1% (w / v), 0.01-0.5% (w / v), 0.05-0.25% (w / v), or 0.1-0.15% ( w / v) is illustrated.
  • the low molecular weight compound is, for example, a compound having a molecular weight of 2000 or less or 1500 or less.
  • the "low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain" contained in the composition of the present invention is an amino group or a nitrogen atom-carbon atom-nitrogen atom in the side chain.
  • a low molecular weight compound containing the above structure is preferable, for example, a structure containing two or more nitrogen atoms with an amino group or a carbon atom sandwiched in the side chain, for example, a basic amino acid, and further, for example, an amino group, a guanidyl group or Basic amino acids having an imidazole group can be preferably used.
  • the basic amino acid for example, it can be selected from the group consisting of lysine, ornithine, arginine, homoarginine and histidine.
  • the low molecular weight compound contained in the composition of the present invention preferably contains only one kind.
  • the low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain those in the form of salts (for example, hydrochloride) or hydrates thereof are used. It may be included, for example, a salt form in which the basicity of the low molecular weight compound is weakened, a salt form in which the water solubility is increased, and the like. Further, the above-mentioned low molecular weight compound may be any of L-form, D-form or DL-form as long as the effect of the present invention can be obtained.
  • composition of the present invention may contain one or more of the low molecular weight compounds.
  • the concentration of the low molecular weight compound contained in the composition of the present invention is not particularly limited as long as it can exert the effect of the present invention, and the type and concentration of HSV, the type and concentration of other components, and It can be appropriately determined in consideration of the storage temperature and the like.
  • histidine which is a basic amino acid
  • its concentration is not particularly limited, but is 0.01 to 2 mg / mL, 0.02 to 1.5 mg / mL, 0.03 to 1 mg / mL, 0.
  • Examples are 04 to 0.7 mg / mL, 0.05 to 0.5 mg / mL, 0.1 to 0.4 mg / mL, 0.15 to 0.3 mg / mL, or 0.2 to 0.25 mg / mL. Will be done.
  • the composition of the present invention is prepared by mixing HSV, hydrolyzed gelatin, and a low molecular weight compound having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain in a solvent.
  • solvents for mixing HSV, hydrolyzed gelatin, and the low molecular weight compounds include, but are not limited to, water and buffers.
  • distilled water, sterilized water, distilled water for injection, or the like is used as the solvent for the composition of the present invention.
  • the composition of the present invention preferably further contains a buffer component.
  • the "buffer component” refers to a compound or mixture having an action of softening fluctuations in the hydrogen ion concentration (pH) of a solution.
  • the buffer component contained in the composition of the present invention is not particularly limited, but for example, tris, bicine, tricine, Hepes, and phosphate (sodium phosphate, potassium phosphate, etc.) can be preferably used.
  • tris, bicine, tricine, Hepes, or phosphate is suitable for the present invention as a buffer component.
  • the concentration of the buffer component contained in the composition of the present invention is not particularly limited as long as it can exert the effect of the present invention, and the type and concentration of herpesvirus, the type and concentration of other components, and storage. It can be appropriately determined in consideration of temperature and the like.
  • the pH of the composition of the present invention is, for example, in the range of pH 6.0 to 10.0, preferably pH 6.5 to 9.5, and more preferably pH 7.0 to 9.0. It is appropriate to be set.
  • the pH of the composition of the present invention is not particularly limited as long as it can exert the effects of the present invention, and is appropriately determined in consideration of the type and concentration of HSV, the type and concentration of other components, the storage temperature, and the like. can do.
  • the composition of the present invention may contain a Tris buffer solution.
  • the preferred composition of Tris buffer in the present invention is Tris-HCl (pH 7.2-8.0) of 1-100 mM, preferably 2-50 mM, more preferably 5-20 mM.
  • the composition of the present invention contains other components in addition to HSV, hydrolyzed gelatin, and a low molecular weight compound having an amino group and a carboxyl group and a nitrogen atom capable of accepting a proton in the side chain. May be good. On the other hand, it is desirable that it does not contain sugar.
  • the composition of the composition of the present invention is not particularly limited as long as it can exhibit the effects of the present invention.
  • the type and concentration of HSV, the type and concentration of hydrolyzed gelatin, the type and concentration of basic amino acids, and the type and concentration of other components can be appropriately determined in consideration of the purpose of use, storage temperature, and the like.
  • composition of the present invention may contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, but are not limited to: antioxidants, preservatives, colorants, stabilizers, diluents, emulsifiers, suspending agents, solvents, buffers, isotonic. Agents and / or pharmaceutical adjuvants.
  • the composition of the present invention may contain glycerol.
  • glycerol When glycerol is contained, the present invention is not particularly limited, but is added to the composition of the present invention at a concentration of 0.1 to 2%, preferably 0.2 to 1%.
  • the composition of the present invention containing glycerol is preferable when the composition is repeatedly frozen and thawed during storage.
  • composition of the present invention can also be dried by means such as freeze-drying.
  • the composition of the present invention preferably does not contain glycerol.
  • the dry composition of the present invention may be redissolved in water or buffer before use.
  • the composition of the invention does not contain proteins and / or sugars other than hydrolyzed gelatin.
  • "not containing" the above-mentioned component means that the above-mentioned component is not contained at a concentration capable of exerting an effect on the storage stability of the virus, and a small amount of the above-mentioned component is unintentionally contained. The inclusion of the above components is acceptable.
  • the present invention is not particularly limited, but the composition of the present invention does not contain a protein other than hydrolyzed gelatin exceeding 0.1 mg / mL, and a sugar exceeding 0.1 mg / mL.
  • sugar refers to any type of sugar, including monosaccharide, disaccharide, or oligosaccharide forms of carbohydrates, as well as sugar alcohols.
  • sugars include, but are not limited to, sugar derivatives such as trehalose, saccharose, sucrose, glucose, lactose, mannitol, and sorbitol, or amino sugars, such as glucosamine or N-acetylglucosamine.
  • composition of the present invention may be any form, and examples thereof include, but are not limited to, injections, injections, and the like.
  • present invention is provided as an injection in the form of a vaccine.
  • composition of the present invention when formulated as a pharmaceutical composition, it can be administered parenterally.
  • the composition can be administered intravenous tissue (including intratumor) or subcutaneously.
  • the composition When administered systemically, the composition may be in the form of a pyrogen-free, acceptable aqueous solution for parenteral administration.
  • Such pharmaceutically acceptable aqueous solutions can be prepared using techniques in the art, subject to considerable attention to pH, isotonicity, stability and the like.
  • the present invention provides a method for storing herpes simplex virus, which comprises carrying out storage in the form of the composition of the present invention described above.
  • the method of the present invention is particularly suitable as a method for stably preserving herpes simplex virus type 1 and its derivatives.
  • the method for preserving the herpes simplex virus of the present invention comprises a step of preparing the composition of the present invention and a step of preserving the prepared composition.
  • the various components constituting the composition of the present invention are used as a general method in the production of a pharmaceutical composition, preferably aseptic preparation of a pharmaceutical composition. It may be carried out by mixing according to the law.
  • the prepared composition may be stored according to a conventional method and may be carried out under either frozen or non-freezing conditions.
  • the composition may be stored in either a dry state or a liquid state.
  • the composition is preferably stored under non-freezing conditions, more preferably in a liquid state.
  • the storage temperature is, for example, but not limited to, 0 ° C. to 37 ° C., preferably 0 ° C. to 30 ° C., and more preferably 0 ° C. to 20 ° C. Particularly preferably, it is over 0 ° C. to 10 ° C.
  • storage is usually carried out in a shaded environment.
  • herpes simplex virus is stored for 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 2 weeks or more. It can be stored for 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.
  • the composition of the present invention is stored in a suitable container.
  • the container is not particularly limited as long as the composition can be aseptically stored, and examples thereof include well-known containers for storing medicines such as vials, ampoules, and infusion bags.
  • the material may also be appropriately selected according to the type and concentration of the components contained in the composition.
  • the storage method of the present invention does not prevent the composition of the present invention from being frozen and stored for a part of the storage period.
  • the herpes simplex virus is stored, for example, for 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more. It can be stored for 2, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.
  • the composition of the present invention that has been cryopreserved after preparation in a liquid state, for example, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, 24 hours or more
  • the method of the present invention is carried out by storing for 1 day or more, 2 days or more, 3 days or more, 5 days or more, or 7 days or more.
  • the storage method of the present invention can be used for storage of final preparations containing HSV and intermediate products in the preparation process.
  • the ability to freeze and thaw a formulation or intermediate product without diminishing its potency (or activity) allows for the manufacturing process, dispensing, labeling and packing of the formulation into containers, the distribution process of the formulation, and the formulation of the formulation. Increased flexibility in user handling. Furthermore, it is possible to refreeze and store a preparation or an intermediate product thawed by mistake or a surplus preparation that has not been administered to a patient, which is also useful from the economical point of view.
  • kits containing hydrolyzed gelatin and a low molecular weight compound having an amino group and a carboxyl group and having a nitrogen atom capable of accepting a proton in the side chain are provided for the storage method of the present invention.
  • the kit may contain other components in addition to hydrolyzed gelatin and low molecular weight compounds having an amino group and a carboxyl group and further having a nitrogen atom capable of accepting a proton in the side chain.
  • Other components are, for example, as described in (1) "Composition" of the present invention.
  • HSV cryopreservation preparation The HSV strain described as HF10 in WO2002 / 092826 International Publication was infected with Vero cells (African green monkey kidney epithelial cells, ATCC No. CRL-1586). HSV was collected and purified from the obtained culture supernatant of infected cells. The resulting HSV was suspended in a solution containing 10% (w / v) glycerol (Merck) and 25 mM Tris (Merck), then dispensed in 1 mL / vial and at -80 ° C. It was cryopreserved. This is referred to as an "HSV cryopreservation preparation". Was dissolved HSV cryopreserved preparations was measured and the infectious titer in TCID50 method, infectious titer was 2.7 ⁇ 10 7 TCID 50 / mL .
  • Example 1 Storage stability test of HSV (1) Preparation and storage of a solution containing HSV 0.13% (w / v) hydrolyzed gelatin (manufactured by Nippi), 0.22 mg / mL L-histidine according to Table 1.
  • Vero cells were grown in RPMI1640 (SIGMA) culture medium supplemented with 10% fetal bovine serum (Bioest) and 100 ⁇ g / mL antibiotic streptomycin and 100 Units / mL penicillin (Thermo Fisher Scientific). Vero cells were seeded on 96-well plates the day before sample infection. The stored sample prepared in Example 1- (1) was serially diluted with 10 mM Tris-HCl buffer pH 7.5 and added to Vero cells for infection. The cells were cultured at a temperature of 37 ° C. and a CO 2 concentration of 5%.
  • FIG. 1 is a graph showing the cell-killing effect without medium washing, the vertical axis shows the survival rate (%) of cells infected with HSV under each storage condition, and the horizontal axis shows the sample name under each storage condition. is there.
  • "Standard” is a Vero cell that has not been infected with a sample. From FIG. 1, as a method of preserving herpes simplex virus (HSV), 2. HSV + gelatin + histidine, 3. HSV + gelatin + histidine + arginine, 4. HSV + gelatin + gelatin + histidine + glycerol, 5. HSV + gelatin + histidine + arginine + glycerol, 6. HSV + gelatin + arginine, 7.
  • the combination of HSV + gelatin + arginine + glycerol had a cell viability of 60% or less after infection.
  • the compositions of 2 to 6 above have a survival rate of 40% or less and are suitable as a preservation solution composition of HSV.
  • Vero cells were grown in RPMI 1640 culture medium supplemented with 10% fetal bovine serum and 100 ⁇ g / mL of the antibiotic streptomycin and 100 Units / mL of penicillin. Vero cells were seeded on 96-well plates the day before sample infection.
  • the stored sample prepared in Example 1- (1) was serially diluted with 10 mM Tris-HCl buffer pH 7.5 and added to Vero cells for infection. Two hours after the addition, the sample was removed, washed once with PBS, replaced with a culture medium, and cultured at a temperature of 37 ° C. and a CO 2 concentration of 5%.
  • the culture broth was removed, the cells were washed once with PBS, and then a preparation solution obtained by diluting Premix WST-1 Cell Proflation Assay 10-fold with 10% fetal bovine serum-containing phenol red-free RPMI1640 was added to the wells. Then, the cells were cultured at 37 ° C. and a CO 2 concentration of 5% for 30 minutes. The absorbance at a wavelength of 450 nm after culturing was determined with an absorptiometer. The result is shown in FIG.
  • FIG. 2 is a graph showing the cell-killing effect of HSV after washing the medium, the vertical axis shows the survival rate (%) of the cells infected with HSV under each storage condition, and the horizontal axis shows the sample under each storage condition. It is a name.
  • "Standard" is a Vero cell that has not been infected with a sample.
  • the result of FIG. 2 is the same as the result of FIG. 1.
  • HSV + gelatin + histidine + arginine 4.
  • HSV + gelatin + histidine + glycerol 5.
  • HSV + gelatin + histidine + arginine + glycerol 5.
  • HSV + gelatin + histidine + arginine + glycerol 6.
  • HSV + gelatin + arginine, 7. It was confirmed that the combination of HSV + gelatin + arginine + glycerol had a cell viability of 40% or less after infection,
  • the formulation containing hydrolyzed gelatin provided excellent stability under all evaluated formulation conditions except rHSA. In particular, it was most prominent when stored in a composition containing herpes simplex virus, hydrolyzed gelatin, histidine, and glycerol.
  • the present invention provides a composition containing a herpes simplex virus and a method for preserving the herpes simplex virus using the composition.

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WO2022158097A1 (ja) * 2021-01-19 2022-07-28 新田ゼラチン株式会社 ウイルス安定化剤、ウイルス安定化剤用ゼラチン加水分解物およびウイルス含有組成物

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JPS57114527A (en) * 1979-10-29 1982-07-16 Merck & Co Inc Vaccine stabilizer
JP2008525444A (ja) * 2004-12-23 2008-07-17 オールクス，インコーポレイテッド ウイルス組成物の安定化

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JPS57114527A (en) * 1979-10-29 1982-07-16 Merck & Co Inc Vaccine stabilizer
JP2008525444A (ja) * 2004-12-23 2008-07-17 オールクス，インコーポレイテッド ウイルス組成物の安定化

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WO2022158097A1 (ja) * 2021-01-19 2022-07-28 新田ゼラチン株式会社 ウイルス安定化剤、ウイルス安定化剤用ゼラチン加水分解物およびウイルス含有組成物

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