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WO2024048570A1 - Composition de vaccin contre l'hépatite b pour administration nasale et système d'administration nasale associé - Google Patents

Composition de vaccin contre l'hépatite b pour administration nasale et système d'administration nasale associé Download PDF

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Publication number
WO2024048570A1
WO2024048570A1 PCT/JP2023/031188 JP2023031188W WO2024048570A1 WO 2024048570 A1 WO2024048570 A1 WO 2024048570A1 JP 2023031188 W JP2023031188 W JP 2023031188W WO 2024048570 A1 WO2024048570 A1 WO 2024048570A1
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Prior art keywords
antigen
hbs
hepatitis
vaccine composition
protein
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English (en)
Japanese (ja)
Inventor
陽一 日浅
理 吉田
道法 小原
恭子 小原
保正 郷
康則 織田
泰造 上下
隆 宮崎
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Toko Yakuhin Kogyo KK
Tokyo Metropolitan Institute of Medical Science
Kagoshima University NUC
Ehime University NUC
Beacle Inc
Original Assignee
Toko Yakuhin Kogyo KK
Tokyo Metropolitan Institute of Medical Science
Kagoshima University NUC
Ehime University NUC
Beacle Inc
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Application filed by Toko Yakuhin Kogyo KK, Tokyo Metropolitan Institute of Medical Science, Kagoshima University NUC, Ehime University NUC, Beacle Inc filed Critical Toko Yakuhin Kogyo KK
Priority to JP2024544278A priority Critical patent/JPWO2024048570A1/ja
Publication of WO2024048570A1 publication Critical patent/WO2024048570A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • A61K39/29—Hepatitis virus
    • 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
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • 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
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M11/00—Sprayers or atomisers specially adapted for therapeutic purposes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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 vaccine composition for nasal administration that can be used for the prevention and treatment of hepatitis B, and a nasal administration system for the vaccine.
  • Hepatitis B is hepatitis caused by infection with hepatitis B virus (HBV), and HBV is transmitted through blood and body fluids.
  • HBV hepatitis B virus
  • Persistent infection of hepatocytes with HBV is a serious infectious disease that causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, but there is no treatment that can eliminate the virus.
  • PEG-IFN pegylated interferon preparations
  • NA nucleic acid analog preparations
  • HBV elimination rate is generally low and the high frequency and variety of side effects are major problems.
  • NA shows a high HBV DNA negative conversion rate of approximately 95% by inhibiting viral replication, it cannot be eliminated and its efficacy quickly disappears upon discontinuation of administration. Therefore, lifelong use is required, which poses major problems in terms of compliance and medical economics, and it has also been reported that there is a possibility of the emergence of resistant viruses with long-term use.
  • preventive vaccines are administered to those at high risk of infection (HBV carrier families, medical workers), and this has achieved some results in significantly reducing the number of HBV carriers.
  • CHB trials of immunotherapy using an HBV vaccine are being conducted.
  • HBs antigens for the purpose of inducing neutralizing antibodies against HBs antigen (hepatitis B virus surface antigen) and inducing acquired immunity against HBc antigen (hepatitis B nucleocapsid antigen) in the treatment of HBV.
  • HBs-S antigen hepatitis B virus surface antigen
  • HBc antigen hepatitis B nucleocapsid antigen
  • phase 1 clinical trial included 29 patients with chronic hepatitis B (CHB) on treatment with nucleic acid analogues (NA) and 42 untreated hepatitis B virus (HBV) carriers, who received once every two weeks.
  • CHB chronic hepatitis B
  • NA nucleic acid analogues
  • HBV hepatitis B virus
  • the composition was intranasally administered 10 times in total.
  • Anti-HBs antibodies were induced in about half of the cases, and the amount of HBs antigen decreased over time. In some cases, HBs antigen disappeared and functional cure was achieved. Moreover, adverse events were minor (Non-Patent Document 1).
  • HBV is a particle composed of DNA at its center and a capsid surrounding it, and further has an envelope structure containing a large number of proteins in the lipid membrane on its surface.
  • the envelope is the outermost structure of the virus and is used for immunological detection of HBV, so it is also called HBV surface antigen (HBs).
  • the full-length protein that forms the surface antigen of HBV is called L protein, and there are three regions, Pre-S1 region, Pre-S2 region, and S region, in order from the N-terminus presented on the outermost part of the particle.
  • the full-length HBs-L antigen protein called the HBs-L antigen protein, lacks the Pre-S1 region, the protein consisting of the Pre-S2 region and the S region is called the M protein, and the HBs-L antigen protein lacks the Pre-S1 and Pre-S2 regions.
  • a protein consisting only of the S region is called an S protein.
  • the various HBV antigen proteins mentioned above can be produced using an expression system using eukaryotic cells such as yeast or animal cells into which genes encoding them have been introduced, and these genetically recombinant surface antigens is widely used as a preventive vaccine against HBV.
  • eukaryotic cells such as yeast or animal cells into which genes encoding them have been introduced
  • S antigen produced in yeast is the mainstream in the current market.
  • the HBs antigen used in the preparation containing HBc antigen, HBs antigen, and CVP used in the above clinical trial is S protein (HBs-S antigen protein).
  • the full-length L protein has, in addition to the S region and Pre-S2 region contained in the M protein, a Pre-S1 region that is the HBV sensor region. Therefore, when the L protein is used as a vaccine, antibodies against the outermost Pre-S1 region are also produced, making it an excellent prophylactic vaccine.
  • Some of the present inventors have confirmed that a vaccine composition that combines HBs-L antigen and HBc antigen exhibits a high ability to activate cellular immunity (Patent Documents 2 and 3).
  • HBV genotypes there are multiple HBV genotypes, A to J, but A, B, C, and D are the main HBV genotypes.
  • S region and Pre-S2 region the relationship between each genotype and phenotype has been clarified.
  • the 124th to 147th amino acid sequence is generally known as the highly immunogenic antigenic determinant "a”.
  • the antigenic determinant "b” is present in common in all genotypes (Non-Patent Document 2). Therefore, with respect to these regions, even if the genotypes are different, it is thought that common antibodies will be produced for at least some of the genotypes.
  • the Pre-S1 region analysis of each genotype and phenotype has not been sufficiently conducted, and the amino acid sequence homology between each genotype of HBV is low. Therefore, even if a vaccine containing the HBs-L antigen protein containing the Pre-S1 region derived from one type of genotype is produced as an active ingredient, the Pre-S1 region is It is not always possible to elicit an immune response through
  • Some of the present inventors have developed an HBs-L antigen protein whose genotype is any of A, B, C, D or variants of each of these genotypes as a vaccine for different genotypes. have proposed virus-like particles containing one or more types of (Patent Documents 1 and 2). Using a combination of virus-like particles having HBs-L antigen protein of one of genotypes A, B, C, or D in one virus-like particle, or using two virus-like particles in one virus-like particle. It has been confirmed that by using virus-like particles containing HBs-L antigen proteins of the above genotypes, it is possible to induce HBs-neutralizing antibodies against multiple types of genotypes.
  • the purpose of this application is to provide a stable vaccine composition for nasal administration and a nasal administration system for the vaccine.
  • the present application provides: (i) a virus-like particle containing hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D;
  • a hepatitis B vaccine composition for nasal administration comprising a base containing a hepatitis B nucleocapsid antigen (HBc antigen) protein, and (ii) a carboxyvinyl polymer treated with external shear force.
  • the present application also provides a hepatitis B vaccine nasal administration system, which comprises filling the hepatitis B vaccine composition for nasal administration of the present application into a syringe-type ejector having a nasal spray nozzle.
  • the present application provides a method for treating hepatitis B, comprising nasally administering an effective amount of the nasally administered hepatitis B vaccine composition of the present application to a subject in need of treatment for hepatitis B.
  • the hepatitis B vaccine composition for nasal administration may be administered using a syringe-type ejector having a nasal spray nozzle.
  • the present application provides (i) hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D.
  • B for nasal administration of a composition comprising a base containing virus-like particles containing virus-like particles, hepatitis B nucleocapsid antigen (HBc antigen) protein, and (ii) a carboxyvinyl polymer treated by applying an external shear force.
  • HBc antigen hepatitis B nucleocapsid antigen
  • carboxyvinyl polymer treated by applying an external shear force.
  • the composition of the present application may be filled into a syringe-type squirt device having a nasal spray nozzle.
  • Virus-like particles containing HBs-L antigen protein have poor stability, and especially when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), the stability further decreases depending on the amount of HBc antigen. At the same time, the present inventors' research revealed that the HBc antigen also becomes unstable.
  • the hepatitis B vaccine composition of the present application contains virus-like particles containing HBs-L antigen protein and HBc antigen protein, it is a stable vaccine in terms of formulation, and can be used as a preventive and therapeutic vaccine for hepatitis B. Suitably used.
  • FIG. 1 is a partially cutaway side view showing a schematic configuration of the entire medical syringe (prefilled device) equipped with a nasal spray nozzle used in Examples.
  • (a) and (b) are exploded perspective views showing the schematic structure of the nasal spray nozzle used in the examples, and show the states before and after the filling rod is inserted into the nozzle body.
  • (a) is a vertical cross-sectional view of the nasal spray nozzle shown in FIG. 3(b) when viewed from a vertical plane, and (b) to (d) are taken along the line BB in FIG. 4(a).
  • Example 3 antibody titers against HBs-LH antigen in mouse serum were examined by ELISA two weeks after nasal administration of formulations 1 to 6 (examples) and formulations 7 and 8 (comparative examples) to mice. Show the results. In Example 3, the results of examining the antibody titer against HBc antigen in mouse serum by ELISA two weeks after nasally administering formulations 1 to 6 (example) and formulations 7 and 8 (comparative example) to mice, respectively. show.
  • virus-like particle containing HBs-L antigen protein contained in the hepatitis B vaccine composition of the present application is a protein having one or more transmembrane domains. These particles are mainly composed of a certain HBs-L antigen protein and a lipid bilayer membrane, and do not contain nucleic acids such as DNA or RNA that control genetic information inside the particles.
  • the genotype of the HBs-L antigen protein contained in the virus-like particle is selected from A, B, C and D. Furthermore, these genotype variants can also be used as the HBs-L antigen protein contained in the virus-like particles used in the present application. Note that the variants defined herein are not limited to variants due to mutation, but also include variants obtained by artificially introducing mutations.
  • the specific amino acid sequence of the HBs-L antigen protein of each genotype may be any known one and is not particularly limited.
  • the amino acid sequence shown in any one of SEQ ID NOs: 1 to 9 as genotype A the amino acid sequence shown in any one of SEQ ID NOs 10 to 18 as genotype B, and the amino acid sequence shown in any one of SEQ ID NOs 29 to 38 as genotype D. Amino acid sequences can be mentioned.
  • amino acid sequences that specify the HBs-L antigen protein of each genotype described above are the amino acid sequences shown in SEQ ID NOs: 1 to 38 that specify the HBs-L antigen protein of each genotype, to the extent that the effects of the present invention are not significantly impaired. It can also be a variant of. Such mutations are not particularly limited, and may include, for example, substitutions, insertions, or deletions.
  • the degree of mutation in the amino acid sequence of each genotype described above is not particularly limited. For example, introducing mutations of about 33 amino acids into the amino acid sequence shown in any one of SEQ ID NOs: 1 to 9 (genotype A), or introducing mutations into the amino acid sequence shown in any one of SEQ ID NOs: 10 to 18 (genotype B). Introduction of mutations of about 26 amino acids, introduction of mutations of about 28 amino acids into the amino acid sequence (genotype C) shown in any of SEQ ID NOS: 19 to 28, and introduction of mutations of about 28 amino acids to the amino acid sequence shown in any of SEQ ID NOS: 29 to 38 ( For genotype D), mutations of about 38 amino acids can be introduced.
  • the degree of mutation introduction into the amino acid sequence of each genotype defined in the present invention is as described in Patent Document 1.
  • a variant if the number of amino acids is 400 among SEQ ID NOs: 1 to 38, a region of 11 amino acids from the N-terminus and/or a region of 6 amino acids from positions 163 to 168 is deleted. Examples include mutants that have Furthermore, among SEQ ID NOs: 1 to 38, when the number of amino acids is not 400, examples include mutants in which the amino acid sequences in the regions corresponding to these are deleted.
  • the three regions of the HBs-L antigen protein, the Pre-S1 region, the Pre-S2 region, and the S region are replaced with regions derived from different genotypes.
  • mutants with deletion can be mentioned.
  • the Pre-S1 region of one genotype is replaced with a Pre-S1 region derived from another genotype
  • the Pre-S1 region and Pre-S2 region of one genotype are replaced with another genotype.
  • substitutions to the derived Pre-S1 and Pre-S2 regions, and further examples include mutants in which the Pre-S2 region of one genotype is replaced with Pre-S2 derived from another genotype.
  • Pre-S2 region it is also possible to replace the Pre-S2 region with a Pre-S1 region derived from another genotype, and also include mutants in which the Pre-S2 region is deleted.
  • mutants in which the Pre-S2 region is deleted.
  • mutations exceeding the number of mutations introduced into the amino acid sequence of each genotype described above.
  • “genotype A,” “genotype B,” “genotype C,” and “genotype D” refer to the respective genes of the HBs-L antigen protein unless otherwise specified. shall mean either the type or a variant thereof.
  • Virus-like particles containing hepatitis B surface L antigen protein (HBs-L antigen protein) of two or more genotypes selected from the group consisting of type A, type B, type C, and type D used in this application may be a combination of particles containing one type of HBs-L antigen protein in each particle, or a combination of particles containing two types of HBs-L antigen protein in one particle.
  • HBs-L antigen a virus-like particle containing one genotype of HBs-L antigen protein per particle will be referred to as "HBs-L antigen”.
  • HBs-L hybrid antigen HBs-Lh antigen
  • the hepatitis B vaccine composition of the present application contains HBs-L antigen proteins of two or more genotypes selected from A, B, C, and D, by appropriately combining HBs-L antigen and/or HBs-Lh antigen. Preferably.
  • Combinations of HBs-L antigen proteins include [type A HBs-L antigen protein and type B HBs-L antigen protein], [type A HBs-L antigen and type D HBs-L antigen protein], [ HBs-L antigen protein of type B and HBs-L antigen protein of type C], [HBs-L antigen protein of type B and HBs-L antigen protein of type D], [HBs-L antigen protein of type C and D HBs-L antigen protein of type B], and HBs-L antigen protein of type B, HBs-L antigen protein of type C, and HBs-L antigen protein of type D].
  • HBs-L antigen protein of type C genotype is a combination of an HBs-L antigen protein of type C genotype and an antigen protein of type D genotype.
  • HBs-Lh antigen which is a virus-like particle containing two types of antigen proteins, an HBs-L antigen protein with genotype C type and an antigen protein with genotype D type, is particularly preferably used.
  • HBs-L antigen and HBs-Lh antigen can be produced using genetically modified yeast (Patent Documents 1 and 2).
  • HBs-L antigen protein has self-assembly ability and can present antigen by assembling on a lipid membrane to form particles.
  • the HBs-L antigen protein has an S region with high lipid affinity, and regardless of the genotype, when produced using biological cells, it sticks into lipid membranes and exists. As a result, the protein takes the form of a virus-like particle, which is a stable antigenic particle structure, and has high immunogenicity due to this particle structure.
  • Hepatitis B nucleocapsid antigen HBc antigen
  • the hepatitis B nucleocapsid antigen (HBc antigen) included in the hepatitis B vaccine composition of the present application is also known as hepatitis B core antigen. Any protein known as an HBc antigen may be used, and is not particularly limited. HBc antigen can be produced using previously reported methods, such as Rolland et al. J Chromatogr B Biomed Sci Appl. 2001 25;753(1):51-65).
  • an HBc antigen expression vector can be obtained by inserting the full-length HBc antigen DNA (ACC# X01587) into a pET-19b vector from which sequences such as His-tag have been removed.
  • the obtained expression vector is introduced into E. coli to obtain an expression strain, the E. coli strain is cultured to obtain bacterial cells, and the HBc antigen can be obtained by purifying the obtained bacterial cells. It is known that the capsid proteins of the HBc antigen bind to each other to form particles, and the HBc antigen produced by Escherichia coli also self-assembles to form particles.
  • the blending ratio of HBs-L antigen and HBc antigen is not limited, and the HBc antigen with respect to the total weight of HBs-L antigen and/or HBs-Lh antigen as virus-like particles
  • the ratio of protein amounts is preferably from 1:0.1 to 1:10, for example, 1: about 0.25, 1: about 0.5, 1: about 1, 1: about 1.25, 1: Examples include cases where the ratio is about 1.5 and 1:about 2.
  • Base containing a carboxyvinyl polymer treated by applying an external shearing force includes, for example:
  • An example is "gel base containing skin/mucosal adhesive" disclosed in WO2007/123193.
  • a carboxyvinyl polymer is a polymer of acrylic acid, and the carboxyl group content of a carboxyvinyl polymer means the ratio (% by mass) of carboxyl groups contained in the polymer to the total amount of the polymer.
  • the method for quantifying the carboxyl group content is not particularly limited, but may be determined using, for example, the method for quantifying carboxyvinyl polymers listed in the Pharmaceutical Excipient Standards.
  • CVP used in the vaccine composition of the present application preferably has a carboxyl group content of 60.0 to 62.0% by mass.
  • the polymer particle diameter of CVP used in the composition of the present invention is such that the mode diameter based on the number of particles is adjusted to 0.05 to 10 ⁇ m (preferably 0.1 to 2.0 ⁇ m) by applying external shear force. is preferably used.
  • mode diameter means the mode diameter based on the number standard.
  • the polymer particle size [particle size distribution] is measured using a number-based particle size distribution that can be measured with a laser diffraction type particle size distribution measuring device, a dynamic light scattering type particle size distribution measuring device, etc. It is preferable that there is no large difference from the distribution results. If the analysis results differ depending on the device, if the mode diameter of the polymer particles is within a predetermined range in at least one device, the mode diameter of the polymer particles can be considered to be within the predetermined range.
  • the mode diameter may be determined by checking the state of the polymer particles using a phase contrast microscope, an opt-SEM (Optical Shadow Effect Mode microscope), or the like. Further, the polymer particle size may be determined by a method using a laser diffraction particle size distribution measuring device according to the method described in the test examples of the present application.
  • the mode diameter of the CVP is adjusted by applying an external mechanical shearing force to the CVP.
  • an external shearing force for example, by applying an external shearing force to commercially available CVP, the mode diameter of the polymer particles can be adjusted to a desired range.
  • the operation of applying shear force is performed by a method known to those skilled in the art.
  • a high-speed rotation emulsifier for example, a high-speed rotation emulsifier, a colloid mill emulsifier, a high-pressure emulsifier, a roll mill emulsifier, an ultrasonic emulsifier, and a membrane emulsifier can be used as a device for applying mechanical shearing force.
  • a high-speed rotation emulsifier for example, a colloid mill emulsifier, a high-pressure emulsifier, a roll mill emulsifier, an ultrasonic emulsifier, and a membrane emulsifier can be used.
  • the hepatitis B vaccine composition of the present application contains a gel base containing CVP that has been treated by applying shearing force from the outside.
  • CVP may be neutralized.
  • the neutralizing agent added to the gel base containing CVP include sodium hydroxide, potassium hydroxide, and L-arginine.
  • CVP is ionized and increases sterically, thereby increasing the viscosity of the gel base.
  • L-arginine its weight ratio to CVP may range from, for example, 1: about 0.5 to 1: about 3, preferably from 1: about 1 to 1: about 2.5, such as 1: about 2. This is an example of what you can do.
  • the hepatitis B vaccine composition of the present application may further contain a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier carriers commonly used in the manufacture of vaccines and intranasal preparations can be used, such as saline, buffered saline, dextrose, water, glycerin, isotonic aqueous buffers, etc. and combinations thereof.
  • preservatives e.g., thimerosal
  • isotonic agents e.g., pH adjusters, surfactants, stabilizers (e.g., sodium edetate hydrate), inactivating agents (e.g., formalin), etc. are added as appropriate. May be blended.
  • At least one cationic surfactant selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride and benzethonium chloride, particularly preferably cetylpyridinium chloride.
  • a cationic surfactant selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride and benzethonium chloride, particularly preferably cetylpyridinium chloride.
  • chloride or cetylpyridinium chloride hydrate When cetylpyridinium chloride hydrate is added, the amount may be determined as appropriate, but for example, the weight ratio with CVP is 1:about 0.01 to 0.03, more preferably 1:about 0.018wt. For example, it may be a section.
  • the concentration of CVP in the hepatitis B vaccine composition of the present application is preferably from 0.1 to 2.0 w/v%, more preferably from 0.3 to 1.0 w/v%, for example about 0.55 w/v%. It is.
  • a base material containing a carboxyvinyl polymer treated by applying an external shearing force of the present application the concentration of the carboxyvinyl polymer treated by applying an external shearing force is about 0.55 w / v %
  • An example is a base containing about 2 parts by weight of L-arginine per 1 part by weight of CVP.
  • a base further containing about 0.018 parts by weight of cetylpyridinium chloride hydrate per 1 part by weight of CVP is also exemplified.
  • the concentration of each antigen in the hepatitis B vaccine composition of the present application is determined by the total amount of HBs-L antigen (total amount of HBs-L antigen and/or HBs-Lh antigen) and the amount of HBs antigen from 0.01 to 0.01, respectively.
  • examples include 10 mg/mL, preferably within the range of 0.05 to 5 mg/mL, for example about 0.1 mg/mL each.
  • the hepatitis B vaccine composition of the present application contains a carboxyvinyl polymer obtained by mixing and stirring stock solutions in which HBs-L antigen and HBc antigen are each suspended separately, and treating the mixed antigen solution by applying a shearing force from the outside. It can be manufactured by mixing with a base. There is no need to add adjuvants other than these ingredients to the compositions of the present application. This mixing and stirring can be achieved in a short time by gentle mixing and stirring without applying stress such as heat or pressure to each antigen.
  • the vaccine composition of the present application is administered by spray into the nasal cavity for the prevention or treatment of hepatitis B.
  • the average particle size of the sprayed preparation is in the range of 50 ⁇ m to 120 ⁇ m (preferably in the range of 50 ⁇ m to 80 ⁇ m), and the particle size distribution of the sprayed preparation is in the range of 10 ⁇ m to 100 ⁇ m (preferably 50% or more, preferably in the range of 10 ⁇ m to 100 ⁇ m). 70% or more), the spray angle from the device is in the range of 30° to 70° (preferably in the range of 40° to 60°), and the spray is administered evenly into the nasal cavity with a full cone spray density.
  • Frull cone which means a uniform spray density with no bias, is one of the spray pattern shapes and means a homogeneous circular area, while the opposite term refers to a donut-shaped area that is localized only on the periphery. It is a "Holocone”.
  • the vaccine composition of the present application is administered by spraying into one or both nostrils intranasally once or multiple times.
  • the dose is determined taking into consideration the subject's age, sex, body weight, etc., but the amount of antigen to be administered is 0.01 to 5 mg of HBc antigen and the total amount of HBs-L antigen (HBs-L antigen).
  • the total amount of antigen and/or HBs-Lh antigen) is preferably in the range of 0.01 to 5 mg, more preferably in the range of 0.05 to 2 mg, respectively.
  • the amount of the formulation sprayed per nostril is preferably 50 ⁇ L to 1000 ⁇ L, more preferably 250 ⁇ L to 1000 ⁇ L.
  • the frequency and period of administration may be determined by taking into consideration the subject's age, sex, weight, etc., and the severity of the disease. For example, administration may be performed multiple times, for example, 3 to 20 times, 5 to 15 times, about 10 times, at intervals of 3 to 21 days, 7 to 17 days, or about 14 days.
  • the hepatitis B vaccine composition of the present application may be administered by being filled into a sprayable device having a nasal spray nozzle.
  • the sprayable device having a nasal spray nozzle is not limited to any commonly used nasal spraying device, and may be a sprayable device without a pump function.
  • an upward pressure airless spray container as a multi-dose spray container, as described for example in WO 2007/123193 and WO 2007/123207, it is possible to administer the spray container at any angle or range of angles without reducing the amount remaining in the container. It can be used without.
  • a disposable type device limited to use for one person, there is a medical syringe equipped with a nasal spray nozzle disclosed in WO2015/199130, and also described in WO2021/066195 and Japanese Patent Application No. 2021-126673.
  • a device having a nasal spray/injection nozzle such as the above can be used.
  • a CVP base, an HBs-Lh antigen stock solution, and an HBc antigen stock solution were prepared by the method shown below and mixed as follows to prepare a nasal hepatitis B vaccine composition.
  • HBs-Lh antigen is produced by feeding yeast transformed to express two HBs-L antigen proteins with genotypes C and D, crushing the resulting bacterial cells, heat treatment, and affinity treatment.
  • a cryopreserved product of virus-like particles produced by gel filtration was thawed and used.
  • the molecular weight of L protein is approximately 45KDa, the purity is over 99%, and the particle size is 60 to 70 nm.
  • the HBs-L antigen stock solution was obtained by suspending the obtained virus-like particles in purified water to which the respective components were added in the amounts listed in Table 2.
  • HBc antigen was obtained by feeding-batch culture of Escherichia coli transformed to express HBc antigen protein (full length), cell disruption, ammonium sulfate precipitation, gel filtration, and endotoxin removal.
  • the molecular weight was approximately 21 KDa, the purity was 99% or more, the particle size was 30 to 35 nm, and the endotoxin concentration was ⁇ 50 EU/mg protein.
  • the obtained nasal hepatitis B vaccine composition was filled into a device for nasal administration, and the droplet size distribution, spray homogeneity, and spray angle were measured.
  • the nasal administration device used was a medical syringe (metered syringe type ejector) equipped with a nasal spray nozzle described in WO2015/199130. The devices used are shown in Figures 2 to 4. The diameter of the ejection port 21 at the tip of the nozzle is 0.26 mm.
  • the average formulation droplet size is in the range of 30 ⁇ m to 80 ⁇ m [63.6 ⁇ m], and 80% or more of the formulation droplet size distribution is in the range of 10 ⁇ m to 100 ⁇ m [85.3%],
  • the spray density is uniform and full cone, and (3) the injection angle is controlled within the range of 30° to 70° [51°], making it an extremely good nasal administration system for hepatitis B vaccine. confirmed.
  • the hepatitis B vaccine nasal administration system of Example 1 is summarized below.
  • Example 2 Mixing ratio of HBs-Lh antigen and HBc antigen stability and effect of CVP HBs-Lh antigen and HBc antigen used in Example 1 were each suspended in PBS, and an HBs-Lh antigen solution (0.1 ⁇ g/mL PBS solution) was prepared. , an HBc antigen solution (0.1 ⁇ g/mL PBS solution) was obtained. The following formulations were prepared using each antigen solution and the CVP base used in the production of Example 1 using PBS (-) containing no antigen.
  • Hepatitis B surface hybrid antigen (HBs-Lh antigen) has poor stability, and especially when used simultaneously with hepatitis B nucleocapsid antigen (HBc antigen), the stability may further decrease depending on the amount of HBc antigen. At the same time, the stability of HBc antigen also decreased. It was confirmed and discovered that the decrease in stability of both antigens can be greatly improved by incorporating carboxyvinyl polymer (CVP).
  • CVP carboxyvinyl polymer
  • Antibody production induction ability test in mice (nasal mucosal inoculation) 1.
  • Preparation of vaccine formulation (1) Preparation of antigen stock solution: Preparation of HBs-Lh/HBc antigen stock solution As the HBs-Lh antigen (type C, type D), the virus particles produced in Example 1 were used. An HBs-Lh antigen stock solution was obtained by suspending 2.857 mg of the obtained virus-like particles in phosphate buffered saline (Gibco PBS, pH 7.4). As the HBc antigen, the antigen produced in Example 1 was used. The HBc antigen stock solution was obtained by suspending 2.857 mg of HBc antigen in phosphate buffered saline (Gibco PBS, pH 7.4).
  • CVP base was produced using the components listed in Table 8 and subjected to shearing force in the same manner as in Example 1. Cetylpyridinium chloride hydrate, polysorbate 80, macrogol 4000, and concentrated glycerin were all added to the CVP base of Example 1 in the amounts shown in the table after obtaining the CVP base. "-" in the table means "not included”.
  • Table 9 shows the pH and viscosity of the mixture obtained by mixing each of Bases 1 to 5 with phosphate buffered saline (Gibco PBS, pH 7.4) at a ratio of 1:1 (volume ratio).
  • Vaccine formulations for nasal vaccination 1 to 5
  • Vaccine formulations 1 to 5 with each antigen concentration of 0.715 mg/mL (10 ⁇ g/14 ⁇ L) were obtained by mixing with Bases 1 to 5 at a ratio of 1:1 (volume ratio).
  • HBs-Lh antigen stock solution (antigen concentration 2.857 mg/mL), HBc antigen stock solution (antigen concentration 2.857 mg/mL), and phosphate buffered saline (Gibco PBS, pH 7.4) at a ratio of 1:0.25:2.75 (volume ratio).
  • vaccine formulation composition [HBs-Lh antigen concentration 0.715 mg/mL (10 ⁇ g/14 ⁇ L), HBc antigen concentration 0.179 mg/mL (2.5 ⁇ g/14 ⁇ L)]
  • vaccine formulation 8 containing no CVP base was obtained.
  • the ingredients of each formulation are shown in Table 10.
  • Antibody production induction ability test in mice (nasal mucosal inoculation) (Method) Vaccine formulations 1 to 8 were injected into both nostrils of BALB/c mice (female, 6 weeks old, 4 mice/group) in 7 ⁇ L doses (total 14 ⁇ L: HBs-Lh antigen amount 10 ⁇ g, HBc antigen amount 10 ⁇ g (Formulations 1 to 5). , 7) or 10 ⁇ g of HBs-Lh antigen and 2.5 ⁇ g of HBc antigen (Preparations 6 and 8).Blood was collected after 2 weeks, and antibody production was induced by measuring the antigen-specific antibody titer in the serum. The results are shown in Table 11 and Figures 5-6.
  • Vaccine formulations 1 and 6 using a CVP base provided in the present application have stronger antibody induction against both HBs-Lh and HBc compared to vaccine formulations 7 and 8 (comparative examples) that do not contain a CVP base. showed ability. Antibody induction ability was enhanced by further adding cetylpyridinium chloride hydrate to the CVP base.
  • carboxyvinyl polymer is the carboxyvinyl polymer produced in Example 1 or 2.
  • HBs-Lh antigen and HBc antigen were produced in Example 1.

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Abstract

La présente invention aborde le problème de la fourniture d'une composition de vaccin pour administration nasale qui est utilisable pour prévenir et traiter l'hépatite B, et un système d'administration nasale du vaccin.　L'invention concerne une composition de vaccin contre l'hépatite B qui comprend : (i) des particules pseudovirales contenant des protéines d'antigène L de surface de l'hépatite B (protéines d'antigène HBs-L) d'au moins deux génotypes choisis dans le groupe constitué des types A, B, C et D, et une protéine d'antigène de nucléocapside de l'hépatite B (antigène HBc); et (ii) un matériau de base contenant un polymère de carboxyvinyle ayant été traité par application externe d'une force de cisaillement. L'invention concerne en outre un système d'administration nasale du vaccin contre l'hépatite B, ledit système comprenant la composition remplie dans un dispositif pulvérisable équipé d'une buse de pulvérisation nasale.
PCT/JP2023/031188 2022-08-30 2023-08-29 Composition de vaccin contre l'hépatite b pour administration nasale et système d'administration nasale associé Ceased WO2024048570A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018225731A1 (fr) * 2017-06-05 2018-12-13 株式会社ビークル Particules de type viral destinées à être utilisées pour provoquer une réponse immunitaire contre le vhb
WO2019070019A1 (fr) * 2017-10-05 2019-04-11 東興薬品工業株式会社 Composition de vaccin nasal contre l'hépatite b et son procédé de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018225731A1 (fr) * 2017-06-05 2018-12-13 株式会社ビークル Particules de type viral destinées à être utilisées pour provoquer une réponse immunitaire contre le vhb
WO2019070019A1 (fr) * 2017-10-05 2019-04-11 東興薬品工業株式会社 Composition de vaccin nasal contre l'hépatite b et son procédé de production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SANADA TAKAHIRO; YAMAMOTO NAOKI; KAYESH MOHAMMAD ENAMUL HOQUE; TSUKIYAMA-KOHARA KYOKO; HASEGAWA HIDEKI; MIYAZAKI TAKASHI; TAKANO J: "Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 520, no. 1, 30 September 2019 (2019-09-30), Amsterdam NL , pages 86 - 92, XP085867964, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2019.09.072 *
SANADA, TAKAHIRO; KAYESH, MOHAMMAD; HASEGAWA, HIDEKI; HIASA, YOICHI; GOH, YASUMASA; MIYAZAKI, TAKASHI; KOHARA, MICHINORI: "HBS-Large Hybrid Protein Vaccination Via Intranasal Route with Carboxyl Vinyl Polymer Rapidly Induced Strong Neutralizing Antibody and Anti-HBS IgA", HEPATOLOGY, vol. 70, no. S1, 1 January 2019 (2019-01-01), US , pages 605A, XP009553573, ISSN: 0270-9139 *

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