Classifications

• • 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
  • A61K39/125—Picornaviridae, e.g. calicivirus
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a stable norovirus vaccine composition.
• Norovirus is a positive-sense, single-stranded RNA virus belonging to the Norovirus genus of the Caliciviridae family.
• the genomic RNA of norovirus has regions encoding three proteins: a nonstructural protein (ORF1), structural protein 1 (VP1): ORF2, and structural protein 2 (VP2): ORF3, and VP1 and VP2 constitute the viral capsid.
• Norovirus is one of the viruses that cause acute gastroenteritis and epidemic diarrhea that occur in winter, and is classified into 10 genogroups. Among them, noroviruses that infect humans belong to Genogroup I (GI), Genogroup II (GII), and Genogroup IV (GIV), and the majority of human infections are GI and GII.
• GI and GII genogroups there are 9 genotypes in GI and 27 genotypes in GII, and it is believed that different genotypes basically have different antigenicity.
• more than 680 million people are infected with norovirus every year around the world, and more than 200,000 people die, mainly in developing countries, but there is still no effective vaccine or treatment.
• VLPs virus-like particles
• Non-Patent Document 1 a Non-Patent Document 1
• chitosan is an allergen, and if you have a chitosan allergy, you may not be able to receive the vaccine.
• norovirus causes many deaths, especially in developing countries, and it is in these countries that the vaccine is most effective.
• the present invention relates to providing a highly heat-stable norovirus vaccine composition that uses VLPs composed of the norovirus VP1 protein as a vaccine antigen.
• the inventors searched for additives that contribute to the thermal stability of the VLP of norovirus VP1 protein and found that the thermal stability of the VLP was improved by combining specific amino acids.
• the present invention relates to the following 1) to 8).
• a norovirus vaccine composition comprising a norovirus VP1 protein, histidine or a salt thereof, and one or more amino acids or salts thereof selected from lysine and glutamic acid.
• the vaccine composition according to 1) wherein the content of one or more amino acids or salts thereof selected from histidine or a salt thereof, lysine, and glutamic acid in the composition is 25 mM or more and 400 mM or less.
• the vaccine composition according to 3) wherein the content of aspartic acid or a salt thereof in the composition is 25 mM or more and 200 mM or less.
• a method for stabilizing a norovirus vaccine containing a norovirus VP1 protein as a vaccine antigen comprising the step of adding histidine or a salt thereof, and one or more amino acids or salts thereof selected from lysine and glutamic acid.
• the method according to 5 wherein one or more amino acids or salts thereof selected from histidine or a salt thereof, lysine and glutamic acid are added to the vaccine so that their concentrations are 25 mM or more and 400 mM or less.
• the method according to 5 further comprising the step of adding aspartic acid or a salt thereof.
• the present invention improves the thermal stability of norovirus vaccines that use VP1 protein as an antigen, enabling the creation of highly distributable and popular norovirus vaccines, which can make a significant contribution to the pharmaceutical industry.
• thermal stability of VP1 protein in various additives Evaluation of the thermal stability of VP1 protein in combination with two types of additives. Histidine concentration and thermal stability of VP1 protein. Evaluation of thermal stability of VP1 protein in combinations of histidine and various amino acids. Evaluation of thermal stability of VP1 protein when mixed with three drugs containing histidine and lysine. Evaluation of thermal stability of VP1 protein when mixed with three drugs containing histidine and glutamic acid.
• a "norovirus vaccine” is a vaccine against human norovirus, and uses norovirus virus-like particles (VLPs) as a vaccine antigen.
• VLPs are recombinant proteins produced by expressing a viral capsid in cells, and are virus-like hollow particles that are morphologically similar to norovirus and do not contain internal genes.
• the norovirus used to prepare the VLP includes all currently known genogroup I (GI), genogroup II (GII) and genogroup IV (GIV) genotypes and genotypes to be isolated and identified in the future, and can be appropriately selected and used.
• the norovirus vaccine of the present invention may be a monovalent vaccine containing only one of the genotypes of GI, GII and GIV, or a multivalent vaccine containing multiple genotypes.
• a multivalent vaccine containing multiple genotypes of GI, GII and GIV is preferable, and a multivalent vaccine containing one or more genotypes of GI and one or more genotypes of GII is more preferable.
• the recombinant protein that forms the VLP is composed of VP1, a structural protein of norovirus, and this VLP can be produced using a baculovirus expression system, a mammalian cell expression system, or a plant expression system, etc.
• the insect cells used in the baculovirus expression system are not particularly limited as long as they are cell lines established from Lepidoptera insects, and examples thereof include silkworm cells (BmN cells, BmN4 cells, BoMo cells, etc.), Antheraea persica cells (Anpe cells), Spodoptera frugiperda cells (Sf9 cells and Sf21 cells, etc.), Antheraea mulberry webworm cells (SpIm cells), and Antheraea gracilis cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.). Improved insect cell lines derived by modifying these cells are also included.
• the norovirus vaccine antigen prepared using the above-mentioned insect cells is, for example, infected with a recombinant baculovirus, cultured at 26 to 28° C. for about 2 to 10 days, and after completion of the culture, the culture supernatant of the infected cells is collected and centrifuged and filtered for clarification. Then, ultrafiltration for concentration and purification can be performed using means such as density gradient centrifugation and chromatography.
• the amount of antigen contained in the norovirus vaccine composition of the present invention is not particularly limited, so long as it is an amount sufficient to induce a specific antibody response, for example, 15 ⁇ g VP1/strain or more, preferably 50 ⁇ g VP1/strain or more.
• the amount of antigen contained in the norovirus vaccine of the present invention can be prepared so that a single dose is, for example, 15 to 150 ⁇ g VP1 (in VP1 equivalent), preferably 50 to 100 ⁇ g VP1 (in VP1 equivalent).
• the norovirus vaccine composition of the present invention contains histidine or a salt thereof, and one or more amino acids or salts thereof selected from lysine and glutamic acid.
• examples of salts of amino acids include salts with inorganic bases, organic bases, inorganic acids, organic acids, etc.
• examples of salts of histidine include preferably sodium salts and hydrochloride salts
• examples of salts of lysine include preferably hydrochloride salts, acetate salts, sulfate salts, etc.
• examples of salts of glutamic acid include preferably sodium salts, potassium salts, hydrochloride salts, etc.
• the thermal stability of the VP1 protein of norovirus that forms VLPs is improved by combining histidine with lysine or glutamic acid, or both. Therefore, in a norovirus vaccine containing the VP1 protein of norovirus as a vaccine antigen, the vaccine can be stabilized by adding histidine or a salt thereof, and one or more amino acids selected from lysine and glutamic acid or a salt thereof.
• stabilization of the vaccine means improving the thermal stability of the VP1 protein of the norovirus that forms the VLP.
• the thermal stability of the protein can be calculated from the inflection temperature (Ti) of the thermal denaturation curve measured using, for example, Tycho NT.6 (NanoTemper).
• the content (concentration) of histidine or a salt thereof, or lysine or a salt thereof, or glutamic acid or a salt thereof used in combination with histidine in the norovirus vaccine composition of the present invention is preferably 25 mM or more, more preferably 50 mM or more, preferably 80 mM or more, and even more preferably 100 mM or more in terms of improving the thermal stability of the VP1 protein, and is preferably 400 mM or less, more preferably 200 mM or less, and more preferably 150 mM or less in terms of solubility.
• the contents of histidine or a salt thereof, lysine or a salt thereof, and glutamic acid or a salt thereof in the composition may be the same or different concentrations as long as the thermal stability of the VP1 protein is improved.
• the ratio (concentration ratio) of the combination of histidine or a salt thereof is 1, and the ratio of lysine or a salt thereof, and glutamic acid or a salt thereof is preferably 0.25 or more, more preferably 0.5 or more, and preferably 2 or less, more preferably 1.5 or less.
• histidine or a salt thereof in order to improve the stability of the VP1 protein of norovirus, it is preferable to use histidine or a salt thereof, one or more amino acids selected from lysine and glutamic acid or a salt thereof in combination with aspartic acid or a salt thereof.
• histidine or a salt thereof a combination of three amino acids, namely histidine or a salt thereof, either lysine or glutamic acid or a salt thereof, and aspartic acid or a salt thereof, can be preferably used.
• salts of aspartic acid include sodium salts, potassium salts, and magnesium salts.
• the content of aspartic acid or a salt thereof in the composition can be appropriately set, but is preferably 25 mM or more, more preferably 50 mM or more, and is preferably 200 mM or less, more preferably 150 mM or less.
• suitable examples of combinations of three types of amino acids include the following: 1) 100-150 mM histidine or a salt thereof, 100-150 mM lysine or a salt thereof, and 100-150 mM glutamic acid or a salt thereof; 2) 100-150 mM histidine or a salt thereof, 100-150 mM lysine or a salt thereof, and 50-150 mM aspartic acid or a salt thereof; and 3) 100-150 mM histidine or a salt thereof, 100-150 mM glutamic acid, and 50-150 mM aspartic acid.
• the norovirus vaccine composition of the present invention can be prepared by dissolving the norovirus VP1 protein, histidine or a salt thereof, one or more amino acids selected from lysine and glutamic acid or a salt thereof, and aspartic acid or a salt thereof, if necessary, in a liquid such as water or a buffer solution, using a known method.
• the liquid can be appropriately selected depending on the application, and examples include water, phosphate buffer, citrate buffer, Tris buffer, physiological saline, and Minimum Essential Medium (MEM).
• the norovirus vaccine composition of the present invention may further contain a medicamentously acceptable carrier.
• Such carriers include carriers that are commonly used in the manufacture of vaccines, such as buffers, emulsifiers, preservatives (e.g., thimerosal), isotonicity agents, pH adjusters, inactivating agents (e.g., formalin), adjuvants other than squalene-containing emulsions, and immunostimulants.
• the volume of the norovirus vaccine composition of the present invention administered at one time is determined by the administration site and administration device, but is usually about 0.05 to 1.0 mL, and preferably 0.2 to 0.5 mL.
• the norovirus vaccine composition of the present invention is administered parenterally, preferably by injection.
• injection include subcutaneous, intramuscular, intradermal, and intravenous administration.
• Subjects to which the norovirus vaccine composition of the present invention is administered include humans and non-human mammals, with humans being preferred.
• non-human mammals include pigs, orangutans, chimpanzees, etc.
• Example 1 Screening of additives that contribute to improving thermal stability
• a total of 21 additives with a proven track record as pharmaceutical additives namely sucrose, trehalose, mannitol, sorbitol, maltose, dextran 40, glycine, arginine, histidine, glutamic acid, aspartic acid, lysine, proline, methionine, N-acetyl-L-cysteine, erythorbic acid, sodium edetate, diethylenetriaminepentaacetic acid, polysorbate 80, polysorbate 20, and poloxalene, were used to evaluate their contribution to the thermal stability of norovirus VP1 protein.
• pharmaceutical additives namely sucrose, trehalose, mannitol, sorbitol, maltose, dextran 40, glycine, arginine, histidine, glutamic acid, aspartic acid, lysine, proline, methionine, N-acetyl-
• an inactivating agent beta propiolactone was added to a final concentration of 0.05%, reacted at 4 ° C. for 12 hours, and the baculovirus particles were further removed with a virus removal filter (0.1 ⁇ m). After removing the virus, the fraction containing VP1 was collected by cesium density gradient centrifugation, concentrated again by ultrafiltration, and replaced with 15 w/w% sucrose-containing phosphate buffered saline (pH 7.2). The purified VLP was ultracentrifuged at 26,000 ⁇ g for 30 minutes, and the supernatant was filtered through a virus removal filter (0.1 ⁇ m) to prepare a vaccine antigen.
• Norovirus VLPs with a protein content of 1600 ⁇ g/mL were diluted with 6.7 mM phosphate buffer to adjust the protein content to 100 ⁇ g/mL. Similarly, norovirus VLPs were diluted with each additive to adjust the protein content to 100 ⁇ g/mL.
• Sucrose, trehalose, mannitol, sorbitol, maltose, and dextran 40 were added to a final concentration of 200, 20, and 2 mM, glycine, arginine, histidine, glutamic acid, aspartic acid, lysine, proline, and methionine to a final concentration of 250, 25, and 2.5 mM, N-acetyl-L-cysteine to a final concentration of 10, 1, and 0.1 mM, erythorbic acid to a final concentration of 100, 10, and 0.1 mM, sodium edetate and diethylenetriaminepentaacetic acid to a final concentration of 0.5, 0.05, and 0.005 mM, and polysorbate 80, polysorbate 20, and poloxalene to a final concentration of 1, 0.1, and 0.01%.
• the thermal stability of the VP1 protein was evaluated using Tycho NT.6 (NanoTemper).
• Tycho NT.6 the inflection point temperature (Ti value) of the thermal denaturation curve was measured, and the thermal stability was evaluated from the ⁇ Ti value, which is the difference from the case where no additive was added (only norovirus VP1 protein). It is considered that the thermal stability is improved by increasing this ⁇ Ti value.
• Example 2 Evaluation of thermal stability of VP1 protein in two-drug mixture
• Two types of amino acids glycine, arginine, histidine, glutamic acid, aspartic acid, lysine, methionine
• Norovirus VLP with a protein content of 1600 ⁇ g/mL was diluted with the additives shown above as a single agent or a two-agent mixture so that the protein content was 100 ⁇ g/mL.
• the additives were added to a final concentration of 100 mM, and the thermal stability of VP1 protein was evaluated using Tycho NT.6 (NanoTemper).
• Example 3 Histidine concentration and evaluation of thermal stability of VP1 protein The relationship between histidine concentration and thermal stability improvement effect was examined for histidine, which contributes greatly to thermal stability. Histidine was added to norovirus VLP with a protein content of 1600 ⁇ g/mL, and diluted to a protein content of 100 ⁇ g/mL. Histidine was added to a final concentration of 25, 50, and 100 mM, respectively. The thermal stability of VP1 protein was evaluated using these prepared solutions using Tycho NT.6 (NanoTemper). The measurement results are shown in Figure 3. Within the set concentration range, the Ti value increased in a concentration-dependent manner. Therefore, it was found that the higher the histidine concentration was up to 100 mM, the greater the effect of improving thermal stability. However, at a concentration of more than 400 mM, the solubility of histidine also deteriorates, and precipitation may occur during storage. In addition, the histidine concentration was set to 100 mM because hypertonic injections may cause pain during administration.
• Example 4 Evaluation of thermal stability of VP1 protein in a mixed formulation of histidine and various amino acids
• glutamic acid, aspartic acid, and lysine which showed a thermal stability improving effect in the mixed formulation, were evaluated for their contribution to the thermal stability of VP1 protein when combined with 100 mM histidine.
• Histidine was added to norovirus VLP with a protein content of 1600 ⁇ g/mL, and glutamic acid or aspartic acid and lysine were further added thereto, and the protein content was diluted to 100 ⁇ g/mL.
• VP1 protein was added to a final concentration of 100 mM, and glutamic acid, aspartic acid, and lysine were added to a final concentration of 25, 50, and 100 mM, respectively.
• the thermal stability of VP1 protein was evaluated using Tycho NT.6 (NanoTemper) for each of the prepared solutions. As a result, as shown in Figure 4, the thermal stability of VP1 protein was further improved by mixing 100 mM histidine with 100 mM glutamic acid, 100 mM aspartic acid, or 100 mM lysine, and the effect of improving thermal stability was particularly high when combined with glutamic acid or lysine.
• Example 5 Evaluation of thermal stability of VP1 protein in a three-drug mixture
• glutamic acid or aspartic acid was further added to the mixed formulation of histidine and lysine, in which improved thermal stability was confirmed, and lysine or aspartic acid was further added to the mixed formulation of histidine and glutamic acid, and the thermal stability was evaluated when the three drugs were mixed.
• Histidine and lysine were added to norovirus VLP with a protein content of 1600 ⁇ g/mL, and glutamic acid or aspartic acid was further added, and the solution was diluted to a protein content of 100 ⁇ g/mL.
• Histidine and lysine were added to a final concentration of 100 mM, and glutamic acid and aspartic acid were added to 25, 50, and 100 mM, respectively. Histidine and glutamic acid were added to norovirus VLP with a protein content of 1600 ⁇ g/mL, and lysine or aspartic acid was further added, and the solution was diluted to a protein content of 100 ⁇ g/mL. Histidine and glutamic acid were added to a final concentration of 100 mM, and lysine and aspartic acid were added to 25, 50, and 100 mM, respectively. Each of the prepared solutions was diluted with Tycho NT. The thermal stability of the VP1 protein was evaluated using NanoTemper 6 (NanoTemper).
• Figure 5 shows the results of adding glutamic acid or aspartic acid to the combination of histidine and lysine.
• Figure 5 confirms that the addition of 100 mM glutamic acid or aspartic acid further improves thermal stability.
• Figure 6 shows the results of adding lysine or aspartic acid to the combination of histidine and glutamic acid.
• Figure 6 shows that the addition of 100 mM lysine or aspartic acid or 50 mM aspartic acid further improves the thermal stability of VP1 protein.
• compositions were three-drug mixture formulations highly effective in improving the thermal stability of VP1 protein: 1) 100 mM histidine, 100 mM lysine, and 100 mM glutamic acid; 2) 100 mM histidine, 100 mM lysine, and 100 mM aspartic acid; and 3) 100 mM histidine, 100 mM glutamic acid, and 100 mM or 50 mM aspartic acid.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Virology (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Communicable Diseases (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Oncology (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Molecular Biology (AREA)
• Medicinal Preparation (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=92842191

Family Applications (1)

Country Status (2)

Citations (4)

• 2024
  • 2024-03-22 WO PCT/JP2024/011464 patent/WO2024195868A1/ja not_active Ceased
  • 2024-03-22 JP JP2025508620A patent/JPWO2024195868A1/ja active Pending

Patent Citations (4)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events