Nasal vaccine-spraying formulation for simultaneously targeting nasal mucosa and nasopharynx

Abstract

The present invention pertains to a formulation that for spraying a nasal vaccine, that contains an antigen and a base agent obtained by adding polyethylene glycol to a crosslinkable polyacrylic acid, and that has an optimized spray pattern for simultaneously targeting the nasal mucosa and the nasopharynx.

Description

A nasal vaccine spray formulation that simultaneously targets the nasal mucosa and nasopharynx

[0001] The present invention relates to a nasal vaccine spray formulation containing an antigen and a base that optimizes the spray pattern to simultaneously target the nasal mucosa and nasopharynx.

[0002] Currently available vaccines are administered by subcutaneous or intramuscular injection. However, intranasal administration has attracted attention as a non-invasive method of administration that promotes immune responses throughout the body and in the nasal mucosa, the first line of defense, and avoids the pain and complicated procedures involved in administration. Much research and development has been conducted on intranasal administration, and some live vaccines have already been put to practical use.

[0003] In nasal administration for the purpose of vaccination, the spray used for ordinary nasal drops has problems with the spreading and retention of the sprayed preparation, and as a formulation base to improve these problems, Patent Document 1 uses a spray administration gel base containing a carboxyvinyl polymer treated by applying external shear force, which improves spreading and retention, thereby achieving effects such as reduced dripping, proper retention of antigens in the nasal mucosa, and improved usability.Furthermore, Patent Document 2 uses this spray administration gel base with inactivated influenza virus whole particles, and succeeds in achieving an effective immune response without the use of an adjuvant, which was difficult to achieve with nasal administration.

[0004] However, with the improved spray technology, the fine spray density is uniform and reaches the nasal mucosa in the first half of the nasal cavity widely and evenly, but not much of the formulation reaches deeper areas, namely the nasopharynx, which is thought to be important for the vaccine immune response. Even if the formulation does not reach the nasopharynx directly, it is expected that the formulation trapped in the nasal mucosa will flow into the inferior nasal meatus and migrate to the nasopharynx by mucosidic clearance or simple dripping. However, the vaccine formulation does not sufficiently reach the pharyngeal tonsils (adenoids), eustachian tube tonsils, palatine tonsils, and posterior pharyngeal lymphoid follicles in the nasopharynx that form the Waldeyer ring, which has been a challenge in achieving a stronger immune response.

[0005] Furthermore, sprayers that spray into the nasopharynx deep within the nasal cavity have also been studied, and Patent Document 3 devised a nasal spray / ejection device that uses a nozzle with a nose rest. However, because the spray here is concentrated deep within the nasopharynx, there is little spray onto the nasal mucosa in the front half, and there are still issues with getting the medication to directly reach both the nasal mucosa and the nasopharynx simultaneously in order to increase effectiveness.

[0006] WO 2007 / 123193 WO 2014 / 103488 WO 2021 / 066195

[0007] The nostrils and nasopharynx are connected linearly through an extremely narrow spatial region in the upper inferior nasal meatus. Therefore, in order to deliver a large amount of formulation to the target site, the nasopharynx (epipharynx), using a spray nozzle inserted through the nostrils, an extremely narrow spray angle and a nearly linear spray were required. However, because of this narrow spray angle, even a slight deviation toward the nasal septum, nasal turbinates, or even up or down significantly reduces the amount of formulation reaching the target site. Furthermore, while spraying the nasopharynx is effective, spraying the nasal mucosa in the anterior part of the nasal cavity is also necessary for an effective immune response. However, narrow spray angles result in little or no adhesion to the nasal mucosa along the way. Therefore, to simultaneously spray the formulation to the nasal mucosa and nasopharynx, a formulation with a spray pattern optimized for a denser central area was required, without an extremely narrow spray angle, so that the center of the sprayed formulation mist reaches the nasopharynx and the outer periphery is captured by the nasal mucosa.

[0008] In view of the above problems, the present inventors conducted extensive research and found that adding polyethylene glycol to cross-linked polyacrylic acid to optimize the spray pattern resulted in a full cone and mountain-shaped spray distribution, confirming that a nasal vaccine base can be obtained that can simultaneously target the nasal mucosa and nasopharynx. Thus, by homogenizing the base and antigen, a nasal vaccine spray can be obtained, which is expected to increase the amount of vaccine that reaches the nasopharynx, which is closely related to immune induction. Specifically, the present invention is as follows:

[0009] (Item 1) A nasal vaccine spray formulation comprising a base in which polyethylene glycol has been added to cross-linked polyacrylic acid, and an antigen.

[0010] (Item 2) The nasal vaccine spray formulation according to Item 1, wherein the polyethylene glycol is Macrogol 400 and / or Macrogol 4000, and the amount of the polyethylene glycol is 0.1 to 10.0% by mass relative to the base.

[0011] (Item 3) The nasal vaccine spray formulation according to Item 1 or 2, wherein the crosslinking position and density of the crosslinked polyacrylic acid are adjusted so that the carboxyl group content is 60.0 to 62.0% by mass, and the crosslinking density is high enough that the crosslinking does not cause spinnability.

[0012] (Item 4) A nasal vaccine spray formulation according to any one of Items 1 to 3, wherein the base is prepared by thickening cross-linked polyacrylic acid with an inorganic base and / or a basic amino acid, adjusting the viscosity of the resulting formulation with a viscosity modifier and / or applying external mechanical shearing force, and then adding polyethylene glycol to optimize the spray pattern, thereby targeting both the nasal mucosa and the nasopharynx simultaneously.

[0013] (Item 5) The nasal vaccine spray formulation according to Item 4, wherein the inorganic base is sodium hydroxide, potassium hydroxide, a sodium salt of an inorganic weak acid, and / or a potassium salt of an inorganic weak acid; the basic amino acid is lysine, arginine, and / or ornithine; and the viscosity modifier is sodium chloride, potassium chloride, and / or calcium chloride.

[0014] (Item 6) The nasal vaccine spray formulation according to any one of Items 1 to 5, wherein the antigen is a live or inactivated bacterium, virus, and / or mycoplasma.

[0015] (Item 7) The nasal vaccine spray formulation according to Item 6, wherein the virus is a novel coronavirus, severe acute respiratory syndrome (SARS), influenza viruses such as influenza A and B, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, adenovirus, herpes virus, human papillomavirus, or rubella virus, and the bacterium is Streptococcus pneumoniae, Mycobacterium tuberculosis, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Vibrio cholerae, or Corynebacterium diphtheriae.

[0016] (Item 8) The nasal vaccine spray formulation according to any one of Items 1 to 5, wherein the antigen is an influenza virus whole particle, split antigen, or subunit antigen.

[0017] (Item 9) The nasal vaccine spray formulation according to any one of Items 1 to 5, wherein the antigen is an inactivated whole particle of the novel coronavirus, a recombinant protein antigen, or any of various viral vectors incorporating a gene encoding an antigen protein.

[0018] (Item 10) The nasal vaccine spray formulation according to any one of Items 1 to 5, wherein the antigen is hepatitis B surface antigen (HBs antigen) and / or hepatitis B nucleocapsid antigen (HBc antigen).

[0019] (Item 11) A base for use in a nasal vaccine spray formulation, which is a base made by adding polyethylene glycol to cross-linked polyacrylic acid.

[0020] (Item 12) A nasal vaccine administration system comprising a spray device in which the nozzle abuts the periphery of the external nostril to fix the spray direction, and the nasal vaccine spray formulation according to any one of Items 1 to 10 is filled in the spray device.

[0021] (Item 13) The nasal vaccine spray formulation according to any one of Items 1 to 5, wherein the nasal vaccine spray formulation is administered into the nasal cavities in a single spray volume of 250 to 750 μL per nostril to simultaneously target the nasal mucosa and nasopharynx.

[0022] The nasal vaccine spray formulation of the present invention is a nasal vaccine spray formulation that simultaneously targets the nasal mucosa and nasopharynx by adding polyethylene glycol to cross-linked polyacrylic acid in order to optimize the spray pattern.

[0023] Furthermore, to optimize spray performance, polyethylene glycol is added to cross-linked polyacrylic acid that has been treated by applying external mechanical shear force, ensuring that the drug reaches the nasal mucosa and nasopharynx as target sites simultaneously. By spreading and remaining in the nasal mucosa and nasopharynx for a long time, it is possible to induce an effective immune response with a smaller amount of antigen.

[0024] According to the present invention, it is possible to provide a nasal vaccine spray formulation with fewer side effects by using an antigen as an active ingredient, inducing an effective immune response with a smaller amount of antigen, and simultaneously targeting the nasal mucosa and nasopharynx.

[0025]

[0023] Figures 1A, 1B, and 1C show a nasal spray / spray nozzle having a tip end with a nozzle outlet formed therein and a nose rest that can contact the periphery of the nostril, and are shown in (A) a perspective view, (B) a side view, and (C) a top view. Figures 1A, 1B, and 1C show a spray device in which a syringe is attached to the nasal spray / spray nozzle shown in Figure 1, and are shown in (A) a perspective view, (B) a side view, and (C) a top view. Figures 1A, 1B, and 1C show a nasal spray / spray nozzle having a pair of sub-nozzles, each of which has an outlet at its tip, and which are connected to each other via a connecting portion, and in which the separation distance between the axis of one sub-nozzle and the axis of the other sub-nozzle increases from the connecting portion toward the tip, and are shown in (A) a perspective view, (B) a side view, and (C) a top view. 3 shows a spray device with a syringe attached to the nasal spray / spray nozzle shown in FIG. 3, with (A) a perspective view, (B) a side view, and (C) a top view. The nasal cavity structure is shown. The nasal cavity model used in the target area reach test is shown, with photographs of the front, side, and rear views. The nasal cavity model shown in FIG. 6 shows the positions of the nasal vestibule, nasal mucosa (anterior), nasal mucosa (posterior), and nasopharynx. A photograph of the trap test paper attached in the target area reach test is shown. The types of shapes used in the spray pattern test are shown. Actual data on the spray pattern of the test specimen used in spray test 1 is shown. Actual data on the spray pattern of the test specimen used in spray test 2 is shown. Actual data on the spray pattern of the test specimen used in spray test 3 is shown. Actual data on the spray pattern of the test specimen used in spray test 4 is shown. Actual data on the spray pattern of the test specimen used in spray test 5 is shown. Actual data on the spray pattern of the test specimen used in spray test 6 is shown. (3) Actual data on the spray pattern of the test sample used in spray test 7 is shown. (4) Photographed spray plume shape and droplet size distribution results from measurement of droplet size distribution are shown. (5) Serum HI antibody titer results from antibody production induction test (2) in mice are shown. The horizontal axis of the graph lists bases A to H in order for each of the four dose volumes (bases containing the existing adjuvant Poly I:C and ODN2006 are shown as G and H for convenience). (5) Nasal wash IgA antibody titer results from antibody production induction test (2) in mice are shown. The explanation for the horizontal axis of the graph is the same as in Figure 18.

[0026] The present invention provides a gel base for nasal mucosa spray administration, which is made by adding polyethylene glycol to cross-linked polyacrylic acid, with the aim of optimizing the spray pattern so that the drug can be delivered to the nasal mucosa and nasopharynx simultaneously.

[0027] The "crosslinked polyacrylic acid" used in the present invention is a hydrophilic polymer obtained by polymerizing acrylic acid as the main component. Examples of crosslinking agents used to produce the crosslinked polymer include, but are not limited to, polyalkenyl ethers such as allylpentaerythritol, allylsucrose, and allylpropylene, and divinyl compounds such as divinyl glycol. A crosslinked polyacrylic acid (carboxyvinyl polymer) with a high crosslink density to the extent that it does not exhibit spinnability is used. Here, "crosslink density" refers to the ratio of the number of crosslinking points (crosslinked structural units) to the total number of structural units in a polymer having a crosslinked structure. The crosslink density affects the polymer's flow characteristics, relative viscosity, relative ion resistance, and other properties. Crosslinked polyacrylic acid refers to polyacrylic acid that has its properties enhanced by adjusting the position and density of crosslinks contained within the polymer. As the crosslink density increases, the carboxyl group content in the crosslinked polymer decreases. "A carboxyvinyl polymer in which the position and density of crosslinking are adjusted so that the carboxyl group content is in the range of 60.0 to 62.0 mass%, and which has a high crosslink density to the extent that it does not cause spinnability" refers to a carboxyl group content in the range of 60.0% to 62.0%, and particularly preferably in the range of 60.0% to 61.0%, and specific examples include products such as Carbopol 940NF, Carbopol 941NF, Carbopol 934NF, Carbopol 980NF, Carbopol 981NF, PW-120, Junron PW-121, Junron PW-312S, Akupec HV-805EG, and Akupec HV-801EG.

[0028] The "polyethylene glycol" used in this invention refers to "macrogol," and those distinguished from the "macrogol" used in pharmaceuticals and pharmaceutical additives are preferred. Because polyethylene glycol is a high-molecular-weight compound, it has a wide molecular weight distribution, but its type is usually determined by its number-average molecular weight. Polyethylene glycols with a number-average molecular weight of approximately 200 to approximately 20,000 are typically used. Depending on the molecular weight, they can be liquid, paste, or solid. In this invention, increasing the blend amount and increasing the molecular weight results in a more centrally focused spray pattern, so the blend amount and molecular weight are controlled according to the target. Macrogol 400 and / or Macrogol 4000 are preferred in this invention, with blend amounts ranging from 0.1 to 10.0% by mass. Macrogol 4000 is particularly preferred in a range of 0.1 to 2.0% by mass.

[0029] The "antigen" that can be included in the present invention refers to various antigens that can act as vaccines for humans or non-human animals, such as poultry (e.g., chickens, ducks, etc.), livestock (e.g., cows, pigs, etc.), and pets (e.g., dogs, cats). Specifically, the antigens include infectious pathogens (e.g., viruses or pathogenic bacteria, etc.), natural products purified from such pathogens, or proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, polynucleotides, or DNA encoding antigens artificially produced by techniques such as genetic recombination. Antigens may take the form of complete virus particles, such as virions, incomplete virus particles, virion-constituting particles, viral nonstructural proteins, proteins or glycoproteins derived from pathogens, protective antigens, or neutralizing epitopes, including those that are infectious and those that have lost their infectious potential (inactivated antigens). In terms of structure, the antigens include conventional live or inactivated vaccines, as well as component vaccines, subunit vaccines, vector vaccines, and gene vaccines. Examples of viral vectors used in nasal vaccines include Sendai virus vectors and human parainfluenza type 2 virus vectors, which are considered to have high affinity for the nasal mucosa. Specific examples of viral antigens include novel coronavirus, severe acute respiratory syndrome (SARS), influenza viruses such as influenza A and B, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), varicella virus, measles virus, mumps virus, poliovirus, rotavirus, adenovirus, herpes virus, human papillomavirus, rubella virus, and the like. Specific examples of pathogenic bacteria include pneumococcus, mycobacterium tuberculosis, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Vibrio cholerae, and Corynebacterium diphtheriae.

[0030] As used in the present invention, "live" bacteria, viruses, and / or mycoplasma refer to pathogenic bacteria, viruses, and / or mycoplasma whose toxicity has been weakened to eliminate pathogenicity, and "inactivated" bacteria, viruses, and / or mycoplasma refer to pathogenic bacteria, viruses, and / or mycoplasma whose ability to infect has been lost.

[0031] The amount of antigen contained in the vaccine preparation of the present invention varies depending on the target disease, the recipient, etc., but is not particularly limited as long as it is an amount sufficient to produce antigen-specific antibodies (IgA, IgG, etc.).

[0032] Examples of the "inorganic base" used to thicken the crosslinked polyacrylic acid include sodium hydroxide, potassium hydroxide, sodium salts of inorganic weak acids, potassium salts of inorganic weak acids, etc. Here, examples of the "sodium salts of inorganic weak acids" include disodium hydrogen phosphate hydrate, and examples of the "potassium salts of inorganic weak acids" include dipotassium hydrogen phosphate.

[0033] "Basic amino acids" include lysine, arginine, ornithine, and the like.

[0034] The "viscosity" as defined in the present invention can be measured, for example, by a capillary viscometer method or a rotational viscometer method, which are listed as viscosity measurement methods in the general testing methods of the Japanese Pharmacopoeia.

[0035] The "viscosity adjusting agent" is an ingredient that is added to adjust the viscosity of the base, and is preferably selected from the group consisting of sodium chloride, potassium chloride, and calcium chloride.

[0036] Possible devices for "externally applying mechanical shearing force" for the purpose of adjusting viscosity and optimizing spray performance include ultrasonic, high-speed rotation, and colloid mill type agitators, and particularly preferred are high-speed rotation agitators such as homomixer, comb-tooth, and intermittent jet flow generating types, but are not limited to these.

[0037] "Optimizing the spray pattern" means controlling the spray pattern to have a full cone and mountain-shaped distribution so that the center of the sprayed formulation mist reaches the nasopharynx and the outer periphery is captured by the nasal mucosa in front, without narrowing the angle too much, so that the formulation can be sprayed simultaneously onto the nasal mucosa and nasopharynx. For example, the full cone / mountain-shaped distribution shown in Figure 9.

[0038] The "target site" refers to (1) the area centered on the nasal vestibule from the external nostrils through the nasal valve to the tip of the inferior turbinate, (2) the first half of the nasal mucosa from the tip of the inferior turbinate to the tip of the superior turbinate, (3) the second half of the nasal mucosa from the tip of the superior turbinate to the choanal cavity, and (4) the nasopharynx (upper pharynx) including the eustachian tube tonsils and pharyngeal tonsils (see Figure 7).

[0039] The nasal vaccine spray formulation of the present invention may further contain a pharmaceutically acceptable carrier in addition to cross-linked polyacrylic acid, polyethylene glycol, and an antigen. The carrier may be a carrier commonly used in the production of vaccines and intranasal formulations. Specifically, isotonicity agents such as dextrose and glycerin, preservatives (e.g., thimerosal), surfactants, stabilizers (e.g., sodium edetate hydrate), and inactivators (e.g., formalin) may be appropriately blended. In addition to the carrier, the nasal vaccine spray formulation of the present invention may be adjusted to an optimal pH depending on the antigen using, for example, an appropriate acidic substance (phosphoric acid, citric acid, hydrochloric acid, etc.), a basic substance (sodium hydroxide, potassium hydroxide, basic amino acids such as lysine and arginine), and various acidic or basic buffer solutions.

[0040] The vaccine of the present invention is administered by spraying into the nasal cavity through the nostrils. The vaccine can be administered using a multi-dose nasal spray container or a device that can spray into both nostrils without a pump function, generally a disposable device that can spray without a pump function.

[0041] The volume of the nasal vaccine spray formulation of the present invention to be administered into the nasal cavity is preferably 250 to 750 μL, more preferably 300 to 500 μL, per nostril in one spray. With regard to the spray volume, in the nasal vaccine spray formulation of the present invention, even when the same amount of antigen is administered, increasing the administration volume of the formulation by a certain amount tends to enhance antibody production. The above preferred spray volume is determined taking into consideration the results in mice in the Examples below in which this effect was verified, and the difference in surface area of ​​the nasal cavity between mice and humans.

[0042] Specifically, devices for spraying the "nasal vaccine spray formulation" of the present invention include a "spray container for gel formulations (upward exhaust airless spray container) that can be sprayed onto the affected area by arbitrarily setting the administration direction of the spray container" as disclosed in Patent Document 1, and a "medical syringe having a tip opening that is in liquid communication with a syringe barrel containing the formulation" as disclosed in WO 2015 / 199130. Preferably, however, a spray device is used in which the nozzle portion of the spray device that is inserted into the nose abuts against the periphery of the external nostril, fixing the directionality of the nozzle, i.e., a "spray device in which the nozzle abuts against the periphery of the external nostril, fixing the spray direction." The disclosures of Patent Document 1 and WO 2015 / 199130 are incorporated herein by reference in their entirety.

[0043] A specific example of a "spraying device in which the nozzle abuts against the periphery of the nostril to fix the spray direction" is a spraying device having a spray / spray nozzle with a nose rest as disclosed in Patent Document 3 (i.e., "a spraying device equipped with a nasal spray / spray nozzle, comprising a tip portion formed with a nozzle outlet and a nose rest that can abut against the periphery of the nostril"). Specifically, this spraying device has a nose rest as shown in Figure 1 provided in the nozzle portion and is attached to a syringe as shown in Figure 2, and is designed so that this nose rest abuts against the entrance of the nostril to fix the entire spraying device and keep the spray direction constant during spraying. The disclosure of Patent Document 3 is incorporated herein in its entirety.

[0044] Another example of a "spraying device in which the nozzle abuts the periphery of the external nostril to fix the spray direction" is a spraying device that has nozzles for both nostrils as shown in Figure 3 and is attached to a syringe as shown in Figure 4. Specifically, it is a "spraying device equipped with a nasal spray / injection nozzle having a pair of sub-nozzles, each of which has an ejection hole at its tip, and which are connected to each other via a connecting part, and in which the separation distance between the axis of one sub-nozzle and the axis of the other sub-nozzle increases from the connecting part toward the tip."

[0045] To further explain the present invention, examples and formulation evaluation tests are shown below, but the present invention is not limited to these. Note that, since the incorporation of an antigen into the formulation of the present invention is not thought to have any particular effect on the spray pattern, detailed studies of spray characteristics were carried out using only a base that does not contain an antigen.

[0046] Example 1: Crosslinked polyacrylic acid (0.55 g) was added to purified water (74.45 g) and stirred until homogeneous. The viscosity was then adjusted by applying mechanical shear using an intermittent jet-generating high-speed stirrer. Sodium hydroxide (0.275 g), sodium chloride (0.45 g), and Macrogol 4000 (1.0 g) were dissolved in purified water (25.55 g), and the resulting mixture was added to the crosslinked polyacrylic acid-containing base and stirred until homogeneous, yielding a colorless, transparent, viscous liquid base for the test specimen.

[0047] Examples 2 to 8 The bases of Examples 2 to 8 were prepared from the formulations shown in Table 1 in the same manner as in Example 1. Note that Example 2, in which mechanical shearing was not applied, was produced without the step of applying mechanical shearing as in Example 1.

[0048] Example 9: A formulation containing influenza virus split antigen as an antigen in a base containing cross-linked polyacrylic acid and polyethylene glycol was prepared as follows. Cross-linked polyacrylic acid (1.1 g) and concentrated glycerin (2.0 g) were added to purified water (71.9 g) and stirred until homogeneous. The viscosity was then adjusted by mechanical shearing using an intermittent jet-generating high-speed mixer. L-arginine (2.4 g) and macrogol 400 (2.0 g) were dissolved in purified water (20.6 g) and added to the cross-linked polyacrylic acid base. The mixture was stirred until homogeneous. The mixture was then autoclaved under appropriate conditions to prepare the base. Influenza virus split antigen (60 μg HA), sodium hydrogen phosphate hydrate (2.5 mg), potassium dihydrogen phosphate (0.4 mg), and sodium chloride (8.1 mg) were mixed with purified water to a volume of 1.0 mL and homogeneously mixed to prepare the antigen stock solution. The obtained base and the antigen stock solution were mixed at a volume ratio of 1:1 to obtain an influenza virus split antigen preparation.

[0049] Examples 10 to 13 In the same manner as in Example 9, antigen preparations of Examples 10 to 13 containing various antigens were prepared from the formulations shown in Table 2.

[0050] Evaluation Test of Example Formulations (1) Target Area Delivery Test (i) Principle and Test Method The nasal cavity has a structure as shown in Figure 5. A spray test was conducted to determine delivery to the target area using a nasal cavity model that realistically reproduces the complex three-dimensional structure of the actual human nasal cavity (spray tests 1 to 7 were conducted using different spray formulations). The nasal cavity model used here was a silicone nasal cavity model device as shown in Figure 6. It accurately reproduces the structure of the human nasal cavity and can be disassembled into individual parts as shown in Figures 7 and 8. The amount of formulation sprayed from the nasal cavity entrance that reached each of the four regions of the nasal cavity (nasal vestibule, anterior nasal mucosa, posterior nasal mucosa, and nasopharynx) was calculated based on the mass that reached each region, and the delivery rate was calculated. Specifically, for the "nasal vestibule," the delivery rate was calculated based on the mass increase after removing the nasal vestibule portion after spraying into the nasal cavity model. For the "nasopharynx" (the innermost region), a test strip was placed in the nasopharynx portion and sprayed into the nasal cavity model. The delivery rate was calculated based on the mass increase of the test strip (see the third photograph in Figure 8). The "nasal mucosa" in the center of the nasal cavity was calculated by subtracting the amount that reached the "nasal vestibule" and "nasopharynx." An additional test was performed by placing a test strip in the center of the "nasal mucosa" (see the second photo in Figure 8) to determine the amount that reached before and after the test strip from the amount trapped on the test strip, and the reach rates for the "nasal mucosa (front)" and "nasal mucosa (back)" were calculated. Note that in this evaluation, only the reach rate to the "nasopharynx" was measured in spray tests 2 to 7.

[0051] (ii) Spray devices used in the spray test In this spray test, the three types of spray devices shown in Table 3 were used appropriately in order to verify the influence of the device.

[0052] (iii) Effect of spray angle To confirm whether a slight deviation in the spray angle when spraying from the nasal entrance affects the rate of delivery to the target area, a test was conducted in which the vertical angle of use [α] was shifted approximately 10° upward from the normal nasal insertion angle, and the horizontal angle of use [β] was shifted approximately 10° toward the nasal concha to confirm the effect on distribution to the target area. This test was only conducted in Spray Test 1.

[0053] (2) Spray Pattern Test To verify the spray pattern of each test formulation (Examples and Comparative Examples), each test formulation was filled into Device B, and 250 mg of the formulation was sprayed vertically upward from the device onto spray pattern test paper (filter paper soaked in bromophenol blue, stained, and then dried) at a distance of 30 mm from the spray hole, and the shape of the discolored area (yellow to blue) at the point of adhesion was observed. The types of spray pattern shapes are shown in Figure 9. This test was performed on the formulations used in all tests in Spray Tests 1 to 7.

[0054] Hereinafter, spray tests 1 to 7 were carried out while changing the test formulation and spray device, and the delivery rate to the target area and spray pattern were confirmed.

[0055] Spray Test 1 In order to evaluate the effects of "crosslinked polyacrylic acid" and "polyethylene glycol," which are essential components of the present invention, the comparative examples shown in Table 4 were prepared in accordance with the method of Example 1, excluding each component. Each was sprayed into a nasal cavity model apparatus using Device B, and (1) a target area reach test was conducted. In addition, (2) a spray pattern test was also conducted.

[0056] (Results) The results of the target area reach test and spray pattern test are shown in the table below. Actual spray pattern data is shown in Figure 10. In Example 1, which contains cross-linked polyacrylic acid and macrogol, the spray was evenly distributed over each target area, and the distribution remained stable even when the spray angle was shifted. In Comparative Examples 1 and 2, the amount that reached the nasopharynx was extremely small, regardless of the spray angle. In Comparative Examples 3 to 5, the amount that reached the nasopharynx was large, but even a slight shift in the spray angle significantly reduced the amount that reached the nasopharynx.

[0057] Spray Test 2 To compare the effects of polyethylene glycol (macrogol) and other alcohol compounds as additives, comparative examples containing various alcohol compounds as shown in Table 6 were prepared in accordance with the method of Example 1, and each was sprayed into a nasal cavity model apparatus using Device A to conduct (1) a target area reach test. In addition, (2) a spray pattern test was also performed.

[0058] (Results) The results of the target area delivery test and spray pattern test are shown in Table 7. The actual spray pattern data are shown in Figure 11. Example 2, which contains macrogol, showed a high nasopharynx delivery rate and a good spray pattern with a full cone / mountain shape distribution. The comparative examples showed a low nasopharynx delivery rate, and Comparative Example 9 showed a relatively high delivery rate among them. However, considering the irritation and safety of isopropanol, it was predicted that it would not be suitable for administration to the mucous membranes by nasal drops.

[0059] Spray Test 3: To examine the effect of differences in the molecular weight of polyethylene glycol (macrogol), the corresponding comparative examples in Table 8 that did not contain polyethylene glycol were prepared in accordance with the method of Example 1, and each was sprayed into a nasal cavity model apparatus using Device A to conduct (1) a target area arrival test. In addition, (2) a spray pattern test was also performed.

[0060] (Results) The results of the target area reach test and spray pattern test are shown in Table 9. The actual spray pattern data are shown in Figure 12. Examples 3 to 5, which contained macrogol with different molecular weights, showed a higher nasopharynx reach rate and a good spray pattern with a full cone / peak distribution compared to the comparative example, which did not contain macrogol.

[0061] Spray Test 4 To examine the effect of different concentrations of polyethylene glycol (macrogol), the corresponding comparative examples in Table 10 that did not contain polyethylene glycol were prepared in accordance with the method of Example 1, and each was sprayed into a nasal cavity model apparatus using Device A to conduct (1) a target area arrival test. In addition, (2) a spray pattern test was also performed.

[0062] (Results) The results of the target area reach test and spray pattern test are shown in Table 11. The actual spray pattern data are shown in Figure 13. Examples 6 to 8, which contained different concentrations of macrogol, showed a higher nasopharynx reach rate and a good spray pattern with a full cone / mountain shape distribution than the comparative example, which did not contain macrogol.

[0063] Spray Test 5 In order to confirm whether formulations containing an actual antigen could achieve the same effects as those in Spray Tests 1 to 4, the comparative examples in Table 12, which did not contain polyethylene glycol (macrogol), were prepared in accordance with the method of Example 9, in contrast to Examples 9 and 10, which used influenza virus split antigen as the antigen, and each was sprayed into a nasal cavity model apparatus using Device B, and (1) a target area reach test was performed. In addition, (2) a spray pattern test was also performed.

[0064] (Results) The results of the target area reach test and spray pattern test are shown in Table 13. The actual spray pattern data are shown in Figure 14. The examples containing antigen (influenza virus split antigen) also showed a higher nasopharynx reach rate and a good spray pattern with a full cone / mountain distribution than the comparative examples not containing macrogol.

[0065] Spray Test 6 In order to confirm whether formulations containing actual antigens could achieve the same effects as those in Spray Tests 1 to 4, the corresponding comparative examples in Table 14, which did not contain polyethylene glycol (macrogol), were prepared in accordance with the method of Example 9 in comparison with Examples 11 and 12, which used hepatitis B virus surface antigen and hepatitis B virus nucleocapsid antigen as antigens, and each was sprayed into a nasal cavity model apparatus using Device C, and (1) a target area reach test was performed. In addition, (2) a spray pattern test was also performed.

[0066] (Results) The results of the target area reach test and spray pattern test are shown in Table 15. The actual spray pattern data are shown in Figure 15. The examples containing antigens (hepatitis B virus surface antigen and hepatitis B virus nucleocapsid antigen) also showed a higher nasopharynx reach rate and a good spray pattern with a full cone / mountain shape distribution than the comparative examples not containing macrogol.

[0067] Spray Test 7 To confirm whether a formulation containing an antigen could achieve the same effects as those in Spray Tests 1 to 4, the comparative examples in Table 16, which did not contain polyethylene glycol (macrogol), were prepared according to the method of Example 9, in contrast to Example 13, which used SARS-CoV-2 S1 protein antigen as the antigen. Each was sprayed into a nasal cavity model device using Device B, and (1) a target area reach test was performed. In addition, (2) a spray pattern test was also performed.

[0068] (Results) The results of the target area reach test and spray pattern test are shown in Table 17. The actual spray pattern data is shown in Figure 16. Even in the example containing the antigen (SARS-CoV-2 S1 protein antigen), a higher nasopharynx reach rate and a good spray pattern with a full cone / mountain distribution were observed compared to the comparative example not containing macrogol.

[0069] (3) Measurement of spray plume shape and droplet size distribution. To confirm the spray plume and droplet size distribution resulting from spraying the formulation of the present invention, the formulation was sprayed into the air along with a comparative formulation. (Method) The test formulation was loaded into Device B, and 250 mg of the formulation was sprayed vertically upward. The shape was photographed from the side using a high-speed camera (High-Speed ​​Microscope VW-9000). The spray plume shape at the time when the maximum spray plume was formed was observed, and the spray angle, etc. were measured. In addition, the droplet size distribution at a distance of 30.0 mm after spraying was measured using a laser diffraction particle size analyzer (Malvern SPRAYTEC). This evaluation was performed on Example 1 and Comparative Examples 1 to 4.

[0070] The results are shown in Figure 17. In Example 1, which contains cross-linked polyacrylic acid and macrogol, it was observed that the spray plume shape had a higher density at the center compared to the Comparative Example. In addition, the droplet size distribution showed an average particle size of 61.5 μm, with 83.7% in the 10 to 100 μm range, confirming that the spray was sufficiently fine.

[0071] (4) Antibody Production Induction Test in Mice (1) The formulations containing the antigens used in Spray Test 7 above were administered intranasally to mice, and their antibody production ability was evaluated in comparison with that of the comparative example. (Method) The formulations of Example 13 and Comparative Examples 17 and 18 used in Spray Test 7 above were administered intranasally to BALB / C mice (female, 6 weeks old) in both nostrils (3 μg of SARS-CoV-2 S1 protein antigen). Three weeks after inoculation, serum, nasal washes, and alveolar lavage fluids were collected, and antibody production induction ability was analyzed by measuring SARS-CoV-2 S1 protein-specific IgA in the nasal washes, SARS-CoV-2 S1 protein-specific IgA in the alveolar lavage fluid, and SARS-CoV-2 S1 protein-specific IgG in the blood. A microsyringe with a polypropylene tube attached to the tip was used as the administration device.

[0072] (Results) The results are shown in the table below. In the example, higher antibody production was observed at each site than in the comparative example.

[0073] (5) Antibody production induction test in mice (2) 1. Preparation of vaccine formulation (1) Preparation of antigen stock solution Influenza split antigen [H1N1] was mixed with physiological saline until homogeneous to prepare antigen stock solutions of the four concentrations shown in the table below.

[0074] (2) Preparation of Bases Each component in the table below was mixed in purified water, neutralized to a pH of about 7 with sodium hydroxide, and mixed until homogeneous to obtain a base composition. For the "mechanical shear added" composition, the resulting composition was subjected to mechanical shear force using an intermittent jet flow generating high-speed agitator (Bases A to C correspond to examples of the present invention, and Bases D to E correspond to comparative examples). Base F was prepared using physiological saline as is.

[0075] (3) Preparation of test samples Test samples No. 01 to No. 24 were prepared by mixing equal amounts of four types of antigen stock solutions with different concentrations and six types of bases A to F with different compositions. In order to compare the effects with those when an existing adjuvant was added, test samples No. 25 to No. 32 were prepared by further adding 10 μg of an existing adjuvant (Poly I:C or ODN 2006) per volume of the formulation to be administered into one nostril to the sample containing base F, which was physiological saline. A total of 32 types of test samples were prepared as shown in the table below.

[0076] (4) Test Method: The 32 test samples listed in the table above were administered into both nostrils of BALB / C mice (female, 8 weeks old) at 2.5 μL, 5.0 μL, 7.5 μL, or 15.0 μL per nostril, taking particular care with viscous samples due to the vehicle. (This corresponds to a 1 μg dose of influenza split antigen per nostril for all test samples.) To avoid choking, the vaccine was administered into one nostril at least two minutes after administration, and then into the other nostril. A microsyringe with a polypropylene tube attached to the tip was used as the administration device. Three weeks after the initial administration, a booster nasal vaccination was administered in the same manner. Three weeks after the second vaccination, serum and nasal washes were collected, and serum HI antibody titers and antigen-specific IgA in nasal washes were measured to analyze antibody production induction.

[0077] (5) Test Results The results are shown in the table below and in FIGS. 18 and 19.

[0078] (6) Discussion In this study, the antigen was adjusted to the same amount (1 μg HA per nostril), but increasing the formulation administration volume to 2.5 μL x 2, 5.0 μL x 2, and 7.5 μL x 2 tended to enhance the production of both HI antibodies in serum and IgA antibodies in nasal washes. The antibody production enhancement rate was particularly large when changing from 5.0 μL x 2 to 7.5 μL x 2. However, changing from 7.5 μL x 2 to 15.0 μL x 2 showed almost no change in antibody production. This is thought to be due to differences in the site of delivery of the formulation depending on the administered volume. 5 μL / body (2.5 μL / nostril): Confined to the nasal cavity. 10 μL / body (5.0 μL / nostril): Reached the nasal cavity and some of the nasopharynx. 15 μL / body (7.5 μL / nostril): Reached the nasal cavity and nasopharynx (nasopharynx) and some of the oropharynx. 30 μL / body (15.0 μL / nostril): Passed through the nasal cavity and pharynx and traveled to the esophagus and gastrointestinal tract. Compared with the test sample using base E, the test samples using bases A to D had a greater effect on enhancing antibody production. This is thought to be due to the effect of cross-linked polyacrylic acid. Compared with the test sample using base D, the test samples using bases A to C, which contain macrogol, tended to have a greater effect on enhancing antibody production, with the change tending to be particularly significant with increasing dosage. Furthermore, the antibody production enhancement effect tended to be greater with increasing molecular weight of macrogol. In particular, when the amount of macrogols was changed from 5.0 μL x 2 to 7.5 μL x 2, the enhancement rate of antibody production was large. Test samples No. 25 to No. 32, which contained two commonly used adjuvants, showed a clear effect of enhancing antibody production compared to the test sample containing base F without adjuvant, but the difference in the enhancement effect when the dosage volume of the formulation was changed tended to be smaller compared to the test samples containing bases A to C, which contained macrogols.

[0079] DESCRIPTION OF SYMBOLS 10: Nasal spray / injection nozzle 11a: Tip 12a: Nozzle outlet 13a: Nose rest 14a: Nozzle main body 11: Sub-nozzle 11A: Tip 12: Injection outlet 13: Opening end 13': Syringe connecting protrusion 14: Boundary line 15: Connecting portion 16: Sub-nozzle axis a: Crotch portion 100: Syringe-type spray / injection device 110: Screw tip 120: Syringe barrel 130: Syringe main body 140: Plunger rod 150: Adapter 160: Piston 170: Finger rest 180: Plunger

Claims

1. A nasal vaccine spray formulation comprising a base of cross-linked polyacrylic acid to which polyethylene glycol has been added, and an antigen.

2. 2. The nasal vaccine spray formulation according to claim 1, wherein the polyethylene glycol is Macrogol 400 and / or Macrogol 4000, and the amount thereof is 0.1 to 10.0% by mass relative to the base.

3. The nasal vaccine spray formulation according to claim 1, wherein the crosslinking position and density are adjusted so that the carboxyl group content in the crosslinked polyacrylic acid is 60.0 to 62.0% by mass, and the crosslinking density is high enough that the crosslinking does not cause spinnability.

4. 2. A nasal vaccine spray formulation according to claim 1, wherein the base is a cross-linked polyacrylic acid thickened with an inorganic base and / or a basic amino acid, the viscosity of the resulting formulation is adjusted by adding a viscosity regulator and / or external mechanical shear force, and then polyethylene glycol is added to optimize the spray pattern, thereby targeting both the nasal mucosa and the nasopharynx simultaneously.

5. the inorganic base is sodium hydroxide, potassium hydroxide, a sodium salt of a weak inorganic acid, and / or a potassium salt of a weak inorganic acid; the basic amino acid is lysine, arginine, and / or ornithine; the viscosity modifier is sodium chloride, potassium chloride, and / or calcium chloride; The nasal vaccine spray formulation according to claim 4.

6. The nasal vaccine spray formulation according to any one of claims 1 to 5, wherein the antigen is a live or inactivated bacterium, virus, and / or mycoplasma.

7. The virus is a novel coronavirus, severe acute respiratory syndrome (SARS), influenza viruses such as influenza A / B, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, adenovirus, herpes virus, human papillomavirus, or rubella virus, The nasal vaccine spray formulation according to claim 6, wherein the bacterium is Streptococcus pneumoniae, Mycobacterium tuberculosis, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Vibrio cholerae, or Corynebacterium diphtheriae.

8. The nasal vaccine spray formulation according to any one of claims 1 to 5, wherein the antigen is a whole particle, split antigen, or subunit antigen of influenza virus.

9. A nasal vaccine spray formulation according to any one of claims 1 to 5, wherein the antigen is an inactivated whole particle of the novel coronavirus, a recombinant protein antigen, or any of various viral vectors incorporating a gene encoding an antigen protein.

10. The nasal vaccine spray preparation according to any one of claims 1 to 5, wherein the antigen is hepatitis B surface antigen (HBs antigen) and / or hepatitis B nucleocapsid antigen (HBc antigen).

11. A base for use in nasal vaccine spray formulations, which is a base made by adding polyethylene glycol to cross-linked polyacrylic acid.

12. A nasal vaccine administration system comprising a spray device in which the nozzle abuts the periphery of the external nostril to fix the spray direction, and the nasal vaccine spray formulation according to any one of claims 1 to 5 is filled in the spray device.

13. A nasal vaccine spray formulation according to any one of claims 1 to 5, characterized in that the nasal vaccine spray formulation is administered into the nasal cavity in a single spray volume of 250 to 750 μL per nostril in order to simultaneously target the nasal mucosa and nasopharynx.