JP2020002117A - Squalene-containing adjuvant composition - Google Patents

Abstract

To provide adjuvant compositions containing squalene, the compositions being able to be a stable emulsion with a wide range of particle size, moreover being able to be a stable freeze-dried preparation if necessary, and additionally having the suppressed oxidation and decomposition of components.SOLUTION: Provided is an adjuvant composition comprising squalene, surfactant, dibutylhydroxytoluene, and/or butylhydroxyanisole, as well as a citrate buffer. The composition can be an oil-in-water emulsion or a lyophilized composition.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an adjuvant composition containing squalene, particularly an adjuvant composition in the form of an oil-in-water emulsion or in a lyophilized form.

  Vaccine preparations such as influenza vaccines are very effective in preventing infectious diseases such as bacteria and viruses, and many vaccine preparations have been produced. For example, Flaud®, an inactivated influenza vaccine for the elderly containing an adjuvant, was first recommended in Italy in 1997 and by 2015, including 15 countries in Canada and Europe. Recommended and used in several countries.

Novartis MF59 (trade name) is used as the base of this influenza vaccine. Its composition is 5 v / v% squalene, 0.5 w / v% polyoxyethylene sorbitan monooleate (trade name: Tween 80), 0.5 w / v% sorbitan trioleate (trade name: Span85), It is contained in a 10 mM citrate buffer at pH 6.5. Furthermore, it is said that this MF59 has an adjuvant effect, and the mechanism is being elucidated (Non-patent Document 1). Further, AS03 (trade name) of GlaxoSmithKline, which contains squalene, polyoxyethylene sorbitan monooleate, α-tocopherol, phosphate buffer, and thimerosal, is also used clinically as an adjuvant (Non-patent Document 2). ).
These squalene-containing adjuvants are in the form of oil-in-water emulsions.

  Adjuvants enhance the immune response to vaccine antigens by a variety of mechanisms of action. The effects of adjuvants include enhancing the immune response to vaccine antigens by improving the retention of vaccine antigens in tissues and translocation to lymphoid tissues, and improving the uptake of vaccine antigens by antigen-presenting cells. Are known to have an immunostimulatory effect that provokes an innate immune response.

The mechanism of action of emulsion-type adjuvants is still being elucidated.For example, it uses a foreign body recognition mechanism to stimulate macrophages to activate cytokine release, and to recruit immune cells such as neutrophils to the vaccine administration site. It has been reported that 0.2 μm or less was selected as a particle size suitable for these (Non-Patent Document 3).
Emulsion-type adjuvants are phagocytosed by antigen-presenting cells such as dendritic cells and macrophages, and elicit various immune responses. It has been reported that the particle diameter that is easily phagocytosed by antigen presenting cells is about 1 to 3 μm (Non-patent Document 4, Non-Patent Document 5).
It also improves the particle size suitable for adjuvants to migrate to the lymph nodes and be transported throughout the body, improves the retention of vaccine antigens in tissues and migration to lymphoid tissues, and improves the uptake of vaccine antigens by antigen-presenting cells. The particle size suitable for the treatment is about 0.05 to 3 μm in order to correspond to each of these action mechanisms (Non-Patent Document 6, Non-Patent Document 7).

As described above, since the optimum particle size of the emulsified particles differs depending on the mechanism of action, the emulsion type adjuvant changes the particle size to about 0.05 to 3 μm in order to have an action according to the purpose. It is desired that the composition be able to maintain a stable emulsion.
However, since the particle diameter of the conventional emulsion of squalene-containing adjuvant is limited to 0.1 to 0.2 μm, an action mechanism depending on the purpose of use may not be obtained in some cases. In addition, the immunostimulatory effect itself is not sufficient, and therefore, the effect of enhancing the immune response to the vaccine antigen cannot be sufficiently exerted.

  In addition, vaccines and adjuvants are available in many forms of administration, including intradermal, subcutaneous, intramuscular, intravenous, intranasal, intravesical, intravaginal, intrarectal, intrathoracic, intraperitoneal, and pulmonary (inhalation) administration. There is something. It is considered that the emulsion type adjuvant has an optimum particle size depending on the administration form. Therefore, an adjuvant capable of forming a stable emulsion with a wide range of particle diameters is required in order to provide an adjuvant in which the administration route can be freely selected according to the purpose.

Furthermore, squalene contained in conventional squalene-containing adjuvants and the surfactants polyoxyethylene sorbitan monooleate and sorbitan trioleate have an unsaturated bond, and therefore are easily oxidized.At extreme pH, polyoxyethylene sorbitan is used. It is known that hydrolysis of monooleate and sorbitan trioleate occurs (Non-Patent Documents 1 and 8). In particular, squalene monohydroperoxide, which is one of squalene oxides, is known to exhibit moderate cytotoxicity to cultured cells (Non-Patent Document 9).
For this reason, the use of emulsion formulations containing squalene, such as MF59 and AS03, has been limited due to the potential toxicity of components considered to be derived from squalene oxide (Patent Document 1, paragraph 0020).

Therefore, in order to utilize a squalene emulsion formulation having an adjuvant effect and use it as a pharmaceutical product, it is necessary to prepare an emulsion formulation in which oxidation of squalene is suppressed.
However, at present, there are many attempts to find the effectiveness of the antigen, and few attempts to improve the stability of the squalene emulsion preparation. For this reason, chemically stable squalane, liquid paraffin, and the like are widely used as oil components of emulsion-type adjuvants, and squalene tends to be avoided even though it has an adjuvant effect (Non-Patent Document 1). .

It is known that MF59 cannot be frozen (Non-Patent Document 1). In vivoGen's AddaVax (trade name) is commercially available as a non-clinical adjuvant having the same composition as MF59. The product manual states that it is stable for 2 years when stored at 4 ° C. It is stated that it must not be done. Also, in fact, freeze-dried formulations of these squalene-containing adjuvants have not been developed.
However, for long-term storage and transport, particularly for long-term storage and transport in hot regions, it is generally desirable that the pharmaceutical composition be in a solid form rather than a liquid state, and a freeze-dried adjuvant is also desired. I have. Further, a more stable adjuvant of a liquid preparation is desired.

Special Table 2013-501756

C.B.Fox, Moleculles 2009, 14, 3286-3312, Rossella Cioncada et al., PLOS ONE, 0185843 October 31, 2017 Frontiers in Immunology, 2017, vol.8, pp1-17, Amanda L. Wilkins et.al. Pharm Biotechnol. 1995; 6: 277-96.MF59.Design and evaluation of a safe and potent adjuvant for human vaccines.Ott G1, Barchfeld GL, Chernoff D, Radhakrishnan R, van Hoogevest P, Van Nest G. Role of Particle Size in Phagocytosis of Polymeric Microspheres Julie A. Champion, Pharm Res.Author manuscript; available in PMC 2009 December 15. Macrophages Recognize Size and Shape of Their Targets Nishit Doshi, Samir Mitragotri, PLOS ONE, 2010, Volume 5, Issue 4 e10051 Intracellular trafficking of particles inside endosomal vesicles is regulated by particle size.J Control Release, 2017 Jun 13; 260: 183-193. KAKENHI Result Report Kazuki Misato "Development of New Infectious Disease Vaccine by Optimal Design of Multifunctional Protein Nanoparticles" The Subunit and Adjuvant Approach; Plenum Press: 1995; pp.141-228 Yoshino Kono, Motoharu Takahashi, Oil Chemistry Vol.44 (4), 248-255 (1995)

  The present invention relates to an adjuvant composition containing squalene, which can be a stable emulsion with a wide range of particle diameters, and can be a stable freeze-dried preparation if necessary, And a composition in which decomposition is suppressed.

In order to solve the above-mentioned problems, the present inventors have conducted repeated studies and obtained the following findings.
(1) An oil-in-water emulsion containing squalene, a surfactant, dibutylhydroxytoluene (hereinafter sometimes referred to as “BHT”) and / or butylhydroxyanisole (hereinafter sometimes referred to as “BHA”), and a citrate buffer Can be an emulsion containing emulsified particles having a wide range of particle sizes from 0.05 to 3 μm, and the emulsion can be used at high temperatures (for example, 40 ° C. to 90 ° C.) to obtain the properties of the emulsified particles. It is very stable with no change in its composition.
(2) When an emulsion containing squalene and a surfactant is not blended with an antioxidant, oxidation of squalene, hydrolysis of the surfactant and oxidation of liberated fatty acids remarkably progress, but the adjuvant composition of the present invention. The above emulsion contains BHT and / or BHA among the antioxidants, and the oxidation and decomposition are effectively suppressed by the synergistic action with the citrate buffer.
(3) An adjuvant composition that is an oil-in-water emulsion containing squalene, a surfactant, BHT and / or BHA, and a citrate buffer has high stability when used as a lyophilized preparation, and has a high temperature (for example, 40 C. to 60.degree. C.) and then re-dissolved, the desired emulsion can be reproduced.
(4) It is known that a conventional oil-in-water emulsion type adjuvant containing squalene has an action of enhancing an immune response to a vaccine and an immunostimulating action. Adjuvant compositions comprising squalene, surfactants, BHT and / or BHA, and citrate buffers also significantly enhance the immune response to the vaccine and have strong immunostimulatory effects themselves.

The present invention has been completed based on the above findings, and provides the following adjuvant compositions.
Item 1. An adjuvant composition comprising squalene, a surfactant, dibutylhydroxytoluene and / or butylhydroxyanisole, and a citrate buffer.
Item 2. Item 2. The adjuvant composition according to Item 1, which is an oil-in-water emulsion or is freeze-dried.
Item 3. Item 3. The adjuvant composition according to item 1 or 2, wherein the surfactant is a fatty acid ester.
Item 4. Item 4. The adjuvant composition according to Item 3, wherein the fatty acid ester is polyoxyethylene sorbitan monolaurate and / or polyoxyethylene sorbitan monooleate.
Item 5. 0.001 to 30 parts by weight of a surfactant, 0.00003 to 0.05 parts by weight of a total of dibutylhydroxytoluene and / or butylhydroxyanisole, and 0.005 to 20 parts by weight based on 1 part by weight of squalene. Item 5. The adjuvant composition according to any one of Items 1 to 4, comprising part by weight of a citrate buffer.
Item 6. Item 6. The adjuvant composition according to any one of Items 1 to 5, which is an oil-in-water emulsion and contains 0.01 to 50% by weight of squalene based on the total amount of the emulsion.
Item 7. Item 6. The adjuvant composition according to any one of Items 1 to 5, which is a freeze-dried preparation and contains 1 to 50% by weight of squalene based on the total amount of the freeze-dried preparation.
Item 8. Item 8. The adjuvant composition according to any one of Items 1 to 7, further comprising an excipient.
Item 9. Item 1 used concurrently, in combination, or in combination with other immunostimulating drugs, or diseases caused by immunosuppression or diseases that can be prevented, ameliorated, or treated by immunostimulation. The adjuvant composition according to any one of claims 1 to 8.
Item 10. The adjuvant composition according to any one of Items 1 to 8, further comprising a vaccine antigen.
Item 11. Item 11. The adjuvant composition according to Item 10, wherein the vaccine antigen is a BCG vaccine antigen.

Adjuvants activate immunity through a variety of mechanisms of action. As described above, the details of the mechanism of action are still being elucidated, but emulsion-type adjuvants, for example, use a foreign body recognition mechanism to stimulate macrophages to activate cytokine release, or to activate neutrophils at the vaccine administration site. It has been reported that immune cells are recruited, and a particle size of 0.2 μm or less has been selected as a suitable particle size. In addition, emulsion-type adjuvants are phagocytosed by antigen-presenting cells such as dendritic cells and macrophages, and elicit various immune responses.However, the particle size that is easily phagocytosed by antigen-presenting cells is about 1 to 3 μm. Have been reported.
It also improves the particle size suitable for adjuvants to migrate to the lymph nodes and be transported throughout the body, improves the retention of vaccine antigens in tissues and migration to lymphoid tissues, and improves the uptake of vaccine antigens by antigen-presenting cells. A suitable particle size is about 0.05 to 3 μm in order to correspond to each of these action mechanisms.

Conventional squalene-containing emulsion adjuvants have a limited action mechanism because the particle size of emulsified particles is limited to about 0.1 to 0.2 μm. Therefore, the immunostimulatory effect and the effect of enhancing the immune response to the vaccine antigen may not be sufficiently obtained.
On the other hand, the adjuvant composition of the present invention can prepare a stable emulsion even when the particle size of the emulsified particles is changed to, for example, about 0.05 to 3 μm. Emulsions containing emulsified particles having these particle diameters are very stable, with no change in the particle diameter and particle size distribution of the emulsified particles even after storage at a high temperature of, for example, 40 to 90 ° C. Therefore, the adjuvant composition of the present invention can variously change or adjust the action mechanism for activating immunity according to the purpose. In addition, if the emulsion contains a plurality of types of emulsified particles having different particle diameters, immunity can be activated by various action mechanisms. That is, according to the present invention, an emulsion design suitable for the purpose has been made possible, and a wide range of applications has been made possible.

In addition, as mentioned above, intradermal, subcutaneous, intramuscular, intravenous, intranasal, intravesical, intravaginal, intrarectal, intrathoracic, intraperitoneal, pulmonary administration (inhalation), etc. Administration by various routes has been developed. Therefore, it is required that the adjuvant can be administered by various routes. The optimal route of administration of the adjuvant depends on the purpose and composition of the components, but in the case of an emulsion type adjuvant, it also depends on the particle size of the emulsified particles. Therefore, in order to form an adjuvant that can be administered by a route that meets needs, it is necessary that the emulsion be a stable emulsion with a wide range of particle sizes.
In this regard, the adjuvant composition of the present invention can be a stable emulsion with a wide range of particle diameters of 0.05 to 3 μm, and thus can be an adjuvant composition corresponding to various administration routes. In addition, this makes it possible to be used as a base for various vaccines or to be administered in combination with various vaccines.

  According to the study of the present inventors, when an emulsion containing squalene does not contain an antioxidant, oxidation of squalene, hydrolysis of a surfactant, and oxidation of a released fatty acid proceed remarkably. The hydrolysis of the surfactant causes the emulsion to disintegrate, and the free fatty acid lowers the pH, making it impossible to maintain a pH suitable for use (during re-dissolution). In addition, peroxides generated by oxidation of squalene and oxidation of surfactant-derived fatty acids oxidize vaccine antigens, thereby reducing the immune response inducing effect. In addition, as described above, squalene monohydroperoxide, which is an oxidation product of squalene, is known to have a moderate level of cytotoxicity in cultured cells, and its production should be minimized.

  In this regard, in the adjuvant composition of the present invention, oxidation of squalene, and hydrolysis of a surfactant and oxidation of a released fatty acid are effectively suppressed. This effect is also obtained when the composition is stored at a high temperature of, for example, 60 ° C. Therefore, the emulsion of the adjuvant composition of the present invention is stable and, for example, when mixed with a vaccine, the oxidation of the vaccine is suppressed, so that even a small amount of the vaccine can maintain a sufficient immunity-inducing effect, and the pH can be reduced. Since it does not fluctuate, it can be used at a predetermined target (within specification) pH. Further, it can be used at a predetermined pH even after freeze-drying and redissolving.

  Further, the emulsion of the adjuvant composition of the present invention particularly contains a citrate buffer among the buffers, whereby the decrease in pH due to the fatty acid generated by the decomposition of the surfactant is effectively suppressed. This effect can also be obtained when the composition is stored at a high temperature of, for example, 40 to 60 ° C. Thus, in the adjuvant composition of the present invention, the decomposition of the surfactant is effectively suppressed by the synergistic action of BHT and / or BHA and the citrate buffer.

An oil-in-water emulsion using squalene as an oil agent has a high adjuvant effect, but there has been no freeze-dried preparation. It has been known that a conventional squalene-containing emulsion adjuvant must not be frozen.
As described above, the adjuvant composition of the present invention is very stable in an emulsion state, but is also very stable even after freeze-drying, and even after the freeze-dried product is stored at a high temperature of 40 to 60 ° C. By re-dissolving, an intended emulsion having a particle size of about 0.05 to 3 μm can be prepared. Therefore, the adjuvant composition of the present invention can be made into a lyophilized preparation, if necessary.

In addition, conventional squalene-containing emulsion-type adjuvants are known to have an immune response enhancing effect and an immunostimulatory effect on vaccines, and the adjuvant composition of the present invention also significantly enhances immune response to vaccines, As such, it has a strong immunostimulatory effect.
For example, C57BL / 6 mice (6-week-old, female) express OVA-expressing E. coli. G7 cell line (mouse lymphoma-derived cell line) was inoculated intradermally, an evaluation sample was intradermally administered on day 14, the tumor diameter was measured up to 3 weeks after cell transplantation, and autopsy was performed on day 21 for tumor weight. In a weighing system, when a re-dissolved solution (median diameter: 2.0 μm) of a freeze-dried oil-in-water emulsion is administered to a mouse, the growth and growth of a transplanted tumor are suppressed. In addition, when a mixture of an emulsion having a median diameter of 2.0 μm and a BCG vaccine was intradermally administered to mice, spleen cells upon stimulation of PPD (Purified Protein Derivative; Mycobacterium tuberculosis culture filtrate purified antigen) were more intense than in the case of vaccine alone. The production of cytokine (IFN-γ) from E. coli was significantly increased.

  According to the present invention, a practically available squalene-containing emulsion-type adjuvant composition that can be changed to a particle size of about 0.05 to 3 μm, which is resistant to lyophilization, is provided for the first time.

It is a figure which shows the particle size distribution of the emulsion of this invention and its lyophilized formulation re-dissolution liquid. Of the emulsion of Example 1-1 (emulsion 1-1a [with mannitol]), the emulsified stock solution-1 used for its production, and the re-dissolved solution of its freeze-dried preparation (lyophilized product 3-1a, Example 3) The particle size distribution is shown. It is a figure which shows the particle size distribution of the small particle emulsion of this invention manufactured by the high pressure emulsification method. It is a figure which shows the small diameter particle emulsion of the emulsion of this invention by SPG film processing. The particle size distribution of each of the emulsion 1-1a (Example 1-1) and the emulsion 2-1 (Example 2) obtained by treating the emulsion 1-1a with an SPG film is shown. It is a figure which shows the pH stabilizing effect of the citrate buffer with respect to PS80 aqueous solution. Comparative evaluations of changes over time in pH of a citrate buffer, a phosphate buffer, and a PS80 aqueous solution without a buffer were performed. It is a figure which shows the stability (90 degreeC, particle size distribution) of the emulsion of this invention. Time-dependent change in particle size distribution of the emulsion of the present invention (emulsion 1-1b, Example 1-1), the known emulsion (reference emulsion 2-1), and the reference emulsion 1-1 containing no BHT at 90 ° C. for 2 days. Is shown. It is a figure which shows the stability (40 degreeC, particle size distribution) of the freeze-dried emulsion of this invention. Re-dissolved solution of the freeze-dried emulsion of the present invention (freeze-dried product 3-1b, Example 3-1) and the freeze-dried preparation of the known formulation emulsion (reference freeze-dried product 2-2b, reference example 2) before and after storage at 40 ° C. Changes in particle size distribution were compared. It is a figure which shows the enhancement of the immunostimulatory effect of the live BCG bacteria by the re-dissolution solution of the freeze-dried emulsion of the present invention. The spleen cells (5 × 10 6) of C57BL / 6J mice (6 weeks old, female) 4 weeks after intradermal administration, mixed with live BCG bacteria alone or mixed with live BCG bacteria and the re-dissolved solution of the freeze-dried preparation of the present invention. After stimulating PPD (10 μg / ml) to cells / mL), the amount of IFN-γ production in the culture supernatant was determined 24 hours later by ELISA.

  The adjuvant composition of the present invention is a composition comprising squalene, a surfactant, BHT and / or BHA, and a citrate buffer. The composition can be an oil-in-water emulsion or a lyophilized composition.

(1) Oil-in- water emulsion The oil-in- water emulsion may be prepared by mixing and emulsifying each component in water, and is obtained by freeze-drying the oil-in-water emulsion and then re-dissolving it. It may be an oil-in-water emulsion.
The adjuvant composition of the present invention is administered to a living body as an oil-in-water emulsion. Therefore, the preferred or appropriate composition and properties required for an oil-in-water emulsion are the same between those prepared by mixing and emulsifying each component in water and those obtained by re-dissolving the lyophilized preparation. is there.

The concentration of squalene in the squalene oil-in-water emulsion, based on the total amount of the emulsion, for example, 0.01 to 50 wt%, preferably 0.1 to 10 wt%, more preferably include 0.5 to 5 wt% be able to. In addition, 1 to 3% by weight is also included. Within the above range, an appropriate adjuvant effect can be obtained.

Surfactant The surfactant can be used without limitation as long as it is a surfactant used in pharmaceutical preparations, and examples thereof include phospholipids and nonionic surfactants.
Examples of the phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lecithin (soy lecithin, egg yolk lecithin) and the like. In addition, hydrogenated phospholipids such as hydrogenated lecithin can also be used.

Examples of the nonionic surfactant include polyoxyethylene sorbitan monolaurate (polysorbate 20), polyoxyethylene sorbitan monopalmitate (polysorbate 40), polyoxyethylene sorbitan monostearate (polysorbate 60), and polyoxyethylene sorbitan monosorbate. Polyoxyethylene sorbitan fatty acid esters such as oleate (polysorbate 80), polyoxyethylene sorbitan monoisostearate;
Sorbitan monolaurate (trade name: Span20), sorbitan monopalmitate (trade name: Span40), sorbitan monostearate (trade name: Span60), sorbitan monoisostearate, sorbitan monooleate (trade name: Span80), Sorbitan fatty acid esters such as sorbitan trioleate (trade name: Span85), diglycerol sorbitan penta-2-ethylhexylate, diglycerol sorbitan tetra-2-ethylhexylate;
Polyoxyethylene hydrogenated castor oil 40 (brand name: NIKKOL HCO-40), polyoxyethylene hydrogenated castor oil 50 (brand name: NIKKOL HCO-50), polyoxyethylene hydrogenated castor oil 60 (brand name: NIKKOL HCO-60) And polyoxyethylene hydrogenated castor oil such as polyoxyethylene hydrogenated castor oil 80 (trade name: NIKKOL HCO-80);
Glycerin monostearate, such as self-emulsifying glyceryl monostearate;
Sucrose fatty acid esters;
Polyoxyethylene (20) polyoxypropylene (20) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (120) polyoxypropylene (40) glycol, polyoxyethylene (124) polyoxy Polyoxyethylene-polyoxypropylene glycol such as propylene (39) glycol and polyoxyethylene (160) polyoxypropylene (30) glycol (trade name: Pluronic F68) are exemplified.

In the adjuvant composition of the present invention, since the hydrolysis of the surfactant and the oxidation of the resulting fatty acid are effectively suppressed, a nonionic surfactant which is a fatty acid ester can be suitably used.
Examples of the nonionic surfactant which is a fatty acid ester include polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, glycerin monostearate, and sucrose fatty acid ester, among which polyoxyethylene sorbitan fatty acid ester is preferable, Oxyethylene sorbitan monolaurate (polysorbate 20) and polyoxyethylene sorbitan monooleate (polysorbate 80) are more preferred, and polyoxyethylene sorbitan monooleate (polysorbate 80) is particularly preferred.
The adjuvant composition of the present invention can be free of sorbitan fatty acid esters, especially sorbitan trioleate (trade name: Span85).

  One surfactant can be used alone, or two or more surfactants can be used in combination.

The concentration of the surfactant in the oil-in-water emulsion is, for example, 0.005 to 10% by weight, preferably 0.01 to 5% by weight, more preferably 0.05 to 3% by weight, based on the total amount of the emulsion. % By weight. In addition, 0.3 to 1% by weight is also included. Within the above range, stable emulsified particles can be obtained.
Further, the content of the surfactant in the oil-in-water emulsion is, for example, 0.001 to 30 parts by weight, preferably 0.01 to 10 parts by weight, more preferably 0 to 10 parts by weight based on 1 part by weight of squalene. 0.05 to 5 parts by weight. In addition, 0.1 to 1 part by weight is also included. Within the above range, stable emulsified particles can be obtained.

BHT / BHA
Among BHT and BHA, BHT is preferable.
The concentration of BHT and / or BHA in the oil-in-water emulsion is, for example, 0.00005 to 0.1% by weight, preferably 0.0001 to 0.03% by weight, more preferably 0.0001 to 0.1% by weight relative to the total amount of the emulsion. 0.0003 to 0.005% by weight. In addition, 0.0005 to 0.003% by weight is also included. Within the above range, oxidation and hydrolysis of the components are effectively suppressed, and stable emulsified particles having various particle diameters can be obtained.
The content of BHT and / or BHA in the oil-in-water emulsion is, for example, 0.00003 to 0.05 parts by weight, preferably 0.00005 to 0.01 parts by weight, based on 1 part by weight of squalene. Parts, more preferably 0.0001 to 0.005 parts by weight. Also, 0.0003 to 0.001 parts by weight may be mentioned. Within the above range, oxidation and hydrolysis of the components are effectively suppressed, and stable emulsified particles having various particle diameters can be obtained.

Buffer Oil-in-water emulsions contain a citrate buffer so as to have a pH of 5.5 to 8.5, especially 6 to 7.5. Citrate buffers include, for example, citric acid and monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, and / or hydrates thereof. And any ones to which sodium hydroxide or potassium hydroxide is added as necessary.
By including a citrate buffer, a decrease in the pH of the emulsion due to hydrolysis of the surfactant is effectively suppressed.

  Further, the oil-in-water emulsion may contain one or more other buffering agents as long as the effects of the present invention are not impaired. Other buffers include carbonate buffer, acetate buffer, phosphate buffer, phosphate buffered saline, citrate phosphate buffer, borate buffer, tartrate buffer, Tris-HCl buffer, amino acid buffer Agents and the like.

The concentration of the buffer in the oil-in-water emulsion may be any amount that is sufficient to exhibit sufficient buffering capacity, but is preferably 5 to 50 mM in total. In particular, it is preferable to include only a citrate buffer as a buffer, in which case the total amount of the citrate buffer is preferably 5 to 50 mM. When a substance having a buffering action such as an amino acid is used as an excipient, the amount of the buffer added may be appropriately adjusted.
The concentration of the citrate buffer in the oil-in-water emulsion is, for example, 0.005 to 5% by weight, preferably 0.01 to 3% by weight, more preferably 0.05 to 1% by weight based on the total amount of the emulsion. % By weight. Further, 0.1 to 0.5% by weight is also included.
The content of the citrate buffer in the oil-in-water emulsion is, for example, 0.005 to 20 parts by weight, preferably 0.01 to 10 parts by weight, more preferably 0.05 to 1 part by weight of squalene. To 1 part by weight. Also, 0.1 to 0.5 parts by weight may be used.

Excipient The oil-in-water emulsion may include an excipient. Excipients can be used to adjust the osmotic pressure ratio of the oil-in-water emulsion. Also, excipients can be used to maintain or improve the stability of the lyophilized formulation when producing the lyophilized formulation from an oil-in-water emulsion.
Excipients include monosaccharides, disaccharides, polysaccharides, sugar alcohols, amino acids, proteins, ureas, inorganic salts and the like. Monosaccharides and disaccharides include glucose, fructose, sucrose, lactose, trehalose and the like. Examples of polysaccharides include dextran, starch, maltodextrin, cellulose, polyvinylpyrrolidone, sodium alginate and the like. Examples of the sugar alcohol include mannitol and sorbitol. As the amino acid, neutral amino acids such as alanine, glycine, and proline can be preferably mentioned, and glycine is more preferable. Preferred examples of the protein include albumin, gelatin, collagen and the like. Examples of the inorganic salt include sodium chloride, potassium chloride, calcium chloride, sodium sulfate, sodium carbonate and the like.
Among them, monosaccharides and sugar alcohols are preferable, and mannitol, sucrose, and trehalose are more preferable.
One excipient can be used alone, or two or more excipients can be used in combination.

The concentration of the excipient in the oil-in-water emulsion may be, for example, 0.05 to 25% by weight, preferably 1 to 10% by weight, and more preferably 2 to 8% by weight. In addition, 3 to 6% by weight is also included. The suitable concentration of the excipient varies depending on the type of the excipient, but can be appropriately adjusted according to the production scale of the emulsion and the concentration of other components.
When the excipient is an amino acid, the concentration of the excipient in the oil-in-water emulsion is preferably 2 to 12% by weight, particularly preferably 7% by weight for glycine. When the excipient is mannitol, for example, 0.05 to 25% by weight, preferably 1 to 10% by weight, more preferably 2 to 8% by weight can be mentioned. In addition, 3 to 6% by weight is also included. By using mannitol as an excipient, mannitol can be used at a concentration that is approximately isotonic with body fluids, so that a preparation with less burden on the living body can be prepared. When the excipient is sucrose, it is preferably 1 to 20% by weight, more preferably 3 to 15% by weight. When the excipient is trehalose, it is preferably from 0.5 to 6% by weight, more preferably from 2 to 5% by weight.
In addition, the content of the excipient (particularly, mannitol) in the oil-in-water emulsion is, for example, 0.1 to 250 parts by weight, preferably 0.2 to 100 parts by weight, more preferably 1 part by weight of squalene. Can be 0.5 to 10 parts by weight. Also, 1 to 5 parts by weight may be mentioned.

  The substances mentioned as excipients can also serve as isotonic agents. Further, a substance different from the excipient may be added as a tonicity agent. The concentration of the tonicity agent is appropriately set according to the content of other components, but may be usually 0.1 to 30% by weight in total, and preferably 1 to 10% by weight.

In the particle size present invention, the particle size of the emulsified particles in the oil-in-water emulsion is to evaluate the particle size distribution using a particle size distribution measuring device corresponds to a median diameter calculated from the particle size distribution. Specifically, it refers to the median diameter obtained using the particle size distribution measuring device (SALD-2200, Shimadzu Corporation) used in the examples. The median diameter is obtained.
The particle size of the emulsified particles can be selected, for example, from 0.05 to 3 μm, preferably from 0.1 to 2 μm. The particle size of the emulsified particles can exceed 0.2 μm, and can be 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, or 1 μm or more. The upper limit of the particle size of the emulsified particles can be about 3 μm.
The emulsified particles in the oil-in-water emulsion may have a single-peak particle size distribution, or may have a plurality of peak particle size distributions. That is, the oil-in-water emulsion may include emulsified particles having a specific particle size, or may include a plurality of types of emulsified particles having different particle sizes.

Production Method An oil-in-water emulsion can be prepared by emulsifying squalene in water in the presence of a surfactant. BHT and / or BHA may be mixed with squalene before emulsification. The surfactant may be mixed with squalene before emulsification, but the emulsion may be further mixed with an aqueous surfactant solution. Further, a citrate buffer may be added to the emulsion, or the emulsion and an aqueous solution containing the citrate buffer may be mixed. Other arbitrarily compoundable components, for example, excipients, tonicity agents, etc., may be added to the emulsion, or the emulsion and excipients, an aqueous solution containing the tonicity agent, etc. may be mixed. .

  In the present invention, “emulsification” refers to making two separated liquids into an emulsion. An emulsion is one in which liquid phases that do not dissolve each other like water and oil are dispersed as fine droplets in the other (W. Clayton, Theory of emulations, 4th ed., Blackstone, New York, 1943). ), The process of forming an emulsion from two or more liquid phases that do not dissolve in each other is called emulsification (P. Becher, Emulsions: Theory and Practice, Reinhold, New York, 1965).

  Emulsification can be performed using a known emulsifier such as a homomixer or a high-pressure emulsifier, and an emulsion having a particle size of 0.05 to 3 μm can be easily prepared by appropriately adjusting the conditions. Further, for example, after an emulsion having a particle diameter of about 2 to 3 μm is produced, the particle diameter can be reduced to about 0.1 to 1 μm by high-pressure emulsification or permeable membrane emulsification. Further, for example, an emulsion containing emulsified particles having a particle size of about 2 to 3 μm obtained by re-dissolving a freeze-dried preparation produced from an emulsion having a particle size of about 2 to 3 μm, for example, by high-pressure emulsification or permeable membrane emulsification, The particle size can be reduced to about 0.1 to 1 μm. At a clinical site, for example, a freeze-dried preparation produced from an emulsion having a particle size of about 2 to 3 μm is re-dissolved, and is subjected to, for example, 0.1 to 1 μm by membrane emulsification using a SPG (Shirasu porous glass) membrane pumping connector or the like. Can be controlled to an appropriate particle size.

Re-dissolved oil-in-water emulsion As described above, an oil-in-water emulsion obtained by re-dissolving, re-suspending, or condensing a freeze-dried preparation is also included in the present invention. Re-dissolution can be performed using an aqueous solvent such as water, physiological saline, a surfactant aqueous solution, or a buffer. However, the physiological saline and the buffer are selected so as not to impair the effects of the present invention, and the concentration thereof is also within a range that does not impair the effects of the present invention, and when administered to a living body, it becomes an isotonic solution. It is desirable to be prepared.

  By re-dissolving, an oil-in-water emulsion containing emulsified particles having a target particle size of about 0.05 to 3 μm is obtained.

(2) Freeze-dried preparation A freeze-dried preparation is obtained by freeze-drying the oil-in-water emulsion described above.
The content of squalene in the freeze-dried preparation is 1 to 50% by weight, preferably 5 to 40% by weight, more preferably 20 to 35% by weight based on the total amount of the composition. The ratio of each content of surfactant, BHT / BTA, and citrate buffer to squalene content is the same as in the oil-in-water emulsion described above.

(3) Method of Use Since the adjuvant composition of the present invention itself has an immunostimulatory effect, it prevents immune disorders, particularly diseases caused by immunosuppression, or diseases which can be prevented, improved or treated by immunostimulation. , Improvement, or therapeutic agent.
In addition, such a disease can be administered to a living body at the same time as a preventive, ameliorating, or therapeutic agent, in combination, or as a mixture. In addition, the adjuvant composition of the present invention can be administered to a living body simultaneously with, or in combination with, other drugs having an immunostimulating effect. The adjuvant composition of the present invention can significantly enhance the efficacy of these drugs.
Diseases due to reduced immunity or diseases that can be prevented, improved or treated by immunostimulation include infectious diseases, cancer, autoimmune diseases, rheumatoid arthritis, spondyloarthritis, vasculitis syndrome, allergic diseases (asthma, hay fever, Allergic rhinitis, etc.), diabetes (PLOS ONE, 7 (8), e41756 (2012)), Parkinson's disease (PLOS ONE, 6 (1), e16610 (2011)) and the like.

  The adjuvant composition of the present invention is particularly suitable for use as a mixture or concomitant drug with an infectious disease vaccine, a cancer vaccine, or a vaccine antigen contained therein, or another immunostimulating agent.

  Currently, the vaccines routinely vaccinated by the Japanese immunization method include live vaccines such as measles-rubella vaccine (MR), polio vaccine, measles vaccine, rubella vaccine, BCG vaccine, and diphtheria pertussis tetanus vaccine (improved DPT-3 vaccine). Inactivated vaccines such as a combined vaccine, a diphtheria tetanus combined vaccine (a DT dual vaccine), an influenza vaccine, and a Japanese encephalitis vaccine have been used. In addition, voluntary inoculation vaccines include mumps (epidemic parotitis) vaccine, varicella vaccine, live vaccines such as cholera vaccine and yellow fever vaccine, which are vaccination vaccines when traveling abroad, rabies vaccine, hepatitis A Inactivated vaccines such as a vaccine, a hepatitis B vaccine, a tetanus toxoid vaccine, an adult diphtheria toxoid vaccine, a pneumococcal vaccine, and a Weil's disease autumn darkness vaccine have been used. Inactivated vaccines such as a pneumococcal vaccine, an influenza b virus vaccine, and a human papilloma virus vaccine have been used as vaccines targeted for the vaccination emergency promotion project. Infectious disease vaccines include, for example, these vaccines. Among them, the BCG vaccine is preferred.

Further, as the immunostimulating agent, mycobacterium genus bacteria such as BCG (Bacillus Calmette-Guerin) -CWS (Cell Wall Skeleton) and CWS of a related bacterium are preferable. BCG-CWS has an immunostimulatory effect and is known to exhibit antitumor activity in immunotherapy of experimental tumors using animal models and human cancer. It is known that an oil-in-water emulsion containing BCG-CWS in an oil has an extremely high antitumor activity due to immunostimulatory action (A. Hayashi et al., Pro. Japan Acad., 70, Ser. B 205-209 (1994), A. Hayashi et al., Pro. Japan Acad., 74, Ser. B 50-56 (1998)).
BCG-CWS can be produced, for example, by the method described in I. Azuma et al., Journal of the National Cancer Institute, Vol. 52, No. 195-101 (1974). Further, if the method described in Japanese Patent No. 5578390 is used, high-purity BCG-CWS can be efficiently produced.

As cancer vaccines, those containing various vaccine antigens are known and can be used without limitation. For example, cancer testis antigens such as MAGE family and SSX family, mutant antigens such as Ras and EGFRvIII, overexpressed antigens such as Her2 / neu, WT1, p53, differentiation antigens such as gp100, Melan-MART-1, HPVE6, HPVE7 and the like Virus antigens, tumor blood vessels or stromal antigens such as VEGFR2 and FAP.
The type of target cancer is not limited, and examples thereof include lung cancer, stomach cancer, liver cancer, pancreatic cancer, colon cancer, uterine cancer, breast cancer, acute myeloid leukemia, tongue cancer, pharyngeal cancer, ovarian cancer, and brain tumor.

  Hereinafter, the present invention will be described more specifically based on Examples and Test Examples, but the present invention is not limited thereto.

(Example 1) Production of squalene emulsion of the present invention
(Example 1-1) Squalene emulsion having a median diameter of 2 μm
(1) Method A. Preparation of Citrate Diluent-1 Polysorbate 80 was mixed so that 6 mg / mL, D (-) mannitol was 90 mg / mL, and the citrate buffer was 20 mM (pH 7.0).
B. Preparation of Citric Acid Diluent-2 Prepared in the same manner as citric acid diluent-1 except that polysorbate 80 and D (-) mannitol were not contained.
C. Approximately 30 mL of n-heptane / ethanol (9: 1, volume / volume) solution was added to 5.4 g of emulsified squalene (manufactured by Kishimoto Special Liver Oil Industry) and 1.5 mg of BHT (Tokyo Kasei Kogyo), and the mixture was stirred and irradiated with ultrasonic waves. At room temperature. Thereafter, the mixture was heated to 60 ° C. in a dry nitrogen stream to distill off the organic solvent. Next, 135 g of a 0.02% by weight aqueous solution of polysorbate 80 was added, and the mixture was preliminarily emulsified at 60 ° C., 7,000 rotations for 5 minutes using a homomixer (Lab Solution, PRIMIX). Further, 8.73 g of a 10% by weight aqueous solution of polysorbate 80 was added, and main emulsification was performed at 60 ° C. and 12,000 rpm for 5 minutes to obtain an emulsified stock solution-1.
D. Emulsion preparation An equal volume of citric acid diluent-1 was added to the above emulsified stock solution-1 to obtain a squalene emulsion of the present invention. The final concentrations (prepared amounts) were 18 mg / mL for squalene, 6 mg / mL for polysorbate 80, 45 mg / mL for D (-) mannitol, and 10 mM for citrate buffer (pH 7.0: 3Na-3. 5 mg / mL, citric acid-0.04 mg / mL) and BHT were adjusted to 10 ppm.

(2) Results The particle size distribution of the emulsified stock solution-1 and the emulsion was measured with a particle size distribution meter (SALD-2200, Shimadzu Corporation) (FIGS. 1a and 1b). A single particle size distribution and appearance with the same particle diameter before and after the addition of the citric acid diluent-1 having a median diameter of 1.9 μm (emulsion stock solution-1) and 1.9 μm (emulsion 1-1a [with mannitol]) respectively A uniform oil-in-water emulsion was obtained (FIG. 1) (squalene content in the emulsion was 18 mg / mL, quantitative; gas chromatography method).
Further, in a 1/30 scale production (emulsion: 24,000 rpm, 5 min, 60 ° C., dilution: using citric acid diluent-2) using the same small experimental emulsifier (Omni-mixer [Omni-International]). Thus, 10 mL of an emulsion having a median diameter of 2.2 μm and a single peak particle size distribution was obtained (emulsion 1-1b).

(Example 1-2) Production of small-diameter particle emulsion of squalene of the present invention
(1) Method A. High pressure treatment of the emulsified stock solution-1 produced in Example 1-1 with a high pressure crusher (BERYU MINI, Biyu Co., Ltd.) (8,000 psi, 3 passes, or 23,000 psi, 20 passes) Thus, a high-pressure processing emulsion-1 and a high-pressure processing emulsion-2 were obtained.
B. Preparation of Small-Diameter Particle Emulsion The high-pressure-treated emulsions-1 and 2 obtained in A were diluted by a factor of 1 with citric acid diluent-1 or citric acid diluent-2 to obtain a squalene small-particle emulsion of the present invention.

(2) Results Emulsion (emulsion 1-2a) exhibiting a single peak particle size distribution of 0.2 μm median and a squalene content of 19.7 mg / mL from the high pressure treated emulsion-1 and the median from the high pressure treated emulsion-2 An emulsion having a single peak of 0.1 μm in diameter (emulsion 1-2b) was obtained (FIG. 2).
There was no difference in median diameter with or without D (-) mannitol. Emulsions (from high-pressure-treated emulsions to emulsions 1-2a (with mannitol)) and from high-pressure-treated products-2 to emulsions 1-2b (with mannitol)).
By adjusting the emulsification conditions in the same manner, an emulsion containing the squalene of the present invention having a particle size of 0.3 to 1.0 μm can be produced.

(Example 2) Emulsion of the present invention into small-diameter particles by the SPG membrane pumping method
(1) Method The emulsion 1-1a (median diameter: 1.9 μm) obtained in Example 1-1 was subjected to a 10-Pass treatment using an SPG membrane pumping connector (SPG Techno Co., Ltd.) having a pore diameter of 1.0 μm.
(2) Results As a result of measurement with a particle size distribution analyzer (SALD-2200, Shimadzu Corporation), emulsion particles (emulsion 2-1) with a single peak having a median diameter of 1.0 μm were obtained (FIG. 3).

(Example 3) Production of freeze-dried preparation of squalene emulsion formulated in the present application
(Example 3-1) Production of freeze-dried squalene emulsion of the present application having a median diameter of 2 μm upon re-dissolution
(1) Method In Example 1-1, an oil-in-water emulsion obtained by diluting with a citric acid diluent-1 (containing mannitol) was filled into a vial for freeze-drying (Φ18.0) in 1 mL portions, and then charged at −80 ° C. The mixture was frozen in a deep freezer and freeze-dried using a freeze dryer (GAMMA2-16LSCPlus, manufactured by MARTIN CHRIST) to obtain a freeze-dried preparation.
(2) After re-dissolving the resulting freeze-dried preparation in water for injection (Otsuka Pharmaceutical), a single peak emulsion having a median diameter of 2.2 μm was obtained as before freeze-drying (freeze-dried product 3-1a) 1) (squalene content, 18.4 mg / mL, gas chromatography method). Similarly, a lyophilized preparation having a median diameter of 2.4 μm upon re-dissolution was obtained (lyophilized product 3-1b).

(Example 3-2) Production of lyophilized preparation of small particle squalene emulsion formulated in the present application
(Example 3-2-1) Production of lyophilized preparation having a median diameter of 0.1 μm upon re-dissolution using sucrose as an excipient
(1) Method Lyophilization was carried out in the same manner as in Example 3-1 using the emulsion of the present application (emulsion 1-2b) having a median diameter of 0.1 μm produced in Example 1-2. However, 90 mg / mL sucrose (Wako Pure Chemical Industries) was used as an excipient.
(2) After re-dissolving the resulting freeze-dried preparation with water for injection, a single-peak emulsion having a median diameter of 0.1 μm was obtained, which was comparable to that before freeze-drying (freeze-dried product 3-2-1). .

(Example 3-2-2) Production of lyophilized preparation having a median diameter of 0.3 μm upon re-dissolution using mannitol as an excipient
(1) Method Using the 0.1 μm emulsion of the present application (emulsion 1-2b) produced in Example 1-2, the mixture was freeze-dried in the same manner as in Example 3-1. The excipient used was 45 mg / mL D (-) mannitol.
(2) After re-dissolving the resulting freeze-dried preparation with water for injection, a single-peak emulsion having a median diameter of 0.3 μm was obtained, which was comparable to that before freeze-drying (freeze-dried product 3-2-2). .

(Reference Example 1) Preparation of emulsion 1 containing no BHT Except that squalene containing no BHT was used, emulsification was performed in the same manner as in Example 1-1 using an Omni-mixer, and on a scale, and a citric acid diluent was used. An emulsion was prepared in the same manner as in Example 1-1 except that it was diluted with -2 and did not contain BHT (Reference emulsion 1-1). An emulsion having a median diameter of 2.0 μm and a single peak particle size distribution similar to that of Example 1-1 was obtained.

(Reference Example 2) Preparation of squalene emulsion and freeze-dried preparation thereof by known formulation (AddaVax) Same formulation (Squalene-10% by weight, Span85-1% by weight, PS80-1% by weight) as a commercial product of squalene emulsion (AddaVax, InvivoGen) ) Was prepared according to the same scale method as in Example 1-1 using Omni-mixer to obtain an emulsified stock solution-2. However, Span85 (Wako Pure Chemical Industries) was previously mixed with squalene and used.
The emulsified stock solution-2 was diluted with citric acid diluent-2 (pH 7.0, final concentration 10 mM) and water for injection so that the squalene concentration became 18 mg / mL, to obtain an emulsion of a known formulation having a particle size of 2.2 μm. (Reference emulsion 2-1).
Next, D (-) mannitol having a final concentration of 45 mg / mL was added to the known formulation emulsion so as to have a final concentration of squalene of 18 mg / mL, and lyophilized according to Example 3, and freeze-dried of the known formulation emulsion. A formulation was prepared. Upon redissolution, a single peak with a median diameter of 1.9 μm was shown (reference freeze-dried product 2-2a). Similarly, an emulsion freeze-dried preparation (reference freeze-dried product 2-2b) showing a single peak with a median diameter of 2.7 μm was obtained.

(Test Example 1) Comparison test of emulsion stability between BHT and other antioxidants
(1) Preparation of Evaluation Sample Example 1 so that the final concentration was squalene-7.2 mg / mL, PS80-7.2 mg / mL, phosphate buffer (pH 7.4) -10 mM, and mannitol-45 mg / mL. Emulsification, dilution, lyophilization, and re-dissolution were performed by the same procedures as in Example-1 and Example 3-1 to prepare a squalene emulsion containing no antioxidant, which was used as Sample A (control). Other samples of B, C, D, E, and F each contained DL-α-tocopherol, tocopherol acetate, ascorbic acid, and 6-O-stearoyl-L as antioxidants in an amount of a final concentration of 10 ppm. -Prepared by adding ascorbic acid, dibutylhydroxytoluene (BHT).

(Antioxidants contained in each sample)
Sample A: without antioxidant Sample B: DL-α-tocopherol sample C: tocopherol acetate sample D: ascorbic acid sample E: 6-O-stearoyl-L-ascorbic acid sample F: dibutylhydroxytoluene (BHT)

(2) Evaluation method Each emulsion sample was stored by standing at 60 ° C., and the pH, the squalene content (determined by the following method A), the oleic acid content (at the time of PS80 degradation) at the start of the test and after 8 days had elapsed. Index, determined by the following method B). The content of free oleic acid was determined by subjecting oleic acid to solvent extraction from the emulsion solution without alkali hydrolysis, converting it into a labeled compound, and quantifying the content by HPLC.
A. Squalene content measuring method The emulsion was extracted with ethyl acetate and quantified by gas chromatography.
B. Free oleic acid content measuring method After extracting a free fatty acid from the emulsion solution with toluene, it was fluorescently labeled with an ADAM reagent (Funakoshi) and quantified by a liquid chromatography method.

ＢＨＴ以外の抗酸化剤を含むエマルションＢ〜Ｅは、抗酸化剤を含まないサンプルＡと同程度にｐＨを減少させ、エマルションを酸性化させたのに対して、ＢＨＴを含むサンプルＦは全くｐＨを減少させなかった。
また、ＢＨＴ以外の抗酸化剤を含むサンプルＢ〜Ｅは、スクアレン減少量が抗酸化剤を含まないサンプルＡと同等であったのに対して、ＢＨＴを含むサンプルＦは全くスクアレン含有量を減少させなかった。
また、抗酸化剤を含まないサンプルＡ、及びＢＨＴ以外の抗酸化剤を含むサンプルＢ〜Ｅは、オレイン酸含量が低下したが、一方で、ＢＨＴを含むサンプルＦは、オレイン酸含有量が見かけ上増加した。これは、前者は生成したオレイン酸が例えば酸化反応など２次的反応によって更に異なった生成物に変化したためであり、後者はその反応がＢＨＴによって抑制されたためと考えられる。
以上の結果、スクアレンと界面活性剤とクエン酸緩衝剤を含む本発明のアジュバント組成物において、各種抗酸化剤の中でＢＨＴが最も優れた抗酸化作用を有することが示された。
なお、上記各サンプルの再溶解前の凍結乾燥製剤を静置保存した場合も、同傾向の結果が得られる。 (3) The pH, the decrease in squalene, and the decrease in oleic acid (an index of PS80 degradation) were calculated by subtracting the measurement after 8 days from the measurement at the start of the results . Table 1 shows the results.

Emulsions B to E containing antioxidants other than BHT reduced the pH to the same extent as Sample A containing no antioxidant and acidified the emulsion, whereas Sample F containing BHT had no pH. Did not decrease.
Samples B to E containing an antioxidant other than BHT had the same squalene reduction amount as Sample A containing no antioxidant, whereas Sample F containing BHT had a squalene content reduced at all. I didn't let it.
In addition, Sample A containing no antioxidant and Samples B to E containing antioxidants other than BHT had a reduced oleic acid content, whereas Sample F containing BHT had an apparent oleic acid content. Increased. This is presumably because the former was changed to a different product by a secondary reaction such as an oxidation reaction, and the latter was suppressed by BHT.
As a result, it was shown that BHT has the most excellent antioxidant effect among various antioxidants in the adjuvant composition of the present invention containing squalene, a surfactant and a citrate buffer.
It should be noted that the same result is obtained when the freeze-dried preparation of each of the above samples before redissolution is stored in a standing state.

(Test Example 2) Surfactant stabilizing effect of citrate buffer
(1) Preparation of Evaluation Sample PS80 was added to the following various buffer solutions to a final concentration of 6 mg / mL to obtain a PS80 solution.
A: No buffer B: 10 mM phosphate buffer (pH 7.0)
C: 10 mM citrate buffer (pH 7.0)
(2) Evaluation method Each evaluation sample was allowed to stand at 40 ° C. for 150 days and at 60 ° C. for 60 days, and the pH was measured over time.
(3) Results The results are shown in FIG. The solution containing no buffer had a higher pH lowering rate than the solution containing the phosphate buffer or the citrate buffer. In addition, the pH holding ability of the citrate buffer was higher than that of the phosphate buffer.
From the above results, the citrate buffer shows higher pH stabilizing ability in the presence of a surfactant than the phosphate buffer, which is widely used among various buffers under the formulation conditions of this formulation. Became clear.

(Test Example 3) Stability of the emulsion of the present invention
(1) Evaluation Sample The emulsion of the present invention produced in Example 1, the BHT-free emulsion of Reference Example 1, and the known formulation emulsion obtained in Reference Example 2 were used.
(2) Evaluation method Each evaluation sample was allowed to stand at 40 ° C. for 2 weeks, 60 ° C. for 2 weeks, and 90 ° C. for 2 days, and changes in particle size distribution, squalene content, and pH from the start of the test were measured.
(3) Result A. Stability of emulsion having a median diameter of 2 μm Under storage conditions of 90 ° C. for 2 days, the emulsion of the present invention (emulsion 1-1b) has almost all of the median diameter, particle size distribution, and squalene content, and has a known formulation emulsion (reference emulsion 2-1). ), The stability was higher. Emulsions of the known formulation resulted in worsened particle size distribution, increased median diameter, and reduced squalene content. In addition, the emulsion containing no BHT (Reference Emulsion 1-1) was unstable due to deterioration of the particle size distribution, increase in the median diameter, and decrease in the squalene content (Table 2, FIG. 5).

B. Emulsion stability of small particle diameter (median diameter 0.1 to 0.2 μm) Under storage conditions of 40 ° C. for 2 weeks and 60 ° C. for 2 weeks, the emulsions of the present invention (emulsion 1-2a, emulsion 1-2a [with mannitol] ], Emulsion 1-2b, and emulsion 1-2b [with mannitol]) had no change in median diameter, particle size distribution, and squalene content. Although an increase in pH was observed in the known formulation emulsion, both the emulsion of the present invention and the known formulation emulsion were stable (Table 3).

(Test Example 4) Lyophilized preparation stability of the emulsion of the present invention
(1) Evaluation sample The freeze-dried emulsion of the present invention of Example 3 (freeze-dried product 3-1b) and the freeze-dried formulation of the known formulation produced in Reference Example 2 (reference freeze-dried product 2-2b) were used.
(2) Evaluation Method Each evaluation sample was allowed to stand at 40 ° C. for 2 weeks and at 60 ° C. for 2 weeks, and changes in particle size distribution, squalene content, and pH from the start of the test were measured.
(3) Results The freeze-dried emulsion of the present invention did not change in the median diameter, particle size distribution, and pH of the re-dissolved solution under any of the storage conditions of 40 ° C. for 2 weeks and 60 ° C. for 2 weeks. On the other hand, the freeze-dried preparation of the known formulation deteriorates the particle size distribution waveform of the re-dissolved solution when stored at 40 ° C. for 2 weeks, and further shows a decrease in pH when stored at 40 ° C. for 2 weeks or 60 ° C. for 2 weeks. Was. In the freeze-dried preparation, the emulsion of the present invention had higher stability than the emulsion of the known formulation (Table 4, FIG. 6).

(Test Example 5) Combination effect of the emulsion preparation of the present invention with a vaccine
(1) Preparation of Evaluation Sample 25 μL of the re-dissolved solution of the squalene emulsion freeze-dried preparation of the present invention (Example 3) was mixed with 1 μg of live BCG bacteria (M. bovis BCG Tokyo 172 (TOKYO 172)), and PBS was further added. To prepare a 110 μL sample.
(2) Evaluation method Evaluation was carried out according to the method known in the literature (J Immunol. 2012 Oct 15; 189 (8): 4079-87.). 110 μL of a sample solution was intradermally administered to the back of the neck of C57BL / 6J mice (female, 6 weeks old) that had been acclimated for one week. Four weeks after the administration, the spleen was collected, a spleen cell dispersion was prepared, and hemolyzed with RBC Lysis Buffer (0.14 M ammonium chloride, 0.017 M Tris solution, pH 7.56). After hemolysis, the cells were suspended in a culture medium (RPMI1640 medium supplemented with penicillin / streptomycin / 10% FBS), erythrocyte debris was removed with a filter, and then centrifuged and washed at 1500 rpm for 5 minutes. The cells were suspended again in the medium to obtain a spleen cell solution. The spleen cell solution was adjusted to 5 × 10 6 cells / mL, PPD was added to a final concentration of 10 μg / mL, and the cells were cultured at 37 ° C. in the presence of 5% CO 2 for 24 hours. After culturing for 24 hours, the supernatant was stored at -80 ° C. Using this culture solution, the amount of IFN-γ was measured using an ELISA kit (manufactured by R & D systems).
(3) Results In the group in which the squalene emulsion formulation of the present invention was mixed and administered with live BCG bacteria, higher IFN-γ production was induced than in the group administered with the live BCG bacteria alone. On the other hand, in the group to which the squalene emulsion preparation alone was administered, IFN-γ production was induced only to the same extent as in the naive group.

  The adjuvant composition of the present invention can be an emulsion containing stable particles having an arbitrary particle size of 0.05 to 3 μm. Therefore, the administration route can be selected according to the needs, and the desired action mechanism can be obtained. In addition, by combining a plurality of emulsions having different particle diameters or a freeze-dried preparation thereof, immunostimulation can be strongly achieved by various mechanisms. It is stable against oxidation. Furthermore, if necessary, a freeze-dried preparation can be prepared. Therefore, it is very useful industrially.

Claims (11)

  An adjuvant composition comprising squalene, a surfactant, dibutylhydroxytoluene and / or butylhydroxyanisole, and a citrate buffer.   2. The adjuvant composition according to claim 1, which is an oil-in-water emulsion or is freeze-dried.   The adjuvant composition according to claim 1 or 2, wherein the surfactant is a fatty acid ester.   The adjuvant composition according to claim 3, wherein the fatty acid ester is polyoxyethylene sorbitan monolaurate and / or polyoxyethylene sorbitan monooleate.   0.001 to 30 parts by weight of a surfactant, 0.00003 to 0.05 parts by weight of a total of dibutylhydroxytoluene and / or butylhydroxyanisole, and 0.005 to 20 parts by weight based on 1 part by weight of squalene. An adjuvant composition according to any of the preceding claims, comprising part by weight of a citrate buffer.   The adjuvant composition according to any one of claims 1 to 5, which is an oil-in-water emulsion and contains 0.01 to 50% by weight of squalene based on the total amount of the emulsion.   The adjuvant composition according to any one of claims 1 to 5, which is a freeze-dried preparation and contains 1 to 50% by weight of squalene based on the total amount of the freeze-dried preparation.   The adjuvant composition according to any one of claims 1 to 7, further comprising an excipient.   Claims used together with, combined with, or mixed with, other immunostimulants, or preventive, ameliorating, or therapeutic agents for diseases that can be prevented, ameliorated, or treated by immunostimulation, or diseases that contribute to immunosuppression. An adjuvant composition according to any one of claims 1 to 8.   The adjuvant composition according to any one of claims 1 to 8, further comprising a vaccine antigen. The adjuvant composition according to claim 10, wherein the vaccine antigen is a BCG vaccine antigen.

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