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WO2018124132A1 - Préparation d'émulsion huile dans eau contenant un composant de squelette à paroi cellulaire bactérienne - Google Patents

Préparation d'émulsion huile dans eau contenant un composant de squelette à paroi cellulaire bactérienne Download PDF

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Publication number
WO2018124132A1
WO2018124132A1 PCT/JP2017/046780 JP2017046780W WO2018124132A1 WO 2018124132 A1 WO2018124132 A1 WO 2018124132A1 JP 2017046780 W JP2017046780 W JP 2017046780W WO 2018124132 A1 WO2018124132 A1 WO 2018124132A1
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cws
bcg
preparation
bacterial
oil
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Japanese (ja)
Inventor
彰洋 中谷
幸広 渋谷
康志 中馬
砂川　洵
仁尤 佐藤
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Japan BCG Laboratory
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Japan BCG Laboratory
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to an emulsion preparation, a suspension preparation, a freeze-dried preparation thereof, and a production thereof containing a cell wall skeleton component (Cell Wallet: CWS) as an active ingredient.
  • CWS has a sugar chain such as arabinogalactan protruding and exposed on the oil particle surface of an emulsion preparation and its lyophilized preparation, reacting with a lectin (for example, concanavalin A), and a preparation showing activity against immune cells and its production It is about the method.
  • Bacterial cell wall skeletal components have an immunostimulatory effect and are known to exhibit antitumor activity in, for example, experimental tumor systems using animal models and human cancer immunotherapy. Furthermore, it is known that when the above bacterial cell wall skeleton component is dispersed and emulsified in an oil component and administered as an oil-in-water emulsion preparation, the antitumor effect due to the immunostimulatory action is remarkably enhanced.
  • BCG-CWS Bacillus Calmette-Guerin
  • the oil-in-water emulsion can be prepared by emulsifying a paste-like composition in which bacteria-CWS is dispersed in oil and adding water containing a surfactant (International Publication Pamphlet No. 2004/012751). No., International Publication Pamphlet No. 2005/102369, Japanese Patent Application No. 2010-271322).
  • oil-in-water emulsion formulations containing bacteria-CWS are unstable, and currently, when using BCG-CWS, a small amount of oil-in-water emulsion is prepared at the time of use in clinical settings.
  • it is always difficult to manually prepare a certain formulation, and practical use as a pharmaceutical is virtually impossible by preparation at the time of use.
  • the present invention relates to bacteria-CWS having high purity and excellent dispersibility in water suitable for the production of various emulsions and suspension formulations, a method for producing the same, and a novel oil-in-water emulsion having excellent storage stability using the same. And it aims at providing a freeze-dried formulation.
  • the present inventors consider that the stability and the like of the preparation are affected by the form and physicochemical properties of bacteria-CWS, and study them, and the form and physical properties of bacteria-CWS are the biological activity and stability of the preparation. Found to be greatly involved in.
  • Bacteria-CWS suitable for the production of various emulsion preparations and a method for producing the same were found, and the buffer solution and the antioxidant were also devised with excellent stability, reacting with a lectin (for example, concanavalin A), and It was possible to find a novel oil-in-water emulsion and its lyophilized preparation characterized by having in vitro activity in immune cells.
  • the sugar chain of the bacterium-CWS protrudes and is exposed on the surface of oil particles (oil droplets), and has reactivity with lectins (for example, concanavalin A) and against immune cells It was possible to produce an oil-in-water emulsion having excellent in vitro activity and improved safety and storage stability, including skin disorders, and a freeze-dried preparation thereof.
  • the present invention has been completed based on the above findings.
  • the gist of the present invention is as follows.
  • [5] The oil-in-water emulsion formulation according to any one of [1] to [4], wherein the potential difference is 4 to 12 mV.
  • [6] The oil-in-water emulsion preparation according to any one of [1] to [5], wherein the bacterium is selected from the group consisting of Mycobacterium and Nocardia.
  • [7] The oil-in-water emulsion formulation according to [6], wherein the bacterium is a BCG bacterium.
  • [8] The oil-in-water emulsion formulation according to any one of [1] to [7], wherein the oil is squalane, squalene or a mixture thereof.
  • BHT dibutylhydroxytoluene
  • the bacterium is BCG, the oil is squalene or squalane, the surfactant is polyoxyethylene sorbitan fatty acid ester, and further contains dibutylhydroxytoluene as an antioxidant and citrate buffer as a buffer. [1] The preparation according to any one of [16]. [18] The bacterium is BCG, the oil is squalene, the surfactant is a polyoxyethylene sorbitan fatty acid ester, and contains a citrate buffer as a buffer, [1] to [16] The formulation described. [19] A freeze-dried preparation of the oil-in-water emulsion preparation according to any one of [14] to [18].
  • a pharmaceutical composition for mammals including humans comprising the emulsion formulation or lyophilized formulation according to any one of [1] to [19].
  • a method for producing an oil-in-water emulsion preparation containing bacterial CWS comprising the following steps: (I) providing a bacterial CWS preparation that satisfies the following conditions: a) Total non-constituent amino acid content in the bacterial CWS preparation is 0.8% by weight or less (0 to 0.8% by weight) b) The fluorescent area ratio of the CWS preparation by auramine staining is 10% or less (0 10%) (Ii) Stirring a mixture containing the bacterial CWS preparation, oil, and organic solvent to disperse the bacterial CWS.
  • the OD value (690 nm) after 60 minutes of dispersion is 0.4 or more compared to the OD value immediately after dispersion.
  • the production method according to any one of [22] to [24], which is (0.4 to 1.0).
  • the OD value (690 nm) after 60 minutes is 0.7 or more (0.7 to 1.0) as compared with the OD value immediately after dispersion. The manufacturing method as described.
  • the bacterial CWS preparation is derived from a bacterium selected from the group consisting of Mycobacterium, Nocardia, Corynebacterium, Rhodococcus, and Gordona. [22] ] To [30].
  • step (iv) of the production method according to any one of [22] to [33] an excipient is further added for emulsification, and the resulting oil-in-water emulsion formulation is freeze-dried
  • step (iv) of the production method according to any one of [22] to [33] an excipient is further added for emulsification, and the resulting oil-in-water emulsion formulation is freeze-dried
  • the method for producing a lyophilized formulation according to [34] wherein the lyophilized formulation is the formulation according to [19].
  • Bacterial cell wall skeletal component (CWS) preparation satisfying: a) The total non-constituent amino acid content in the CWS preparation is 0.8 wt% or less (0 to 0.8 wt%), and b) the fluorescent area ratio of the CWS preparation by auramine staining is 10% or less (0 ⁇ 10%). [37] The CWS-adjusted product according to [36], wherein the CWS preparation has a fluorescence area ratio of 5% or less (0 to 5%). [38] The CWS-adjusted product according to [37], wherein the CWS preparation has a fluorescence area ratio of 2% or less (0 to 2%).
  • the above-mentioned bacterium is selected from the group consisting of Mycobacterium, Nocardia, Corynebacterium, Rhodococcus, and Gordona, [36] to [47] A bacterial CWS preparation according to any one.
  • the bacterial CWS preparation according to [48], wherein the bacterium is selected from the group consisting of Mycobacterium and Nocardia.
  • the bacterial CWS preparation according to [49], wherein the bacterium is BCG or Nocardia.
  • the bacterial CWS preparation according to [50] wherein the bacterium is a BCG bacterium.
  • a pharmaceutical composition comprising the bacterial CWS preparation according to any one of [36] to [51].
  • a method for producing a bacterial CWS preparation comprising a step of iii) carrying out an enzyme treatment of a purified crushed cell product, and iv) a step of washing after the enzyme treatment.
  • the remaining amounts of PGL and glycerol monomycolic acid ester (GroMM) in the purified cell preparation are 10% by weight or less (0 to 10% by weight) of the original content of the raw material cells,
  • [59] The method for producing a bacterial CWS preparation according to any of [56] to [58], wherein the bacterium comprises fatty acid, sugar chain, and peptidoglycan in CWS.
  • the method for producing a bacterial CWS preparation according to [60] wherein the bacterium is selected from the group consisting of Mycobacterium and Nocardia.
  • [66] The method for producing a bacterial CWS preparation according to any one of [56] to [65], wherein the enzyme-treated enzyme is a proteolytic enzyme.
  • [69] The method for producing a bacterial CWS preparation according to any of [56] to [68], wherein a halogen-based organic solvent is not used.
  • the above-mentioned bacterium is selected from the group consisting of Mycobacterium, Nocardia, Corynebacterium, Rhodococcus, and Gordona, [71] to [74] A purified cell preparation of the bacterium according to any one of the above.
  • the purified bacterial cell preparation according to [76] wherein the bacterium is a BCG bacterium.
  • the purified bacterial cell preparation according to any of [71] to [78], wherein the purified bacterial cell preparation has a CWS content of 35 to 47% by weight.
  • the purified cell preparation can be collected by filtration [71] to [79] A purified cell preparation of the bacterium according to any one of [79].
  • a method for producing a purified bacterial cell preparation comprising a step of heating a live or dead bacterium in a mixed solution of water and a hydrophilic organic solvent under reflux.
  • the above-mentioned hydrophilic organic solvent is one or more selected from methanol, ethanol, 1-propanol, isopropanol, tetrahydrofuran, dioxane, acetonitrile, acetone, methyl ethyl ketone, [81] or [82] A method for producing a purified cell preparation of the bacterium described in 1.
  • bacterial cytoskeleton component (CWS) preparation means a complex that may contain various substances derived from raw materials and manufacturing processes, rather than containing only bacterial CWS and purified bacterial cell components.
  • preparation means a complex that may contain various substances derived from raw materials and manufacturing processes, rather than containing only bacterial CWS and purified bacterial cell components.
  • bacterial cytoskeletal component (CWS) preparation means a complex that may contain various substances derived from raw materials and manufacturing processes, rather than containing only bacterial CWS and purified bacterial cell components.
  • CWS cytoskeletal component
  • purification purification
  • the oil-in-water emulsion of the present invention and its lyophilized preparation have sugar chains protruding and exposed on the surface of oil particles, exhibiting reactivity with lectins and excellent activity against immune cells, and excellent storage stability. Yes. Therefore, the emulsion of the present invention and its lyophilized preparation can be used as pharmaceuticals for immunomodulators such as cancer immunotherapeutic agents, hay fever and asthma.
  • FIG. 6 is a diagram (FIG. 6 of Microbiology Spectrum, 2 (3), 1-19 (2014)) showing how the basic structure of the cell walls of mycobacteria (tuberculosis) is in the substance level.
  • the main compounds contained in the cell wall including extracellular adhesion components
  • lipids extracted with an organic solvent are represented among the components of the cell walls of acid-fast bacteria. It is described that the peptidoglycan-arabinogalactan-mycolic acid portion (cell wall skeleton) cannot be extracted with an organic solvent or the like among cell wall components.
  • FIG. 1 A schematic diagram A) of a BCG-CWS oil paste in water is an oil paste of BCG-CWS of Patent Document 3.
  • BCG-CWS and BCG-CWS of Patent Document 3 a) Induction effect of blood IFN- ⁇ production upon intraperitoneal administration of BCG-CWS oil-in-water emulsion to SJL / J mice. b) OVA gene transfer of BCG-CWS oil-in-water emulsion Tumor growth inhibitory effect upon intratumoral administration to G7 cell line cancer-bearing mice (C57BL / 6 mice).
  • FIG. 5 is an image diagram illustrating differences in physical properties (dispersibility in water, particle size distribution in n-heptane, etc.) regarding the BCG-CWS of the present invention and BCG-CWS of Patent Document 3 by changes in the surface morphology of BCG-CWS.
  • (A) is a dispersed state
  • (b) is a normal reversible hydrophobic interaction of the mycolic acid moiety
  • (c) is a form in which the mycolic acid moiety is strongly irreversibly aggregated or fused.
  • hTLR2 reporter activity of BCG cells (dead cells) and BCG purified cells of the present invention a) hTLR2 reporter activity of BCG cells (dead cells) and BCG purified cells of the present invention.
  • hTLR2 reporter activity of the purified BCG cells of the present invention and their disrupted products In vitro reporter activity in various hTLR gene-introduced HEK cell lines of BCG cells (dead cells), purified BCG cells of the present invention, and disrupted products thereof.
  • hTLR4 reporter activity of BCG cells (dead cells) and BCG purified cells of the present invention d) hTLR4 reporter activity of the purified BCG cells of the present invention and the disrupted product thereof.
  • the first aspect of the present invention relates to an oil-in-water emulsion containing bacteria-CWS and a freeze-dried preparation thereof.
  • the “oil-in-water emulsion and lyophilized preparation thereof” of the present invention can be produced, for example, as follows using the bacterium-CWS of the present invention. That is, (1) A mixed oil (oil paste) with bacteria-CWS and oils such as squalene and squalane is prepared using an organic solvent, (2) polysorbates are added as a surfactant, and (3) The above (2) is emulsified in water to obtain an emulsion.
  • the emulsion can be diluted with a buffer mixed with mannitol or the like as an excipient, and used as a diluent to produce an oil-in-water emulsion rich in uniformity.
  • a freeze-dried preparation can be obtained by drying.
  • BCG-CWS produced by a known method Patent Document 3
  • the minimum amount of oil required for preparation of an oil-in-water emulsion is large, the storage stability of the lyophilized preparation is low, and the storage stability It was found that the reproducibility of was low. From these results, it was found that the form of BCG-CWS and its physical properties are greatly related to the storage stability of the emulsion and its lyophilized preparation.
  • the “bacterial cell wall skeleton component” of the present invention represents an arbitrary cell-derived component including a cell wall skeleton component derived from bacteria (Cell Wall Skeleton; CWS), and is abbreviated as Bacteria-CWS.
  • Bacteria-CWS includes, for example, bacterial bodies themselves such as Mycobacterium genus such as Mycobacterium tuberculosis, Lactobacillus genus such as lactic acid bacteria, and dead bacteria of Saccharomyces genus such as yeast, and to some extent Mention may be made of separated cell wall skeleton components.
  • the bacteria include Gram-positive bacilli, Mycobacterium bacteria, Nocardia bacteria, Corynebacterium bacteria, Rhodococcus bacteria, and Gordona bacteria.
  • Mycobacterium bacteria specifically include Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis [bovine tuberculosis, BCG (Bacillus Calmette-Guerin)] , Mycobacterium smegmatis, Mycobacterium africanum (African fungus), Mycobacterium microti (Mycobacterium tuberculosis), Mycobacterium leprae (Rye bacterium), Mycobacterium kansasii, Mycobacterium avium, Mycobacterium phlei, etc.
  • “Lactobacillus bacterium” includes Casei strain Shirota, among which BCG bacterium, a kind of Mycobacterium bovine tuberculosis, is preferred. And Nocardia Rubra, a kind of Nocardia bacterium.
  • the “cell wall skeleton (CWS)” of the present invention refers to a component obtained by removing components other than fatty acid (mycolic acid), sugar chain (arabinogalactan) and peptidoglycan from the cell wall of Mycobacterium tuberculosis shown in FIG. Show.
  • CWS is an insoluble residue substantially free of soluble components such as proteins, nucleic acids and fatty acids, obtained through purification processes such as crushing of mycobacterial cells, nucleolytic enzyme treatment, proteolytic enzyme treatment, and solvent washing (J. Nat. Cancer Inst., 52, 95-101 (1974)).
  • BCG-CWS Patent Document 3
  • the production method and analytical values were shown and the purity or impurity content is clearly specified
  • the water dispersibility was very poor and the oil-in-water
  • the stability of the freeze-dried preparation of the emulsion, suspension and oil-in-water emulsion is low or not reproducible.
  • BCG-CWS having excellent dispersibility in water could be obtained by changing the production method using the improvement in physical properties as an index.
  • fatty acid examples include mycobacteria derived from the genus Mycobacterium and fatty acids possessed by the genus Nocardia. Preferred is mycolic acid.
  • the “sugar chain” of the present invention refers to a sugar chain bonded to CWS or the like of the genus Mycobacterium or Nocardia. Preferably, arabinogalactan can be used.
  • An emulsion was prepared and compared using the BCG-CWS produced by the known method (Patent Document 3) and the BCG-CWS of the present invention excellent in dispersibility in water.
  • the oil-in-water emulsion of the present invention is 1) Reactivity with lectin (concanavalin A): Although it reacts with lectin and aggregation is confirmed, the emulsion using known BCG-CWS shows no reaction (FIG. 2). 2) Zeta potential: Regarding the zeta potential, an oil-in-water emulsion using 15 times the weight ratio of oil relative to BCG-CWS shows a strong zeta potential ( ⁇ 15.2 mV), while BCG-CWS of Patent Document 3 is used. The resulting emulsion showed a zeta potential (-7.8 mV) equivalent to the BCG-CWS-free vehicle emulsion (-7.1 mV).
  • the conversion efficiency (content efficiency), the stability of the emulsion, and the storage stability of the lyophilized preparation are all significantly superior to the known BCG-CWS of the BCG-CWS of the present invention. It has become possible to produce an oil-in-water emulsion having a high BCG-CWS content and a lyophilized preparation thereof.
  • the “storage stability” of the present invention refers to CWS content etc. after several days to 2 weeks have passed under conditions of room temperature for emulsions and 40 ° C. for lyophilized preparations. Say that.
  • the weight loss is preferably 10% or less over 3 days, and for emulsion lyophilized formulations, it is preferably 2%.
  • the “reactivity with lectin” in the present invention means that the lectin binds to, for example, concanavalin A to cause aggregation.
  • a lectin is a protein having a property of specifically binding to a sugar chain, and has been separated from plants exclusively.
  • Concanavalin A is a sugar chain-binding protein or glycoprotein obtained from nata beans.
  • the fact that the oil-in-water emulsion of the present invention and the lyophilized preparation thereof show reactivity with lectin indicates that arabinogalactan protrudes and is exposed on the surface of oil droplets (oil particles) in the emulsion.
  • the lectin is not particularly limited, and for example, rhodamine-labeled concanavalin A can be used.
  • the “zeta potential” of the present invention indicates the charged state of the oil particle surface in the oil-in-water emulsion measured using a zeta sizer.
  • the difference in zeta potential from the zeta potential of the vehicle emulsion is large (6 to 9 mV), and BCG-CWS of Patent Document 3 is used.
  • the difference in zeta potential from the zeta potential of the vehicle emulsion was hardly observed (1 mV or less) (Table 10).
  • the emulsion using the BCG-CWS of the present invention has a zeta potential difference of 3 to 14 mV with respect to the vehicle emulsion, preferably 4 to 12 mV. Particularly preferred is 4 to 11 mV.
  • zeta potential difference 3 to 14 mV with respect to the vehicle emulsion, preferably 4 to 12 mV. Particularly preferred is 4 to 11 mV.
  • the difference in the above and the reactivity with lectin indicate a structure / form in which sugar chains, that is, arabinogalactan protrude and are exposed on the surface of the oil particles (oil droplets) of the emulsion.
  • the oil-in-water emulsion of the present application is a novel oil-in-water emulsion that is completely different from the known emulsion (Patent Document 3) characterized in that the reaction with the lectin known so far is negative. Was confirmed (FIG. 3).
  • the oil-in-water emulsion using the BCG-CWS of the present invention is more stable and has a higher conversion efficiency than the oil-in-water emulsion of Patent Document 3, that is, an emulsion having a high BCG-CWS content with respect to oil droplets.
  • Patent Document 3 that is, an emulsion having a high BCG-CWS content with respect to oil droplets.
  • a possible reason is that sugar chains protrude and are exposed on the surface of the oil droplets, that is, the BCG-CWS of the present invention functions as a surfactant.
  • the particle size distribution of BCG-CWS of the present invention using 7.5 times the weight ratio of oil relative to BCG-CWS and BCG-CWS of Patent Document 3 Is shown in FIG.
  • the median diameters thereof were 2.4 ⁇ m for the BCG-CWS emulsion of the present invention and 2.7 ⁇ m for the BCG-CWS emulsion of Patent Document 3, indicating substantially the same median diameter.
  • the oil amount is 3.8, 6.0, and 7.5 times the weight of the BCG-CWS
  • the same median diameter and The particle size distribution waveform was shown (FIG. 4a).
  • the BCG-CWS oil-in-water emulsion of the present invention can have a median diameter of 2.5 ⁇ m or less depending on emulsification conditions and lyophilization conditions.
  • the biological activity of the BCG-CWS of the present invention exhibited excellent activities such as 1) in vitro activity against immune cells, 2) migration to lymph nodes, 3) in vivo activity in animal experiments, and the like.
  • 1) In vitro activity against immune cells Little is known about the in vitro activity of oil-in-water emulsions of BCG-CWS, but in-vivo against general-purpose immune cells using oil-in-water emulsions prepared from BCG-CWS of Patent Document 3 and BCG-CWS of the present invention. Vitro activity was examined. As a result, the emulsion prepared from the BCG-CWS of the present invention showed unexpectedly significant activity.
  • the raw 264.7 cell line is about 16 times
  • the TLR2 gene-introduced HEK cell line is about 22 times
  • the BC-1 cell line is about 6 times the in vitro activity.
  • the in vitro activity of the oil paste (mixed oil) composed of BCG-CWS, oil and a surfactant before preparation of the emulsion was examined.
  • the oil paste prepared from BCG-CWS of the present invention showed in vitro activity of Raw 264.7 cells, but the oil paste prepared from BCG-CWS of Patent Document 3 showed almost no activity.
  • the activity of the BCG-CWS oil paste of the present invention decreased as the amount of oil increased (FIG. 8). This phenomenon was considered to correlate with a decrease in the amount of arabinogalactan exposed to the oil droplet surface as the oil amount increased (FIG. 9).
  • the BCG-CWS emulsion of the present application showed stronger tumor growth inhibitory activity than the BCG-CWS emulsion of Patent Document 3 (FIG. 10b).
  • the BCG-CWS emulsion of the present invention strongly suppressed tumor engraftment (Table 13).
  • the “oil-in-water emulsion” of the present invention can be produced using a method according to Patent Document 3 described above. That is, 1) a step of mixing and stirring bacteria-CWS and oil in an organic solvent as a dispersion auxiliary solvent; 2) a step of distilling off the organic solvent of 1) to prepare an oil paste (mixed oil); 3) A step of emulsifying a mixed oily product of bacteria-CWS and oil in water in the presence of a surfactant (emulsified solution). 4) A step of diluting with an aqueous solvent containing a buffer, excipient and the like (diluent).
  • the bacteria-CWS concentration can be 0.1-4.0 mg / mL.
  • Emsification in the present invention refers to emulsification of two separated liquids into an emulsion.
  • a substance having an emulsifying action is called an emulsifier, and a surfactant is used. That is, an emulsion is a liquid in which one of liquid phases, such as water and oil, that is not soluble in each other is dispersed as fine droplets in the other (W. Clayton, Theory of emulsions, 4th ed., Blackstone, New).
  • oil-in-water emulsion freeze-dried preparation is a product obtained by freeze-drying the above oil-in-water emulsion by a usual method.
  • oils examples include mineral oils and animal and vegetable oils as described in known literature (Immunology Vol. 27, 311 to 329 (1974)).
  • mineral oil examples include liquid paraffin (Drecall 6VR, Moresco Bioless U-6, Moresco Bioless U-8, etc.), Biol (Bayol F), and the like.
  • animal and vegetable oils include soybean oil, synthelan 4, ethyl oleate, peanut oil, coconut oil, sesame oil, AD-65 (a mixture of peanut oil, aracel and aluminum monostearate), terpenoid derivatives such as squalane and squalene. .
  • the mixture of the some oil chosen from these animal and vegetable oils and mineral oil can be mentioned.
  • Preferable examples include squalane, squalene, or a mixture of vegetable oil (or oil derived therefrom) such as soybean oil, ethyl oleate or oleic acid and squalane, squalene, for example, minerals such as Drecall 6VR and various liquid paraffins.
  • a mixture of oil, squalane and squalene may be mentioned. More preferably, squalane, squalene, drecall 6VR or a mixture thereof can be mentioned. Even more preferred are squalane and squalene.
  • the concentration of oil is 3 to 30 times the amount of oil as a weight ratio with respect to bacteria-CWS.
  • the oil paste used for the production of the emulsion is the following 1) and 2): 1) A step of mixing and stirring bacteria-CWS and oil in an organic solvent as a dispersion auxiliary solvent; 2) A step of distilling off the organic solvent of 1) above; Can be prepared.
  • organic solvent to be used include hydrocarbon solvents such as hexane, heptane, and toluene, alcohol solvents such as 5 to 20% ethanol, and hydrocarbon solvents including ketone solvents such as acetone.
  • the “surfactant” of the present invention refers to a surfactant generally used in pharmaceutical preparations, and is not particularly limited. Examples thereof include phospholipids and nonionic surfactants. Examples of phospholipids include phosphatidylamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, and lecithin. Hydrogenated phospholipids can also be used. Examples of nonionic surfactants include polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene hydrogenated castor oil derivatives, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters, and the like.
  • polyoxyethylene sorbitan fatty acid ester examples include polyoxyethylene sorbitan monolaurate (polysorbate 20), monopalmitate (polysorbate 40), monostearate (polysorbate 60), or monooleate (polysorbate 80). It is done.
  • sorbitan fatty acid esters include sorbitan monolaurate (Span20), monopalinate (Span40), monostearate (Span60), monooleate (Span80), and the like.
  • Preferred surfactants include egg yolk phosphatidylcholine, egg yolk lecithin, soybean lecithin, polysorbate 80, polysorbate 20, polyoxyethylene hydrogenated castor oil 60 (HCO-60), polyoxyethylene hydrogenated castor oil 50 (HCO-50), polyoxy Mention may be made of ethylene (160) polyoxypropylene (30) glycol (Pluronic F68). More preferred are polyoxyethylene sorbitan fatty acid esters (polysorbates) such as polysorbate 80, polysorbate 40, polysorbate 60, and more preferred are polysorbate 20, polysorbate 80, and mixtures thereof, and particularly preferred surfactant is polysorbate. 80.
  • the concentration of the surfactant is suitably in the range of 0.01 to 5% by weight in the oil-in-water emulsion, preferably 0.01 to 3% by weight, more preferably 0.05 to 2% by weight. it can.
  • These surfactants are not limited to one type, and several types can be used in combination as appropriate.
  • the “antioxidant” of the present invention represents a compound having a radical inhibitory function for suppressing a chain reaction of auto-oxidation or a peroxide decomposing function for inactivating a peroxide by non-radical decomposition
  • the antioxidant is not particularly limited as long as it can be used. Specifically, tocopherols, dibutylhydroxytoluene (hereinafter referred to as BHT), butylhydroxyanisole (hereinafter referred to as BHA), nordihydroguaiaretin, propyl gallate, vitamin C (VC) and fatty acid esters thereof (V.C.E.) or sorbic acid can be exemplified.
  • Preferred examples of the antioxidant include BHT, ⁇ -tocopherol (VE), and ⁇ -tocopherol ester derivative (VEEE). Particularly preferred is BHT.
  • the blending amount of these antioxidants in the present invention is not particularly limited, but in order to sufficiently prevent the oxidative degradation of the drug substance, it is 0.0001 to 5% by weight based on the whole composition. More preferred is 0.0001 to 3% by weight.
  • the antioxidant is not limited to one type, and can be used in combination of several types as appropriate.
  • the antioxidant may be added at any step in the production process of the oil-in-water emulsion, but it can be preferably added at the time of producing a mixed oil (oil paste) of bacteria-CWS and oil.
  • ⁇ -tocopherol is generally used, an antioxidant was investigated with the aim of further improving the stability of the emulsion which is the oil-in-water emulsion of the present invention and the diluted solution thereof.
  • BHT was the most excellent in peroxidation of polysorbate 80 decomposition product (oleic acid) and squalene oxidation, and the fluctuation in pH associated therewith was also suppressed. From the above results, it was found that BHT was most excellent in the antioxidant effect as compared with ⁇ -tocopherol, vitamin C and its ester.
  • excipient of the present invention is a component used for the purpose of maintaining and improving the stability of the emulsion as an emulsion or the stability as a lyophilized preparation.
  • excipients that can be used in the present invention include monosaccharides, sugar alcohols, polysaccharides, amino acids, proteins, urea, and inorganic salts.
  • monosaccharides and disaccharides include glucose, fructose, sucrose, lactose, and trehalose.
  • sugar alcohols include mannitol and sorbitol.
  • Preferred polysaccharides include dextran, starch, maltodextrin, cellulose, polyvinyl pyrrolidone, sodium alginate and the like.
  • amino acid neutral amino acids such as alanine, glycine and proline are preferable, and glycine can be mentioned as a more preferable neutral amino acid.
  • Preferred examples of the protein include albumin, gelatin, collagen and the like.
  • inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium sulfate, sodium carbonate and the like.
  • Preferred excipients include monosaccharides or sugar alcohols, and particularly preferred excipients include mannitol.
  • excipients are not limited to one type, and can be used in combination of several types as appropriate.
  • concentration of the excipient is suitably in the range of 0.1 to 20% by weight, more preferably 0.1 to 10% by weight in the oil-in-water emulsion.
  • suitable concentration of the excipient varies depending on the type of the excipient, but can be appropriately adjusted according to the production scale and the content of each component.
  • an amino acid for example, it is 2.25 to 11.25 wt% (300 to 1500 mM), preferably 6.75 wt% (900 mM) for glycine.
  • the concentration in the oil-in-water emulsion is preferably 1 to 10% by weight, more preferably 1 to 8% by weight, and more preferably 3 to 6% by weight.
  • mannitol By using mannitol as an excipient, it can be used at the same concentration as isotonicity, so that a stable preparation with less burden on the living body can be prepared.
  • the substances mentioned as the excipient may be used as an isotonic agent as necessary. These excipients or tonicity agents are not limited to one type, and several types can be used in combination as appropriate. Usually, the excipient also serves as an isotonic agent, but a substance different from the excipient may be selected as the isotonic agent.
  • the concentration of the tonicity agent is appropriately set according to the content of other components, but is usually in the range of 0.1 to 30% by weight, preferably 1 to 10% by weight.
  • the “buffer” of the present invention is a buffer used for the purpose of keeping the pH of the oil-in-water emulsion of the present invention (including an emulsion obtained by resuspending a lyophilized preparation with water) constant. Represents an aqueous solution containing.
  • the buffer there are no particular limitations on the buffer that can be used in the present invention, but any buffer that can be used in pharmaceutical preparations is preferred.
  • amino acids such as glycine, alanine, arginine, glutamine, cysteine, serine, threonine, valine, histidine, phenylalanine, methionine, aspartic acid, glutamic acid, ⁇ -aminocaproic acid, lysine, leucine, etc., or hydrochloride, sodium salt thereof , Salts such as potassium salts, ampholytes such as nicotinamide, aminobenzoic acid or sodium salts thereof, citric acid, phosphoric acid, lactic acid, acetic acid, boric acid, propionic acid, butyric acid, valeric acid, glycolic acid, Organic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phthalic acid, tartaric acid, salts such as sodium salt, potassium salt, etc.
  • the phosphate phosphate buffer includes a buffer solution composed of 5 to 50 mM sodium dihydrogen phosphate and disodium hydrogen phosphate, and the ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is usually 1 : It may be dissolved at 2.3 (weight ratio), and the pH may be finely adjusted with an acid such as hydrochloric acid or a base such as sodium hydroxide as appropriate.
  • the Tris buffer include a buffer consisting of a 5 to 50 mM Tris solution, and the pH can be finely adjusted as necessary with, for example, 0.1 to 4N hydrochloric acid.
  • the citrate buffer include a buffer consisting of a 5 to 50 mM citrate solution.
  • the pH can be finely adjusted as appropriate with 0.01 to 4 N citric acid, hydrochloric acid, sodium citrate, sodium hydroxide, or the like.
  • Preferred examples of the buffer solution include a citrate buffer solution and a phosphate buffer solution, and particularly preferred is a citrate buffer solution.
  • the oil-in-water emulsion preparation of the present invention is preferably adjusted to pH 5.5 to 8.5, preferably pH 6.0 to 7.5 with a buffer.
  • the amount of these buffering agents in the present invention is not particularly limited as long as it is a sufficient amount to exhibit a sufficient buffering capacity, but is preferably 5 to 50 mM.
  • a substance having a buffering action such as an amino acid is used as an excipient
  • the addition amount of the buffering agent is appropriately adjusted.
  • a phosphate buffer is generally used as a buffering agent, in the case of the oil-in-water emulsion of the present invention, the stability of polysorbate 80, which is considered to be most easily hydrolyzed among the formulation components, is improved. As a result of examination as an index, as shown in FIG. 12, it was found that the citrate buffer solution is clearly superior in stability and buffer capacity as compared with the phosphate buffer solution.
  • An oil-in-water emulsion formulation can be provided by ascites (resuspending) the lyophilized formulation of the present invention with an aqueous solvent.
  • Such oil-in-water emulsion formulations are also included in the present invention.
  • the aqueous solvent used for resuspending the lyophilized preparation in the present invention is a dispersion medium for the emulsion particles, and examples thereof include distilled water for injection, physiological saline, and buffer solution. It is not particularly limited as long as it is a possible aqueous solvent.
  • the aqueous solvent used to resuspend the lyophilized preparation is an aqueous solvent containing an inorganic salt such as physiological saline or a buffer
  • the composition of the aqueous solvent is an oil-in-water type after resuspension. What is necessary is just to select suitably in consideration of stability etc. of an emulsion formulation.
  • the concentration of inorganic salt (inorganic salt added as a buffering agent or excipient) in the oil-in-water emulsion formulation after resuspension is adjusted to be an isotonic solution when administered to humans. It is preferable.
  • oil-in-water emulsions emulsions and diluents
  • oil-in-water emulsions which are production intermediates for preparing the lyophilized preparation of the present invention
  • the “oil-in-water emulsion” may further contain excipients, buffering agents and the like. That is, the oil-in-water emulsion preferably contains an excipient such as mannitol, and is further adjusted with a buffer to adjust the pH to 5.5 to 8.5.
  • a freeze-dried preparation can be produced by lyophilizing the oil-in-water emulsion described above.
  • the freeze-dried preparation of the present invention can be obtained by freeze-drying an oil-in-water emulsion, and finally replacing the inside of the vial with nitrogen and capping.
  • the freeze-drying temperature and time are not particularly limited.
  • lyophilization of, for example, 0.05 to 4 mL / vial is possible.
  • the oil-in-water emulsion preparation of the present invention is administered parenterally such as by injection.
  • the administration form may be subcutaneous or intradermal administration or intrathoracic administration for therapeutic / preventive purposes.
  • the amount of bacteria-CWS in the emulsion is usually at a concentration of 0.01 to 2.0 mg / ml.
  • the lyophilized preparation of the present invention is resuspended in the oil-in-water emulsion of the present invention in an appropriate amount of an aqueous solvent such as water, physiological saline, or a buffer solution.
  • an aqueous solvent such as water, physiological saline, or a buffer solution.
  • concentration of each component in the oil-in-water emulsion when the lyophilized preparation is resuspended and administered to a living body may be different from the concentration of each component in the oil-in-water emulsion as a production intermediate before lyophilization.
  • the amount of the aqueous solvent can vary depending on the administration concentration and the like.
  • the dose and frequency of administration vary depending on the target disease, patient's disease, symptoms, age, weight, sex, etc., for example, the dose of bacteria-CWS per administration for adults once a week or once every 4 weeks As an example, administration in the range of 3 to 200 ⁇ g, preferably in the range of 5 to 120 ⁇ g can be mentioned.
  • the freeze-dried preparation of the present invention and the oil-in-water emulsion preparation obtained by condensing the same are useful as cancer therapeutic agents or preventive agents, specifically as immunotherapeutic agents.
  • cancer therapeutic agents include an embodiment as a cancer metastasis inhibitor. It can also be used as a concomitant agent with various cancer immunotherapeutic agents.
  • it can be used as a therapeutic agent or preventive agent for various immune diseases, for example, for the following diseases alone or in combination.
  • the BCG-CWS oil-in-water emulsion of the present invention has a strong in vitro activity on immune cells, a strong adjuvant effect in animal experiments in vivo, and a lymph node after intradermal administration that is thought to lead to clinical effects.
  • An oil-in-water emulsion and lyophilized preparation thereof which has excellent features such as excellent transferability and the like, has high safety including skin disorders due to dose reduction, etc., and excellent storage stability It is made.
  • the second aspect of the present invention relates to “bacteria with excellent dispersibility in water—CWS”. That is, the present invention relates to high-purity bacteria-CWS suitable for production of various emulsions, suspensions and lyophilized preparations thereof.
  • high purity in the present invention refers to bacteria—CWS from which impurities such as proteins bound to bacteria—CWS have been removed. Specifically, the content of non-constituent amino acids and impure proteins is 1.0% or less. It is desirably 0.8% or less. It also shows that it contains no surfactant and no halogenated organic solvent as a residual solvent.
  • total non-constitutive amino acids refers to the total amount of amino acids derived from impurities other than the constituent amino acids of peptidoglycan contained in bacteria-CWS.
  • the content of all non-constituent amino acids derived from impurities is preferably 1.0% or less of the total bacterial-CWS composition. More preferably, it may be 0.8% or less.
  • the “impure protein” of the present invention refers to the total amount of protein derived from impurities contained in bacteria-CWS.
  • the content of protein derived from impurities is preferably 1.0% or less of the bacterial-CWS composition. More preferably, it may be 0.8% or less.
  • the BCG-CWS of the present invention has a higher purity than the BCG-CWS of Patent Document 3.
  • Halogen-free organic solvent and surfactant-free in the present invention means “halogenated hydrocarbon and any surface activity used in the production of bacteria-CWS such as dichloromethane, dichloroethane, chloroform, carbon tetrachloride”. It means not containing any agents. This represents that the halogen-based organic solvent and the surfactant do not remain in the bacteria-CWS by not using the halogen-based organic solvent in the production process of the present invention.
  • “Excellent dispersibility in water” of the present invention means that, based on Test Example 5, the OD value (absorbance at a wavelength of 690 nm) from immediately after dispersion of the bacterium-CWS of the present invention in physiological saline to 60 minutes later. Measure the change in, and set the value immediately after 1 to evaluate the dispersibility by the OD relative value (physical property value) after 60 minutes of dispersion, and the OD relative value (physical property value) is 0.4 or more after 60 minutes.
  • the OD relative value (physical property value) after 60 minutes of dispersion is 0.7 or more, and more preferably 0.9 or more (FIG. 13).
  • BCG-CWS of Patent Document 3 BCG-CWS of Patent Document 3
  • the OD relative value (physical property value) after 60 minutes of dispersion decreased to 0.29, and sedimentation of BCG-CWS was observed. It was.
  • the physical properties of the bacterium-CWS (BCG-CWS) of the present invention are different from those of the BCG-CWS of Patent Document 3 as indicated by the OD relative value (physical property value) after 60 minutes.
  • BCG-CWS of Patent Document 3 irreversible strong aggregation and fusion of bacteria-CWS mycolic acids occur due to the treatment in the presence of a surfactant or high temperature treatment in the production process, and the density of the mycolic acid portion is reduced. Since it has the form which formed the high laminate structure, it is thought that the dispersibility to water is falling.
  • BCG-CWS is composed of mycolic acid, arabinogalactan, and peptidoglycan, and has a surfactant-like structure that allows arabinogalactan to move relatively freely in water.
  • the BCG-CWS of Patent Document 3 does not change the dispersibility in water even when heated or re-crushed in a fat-soluble organic solvent (FIGS. 16a and 17).
  • the strong aggregation and fusion observed in BCG-CWS was found to be irreversible.
  • the BCG-CWS of the present invention as shown in FIG. 14 (a) or (b) using the dispersibility (OD relative value; physical property value) of BCG-CWS as an index is a surfactant in the manufacturing process.
  • the form, surface structure, etc. are clearly different from BCG-CWS of Patent Document 3, which shows a form in which the mycolic acid portion is irreversibly aggregated and fused as shown in FIG. It was shown that it can be determined as.
  • the “bacteria-CWS suitable for production of various emulsions and freeze-dried preparations” of the present invention has high storage stability, not only emulsions such as oil-in-water emulsions and water-in-oil emulsions, but also dispersibility in water. Because of its superiority, it is a bacteria-CWS that can be produced efficiently and with high yield in the production of various emulsions such as suspension of bacteria-CWS, aqueous suspensions and freeze-dried preparations thereof.
  • BCG-CWS of the present invention and BCG-CWS of Patent Document 3 are related to 1) antibody, 2) auramine staining, 3) zeta potential, and so on.
  • BCG-CWS disclosed in Japanese Patent Application Laid-Open No. H10-228867 show that the difference in the form and surface structure of BCG-CWS is remarkable.
  • BCG-CWS of Patent Document 3 did not show a binding reaction.
  • the difference in morphology and surface structure between the BCG-CWS of the present invention and the BCG-CWS of Patent Document 3 can be clearly distinguished using the anti-BCG antibody.
  • the difference in morphology and structure between the BCG-CWS of the present invention and the BCG-CWS of Patent Document 3 is evaluated by the fluorescence intensity of the auramine staining method shown in (a) and (b) of FIG. can do.
  • the auramine staining method is a method of staining by attaching auramine to the mycolic acid portion of the cell wall, and therefore, when the density of the mycolic acid portion is reduced, it is easily decolored by washing treatment (microscope Vol. 48, No. 1, 51-). 56, (2013)). Also in this method (Test Example 8), the BCG-CWS of the present invention is not stained by the auramine staining method (FIG.
  • the BCG-CWS of the present invention has a fluorescence area ratio of auramine staining of 10% or less, preferably 5% or less.
  • the BCG-CWS of Patent Document 3 has a laminate structure in which the mycolic acid portions are strongly aggregated and fused, and it is clear that most of the mycolic acid and arabinogalactan are blocked in the laminate. It was.
  • the particle size distribution in the n-heptane solvent showed a single peak in the BCG-CWS of the present invention due to the difference in form and structure, but showed a bimodality in the BCG-CWS of Patent Document 3 (FIG. 20).
  • FIG. 14 a schematic view of the form of the BCG-CWS of the present invention focusing on the aggregation of mycolic acid of BCG-CWS of Patent Document 3 is mainly shown.
  • the “single peak particle size distribution” of the present invention is a particle size distribution having a single particle size distribution measured by a laser diffraction method when bacteria-CWS is suspended in physiological saline or n-heptane. It means that.
  • FIG. 14 (a) or (b) which is not strongly aggregated and fused in the BCG-CWS of the present application, maintains its shape and structural difference, and the structure in which arabinogalactan can move relatively freely in water is disclosed in Patent Document 3.
  • the mycolic acid portion is irreversibly strongly aggregated and fused as shown in FIG. 14 (c), and many arabinogalactans are strongly agglomerated and fused with each other and blocked in the laminated structure. it seems to do.
  • the zeta potential is evaluated in an emulsified state in order to obtain information on the zeta potential under more relaxed conditions that do not affect the morphology and surface structure.
  • the zeta potential difference was 4 to 10 mV, and in the BCG-CWS of Patent Document 3, the zeta potential difference was 1 mV or less.
  • the BCG-CWS of the present invention and the BCG-CWS of Patent Document 3 were easily discriminated by the zeta potential difference measurement of this method (Test Example 15, Table 10). Further, in the in vitro activity evaluation using Raw264.7 cells, a correlation with the zeta potential difference was also observed.
  • the induction amount of TNF- ⁇ is about 1000 pg / mL or more, whereas the oil-in-water type of BCG-CWS of Patent Document 3 with little potential difference
  • the emulsion was about 300 pg / mL ( Figure 15). That is, BCG-CWS in the range of this potential difference (4 to 11 mV) is the BCG-CWS of the present invention that can produce a stable oil-in-water emulsion that reacts with lectin and has in vitro activity against immune cells.
  • the BCG-CWS of the present invention which is excellent in water dispersibility, is suitable for the production of oil-in-water emulsions and water-in-oil emulsions, and BCG-CWS itself, which is difficult to produce with the BCG-CWS of Patent Document 3, is difficult.
  • BCG-CWS is also suitable for the production of suspension formulations.
  • BCG-CWS of the present invention has a higher in vitro activity against immune cells compared to the BCG-CWS of Patent Document 3, for example, as shown in FIG. 21, a mouse immature dendritic cell line (BC-1 cell)
  • the activity of the BCG-CWS of the present invention was about 12.5 times higher than that of the BCG-CWS of Patent Document 3. Further, as shown in FIG.
  • the BCG-CWS of the present invention is about 3 times more than the BCG-CWS of Patent Document 3.
  • the induction activity of IL-12 was 2 times higher.
  • BCG-CWS of the present invention is about 2 more than BCG-CWS of Patent Document 3. .8-fold higher TNF- ⁇ -inducing activity.
  • TLR2 agonist activity against Toll-Like Receptor (TLR)
  • TLR2 showed almost the same activity, and none showed activity against TLR4 (FIG. 24).
  • the bacterium-CWS of the present invention can be applied not only to cancer but also to various immune diseases. Bacteria-CWS reduces the side effects that can occur with viable bacteria and can be used as a substitute, so clinical studies for various cancers have already been conducted with known bacteria-CWS.
  • the conventional production method is a multistage and low rational production process, and the obtained bacteria-CWS is difficult to formulate due to problems of form and physical properties, and it is difficult to supply it as a pharmaceutical product.
  • the bacterium-CWS of the present invention can stably supply high-quality products by a rational and economical production method and can be easily formulated into various formulations. High bacteria-CWS can be provided for the first time.
  • the “medicament” containing the bacterium-CWS of the present invention as an active ingredient refers to a composition used for pharmaceutical (animal medicine) use, for example, an anticancer agent, an antiviral agent, an antiinfective agent, Used for applications such as immunostimulants (adjuvants), immune function regulators, etc.
  • a composition used for pharmaceutical (animal medicine) use for example, an anticancer agent, an antiviral agent, an antiinfective agent, Used for applications such as immunostimulants (adjuvants), immune function regulators, etc.
  • Various known dosage forms can be used as the pharmaceutical dosage form of the present invention.
  • various emulsion preparations or suspension preparations can be mentioned.
  • the various emulsion preparations or suspension preparations of the present invention refer to bacterial-CWS emulsions or aqueous suspensions used for pharmaceutical applications.
  • the emulsion includes an oil-in-water (o / w) emulsion, Water-in-oil type (w / o) emulsion, water-in-oil-in-water type (w / o / w) emulsion, etc. are mentioned.
  • An oil-in-water emulsion is preferable.
  • the emulsion preparation using BCG-CWS of the present invention is an oil-in-water emulsion using BCG-CWS of Patent Document 3, such as reacting with concanavalin A and showing strong in vitro activity against immune cells.
  • An oil-in-water emulsion having different and unprecedented characteristics can be provided.
  • the BCG-CWS of the present invention is excellent in water dispersibility, the production of an aqueous suspension of BCG-CWS which is difficult to produce with BCG-CWS of Patent Document 3 having poor water dispersibility. Is also possible if necessary. For example, it can be applied to urinary bladder therapy for bladder cancer.
  • the application as an oral solid agent like a tablet or intestinal solvent which paid attention to intestinal immunity can be mentioned.
  • the content of bacteria-CWS is in the range of about 1 to 200 ⁇ g / dose, preferably in the range of about 5 to 120 ⁇ g / dose in order to elicit an immune response in the case of a prescription agent.
  • the pharmaceutical composition of the present invention can be administered by an appropriate means, and includes injection, intrathoracic administration, oral administration, intranasal administration, intravesical administration, etc. It is not limited.
  • One preferred embodiment is intradermal, subcutaneous and intrathoracic injection.
  • the term common to the 1st aspect of this invention represents the same meaning as the term of the 1st aspect.
  • the third aspect of the present invention relates to a method for producing a bacterium-cell wall skeleton (bacteria-CWS) having physical properties suitable for the production of various emulsion formulations and suspension formulations excellent in water dispersibility.
  • the “purification of purified cells” of the present invention can use dry purified cells, but usually uses wet purified cells and is suspended in water or a mixed solution of water and a hydrophilic organic solvent. Crush.
  • the means for crushing is not particularly limited, but a crusher such as a high-pressure crusher, a bead mill, an ultrasonic irradiation device, or a homomixer can be used, and a high-pressure crusher is preferably used.
  • the particle size distribution range of the crushed material is approximately 0.1 to 5.0 ⁇ m, The thickness is preferably 0.1 to 2.0 ⁇ m. Since heat generation occurs in the crushing step, it is desirable to perform the treatment temperature in the range of 4 to 65 ° C. in order to avoid a change in form due to heat generation of CWS. A range of 4 to 45 ° C. is more preferable. If necessary, the disrupted material is centrifuged at 1,000 to 9,000 ⁇ g for 5 to 30 minutes, preferably 3,000 to 8,000 ⁇ g for 5 to 20 minutes.
  • the “hydrophilic organic solvent” in the present invention is an organic solvent miscible with water, which is a lower alcohol such as methanol, ethanol or isopropanol, tetrahydrofuran or acetone, but 1-propanol, dioxane, acetonitrile or methyl ethyl ketone. Can be added.
  • the content of the hydrophilic organic solvent is 0 to 30% by volume in the crushing treatment, preferably 5 to 15% by volume. Is 0 to 20% by volume, preferably 0 to 10% by volume. In the cleaning treatment, it is 0 to 30% by volume or 70 to 100% by volume, preferably 0 to 30% by volume.
  • the “purification treatment” of the present invention means enzyme treatment after crushing purified bacterial cells of bacteria, washing with water, a hydrophilic organic solvent or a mixed solution of water and hydrophilic organic solvent. The number of times and the order are not limited to this.
  • Enzymatic treatment includes continuous treatment of nucleolytic enzyme and proteolytic enzyme. In this production method, high purity and water can be obtained only by a short time treatment of proteolytic enzyme by using highly purified cells.
  • One of the features is that BCG-CWS with excellent dispersibility can be produced.
  • the nucleolytic enzyme and the treatment method are not particularly limited, and enzymes and treatment methods well known to those skilled in the art can be appropriately used. Examples of the nuclease include DNase and RNase. Preferably, DNase can be mentioned.
  • Specific examples include endonucleases derived from Serratia bacteria (benzonase, manufactured by Merck), double-strand specific endonucleases derived from bovine pancreas (DNase I, manufactured by Roche), and preferably benzonase.
  • the range of nucleolytic enzyme treatment temperature and time can be appropriately selected according to the type of enzyme, and the range of treatment temperature is 20 to 40 ° C., preferably 20 to 30 ° C.
  • the proteolytic enzyme and the treatment method are not particularly limited, and enzymes and treatment methods well known to those skilled in the art can be appropriately used. Specific examples include pronase (Sigma-Aldrich), papain, trypsin, chymotrypsin and the like.
  • Preferable examples include pronase and papain. More preferred is pronase.
  • the proteolytic enzyme treatment temperature and time range can be appropriately selected according to the type of enzyme, and the treatment temperature range is 30 to 45 ° C, preferably 30 to 40 ° C.
  • the feature of the above-mentioned purification treatment is to avoid the irreversible strong aggregation of BCG-CWS, the form and physical property change caused by the fusion, and the patent document that does not pay attention to the conventional CWS form and physical property change. 3 is greatly different from the BCG-CWS manufacturing method.
  • the process after crushing BCG cells is multistage (Patent Document 3, Non-Patent Documents 2 to 4), and a surfactant is also used (Patent Document 3, Non-Patent Document 4). ).
  • BCG-CWS has no physical properties suitable for the production of various emulsions, suspensions, and lyophilized preparations that do not contain a surfactant and a halogen-based organic solvent and have high purity and excellent dispersibility in water. was successfully produced in high yield.
  • the conventional bacteria-CWS production method is a multi-stage and low rational production process, and the obtained bacteria-CWS is difficult to formulate due to problems in form and physical properties, and it is difficult to supply it as a pharmaceutical product.
  • the bacterium-CWS of the present invention can stably supply high-quality products by a rational and economical production method and can be easily formulated into various formulations. High bacteria-CWS can be provided for the first time.
  • the term which is common in the 1st and 2nd aspect of this invention represents the same meaning as the term of the 1st and 2nd aspect.
  • the fourth aspect of the present invention relates to a purified microbial cell that is an intermediate for the production of the bacterium-CWS of the present invention and a method for manufacturing the same.
  • the “extracellular component” of the present invention is a component that exists in the outer lipid layer of acid-fast bacteria or adheres to the cell wall of the inner lipid layer, such as proteins, free lipids, glycolipids, saccharides, inorganic substances, etc. (Microbiology Spectrum, 2 (3), 1-19 (2014), Molecular Microbiology, 31 (5), 1561-1572 (1999)).
  • extracellular adhesion components include glycolipids such as TDM, TMM (trehalose monomycolate), GMM (glucose monomycolate), PIM (phosphatidylinositol mannoside), PDIM, and the like. , PGL, GroMM, wax D component, and the like.
  • examples of water-soluble substances include proteins such as MPB64 (Mycobacterial protein fractionation Rmof 0.64 in vitrophoresis) and MDP1 (mycobacterial DNA-binding protein 1), LM (Lipomannan), LAM (Lipomannan) Ingredients and remaining culture fluid components
  • the “purified cell” of the present invention is a bacterial cell from which an extracellular adhesion component (extracellular component) has been removed.
  • the removal rate of the extracellular adhesion component is not particularly limited, but it is desirable to remove it as much as possible.
  • a microbial cell (degreasing microbial cell) from which a fat-soluble extracellular adhesion component is removed using an organic solvent such as a chloroform / methanol mixed solvent Japanese Patent Laid-Open No. 2006-31604
  • water and a hydrophilic organic solvent Bacteria from which not only the fat-soluble extracellular adhesion component but also the water-soluble extracellular adhesion component are removed using the above mixed solution is suitable.
  • the BCG purified microbial cell of the present invention has an improved BCG-CWS content in the purified microbial cell since the extracellular adhesion component has been removed, and has a high content of 35 to 47% by weight.
  • PGL and GroMM are examples of components having strong affinity for BCG-CWS and low removal efficiency. Therefore, it has been found that these two types of extracellular adhesion components can be used as an index indicating how much the extracellular adhesion components have been removed from the BCG cells.
  • FIG. 25a when BCG killed bacteria are used, the contents of PGL and GroMM contained in the purified cells are 10% by weight or less of the contents contained in the raw BCG killed bacteria. Confirmed by TLC. Next, when the PGL content was measured by HPLC, it was confirmed that the content was 0.4% by weight or less of the dried purified cells. Accordingly, by confirming the end point of washing using PGL or GroMM, it is possible to obtain a purified BCG microbial cell containing almost no extracellular adhesion component (extracellular microbial component).
  • the “bacteria-CWS content” in the present invention refers to the content of bacteria-CWS in the purified cells, and the content of bacteria-CWS constitutes bacteria-CWS.
  • the amount of fatty acid (for example, mycolic acid) is evaluated.
  • the purified cells were subjected to alkaline hydrolysis, and the amount of liberated fatty acid was evaluated by comparison with the sample.
  • the fatty acid (eg, mycolic acid) portion and sugar chain (eg, arabinogalactan) portion are the cell surface.
  • the purified microbial cell of the present invention or a crushed product thereof is used as a pharmaceutical composition
  • administration in the range of about 10 to 500 ⁇ g / dose is performed in order to elicit an immune response. It is preferable.
  • the pharmaceutical composition of the present invention can be administered by an appropriate means, and includes injection, intrathoracic administration, oral administration, intranasal administration, intravesical administration, etc. It is not limited to them.
  • intradermal and subcutaneous injection can be mentioned.
  • the purified cells of the present invention can be applied in the same manner as described in Bacteria-CWS.
  • the method for removing extracellular adhesion components used in the method for producing purified bacterial cells is not particularly limited.
  • bacterial cells are washed and purified with water, an organic solvent, or a mixed solution of water and an organic solvent.
  • a method for recovering microbial cells is mentioned.
  • Solvents used include water, lower alcohols such as methanol, ethanol, 1-propanol and isopropanol, saturated hydrocarbons such as n-hexane, n-heptane and cyclohexane, aromatic hydrocarbons such as benzene and toluene, dichloromethane, dichloroethane and chloroform.
  • halogenated hydrocarbons such as carbon tetrachloride, ethers such as tetrahydrofuran and dioxane, acetonitrile, acetone, methyl ethyl ketone, and ethyl acetate can be used in appropriate combinations.
  • water, lower alcohols such as methanol, ethanol, 1-propanol and isopropanol
  • saturated hydrocarbons such as n-hexane and n-heptane
  • aromatic hydrocarbons such as toluene, tetrahydrofuran, dioxane, acetonitrile, acetone, methyl ethyl ketone, etc. They can be used in appropriate combinations.
  • a mixed solution of water and a hydrophilic organic solvent such as methanol, ethanol, isopropanol, tetrahydrofuran, or acetone is used.
  • a mixed solution of water and tetrahydrofuran is particularly preferred.
  • a preferred ratio of the hydrophilic solvent to water is 10 to 40% by weight, which has a high cleaning effect and enables filtration and cleaning.
  • Examples of the method for collecting the cells include a centrifugal separation method and a filtration method, but are not limited at all. Preferably, a filtration method is used. Examples of washing and purification methods include room temperature or warming stirring and heating under reflux. Preferably heating reflux is mentioned.
  • filterable means that it can be easily treated by a general filtration operation without using a centrifugal separation treatment in the separation treatment of the washing liquid and the bacterial cells.
  • the cells can be isolated by filtering the washing liquid and the cells.
  • compression filtration is desirable. Therefore, it is easy to separate the cleaning liquid, and the end point of the cleaning can be easily managed by checking the separated cleaning liquid.
  • Conventional untreated BCG killed bacteria and publicly known Japanese Patent Application Laid-Open No. 2006-31604 BCG defatted bacterial cells are difficult to filter, and have been mainly used for separation from a washing solution.
  • the purified BCG cells of the present invention have different physical properties from the raw BCG killed bacteria and BCG defatted cells due to the removal of extracellular adhesion components. Can now be separated.
  • acid-fast bacteria such as Mycobacterium tuberculosis and BCG bacteria tend to aggregate and are difficult to filter, but it is known that extracellular adhesion components and adhesion factors such as MDP1 are involved in the aggregation.
  • MDP1 extracellular adhesion components and adhesion factors
  • the purified microbial cell of the present invention the cell adhesion component which is one of the causes of aggregation has been removed, and as a result, it is considered that filtration has become easy.
  • the filterability of the purified bacterial cell of the present invention can be filtered without clogging when, for example, a filter having a nominal filtration accuracy of 3 ⁇ m or more is used for filtration, and even when a kilogram-scale wet bacterium is used. Filtration is possible within ⁇ 3 hours. As a result, since separation processing such as centrifugation can be avoided, scale-up is possible and processing time can be greatly shortened. Thus, the filterability of the purified microbial cells of the present invention is greatly different from BCG dead bacteria and known BCG defatted microbial cells.
  • the “filter” of the present invention refers to a separation membrane used for filtration such as filter paper, nonwoven fabric, filter cloth, and mesh sheet.
  • a filter having a desired nominal filtration accuracy can be used as necessary.
  • a filter having a nominal filtration accuracy of 0.45 to 5 ⁇ m is desirable, preferably One having a thickness of 1 to 3 ⁇ m can be mentioned.
  • End point management of the present invention is to confirm the elution amount of a low-efficiency lipid-soluble component present in purified cells, such as PGL and / or GroMM, or the presence or absence thereof, It is to confirm by using chromatography (TLC) or high performance liquid chromatography (HPLC).
  • the end point of washing for example, when BCG killed bacteria are used and PGL is evaluated by HPLC, impurities are extracted from the purified cells using a hydrophobic organic solvent, and the extract and PGL standard are analyzed by HPLC. . If the PGL content in the purified cells is reduced to 0.5% by weight (vs. dry purified cells) or less, the end of the washing step is determined. Preferably, it is reduced to 0.4% by weight (vs. dry purified cell weight) or less.
  • Both the purified BCG cells and the dead BCG of the present invention do not show agonist activity for TLR4, but show agonist activity only for TLR2. Furthermore, the purified microbial cells of the present invention showed about 1.6 times higher activity in TLR2 as compared with dead bacteria of acid-fast bacteria (BCG bacteria) (FIG. 27), and TNF- ⁇ producing activity. Also showed about 1.3 to 1.4 times higher activity (FIG. 28) and 4 times higher activity in JAWSII cells (FIG. 29).
  • the purified BCG cells of the present invention are shown to have a high immunostimulatory activity even when compared with the BCG killed bacteria as a raw material.
  • the extracellular adhesion component includes an immunostimulatory substance such as TDM.
  • the extracellular adhesion component having immunostimulatory activity such as TDM is removed in the purified microbial cell of the present invention, the immunostimulatory activity of the purified microbial cell of the present invention is higher than the dead microbial cell containing them. Is excellent.
  • the purified cell of the present invention is excellent in immunostimulatory activity, and based on the effect, the immunostimulant and anti-adjuvant that avoids side effects using the purified cell of the present invention. It was shown that the purified microbial cells of the present invention and the crushed material thereof can be used for pharmaceutical purposes.
  • an immunomodulator such as a vaccine using the purified bacterial cell of the present invention is used. Using it, pollen and mite allergy suppressors that avoid side effects became possible.
  • the fifth aspect of the present invention relates to a method for producing mycolic acid of acid-fast bacteria using the purified acid-fast bacteria of the present invention.
  • the “alkaline hydrolysis” of the present invention uses, for example, an alkali metal hydroxide such as lithium hydroxide, sodium hydroxide or potassium hydroxide, and is added as an aqueous solution or an alcohol solution, either alone or as a mixture, and then hydrolyzed. To do.
  • Preferred alkalis include sodium hydroxide and potassium hydroxide. More preferably, potassium hydroxide can be mentioned.
  • the alkali content of the aqueous solution and the alcohol solution can be appropriately selected according to the purpose, and examples thereof include 5 to 15% (weight / volume).
  • the term “neutralization and acidification” in the present invention refers to neutralizing and acidifying an alkaline solution with an aqueous solution of an inorganic acid such as hydrochloric acid or sulfuric acid after completion of alkaline hydrolysis.
  • the “liquid-liquid extraction” of the present invention refers to solvent extraction from a solution neutralized and acidified with an aqueous solution of an inorganic acid with a hydrophobic organic solvent, washing the extracted organic solvent layer with water, washing with an aqueous solution containing alcohol.
  • organic solvent used for solvent extraction include saturated hydrocarbons such as n-hexane, n-heptane, and cyclohexane, aromatic hydrocarbons such as benzene and toluene, acetate esters such as ethyl acetate, such as dichloromethane, and dichloroethane. And halogenated hydrocarbons such as chloroform and carbon tetrachloride.
  • hydrophobic organic solvent examples include n-hexane, n-heptane, cyclohexane, benzene and toluene. More preferred are n-hexane and n-heptane.
  • the alcohol-containing aqueous solution used for washing the extracted organic solvent layer include a mixed solution of methanol, ethanol, 1-propanol, isopropanol and the like and water. The alcohol ratio may be 10 to 95% by volume, preferably 70 to 90% by volume.
  • Example 1 Production of purified mycobacteria (BCG bacteria) (1) Starting material (BCG bacteria: M. bovis BCG Tokyo 172 (ATCC 35737)) The BCG bacterium was cultured as a fungal membrane on the surface of the culture medium at 37 ° C. in a sorton medium until the initial stationary phase. Cultured cells were inactivated by heating to 80 ° C. for about 30 minutes and centrifuged. Purified cells were produced using BCG killed bacteria obtained as a precipitate as a raw material.
  • BCG bacteria M. bovis BCG Tokyo 172 (ATCC 35737)
  • Test Example 1 Purity of purified mycobacteria (BCG fungus) Purified cells of the present invention were evaluated by the following method to determine the remaining amount of extracellular adhesion components.
  • (1) Measurement of residual amount of PGL evaluation by high performance liquid chromatography (HPLC)
  • HPLC high performance liquid chromatography
  • the residual amount of PGL was 9.3% (Example 1 (2) -a)).
  • HPLC conditions column temperature: 40 ° C., detection wavelength: 275 nm, flow rate: about 1.0 mL / min (PGL retention time: about 8 minutes), injection volume: 10 ⁇ L, measurement time: 35 minutes, mobile phase: chloroform (containing ethanol) , Methanol (gradient), column: Nacalai Tesque Cosmosil 5SL-II 4.6I. D. ⁇ 150mm
  • TLC thin layer chromatography
  • Test Example 2-1 Measurement of BCG-CWS content in purified cells of the present invention
  • Method 4 mg of the purified cells (dried material) of Example 1 (2) -a) were weighed and 0.5 M hydroxylated. Heating was performed at 65 ° C. for 3 hours in 1 mL of a potassium solution. Further, the CWS sample was weighed and subjected to the same operation, and the mycolic acid liberated in each solution was fluorescently labeled with an ADAM (9-Anthryldiazotane, trademark, Funakoshi) reagent and analyzed by HPLC.
  • ADAM 9-Anthryldiazotane, trademark, Funakoshi
  • HPLC conditions column temperature: 50 ° C., excitation wavelength: 365 nm, measurement wavelength: 412 nm, flow rate: about 1.0 mL / min, injection amount: 10 ⁇ L, measurement time: 60 minutes, mobile phase: methanol, toluene (gradient), column : Develosil C30-UG-3 (3 ⁇ m, 4.6 ⁇ 150 mm) manufactured by Nomura Chemical
  • Test Example 2-2 Measurement of PGL content in purified cells (1) Method In the same manner as in Test Example 1 (1), measurement was performed using a PGL sample. However, PGL isolated and purified by silica gel column chromatography from the 60 vol% THF aqueous solution of Example 1 (2) was used as a standard. (2) Results As a result, the PGL contents in BCG dead cells and purified cells were 2.0% by weight and 0.3% by weight, respectively.
  • Reference Example 1 Washing effect of mixed solution of water and hydrophilic organic solvent of the present invention
  • Special Table 2011-500540 The washing effect of the chloroform / methanol mixed solvent (1: 1, volume / volume) often used as the washing solution for bacterial cells described in 1) and the washing effect of the present invention were compared.
  • washing method a Known chloroform / methanol washing According to the above-mentioned known literature (special table 2011-500540), 10 g of BCG dead cells (wet, about 2 g of dry matter) were mixed with chloroform / methanol mixed solvent (1: 1 , Volume / volume) was added and stirred for 60 minutes under a nitrogen atmosphere. The suspension was subjected to suction filtration, and the supernatant was washed with 10 mL of a chloroform / methanol mixed solvent (1: 1, volume / volume). The washing solution up to this operation was concentrated to obtain 334 mg of an extract.
  • Test Example 3 Observation of the binding activity of purified acid-fast bacilli (BCG bacteria) to saccharide-binding protein / lectin (Concanavalin A; ConA) a) Preparation of evaluation sample BCG cells (dead bacteria) dried by heating under reduced pressure Alternatively, 5 mg of each purified cell was weighed into a homogenizer vessel, and about 2 mL of physiological saline was added. Using a potter homogenizer, homogenization was performed at 1,200 rpm for 5 minutes to obtain a suspension of BCG cells (dead cells) or purified cells.
  • Test Example 4 Evaluation of biological activity of purified mycobacteria (BCG bacteria) (1) Measurement of TNF- ⁇ production amount a) Preparation of evaluation sample About 4 mg of BCG cells (dead), purified cells and About 1 mL of 0.01 wt% polysorbate 80-containing physiological saline was added to the purified crushed cell product (dried product), and suspended in a potter homogenizer at 3,000 rpm for 5 minutes at room temperature. The CWS content of each suspension was measured (similar to Test Example 2), adjusted to 10 ⁇ g CWS / mL with 10% FBS-containing DMEM medium, and used as an evaluation sample. b) Measurement method According to a known method (Drug Discov.
  • RAW264.7 cells (trademark, ATCC No. TIB-71) were placed on a 96-well microplate at 5 ⁇ . Seeding at 10 ⁇ 4 cells / well. After culturing and adhering in DMEM medium containing 37%, 5% CO2, 10% FBS for 5 hours, an evaluation sample is added, and after culturing for 20 hours, the TNF- ⁇ concentration in the culture supernatant is measured by cytokine ELISA did.
  • c) Measurement results As shown in FIG. 28a, the purified microbial cells of the present invention showed about 1.3 times higher TNF- ⁇ -inducing activity than the BCG microbial cells (dead cells) as a raw material. Furthermore, as shown in FIG. 28b, the crushed product of the purified microbial cells of the present invention also showed a high TNF- ⁇ -inducing activity similar to that of the purified microbial cells of the present invention.
  • the purified microbial cells of the present invention showed an activity to TLR2 that was about 1.6 times higher than the BCG microbial cells (dead bacteria) as a raw material. Furthermore, as shown in FIG. 27b, the purified microbial cell product of the present invention also showed about 0.7 times the activity to TLR2 of the purified microbial cell of the present invention.
  • HEK-Blue-hTLR4 cells (trademark, INVIVOGEN) are placed on a 96-well microplate at 5 ⁇ 10 ⁇ 4 cells / well. Sowing. After 24 hours of incubation in DMEM medium containing 5% CO2, 10% FBS at 37 ° C., an evaluation sample was added. After 24 hours of culture, the culture supernatant was incubated with QUANTI-Blue (trademark, INVIVOGEN) at 37 ° C. After reacting for 60 minutes, absorbance at 655 nm was measured using a microplate reader. c) Measurement results As shown in FIG.
  • the purified microbial cells of the present invention did not show activity against TLR4, similarly to the BCG microbial cells (dead bacteria) as a raw material.
  • the purified microbial cell disruption product of the present invention did not show the activity against TLR4 as the purified cell product of the present invention.
  • Example 2 Production of purified cells of Nocardia rubia fungus
  • Nocardia rubia dead cells JCM2156 strain 5 g (wet) were sequentially heated and refluxed with 25 g of 65 wt% tetrahydrofuran aqueous solution and 50 g of 60 wt% tetrahydrofuran aqueous solution for 60 minutes, The resultant was washed with a 90% by weight aqueous acetone solution and subjected to hot filtration to obtain 0.7 g (dry) of purified cells.
  • Example 3 Production of purified crushed cells (1) Raw materials BCG purified cells produced in the same manner as in Example 1 were used. (2) Manufacture of crushed material The above-mentioned BCG purified bacterial cell 1.1 g (dried product) was suspended in 40 g of 10% by volume isopropanol aqueous solution and stirred with an electric homogenizer (Omni TH, Omni-international) (20,000 rpm, 60 ° C., 5 minutes) and then crushed at 20,000 psi at room temperature using BERYU MINI (beautiful granules). A part of the crushed liquid was freeze-dried using a freeze dryer (ALPHA 2-4, MARTIN CHRIST) to obtain 50 mg of a dried product of purified crushed cells. CWS content: 38.2% (HPLC)
  • Example 4 Production of mycolic acid from purified cells (1) Production of mycolic acid 140 g of purified cells (dried body) in 1.4% of 10% (weight / volume) potassium hydroxide-50 volume% isopropanol aqueous solution After heating under reflux for 2 hours, 1.4 L of water was added under cooling and further acidified with 6M hydrochloric acid. Extraction was performed twice with 2.1 L of n-heptane, and the n-heptane layer was washed twice with 1.4 L of water, then washed twice with 1.4 L of 90% by volume ethanol aqueous solution, and the obtained n-heptane layer Was concentrated under reduced pressure to obtain 14.6 g of white powder of mycolic acid.
  • Example 5 Production of CWS of BCG bacteria (BCG-CWS) (1) Raw material Example 1G purified bacterial cells were used. (2) Production of CWS a) Disruption of purified bacterial cells 223.6 g of BCG purified bacterial cells (wet, dry weight about 71.6 g) was suspended in a 10% by volume aqueous isopropanol solution, and a high-pressure homogenizer DeBEE2000 (registered trademark, BEE International). Was crushed at 35 kpsi and centrifuged at 25 ° C. at 6800 ⁇ g for 10 minutes. Next, the supernatant was centrifuged at 18000 ⁇ g for 60 minutes at 25 ° C.
  • DeBEE2000 registered trademark, BEE International
  • Composition analysis data Table 1 shows the analysis results of the composition of the BCG-CWS of the present invention.
  • the analysis value of the composition of BCG-CWS (Reference Example 2) produced according to Patent Document 3 and the document value of BCG-CWS described in Patent Document 3 are also shown.
  • Example 6 Repurification of CWS (BCG-CWS) of the present invention
  • BCG-CWS CWS
  • Example 5 100 mg of BCG-CWS obtained was suspended in 5 mL of 20 vol% isopropanol aqueous solution, stirred at 40 ° C for 30 minutes, and then centrifuged at room temperature. To obtain a precipitate. This was repeated to obtain 99.5 mg of repurified BCG-CWS.
  • the obtained repurified BCG-CWS was quantified for protein content using Micro BCA Protein Assay Kit (trademark, Thermo Fisher Scientific). As shown in Table 2 below, the protein content derived from impurities was further reduced by performing the purification again.
  • Reference Example 2 Production of BCG-CWS according to a known method (Patent Document 3) a) Crushing treatment 578 g of dead BCG (wet, dry weight about 111 g) was suspended in 3 L of water, and MiniDeBee (registered trademark, BEE) was suspended. International)) and crushed at 35 kpsi. They were centrifuged at 25 ° C. at 6,760 ⁇ g for 10 minutes. The supernatant was then centrifuged at 18,000 ⁇ g for 60 minutes at 25 ° C. to obtain a precipitate. b) Nucleolytic enzyme treatment and proteolytic enzyme treatment Benzonase (Merck Ltd.) was added to the above precipitate and reacted at 25 ° C for 17 hours.
  • the precipitate was recovered by centrifugation, and suspended / centrifugated (precipitate recovery) with a 1 wt% Triton X-100 aqueous solution was repeatedly washed 5 times, and then pronase (Sigma-Aldrich) was added and reacted at 37 ° C. for 17 hours.
  • the precipitate was collected by centrifugation at 18000 ⁇ g for 20 minutes at 25 ° C., resuspended in a 1 wt% Triton X-100 aqueous solution, stirred at 60 ° C. for 2 hours, and centrifuged to obtain a precipitate.
  • Test Example 5 Dispersibility of BCG-CWS of the present invention in water (OD relative value: physical property value) (1) Measurement sample A: BCG-CWS of the present invention (sample of Example 5) B: BCG-CWS of Patent Document 3 (sample of Reference Example 2) (2) Dispersibility measurement method: 5 mg of the above BCG-CWS was weighed in each homogenizer vessel, and about 2 mL of physiological saline was added. Using a three-one motor, the suspension was homogenized at 1,200 rpm for 5 minutes. This solution was adjusted to 1 mg / mL, and 1.5 mL was added to a 3 mL plastic cuvette. Absorbance (690 nm) was measured over time for 60 minutes using a spectrophotometer (U-5100, Hitachi High-Tech Science). The relative value (physical property value) was calculated with the absorbance at 0 minute of measurement as 1.
  • the OD relative values (physical properties) of the BCG-CWS of the present invention are 0.9 or more, 0.75, 0.58, and 0.48 for the samples of Example 5, respectively, A, B, and C. , D, and the sample of Reference Example 2 as E.
  • Test Example 6 Antibody reactivity of the BCG-CWS of the present invention Sandwiching the binding between the BCG-CWS of the present invention (Example 5) and the anti-BCG killed antibody of the BCG-CWS (Reference Example 2) of Patent Document 3 -Evaluated by ELISA method.
  • Reagent A rabbit (Japan white) anti-BCG killed IgG antibody was prepared and used.
  • Method Rabbit anti-BCG killed IgG antibody was solidified on Nunc-immuno plate II (trademark, Nunc). After each reaction with the above BCG-CWS, a biotin-labeled anti-BCG killed bacterial antibody was added and allowed to stand at room temperature for 60 minutes.
  • Streptavidin-HRP Conjugate (Vector Lab.) was added and allowed to stand at room temperature for 60 minutes. A coloring reagent (Stabilized hydrogen peroxide, R & D) was added, and after reaction at room temperature, 1N sulfuric acid was added to stop the reaction. Absorbance at 450 nm was measured using a microplate reader. (3) Results The measurement results are shown in FIG.
  • the BCG-CWS of the present invention is highly reactive with anti-BCG killed bacteria antibodies, whereas the BCG-CWS of Patent Document 3 has very low reactivity with anti-BCG killed antibodies. When an anti-BCG antibody was used at 1000 ng / mL, the BCG-CWS of the present invention showed a binding reaction (OD450 value: 0.34).
  • BCG-CWS of the present invention showed a binding reaction.
  • BCG-CWS of Patent Document 3 did not show a binding reaction at any antibody addition amount (OD450 value: 0.01).
  • OD450 value: 0.01 the anti-BCG killed bacterial antibody.
  • the surface structure of BCG-CWS recognized by the antibody is greatly different between the case of BCG-CWS of the present invention and the case of BCG-CWS of Patent Document 3.
  • Test Example 7 Particle size distribution of BCG-CWS of the present invention and BCG-CWS of Patent Document 3
  • the particle size distributions of BCG-CWS of the present invention and BCG-CWS of Patent Document 3 were compared.
  • (1) Method The above-mentioned CWS is suspended in n-heptane using a potter type homogenizer using physiological saline or an ultrasonic generator, and the particle size is measured using a particle size distribution analyzer (SALD-2200, Shimadzu Corporation). Distribution was evaluated.
  • SALD-2200 particle size distribution analyzer
  • the BCG-CWS of the present invention showed a single particle size peak, and the BCG-CWS of Patent Document 3 showed two particle size peaks (Fig. 20 (b)).
  • the BCG-CWS of the present invention and the BCG-CWS of Patent Document 3 were greatly different in particle size distribution behavior in physiological saline and n-heptane.
  • Test Example 8 Morphological evaluation of CWS by auramine staining method
  • the morphological difference between BCG-CWS of the present invention and BCG-CWS of Patent Document 3 was compared by auramine staining method.
  • BCG-CWS: E (Sample name is based on Test Example 5)
  • BCG-CWS (B) of Patent Document 3 shows that the density of the mycolic acid portion is high and strongly aggregated or fused. Further, it was revealed that auramin staining was negative when the dispersibility in water (physical property value) was 0.4 or more. (Note 1: Observation with a field of view of 100 times magnification of a laser scanning confocal microscope (FV-1000, Olympus))
  • Test Example 9 Comparison of reactivity between BCG-CWS of the present invention and lectin (Concanavalin A) in water of Patent Document 3 (1) Evaluation sample BCG-CWS (A) of the present invention BCG-CWS (E) of Patent Document 3 (Use the sample of Test Example 5) (2) In the same manner as in Method Test Example 5, the BCG-CWS of (1) was suspended in physiological saline using a potter type homogenizer. 2 ⁇ L of rhodamine-labeled concanavalin A solution (Funakoshi) was added to 50 ⁇ L of each evaluation sample suspension, lightly pipetted, dropped in an appropriate amount onto a slide glass, air-dried, and observed with a confocal laser scanning microscope.
  • Test Example 10 Evaluation of zeta potential in suspension of BCG-CWS (1) Evaluation sample BCG-CWS (A) of the present invention BCG-CWS (E) of Patent Document 3 (Use the sample of Test Example 5) (2) Method 5 mL of isopropanol was added to 50 mg of BCG-CWS in (1) above, and the mixture was dispersed by ultrasonic irradiation for 5 minutes. Using a high-pressure crusher (BERYU-MINI, fine granules), the sample dispersion was crushed at 20,000 psi.
  • Test Example 11 Measurement of TNF- ⁇ production by BCG-CWS of the present invention Based on the morphological difference between BCG-CWS of the present invention and BCG-CWS of Patent Document 3, biological activity (of TNF- ⁇ Production) was compared.
  • (1) Preparation of evaluation sample To about 4 mg of BCG-CWS of the present invention and BCG-CWS of Patent Document 3, about 2 mL of 0.01 wt% polysorbate 80-containing physiological saline was added, and 3,000 rpm with a potter type homogenizer. Suspended for 5 minutes at room temperature.
  • the CWS content of each suspension was measured (similar to Test Example 2), adjusted to 10 ⁇ g CWS / mL with 10% FBS-containing DMEM medium, and used as an evaluation sample.
  • (3) Measurement Results As shown in FIG. 23, the BCG-CWS of the present invention showed about 2.8 times higher TNF- ⁇ inducing activity than the BCG-CWS of Patent Document 3.
  • Test Example 11 Evaluation of affinity of BCG-CWS for TLR2 and TLR4 Based on the morphological difference between BCG-CWS of the present invention and BCG-CWS of Patent Document 3 to biological activity (TLR2 and TLR4) The effects were compared.
  • An evaluation sample was prepared using BCG-CWS of the present invention or BCG-CWS of Patent Document 3 in the same manner as in Test Examples 4 (2) and (3).
  • lipopolysaccharide Standard LPS from E. coli O111: B4, INVIVOGEN
  • Measuring method It measured according to the method of Test Example 4 (2) and (3). Both BCG-CWS of the present invention and BCG-CWS of Patent Document 3 react with TLR2 as shown in FIG. 24-a), but did not react with TLR4 as shown in FIG. 24-b).
  • IL-12 measurement method a) Activity evaluation using a mouse immature dendritic cell line (BC-1 cell): This was carried out according to a known method (J Leukoc Biol. 2002 Jan; 71 (1): 125-32). BC-1 cells were seeded on a 96-well microplate at 5 ⁇ 10 4 cells / well. After 5 hours of incubation in MEM ⁇ medium containing 37%, 5% CO2, 20% FBS and adhering, an evaluation sample was added, and after culturing for 20 hours, the IL-12 concentration in the culture supernatant was measured by cytokine ELISA did. b) Activity evaluation using mouse dendritic cell line (JAWSII cell): It measured according to the method of Test Example 4 (4).
  • FIG. 14 (a) or (b) It has become clear that the present invention does not return to the BCG-CWS of the present invention. This indicates that the morphological change from FIG. 14 (a) or (b) to FIG. 14 (c) and the accompanying physical property change are irreversible in BCG-CWS.
  • Reference Example 4 Effect of Surfactant on Form and Physical Properties of BCG-CWS of the Present Invention
  • the method for producing BCG-CWS of the present invention is characterized by not using a surfactant.
  • a surfactant is used (Reference Example 2). Therefore, in order to clarify the influence of the surfactant on the form and physical properties of the BCG-CWS of the present invention, the BCG-CWS of the present invention is heated directly in the absence of the surfactant or the BCG-CWS powder as described below. Changes in physical properties were investigated by heating in water in the presence of a surfactant.
  • BCG-CWS is considered to form an ultra-thin film-like three-layer structure composed of mycolic acid-arabinogalactan-peptidoglycan (Microbiology Spectrum, 2 (3), FIG. 1-19 (2014) 6). Therefore, in water, it is considered that the peptidoglycan layer faces outward and the mycolic acids face each other (FIGS. 14 (b) and (c)) (J. Microbiol. Methods, 72 (2), 149-156 (2008)). It is considered that the hydrophobic interaction between mycolic acids as shown in FIG. 14B is a weak bond that is dissociated by a physical process such as a dispersion operation.
  • BCG-CWS was produced without the use of surfactants or high-temperature treatment, and was excellent in water dispersibility. For the first time, it has become possible to produce bacteria-CWS having physical properties suitable for the production of various emulsion and suspension formulations.
  • Example 7 Production of Emulsion Using BCG-CWS
  • BCG-CWS of Patent Document 3 The effect of the difference in form and physical properties between BCG-CWS of the present invention and BCG-CWS of Patent Document 3 on the emulsion was examined.
  • a known oil-in-water emulsion formulation and production method Proc. Japan Acad., 70, Ser. B, 205-209 (1994), Japanese Patent Application Laid-Open No. 2010-271322
  • the above two BCG-CWS are used in water.
  • An oil-type emulsion was produced.
  • BCG-CWS 360 mg about 30 mL of n-heptane / ethanol (9: 1, volume / volume) solution, squalene (manufactured by Kishimoto Special Liver Oil Industry) 5.4 g (15 times weight ratio to BCG-CWS), BHT (Tokyo Chemical Industry Co., Ltd.) 1.5 mg was added and stirred, and then dispersed at room temperature by ultrasonic irradiation. Then, it heated at 60 degreeC under dry nitrogen stream, and the organic solvent was distilled off.
  • Example 8 Production of freeze-dried preparation (1) Method Emulsification was carried out in the same manner as in Example 7 to obtain an oil-in-water emulsion emulsion.
  • An equal weight (53.5 g) of 6 mg / mL polysorbate 80/90 mg / mL mannitol / 20 mM citrate buffer (pH 7.0) aqueous solution is added to and mixed with the emulsion, and the final concentration of polysorbate 80 is 6 mg / mL.
  • a 45 mg / mL oil-in-water emulsion (diluted solution) was obtained.
  • the diluted solution is filled into vials for freeze-drying ( ⁇ 18.0 ⁇ 33.0 mm, CS, Fuji Glass) 1 mL at a time, frozen in a deep freezer at ⁇ 80 ° C., and then freeze-dried to obtain the freeze-dried preparation of the present invention. Obtained. Freeze drying was performed using a freeze dryer (GAMMA 2-16 LSCplus, manufactured by MARTIN CHRIST). The particle size distribution of the lyophilized redissolved solution was measured with a particle size distribution meter (SALD-2200, Shimadzu Corporation). A part of the redissolved solution was sampled, and the BCG-CWS content was measured according to Test Example 2.
  • Example 9 Production of emulsion and lyophilized preparation with different oil amount relative to BCG-CWS
  • emulsion production was carried out in the same manner as in Examples 7 and 8, except that the weight ratio of oil to BCG-CWS was changed.
  • the emulsifier was implemented on a small scale using an electric homogenizer as in Example 3.
  • the emulsification conditions at that time are as follows.
  • Test Example 12 Stability Evaluation of Emulsion (Dilution Solution) (1) Preparation of Evaluation Sample An emulsion dilution solution was prepared according to Example 7. However, the final amount (preparation weight) of squalene and PS80 in each emulsion preparation was made 15 times and 5 times the BCG-CWS preparation weight, respectively, and the citrate buffer was used as the buffer. . Emulsion diluent using BCG-CWS of the present invention Emulsion diluent using BCG-CWS of Patent Document 3
  • Example 10 Production of 0.1 mL, 0.2 mL Low Volume Filled Emulsion Lyophilized Formulation Emulsions prepared as in Example 7 were added to an equal weight (287.7 g) of 6 mg / mL polysorbate 80/90 mg / mL.
  • the emulsion diluted solution is filled into lyophilization vials ( ⁇ 18.0 ⁇ 33.0 mm, CS, Fuji Glass) in 0.1 mL or 0.2 mL portions using a piston pump (DIGISPENSE3009, manufactured by IVEK) at ⁇ 80 ° C.
  • a piston pump DIGISPENSE3009, manufactured by IVEK
  • MDF-U73V deep freezer
  • the product was freeze-dried with a freeze dryer (GAMMA 2-16 LSCplus, manufactured by MARTIN CHRIST) to obtain a freeze-dried preparation.
  • the BCG-CWS content in the vial is 108 ⁇ g CWS / vial and 203 ⁇ g CWS / vial, respectively, and the squalene content in the vial is 1. 58 mg squalene / vial, 3.02 mg squalene / vial. It was confirmed that the BCG-CWS emulsion of the present invention can produce a small-capacity oil-in-water emulsion freeze-dried preparation.
  • Test Example 13 Surface morphology of BCG-CWS and emulsion formulation of the present invention (confirmation of presence of arabinogalactan)
  • BCG-CWS of the present invention and its emulsion preparation it was confirmed by concanavalin A binding and agglutination reaction that arabinogalactan was exposed on the surface of CWS and emulsion oil particles (oil droplets).
  • Test Example 14 Evaluation of zeta potential of BCG-CWS emulsion of the present invention (1) Preparation of evaluation sample The following emulsion dilution liquid prepared according to Example 7 was used. However, the final amount (preparation weight) at the time of production of squalene and PS80 in each emulsion preparation was 15 times and 5 times, respectively, with respect to the CWS preparation weight.
  • ⁇ Emulsion dilution using BCG-CWS (A) of the present invention ⁇ Emulsion dilution using BCG-CWS (E) of Patent Document 3 ⁇ Vehicle emulsion dilution (2) Measuring method Zeta according to Test Example 10 The potential was measured.
  • Test Example 15 Correlation between Physical Property Value and Zeta Potential Difference Using BCG-CWS Oil-in-Water Emulsion with Different Physical Property Values (1) Preparation of Evaluation Sample The following BCG-CWS oil-in-water type prepared according to Example 7 An emulsion dilution was used. However, the amount of squalene and PS80 in each BCG-CWS oil-in-water emulsion was 15 times and 5 times the BCG-CWS charged weight, respectively. A citrate buffer (pH 7.0) was used.
  • Emulsion diluent prepared using BCG-CWS samples A, B, C, and D of the present invention ⁇ Emulsion diluent prepared using BCG-CWS sample E of Patent Document 3 ⁇ Vehicle emulsion diluent (2 ) Measuring method The zeta potential was measured according to Test Example 10. (3) The resulting zeta potential and the zeta potential obtained from various BCG-CWS emulsion dilutions were evaluated as the zeta potential difference calculated from the difference in zeta potential obtained from the vehicle emulsion dilution. The results are shown in Table 10.
  • the BCG-CWS emulsion dilution of the present invention clearly had a larger potential difference than the BCG-CWS emulsion dilution of Patent Document 3. That is, the potential difference of the BCG-CWS emulsion dilution of the present invention was 6-9 mV. On the other hand, in the emulsion diluent of Patent Document 3, the value was 1.0 or less.
  • Test Example 16 Correlation between oil amount and zeta potential difference with respect to BCG-CWS (1) Preparation of evaluation sample According to Example 7, the weight ratio of BCG-CWS to 7.5, 15, and 22.5 times squalene was used. The BCG-CWS oil-in-water emulsion dilutions prepared above and their vehicle emulsions were used. In each case, an amount of PS 80 of 1/3 weight of squalene and a citrate buffer (pH 7.0) were used. (2) Measuring method The zeta potential difference was measured in the same manner as in Test Example 10. (3) The results are shown in Table 11. The zeta potential difference decreased in correlation with the increase in oil amount.
  • Test Example 17 Confirmation of co-localization of BCG-CWS and oil in the preparation The following evaluation was performed in order to confirm the presence state of BCG-CWS in the preparation.
  • (2) Results The fluorescence of BCG-CWS and squalene colocalized, and it was confirmed that BCG-CWS was present in the oil droplets of the emulsion emulsion.
  • Test Example 18 Comparative verification of effects of various antioxidants Antioxidants were investigated for the purpose of improving the stability of freeze-dried BCG-CWS oil-in-water emulsions.
  • An emulsion freeze-dried preparation was prepared according to Example 8. However, the final amount (prepared weight) of squalene and PS80 in each emulsion preparation was made to be 6 times or 2 times as the BCG-CWS charged weight ratio, respectively. After re-dissolving the lyophilized preparation, a solution containing 10 ppm of each antioxidant (A to F) was used.
  • B DL- ⁇ -tocopherol (VE) C: Tocopherol acetate (V.E E) D: Ascorbic acid (VC) E: 6-O-stearoyl-L-ascorbic acid (VC) F: Dibutylhydroxytoluene (BHT) (2) Evaluation method Each evaluation sample was stored at 60 ° C. under static conditions. At the start of the test, after 2 days, after 4 days and after 8 days, pH, squalene content, and olein as an index of PS80 degradation The acid content was measured.
  • Test Example 19 Comparative verification of the effect of the buffering agent The buffering agent was examined for the purpose of improving the stability of the BCG-CWS oil-in-water emulsion. In order to compare the effect on the preparation of the present invention, the pH retention ability of various buffers was compared in the presence of PS80.
  • PS80 Preparation of evaluation sample PS80 was added to the following various buffers so that it might become 6 mg / mL, and PS80 solution was obtained.
  • B Phosphate buffer (pH 7.0)
  • C Citrate buffer (pH 7.0)
  • Evaluation Method Each evaluation sample was allowed to stand at 40 ° C. or 60 ° C., and the pH from the start of the test to about 60 and 150 days later was measured.
  • Test Example 20 In Vitro Bioactivity Evaluation by Emulsion Using BCG-CWS of the Present Invention (1) Preparation of Evaluation Sample An emulsion manufactured according to Example 7 was used. However, a BCG-CWS weight ratio of 15 times the amount of squalene, 5 times the amount of PS80, and citrate buffer (pH 7.0) were used.
  • b) Evaluation of IL-12 activity using BC-1 cells It implemented similarly to Experiment 11 (3).
  • c) Evaluation of reporter activity of hTLR2 using HEK-Blue-hTLR2 cells It measured according to the method of Test Example 4 (2).
  • TNF- ⁇ activity evaluation As shown in FIG. 5, the emulsion preparation using BCG-CWS of the present invention had about 16 times higher TNF- ⁇ -inducing activity than the emulsion preparation using BCG-CWS of Patent Document 3.
  • IL-12 activity assessment As shown in FIG. 6, the emulsion preparation using the BCG-CWS of the present invention had an IL-12 induction activity about 6 times higher than the emulsion preparation using the BCG-CWS of Patent Document 3.
  • Evaluation of TLR2 reporter activity As shown in FIG. 7, the emulsion formulation using the BCG-CWS of the present invention had about 22 times higher hTLR-2 reporter activity than the emulsion formulation using BCG-CWS of Patent Document 3.
  • Test Example 21 Correlation between oil amount of BCG-CWS oil paste and in vitro activity As part of observing the correlation between the in vitro activity of BCG-CWS oil-in-water emulsion and the amount of oil used, an intermediate oil paste was used. The correlation between the in vitro activity and the oil amount was examined.
  • Test Example 22 Comparison of TNF- ⁇ production during stimulation of Raw 264.7 cells with oil-in-water emulsion of BCG-CWS having dispersibility (physical property values) in various waters (1)
  • Various BCG-CWS samples in Test Example 5 Was used to prepare an oil-in-water emulsion according to Example 7. However, 15 times the amount of squalene, 5 times the amount of PS80 and citrate buffer (pH 7.0) were used with respect to the BCG-CWS weight ratio used.
  • Results As shown in FIG.
  • Test Example 23 In vivo behavior of BCG-CWS during intradermal administration of pharmaceutical preparation CWS behavior evaluation experiment after known intraguinea pig intradermal administration (Drug Discov. Ther., 2 (3), 168-177 (2008), Drug Discov Ther., 2 (3), 178-187 (2008)), a fluorescent BCG-CWS oil-in-water emulsion produced according to Test Example 17 was prepared, and the lymph node was measured using the fluorescence intensity as an index. A method for evaluating the transferability of the BCG-CWS emulsion of the present invention and the BCG-CWS emulsion of Patent Document 3 were compared using the method.
  • Test Example 24 Comparative known literature (Cancer Sci., 99 (7), 1435-1440 (2008))
  • the IFN ⁇ production-inducing activity in blood upon intraperitoneal administration of mice was evaluated.
  • (1) Evaluation Sample An oil-in-water emulsion containing the BCG-CWS of the present invention or the BCG-CWS of Patent Document 3 was produced according to Example 7.
  • oil paste was prepared using 4 mg of BCG-CWS, 27 times the weight of BCG-CWS squalane, and PS80 of 18 times the weight of BCG-CWS, and 4.5 mL of 45 mg / mL D ( -) Mannitol / 8 mM phosphate buffer (pH 7.4) was added and emulsified with a potter homogenizer at 1,200 rpm, 60 ° C. for 12 minutes. Thereafter, it was diluted with 45 mg / mL D ( ⁇ ) mannitol / 8 mM phosphoric acid to prepare an emulsion for administration of 1 mg CWS / mL.
  • the vehicle emulsion was prepared in the same manner as described above without using BCG-CWS.
  • Test Example 25 E.I. Evaluation of Antitumor Activity of G7-OVA Transplanted Tumor Mice
  • G7 cell line (mouse lymphoma-derived cell line) is inoculated intradermally at 1 ⁇ 10 ⁇ 6 cells / 50 ⁇ L, and then the emulsion for each evaluation becomes 50 ⁇ g CWS / 50 ⁇ L / mouse on days 1, 4, 7 and 14 respectively.
  • autopsy was performed and the tumor weight was weighed.
  • Example 26 Evaluation of Antitumor Activity of MethA Cell Transplanted Mice
  • an oil-in-water emulsion containing BCG-CWS of the present invention or BCG-CWS of Patent Document 3 was produced. Specifically, an oil paste was prepared from 12 mg of BCG-CWS and squalane of 27 times the weight of BCG-CWS, and about 4 mL of 8 mM phosphate buffer containing PS80 of 18 times the weight of BCG-CWS. Was added and emulsified with an electric homogenizer to obtain an emulsion.
  • the MethA cell line was washed 3 times with phosphate buffered saline and resuspended to 2 ⁇ 10 ⁇ 6 cells / mL.
  • b) Cell transplantation and emulsion administration BALB / c mice (female, 6 weeks old, Charles River) acclimatized for about one week
  • (3) Results The results obtained are shown in Table 13.
  • the BCG-CWS emulsion of the present invention strongly inhibited tumor survival compared to the vehicle emulsion.
  • the oil-in-water emulsion preparation and lyophilized preparation containing the bacterial CWS of the present invention are novel preparations in which arabinogalactan is exposed on the surface of the oil particles, and thus are excellent in reactivity with lectins and storage stability. Furthermore, it shows excellent activity against immune cells. Therefore, the emulsion preparation of the present invention and its lyophilized preparation can be used as pharmaceuticals for immunomodulators such as cancer immunotherapeutic agents, hay fever and asthma.
  • the bacterium CWS of the present invention uses a purified microbial cell from which the extracellular adhesion component of the bacterium has been removed and the microbial cell surface is exposed to a part of the arabinogalactan, and the use of a surfactant or high temperature It is CWS produced by performing the crushing process and the refinement
  • the purified bacterial cell of the present invention has a high degree of purification because the extracellular adhesion components of the bacteria are removed and the mycolic acid portion and a part of the arabinogalactan are exposed on the cell surface. ing. Therefore, using bacteria containing mycolic acid, sugar chain, and peptidoglycan such as BCG bacteria, bacterial CWS such as mycolic acid and BCG-CWS can be produced with high purity and high yield by a simpler production process. It was.

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Abstract

Dans le cadre de la présente invention, un squelette à paroi cellulaire bactérienne ayant une fraction d'acide mycolique et certains arabinogalactanes exposés sur la surface a été préparé, moyennant quoi, lorsque le squelette à paroi cellulaire bactérienne préparé a été utilisé pour préparer une émulsion huile dans eau, il était possible de préparer une nouvelle émulsion huile dans eau ayant l'arabinogalactane du squelette à paroi cellulaire bactérienne faisant saillie et exposée sur la surface de particules d'huile et une préparation lyophilisée de l'émulsion huile dans eau. Cette préparation d'émulsion présente une activité immunostimulatrice supérieure et une stabilité exceptionnelle. Par conséquent, de nouveaux agents immunothérapeutiques pour le cancer peuvent être préparés à l'aide de cette préparation d'émulsion.
PCT/JP2017/046780 2016-12-26 2017-12-26 Préparation d'émulsion huile dans eau contenant un composant de squelette à paroi cellulaire bactérienne Ceased WO2018124132A1 (fr)

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JPWO2021141025A1 (fr) * 2020-01-10 2021-07-15
CN116407523A (zh) * 2023-03-01 2023-07-11 福建省微生物研究所 一种含有红卡壁骨架的载药膜制备方法及其产品和应用

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JP2008214266A (ja) * 2007-03-05 2008-09-18 Dainippon Sumitomo Pharma Co Ltd 細菌細胞壁骨格成分の製造方法
JP2009055812A (ja) * 2007-08-30 2009-03-19 Tosoh Corp 試料からの抗酸菌の分離回収方法
WO2012157479A1 (fr) * 2011-05-13 2012-11-22 国立大学法人 東京大学 Ctrp6 qui peut être utilisée à titre d'agent thérapeutique et prophylactique pour les maladies auto-immunes
WO2014034669A1 (fr) * 2012-08-28 2014-03-06 国立大学法人北海道大学 Structure membranaire lipidique comprenant un composant de cellule bactérienne ayant une dispersibilité dans un solvant non polaire, et son procédé de fabrication
JP2014512363A (ja) * 2011-04-08 2014-05-22 ユニバーシティー オブ レスター Ｍａｓｐ−２依存性補体活性化に関連した状態を治療するための方法

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WO2005102369A1 (fr) * 2004-04-22 2005-11-03 Dainippon Sumitomo Pharma Co., Ltd. Préparation pharmaceutique contenant un composant du squelette de la paroi de cellules bactériennes
JP2008214266A (ja) * 2007-03-05 2008-09-18 Dainippon Sumitomo Pharma Co Ltd 細菌細胞壁骨格成分の製造方法
JP2009055812A (ja) * 2007-08-30 2009-03-19 Tosoh Corp 試料からの抗酸菌の分離回収方法
JP2014512363A (ja) * 2011-04-08 2014-05-22 ユニバーシティー オブ レスター Ｍａｓｐ−２依存性補体活性化に関連した状態を治療するための方法
WO2012157479A1 (fr) * 2011-05-13 2012-11-22 国立大学法人 東京大学 Ctrp6 qui peut être utilisée à titre d'agent thérapeutique et prophylactique pour les maladies auto-immunes
WO2014034669A1 (fr) * 2012-08-28 2014-03-06 国立大学法人北海道大学 Structure membranaire lipidique comprenant un composant de cellule bactérienne ayant une dispersibilité dans un solvant non polaire, et son procédé de fabrication

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Publication number Priority date Publication date Assignee Title
JPWO2021141025A1 (fr) * 2020-01-10 2021-07-15
JP7691937B2 (ja) 2020-01-10 2025-06-12 株式会社ヤクルト本社 乳酸菌細胞壁破砕物及び乳酸菌細胞壁破砕物の製造方法
CN116407523A (zh) * 2023-03-01 2023-07-11 福建省微生物研究所 一种含有红卡壁骨架的载药膜制备方法及其产品和应用

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