JPH0556326B2 - - Google Patents

Abstract

A composition which is capable of producing an effective adjuvant response or stimulating the immune response of a warm blooded animal which comprises an effective amount of a refined detoxified endotoxin material, in combination with a pharmaceutically acceptable carrier.

Description

[Detailed description of the invention]

The present invention relates to the use of refined detoxified endotoxin (RDE) products as potent immune stimulants and as adjuvants. Used in the present invention
RDE is characterized as having no detectable 2-keto-3-deoxyoctonic acid and containing about 350-475 nmol/mg of phosphorus and about 1700-2000 nmol/mg of fatty acids. Endotoxin extracts obtained from Enterobacteriaceae, including the parent organism and mutants, are known. These extracts are
It has been used for immunotherapy of various immunogenic tumors [Ribi et al. "Cancer Immunol. Immunother." No. 7]
vol. 43-58 (1979).
Requirement for Immunotherapy of the
Guinea−Pig Line−10 Tumor with
[See ``Endotoxin'']. However, endotoxin extracts are known to be highly toxic and therefore
It has limited use in the treatment of cancerous tumors. Efforts have been made to detoxify endotoxin while preserving its tumor regression potential. As shown in Ribi et al., supra, known chemical means to detoxify endotoxins while retaining adjuvant activity, such as succinylation and phthalylation, result in loss of endotoxin toxicity and loss of tumor regression potential. brought about both. Therefore, prior art attempts to obtain purified and detoxified endotoxin have been far from successful. Endotoxin extracts of the type used as starting material for producing the RDE used in the present invention can be obtained from any Enterobacteriaceae, including the parent organism and mutants. The following genera are intended to illustrate and illustrate the types of microorganisms that can be used. Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomonas, Pasturella, Neisseria, Proteus ,
Klebsiella and Serratia. The following species are typically used. S. minnesota, S. typhimurium, B. pertussis,
B. abortus, S. enteritidis, E. coli, S. typhi, S. mareescens, S. typhosa, Shigella flexni
and S. abortus equi. The endotoxin extract used as starting material can be prepared by one of several known methods. For example, MEWebster, JFSagin, M.
Landy, and AG Johnson, J. Immunol.1955,
744, 55; O. Westphal, O. Luderitz and F.
Bister, Z. Naturforsch, 76, 148 (1952); O.
Westphal, Pyrogens Polysaccharides in
Biology，Tr.Second Macy Conference
(edited by George F. Springer) New Jersey
Madison Printing Co., Madison, 1957, 115; C.
Galanos, O. Luderitz, O. Westphal, Eur.J.
Biochem.9, 245 (1969); CHChen, AG
Johnson, N. Kasai, BAKey, J. Levin, A.
Nowotny, J. Infect. Dis. 128 543 (1973); E. Ribi,
W T Haskins, M. Landy, KCMilner, The
Journal of Experimental Medicine114, 647
(1961); L.Leive, Biochem.Biophys.Res.
Comm.21, 290 (1965); and E.Ribi, KC.
Milner and T. Perrine, J. Immunol. 82, 75 (1959)
Please refer to The most suitable way to obtain endotoxin extract is by Chen
et al., methanol-chloroform precipitation. Methanol chloroform precipitate (MCP:
methanolchloroform precipitate) with organic or inorganic acids, followed by lyophilization to produce hydrolyzed crude lipid A with reduced toxicity and pyrogenicity compared to the endotoxin starting material. do. The product obtained is then treated with a solvent that is particularly capable of dissolving fatty acids and other impurities without dissolving the crude lipid A described above. A suitable solvent for this purpose is acetone.
The phosphate content of detoxified and purified lipid A is approximately 1/2 of that observed for the toxic equivalent suggesting that the phosphate content is related to the toxic effects of endotoxin. Suitable inorganic acids used to react with MCP are hydrochloric acid, sulfuric acid or phosphoric acid; suitable organic acids are toluenesulfonic acid or trichloroacetic acid.
The reaction may suitably be carried out at a temperature of about 90 DEG to 130 DEG C. for a time sufficient to achieve complete hydrolysis, usually about 15 to 60 minutes. Preparation of detoxified crude endotoxin may also be carried out by reacting the starting material with the selected acid in the presence of organic solvents such as chloroform, methanol and ethanol or combinations thereof. I can do it. The crude lipid A obtained is dissolved in acetone, which is particularly suitable for removing fatty acid components. The solvent is then removed to produce crude detoxified endotoxin. The crude detoxified endotoxin is then dissolved in a solvent and passed through a suitable chromatography column, such as a molecular exclusion (or exclusion) chromatography column, to separate the RDE fraction.
This is then combined after removal of the solvent. In one embodiment, the crude detoxified endotoxin solution is passed through a Sephadex column in the presence of a solvent such as chloroform, methanol, acetone, pyridine, ether or acetic acid or a combination thereof. Column pressure can vary, but typically ranges from about atmospheric pressure to about
The pressure is 6.8 atmospheres (100 b/in ² ) and the flow rate is about 0.1-10 ml/min. Alternatively, the crude detoxified endotoxin solution is passed through a DEAE-cellulose column under the same pressure conditions as described for the Sephadex column above. The flow rate can be maintained at about 2-15 ml/min. The solvent used is also the same as that used for the sesadex column, but water and/or diethylamine can be added to the total mixture at a concentration of up to about 1%. Another method of producing RDE from crude detoxified endotoxin is to pass the solution through a low pressure silica gel 60 column with a particle size of about 15-63μ and a suitable solvent such as chloroform, methanol, water or water. Including the use of ammonium oxide. The preferred volume ratio of the solvent mixture is about 50:25:4:2. It is therefore an object of the present invention to use purified and detoxified endotoxin products that can be used effectively to stimulate the immune system of warm-blooded animals. Specifically, using RDE as a B-cell mitogen (or mitogen),
Lymphokine production can be stimulated, macrophages can be stimulated, and RDE can be used as an adjuvant to enhance the immune response of warm-blooded animals. The present invention relates to the use of purified and detoxified endotoxin (RDE) as an immune system stimulator. The RDE used in the present invention can be detected by
- free of keto-3-deoxyoctonic acid, about 350-475 nmol/mg of phosphorus and about 1700-2000 n of fatty acids
It has mol/mg. The RDEs of the invention are used as stimulators of the immune response of warm-blooded animals against antigens such as bacterial and fungal cells, bacterial cell fragments, viruses, or viral subunit synthetic peptides that resemble cellular and viral subunits. be able to. Specific antigens affected by the increased immune response upon RDE administration include cryptococcus neoformans candida spp., chlamydia spp., legionella spp., clostridia spp. ,
Hepatitis meningitis
meningitis), streptococcus spp.
Staphylococcus species, Klebsiella species, herpes virus,
brucella spp., borditella spp., salmonella spp.
Shigella species, campylobacter
seeds, yersinia spp., pasturella spp., francisella
species, Listeria species, etc. The RDE used in the present invention exhibits B cell mitogenic activity. That is, RDE activates B lymphocytes, the cells that respond to antibody production, when administered to warm-blooded animals in an appropriate dosage regimen. Production Example 1 Preparation of Crude Detoxified Endotoxin In a three-necked round-bottomed flask equipped with a condenser, Chen
A sample of methanol-chloroform precipitate prepared according to the method of J. Infect. Dis. 128, 543 (1973)
650 mg were suspended in 150 ml of 0.1N HCl and immersed in a sonicator. After the sonication treatment, the glass apparatus was lowered into an oil bath maintained at 120° C. so that the internal temperature of the flask approached or exceeded the boiling point of the solution. Overheating of the solution was minimized by equipping the flask with a capillary tube coupled to a nitrogen gas source through one of the necks. A continuous flow of nitrogen was maintained during the hydrolysis operation. Hydrolysis was continued for 30 minutes, after which the solution was cooled in an ice bath and sonicated to disperse the solid material into a collection tube. The flask was rinsed with distilled water to remove any solid material stuck to the sides of the flask, and the wash was added to the suspension in the collection tube. Centrifugation was performed at 12000 rpm for 80 minutes.
The supernatant was decanted and discarded. The solid residue was resuspended in distilled water, sonicated until the suspension was well dispersed, and centrifuged again. The centrifugation step was repeated. The residue is taken up in distilled water, shell-frozen, and lyophilized to produce purified lipids.
382mg of A was obtained. 150 mg of this material was treated with cold acetone (0°C) to remove fatty acids, sonicated, and passed through a Whatman No. 1 gravity filtration device for 50 minutes.
Filtered at °C. Crude detoxified endotoxin when dried
100mg remained. Production example 2 Preparation of crude detoxified endotoxin MCP (methanol-chloroform precipitate) sample
120 mg was suspended in 12 ml of absolute methanol and sonicated to disperse the solid material.
cm) in a bottle with a screw cap. in each tube
2 ml of 0.2N HCl were added and the resulting suspension was incubated in a boiling water bath for 45 minutes. After hydrolysis, each tube was cooled in an ice water bath for approximately 10 minutes.
Centrifuged at 2500 rpm. The supernatant was decanted and the residue was diluted with chloroform/methanol (2:
1) Add and dissolve 5 ml of the mixture. 2 water in each tube
ml and mixed the solution. Two-phase solution at 2500 rpm
Centrifuged again for 10 minutes. The upper aqueous phase was decanted and 1 ml of a chloroform/methanol (4:1) mixture was added to each tube to obtain a clear solution.
The solutions were combined and the solvent was evaporated on a rotary evaporator. The residue was dried under high vacuum and lyophilized to yield 45 mg of crude lipid A. 20 mg of this material was treated with cold acetone (0°C), sonicated,
Filtered through a Whatman No. 1 gravity filter at 5°C. Upon drying, 13 mg of crude detoxified endotoxin remained. Production example 3 Preparation of purified detoxified endotoxin LH-20-100 (particle size 25-100 μm: Pharmacia)
110 g and 600 ml of chloroform/methanol (2:1) mixture were combined and allowed to stand for 30 minutes. The resulting slurry was added to a 25 x 1000 mm glass chromatography column (BRL Laboratories) equipped with a pressure fitting. After filling, the column was attached to an ISCO Model 132 pump through Teflon pressure tubing. 400 ml of a chloroform/methanol (4:1) mixture was pumped through the column at a rate of 3 ml/min. 100 mg of the crude detoxified endotoxin prepared according to Production Example 1 was dissolved in chloroform/
The sample loop was applied to the column in 2.5 ml of methanol (4:1) mixture. After reducing the flow to 1 ml/min and collecting 150 ml of eluate, the eluate was frozen in a fraction collector. 4 ml fractions were collected and purified detoxified endotoxin fractions were determined by thin layer chromatography analysis of each fraction [E. Merck, 0.25 mm thickness, eluent: chloroform/methanol/H ₂ O/NH ₄ OH (50:
25:4:2)]. Purified detoxified endotoxin fractions are combined;
Upon removal of the solvent, 30 mg of purified detoxified endotoxin remained as a white powder. Production example 4 Preparation of purified detoxified endotoxin DEAE-cellulose (Whatman DE-32) 33
g was suspended in 150 ml of glacial acetic acid and gently stirred for 10 minutes to obtain a slurry powder. The mixture was left overnight. The slurry was poured into a 25 x 400 mm column and settled by tapping, followed by discarding the excess acid.
The column was washed with 2000 ml of methanol and 200 ml of a chloroform/methanol (4:1) mixture. 100 mg of the crude detoxified endotoxin sample prepared in Production Example 1 was added to chloroform/methanol (4:1).
The mixture or chloroform/methanol/water (80:20:1) mixture was added to the column in 3 ml.
The column was eluted with 350 ml of a chloroform/methanol (4:1) mixture followed by 300 ml of a methanol/water (99:1) mixture. Using a linear gradient apparatus, the column was eluted with a 2000 ml linear gradient starting with 100% methanol and ending with 0.2M acetic acid in methanol. The column was eluted at a rate of 6 ml/min and 15 ml fractions were collected. The analysis of the total phosphorus content of every other fraction was
Bartlett, GRJBiol.Chem.234, 466−471.
(1959). The fractions were combined and distilled to near dryness on a rotary evaporator with 10 ml of a chloroform/methanol (2:1) mixture and 40 ml of 0.001M acetic acid in a separating funnel.
I took it inside. Separate the lower phase and use Whatman No.2
After filtration through filter paper and evaporation to dryness, 19.2 mg of purified detoxified endotoxin was obtained. Assays (1) B cell mitogenic activity - RDEs are characterized by their ability to activate B lymphocytes, cells capable of responding to antibody production, as follows. Mitogenic activity of purified and detoxified endotoxin

[Table] [Experimental observation record: 1 × 10 ⁶ cells/ml of each strain were cultured in 0.2 mg of RPM1-1640 (5% FCS) in 96 well microtiter plates with or without mitogen. did. During the last 18 hours of the 48-hour incubation, add 1 µl of ^3H -thymidine to each well and add ^3H -thymidine to each well.
Uptake was measured by standard scintillation method. ^* CPM - counts per minute
minute) ^** SI - stimulation index〕 (2) Interleukin - production;
RDE can be used to stimulate lymphokine production released by macrophages. Interleukins are soluble immune regulators released by macrophages that respond to the activation of lymphocytes at the site of infection. Mouse macrophages were attached to microtiter plates (1×10 ⁶ /well), and each measurement was performed in triplicate. The first well received 1 ml of a 50 μg/ml solution of standard endotoxin. A second set of wells contained 50 μg/ml of RDE according to the invention.
ml solution was added. Contains 1 ml of 5% FCS (fetal calf serum) for each drug.
Dissolved in M199 medium. M199 with 5% FCS
Media was added to a third set of wells to serve as a control. The plates were incubated at 37°C for 20 hours. The upper layer liquid was taken out and diluted to obtain a solution with a ratio of upper layer liquid to distilled water of 1:10. Regarding this upper layer liquid, Cellular Immun.64 of Gery et al.
293 (1981), interleukin production was tested. Cells remaining after removing the supernatant were lysed using 0.1% Triton X-100.
did. The resulting solution was diluted to 5% with a dilution ratio of 1:10.
Diluted with RPMI1640 medium containing FCS. For diluted solutions, the increase in ^3H -thymidine uptake of PHA-induced macrophages was
Interleukin production was tested by measuring as described in Gery et al. The results are shown in the table.

[Table] As shown in the table, interleukin from the RDE of the present invention in lysed cells.
Production significantly exceeded interleukin production from standard endotoxin extracts. A similar analysis was performed using human monocytes instead of mouse macrophages. The results are as follows.

(3) Macrophage Activation The PDEs of the present invention also stimulate macrophages, as measured by their ability to eat (engulf) fluorescent beads. Three test solutions each were prepared by mixing 1×10 ⁶ cells of peritoneal exudate with 5 μ of test material solution containing standard endotoxin or RDE according to the invention or control saline. Each solution contains 1μg of test material
and 20μ of a fluorescent bead suspension. Test solutions were placed on individual microscope slides and incubated for 90 minutes at 37°C. After incubation, the slides were observed under a fluorescence microscope. The number of cells that ate the fluorescent beads was determined by visual observation, along with the number of beads/cell. The phagocytosis coefficient was calculated using the following formula. Phagocytosis coefficient=
% Phagocytic Cells x Number of Beads/100 Cells/1000 The results of two experiments are shown in the table below. Table: Phagocytosis coefficient ^* Experiment 1 Experiment 2 Standard endotoxin 5.0 3.6 RDE 5.4 4.1 Control 1.0 0.5 As is clear from the table, the phagocytosis coefficient for RDE of the present invention was significantly larger than that for the standard endotoxin. This demonstrated that RDE is a very effective stimulator of macrophages. (4) Adjuvant Activity To further substantiate the use of RDE as a stimulator of the immune system of warm-blooded animals, we determined the ability of the antibody to lyse sheep red blood cells (SRBC). The adjuvant activity was tested as determined by the ability of RDE to increase the immune response to. Three test materials were prepared. Each material contained an amount of 1×10 ⁷ SRBC. Test material No. 2 further contained SRBC plus 20 mg of standard endotoxin, and test material No. 3 contained SRBC plus 20 mcg of RDE according to the invention. Test materials were injected intraperitoneally into BALB/C strain mice. After 4-5 days, the test animals were sacrificed and the spleens were removed and crushed in a tissue grinder. Standard cell suspensions for each spleen were prepared using a hemocytometer and each suspension was placed on a slide with target SRBC, media and agar. The slides were incubated for 2 hours at 37°C, and guinea pig serum was added to each slide as a source of complement and incubated for 30 minutes at 37°C. The slides are then examined and antibodies are developed with the help of complement found in guinea pig serum.
The amount of plaque-forming cells, which are areas where SRBC cells were destroyed, was measured. The results are shown in the table. Table Test Materials PFC/2 x 10 ⁵ Spleen Cells SRBC 88 SRBC + Standard Endotoxin 165 SRBC + RDE 219 As it is clear from the table, the number of plaque-forming cells obtained using RDE is significantly higher than with standard endotoxin. This proves that RDE is an effective adjuvant. The RDE used in the invention can be administered in combination with a pharmaceutically acceptable vehicle such as saline or an oil droplet emutsion. The composition can be stabilized, for example by a freeze-drying operation, and then reconstituted without loss of efficacy. As mentioned above, compositions for the treatment of warm-blooded animals and humans can be used in the form of oil drop emulsions. The amount of oil used is approximately based on the total volume of the composition.
It ranges from 0.5 to 3.0% by volume. Approximately 0.75-1.5% by volume
It is preferable to use this oil. Examples of suitable oils include light mineral oil, squalene, 7-n-hexyl octadecane, Conoco Super Oil, and Drakeol 6VR Mineral Oil (Pennreco, Bulter, Pennsylvania).
is included. The homogeneous oil-containing mixture is then combined with a cleaning agent, which may optionally be dissolved in a saline solution prior to mixing. The amount of detergent is typically about 0.02-0.20% by volume, preferably about 0.10-0.20% by volume, based on the total volume of the composition. Any conventional cleaning material can be used, including Tween-80 and Arlacel
[Manufactured by Atlas Chemical] is included. The mixture obtained by adding the detergent is then homogenized to form a suspension with a high percentage of oil droplets coated with RDE (determined by observation under a microscope). The amount of RDE in one injection is between 5 and 500 μg, preferably between 10 and 100 μg for a total human adult body weight of 70 Kg. One to three infusions are administered over a period of about two months. The RDE compositions used in the present invention exhibit significantly lower pyrogenic activity than compositions containing standard endotoxins, as evidenced by the examples below. Three New Zealand rabbits were injected via the ear vein with a composition containing a standard endotoxin. The amount of standard endotoxin required to produce a body temperature increase of at least 0.46°C in 50% of the test subjects was measured over a 3 hour period using a rectal thermometer. Three other test rabbits were also injected with the RDE-containing composition and subjected to similar pyrogenic activity tests. The results are shown in the table below.

[Table] Dosage required to produce a greater febrile response As shown in the table, the standard endotoxin is 0.012μg/Kg
produced a febrile response in 50% of the test animals.
However, RDE did not cause an exothermic response even at concentrations exceeding 10 μg/Kg. This value is 850 times higher than the standard bacterial endotoxin dosage level.

Claims (1)

[Claims] 1. A method for producing an immune response enhancer, comprising: (1) hydrolyzing an endotoxin extract derived from Enterobacteriaceae bacteria with an acid, and drying the hydrolyzate to obtain crude lipid A; (2) obtaining an insoluble product by treating the crude lipid A with a solvent capable of dissolving fatty acids, (3) obtaining a solution by dissolving the insoluble product in another solvent, ( 4) obtaining a purified endotoxin-containing fraction having immunoenhancing activity by treating the solution with column chromatography; and (5) mixing the obtained purified endotoxin with a pharmaceutically acceptable excipient. , a method characterized by: 2. Claim 1 produces a unit dosage form of immunostimulant containing 5 to 500 μg of purified endotoxin.
The method described in section. 3. Claim 2 produces a unit dosage form of immunostimulant containing 10-100 μg of purified endotoxin.
The method described in section. 4. The method according to claim 1, for producing a lyophilized immunopotentiator. 5. The method according to claim 1, for producing an immunostimulant in the form of an oil extract.