CH660125A5 - CLEANED, DETOXICATED ENDOTOXIN COMPOSITION. - Google Patents

Description

The present invention relates to a composition containing purified, detoxified endotoxin. This purified, detoxified endotoxin is a powerful immunostimulator for warm-blooded animals and can also have an adjuvant effect. The endotoxin has no detectable 2-keto-3-deoxyoctanoate between 350 and 475 n mol / mg phosphorus and between 1700 and 2000 n mol / mg fatty acids.

The endotoxin-containing extracts can be obtained from duck robacteriaciae, and Enterobacteriaciae includes both known parent organisms and mutants thereof. These extracts have previously been used for the immunotherapy of various immunogenic tumors (see “Peptides as Requirement for Immunotherapy of the Guinea-Pig Line 10 Tumor with Endotoxins; Ribi, et al Cancer Immunol. Immunother., Volume 7, pages 43-58 (1979) However, efforts have been made to "detoxify" endotoxins while maintaining the capacity of the endotoxins to regress to tumors.

As described by Ribi et al in Cancer Immunol. Immunother. endotoxins, when subjected to known chemical processes such as the formation of salts of succinic acid or the production of phthalates, lost their endotoxicity and ability to regress ulcers while maintaining their effectiveness as adjuvants. Therefore, the known methods for producing detoxified endotoxins have so far been unsuccessful.

An endotoxin extract of the type which can be used as a starting material for the production of the purified, detoxified endotoxin which is contained in the composition according to the invention is usually obtained from Enterobacteriaciae, including the parent organisms and the corresponding mutants. The genera of microorganisms listed below can, for. B. for the production of the purified, detoxified endotoxin: Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomonas, Pastorella, Neisseria, Proteus, Klebsiella and Serratia.

The following types are typically used: S. minnesota, S. typhimurium, B. pertussis, B. abortus, S. enteritidis, E. coli, S. typhi, S. marcescens, S. typhosa, Shigella flexni and S. abortus equi .

The endotoxin extracts used as starting materials can be prepared by known methods, e.g. following the procedures of M.E. Webster, J.F. Sagin, M. Landy and A.G. Johnson, J. Immunol. 1955,744, p. 55; O. Westphal, O. Luderitz and F. Bister, Z. Naturforsch, 76 148 (1952); O. Westphal, Pyrogens, Polysaccharides in Biology, Tr. Second Macy Conference (George F. Springer, ed.), N.J. Madison, Madison Printing Co., 1957, 115; C. Ga-lanos, O. Luderitz, O. Westphal, Eur. J. Biochem. 9.245 io (1969); C.H. Chen, A.G. Johnson, N. Kasai, B.A. Key, J. Levin, A. Nowotny, J. Infect. Dis. 128,543 (1973); E. Ribi, W.T. Haskins, M. Landy, K.C. Milner, The Journal of Experimental Medicine 114,647 (1961); L. Leive, Biochem. Bio-phys. Res. Comm. 21,290 (1965); and E. Ribi, K.C. Milner i5 and T. Perrine, J. Immunol. 82 75 (1959).

A very suitable method for the production of the endotoxin extract was disclosed by Chen et al, namely the methanol-chloroform precipitation.

The methanol-chloroform precipitate is usually reacted with an organic or inorganic acid and then lyophilized to give the hydrolyzed crude lipid A, which has reduced toxicity and pyrogenicity compared to the endotoxin starting material used. The product 25 obtained can then be treated with a solvent which has the ability to dissolve in particular fatty acids and other impurities without dissolving the crude lipid A. A suitable solvent for this purpose is acetone. The phosphate content of the detoxified, purified Li-30 pid A is half of the content observed for the toxic equivalent, assuming that the phosphate content is related to the toxic effects of the endotoxins.

Suitable organic acids which can be used for the reaction with the methanol-chloroform precipitate are hydrochloric acid, sulfuric acid or phosphoric acid and suitable organic acids are toluenesulfonic acid or trichloroacetic acid. The reaction may suitably be carried out at temperatures between about 90 ° 40 and 130 °, typically allowing to react until complete hydrolysis has taken place, usually for a period of about 15 to 60 minutes.

The preparation of the detoxified crude endotoxin can also be carried out by mixing the starting material with the selected acid in the presence of an organic solvent, e.g. Chloroform, methanol and ethanol and mixtures thereof.

The crude lipid A obtained can then be dissolved in acetone, acetone being particularly suitable for removing the fatty acid constituents. The solvent is then generally removed to obtain the crude detoxified endotoxin.

The crude, detoxified endotoxin can then be dissolved in a solvent and passed through a suitable chromatography column, e.g. a molecular exclusion chromatography column to separate the purified, detoxified fractions, which are then combined again after removal of the solvent. In a preferred embodiment of this process, the crude, detoxified endotoxin solution is passed through a Sepha-dex column in the presence of a solvent such as e.g. Chloroform, methanol, acetone, pyridine, ether or acetic acid or mixtures thereof. The pressure of column 65 can typically range from about atmospheric pressure to about 7 kPa and the flow rate is generally about 0.1 to 10 ml / min.

Alternatively, the crude, detoxified endotoxin solution can be passed through a DEAE cellulose column using the same pressure conditions as those given above for the Sephadex column. The throughput can be maintained in a range between about 2 and 15 ml / min. The solvents used are also the same as those used for the Sephadex column, although water and / or diethylamine can be added to all of these mixtures at a concentration up to about 1%.

Other methods of making crude, purified endotoxin include e.g. passing the solution through a low pressure silica gel 60 column in which the particle size is in the range of about 15 and 63 microns using a suitable solvent such as e.g. Chloroform, methanol, water or ammonium hydroxide. The preferred volume ratio of the solvent mixture mentioned above is in the range of about 50: 25: 4: 2.

It is therefore an object of the present invention to provide a composition capable of inducing an effective adjuvant or stimulating the immune response of a warm-blooded animal. This composition is characterized in that it contains an effective amount of a composition containing a purified, detoxified endotoxin, the endotoxin being no detectable 2-keto-deoxyoctanoate, between 350 and 475 n mol / mg phosphorus and between 1700 and 2000 n mol / mg fatty acids has, and contains a pharmaceutically acceptable carrier material.

In particular, the purified, detoxified endotoxin can be contained in the composition as a B cell mitogen. It is used to stimulate the production of lymphocytes (lymphokines) and macrophages and it also serves as an adjuvant, which increases the immune response in a warm-blooded animal.

The purified, detoxified endotoxin described can be used as a stimulant for the immune action in warm-blooded animals against antigens, e.g. Microbial cells and cells of fungi, microbial cell fragments, viruses, synthetic sub-virus peptide units, with mimic cellular and viral sub-units (mimic cellular and viral sub-units. Specific antigens, which are enhanced by the immune action, through the administration of purified , detoxified endotoxin, are attacked, for example, cryptococcus neoformans candida spp., Chlamydia spp., legionella spp. Clostridia, hepatitis me-ningitis, streptococus spp., staphylococus spp., klebsiella spp., herpes virus, brucella spp., ., Salmonella spp., Shigella spp., Camphylobacter spp., Yersinia spp., Pastorella spp., Francisella spp., Listeria spp., And the like.

The purified, detoxified endotoxin contained in the composition according to the invention shows B-cell mitogenicity, which means that when the purified, detoxified endotoxin is administered to a warm-blooded animal, B-lymphocytes are activated with a suitable administration instruction, these B- Lymphocytes are the cells that are responsible for the production of antibodies.

Experiments (1) B cell mitogenicity

The purified, detoxified endotoxin is characterized by its ability to activate B-lymphocytes, which B-lymphocytes are cells that are responsible for the production of antibodies as follows:

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Mitogenic activity of purified, detoxified endotoxin

Can of 3H thymidine incorporation (Hg / ml)

CPM * SI *

BALB / c nu / + 10 34,500 5.0

BALB / c nu / nu 10 38.693 5.6

Protocol - 1 x 106 cells / ml of each strain was grown with or without mitogen in 0.2 mg RPM1-1640 (5% FCS) in 96 hollowed out microtiter plates. 1 µcl of H-thymidine was added to each well during the last 18 hours of 48-hour incubation, and H uptake was measured using standard scintillation techniques.

* CPM - number per minute ** SI - stimulation index

(2) Production of Interleukin I The purified, detoxified endotoxin can be used to stimulate the production of lymphokines released by macrophages. The interleukin-I is a soluble immunoregulator that has been released by macrophages and is responsible for the activation of the lymphocytes at the site of an infection. Interleukin-I plays a critical role as an enhancer in the immune effect.

Mouse-like macrophages were adhered to microtiter plates (1 x 106 / well) and each determination was carried out three times. The first cavities received 1 ml of a 50 ng / ml solution of standard endotoxin, which was prepared in accordance with the procedure described in the above-mentioned parallel application. A 50 ml / ml solution of the purified, detoxified endotoxin in accordance with the present invention was added to the second group of hollows. Each agent was solubilized in M 199 medium with 5% FCS (fetal calf serum) and 1 ml of one M 199 medium with 5% FCS was added to the third group of wells to serve as a control.

The plates were incubated at a temperature of 37 CC for 20 hours. The supernatant was removed and diluted to give a solution having a 1:10 ratio of the supernatant to distilled water. The supernatant was assayed for interleukin I production by the method of Gery et al. Cellular immune. 64, 293 (1981).

The cells remaining after removal of the supernatant fluid were lysed using 0.1% Triton X-100. The resulting solution was diluted with RPMI-1640 medium containing 5% fetal calf serum at a 1:10 dilution ratio.

The diluted solution was examined for interleukin I production by measuring an increase in PHA-induced macrophage uptake of H3-thymidine as described by Gery et al supra. The results obtained are summarized in Table I below.

Table I

Test material (50 | ig / ml) effect of the lysate effect

Supernatant, (l: 10 dilution (l: 10 dilution))

Endotoxin standard 31.137 ± 1.721 51.695 ± 2797 purified, detoxified endotoxin 16.782 ± 1.295 66.178 ± 2332 control 3.323 ± 31 12.153 ± 497

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As shown in Table I, the interleukin I production from the described purified, detoxified endotoxin in the lysed cells largely exceeds the interleukin I production from the standard endotoxin extract.

The same study was carried out using human monocytes instead of mouse macrophages.

The following results were obtained:

Table IA

Test material (10 jig / ml) effect of the lysate effect

Supernatant, (1:50 dilution (1:50 dilution) dilution)

Endotoxin standard 42.418 + 1763 51.821 + 1348 purified, detoxified endotoxin 17.910 + 1983 50.626 + 813 control 1.693 + 289 15.173 + 1.292

(3) Macrophage Activation The purified, detoxified endotoxin contained in the composition of the invention also stimulates the macrophages as measured by the macrophage's ability to phagocytose (devour) fluorescent beads. 1 x 106 peritoneal exudate cells were mixed with 5 ^ 1 of a test material which contained standard endotoxin, purified, detoxified endotoxin according to the present invention and saline as a controil solution, and three test solutions were formed in this way. Each of these solutions contained 1 μl of the test material and 20 μl of a fluorescent bead suspension. The test solutions were placed on individual microscope slides and then incubated at a temperature of 37 ° C for 90 minutes.

After incubation, the slides were observed under a fluorescence microscope. The number of cells which phagocytosed the fluorescent beads was determined by visual observation and the number of beads per cell was also determined.

The phagocytic index was calculated using the following formula:

% phagocytizing cells x

•, t j number of beads / 100 cells phagocytic index = - '■

and the results of the two experiments are given in Table II below.

Table II

Phagocytic index * Example 1 Example 2

Endotoxin standard 5.0 3.6

RDE 5.4 4.1

Control 1.0 0.5

From Table II it can be seen that the phagocytic index for the purified, detoxified endotoxin according to the present invention is significantly larger than the corresponding index for standard endotoxin and from this fact it can be seen that the purified, detoxified endotoxin is an extraordinarily powerful stimulator of macrophages.

(4) Adjuvant activity In order to determine the use of purified, detoxified endotoxin as a stimulator of the immune system of warm-blooded animals, the purified and detoxified endotoxin was examined for its adjuvantity, as well as for its ability to immunize the red blood cells of sheep (SRBC) was determined by measuring the ability of antibodies to lyse sheep red blood cells. Three test materials were made. io Each of these materials contained 1 x 107 of SRBC. Test material # 2 also contained 20 mcgs of standard endotoxin + SRBC, while test material # 3 contained 20 mcgs of purified, detoxified endotoxin in accordance with the present invention + SRBC. i5 The test materials were injected interperitoneally into a strain of BALB / C mice. After 4-5 days, the test animals were sacrificed and from these test animals the spleen was removed, which was ground in a device for grinding tissues. Using a Haemocy-20 tometer, a standard cell suspension of each spleen was made and each of these suspensions were placed on lens carriers along with SRBC, media and agar.

The slides were incubated for 2 hours at a temperature of 37 ° C and sera from 25 guinea pigs were added as a supplemental source to each of the slides, followed by incubation at a temperature of 37 ° C for 20 minutes.

The slides were then examined to determine the amount of coated cells, which are zones in which the antibodies destroyed the SRBC cells using the additive found in the guinea pig sera.

The results obtained are shown in Table III below.

35 Table III

Test material PFC / 2 x 105 spleen cells

SRBC 88

40 SRBC + endotoxin standard 165

SRBC + RDE 219

As can be seen from Table III, the number of coating cells resulting from the use of purified, detoxified endotoxin is significantly higher compared to the standard endotoxin, which can be determined to be the purified, detoxified one Endotoxin is a powerful adjuvant. The purified, detoxified endotoxin used in accordance with the present invention can be administered together with a pharmaceutically acceptable medium such as e.g. Saline or an oil droplet emulsion. The composition mentioned can e.g. B. can be stabilized by lyophilization and then they can be re-constructed without losing their effectiveness.

As described above, this composition can be used to treat warm-blooded animals and humans in the form of an oil droplet emulsion. The amount of oil used is generally in the range of about 0.5 and 3% by volume based on the total volume of the composition. It is preferred to use about 0.75 to 1.5% by volume of the oil. Examples of suitable oils are light mineral oils, squalane, 7-n-hexyloc-tadecane, coconut super oil and Drakeol 6 VR mineral oil 65 (manufactured by Pennreco Company, Bulter, Pennsylvania).

The homogenized, oil-containing mixture can then be combined with a surfactant, preferably in a salt solution before adding

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mix is resolved. The amount of surfactant is typically in the range of about 0.02 to 0.20 volume percent, and more preferably between about 0.10 and 0.20 volume percent, based on the total volume of the composition. Any of the usual surface active materials including Tween-80 and Arlacel (manufactured by Atlas Chemical Company) can be used.

The mixture obtained by adding the surfactant is then preferably homogenized to form a suspension which has a high percentage of oil droplets coated with the purified, detoxified endotoxin, which could be determined by microscopic observation.

The amount of purified, detoxified endotoxin in a single injection is generally in the range of 5 to 500 ng, preferably between 10 and 100 μg (per total body weight of an adult human weighing 70 kg).

1-3 injections can be given over a period of about 2 months.

The purified, detoxified endotoxin-containing composition of the present invention has an extremely lower pyrogenic activity than the composition containing the standard endotoxin, which can be demonstrated by the following example.

Three rabbits from the New Zealand strain were injected (into the vein of one ear) with a composition containing the standard endotoxin. The amount of standard endotoxin required to raise the body temperature at least 0.46 C in 50% of the test animals was determined for 3 hours using a rectal thermometer.

The other three test rabbits were injected with a composition containing purified, detoxified endotoxin and the same pyrogenic activity test was performed. The results obtained are summarized in Table IV below.

io Table IV

Pyrogenicity

Test material activity in rabbits * (in (ig 1 kg)

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Standard endotoxin 0.012

purified, detoxified endotoxin 10 (i.e. no fever at the highest dose = 10 | ig)

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* The dose necessary to produce a fever higher than 0.46 ° C in 50% of the test animals.

As shown in Table IV, the standard 25 endardin causes fever in 50% of the test animals at a dose of 0.012 ng / kg. However, the purified, detoxified endotoxin causes fever with a dose of> 10 (ig kg, which is 850 times the dose level of the standard endotoxin.

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Claims (5)

660 125 1. Composition capable of producing an effective adjuvant effect or stimulating the immune effect of a warm-blooded animal, characterized in that it contains an effective amount of a composition containing a purified, detoxified endotoxin, the endotoxin being no detectable 2 Keto-deoxyocta-noat, between 350 and 475 n mol / mg phosphorus and between 1700 and 2000 n mol / mg fatty acids, and contains a pharmaceutically acceptable carrier. 2. Composition according to claim 1, characterized in that the effective amount is between 5 and 500 jag / total body weight of a 70 kg person. 2nd PATENT CLAIMS 3. Composition according to claim 2, characterized in that the effective amount is between 10 and 100 ng / total body weight of a 70 kg person. 4. Composition according to claim 1, characterized in that it is in lyophilized form. 5. Composition according to claim 1, characterized in that it is an oil droplet emulsion.