NL8402515A - PREPARATION WITH IMMUNE-STIMULATING EFFECT. - 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

1 h 'λ - 1 -

Preparation with an immunostimulating effect.

The invention relates to the use of a purified, detoxified endotoxin (refined detoxified endotoxin or abbreviated RDE, as indicated hereinafter) product as a potent immune stimulator and also as an adjuvant. The RDE used in the present invention is characterized as having no detectable 2-keto-3-deoxyoctanate between about 350 and 475 nmoles / mg phosphorus and between about 1700 and 2000 nmoles / mg fatty acids.

Endotoxin extracts obtained from Enterobacteriaciae, including parent organisms and mutants, are known. These extracts have been used for 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., 43-48 (1979)). However, it is known that the endotoxin extracts are highly toxic and can therefore be used to a limited extent for the treatment of cancerous tumors. They have made attempts to "detoxify" endotoxins while maintaining their tumor regressive capacity. As shown in Ribi, et al, supra, known chemical methods of "detoxifying" endotoxins while retaining their auxiliary activity, such as succinylation and phthalylation, have resulted in both loss of endotoxicity and tumor regressive potency. Therefore, past attempts to obtain a purified, detoxified endotoxin product have so far been unsuccessful.

Endotoxin extracts of the type used as a starting material for preparing the RDE used in the present invention can be obtained from a variety of Enterobacteriaciae including parent organisms and mutants. For example, the following genera are illustrative of the type of microorganism that can be used: 35 Salmonella, Shigella, Escherichia, Brucella, 8402515 r ', · ΐ - 2 -

Bordetalla, Citrobacter, Pseudomonas, Pasturella, Neisseria, Proteus, Klebsiella and Serratia.

In particular, the following species are used: S.minnesota, S.typhimurium, B.pertussis, 5 B.abortus, S.enteritidis, E.coli, S.typhi, S.mareescens, S.typhosa, Shigella flexni and S.abortus equi.

The endotoxin extracts used as starting material can be prepared by one or more known methods (see, for example, Webster, ME, Sagin, JF, Landy, M. and Johnson, AG, J. Immunol. 1955, 744, 55; Westphal, 0., Luderitz, 0., and Bister, F., Z. Naturforsch, 76 148 (1952); Westphal, 0., Pyrogens, Polysaccharides in Biology, Tr. Second Macy Conference (George F. Springer, ed. ), Madison, NJ Madison Printing Co., 1957, 115; Galanos, C., Luderitz, 0., Westphal, 15, Eur. J. Biochem. 9, 245 (1969); Chen, CH, Johnson, AG,

Kasai, N. Key, B.A., Levin, J., Nowotny, A., J. Infect. Piss.

128 543 (1973); Ribi, E., Haskins, W.T., Landy, M., Milner, K. C., The Journal of Experimental Medicine 114 647 (1961); Leive, L., Biochem. Biophys. Res. Comm. 21 290 (1965) and Ribi, E, 20 Milner, K.C., and Perrine, T., J. Immunol. 82 75 (1959)).

A very suitable method for obtaining the endotoxin extract is that described by Chen, et al, namely, methanol-chloroform precipitation.

The methanol-chloroform precipitate (MCP) is reacted with an organic or inorganic acid and then freeze-dried to obtain a hydrolysed crude lipid A of reduced toxicity and pyrogenicity compared to the starting endotoxin material. The resulting product is then treated with a solvent which can specifically dissolve fatty acids and other impurities without dissolving the crude lipid A. A solvent suitable for this purpose is acetone. The phosphate content of the detoxified purified lipid A is. about half of what has been observed in the toxic counterpart, suggesting that the phosphate content is related to the toxic effects of 8402515-3-endotoxins.

For reaction with MC3? suitable inorganic acids are hydrochloric, sulfuric or phosphoric acid and the suitable organic acids are toluenesulfonic or trichloroacetic acid.

The reaction can be carried out well at a temperature of 90-130 ° C for a time sufficient to complete hydrolysis, usually 15-60 minutes.

Raw, detoxified endotoxin can also be prepared by reacting the starting material with the selected acid in the presence of an organic solvent, such as chloroform, methanol, ethanol or combinations thereof.

The resulting crude lipid A is dissolved in acetone, which is particularly suitable for the removal of the fatty acid component. The solvent is then removed to yield crude, detoxified endotoxin.

The crude, detoxified endotoxin is then dissolved in a solvent and the solution is passed through a suitable chromatography column, for example a molecular exclusion chromatography column, to separate the EDE fractions, which are 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 combinations thereof. The pressure of the column can vary, but typically atmospheric pressure is up to 100 pounds / in2 and the flow rate is 0.1-10 ml / min.

Optionally, the crude detoxified endotoxin solution is passed through a DEAE cellulose column under the same pressure as mentioned above for the Sephadex column. The flow rate can be kept at 2-15 ml / min. The solvents used are also the same as for the Sephadex column, although water and / or diethylamine can be added to all mixtures at a concentration of up to 1%.

35 Other methods of preparing EDE

8402515 * 3 - 4 - from crude, detoxified endotoxin are passing the solution through a low pressure silica gel 60 column with a particle size of 15-63 microns and using a suitable solvent such as chloroform, methanol, water or ammonium hydroxide. The volume ratio of the above solvent is preferably 50: 25: 4: 2.

The invention therefore contemplates the use of a purified, detoxified endotoxin product which can be used effectively to stimulate the immune system of a warm-blooded animal. EDE can be used primarily as a B-cell mitogen to stimulate the production of lymphokines, to stimulate microphages and to aid in enhancing the immune response of a warm-blooded animal.

The present invention relates to. the use of purified, detoxified endotoxin (EDE) as a stimulant of the immune system. The EDE used in the invention has no detectable 2-keto-3-deoxyoctanate at 350-475 nmoles / mg phosphorus and 1700-2000 nmoles / mg fatty acids.

"The EDE of the present invention can be used as a stimulator of the immune response in warm-blooded animals against antigens, such as microbacterial and fungal cells, microbial cell fragments, viruses, virus components and synthetic peptides, which mimic cell and virus components. Antigens particularly affected by the immune response, which is enhanced by the administration of EDE, are cryptococcus neoformans Candida spp., Chlamydia spp. ,. legionella spp. clos-tridia, hepatitis meningitis, streptococus spp., staphyloeocus spp., klebsiella spp., herpes virus, brucella spp.,. borditella spp., salmonella spp., shigella spp., camphylobacter spp., yer-30 sinia spp., pasturella spp., francisella spp., listeria spp. and such.

The EDE used in the invention exhibits B cell mitogenicity, that is, EDE activates when administered to a warm-blooded animal in an appropriate dose of B lymphocytes, which are the cells used for the production of antibodies 8402515 * - 5 - 5 - be responsible.

Taste.

5 (1) B cell mitogenicity. EDE is characterized by its ability to activate B lymphocytes, which are the cells responsible for the production of antibodies, as follows: Hitogenic activity of purified, detoxified endotoxin.

3. .

D dose of H-thymx dx uptake

CyUg / ml) CPM * SI ** 15 'BALB / c nu / + 10 34,500 5.0 - * BALB / c nu / nu 10 ~ 38,693 5.6

Protocol: 1 x 10 ^ cells / ml from each strain with or without 20 mytogens grown in 0.2 mg RPM1-1640 (5% FCS) in 96 well microtiter plates. 1 µl H-thymidine was added to each well during the last 18 hours of a 48-hour incubation and 3. .

H uptake was measured by standard scxntxllatx methods.

* CPM: counts per minute. ** SI: stimulation index.

(2) Interleukin I production.

The EDE can be used to stimulate the production of lymphokines released by macrophages. Interleukin-I is a soluble immune regulator released by macrophages responsible for the activation of lymphocytes at the site of an infection. Inter-leukin-I plays a critical role in enhancing the immune response. 35 Murine macrophages were attached on micro-8402515-6 g titre plates (1 x 10 / well), each assay made in triplicate. The first wells received 1 ml of 50 µg / ml solution of standard toxin prepared according to the procedure described in the aforementioned patent application. To the second series of wells 1 ml of 50 µg / ml solution of RDE according to the invention was added. Each agent was solubilized in M 199 medium with 5% FCS (fetal calf serum) 1 ml. M 199 medium with 5% FCS was added to the third series of wells to serve as a control.

The plates were incubated at 37 ° C for 20 hours. The supernatant was removed and diluted to give a solution with a 1:10 ratio of the supernatant to distilled water. The supernatant was tested for interleukin I production according to the method of Gery, et al. Cellular Immun. 64, 293 (1981).

The cells remaining after removal of the supernatant were lysed using 0.12 Triton X-100. The resulting solution was diluted with RPMI 1640 medium containing 5% fetal calf serum at a 1:10 dilution ratio.

The dilute solution was tested for Interleukin I production by measuring an increase in PHA

3 elicited macrophage uptake of H-thymidine as described in Gery et al above. The results are shown in Table A.

25

TABLE A

Test Material Supernatant Lysate (50 µg / ml) Response Response 30 __ (1:10 dilution) _ (1:10 dilution)

Endotoxin standard 31,137 ± 1,721 51,695 ± 2,797 SDE 16,782 ± Ί,295 66,178 ± 2,332

Control 3,323 ± 31 12,153 + 497 35 8402 515 - 7 -

As shown in Table A., the Interleukin I production from EDE of the invention in the lysed cells greatly exceeded the Interleukin I production from the standard doxin extract.

The same experiment was performed with human monocytes instead of murine macrophages.

The results are as follows: TABLE Aa 10

Test Material Supernatant Lysate (10 µg / ml) Response Response _ (1:50 dilution) _ (1:50 dilution)

Endoxine 15 standard. 42,418 ± 1,763 51,821 ± 1,348 EDE 17,910 ± 1,983 50,626 ± 813

Control 1,693 ± 289 15,173 ± 1,292 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 8 402 515

Makraphage activation.

The BDE of the invention also stimulates macrophages as measured by the ability of macrophages to phagocytes (engulf) fluorescent beads.

5 x 10 peritoneal exudate cells were mixed with 5 µl solution of a test material, which consisted respectively of standard toxin, RDE of the invention 8 and salt to control, to form three test solutions.

9

Each solution contained 1 µg of test material and 20 µl of suspension of fluorescent beads. The test solutions were placed on 11 separate microscope slides and then incubated at 37 ° C for 90 minutes.

13

After incubation, the slides were observed under a fluorescent microscope. Visual observation determined the number of cells that phagocytosed the fluorescent beads 16 as well as the number of beads / cell.

17 35 The phagocytic index was calculated according to / '<v- - 8 - s' the formula:% phagocytosing cells x number of beads / PHAGOCYTIC INDEX --100 .c.? LleS-- 1000 5 and the results of the two tests are given in table B.

TABLE B

Phagocytic index * 10 Test 1_Test 2

Endotoxin standard 5.0 3.6 EDE 5.4 4.1

Control 1.0 0.5 As shown in Table B, the phagocytic index for EDE of the invention was significantly greater than that of standard toxin, thus establishing that EDE is an extremely potent macrophage stimulator. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 8 402 515 (4) Auxiliary operation.

2

In order to further investigate the use of EDE as stimulator 3 of the immune system of warm-blooded animals, the EDE is tested for auxiliary activity as determined by its ability to enhance the immune response to sheep red blood cells (SEBC) by measuring the ability. from antibodies to lysing. the sheep red blood cells. Three test materials were prepared, each containing 1 x 10 9 SEBC. Test material number 2 additionally contained 20 mcg 10 standard toxin + SEBC, while test material number 3 11 contained 20 mcg EDE of the invention + SEBC.

12

The test material was injected interperitoneally into mice of the BALB / C strain. After 4-5 days, the 14 test animals were sacrificed and the spleens removed and finely ground in a tissue mill. A standard cell suspension of each spleen was prepared using a hemocytometer * - 9 - and each of the above suspensions was placed on microscope slides along with the target SKBC, medium and agar.

Slides were incubated for 2 hours at 37 ° C and serum was applied to each slide as complement source of guinea pig, followed by incubation at 37 ° C for 30 minutes.

Slides were then examined to determine the amount of plate-forming cells, which are regions in which antibodies destroyed the SRBC cells using the complement found in the serum of the guinea pig.

The results are shown in Table C.

TABLE C

15 Test material PFC / 2x10 ~ * spleen cells SRBC 88 SKBC + endotoxin standard 165 SKBC + RDE 219 20

As shown in Table C, the number of plate-forming cells resulting from the use of RDE compared to standard toxins is significantly higher, showing that EDE is a potential vehicle.

The RDE of the invention can be administered in combination with a pharmaceutically acceptable medium, such as salt or an oil droplet emulsion. For example, the above preparation can be stabilized by a freeze-drying method and then reconstituted without loss of potency.

As described above, the preparation for the treatment of warm-blooded animals and humans can be used in the form of an oil droplet emulsion. The amount of oil used is 0.5-3.0% by volume, based on the total volume of the preparation. It is preferable to use 0.75-1.5 vol% oil. Examples of suitable oils are light mineral 8402515 lichte - 10 - oil, squalane, 7-n-hexyloctadecane, Conoco superoil and Drakeol 6 VR mineral oil (produced by Pexmreco Company, Bulter, Pennsylvania).

The homogenized oil-containing mixture is then combined with a detergent, which can optionally be dissolved in a salt solution before mixing. The amount of detergent is in particular 0.02-0.20 vol% and preferably 0.10-0.20 vol%, based on the total volume of the preparation. One can. use any regular detergent, including Tween-80 and Arlacel (manufactured by Atlas Chemical Company).

The mixture is then homogenized, which results after the addition of detergent to a suspension with a high percentage of oil droplets coated with EDE as. determined by observation under a microscope.

The amount of RDE in a single injection is 5-500 µg, preferably 10-100 µg (per 70 kg total body weight of an adult human.

1-3 injections were administered over a 2 month period.

The RDE preparation of the invention exhibits significantly less pyrogenic activity than. a formulation containing standard end o oxine as shown in the following example.

Three rabbits of the New Zealand strain were injected (into an ear vein) with a standard toxin-containing preparation. The amount of standard endotoxin required to cause a body temperature elevation of at least 0.46 ° C in 50% of the test population was determined over a 3 hour period using a rectal thermometer. Three other guinea pigs were also injected with an EDE-containing preparation and the same fumigation test was performed. The results are shown in the following table D. 1 8402515

O

- 11. TABLE D

Pyrogenicity 5 Test material Rabbit activity * (in / txg 1 / kg)

Endotoxin standard 0.012 EDE 10 (i.e., no fever at highest dose = 10 µg) * The dose required to produce a fever response of more than 0.46 ° C in 50% of a test population.

As shown in Table D, standard do-toxin gave a fever response in 50% of the test animals at a dose of 0.012 µg / kg. However, EDE did not produce a fever response at> 10 µg / kg, which is 850 times the dose level of standard toxin.

8402515

Claims (6)

1. A preparation which can give an effective auxiliary response or stimulate the immune response in a warm-blooded animal, characterized in that it contains an effective amount of a purified, detoxified endotoxin-containing product without detectable 2-ketodeoxyoctanate at 350-475 nmoles / mg of phosphorus and 1700-2000 nmoles / mg of fatty acid, whether or not in combination with a pharmaceutically acceptable carrier. Preparation according to claim 1, characterized in that the effective amount is 5-500 µg / kg 10. Preparation according to claim 2, characterized in that the effective amount is 10-100 µg / kg. Preparation according to claim 1, characterized in that it is in freeze-dried form. Preparation according to claim 1, characterized in that it is an oil droplet emulsion. 6. New preparation as described in the description. 20 8402515