DE3431058A1 - PURIFIED, DETOXICATED ENDOTOXIN - Google Patents

Description

PURIFIED, DETOXED ENDOTOXIN

The invention relates to the use of a purified, detoxified endotoxin (RDE) product as being more effective Immune stimulator as well as adjuvant. The purified, detoxified used according to the invention Endotoxin (RDE) is characterized in that there is no detectable 2-keto-3-deoxyoctanoate, between about Contains 350 and 475 nM / mg phosphorus and between about 1700 and 2000 nM / mg fatty acids.

Endotoxic extracts obtained from Enterobacteriaceae, including related organisms and mutants are known. Such extracts are used in immunotherapy in various immunogenic tumors (see Peptides as Requirement for Immunotherapy of the Guinea-Pig Line-10 Tumor with Endotoxins; Ribi, et al., Cancer Immunol. Immunother., Vol. 7, pp. 43-58, 1979) was used been. However, these endotoxin extracts have been shown to be highly toxic, which is why they are used in treatment limited by cancerous tumors. Attempts have been made to "detoxify" the endotoxins without to impair their tumor-rebuilding property. As shown in Ribi et al., Supra, these led to detoxification of endotoxins while maintaining their adjuvant character, known chemical processes such as e.g. succinylation and phthalylation, to the loss of both endotoxicity and tumor-reducing effectiveness. Thus, the attempts heretofore made to produce a purified, detoxified endotoxin product remain without success.

Endotoxin extracts of the RDE used as a starting material for the production of the RDE according to the invention The type used can be from all Enterobacteriaceae

including related organisms and mutants. The following genera are examples of usable microorganisms:

Salmonella, Shigella, Escherichia, Bruceila, Bordetella, Citrobacter, Pseudomonas, Pasturella, Neisseria, Proteus, Klebsiella and Serratia.

The following species are particularly suitable for use in the present invention:

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 endotoxic extracts used as the starting material can be prepared, for example, by one of the following; known procedures getting produced:

1) Webster, ME, Sagin, JF, Landy, M., and Johnson, AG, J. Immunol. 1955, 744, 55.

2) Westphal, O., Luderitz, O., and Bister, F., Z. Naturforsch , 76. 148 (1952).

3) Westphal, O., Pyrogens, Polysaccharides in Biology, Tr. Second Macv Conference (George F. Springer, ed.), Madison, NJ Madison Printing Co., 1957, 115.

4) Galanos, C, Luderitz, Ο., Westphal, Ο ,, Eur. J. Biochem. 9, 245 (1969).

5) Chen, CH., Johnson, A.G., Kasai, N., Key, B.A.,

Levin, J., Nowotny, A., J. Infect. Pis. 128 543 (1973).

6) Ribi, E., Haskins, WT, Landy, M., Milner, K.C, The Journal of Experimental Medicine 114,647 (1961).

7) Leive, L., Biochem. Biophys. Res. Comm. 21 290 (1965).

8) Ribi, E., Milner, KC, and Perrine, T., J. Immunol. 8J2 75 (1959).

The Chen et al. described method, namely the

Methanol-chloroform precipitation, is the most suitable Method for obtaining the endotoxic extract.

The methanol-chloroform precipitate (MCP) is with a organic or inorganic acid reacted and then lyophilized to a hydrolyzed crude lipid A with reduced toxicity and pyrogenicity compared to the starting material endotoxin. The received Material is then treated with a solvent which is capable of removing particularly fatty acids and other contaminants to be solved without the crude lipid Ά being dissolved. A suitable solvent for this purpose is acetone. The phosphate content in the detoxified, purified lipid A is about half that found in the toxic counterpart Concentration suggesting that the phosphate content is related to the toxic effects of endotoxins is related.

The inorganic acids suitable for the reaction with MCP are hydrochloric acid, sulfuric acid or phosphoric acid, while the toluenesulfonic acid and the trxchloressxgsaure suitable organic acids are. The reaction can be carried out at any temperature between about 90.degree. C. and 130.degree for a period of time sufficient to carry out the hydrolysis, usually between about 15 and 60 minutes, be carried out appropriately.

The production of raw, detoxified endotoxin can also be done by reacting the starting material with the selected Acid in the presence of an organic solvent such as chloroform, methanol, ethanol or mixtures thereof take place.

The crude lipid A obtained is dissolved in acetone, which becomes entr removal of the fatty acid components is particularly suitable, dissolved. The solvent is then removed to remove crude, to get detoxified endotoxin.

The crude, detoxified endotoxin is then dissolved in a solvent and passed through a suitable chromatography column, such as a molecular exclusion chromatography column, to separate the RDE fractions, which are 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, acetic acid, or mixtures thereof. The column pressure can vary, but is usually approximately in the range between atmospheric pressure and 70 N / cm ² , while the flow rate is approximately between 0.1 and 10 ml / min.

According to another embodiment, the raw, detoxified Endotoxin solution under the same pressure conditions as in the Sephadex column described above through a DEAE cellulose column sent. The flow velocity can be maintained between about 2 and 15 ml / min. The solvents are also the same as in the Sephadex column can be used, although all mixtures are water and / or diethylamine in a concentration up to about 1% can be added.

In other processes for the production of RDE from raw, detoxified endotoxin, among other things, the solution is through a low pressure silica gel 60 column with a particle size of between about 15 µm and 63 µm using a suitable solvent such as chloroform, methanol, water or ammonium hydroxide. The preferred volume ratio of the above solvent mixture is about 50: 25: 4: 2.

The invention therefore relates to the use of a purified, detoxified endotoxin product which is effective Can be used to stimulate the immune system in warm-blooded animals. The purified, detoxified endotoxin (RDE) can

especially as a B-cell mitogen to stimulate lymphokine production, for stimulating macrophages, as well as as a the immune response enhancing adjuvant of warm-blooded animals can be used.

The invention relates to the use of purified, detoxified endotoxin (RDE) as a stimulant of the immune system. The RDE used in the invention, which has no detectable 2-keto-3-deoxyoctanoate, contains between about 350 and 475 nM / mg phosphorus and between about 1700 and 2000 nM / mg fatty acids.

The RDE according to the invention can be used as a stimulant of the immune response of warm-blooded animals against antigens such as microbacteria and fungal cells, microbacterial cell fragments, viruses, synthetic Peptides (subunits of viruses) mimicking cell and virus subunits. Both special antigens that are affected by the increased immune response due to the administration of RDE it is Cryptococcus neoformans candida spp., Chlamydia spp., Legionella spp. clostridia, hepatitis meningitis, Streptococcus spp., Staphylococcus spp., Klebsiella spp., Herpesvirus, Bruceila spp., Borditella spp., Salmonella spp., Shigella spp., Camphylobacter spp., Versinia spp., Pasturella spp., Francisella spp., Listeria spp. and the same.

The RDE used in the present invention has a B cell mitogen Activity, i.e. when administered to warm-blooded animals at an appropriate dose it activates the B lymphocytes, which are responsible for the production of antibodies responsible cells are.

try

(1) B-cell mitogenic activity

RDE is characterized by its ability to activate B lymphocytes, which are the cells responsible for producing antibodies, as follows
is performed.

Mitogenic activity of purified, detoxified

nu / + dose Endotoxin SI ** nu / nu ^ g / ml) 5.0 ³ H-thymidine uptake 5.6 10 10 BALB / c 10 CPM * BALB / c 34,500 38,693

Protocol:

1x10 cells / ml of each strain were with or without mitogens in 0.2 ml RPMI-1640 (5% fetal calf serum) in microtiter plates grown with 96 wells. During the last 18 hours, the 48-hour

4 -1
digen incubation time ³ H-thymidine with 3.7-1Os Πμϋχ)

introduced and the ³ H uptake was measured using conventional scintillation methods.

* CPM = counts per minute
** SI = stimulation index

(2) Production of Interleukin-I

The RDE can be used to stimulate the production of the lymphokines released by macrophages. Interleukin-I is a soluble, macrophage-released immune regulator that is responsible for the activation of lymphocytes in the Source of infection is responsible. Interleukin plays an important role as an enhancer of the immune response.

Mouse macrophages were placed on microtiter plates (1x10 per Well), each determination being carried out three times. In the first wells 1 ml of a 50 ^ g / ml solution of the according to that in the aforementioned pending application described procedure introduced standard endotoxins produced. The second series of Wells was 1 ml of a 50 ^ g / ml solution of the invention RDE admitted. Each agent was solubilized in M 199 medium with 5% FCS (fetal calf serum). to For control purposes, 1 ml of M 199 medium was added to the third series of wells.

The plates were incubated ^{at 37 ° C. for 20 hours.} The supernatant was removed and diluted, after which a solution with a ratio of 1:10 of supernatant to αεί 5 of still water was obtained. The supernatant was prepared according to the method of Gery et al., Cellular Immun. 64 , 293 (1981) examined for interleukin-I production.

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

The diluted solution was tested for interleukin-I production by measuring the increase in PHA-induced macrophage ^{uptake of H 3} -thymidine, as described in the above method by Gery et al. The results are shown in Table I.

Table I.

Test material Reaction of the supernatant Reaction of the lysate (50 µg / ml) (1:10 dilution) (1:10 dilution)

Default-
Endotoxin 31 .137 + 1 .721 51 .695 + _ 2797 RDE 16 .782 ± ¹ .295 66 .178 + _ 2332 control 3 .323 + 3 1 12th .153 + 497

As can be seen from Table I, the interleukin-I production was from RDE according to the invention in the dissolved cells far higher than the production of interleukin-I from the standard endotoxin extract.

The same experiment was carried out using human monocytes in place of the mouse macrophages. The results are shown in the table below:

Table IA

Test material Reaction of the supernatant Reaction of the lysate (10 μg / ml) (dilution ratio: (dilution ratio-

1:50) nis: 1:50)

Default-

Endotoxin 42,418 _ + 1763 51,821 _ + 1348

RDE 17.910 + 1983 50.626 + _ 813

Control 1,693 + 289 15,173 + _ 1292

(3) activation of macrophages

The RDE according to the invention also stimulates the macrophages, such as by the ability of macrophages to produce fluorescent vesicles to phagocytize, could be detected.

To prepare three test solutions, 1 × 10 peritoneal exudate cells were mixed with 5 μl each of a solution of a test material that contained normal endotoxin, RDE according to the invention and, as a control, physiological saline solution. Each of the solutions contained 1 μg of the test material and 20 ng of a suspension of fluorescent bubbles. The test solutions were applied to individual microscope slides and then ^{incubated at 37 ° C. for 90 minutes.}

After incubation, the slides were examined under a fluorescence microscope and visualized

achtung determines the number of cells that have phagocytosed fluorescent vesicles, as well as the number the vesicles per cell are determined.

The phagocytic index was calculated according to the following formula:

Phagocytic index =% of phagocytic cells χ

Number of vesicles per 100 cells

1000

The results of two tests are given in Table II on shown.

Table II

Phagocytosis Index * Trial 1 Trial 2

Standard endotoxin 5 , 0 3 , 6 15 RDE. 5 , 4 4th ,1 control 1 , 0 0 , 5

As can be seen from Table II, the phagocytosis index of the RDE according to the invention was significantly higher than that of Standard endotoxin, so that evidence is provided that the purified., “Detoxified endotoxin (RDE) is an extraordinary is an effective macrophage stimulator.

(4) Effectiveness as an adjuvant

To further confirm the fact that the use of RDE stimulates the immune system in warm-blooded animals, the RDE examined for adjuvant activity

iCriterium of its ability to increase the immune response against sheep red blood cells (SRBC) by measuring the ability of antibodies to produce to dissolve sheep red blood cells.

Three test materials were prepared, each with SRBC (sheep red blood cells), in an amount of 1 χ 10 contained. Test material # 2 continued to contain 20 pg standard endotoxin, + SRBC, while the test material # 3 contained 20 μg of the RDE + SRBC according to the invention.

The test substances were injected interperitoneally into mice of the BALB / C strain. After 4 to 5 days, the Test animals killed and their spleen removed and ground in a tissue grinder. Using a hemocytometer a standard cell suspension was prepared from each spleen and each of these suspensions together with the test object SRBC, the media and agar on microscope slides upset.

The slides were incubated for 2 hours at 37 ° C, and complement was added to each slide Guinea pig serum was applied before a further incubation for 30 minutes at 37 ° C followed.

The slides were then examined to determine the number of plaque-forming cells, i.e. the areas in which the antibodies destroyed the SRBC cells with the help of the complement found in the guinea pig serum.

The results are shown in Table III.

Table III

Test material PFC * / 1 χ IQ ⁵ spleen cells

SRBC 88

SRBC + standard endotoxin 165

SRBC + RDE 219

* PFC = plague-forming cells

As can be seen from Table III, the number of plague producing cells was significant when using RDE higher compared to the number using standard endotoxin, confirming that RDE is a highly potent Is adjuvant.

The RDE used in the present invention can be used in conjunction with a pharmaceutically acceptable agent such as e.g. physiological saline solution or an oil droplet emulsion administered. The above-mentioned composition can, for example by means of a lyophilization process, can be stabilized and then restored to their original state without any loss of effectiveness.

As described above, the composition can be used in the treatment of warm-blooded animals and humans in the form of an oil droplet emulsion be used. The amount of oil used is between about 0.5 and 3.0% by volume, based on the Total volume of the composition. The preferred amount of oil to be used is between about 0.75 and 1.5% by volume. Examples of suitable oils are light mineral oil, sgalan, 7-n-hexyloctadecane, Conoco super oil and Drakeol 6 VR Mineral Oil (manufactured by Pennreco Company, Bulter, Pennsylvania).

The homogenized, oil-containing mixture is then with a detergent, which is optionally dissolved beforehand in a physiological saline solution. the the average amount of detergent is usually between about 0.02 and 0.20 volume percent and the preferred The amount is between about 0.10 and 0.20% by volume, based on the total volume of the composition. Everyone can do it common detergents such as Tween-80 and Arlacel (manufactured by Atlas Chemical Company) can be used.

The mixture obtained after the addition of the detergent is then homogenized, creating a suspension with a high percentage of oil droplets coated with RDE, which can be demonstrated under the microscope leaves.

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

1 to 3 injections were given over a period of approximately two months.

The RDE composition used according to the invention has has a significantly lower pyrogenic activity than a standard endotoxin-containing composition, such as the following example shows.

Three New Zealand breed rabbits were injected with a standard endotoxin-containing composition (into the ear vein). The required to increase the body temperature by at least 0.46 ⁰ C in 50% of all test animals amount of standard endotoxin was determined using a rectal thermometer for a period of 3 hours.

Three other guinea pigs were also injected with a composition containing RDE, followed by the same Pyrogenic activity test was performed. The results are shown in Table IV below.

Pyrogenic activity

Test material

Table IV

Activity in rabbits * (in iig 1 / kg)

5 standard endotoxin RDE

0.012

10 (i.e. no fever at the highest dose = 10 μg)

* The dose that is required to have a fever response over 0.46 ⁰ C at 50% of the total number of animals produce.

As shown in Table IV, standard endotoxin caused 50% of the test animals at a dose of 0.012 µg / kg a febrile reaction, while RDE at a dose> 10 μg / kg, i.e. a dose 850 times higher than with standard endotoxin, did not cause a fever reaction.

Claims (5)

ν. FÜ NER EB BI * JG "-H" a: u S FINCK PATENTANWÄLTE EUROPEAN PATENT ATTORNEYS MARIAHILFPLATZ 2 & 3, MUNICH ΘΟ POSTAL ADDRESS: POSTBOX 95 O1 60, D-8OOO MUNICH 95 3431058 2 Aug. 3, 1984 RIBI IMMUNOCH, INC. DEAA-32169.7 PURIFIED, DETOXIFIED ENDOTOXIN claims 1. To generate an effective adjuvant's reaction or composition capable of stimulating the immune response of warm-blooded animals, characterized that they are an effective amount of a composition of purified, detoxified endotoxin with no detectable 2-keto-deoxyoctanoate, between about 350 and 475 nM / mg phosphorus and between about 1700 and 2000 nM / mg fatty acids in association with a pharmaceutically acceptable carrier. 2. Composition according to claim 1, characterized in that the effective amount is between about 5 and 500 iig / kg. 3. Composition according to claim 2, characterized in that the effective amount is between about 10 and 100 yug / kg. 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 in the form of an oil droplet emulsion.