CA1225592A - Refined detoxified endotoxin - Google Patents

Abstract

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

122559~

~p 2961 BACKGROUND_OF_T~E_INV NTION

The present invention is directed to the use of a refined detoxified endotoxin (RDE) product as a potent immuno-stimulator and also as an adjuvarlt. The R~E used in the present invention is characterized as having no detectable

2-keto-3-~eoxyoctanoate, between about 350 and 475 nmoles/mg of phosphorus and between about 1700 and 2000 nmoles/mg of fatty acids.
Rndotoxic extracts obtained from Enterobacteriaciae including parent organisms and mutants are known. These extracts have been used for immunotherapy of various immuno-genic tumors [see PePtides as R_gLuirement for Imm _ th~ 3~ p~
the Guinea-Pig Line-10 Tumor_with Endotoxins; Ribi, et al Cancer Immunol. Immunother., Vol. 7, pgs. 43-58 (1979).
~ owever, the endotoxin extracts are known to be highly toxic and, therefore, of limited use in the treatment of cancerous tumors. Rfforts have becn made to "detoxify" the endotoxins while retaining its tumor regressive capacity. As shown, in Ribi, et al, supra, chemical procedures known to "detoxify" endotoxins wh;le retaining adjuvanticity, such as succinylation and phthalylation resulted in both loss of endotoxicity and tumor regressive potency. Therefore, prior lX~S~
art attempts to obtain a refined d~toxi~ied endo~oxin product have thus far not been successful.
Endotoxin extracts of the type used as a starting mateeial to produce ~he RDE used in the present invention may be obtained from any Enterobacteriaciae including parent organisms and mutants. By way of exampl~, the following genera are illustrative of the type of microorganisms that may be used:
Salmonella, Shiqella t Escherichia, Brucella, Bordetella, Citrobacter, Pseudomonas, Pasturella, Neisseria, Proteus, Klebsiella, and Serratia.
The following species are typically employed:
S.minnesota, S.typhimurium, B pertussis, B.abortus, S.enteritidis, E.coli, S.ty~ S.marcescens, S.tynhosa, Shiqella flexni, and S.abortus ~g~.
The endotoxic extracts used as a starting material may be prepared by one of several known methods [see, for example, Webster, M.E., Sagin, J.F., Landy, M., and Johnson, A.G., J. Immunol. 1955, 744, 55; Westphal, O., Luderitz, O., and Bister, F., Z. Naturforsch, 76 148 (1952); Westphal, O., Pyrogens, PolYsaccharides in Bioloqy, Tr. Second Macy Conference (George F. Springer, ed.), Madison, N.J. Madison Printing Co., 1957l 115; Galanos, C., Luderitz, O., Westphal, O., Eur. J. Biochem. 9, 245 (1969); Chen, C.H., Johnson, A.G., Kasai, N., Key, B.A., Levin, J., Nowotny, ~., J. Infect. Dis.

~2~5g2 128 543 (1973); Ribi~ B., 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., Milner, K.C., and Perrine, T. r J Immunol~ 82 75 (195g)] .
A most suitable method of obtaining the endotoxic extract is that disclosed by Chen, et al; namely, methanol-chloroform precipitation.
The methanol-chloroform precipitate (MCP) is reacted with an organic or inorganic acid and then lyophilized to produce a hydrolyzed crude lipid A with reduced toxicity and pyrogenicity as compared with the starting endotoxin material.
The resulting product is then treated with a solvent which is capable of specifically dissolving fatty acids and other impurities without dissolving the crude lipid A. A sui~able solvent for this purpose is acetone. The phosphate content of the detoxified, refined lipid A is about one-half that observed for the toxic counterpart suggesting that the phosphate content is related to the toxic effects of endotoxins.
Suitable inorganic acids used to react with MCP are hydrochloric acid, sulfuric acid or phosphoric acid and the suitable organic acids are toluene sulfonic acid or trichlo-roacetic acid. The reaction may be suitably conducted at a temperature between about 90 and 13nC. for a time sufficient to complete hydrolysis usually between about 1~ and 60 minutes.

12~5592 The preparation of crude detoxified endotoxin may also be accomplished by reacting the starting material with the selected acid in the presence of an organic solvent such as chloroform, methanol, and ethanol or combinations thereof.
The resulting crude lipid A is dissolved in acetone which is particularly suited to remove the 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 chromatographic column such as, for example, a molecular exclusion chromatographic column, to separate the RDE fractions which are then combined after removal of the solvent. In one embodiment, the crude detoxified endotoxin solution is passed through a Sepiladex 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 may vary but is typically in the range of between about atmospheric and 100 lbs/in2 and the flow rate is between about 0.1 and 10 ml/min.
Alternatively, the crude detoxified endotoxin solution is passed through a DEAE-cellulose column under the same pressure conditions as mentioned above for the Sephadex column. The flow rate may be maintained between about 2 and 15 ml/min. The solvents used are also the same as used for the Sephadex column although water and/or diethylamine can be added ~5~ii92 to all mixtures at a concentration of up to about 1%.
Other methods of producinq RDE ~rom crude detoxified endotoxin include passinq the ~olution throuqh a low pressure silica-gel ~0 column having a particle size of between about 15 and 63 microns and using a suitable solvent such as chloroform, methanol, water or ammonium hydroxide. The preferred volume ratio of the aforementioned solvent mixture is about 50:25:4:2.
It is, therefore, an object of the present invention to employ a refined detoxified endotoxin product which can be effectively used to stimulate the immune system of a warm blooded animal. Specifically, RDE can be used as a B-cell mitogen, to stimulate the production of lymphokines, stimulate macrophages, and as an adjuvant which enhances the immune response of a warm blooded animal.

SUMMARY OF THE INVENTION
-The present invention is directed to the use of refined detoxified endotoxin tRDE) as a stimulant of the immune system. The RDE used in this invention has no detectable 2-keto-3-deoxyoctanoate, between about 350 and 475 nmoles/mg of phosphorus and between about 1700 and 2000 nmoles/mg of fatty acids.
The RDE of the present invention can be used as a stimulant of the immune response in warm blooded aminals against antigens such as microbacterial and fungal cells, microbacterial cell fraqments, viruses, virus sub units 12~55~2 synthetic peptides which mimic cellular and viral sub units.
Specific antigens which are affected by the immune response augmented by the administration of RDE include cryptococcus neoformans candida spp., chlamydia spp., legionella spp.
clostridia, hepatitis meningitis, streptococus spp, staphy-lococus spp., klebsiella spp., herpes virus, brucella spp., borditella spp., salmonella spp., shigella spp., camphylobacter spp, yersinia spp., pasturella spp., francisella spp., listeria spp., and the like.
The RDE used in the present invention exhibits B-cell mitogenicity that is, RDE when administered to a warm blooded animal under a suitable dosage regimen activates B-lymphocytes which are the cells responsible for the manufacture of antibodies.

ASSAYS

(1) B CELL MITOGENICITY - RDE is characterized by its ability to activate B lymphocytes which are the cells responsible for the manufacture of antibodies, as follows:

12~559Z
MITOGENIC ACTIVITY OF REFINED DETOXIFIED ENDOTOXIN

Dose 3H-thymidine incorporation (~g/ml)CPM* SI**
.
BALB/c nu/+ 1034,500 5.0 BALB/c nu/nu 1038,693 5.6 Protocol-lx106 cells/ml of each strain cultured with or with-out mitogens in 0;2 mg of RPMl-1640 (5~ FCS) in 96 well micro-titer plates. 1 ucl 3H-Thymidine was added to each well the last 18 hours of a 48-hour incubation and 3H-incorporation was measured by standard scintillation techniques.
*CPM - counts per minute **SI - stimulation index .
-(2) INTERLE~IN-I-PROD~eTION
The RDE can be used to stimulate the production of lymphokines released by macrophages. Interleukin-I is a soluble immunoregulator released by macrophages which is responsible for the activation of lymphocytes at the site of an infection.
Interleukin-I plays a critical role as an amplifier of the immune response.
Murine macrohages were adhered to microtiter plates (1 X 106/well), each determination was made in triplicate.
The first wells received 1 ml. of a 50 ~ug/ml solution of standard endotoxin produced in accordance with the known procedure.
1 ml. of a 50 ~ug/ml solution of RDE in accordance with the present invention was added to the second set of wells.

Each agent was solubilized in M 199 medium with 5% FCS (fetal 1, ` ~\
12~5592 calf serum) 1 ml. M 199 medium with 5~ FCS was added to the third set of wells to serve as a control.
The plates were incubated at 37C for 20 hours. The supernatant was removed and diluted to obtain a solution having a 1:10 ratio of supernatant to distilled water. The supernatant was tested for interleukin-I production by the method of Gery, et al. eell~lar-~mmun.-64, 293 (1981).
The cells remaining after removal of the supernatant were lysed using 0.1% Triton* X-100. The resulting solution was diluted with RPMI 1640 medium with 5~ fetal calf serum at a 1:10 dilution ratio.
The diluted solution was tested for Interleukin-I pro-duction by measuring an augmentation of PHA inducted macrophage uptake of H3-thymidine as described in Gery et al. supra.
The results are shown in Table I.

TABLE I

Test Supernatant Lysate Material Response Response (50 yg./ml) (1:10 Dilution) (1:10 Dilution) . .
Endotoxin Standard 31,137 ~ 1,721 51,695 ~ 2797 RDE 16,782 ~ 1,295 66,178 ~ 2332 Control 3,3~3 ~ 31 12,153 ~ 497 *a Trademark 122~i592 As shown in Table I, Interleukin-I production from RDE of the present invention in the lysed cells greatly exceeded ~he Interleukin-I production from the standard endotoxin extract.
The same assay was conducted using human monocytes instead of murine macrophages.
The results are as follows:

TABLE IA

-Test Supernatant Lysate Material Response Response (10Jug/ml.) (1:50 Dilution) (1:50 Dilution) Endotoxin Standard 42,418 + 1763 51,821 + 1348 RDE 17,gl0 + 1983 50,626 + 813 Control 1,693 + 289 15,173 + 1,292

(3) MACROPHAGE ACTIVATION
RDE of the present invention also stimulates macro-phages as measured by the ability of macrophages to phagocytize (engulf) fluorescent beads.
1 X 106 peritoneal exudate cells were mixed with 5 ~u of a solution of a test material containing standard endotoxin, RDE of the present invention and saline as a control to respectively form three test solutions. Each of the solutions contained 1 ~g of the tes~ material and 20 ~ ~ of a fluorescent bead suspension. The test solutions were placed on individual microscope slides and then incubated for 90 minutes at 37C.
After incubation, the slides w~re observed under a fluorescent microscope. By visual observation, the number of cells which phagocytosed the fluorescent beads was determined as well as the number of beads/cell.
The phagocytic index was calculated in accordance with the formula:

HAGOCYTIC INDEX = ~ cells phagocytosing x number of beads/100 cells and the results of two experiments are shown in Table II.

TABLE II

Phagocytic Index *
Expt. 1 Expt. 2 Endotoxin Standard 5.0 3.6 RDE 5.4 4.1 Control 1.0 0.5 As can be seen from Table II, the phagocytic index for RDE of the present invention was siqnificantly greater than that of standard endotoxin~ thereby establishing that RDE is an exceedingly potent stimulator of macrophages.

-ln-~2~i9Z

(4) ADJUVANT ACTIVITY
To further establish the use of RDE as a stimulator of the immune system of warm blooded animals, RDE was ~ested for adjuvanticity as determined by its ability to augment the immune response to sheep red blood cells (SRBC) by measuring the ability of antibodies to lyse the sheep red blood cells.
Three test materials were prepared. Each contained 1 x 10 amount of SRBC. Test material #2 furthe~ contained 20 mcgs. of standard endotoxin + SRBC while test material #3 contained 20 mcgs. of RDE in accordance with the present inven-tion + SRBC.
Test materials were injected interperitoneally into strain BALB/C mice. After 4-5 days, ~he test animals were sacrificed and the spleens removed and ground in a tissue grinder. A standard cell suspension of each spleen was made using a haemocytometer and each of the above suspensions were placed on slides along with the target SRBC, media, and agar.
The slides were incubated for 2 hours at 37C and Guinea Pig sera was added as a complement source to each of the slides followed by incubation for 30 minutes at 37C.
Thereafter, the slides were examined to determine the quantity of plaque forming cells, which are areas in which antibodies destroyed the SRBC cells with the aid of the complement found in the Guinea Pig sera.
The results are shown in Table III.

TABL~ IIT

Test Material P~C~2xlO Spleen Cells SRRC 8~
SRBC + Endotoxin Standard 165 SRBC + RDE 219 As can be seen from Table III, the number of plaque forming cells resulting from the use of RD~ compared with standard endotoxin was signi~ic~ntly higher which establishes that RnE is a poterlt adjuv~nt.
The R~E as used in the present inverltiorl may be administere~ in combination with a pharmaceutica1ly acceptable medium such as saline or an oil-droplet emulsion. The afore-said composition may be stabilized as, ~or example, by a lyophilization procedure and then reconstituted without loss of potency.
As described ~bove, the composition for treatment of warm blooded animals and humans may be used in the form of an oil droplet emulsion. The amount of oil used is in the range of between about 0.5 and ~.0 percent by vo1ume based on the total volume of the composition. It is preferred to use between about ~.75 and 1.5 percent by volume of the oil.
~xamples of suitable oils inclu~e lil3ht mineral oil, squalane, 7-n-hexylocta~ecane, Conoco superoil and Drakenl ~ VR mineral * trademarks `" 12~i592 oil (produced by the Pennreco Company, Bulter, Pennsylvania).
The homogenized oil containing mixture is then combined with a detergent which may optionally be dissolved in a saline solution prior to mixing. The amount of detergent is typically between about 0.02 and 0.20 percent by volume and preferably between about 0.10 and 0.20 percent by volume based on the total volume of the composition. Any common detergent material may be used including Tween-80*, and Arlacel*
tproduced by the Atlas Chemical Company).
The mixture resulting from the addition of detergent is then homogenized to form a suspension which has a high percentage of oil droplets coated with RDE as determined by observation under a microscope.
The amount of RDE in a single injection is between 5 and 500 ~g, preferably between 10 and 100 ~g (per total body weight of a 70 kg human adult.
1-3 injections are administered over a period of about 2 months.
The RDE composition used in the present invention exhibits significantly less pyrogenic activity than a composition containing standard endotoxin as evidenced by the following example.
Three New Zealand strain rabbits, were injected (into an ear vein) with a composition containing standard endotoxin.
The amount of standard endotoxin necessary to cause an increase * trademarks ~2~5':3'~:

in body temperature of a~ least 0.46C in 50% of the tes~
popula~ion was determined over a period of 3 hours using a rectal thermometer.
Three other test rabbits were also injected with a composition containing RDE and the same pyrogenic activity test was made. The results are shown in the following Table IV.
TABLE IV

PYROGENICITY

Test Material Rabbit Activity*
(in ~q 1/kq Endotoxin Standard 0.012 RDE lO (i.e. no fever at highest dose = 10 ~9) _ _.
*The dose necessary to cause a febrile ~fever) response of greater than 0.46C in 50% of a test population.

As shown in Table IV, standard endotoxin produced a fever response in 50% of the test animals at a dosage of 0.012 Jug/kg. RDE however, did not produce a fever response at>
lO~ug/kg, which is 850 times the dose level of the standard endotoxin.

Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition capable of producing an effective adjuvant response or stimulating the immune response of a warm blooded animal comprising an effective amount of a com-position comprising refined detoxified endotoxin having no detectable 2-keto-deoxyoctanoate, between about 350 and 475 n moles/mg of phosphorus and between about 1700 and 2000 n moles/mg of fatty acids, in combination with a pharmaceuti-cally acceptable carrier.

2. A composition according to claim 1 wherein said effective amount is between about 5 and 500 µg/kg.

3. A composition according to claim 2 wherein said effective amount is between about 10 and 100 µg/kg.

4. A composition according to claim 1 which is in lyophilized form.

5. A composition according to claim 1 which is an oil droplet emulsion.