Process for preparing refined detoxified endotoxin

(42) Date of commencement of the patent: 15. 1.1988 (45) Date of issue: 10. 8.1988

<td>(30) Priority:</td><td>(73) Patent owner:</td>

<td>26. 5. 1982 US 382404 claims.</td><td>RIBI IMMUNOCHEM RESEARCH, INC. HAMILTON (US).</td>

<td>(56) Documents:</td><td></td>

<td>DE-PS 472221 DE-PS 185031</td><td></td>

(54) METHOD OF PRODUCING A REFINED ENTGETTED ENDOTOXIN PRODUCT

CO

AT 386,337

WR «78018

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The invention relates to a process for the preparation of a detoxified endotoxin product (RDE) which, when contacted with a cell wall scaffold (CWS), is a therapeutically active composition for the treatment of cancerous tumors without the deleterious side effects normally associated with endotoxins. The RDE present in such composition according to the present invention is characterized by having no detectable levels of 2-keto-3-deoxyoctanoate, between about 350 and 475 nmol / mg phosphorus and between about 1700 and 2000 nmol / mg fatty acids.

Endotoxic extracts obtained from Enterobacteriaciae, including parent organisms and mutants, are known. These extracts have been used for the immunotherapy of various immunogenic tumors [s. Peptides as Requirement for Guinea-Pig Line-10 Tumor with Endotoxins; Ribi et al., Cancer Immunol. Immunother., Vol. 7, pp. 43-58 (1979)]. This publication is hereby incorporated by reference. However, it is further known that the endotoxin extracts are highly toxic and therefore can be used only to a limited extent in the treatment of cancerous tumors. Efforts have been made to detoxify the endotoxins while maintaining their tumor-forming properties. As set forth in Ribi et al., The known chemical methods for detoxifying endotoxins, while maintaining the auxiliary action, such as succinylation and phthalylation, result in both loss of endotoxicity and tumor regression effectiveness. Neither have detoxified endotoxin products been obtained by treatment of bacterial toxins with acids. Thus, previous attempts to obtain an endotoxin product with high tumor regression efficacy and little or no toxicity have not been successful.

The cell wall framework is essentially a cell wall where much of the protein and lipids normally found in the cell wall are removed. It is a polymeric mycollate arabinogalactan mucopeptide containing traces of trehalose mycolate (P.₃ ) and undigested tuberculoproteins. The cell wall skeleton is obtained from any fungal bacterium, such as M.smegmatis, M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M. kansasii, M.tuberculosis (strain H 37 RV and Ayoma B), and M .bovis, strain BCG. Furthermore, cell wall skeletons can be obtained from bacteria other than fungal bacteria, such as E. coli, B. abortus and Coxiella burnettii.

The cell wall skeleton is obtained by first breeding and harvesting a bacterium such as M.bovis, strain BCG (Bacillus Calmette-Guerin). The resulting whole cell debris is worked up by a cell fractionator [Ribi Cell Fractionator (Sorvall, model RF-1)] in which the cells are disrupted and the outer envelope or cell wall is separated from the protoplasmic contaminants. The resulting cell walls are then subjected to a series of solvent extraction and enzymatic treatments (e.g., trypsin and / or chymotrypsin) to obtain a purified cell wall skeleton.

An object of the invention, therefore, is the production of a superior refined and detoxified endotoxin product which has a high tumor regression effect, without the toxic side effects normally occurring as a concomitant. This refined detoxified endotoxin in combination with cell wall skeleton is a highly effective composition for the treatment of cancerous tumors.

More particularly, the invention relates to a process for the production of refined, detoxified endotoxin with undetectable 2-keto-3-deoxyoctanoate, between about 350 and 475 nmol / mg phosphorus and between about 1700 and 2000 nmol / mg fatty acid, characterized in that you

a) an endotoxin extract obtained from microorganisms of the family Enterobacteriaciae with an acid such as hydrochloric acid, sulfuric acid, phosphoric acid, toluenesulfonic acid or trichloroacetic acid, hydrolyzed;

d) freezing the hydrolyzed product to obtain crude lipid A;

c) treating the crude lipid A with a first solvent such as acetone to dissolve fatty acids contained therein;

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d) dissolving the resulting insoluble product in a second solvent such as methanol, chloroform, acetone, pyridine, ether and acetic acid or mixtures of these solvents; and

e) passing the resulting solution through a chromatographic column to obtain the desired

To receive product.

Endotoxin extracts of the type used as starting material for the production of RDE can be obtained from any of the Enterobacteriaciae, including parent organisms and mutants. The following types can serve as examples of microorganisms that can be used:

Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomonas, Pasturella,

Neisseria, Proteus, Klebsiella and Serratia.

The following types are typically usable:

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 any known method [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, 0., Eur. J. Biochem. 9, 245 (1969); Chen, CH, Johnson, AG, Kasai, N., Key, BA, Levin, J., Nowotny, A., J. Infect. 128 543 (1973); Ribi, E., Haskins, WT, Landy, M.,

Milner, KC, The Journal of Experimental Medicine 114, 647 (1961), Leive, L., Biochem. Biophys. Res. Comm. 21,290 (1965); and Ribi, E., Milner, KC, and Perrine, T., J. Immunol. 82 75 (1959)].

The preferred method for obtaining the endotoxic extract is that described by Chen, et al., Namely methanol-chloroform precipitation.

The methanol-chloroform precipitate (MCP) is then reacted with an organic or an inorganic acid and then freeze-dried to a hydrolyzed crude lipid A which has reduced toxicity and pyrogenicity compared to the starting endotoxin. The resulting product is then treated with a solvent capable of specifically dissolving the fatty acids and other impurities without dissolving the crude lipid A. The preferred solvent for this purpose is acetone. The phosphate content of the detoxified refined lipid A is about one-half that observed with the toxic counterpart, suggesting that the phosphate content is related to the toxic effects of the endotoxins.

The preferred inorganic acids used for reaction with MCP are hydrochloric acid,

Sulfuric acid or phosphoric acid, preferred organic acids are toluenesulfonic acid or trichloroacetic acid. The reaction may conveniently be carried out at a temperature between about 90 and 130 ° C for a time sufficient to terminate the hydrolysis and which is usually between about 15 and 60 minutes.

The preparation of the crude detoxified endotoxin may also be accomplished by reacting the starting material with the 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 suitable for removing the fatty acid components. The solvent is then removed to yield the crude detoxified endotoxin.

This crude detoxified endotoxin is then dissolved in a solvent and passed through a suitable chromatographic column, such as a molecular exclusion chromatography column (molecular exclusion chromatography), to separate the RDE fractions, which are then combined after removal of the solvent. According to one embodiment 50, 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 may vary, but it is typically in a range between about normal pressure and 6.7 bar, the flow rate is between about

0.1 and 10 ml / min.

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In another embodiment, the crude detoxified endotoxin solution is passed through a DEAE-cellulose column under the pressure conditions given above for the Sephadex column. The flow rate can be maintained between about 2 and 15 ml / min. The solvents used are the same as those in the Sephadex column, although to all mixtures water and / or diethylamine can be added in a concentration of up to about 1%.

Other methods of producing RDE from crude detoxified endotoxin are by passing the solution through a low pressure silica gel 60 column having a particle size between about 15 and 63 microns and using a solvent selected from chloroform, methanol, water and ammonium hydroxide. The preferred volume ratio of the aforementioned solvent mixture is about 50: 25: 4: 2.

The RDE produced by the invention is combined with CWS to form a composition having potent anti-tumor activity. Tumors that can be treated with this RDE-CWS composition are animal tumors, such as scaly cell carcinoma in cattle, bovine fibrosarcoma, horse sarcoid, equine melanoma, scaly cell carcinoma in horses, mammary gland tumor in pigs, canine adenoma, canine melanoma, human tumors such as breast tumors , Lung tumors, rectal tumors, malignant melanoma, scaly cell carcinoma and ovarian tumors.

The composition is preferably administered by injection into a pharmaceutically acceptable medium, such as an oil droplet emulsion, directly into the tumor under the conditions described in detail below. For example, the foregoing composition can be stabilized by a lyophilization method and then reconstituted without loss of effect.

The amount of RDE in a single injection for the treatment of animals is between about 6.25 and 250 pg / ml. The amount of CWS is between about 125 and 750 pg / ml. The number of ml of biological substance injected into the tumor is determined by the size of the tumor in accordance with the following table:

Dosage in animals according to tumor size

<td>Tumor diameter (cm)</td><td>Amount of injected biological substance</td>

<td>0-1</td><td>up to 0.5 ml</td>

<td>1-2</td><td>0.5 - 2.5 ml</td>

<td>2-3</td><td>2.5-5 ml</td>

<td>3-5</td><td>5 - 10 ml</td>

<td>5-8</td><td>10 - 15 ml</td>

<td>greater than 8</td><td>15-20 ml</td>

The maximum dose per injection is about 1500 pg for RDE and about 4500 pg for CWS. The treatment duration is 5 injections given at 1 week intervals.

The RDE-CWS composition in a suitable injectable medium, such as in an oil droplet emulsion, is administered directly into human tumors. The amount of RDE or CWS in a single injection is between about 50 and 1000 pg, with the preferred single dose being between about 275 and 325 pg for each component in an adult patient of typically 70 kg weight. The injections are given about once a week for a total of 15 injections. It is usually advantageous to administer a composition containing between about 50 and 500 pg / ml RDE and between about 50 and 500 pg / ml CWS per injection.

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As noted above, the composition for treatment can be warm-blooded

Animals and humans are administered in the form of an oil droplet emulsion. The amount of oil used ranges between about 0.5 and 3.0% by volume, based on the total volume of the oil

Composition. Preferably, about 0.75 to 1.5% by volume of oil is used. Examples of such

Oils are light mineral oils, squalane, 7-n-hexylocatadecane, Conoco superoil and Drakeol 6 VR mineral oil (manufactured by Pennreco Company, Bulter, Pennsylvania).

The mixture containing the homogenized oil is then combined with a detergent, which may conveniently be dissolved in a saline solution prior to mixing. The amount of detergent is typically between about 0.02 and 0.20% by volume, preferably between about 0.10 and 0.20% by volume₀, based on the total volume of the composition. Any known detergent material may be used, including Tween-80 and Arlacel (manufactured by Atlas Chemical Company).

The mixture obtained after the addition of the detergent is then homogenized to form a suspension which has a high percentage of oil droplets coated with RDE and CWS as observed by observation under a microscope.

The invention will be further illustrated by the following examples, without limiting the invention in any way.

Example 1: Preparation of Crude Detoxified Endotoxin

A 650 mg sample of a methanol-chloroform precipitate prepared by the method of Chen et al., J. Infect. 128 543 (1973) was suspended in 150 ml of 0.1 N HCl in a 3-necked round bottom flask equipped with a condenser immersed in a sonicator. After sonication, the glass apparatus was lowered into an oil bath maintained at 120 ° C in which the internal temperature of the flask was brought close to or above the boiling point of the solution. Overheating of the solution was minimized by equipping the flask with a capillary tube connected through one of the necks to a nitrogen gas source. During the hydrolysis, a continuous stream of nitrogen was maintained.

The hydrolysis was carried out for 30 minutes, after which the solution was cooled in an ice bath, sonicated to disperse the solid materials, and then distributed in Corex tubes. The flask was washed with distilled water to remove any solids adhering to the sides of the flask, and the washings were added to the suspension in the Corex tubes. It was centrifuged at 12,000 rpm for about 80 minutes.

The supernatant was decanted and discarded. The solid residue was resuspended in distilled water, sonicated until well dispersed in the suspension and recentrifuged. The centrifuging process was then repeated. The residue was taken up in distilled water, frozen in the shell and lyophilized to give 382 mg of crude lipid A. 150 mg of this material was treated with cold (0 ° C) acetone to remove fatty acids, sonicated, and filtered through a Whatman No. 1 gravity filtration apparatus at 5 ° C. After drying, 100 mg of crude detoxified endotoxin was obtained.

Example 2: Preparation of crude, detoxified endotoxin

A sample of 120 g of MCP (methanol-chloroform precipitate) was suspended in 12 ml of absolute alcohol, sonicated to disperse the solid material, and distributed into 6 (1 x 10 cm) 45 screw cap vials. 2 ml of 0.2N HCl was added to each tube and the resulting suspension was incubated in boiling water bath for 45 minutes. After hydrolysis, the tubes were cooled in an ice water bath and centrifuged for about 10 minutes at 2500 rpm. The supernatant was decanted and the residue was added to its solution with a 2: 1 mixture of chloroform / methanol. The 50-phase solution was again centrifuged at 2500 rpm for 10 minutes. The upper aqueous phase was discarded and to each tube was added 1 ml of a 4: 1 mixture of chloroform / methanol to give a clear solution. The solutions were combined and the solvent evaporated on a rotary evaporator. The residue became

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- 6 dried under high vacuum and then freeze-dried to give 45 mg of the crude lipid A. 20 mg of this material was treated with cold (0 ° C) acetone, sonicated and filtered through a Whatman No. 1 gravity filtration apparatus at 5 ° C. After drying, 13 mg of crude detoxified endotoxin remained.

Example 3: Preparation of refined detoxified endotoxin

110 g LH-20-100 (25 to 100 pm particle size: Pharmacia) were combined with 600 ml of a 1: 1 mixture of chloroform / methanol, which mixture was then allowed to stand for 30 minutes. The resulting slurry was placed in a pressure port equipped 25^x 1000 mm glass chromatography column (BRL Laboratories). After the package was complete, the column was connected to an ISCO Model 132 pump via a Teflon pressure hose. 400 ml of a 4: 1 mixture of chloroform / methanol were pumped through the column at a rate of 3 ml / min. 100 mg of the crude detoxified endotoxin, prepared according to the procedure of Example 1, were applied to the column in 2.5 ml of a 4: 1 chloroform / methanol mixture via a sample line. The flow rate was reduced to 1 ml / min, and after receiving 150 ml of eluent, the column effluent was connected to a fraction collector. Four fractions were collected and refined detoxified endotoxin fractions were determined by thin layer chromatography analysis of the fractions [E. Merck, 0.25 mm thick, chloroform / methanol / H₂O / NH₄OH (50: 25: 4: 2) as eluent].

The refined detoxified endotoxin fractions were pooled and the solvent was evaporated to yield 30 mg of refined detoxified endotoxin as a white powder.

Example 4: Preparation of refined detoxified endotoxin g DEAE cellulose (Whatman DE-32) was suspended in 150 ml of glacial acetic acid and stirred gently for 10 minutes to obtain a powder slurry. The mixture was allowed to stand overnight.

The slurry was poured into a 25 x 400 mm column and allowed to settle tapped, after which the excess acid was removed. The column was washed with 2000 ml of methanol and then with 200 ml of a 4: 1 mixture of chloroform / methanol. A

100 mg sample of the crude detoxified endotoxin prepared according to the procedure of Example 1 was added to the column in 3 ml of a 4: 1 mixture of chloroform / methanol or a 20: 1 mixture of chloroform, methanol and water. The column was eluted with 350 ml of a 4: 1 mixture of chloroform / methanol followed by 300 ml of a 99: 1 mixture of methanol / water. Using a linear gradient elution apparatus, the column was eluted with 2000 ml with a linear gradient beginning with 100% methanol and ending with 0.2M acetic acid in methanol. The elution of the acid was carried out at a rate of 6 ml / min and 15 ml fractions were taken. Each other fraction was assayed for total phosphorus content by the method of Bartlett GR, J.Biol.Chem. 234, 466-471 (1959). The fractions were combined and evaporated almost to dryness on a rotary evaporator and taken up in 10 ml of a 2: 1 mixture of chloroform / methanol and 40 ml of 0.001M acetic acid in a separatory funnel. The lower layer was separated, filtered through Whatman # 2 filter paper, and evaporated to dryness to yield 19.2 mg of refined detoxified endotoxin.

Example 5:

Thirteen guinea pigs of the genus 2, which showed a tumor growth of the cell line 10, which was about 9 mm in size, were once with 0.4 ml of a sterile oil droplet emulsion, u.zw. Drakeol 6 VR mineral oil (Pennsylvania Refining Company, Butler, Pennsylvania) containing 50 pg RDE and 50 pg CWS injected directly into the tumor tissue.

At the end of a three-month period, the animals were examined and found to have undergone a total regression of tumor growth in 12 out of 13 animals. In a control experiment, 6 species were 2 of guinea pigs, which had a cell line 10 tumor growth of about 9 mm^-once injected with 0.4 ml containing 50 pg of RDE alone. The injections were made directly into the tumor tissue. None of the 6 tumors showed signs of regression after 3 months.

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