Method of Preparing a Component Consisting of Sugar, Lipid and Protein Derived From Pseudomonas Aeruginosa Which Possess Anti-Tumor and Interferon Inducing Properties

Pseudomonas aeruginosa is known to be naturally resistant against most of the antibiotics in general use. Despite the fact that some few antibiotics are effective, the use of antibiotics is not effective in such cases as neonates who are physiologically immature in immune response or in cases when immunity depressant is administered. On the other hand, both adult human beings and animals possessing normal immunity could hardly be victims of natural infection by this bacterial genus, and even when infected, antibiotics are effective for them.

In view of the above, it can well be realized that immunity against this bacterial genus is effective for infection caused by the same bacillus. In fact, for a certain type of infection by this bacterial genus, either active or passive immunity (serum or plasma) is effective.

The thermostabile O antigen, a lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa is known as an antigen originating in Pseudomonas aeruginosa and possessing the capability of protecting human, mouse and rabbit from infection caused by the homologous serotype strains of Pseudomonas aeruginosa.

However, because this O antigen is immunologically specific in its protective activity, it has the disadvantage that humans and animals immunized by a particular O antigen can be protected from infections caused only by the bacterial strains belonging to the same homologous serotype strain as the immunogen but it can hardly be protective against such infections as are due to bacterial strains whose serological specificity is heterologous.

Nevertheless, according to a method of isolating OEP from Pseudomonas aeruginosa disclosed in the British Pat. No. 1,365,950, and the product referred to therein as CWP in the above U.K. patent being equivalent to the OEP referred to in connection with the present invention, OEP almost completely free of O antigen can be isolated from Pseudomonas aeruginosa, and this OEP has been proved to be an antigen which can also protect humans, mice and rabbits from infection by different serotype strains of Pseudomonas aeruginosa to a great extent.

A dissociant strain of Pseudomonas aeruginosa was inoculated in a fermenter and cultured at 37.degree. C. A quantity of an autolyzing agent such as toluene or chloroform was added to the bacterial culture several hours after the stationary state had been reached, so that it could be autolyzed. A component (OEP) consisting mainly of protein, and a small amount of lipid and sugar was isolated from the culture filtrate and purified by physicochemical methods until it was homogeneous. Then, the OEP thus obtained was processed by either proteolytic enzyme or reductant, or after the processing by reductant further digested by proteolytic enzymes derived from animal, plant or microorganism thereby preparing the bacterial component of Pseudomonas aeruginosa, possessing extremely strong anti-tumor and interferon inducing activities. This component possesses a specific preventive property against infection due to Pseudomonas aeruginosa and non-specific protective property against infections by Pseudomonas aeruginosa as well as the other bacterial strains. When immunized with the bacterial component of Pseudomonas aeruginosa mentioned above, an excellent preventive activity against virus and invasion of tumor cold be obtained, and this bacterial component could also prevent infections by certain serotype strains of Pseudomonas aeruginosa.

Accordingly, it is an object of the present invention to provide a method of obtaining OEP from Pseudomonas aeruginosa which is an improvement over the method disclosed in British Pat. No. 1,365,950.

Another object of the present invention is to provide a method of preparing a bacterial component of Pseudomonas aeruginosa possessing a more powerful biological activity (anti-tumor and interferon-inducing activities) by further processing OEP obtained according to the British Pat. No. 1,365,950 with either reductant or with proteolytic enzyme or with proteolytic enzyme after treatment with reductant.

An important object of the present invention is a bacterial component of Pseudomonas aeruginosa possessing anti-tumor and interferon-inducing activities.

Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.

The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to each of the others, and the composition possessing the features, properties, and the relation of constituents which are exemplified in the following detailed disclosure, and the scope of the invention will be indicated in the claims.

For a fuller understanding of the invention, reference is had to the following description taken in connection with the accompanying drawings, in which:

FIG. 1 is a graph showing the elution pattern of 2-mercaptoethanol-treated OEP resulting from a column chromatography on Sephadex G75; and

FIG. 2 is a graph showing the elution pattern of the substance of peak I in FIG. 1 treated with alkaline protease resulting from a column chromatography on Sephadex G75.

A dissociant strain of Pseudomonas aeruginosa was inoculated in a fermenter and cultured at 37.degree. C. A quantity of an autolyzing agent such as toluene or chloroform was added to the bacterial culture several hours after the stationary state had been reached, so that it could be autolyzed. A component consisting mainly of protein, and a small amount of lipid and sugar (hereunder abbreviated as OEP), was isolated from the culture filtrate and purified by physicochemical methods until it was homogeneous.

Then, the OEP thus obtained was processed by either proteolytic enzyme or reductant, or after the treatment by reductant further digested by proteolytic enzymes derived from animal, plant and microorganism thereby preparing the bacterial component of Pseudomonas aeruginosa, possessing extremely strong anti-tumor and interferon-inducing activities.

The bacterial component of Pseudomonas aeruginosa obtained by the above method possesses an extremely powerful biological activity. As for the method of isolating OEP composed of highly purified protein, further details are given in the description in the British Pat. No. 1,365,950 quoted hereabove.

More concretely, the "powerful biological activity" means anti-tumor and interferon-inducing activities as well as non-specific protection against bacillus and further vaccine activity preventing infection due to Pseudomonas aeruginosa strains regardless of their serotypes.

The present invention aims at the isolation of a bacterial component possessing an extremely powerful biological activity referred to hereabove, the isolation being effected by processing OEP having a powerful biological activity with reductant and digesting it thereafter with proteolytic enzyme.

Using the above method, OEP was obtained by a mass culture of bacterial strains of Pseudomonas aeruginosa. This OEP was proved to have a significant biological activity. When expressed in terms of inhibition of the growth of ascites tumor (sarcoma 180), the dose of OEP of 50% inhibition (ED.sub.50) was 1 .mu.g/kg mouse/day determined according to the method of Kuretani and his associates.

When this OEP is processed by reductants such as sodium borohydride, 2-mercaptoethanol, sodium monothiophosphate and dithiothreitol and fractionated by one of the fractionating methods such as gel filtration, ion exhange resin, salting out by ammonium sulfate and electrophoresis as well as the method using organic solvents such as acetone and ethanol or the combination thereof, a bacterial component possessing a biological property more effective in anti-tumor and interferon-inducing activities can be isolated (refer to Example 1).

As shown in one of the examples, its activity against sarcoma 180A expressed in ED.sub.50 value was 0.45 .mu.g/kg mouse/day and interferon-inducing activity was 0.01 .mu.g/ml in the concentration representing 18 units. Then, the substance isolated by reductant and possessing a powerful biological activity was digested by proteolytic enzyme. For the above process, proteolytic enzymes derived from aminals, plants and microorganisms are applicable. Suitable enzymes are alkaline protease derived from Bacillus subtilis, trypsin and chymotrypsin, and alkaline protease derived from Pseudomonas aeruginosa.

A bacterial component possessing the extremely powerful biological activity referred to hereabove can be isolated by one of the conventional fractionating methods such as gel filtration, diaphragm, ion exchange resin, electrophoresis and salting out of ammonium sulfate as well as the method using organic solvents such as acetone and ethanol or the combination thereof after the digesting process has been made by one of or a mixture of these proteolytic enzymes, as will be shown in Examples 2 and 3.

The substance obtained through the treatment by enzyme consists of such components as protein, sugar, aminosugar and lipid whose chemically analyzed values are 15-50%, 10-20%, 10-20%, and 25-40% respectively.

(1) Details on the chemically analyzed values of these substances and chemical properties are given in the relative examples (Examples 1, 2 and 3).

(2) Biological activity (although this activity can be produced by means of injection, it can also be produced by oral administration).

(i) Activity as an antigen (vaccine) possessing specific or non-specific preventive property against infection.

As a non-specific preventive effect actually displayed, mouse infections caused by Klebsiella, Salmonella typhi and Salmonella typhimurium were successfully prevented 24 hours after the mice were immunized with OEP (prior to the treatment by proteolytic enzyme).

Mice were protected against challenges carried out with Fisher's 7 immunotype strains and 14 kinds of Homma's serotype strains shown in Tables A and B.

Mice were immunized by intraperitoneal administration of OEP, and a week after the last injection, each mouse was intraperitoneally injected with 0.5 ml of live bacilli suspended in 2-5% mucin solution.

S: significant.

Mice were injected with OEP intraperitoneally, and a week after the last injection, they were challenged intraperitoneally with live bacilli suspended in 0.5 ml of 2-5% mucin solution.

As shown in Example 5, the experimental results on protective immunity conferred by the component which had been treated with proteolytic enzyme and isolated at peak 1, revealed that it could not protect mice from all the serotype strains belonging to Fisher's immunotypes, though the OEP was protective before the treatment with proteolytic enzyme (refer to Table C).

Mice were injected with protease-treated OEP intraperitoneally, and in a week they were challenged intraperitoneally with live bacilli suspended in 0.5 ml of 2.5% mucin solution.

S: significant

Ns: not significant

Therefore, although a vaccine prepared from protease-treated OEP is not quite as effective as the intact OEP which was not given by the enzyme treatment, it still has a powerful activity to a broad extent as an immunopotentiator in the in vivo protection mechanism.

(ii) Anti-tumor activity:

As for the measurement of anti-tumor activity, the effect on the inhibition of the growth of tumor against mouse ascites tumor (sarcoma 180) was investigated using the method employed by Kuretani et al. (Chem. Pharm. Bull, 17,848, 1969).

Female mice weighing 18-20 g, ddN, each group divided into 6 mice, were used for the experiment. The method adopted was the intraperitoneal transplantation of 0.05 ml of seven-day-old ascites tumor cells (1 .times. 10.sup.7) into each mouse; from the next day on, 0.05 ml of a saline solution containing the test sample was intraperitoneally injected into a mouse once daily for 5 days. A specimen of ascites fluid was removed 7 days after the tumor transplantation, and the total cell volume contained therein was expressed as the percentage of the total cell volume of the control groups (groups given physiological saline solution.

When investigated by this method, the OEP before the treatment by proteolytic enzyme proved to be a substance whose dose (ED.sub.50) required for inhibiting the growth of 50% tumor cells corresponded to 1 .mu.g/kg mouse/day and exhibit almost no deviation due to the difference of lots used for the culture as shown in Table D.

It was also found out that OEP was a substance possessing an extremely powerful activity when compared with bacterial components derived from other microorganisms and the components other than OEP derived from Pseudomonas aeruginosa as shown in Table E. Furthermore, as shown in Examples 1, 2, and 3, it was confirmed that the above ED.sub.50 value of OEP had increased up to 0.2 .mu.g/kg mouse/day after chemical treatments, and that these anti-tumor activities could also be expected by oral administration.

(iii) Interferon-inducing activity:

The interferon-inducing activity (IF) was measured by the method employed by Kojima et al. ("Interferon", Igakushoin, 74-83, 1970, Kitasato Arch. Exp. Med., 43, 1970, Japan J. Microbiol., 18, 217-222, 1974).

A culture solution containing 0.005% calcium chloride and 10% calf serum was used as the buffer solution of 1/200 mol. 10 fold diluent of the test solution was then prepared, and this test solution was mixed with 1-2 .times. 10.sup.7 cells of young rabbit (spleen cells or lymphnode cells) weighing 800-1,000g. This mixture was cultivated in a tube at 25.degree. C for 24 hours. As for the centrifuged supernatant, the productivity of interferon-inducing property was determined by use of RK13 cell line of rabbit kidney and Vesicular Stomatitis virus.

Interferon unit (IF value) was expressed by a reciprocal of the dilution multiple of the above culture supernatant required for reducing the Plaque count (approximately 100) of the control group down to 50%. As shown in Examples 1, 2 amd 3 as well as Table F, it was found out that the IF value of OEP could be increased up to a potency of approximately 10 times higher when OEP was treated with enzyme.

The production of serum interferon was recognized when OEP was intravenously injected into rabbits. The development of vaccina cirus on skin was inhibited by orally administering OEP to rabbits (see Example 4).

90 mg of OEP was dissolved into a buffer solution of pH 8.0, 2-mercaptoethanol was added so that a 4% solution (volume/volume) was obtained. The solution after reacting at room temperature for 18 hr. ws fractionated by column chromatography on Sephadex G75 equilibrated with the buffer solution in the presence of 4% 2-mercaptoethanol, and then separated into 2 fractions as shown in FIG. 1.

In the fraction at Peak I, 42 mg, dry weight, representing 46% of the original sample was collected and the rest was collected in the fraction isolated at Peak II. An investigation carried out on the activity of these two fractions revealed that ED.sub.50 at Peak I against ascites tumor (sarcoma 180) was 0.45 .mu.g/kg mouse/day, ED.sub.50 for the Peak II fraction was more than 30 .mu.g. The IF activity represented 18 units with the concentration of 0.01 .mu.g/ml, while the same at Peak II was higher than 1 .mu.g/ml. As is proved by the above result, most of the active portion of OEP (anti-tumor and IF-inducing activities) could be fractionated out because of the correlation between the activity and yield at Peak I.

When measured by the method moified by Lowry et al., using bovine serum albumin as the standard protein, a substance obtained from a fraction at Peak I possessing a powerful activity contained 77% protein, and when measured by an amino-acid analyzer and expressed in molar ratios, its amino-acid composition was 6.6% lysine, 2.3% histidine, 4.3% arginine, 8.4% aspartic acid, 5.7% threonine, 5.4% serine, 11.7% glutamic acid, 4.6% proline, 8.3% glycine, 11.6% alanine, 7.3% valine, 2.1% methionine, 4.8% isoleucine, 8.9% leucine, 2.8% tyrosine, 3.9% phenylalanine, 1.2% tryptophan, and 1.2% cystine.

The sugar content was found to be 4.5% glucose when measured by a method using anthrone reagent, and the measurement made by gas chromatography revealed that the sugar was composed of 13.9% rhamnose, 4.2% mannose and 61.8% glucose, the rest of the composition being unidentified.

The amino-acid content was 4.5% when measured by the Elson-Morgan method for which glucosamine was used as the standard sample. 2-keto-3-deoxyoctonate (KDO) was 1.3% when determined by the method using thiobarbiturate, and phosphorus was 1.2% by Allen's method. As the result of chemical analyses, lipid was proved to be 8.8%, while the composition of fatty acid as determined by gas chromatography was as follows:

C16:0 43.5%; c16:1 5.8%; c18:0 4.8%; c18:1 22.2%.

an electro-focussing method had revealed an isoelectric point of this substance at pH 4.35 and the maximum absorption in its ultraviolet region at 278 nm.

20 mg of the substance at Peak I in Example 1 which possessed a powerful activity was dissolved in a buffer solution of pH 8.0. To this solution 1 mg (5% weight/weight) of alkaline protease derived from B. subtilis (Nagase Sangyo K.K. in Japan) was added and kept at 37.degree. C for 18 hours.

Then the mixture was subjected to column chromatography on Sephadex 75 and was separated into 3 fractions (elution pattern by column chromatography is shown in FIG. 2). An investigation carried out on the activities of these 3 fractions revealed that ED.sub.50 at Peak I was 0.2 .mu.g/kg mouse/day against sercoma 180A and the same at Peak II and III was more than 10 .mu.g.

On the other hand, the interferon-inducing activity was shown in the concentration of 0.01 .mu.g/ml with 184 units at Peak I and 10 .mu.g/ml with 45 units at Peak II and no activity was exhibited at lower concentrations. In other words, a substance possessing an extremely powerful activity was fractioned at Peak I. When measured by the method employed for Example 1, the chemical composition of this substance fractionated at Peak I was 17% protein, 14.5% sugar, 12.5% aminosugar, 3.8% KDO, 2.7% P and 31% lipid, and the compositions of both sugar and fatty acid were the same as those shown in Example 1.

20 mg of the substance at Peak I in Example 1 which possessed a powerful activity was dissolved in a buffer solution of pH 7.8. To this solution 1 mg of trypsin (Sigma Co.) was added and the solution was kept at 37.degree. C for 18 hours, and then fractionated by column chromatography on Sephadex G75 thereby obtained 3 fractions just as in Example 2. 8 mg (dry weight) of the substance was collected from the fraction which was first eluted. When the activity was investigated, the fraction at Peak I showed 0.25 .mu.g/kg mouse/day against sarcoma 180A and interferon-inducing activity showed the concentration of 0.01 .mu.g/ml with 214 units. A bacterial component with a powerful activity was thus isolated at Peak I. It was also found out that the substance fractionated at this Peak I contained 47% protein.

OEP was orally administered to young rabbits weighing 600-900 g for a total of 7 times in a single dose of 1 mg/kg 6, 4, 2 and 0 days and 1, 3 and 5 days, respectively, before and after the challenges were made against virus. On the day OEP was administered, 0.1 ml of various dilutions of virus solution was given to the rabbits intracutaneously. Determination was made 7 days after the challenge against virus was carried out, and vaccine lesion in rabbit skin was successfully inhibited by the component as shown in Table G.

Mice divided into groups of 5 were used. The fraction isolated at Peak I described in Example 2 was used for immunization by intraperitoneal administration twice a week for 3 weeks. A week after the final day of injection, live bacilli of each of 7 immunotype strains of Fisher et al. were suspended in a sterile solution, and the suspension was used for the intraperitoneal challenge. After the challenge was carried out, the mice were observed for 1 week, and a significant difference in the mortality between immunized and non-immunized groups was determined by probit analysis diagrammatic method. As shown in Table C, mice immunized with the fraction isolated at Peak I in Example 2, were protected against challenge with 5 out of 7 Fisher's immunotypes.

The amino acid composition mol fraction in % before and after the treatment with proteolytic enzyme, is as follows:

Chemical analyses of OEP before and after protease digestion (Alkaline protease, opt pH 8, B. subtilis) are as follows:

It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in carrying out the above method and in the composition set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention, which, as a matter of language, might be said to fall therebetween.