JPS6253485B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION This invention relates to pyridine-soluble extracts derived from microorganisms, which extracts have antitumor properties when combined with cell wall skeletons and trehalose dimycolate, and to uses thereof. Bacteria such as Corynebacterium parvum
It has been the subject of experiments to isolate and characterize components that contribute to the inhibition of tumor growth [e.g., Cantrell et al.
Research 39, pp. 3554-3563 (September 1979), Anti
Tumor Activity and Lymphoreticular
Stimulation Properties of Fractions Isolated
from C.parvum]. Apart from antitumor activity, C. parvum has been shown to be an effective stimulator of the lymphoreticular system and embryonic development resulting in an unfavorable increase in the weight of the spleen and liver. Applicants have found that pyridine-soluble extracts of microorganisms have effective anti-tumor properties without the undesirable toxicity of prior art products. The cell wall skeleton is essentially a cell wall that has many of the proteins and lipids normally found in isolated cell walls. This is a polymerized mycolic acid arabinogalactan mucopeptide containing trehalose dimycolate (P3) and residues of terminally digested Mycobacterium tuberculosis proteins. Cell wall skeleton is M. smegmatis, M. phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheria, Corynebacterium・Parvum (Corynebacterium)
parvum), M. kansasii, M. tuberculosis H37RV strain and
It can be obtained from any microorganism including, but not limited to, AyomaB strain, and M. bovis BCG strain. To produce the cell wall skeleton, the M. bovis BCG strain [Bacillus calmetsteigerin]
Bacteria such as Calmette-Guerin are first grown and then collected. The whole cell residue thus obtained is converted into a cell fractionator (Ribi Cell Fractionator) that crushes the cells.
Fractionator) Sorvall, model RF−
1] to separate the outer coat or cell wall from cytoplasmic impurities. The cell wall thus obtained is subjected to a series of solvent extractions and enzymatic treatments (eg trypsin and/or chymotrypsin treatment) to obtain a purified cell wall skeleton. Trehalose dimycolate (TDM) can be used for example in M. avium, M. frey, M. tuberculosis H37RV strain and Ayoma B strain, M. bovis.
BCG, obtained from microorganisms such as M. smegmatis, M. kansasii, Nocardia rubra, M. bovinis and Corynebacterium diphtheriae. Bacteria such as M. avium are grown, collected, and heat sterilized. The cells are then extracted with several solvents and the active solvent soluble fraction is extracted. This extract is further purified by a series of solvent extractions to obtain crude TDM [Azma et al., Journal of
the National Cancer Institute, Volume 52, 95–
101 pages (1974) Bioloyally active components
from mycobacterial cell walls.i.isolation and
composition of cell wall skeleton and
See component P ₃ ]. This description is incorporated into the specification by reference. Purified TDM can be obtained by further purifying crude TDM by centrifugal fine silica gel chromatography, as described by Azma et al. The invention therefore aims to provide a pharmaceutical composition comprising a pyridine-soluble extract of a microorganism together with a cell wall skeleton and trehalose dimycolate. Another object of this invention is to provide a method for treating tumors in non-human warm-blooded animals using a composition comprising a pyridine-soluble extract of a microorganism, a cell wall skeleton, and trehalose dimycolate. (Summary of the Invention) This invention contains about 7% to about 20% protein, about 10% to about 20% by weight protein,
a pyridine soluble extract from a microorganism comprising about 16% by weight sugars and about 35 to about 55% fatty acids;
A pharmaceutical composition comprising a cell wall skeleton (CWS) and trehalose dimycolate (TDM). Preferably, the extract contains about 12% by weight each of proteins and sugars and about 45% by weight of fatty acids. For example, M. bovis BCG, M. phlei, M. smegmatis,
M. kansasii, Nocardia rubra, Corynebacterium
Any microorganism can be used to produce pyridine soluble extracts, including Corynebacterium diphtheriae and Corynebacterium parvum. Corynebacterium parvum is particularly preferred. Whole cells of the microorganism, preferably in paste form, are mixed with pyridine. The mixture thus obtained is separated to obtain a supernatant fraction containing a pyridine-soluble extract and a pyridine extraction residue. Optionally, the pyridine residue is again subjected to a separation operation using pyridine as described above to separate an additional amount of the desired extract. The pyridine is then removed from the extract and the dried extract is dialyzed against a suitable liquid such as distilled water. Check microscopically for the absence of whole cell or cell fragment contaminants. The purified extract thus obtained can then be lyophilized by known methods to obtain a stable product. The pyridine soluble extract produced according to the method of this invention is combined with CWS and TDM to produce a composition that has effective antitumor activity without stimulating the induction of splenic and liver hypertrophy. Cancers that can be treated with this composition include animal tumors such as bovine squamous cell carcinoma, bovine fibrosarcoma, equine sarcoma, equine melanoma, equine squamous cell carcinoma, canine mammary carcinoma;
canine nematomas and canine melanomas, and human tumors;
Examples include breast cancer, lung cancer, colon cancer, malignant melanoma, squamous cell carcinoma and ovarian tumors. The composition is preferably administered directly to the tumor by injection in a pharmaceutically acceptable vehicle such as an oil emulsion under conditions detailed below. The compositions described above can be stabilized, for example by lyophilization, and reconstituted without loss of potency. For animal treatment, the amount of pyridine soluble extract in one injection is about 375 to about 2500 micrograms/ml. The amount of CWS and TDM is 125-375
Micrograms/ml. The number of milliliters of biologic to be injected into the tumor is determined by the size of the tumor according to the following table.

Table: The maximum dose per injection is approximately 40 mg for each of pyridine soluble extract and CWS, and that of TDM is 6 mg. The course of treatment consists of no more than six injections at intervals of about two weeks. The compositions of this invention in a suitable injection vehicle, such as an oil emulsion, are administered directly to a human tumor. The amount of pyridine soluble extract in one injection is approximately 200 ~
5000 micrograms, preferably about 800-1200 micrograms, the amount of CWS is about 50-2000 micrograms, and the amount of TDM is about 50-1200 micrograms.
It is 1000 micrograms. A preferred unit dose of CWS and TDM is about 475-525 micrograms. All above dose levels are typical 70Kg
based on adult patients. Injections are given approximately once a week, no more than 15 times in total. As mentioned above, compositions for the treatment of warm-blooded animals and humans can be used in the form of oil drop emulsions. The amount of oil used ranges from about 0.5 to about 3.0% by volume based on the total volume of the composition. Approximately 0.75 to approx.
Preferably, 1.5% by volume of oil is used. Examples of such oils include light mineral oil, squalene, squalane, 7-n-hexyl octadecane, Conoco superoil and Drakeol 6VR mineral oil (Penreco, Butler, Made in Pennsylvania]. The homogenized oil-containing mixture is then mixed with a detergent, optionally dissolved in a salt solution prior to mixing. The amount of detergent is about 0.02-0.25% by volume, preferably about 0.10-0.10% by volume based on the total volume of the composition.
0.20% by volume. Tween-80,
Any common detergent can be used, such as Arlacel (manufactured by Atlas Chemical). The mixture obtained by addition of detergent is then homogenized to produce a suspension which, when observed microscopically, contains a high proportion of oil droplets coated with the active ingredient. The invention will now be explained in more detail by way of example. However, this does not limit the scope of the invention. Example 1 Preparation of pyridine-soluble extract from Corynebacterium parvum Corynebacterium parvum (P. acnes, strain 4182) was incubated at 37°C for 48-72 hours in NIH thioglycollate medium. The cells were allowed to grow and harvested to obtain a whole cell paste. This paste was then washed with 500ml of distilled water. 90 g (wet weight) of the wash paste was mixed with 200 ml of pure pyridine and centrifuged at 1700 xg for 1 hour at 4°C. The pyridine soluble extract was taken as the supernatant fraction. The remaining residue was extracted with additional pyridine under the same conditions as above. The strained pyridine extract was collected using Whatman No. 1 paper and placed in a Buchi Rotavapor (manufactured by Brinkman Instruments, Westbury, New York) at 50°C. The solvent was removed by evaporation. The dried pyridine extract was extensively dialyzed against distilled water and lyophilized. The resulting purified pyridine extract contained about 12% protein, about 12% sugar and about 45% fatty acid by weight. Observe the extract with an electron microscope,
It was found that there was no contamination with whole cells and cell wall fragments. The yield of pyridine soluble extract was 9% (8.1 g). Example 2 Preparation of Pyridine Soluble Extract from M. bovis BCG Strain M. bovis BCG strain was grown for 3-4 weeks at 37°C in Sautons medium, harvested and washed whole cell paste. Obtained. 50 g (wet weight) of the cleaning paste was then processed as in Example 1 to obtain 7% (3.5 g) of pyridine soluble extract. This extract contained 15% protein, 10% sugar and about 52% fatty acids by weight. Example 3 Testing against strain-10 tumors in guinea pigs 300 micrograms of the pyridine-soluble extract prepared according to example 1 was applied directly to the tumor tissue of 6 strain-2 guinea pigs bearing strain-10 tumors grown to approximately 9 mm in diameter; A single injection of 0.4 ml of a sterile oil drop emulsion (Drakeol 6VR mineral oil (Pennsylvania Refining Co., Butler, Pennsylvania) containing 50 micrograms each of cell wall skeleton and trehalose dimycolate was given. Animals were examined after 3 months. 5 out of 6 animals
Complete degeneration had occurred in the animals. As a control experiment, a strain that grew to about 9 mm in diameter -
10 Tumor-bearing system - 2 Directly apply the above oil droplet emulsion without pyridine extract or cell wall skeleton and trehalose dimycolate to the tumor tissues of 6 guinea pigs.
A single injection of 0.4 ml was given. After 3 months, none of the 6 tumors showed signs of regression.

Claims (1)

[Scope of Claims] 1. A pyridine-soluble extract from a microorganism, cell wall, comprising about 7 to about 20% by weight protein, about 10 to about 16% by weight sugar, and about 35 to about 55% by weight fatty acid. A pharmaceutical composition comprising a pharmaceutically effective amount of each of skeletal and trehalose dimycolate together with a pharmaceutically acceptable carrier. 2 The microorganisms include M. bovis BCG, M.
M.phlei, M. smegmatis.
smegmatis), M. Kansasii, Nocardia rubra, Corynebacterium
2. A composition according to claim 1, wherein the composition is Corynebacterium parvum, and preferably Corynebacterium parvum. 3. The extract contains about 12% by weight of each protein and sugar and about 45% by weight of fatty acids, and the amount of pyridine-soluble extract and cell wall skeleton is about 40 mg or less each, and the amount of trehalose dimycolate is about 40 mg or less. 6 mg or less and the composition is in lyophilized form or in the form of an oil emulsion. 4 Oil is squalene, light mineral oil squalane, 7-n
- hexyl octadecane, Conoco superoil or Drakeol 6VR mineral oil, and in the composition,
4. The composition of claim 3, present in an amount of about 0.5 to 3.0% by volume based on the total volume of the composition. 5 Add detergent to the total volume of the composition from about 0.02 to
Contains 0.25% by volume, with pyridine soluble extract of approx.
200-5000 micrograms, the amount of cell wall skeleton is about 50-2000 micrograms, and the amount of trehalose dimycolate is about 50-1000 micrograms. 6. A pyridine-soluble extract derived from a microorganism comprising from about 7 to about 20% by weight protein, from about 10 to about 16% by weight sugar, and from about 35 to about 55% by weight fatty acid, each of a cell wall skeleton and trehalose dimycolate. A method for treating a tumor in a warm-blooded non-human animal, comprising directly injecting into the tumor of a warm-blooded non-human animal a pharmaceutical composition comprising a pharmaceutically effective amount of a pharmaceutically acceptable carrier. 7. A patent claim characterized by the direct injection into a tumor of a composition containing about 375 to 2500 micrograms/ml of pyridine soluble extract and about 125 to 375 micrograms/ml of cell wall skeleton and trehalose dimycolate, respectively. The method described in item 6. 8. The method of claim 6 or 7, wherein the composition is injected directly into the tumor no more than six times at intervals of about two weeks. 9 about 200 to 5000 micrograms, preferably about
800-1200 micrograms of pyridine soluble extract, about 50-2000 micrograms, preferably about
475-525 micrograms of cell wall skeleton, ca.
Claim 8 characterized in that a composition containing 1000 micrograms, preferably about 475 to 525 micrograms of trehalose dimycolate is injected directly into the tumor no more than 15 times at intervals of about one week. Method described.