JPS6254774B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION The present invention relates to a pyridine soluble extract derived from microorganisms, which extract contains trehalose dimycolate (TDM) and acetone precipitation of endotoxin glycolipids extracted with chloroform-methanol. Provides a pharmaceutical composition that has antitumor properties when combined with a by-product (ACP). Bacteria such as Corynebacterium parvum have 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 spleen and liver weight. Applicants have found that pyridine-soluble extracts of microorganisms have effective anti-tumor properties without the undesirable toxicity of prior art products. Trehalose dimycolate ( TDM ) can be used for example in M. avium , M. frey , M. tuberculosis H37RV strain and Ayoma B strain, M. bovis BCG, M. smegmatis , M. kansasii , Microorganisms such as Nocardia rubra, M. bovinis and Corynebacterium diphtheriae are obtained. 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 25, 95~
101 pages (1974) Bioloyally active components
from mycobacteria cell walls.i.isolation and
composition of cell wall skeleton and
See component P3]. This description is incorporated herein by reference. Purified TDM can be obtained by further purifying crude TDM by centrifugal fine silica gel chromatography, as described by Azuma et al. The acetone precipitated byproduct (ACP) of endotoxin glycolipid extracted with chloroform-methanol has no tumor-degrading ability when used alone or in combination with trehalose dimycolate. For a more complete review of ACP, its properties and methods of preparation, see Ribi et al., Cancer Immunol, Immuother, Vol. 7, pp. 43-58 (1979) Peptides as
Requirement for Immunotherapy of the Guinea
-Pig Line-10 Tumor with Endotoxins is incorporated herein by reference. ACP is associated with the following genera: Salmonella , Shigella , Escherichia , Brucella , Bordetella , Citrobacter , Pseudomonas, Pasturella , and Neisseria . ( Neisseria ), Proteus ( Proteus ),
It can be prepared from any Enterobacteriaceae including, but not limited to , Klebsiella , and Serratia. Typically the following species i.e. S. minnesota ( S.
minesota), S. teipimurium ( S.
typhimurium), B. pertussis ,
B. abortus , S. enteritidis , E. coli , S. teipi .
typhi) , S.marcescens , S.
S. typhosa, Shigella flexni and S. abortus equi are used. The present invention therefore aims to provide a pharmaceutical composition comprising a pyridine-soluble extract derived from a microorganism together with trehalose dimycolate and an acetone precipitation by-product (ACP) of endotoxin glycolipid extracted with chloroform-methanol. shall be. Another object of this invention is to provide a method of treating tumors in warm-blooded animals and humans using a composition comprising a pyridine-soluble extract of a microorganism, TDM and ACP. (Summary of the Invention) This invention provides about 7% to about 20% by weight of protein, about 10% by weight of protein;
The present invention relates to a pharmaceutical composition comprising a pyridine soluble extract from a microorganism comprising ~16% by weight of sugars and from about 35% to about 55% by weight of fatty acids, together with TDM and ACP. 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 Corymebacterium 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. Verify the absence of whole cell or cell fragment contaminants by electron microscopy. 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 TDM and ACP 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 TDM and ACP is 375-1250
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 doses per injection are approximately 40 mg for pyridine soluble extract, approximately 6 mg for TDM, and approximately 20 mg for ACP. 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 TDM is about 50-1000 micrograms and the amount of ACP is about 150-1000 micrograms. Preferred unit doses of both TDM and ACP are about 475-525 micrograms. All of the above dose levels are typical 70
Based on adult patients with Kg. 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 Instrument, Westbury, NY) 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 approximately 52% fatty acids by weight. TDM and ACP in a pharmaceutically acceptable medium
Pyridine soluble extract in combination with was significantly effective in treating tumors and caused complete regression of tumors in most cases.

Claims (1)

[Claims] 1. Trehalose, a pyridine-soluble extract derived from a microorganism, 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 dimycolate, an acetone precipitated by-product of endotoxin glycolipid extracted with chloroform-methanol, together with a pharmaceutically acceptable carrier. 2 The microorganisms include M. bovis BCG, M.
M.phlei, M. smegmatis.
smegmatis), M.kansasii, Nocardia rubra, Corynebacterium
The composition according to claim 1, which is Corynebacterium parvum. 3. the extracts each contain about 12% by weight of proteins and sugars and about 45% by weight of fatty acids, and the amount of pyridine-soluble extract is about 40 mg or less;
Claim 1 or 1, wherein the amount of trehalose dimycolate is about 6 mg or less, the amount of acetone precipitation by-product is about 20 mg or less, and the composition is in lyophilized form or oil droplet emulsion. Composition according to item 2. 4 The oil is squalene, light mineral oil, squalane, 7-
n-hexyl octadecane, Conoco superoil or Drakeol 6VR mineral oil, and in the composition:
present at about 0.5 to 3.0% by volume based on the total volume of the composition, and about 0.02 to 3.0% based on the total volume of the composition
Claim 3 in which 0.25% by volume detergent is present
Compositions as described in Section. 5 The amount of extract is about 200 to 5000 micrograms, and the amount of trehalose dimycolate is about 50 to 5000 micrograms.
1000 micrograms and the amount of acetone precipitation by-product is about 150 to 1000 micrograms. 6. With a pyridine-soluble extract from a microorganism comprising from about 7 to about 20% by weight protein, from about 10 to about 16% by weight sugars, and from about 35 to about 55% by weight fatty acids, trehalose dimycolate and chloroform-methanol. The treatment of tumors in warm-blooded animals by direct injection into the tumor, comprising a pharmaceutically effective amount of each of the acetone-precipitated by-products of the extracted endotoxin glycolipids, together with a pharmaceutically acceptable carrier. A composition according to claim 1 for use in a method. 7. The composition of claim 6 containing about 375 to 2500 micrograms/ml of pyridine soluble extract, and about 375 to 1250 micrograms/ml each of trehalose dimycolate and acetone precipitation byproduct.