JPS5917990A - Purified pyridine soluble extractderived from microorganism, production thereof and drug containing same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION This invention relates to pyridine-soluble extracts derived from microorganisms which, when combined with cell wall skeletons (CWS), provide pharmaceutical compositions with anti-tumor properties. .

Corynebae
Bacteria such as C. terium parvum have been the subject of experiments to isolate and characterize components that contribute to inhibition of tumor growth [e.g.
ntrell) etc., Cancer Re1Iearch
39X, pp. 3554-3563 (September 1979), has been shown to be an effective stimulator of the phosphorus A reticular system and embryonic development resulting in an unfavorable increase in tile and liver weights. Applicant has disclosed that a pyridine-soluble extract of a microorganism is
It has been found that it has 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 excised cell walls. It is obtained from, but not limited to, a polymerized mycolic acid arabinogalactan mucomicroorganism containing trenolose dimycolate (P3) and undigested Mycobacterium tuberculosis protein residues.

To produce the cell wall skeleton, use the M, &bisBCG strain [Pacillus calmetsutieferin (Bae111usCa
Bacteria such as L. lmette -Gu@rln) are first grown and then harvested. The whole cell residue obtained in this way is used in a cell fractionator [Lipi Cell 7 Luxionator (fHbiC@11 Frac
tronator) Sorvall.

model RF-1) to separate the envelope 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 chymotrigsin treatment) to obtain a purified cell wall skeleton.

The invention therefore aims to provide a pharmaceutical composition comprising a pyridine-soluble extract of a microorganism together with a cell wall skeleton.

Another object of this invention is to provide a method for producing pyridine-soluble extracts of microorganisms.

Another object of Jin's 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 and a cell wall skeleton.

V, bottom margin (Summary of the invention) This invention contains about 7 to about 20 weight, 1 t% protein, about 10
The present invention relates to a pharmaceutical composition comprising a pyridine soluble extract from a microorganism comprising up to about 16% by weight of sugars and about 35 to about 55 M fatty acids together with a cell wall skeleton (CWS). Preferably, the extract contains about 12% by weight each of proteins and sugars and about 45% by weight of fatty acids.

For example, M, bovig BCG,
Any microorganism can be used to produce pyridine-soluble extracts, including M., Freyi, and M. Frey. Particularly preferred is Gocterium pulpum.

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 dipyridine-soluble extract and a pyridine extraction residue. Optionally, the viridin residue is again subjected to a separation operation using viridin 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 prepared according to the method of this invention is combined with CWS to produce a composition with effective antitumor activity without stimulating the induction of tile 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 adenoma, and Includes canine melanoma, as well as human tumors such as breast cancer, lung cancer, colon cancer, malignant melanoma, squamous cell carcinoma and ovarian tumors.

Preferably, the composition is administered directly to the tumor by injection in a medicinal medium such as an oil emulsion, under conditions detailed below.

The composition is stabilized, for example by lyophilization,
and can be reconstituted without loss of potency.

For animal treatment, the pyridine soluble extract violet in one injection may range from about 375 to about 2500 micrograms/d.
It is. The amount of CWSO 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.

The maximum dose for injection is approximately 40 II #9 for each of the pyridine soluble extract and G.

The course of treatment consists of no more than six injections at intervals of approximately 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 about 200-5000 micrograms, preferably about 800-1200 micrograms, and the amount of CVI/S is about 50-2000 micrograms. A preferred unit dose of CWS is about 475-5
It is 25 micrograms. All of the above dosage levels are based on a typical 70 hour adult patient.

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 is approximately 0.5 based on the total volume of the composition.
~3.0 capacity. Approximately 0.75 to approximately 1.5
Preferably, a volume of oil is used. Examples of such oils include light mineral oil, squalene, squalane, 7-n-hexyl octadecane, Conoco/4-oil.
oeo sup@roil) and Dra
k@ol) 6 V R mineral oil [Penraco
), Butler, 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 approximately 0.02 to 0.0 based on the total volume of the composition.
．． 25 by volume, preferably about 0.10 to 0.20 by volume. Any common detergent can be used, such as Tween-80, Arlacal (manufactured by Atlas Chemical), etc.

The mixture obtained by addition of the detergent is then homogenized to produce a suspension which, when viewed microscopically, contains a high proportion of oil droplets coated with the active ingredient.

Next, this invention will be explained in more detail by way of example; however, the scope of Jin's invention is not limited thereby.

Corynebacterium nogluvum [P, acnes (P,
aen@s), strain 4182] was grown in NIH thioglycollate medium for 48 to 72 hours at 37°C,
Then, bacteria were collected to obtain a whole cell paste.

This paste was then washed with soomg distilled water.

90g (wet weight) of the cleaning paste was mixed with 200d of pure pyridine and centrifuged at 1700X, f for 1 hour at 4°C. The pyridine soluble extract was collected as a supernatant fraction. The remaining residue was extracted using additional gin under the same conditions as above.
an) Collect the pyridine extract after filtration using AIF paper and
hi Rotavipsr) [Printerman I/Struments (BrinkmanInstrum@nts)
The solvent was removed by evaporation at 50° C. in a 100 ml (Made in Westbury, New York). The dried pyridine extract was extensively dialyzed against distilled water and lyophilized. The obtained purified pyridine extract contains about 12% protein by weight, about 12
It contained % sugar by weight and about 45% fat, acid by weight. The extract was observed under an electron microscope and found to be free of contamination by whole cells and cell wall fragments. The yield of pyridine soluble extract was 9% (8,1,9).

Preparation of the M, &bis BCG strain by Sautons
) The cells were grown in culture medium at 37° C. for 3 to 4 weeks and harvested to obtain a washed whole cell paste. 50 g (wet weight) of the cleaning paste was then treated as in Example 1, containing 7% (
3, 5,! A pyridine soluble extract of i+) was obtained. This extract contained 15% by weight of protein, 10% by weight of sugars and approximately 52% by weight of fatty acids.

Example 3 Testing on Line 10 Tumors in Guinea Pigs Three hundred micrograms of pyridine soluble extract prepared according to Example 1 and 50 micrograms of the pyridine soluble extract prepared according to Example 1 were applied directly to the tumor tissue of Line 2 Guinea pigs with Line 10 tumors grown to approximately 9 m in diameter. Sterile oil droplet emulsion containing micrograms of cell wall skeleton [Drakeol 6VR mineral oil (Pennsylvania Refining Co., Patra, Pennsylvania) 0.4 dt single injection L
7'c.

When the animals were examined after 3 months, complete regression of the tumor had occurred in 6 of 7 animals.

As a control experiment, the above oil droplet emulsion 9.4d containing no pyridine extract or cell wall skeleton was directly injected once into the tumor tissue of a line 2 guinea pig 6 having a line 10 tumor grown to about 9 mm in diameter. After 3 months, none of the 6 tumors showed signs of regression.

Patent applicant Livi Imnochem Research, Incorporated Patent agent Akira Ikugi Patent attorney Kazuyuki Nishidate Patent attorney Fukumoto Seki patent attorney Akiyuki Yamaguchi 501-

Claims (1)

[Claims] 1. (a) Prepare a bacterial whole cell paste, (
b) washing the paste; (,) reacting the paste with pyridine to form an extract and a residue; (d) removing gin from the extract; and (.) reacting the paste with pyridine. A method for producing a purified pyridine-soluble extract derived from a microorganism, which comprises obtaining a purified pyridine-soluble extract by dialyzing the obtained dry extract. 2. The method according to claim 1, wherein the purified pyridine-soluble extract is further freeze-dried. 3. Microorganisms are M, &bis(M, bovls)BC
G. M, 7 lei (M, phl*i→, M, smegmatism (Coryn*bseterlum parvum)
1 and preferably Coryn@bael@rium parvum
) The method according to claim 1 or 2. 4. Obtaining an additional amount of pyridine-soluble extract by reacting said residue with pyridine, claim 1.
The method according to any one of Items 1 to 3. 5. A pyridine soluble extract from a microorganism comprising from about 7 to about 20 weight percent protein, from about 10 to about 16 weight percent sugar, and from about 35 to about 55 weight percent fatty acids. 6. The amount of protein and sugar is about 12% by weight each,
The extract according to claim 5, wherein the amount of fatty acids is about 45% by weight. 7. A pharmaceutically effective amount of a microorganism-derived gyricin soluble extract comprising from about 7 to about 20% protein, from about 10 to about 16% sugar, and from about 35 to about 55% fatty acid. together with a cell wall scaffold and a pharmaceutically acceptable carrier. 8. The composition according to claim 7, wherein the carrier is an oil droplet emulsion, and the oil is in lyophilized form with a volume of 0.5 to 3.0 with respect to the total volume of the composition. . 9. The oil is squalene, light mineral oil, squalape 7-n-hexyl octadecane, Conoco super oil (Conoco
eo auperoil) or Drak
eol) 6 VR mineral oil, and there is present in the composition about 0.02 to 0.25 volumes of detergent relative to the total volume of the composition. Composition of. 10. The amount of the pyridine-soluble extract and the cell wall skeleton are each less than 40rn9, and preferably the amount of the pyridine-soluble extract is about 200 to 5000 micrograms, and the amount of the cell wall skeleton is about 50 to 5000 micrograms. 8. The composition of claim 7, wherein the composition is 2000 micrograms. 11. A pharmaceutically effective amount of 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 sugar, and from about 35 to about 55% by weight fatty acid. , preferably about 375-2500 micrograms/a of pyridine soluble extract and about 125-37 micrograms/a, together with a cell wall skeleton and a pharmaceutically acceptable carrier.
A method of treating a tumor in a warm-blooded animal, comprising directly injecting a pharmaceutical composition comprising 5 milligrams/ml of cell wall skeleton into the tumor no more than 6 times at intervals of about 2 weeks. 12. Applying to the tumor a composition comprising about 200-5000 micrograms, preferably about 800-1200 micrograms of pyridine soluble extract and about 50-2000 micrograms, preferably about 475-525 micrograms of cell wall scaffold. 12. A method according to claim 11, characterized in that the method comprises direct injection. Margin below