GB2122897A - Treatment of cancer with microorganism obtained products - Google Patents
Treatment of cancer with microorganism obtained products Download PDFInfo
- Publication number
- GB2122897A GB2122897A GB08317744A GB8317744A GB2122897A GB 2122897 A GB2122897 A GB 2122897A GB 08317744 A GB08317744 A GB 08317744A GB 8317744 A GB8317744 A GB 8317744A GB 2122897 A GB2122897 A GB 2122897A
- Authority
- GB
- United Kingdom
- Prior art keywords
- composition
- amount
- weight
- extract
- pyridine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A pharmaceutical composition comprising a purified pyridine-soluble extract obtained from a microorganism which contains 7 and 20% by weight of protein, 10 and 16% by weight of sugar, and 35 and 55% by weight of fatty acids which when combined with trehalose dimycolate and an acetone precipitated by- product of endotoxic glycol lipids extracted with chloroform-methanol in a pharmaceutically acceptable medium is useful as an anti-tumor agent in the treatment of animals and humans.
Description
SPECIFICATION
Pyridine soluble extract of a microorganism
The present invention is directed to a pyridine-soluble extract of a microorganism which, when combined with trehalose dimycolate (TDM) and an acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol (ACP), provides a pharmaceutical composition possessing anti-tumor properties.
Bacteria such as Corynebacterium parvum have been the subject of experimental work to isolate and characterise the component responsible for inducing inhibition of tumor growth (see, for example,
Anti Tumor Activity andLymphorectular Stimulation Properties of Fractions Isolated from C. parvum;
Cantrell, et al., Cancer Research 39, pgs. 3554-3563 (September, 1979)). Apart from anti-tumor activity, C. parum has shown to be a potent stimulator of the lymphorecticular system resulting in undesirable increases in spleen and liver weights and blastogenesis. Applicant has discovered that a pyridine-soluble extract of a microorganism possesses potent anti-tumor properties without the undesirable toxic effects associated with the prior art products.
Trehalose dimycolates (TDM), may be obtained from organisms such as, for example, M. avium,
M. phlei, M. tuberculosis (Strain H 37 RV and Ayoma B), M. bovis BCG, M. smegmatis, M. kansasii,
Nocardia rubra, M. bovinis and Corynebacterium diphtheriae.
Bacteria such as M. avium are grown, harvested and then heat killed. The cell mass is then extracted with several solvents and then an active, solvent soluble, fraction is extracted. This extract is further purified by a series of solvent extractions to provide crude TDM (see Biologically Active
Components from Mycobacterial Cells Walls. I. Isolation and Composition of Cell Wall Skeleton and
Component P3; Azuma, et al., Journal of the National Cancer Institute, Volume 52, pgs. 95-101, 1974) incorporated herein by reference. As disclosed in Azume et al., crude TDM may then be further purified by centrifugal microparticulate silica gel chromatography to give purified TDM.
An acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol (ACP) does not possess tumor-regressive properties when used alone or in combination with trehalose dimycolate. For a more complete discussion of ACP, its properties and methods of production, reference is made to Pep tide as Requirement for Immunotherapy of the Guinea-Pig Line- 10 Tumor with
Endotoxins; Ribi, et al., Cancer Immunol. Immunother., Volume 7, pgs. 43058 (1979) incorporated herein by reference. ACP can be prepared from any Enterbacteriaciae including, but not limited to, the following genera:
Salmonella, Shigella, Escherichia, Brucella, Bordetella, Citrobacter, Pseudomnas, Pasturella,
Neisseria, Proteus, Klebsiella, and Serratia.
The following species are typically employed:
S. minnesota, S, typhimurium, B. pertussis, B. abortus, S. enteritidis, E. coli, S. typhi,
S. marcescens, S. typhosa, Shigella flexni, and S. abortus equi.
According to the invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a purified pyridine-soluble extract obtained from a microorganism, said extract containing between about 7 and 20% by weight of protein, between about 10 and 16% by weight of sugar, and between about 35 and 55% by weight of fatty acids, trehalose dimycolate, and an acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol and a pharmaceutically acceptable carrier.
The pyridine soluble extract preferably contains about 12% by weight of each of protein and sugar and about 45% by weight of fatty acids.
Any microorganism may be used to obtain the pyridine-soluble extract including, for example,
M. bovis BCG, M. phlei, M. smegmatis, M. kansasii, Nocardia rubra, Corynebacterium diphtheriae and
Corynebacterium parvum. Corynebacterium parvum is especially preferred.
Whole cells of the bacteria preferably in the form of a paste, are mixed with pyridine. The resulting
mixture is separated to obtain a supernatant fraction which contains the pyridine-soluble extract and a pyridine residue. Optionally, the pyridine residue may be subjected to repeated separation procedures as described above using pyridine to remove further quantities 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. The absence of whole cells or cell fragment contaminants is confirmed by electron microscopy. The resulting purified extract may then be lyophilized by known methods to obtain
a stable product.
The pyridine-soluble extract produced in accordance with this invention may be combined with
TDM and ACP to produce a composition having potent anti-tumor activity without stimulating the
induction of spleen and liver enlargements. The cancers which may be treated by this composition
include animal tumors such as bovine squamos cell carcinoma, bovine fibrosarcoma, equine sarcoid,
equine melanoma equine squamous cell carcinoma, canine mammary tumors, canine adenoma and
canine melanoma and human tumors such as breast tumors, lung tumors, colon tumors, malignant
melanoma, squamous cell carcinomas and ovarian tumors.
The composition is preferably administered by injection in a pharmaceutically acceptable medium
such as an oil-droplet emulsion directly into the tumor under conditions more particularly described below. The aforesaid composition may be stabilized as, for example, by a lyophilization procedure and then reconstituted without loss of potency.
The amount of the pyridine-soluble extract in a single injection for the treatment of animals is between about 375 and 2500 micrograms/milliliter. The amount of each TDM and ACP is between 375 and 1250 micrograms/milliliter.
The number of milliliters of the biologic injected into the tumor is determined by the size of the tumor in accordance with the following table:
Animal Dosage According to Tumor Size
Amount of Biologic
Diameter of Tumor (cm) Injected (ml) 0--1 up to 0.5
1-2 0.5 to 2.5 2-3 2.5to5
3-5 5to 10
5-8 10 two 15 greater than 8 15 to 20 The maximum dose per injection is about 40 milligrams for the pyridine-soluble extract, about 6 milligrams for TDM and about 20 milligrams for ACP. The course of treatment comprises up to six injections administered at about two week intervals.
The present composition in a suitable injection medium such as an oil-droplet emulsion is administered directly onto human tumors. The amount of the pyridine-soluble extract in a single injection is between about 200 and 5000 micrograms, preferably between about 800 and
1200 micrograms. The amount of TDM is between about 50 and 1000 micrograms and the amount of
ACP is between about 1 50 and 1000 micrograms. The preferred single dosage level for each of TDM and ACP is between about 475 and 525 micrograms. All of the above-mentioned dosage levels are based on a typical 70 kilogram adult patient. The injections are administered about once every week for up to a total of 1 5 injections.
As described above, the composition for treatment of warm blooded animals and humans may be
used in the form of an oil droplet emulsion. The amount of oil used is in the range of between about 0.5
and 3.0 percent by volume based on the total volume of the composition. It is preferred to use between
about 0.75 and 1.5 percent by volume of the oil. Examples of such oils include light mineral oil,
squalane, squalene, 7-n-hexyloctadecane, Conoco superoil and Drakeoii 6 VR mineral oil (produced by
the Penreco Company, Butler, Pennsylvania).
The homogenised oil containing mixture is then combined with a detergent which may optionally
be dissolved in a saline solution prior to mixing. The amount of detergent is typically between about
0.02 and 0.25 percent by volume and preferably between about 0.10 and 0.20 percent by volume
based on the total volume of the composition. Any common detergent material may be used including
Tween-80 and Arlacel (produced by the Atlas Chemical Company).
The mixture resulting from the addition of detergent is then homogenised to form a suspension
which has a high percentage of oil droplets coated with the active components as determined by
observation under a microscope.
The following examples are for illustrative purposes only and are not intended to limit or in any
way redefine the invention as claimed in the claims appended hereto.
EXAMPLE 1
Preparation of Pyridine-Soluble Extract from Corynebacterium Parvum
Corynebacterium parvum (P. acnes, Strain 4182) was grown and harvested at 370C in NIH
thioglycolate broth for between 48 and 72 hours to obtain a whole cell paste. The paste was then
washed with 500 ml of distilled water. 90 grams (wet weight) of the washed paste was mixed with
200 ml of neat pyridine and centrifuged at 1 700 x g for one hour at 4"C. A pyridine-soluble extract was
removed as a supernatant fraction. The remaining residue was extracted with additional pyridine under
identical conditions as described above. Following filtration, using Whatman No. 1 paper, the pyridine
extracts were pooled and the solvent was removed by evaporation at 50 C in a Buchi Rotavapor
(Brinkmann Instruments, Westbury, New York). The dried pyridine extract was extensively dialyzed
against distilled water and then lyophilized. The resulting purified pyridine extract contained about 12% by weight of protein, about 12% by weight of sugar and about 45% of fatty acids. The extract was examined under an electron microscope and found to be free of contaminating whole cells and cell wall fragments. The yield of the pyridine-soluble extract was 9% (8.1 g).
EXAMPLE 2
Preparation of Pyridine-Soluble Extract from M. bovis Strain BCG
M. bovis Strain BCG was grown and harvested in Sautons medium at 370 for 3 to 4 weeks to obtain a washed whole cell paste produced in the same manner disclosed in Example 1. 50 grams (wet weight) of the washed paste was then treated in the same manner as Example 1 to produce a yield of the pyridine-soluble extract of 7% (3.5 g). The extract contained 15% by weight of protein, 10% by weight of sugar and 52% by weight of fatty acids.
The pyridine soluble extract in combination with TDM and ACP in a pharmaceutically acceptable medium is significantly effective in the treatment of tumors to obtain total regression of the tumor in most cases.
Claims (6)
1. A pharmaceutical composition comprising a therapeutically effective amount of a purified pyridine-soluble extract obtained from a microorganism, said extract containing between about 7 and 20% by weight of protein, between about 10 and 1 6% by weight of sugar, and between about 35 and 55% by weight of fatty acids, trehalose dimycolate, and an acetone precipitated by-product of endotoxic glycolipids extracted with chloroform-methanol and a pharmaceutically acceptable carrier.
2. The composition of claim 1 wherein the microorganism is M. bovis BCG, M. phlei,
M. smegmatis, M. Kansasii, Nocardia rubra, Corynebacterium diphtheriae or Corynebacterium parvum and preferably Corynebacterium parvum.
3. The composition of claim 1 or 2 wherein the extract contains about 12% by weight of each of protein and sugar a.nd about 45% by weight of fatty acids, the amount of the extract is up to about 40 milligrams, the amount of trehalose dimycolate is up to about 6 milligrams, and the amount of said acetone precipitated by-product is up to about 20 milligrams, and the composition is in lyophilized form or in the form of an oil droplet emulsion.
4. The composition of claim 3 wherein the oil is a light mineral oil, squalane, squalene, 7-n-hexyloctadecane, Conoco superoil or Drakeol 6 VR mineral oil, the oil being present in an amount between about 0.5 to 3.0% by volume based on the total volume of the composition and there may be present in the composition a detergent in an amount between about 0.02 and 0.25% by volume based on the total volume of the composition.
5. The composition according to any of the preceding claims wherein the amount of the extract is between about 200 and 5000 micrograms, the amount of trehalose dimycolate is between about 50 and 1000 micrograms and the amount of the acetone precipitated by-product is between about 1 50 and 1000 micrograms.
6. A composition according to claim 1 substantially as described herein with reference to
Example 1 or Example 2.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US39382382A | 1982-06-30 | 1982-06-30 |
Publications (3)
Publication Number | Publication Date |
---|---|
GB8317744D0 GB8317744D0 (en) | 1983-08-03 |
GB2122897A true GB2122897A (en) | 1984-01-25 |
GB2122897B GB2122897B (en) | 1986-03-26 |
Family
ID=23556394
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB08317744A Expired GB2122897B (en) | 1982-06-30 | 1983-06-30 | Treatment of cancer with microorganisms obtained products |
Country Status (6)
Country | Link |
---|---|
JP (1) | JPS5917991A (en) |
CA (1) | CA1206415A (en) |
DE (1) | DE3323092C2 (en) |
FR (1) | FR2529462B1 (en) |
GB (1) | GB2122897B (en) |
IT (1) | IT1163624B (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2135577A (en) * | 1983-01-27 | 1984-09-05 | Taisho Pharmaceutical Co Ltd | Hydrocortisone butyrate propionate ointments |
FR2552326A1 (en) * | 1983-09-23 | 1985-03-29 | Ribi Immunochem Research Inc | COMPOSITION OF SOLUBLE EXTRACT OF PYRIDINE AND REFINED DETOXIFIED ENDOTOXIN AND USE THEREOF |
CN100341574C (en) * | 2005-07-08 | 2007-10-10 | 中国药科大学 | Novel use of cell wall skeleton of red nocar-ray-fungus for treating liver diseases |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BE1007823A3 (en) * | 1993-12-10 | 1995-10-31 | Anda Biolog Sa | Use of a composition containing at least one antigen and / or one or more fragments of this antigen for obtaining a drug for treating and / or preventing cancer. |
ZA984650B (en) * | 1997-06-19 | 1998-12-08 | Orion Corp | Intratumoral administration of triphenylethylenes for the treatment of cancer |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2189021A1 (en) * | 1972-06-20 | 1974-01-25 | Anvar | Non-arthrogenic immunological adjuvants - obtained by extraction of lipid-free mycobacterial residues |
US3976544A (en) * | 1973-06-19 | 1976-08-24 | The Agence Nationale De Valorisation De Le Recherche | Water-soluble immunological adjuvants, in particular for vaccines, obtained from mycobacteria and related microorganisms and process for their extraction |
JPS5428813A (en) * | 1977-08-09 | 1979-03-03 | Yuuichi Yamamura | Solid preparation containing cell membrane extract substance used as suspension when using same |
-
1983
- 1983-06-17 CA CA000430665A patent/CA1206415A/en not_active Expired
- 1983-06-27 DE DE3323092A patent/DE3323092C2/en not_active Expired
- 1983-06-28 FR FR8310673A patent/FR2529462B1/en not_active Expired
- 1983-06-29 IT IT21854/83A patent/IT1163624B/en active
- 1983-06-29 JP JP58116250A patent/JPS5917991A/en active Granted
- 1983-06-30 GB GB08317744A patent/GB2122897B/en not_active Expired
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2135577A (en) * | 1983-01-27 | 1984-09-05 | Taisho Pharmaceutical Co Ltd | Hydrocortisone butyrate propionate ointments |
FR2552326A1 (en) * | 1983-09-23 | 1985-03-29 | Ribi Immunochem Research Inc | COMPOSITION OF SOLUBLE EXTRACT OF PYRIDINE AND REFINED DETOXIFIED ENDOTOXIN AND USE THEREOF |
GB2149301A (en) * | 1983-09-23 | 1985-06-12 | Ribi Immunochem Research Inc | Anti-cancer compositions containing bacterial extracts |
CN100341574C (en) * | 2005-07-08 | 2007-10-10 | 中国药科大学 | Novel use of cell wall skeleton of red nocar-ray-fungus for treating liver diseases |
Also Published As
Publication number | Publication date |
---|---|
IT1163624B (en) | 1987-04-08 |
GB2122897B (en) | 1986-03-26 |
DE3323092A1 (en) | 1984-01-05 |
IT8321854A0 (en) | 1983-06-29 |
JPS5917991A (en) | 1984-01-30 |
DE3323092C2 (en) | 1987-02-05 |
CA1206415A (en) | 1986-06-24 |
FR2529462A1 (en) | 1984-01-06 |
FR2529462B1 (en) | 1987-02-13 |
JPS6254774B2 (en) | 1987-11-17 |
GB8317744D0 (en) | 1983-08-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA1217136A (en) | Refined detoxified endotoxin product | |
US4877611A (en) | Vaccine containing tumor antigens and adjuvants | |
US4436727A (en) | Refined detoxified endotoxin product | |
US4505900A (en) | Refined detoxified endotoxin product | |
CA1225591A (en) | Refined detoxified endotoxin product | |
DE3434766C2 (en) | Pharmaceutical preparation | |
US4504473A (en) | Pyridine soluble extract of a microorganism | |
US4505899A (en) | Refined detoxified endotoxin product | |
GB2122897A (en) | Treatment of cancer with microorganism obtained products | |
US4505903A (en) | Pyridine soluble extract of a microorganism | |
US4503048A (en) | Pyridine soluble extract of a microorganism | |
Butler et al. | Combined immunostimulation in the prevention of tumor take in mice using endotoxins, their derivatives, and other immune adjuvants | |
SE447629B (en) | FOR FAST FORM OVERFORD PHARMACEUTICAL COMPOSITION CONTAINING CELL WALL SKELETON OF NOCARDIA RUBRA | |
CA1206416A (en) | Pyridine soluble extract of a microorganism | |
US4613504A (en) | Pyridine-soluble extracts of microorganisms | |
CA1209504A (en) | Pyridine soluble extract of a microorganism | |
CA1202903A (en) | Stable composition and preparation thereof | |
HU191713B (en) | Process for producing composition containing purified detoxicated endotoxin | |
KR820000420B1 (en) | Soldified pharmaceutical composition comprising cell wall skeleton |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PCNP | Patent ceased through non-payment of renewal fee |