CA1206416A - Pyridine soluble extract of a microorganism - Google Patents
Pyridine soluble extract of a microorganismInfo
- Publication number
- CA1206416A CA1206416A CA000430716A CA430716A CA1206416A CA 1206416 A CA1206416 A CA 1206416A CA 000430716 A CA000430716 A CA 000430716A CA 430716 A CA430716 A CA 430716A CA 1206416 A CA1206416 A CA 1206416A
- Authority
- CA
- Canada
- Prior art keywords
- weight
- composition
- pyridine
- amount
- soluble extract
- 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.)
- Expired
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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Molecular Biology (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)
Abstract
ABSTRACT OF THE DISCLOSURE
A pharmaceutical composition is disclosed comprising a purified pyridine-soluble extract obtained from a microorganism which contains between about 7 and 20% by weight of protein, be-tween about 10 and 16% by weight of sugar, and between about 35 and 55% by weight of fatty acids which when combined with cell wall skeleton and trehalose dimycolate in a pharmaceutically acceptable medium is useful as an anti-tumor agent in the treatment of animals and humans.
A pharmaceutical composition is disclosed comprising a purified pyridine-soluble extract obtained from a microorganism which contains between about 7 and 20% by weight of protein, be-tween about 10 and 16% by weight of sugar, and between about 35 and 55% by weight of fatty acids which when combined with cell wall skeleton and trehalose dimycolate in a pharmaceutically acceptable medium is useful as an anti-tumor agent in the treatment of animals and humans.
Description
~2~64~i U-Wp-26~1-(2) B~CKGROUND OF THE INV~NTION
The present invention is directed to a pyridine-soluble extract of a microorganism which! when`combined with cell wall skeleton (CWS) and trehalose dimycolate (TDM~ provides a pharmaceutical composition possessing anti-tumor properties.
Bacteria such as CorYnebacteri-um parvum have been the subject of experimental work to isolate and characterize the component responsible for inducing inhibition of tumor growth [see, for example, nti Tumor Activity and Lymphoreticular Stimulation Properties of Fractions Isolated from ~. pa_vum;
Cantrell, et al, Cancer Research 39, pgs. 3554-3563 (September, 1979)3. Apart from anti-tumor activity, C. parvum has shown to be a potent stimulator of the lymphoretlcular sy~tem resultlng in undesirable increases in spleen and liver weights and blastogenesis. Appll`icant 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.
Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan muco-peptid~ contalning remnants of trehalose mycolates ("P3") and undigest~d tubercu~oproteins. Ce~l wall skeleton is obtained from any microorganism including, ~ut not limited to, M.smegmatis, M~phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium d_phtheria, Corynebacterium_parvum, kansasiî, .
;
~r`` ~
~z~
M tuberculosis (S~rain ll 37 RV and Ayoma J3~, and M.bovis S~rain BCG. Additionally, cell wall skeleton may be obtained from such other organisms as E.coli, B.abortus NS _oxiella burnettii.
Cell wall skeleton may be produced by first growing and harvesting bacteria such as M.bovis strain BCG (Bacillus Calmette - Guerin). The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic irnpurities.
The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g., trypsin and/or chymotrypsin) to give purified cell wall skeleton.
Trehalose dimycolates (TDM), may be obtained from the 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.bovinls and orynebacterium 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 biolo~ically active components from mycobacterial cell walls. i. isolation and composition of cell wall skeleton and component p3: Asuma, et al, Journal of the National Cancer Institute, Volume 52, pgs.
95-101, 1974). As disclosed in Aæuma er al, crude TDM may then be further purified by centri-fugal microparticulate silica gel chromatography to give purified TDM.
It is, therefore, an obiect of the present invention to provide a pharmaceutical composition containing a pyridine-~' ~Z~6416 soluble extract of a microorganism in combination with cell wallskeleton and trehalose dimycolate.
It is another object of the invention to provide a method of treating tumors in warm blooded animals and humans using the composition containing ~he pyridine-soluble extract of a micro-organism, cell wall skeleton and trehalose dimycalate.
. , ' ' , SUMMARY OF THE INVENTIC)N
, The present invention relates to pharmaceutical composi-tions comprising a pyridine-soluble extract of a microorganism containing between about 7 and 20% by weight of protein, about 10 and 16% by weight of sugar and about 35 to 55~ by weight of fatty acids in combination with cell wall skeleton (CWS) and trehalose dimycolate (TDM). The extract preferably contains about 12~ by weight of each of protein and sugar and about 45%
~y weight of fatty acids.
Any microorganism may be used to obtain the pyridine-soluble extract includ~ng, for e~ample, M. bovis BCG, M. Phlei, M. sme~matis, M. kansasii, Nocardia rubra, Corynebacterium di~htheriae and CorYnebacterium parvum. Corynebacterium parvum is e~pecially preferred.
Whole cells of the microorganism, 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 ~uantities of the desired extract.
The pyridine is ~hen removed from the extract and the dried extract is dialyzed against a suitable liquid such as distilled water. The absence of whole cell and cell fraymen~
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 CWS and TDM to produce a composition having potent anti tumor activity without stimula-ting the induction of spleen and liver enlargements. The c~ncers which may be treated by this composition include animal tu~or~ such as bovine squamous cell carcinoma, bovine ~ibro-sarcoma, 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 partic-ularly described below. The aforesaid composition may be stabilized as for èxample, by a lyophilization procedure and then reconstituted without loss of potency.
The amount of the pyridine-soluble extract in a single ~n~e~tion for the treatment of animals is hetween about 375 and 2500 microgram~/milliliter. The amount of each of CWS and TDM
is between about 125 and 375 micrograms/milliliter.
The number of milliliters of the blologic injected into the tumor is determined by the size of the tumor in accordance with the following table:
Animal Dosa~e Accordinq to Tumor S~ze Diameter of Tumor (cm) Amount of Biologic Injected (ml~
0-l up to 0.5 1-2 0.5 to 2.5
The present invention is directed to a pyridine-soluble extract of a microorganism which! when`combined with cell wall skeleton (CWS) and trehalose dimycolate (TDM~ provides a pharmaceutical composition possessing anti-tumor properties.
Bacteria such as CorYnebacteri-um parvum have been the subject of experimental work to isolate and characterize the component responsible for inducing inhibition of tumor growth [see, for example, nti Tumor Activity and Lymphoreticular Stimulation Properties of Fractions Isolated from ~. pa_vum;
Cantrell, et al, Cancer Research 39, pgs. 3554-3563 (September, 1979)3. Apart from anti-tumor activity, C. parvum has shown to be a potent stimulator of the lymphoretlcular sy~tem resultlng in undesirable increases in spleen and liver weights and blastogenesis. Appll`icant 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.
Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan muco-peptid~ contalning remnants of trehalose mycolates ("P3") and undigest~d tubercu~oproteins. Ce~l wall skeleton is obtained from any microorganism including, ~ut not limited to, M.smegmatis, M~phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium d_phtheria, Corynebacterium_parvum, kansasiî, .
;
~r`` ~
~z~
M tuberculosis (S~rain ll 37 RV and Ayoma J3~, and M.bovis S~rain BCG. Additionally, cell wall skeleton may be obtained from such other organisms as E.coli, B.abortus NS _oxiella burnettii.
Cell wall skeleton may be produced by first growing and harvesting bacteria such as M.bovis strain BCG (Bacillus Calmette - Guerin). The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic irnpurities.
The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g., trypsin and/or chymotrypsin) to give purified cell wall skeleton.
Trehalose dimycolates (TDM), may be obtained from the 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.bovinls and orynebacterium 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 biolo~ically active components from mycobacterial cell walls. i. isolation and composition of cell wall skeleton and component p3: Asuma, et al, Journal of the National Cancer Institute, Volume 52, pgs.
95-101, 1974). As disclosed in Aæuma er al, crude TDM may then be further purified by centri-fugal microparticulate silica gel chromatography to give purified TDM.
It is, therefore, an obiect of the present invention to provide a pharmaceutical composition containing a pyridine-~' ~Z~6416 soluble extract of a microorganism in combination with cell wallskeleton and trehalose dimycolate.
It is another object of the invention to provide a method of treating tumors in warm blooded animals and humans using the composition containing ~he pyridine-soluble extract of a micro-organism, cell wall skeleton and trehalose dimycalate.
. , ' ' , SUMMARY OF THE INVENTIC)N
, The present invention relates to pharmaceutical composi-tions comprising a pyridine-soluble extract of a microorganism containing between about 7 and 20% by weight of protein, about 10 and 16% by weight of sugar and about 35 to 55~ by weight of fatty acids in combination with cell wall skeleton (CWS) and trehalose dimycolate (TDM). The extract preferably contains about 12~ by weight of each of protein and sugar and about 45%
~y weight of fatty acids.
Any microorganism may be used to obtain the pyridine-soluble extract includ~ng, for e~ample, M. bovis BCG, M. Phlei, M. sme~matis, M. kansasii, Nocardia rubra, Corynebacterium di~htheriae and CorYnebacterium parvum. Corynebacterium parvum is e~pecially preferred.
Whole cells of the microorganism, 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 ~uantities of the desired extract.
The pyridine is ~hen removed from the extract and the dried extract is dialyzed against a suitable liquid such as distilled water. The absence of whole cell and cell fraymen~
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 CWS and TDM to produce a composition having potent anti tumor activity without stimula-ting the induction of spleen and liver enlargements. The c~ncers which may be treated by this composition include animal tu~or~ such as bovine squamous cell carcinoma, bovine ~ibro-sarcoma, 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 partic-ularly described below. The aforesaid composition may be stabilized as for èxample, by a lyophilization procedure and then reconstituted without loss of potency.
The amount of the pyridine-soluble extract in a single ~n~e~tion for the treatment of animals is hetween about 375 and 2500 microgram~/milliliter. The amount of each of CWS and TDM
is between about 125 and 375 micrograms/milliliter.
The number of milliliters of the blologic injected into the tumor is determined by the size of the tumor in accordance with the following table:
Animal Dosa~e Accordinq to Tumor S~ze Diameter of Tumor (cm) Amount of Biologic Injected (ml~
0-l up to 0.5 1-2 0.5 to 2.5
2-3 2.5 to 5
3 5 5 to 10 5-8 10 to 15 greater than 8 15 to 20 The maximum dose per injection is about 40 milligrams for the pyridine-soluble extract, 40 milligrams for CWS, and 6 milligrams for TDM. The course of treatment comprises up to six injections administered at abou~ two week intervals.
The present composition in a suitable injection medium such as an oil-droplet emulsion is administered directly into human tumors. The amount of the pyridine-soluble extract in a single injection is between about 200 and S000 micrograms, preferably be~ween about B00 and 1200 micrograms. The amount of CWS i8 between about 50 and 2000 micrograms while-the amount of TDM is between about 50 and lO00 micrograms. The preferred ~ingle dosage level for each of CWS and TDM is between about 475 and 525 microgramsO All of the above-mentioned do~age levels are ba~ed on a typical 70 kilogram adult patient. The injec-tions are administered about once every week for up to a total of 15 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 volwne of the composition. It is preferred to use bet.Jeen abo 0.75 and 1.5 percent by volume of the oil. Rxamples of such oils include light mineral oil, squalane, squalene, 7-n-hexyl-octadecane, Conoco ~a trademark) superoil and Drakeol 6 VR (a trademark) mineral oil (produced by -the Penreco Company, Butler, Pennsylvania).
The homogenized 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 ~ercent by ~olume 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 (a -tradernark) and Arlacel (a trademark) (produced by the A-tlas Chemical Company).
The mixture resulting from the addition of detergent is then homogenized to form a suspension which has a high percen-tage of oil droplets coated with the active components as detexmined by observat,ion under a microscope.
The following examples are for illustrative purposes only and are not intended to limit or in any way redefine the inven-tion as claimed in the claims appended hereto.
EXAMPLE I - Preparation of Pyridine-Soluble Ex-tract from Corynebacterium Parvum Corynebacterium parvum (P.acnes, Strain 4182) was grown and harvested at 37C. in NIH thioglycolate broth for between 48 and 72 hours to obtain a whole cell paste. The paste was washed with 500 mg. of di,stilled waterO 90 grams (wet weight) of the washed paste was mixed with 200 ml. of neat pyridine and centrifuged at 1700 x g for one hour at 4C. A pyridine-soluble extract was removed as a superna-tant fraction. The ~, 6~:~6 remaining residue was extrac~ed 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 wa~ extensively dia~yzed against dis~
tilled water and then lyophilized. ~he resul~ing purified p~ri~ine extract contained about 12% by weight of p~otein, about 1~% by weight of sugar and 45~ by weight of fatty acids. The ex~ract was examined under an electron microscope and found to b~ free of contaminat~ng whole cells and cell wall fragments.
The yield of the pyridine-soluble extract was 9~ ~8.1 9.).
EXAMPLE 2 - Pre~aration of Pyridine-Soluble Extract from M.bovis Strain BCG
M. bovis Strain BCG was grown and harvested in Sautons medium at 37~ for about 3 to 4 weeks to obtain a washed whole cell paste. 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% (3.59). The extract contained 15% by weight of protein, 10~ by weight of sugar and 52% by weight of fat~y acids.
EXAMPLE 3 - Guinea-Pig Line-10 Tumor Tests Six s~rain 2 guinea pigs having Line-10 tumor growths of about 9mm. in diameter were injected once with 0.4 ml of a sterile oil droplet emulsion, i.e., Drakeol 6 VR mineral oil (Pennsylvania Refining Companyr Butler, Pennsylvania), containing 300 micrograms of the pyridine-soluble extract ~2~6~
prepared in accordance with Example 1 and 50 micrograms of each of cell wall skeleton and trehalose dimycolate, directly into the tumor tissue.
At the end of three months, the animals were examined and in 5 of the 6 animals, total regression had occurred.
In a control experiment, six strain 2 guinea pigs having Line-10 tumor growths of about 9mm. in diameter were injected once with 0.4 mI of the sterile oil drople~ emulsion described above without the pyridine extract or cell wall skeleton and trehalose dimycolate. The injections were made directly into the tumor tissue. None of the six tumors showed any signs of regression after three months.
The present composition in a suitable injection medium such as an oil-droplet emulsion is administered directly into human tumors. The amount of the pyridine-soluble extract in a single injection is between about 200 and S000 micrograms, preferably be~ween about B00 and 1200 micrograms. The amount of CWS i8 between about 50 and 2000 micrograms while-the amount of TDM is between about 50 and lO00 micrograms. The preferred ~ingle dosage level for each of CWS and TDM is between about 475 and 525 microgramsO All of the above-mentioned do~age levels are ba~ed on a typical 70 kilogram adult patient. The injec-tions are administered about once every week for up to a total of 15 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 volwne of the composition. It is preferred to use bet.Jeen abo 0.75 and 1.5 percent by volume of the oil. Rxamples of such oils include light mineral oil, squalane, squalene, 7-n-hexyl-octadecane, Conoco ~a trademark) superoil and Drakeol 6 VR (a trademark) mineral oil (produced by -the Penreco Company, Butler, Pennsylvania).
The homogenized 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 ~ercent by ~olume 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 (a -tradernark) and Arlacel (a trademark) (produced by the A-tlas Chemical Company).
The mixture resulting from the addition of detergent is then homogenized to form a suspension which has a high percen-tage of oil droplets coated with the active components as detexmined by observat,ion under a microscope.
The following examples are for illustrative purposes only and are not intended to limit or in any way redefine the inven-tion as claimed in the claims appended hereto.
EXAMPLE I - Preparation of Pyridine-Soluble Ex-tract from Corynebacterium Parvum Corynebacterium parvum (P.acnes, Strain 4182) was grown and harvested at 37C. in NIH thioglycolate broth for between 48 and 72 hours to obtain a whole cell paste. The paste was washed with 500 mg. of di,stilled waterO 90 grams (wet weight) of the washed paste was mixed with 200 ml. of neat pyridine and centrifuged at 1700 x g for one hour at 4C. A pyridine-soluble extract was removed as a superna-tant fraction. The ~, 6~:~6 remaining residue was extrac~ed 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 wa~ extensively dia~yzed against dis~
tilled water and then lyophilized. ~he resul~ing purified p~ri~ine extract contained about 12% by weight of p~otein, about 1~% by weight of sugar and 45~ by weight of fatty acids. The ex~ract was examined under an electron microscope and found to b~ free of contaminat~ng whole cells and cell wall fragments.
The yield of the pyridine-soluble extract was 9~ ~8.1 9.).
EXAMPLE 2 - Pre~aration of Pyridine-Soluble Extract from M.bovis Strain BCG
M. bovis Strain BCG was grown and harvested in Sautons medium at 37~ for about 3 to 4 weeks to obtain a washed whole cell paste. 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% (3.59). The extract contained 15% by weight of protein, 10~ by weight of sugar and 52% by weight of fat~y acids.
EXAMPLE 3 - Guinea-Pig Line-10 Tumor Tests Six s~rain 2 guinea pigs having Line-10 tumor growths of about 9mm. in diameter were injected once with 0.4 ml of a sterile oil droplet emulsion, i.e., Drakeol 6 VR mineral oil (Pennsylvania Refining Companyr Butler, Pennsylvania), containing 300 micrograms of the pyridine-soluble extract ~2~6~
prepared in accordance with Example 1 and 50 micrograms of each of cell wall skeleton and trehalose dimycolate, directly into the tumor tissue.
At the end of three months, the animals were examined and in 5 of the 6 animals, total regression had occurred.
In a control experiment, six strain 2 guinea pigs having Line-10 tumor growths of about 9mm. in diameter were injected once with 0.4 mI of the sterile oil drople~ emulsion described above without the pyridine extract or cell wall skeleton and trehalose dimycolate. The injections were made directly into the tumor tissue. None of the six tumors showed any signs of regression after three months.
Claims (5)
1. A pharmaceutical composition comprising a thera-peutically 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, cell wall skeleton and trehalose dimycolate, and a pharmaceutically acceptable carrier.
by weight of fatty acids, cell wall skeleton and trehalose dimycolate, and a pharmaceutically acceptable carrier.
2. The composition of claim 1 wherein the micro-organism is M. bovis BCG, M. phlei, M. smegmatis, M.Kansasii, Nocardia rubra, Corynebacterium diphtheriae or Corynebacter-ium parvum and preferably Corynebacterium parvum.
3. The composition of claim 1 wherein the extract contains about 12% by weight of each of protein and sugar and about 45% by weight of fatty acids, and further, the amount of each of the pyridine-soluble extract and cell wall skeleton is up to about 40 milligrams, and the amount of trehalose dimy-colate is up to about 6 milligrams, the composition being 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 6VR mineral oil, the oil being present in an amount between about 0.5 and 3.0% by volume based on the total volume of the composition.
5. The composition of claim 3 characterized in that it includes a detergent in an amount between about 0.02 and 0.25% by volume based on the total volume of the composi-tion and the pyridine-soluble extract product is between about 200 and 5000 micrograms, the amount of cell wall skeleton is between about 50 and 2000 micrograms and the amount of trehalose dimycolate is between about 50 and 1000 micrograms.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US39382282A | 1982-06-30 | 1982-06-30 | |
| US393,822 | 1982-06-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1206416A true CA1206416A (en) | 1986-06-24 |
Family
ID=23556390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000430716A Expired CA1206416A (en) | 1982-06-30 | 1983-06-20 | Pyridine soluble extract of a microorganism |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS601133A (en) |
| CA (1) | CA1206416A (en) |
| DE (1) | DE3323093C2 (en) |
| FR (1) | FR2536280B1 (en) |
| GB (1) | GB2122896B (en) |
| IT (1) | IT1163623B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4663306A (en) * | 1983-09-23 | 1987-05-05 | Ribi Immunochem Research, Inc. | Pyridine-soluble extract-refined detoxified endotoxin composition and use |
| US4803070A (en) * | 1986-04-15 | 1989-02-07 | Ribi Immunochem Research Inc. | Immunological emulsion adjuvants for polysaccharide vaccines |
| 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. |
| EP1097715A4 (en) * | 1998-07-16 | 2004-12-29 | Hayashi Akira | Preparations for immunotherapy for cancer having bacterial somatic constituent as the active ingredient |
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
- 1983-06-20 CA CA000430716A patent/CA1206416A/en not_active Expired
- 1983-06-27 DE DE3323093A patent/DE3323093C2/en not_active Expired
- 1983-06-29 JP JP58116249A patent/JPS601133A/en active Granted
- 1983-06-29 IT IT21853/83A patent/IT1163623B/en active
- 1983-06-29 FR FR8310788A patent/FR2536280B1/en not_active Expired
- 1983-06-30 GB GB08317742A patent/GB2122896B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE3323093C2 (en) | 1986-10-16 |
| IT8321853A1 (en) | 1984-12-29 |
| JPS6253485B2 (en) | 1987-11-10 |
| FR2536280A1 (en) | 1984-05-25 |
| FR2536280B1 (en) | 1987-02-20 |
| GB2122896A (en) | 1984-01-25 |
| GB2122896B (en) | 1986-03-26 |
| JPS601133A (en) | 1985-01-07 |
| IT1163623B (en) | 1987-04-08 |
| GB8317742D0 (en) | 1983-08-03 |
| DE3323093A1 (en) | 1984-01-05 |
| IT8321853A0 (en) | 1983-06-29 |
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