CA1202903A - Stable composition and preparation thereof - Google Patents
Stable composition and preparation thereofInfo
- Publication number
- CA1202903A CA1202903A CA000426843A CA426843A CA1202903A CA 1202903 A CA1202903 A CA 1202903A CA 000426843 A CA000426843 A CA 000426843A CA 426843 A CA426843 A CA 426843A CA 1202903 A CA1202903 A CA 1202903A
- Authority
- CA
- Canada
- Prior art keywords
- tdm
- composition
- cws
- oil
- percent
- 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
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Colloid Chemistry (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A stable composition containing cell wall skeleton (CWS) and trehalose dimycolates (TDM) which may be effectively reconstituted after lyophilization. The composition is useful in the treatment of various cancerous tumors without side effects. Also disclosed is a method of producing the composition and a method of treating cancerous tumors using said composition.
A stable composition containing cell wall skeleton (CWS) and trehalose dimycolates (TDM) which may be effectively reconstituted after lyophilization. The composition is useful in the treatment of various cancerous tumors without side effects. Also disclosed is a method of producing the composition and a method of treating cancerous tumors using said composition.
Description
~L202~101~311 BACKGROUND OF THE INVENTION
The subject matter of the present invention is directed to a stable, therapeutically efective composition containing cell wall skeleton (CWS~ and purified trehalose dimycolates (TDM). Both substances are isolates of bacteria and when used together as a composition, are effective in obtaining suppres-sion and regression of tumor cells. The present invention also includes a method of preparing the composition as well as use of the composition in treating cancerous tumors and as an ad-juvant.
~ he comoination of CWS and TDM is known in the art (SeeBiologically Active Compounds from Mycobacterial Cell Walls.
II. Suppression and Regression of Strain-2 Guinea Pig Hepatoma;
Meyer et al, Journal of the National Cancer Institute, Volume 52, No. 1, January, 1974; and Mycobacterial Cell Wall Components in Tumor Suppression and Regression; Ribi et al, National Can-cer Institute Monograph No. 39, pgs. 115-120, October, 1972) 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 mucopeptide containing remnants of trehalose mycolates ("P3") and undigested tuberculoproteins. Cell wall skeleton is ob-tained from any mycobacteria including, but not limited to, M.smegmatis, M.phlei, M. avium, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M. bovinis, M. Kansasii, M. Tuberculosis (Strain H 37 RV and Ayoma B), and M. Bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abor-tus and Coxiella Burnettii.
,~
, .
Z9~3 The process of producing cell wall skeleton is time consuming. The bacteria such as M. Bovis Strain BCG (Bacillus Calmette - Guerin) is grown and harvested. The resulting ~-hole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-l)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic impurities. 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.
The second component of the instant composition, tr~halose dimycolates (TDM), may be obtained from any mycobacteria as, for example, _. 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 is 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 ~cobacte~ial Cell 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). As disclosed in A~uma, et al, crude TDM may then be further purified by centrifu~al microparticulate silica gel chromatography to give purified TDM.
CWS and TDM produced as described above have been combined in an oil droplet emulsion. The non-iiving components are ground with a small amount of mineral oil and emulsified in saline to produce an anti-tumor composition suitable for ~.' injection (See Immunotherapy with Non-viable Micro~ial Components, Ribi, et al; Annals of the National Academy o~
Siences, Volume 227, pgs. 228-238, September 20th, 1976).
However, the prior art oil in saline emulsions con-taining CWS and TDM suffer from a major disadvantage. The emul-sion has a relatively short shelf life at room temperature and, therefore, must be used shortly after preparation to produce the desired results.
It is well-known in the art that lyophilizing a pharmaceutical preparation can extend shelf life considerably (see, for example, USP 3,932,943; USP 3,594,471; and USP
4,134,21~). To be successul, the lyophilized product must be able to be reconstituted at a later time without any loss in potency, that is, with the same potency as the pre-lyophilized product. However, the prior art CWS-TDM oil in saline emul-sions have not been effectively lyophilized. Applicants have discovered that if these emulsions are not stabilized without delay, as, for example, by lyophili~ation, they will begin a process of degradation resulting in a significant percent of oil droplets becoming uncoated. The uncoated material is not active in tumor regression. The therapeutically effective emul-sion of the present invention is stabilized by a lyophilization procedure; other stabilization procedures can also be utilized.
It is therefore an object of the invention to provide a stable CWS-TDM composition which may be effectively recon-stituted to produce an effective anti-tumor preparation having a superior shelf life that is, a shelf life o~ a year and even longer. It is another object of the invention to provide a CWS-TDM composition having a large number of coated oil drop lets which are very effective in tumor regression, without side ~2~
effects.
It is still another object of the invention to provide a method of treating various cancer tumors with a stable and potent CWS-TDM composition. It is another object of the invention to employ the CWS-TDM composition as an adjuvant as for example, to increase the immune response to immunogens including, for example, microorganisms r proteins, carbohydrates, allergens, viruses and the like.
THE INVENTION
The present invention is directed to a stable composition comprising CWS and TDM in which the active materials are coated on oil droplets. The composition may be effectively reconstituted in an aqueous solution with the same potency as in the pre-lyophilized state.
The process of producing the instant composition comprises mixing CWS and TDM for a time sufficient to form a uniEorm suspension. If desirable, the TDM may be dissolved in a suitable solvent known to those skilled in the art. For example, such solvents include chloroform, ether, methanol, ethanol, combinations thereof and the like. The weight r~tio of C~S to TDM is in the range of between about 1.0 and 6:1, preferably between about 2.5 and 3.0:1.
A light, non-biodegradable oil is then added and the resulting mixture is homogeni3ed to form a paste-like substance.
The use of a light hydrocarbon non-biodegradable oil is an essential element of the process since biodegradable oils do not achieve the objects of the invention. Furthermore, the oil must be light weight typically having a viscosity of between about 8 and 12 centistokes measured at 100 F. Preferably~ the viscosity is in the range of between about 10 and 10.6 centistokes.
The amount of oil used in the proccss is in the range of between about 0.5 and 3.0 pcrcent by volumc based on the total volume of the composition. It is preferred to use between about 0.75 and 1.5 percent by volume of thè oil. Examples oE such oils include light mineral oil, squalane, 7-n-hexyloctadecane, Conoco (a trademark) superoil and Drakeol 6 VR la trademark) mineral oil (produced by -the Pennreco Company, Butler, Pennsylvania).
The homogeni~ed oil containing mi~t~re i5 then combined with a detergent ~hich may optionally be dissolved in a saline solution prior to oixing. The amount of detexgent is typically between about 0.02 and 0.20 percent by volume and preferably between about 0.10 and 0.20 percent by volume based on the total volume of the composition. ~ny common detergent material may be used including Tween-80 (a trademark), and ~rlacel, ta trademarX) (pxoduced by the Atlas Chemical Company~.
The mixture resultinc3 from the addition of detergent is then homogeni~ed -to form a suspension which has a high percentage of oil drople-ts coated ~ith CWS and TDM as determined by observation under a microscope.
The novel composition of thc present invention is an effective a~cnt in the treatmcnt of cancerous tumors in humans ancl animals and the composi-tion, in thc form of an oil droplet emulsion, is injected directly into thc -tumor tissue. Cancers which can be treated includc bovinc fibrosarcoma, equine sarcoid, equine melanoma, canine melanoMa, bovine ocular sarcoma, etc. The daily dosage of the composition to a typical adult patient weic3h1ng about 70 kiloc3rams is sufficient to provide about 150-300 ~cJ of CWS and 50-100 ~cJ oE TDM. Therapy may be continued for approximately two -to six months providing a ~202~3 total dosage of up to 15 mg of CWS and 15 mg. of TDM.
The instant composi-tion, when used to increase a response to immunogens, is administered at a dosage of between about 10 and 500 micrograms. CWS and TDM can also be used separately as adjuvants; each in amounts of about 10 to about 500 micro-grams.
Description of the Preferred Embodiments The following examples are for illustrative purposes only and are not intended to limit or in any way redefine the invention as claimed i~ the claims appended hereto.
Example 1 - Preparation of CWS-TDM Composition 150 mg of CWS are introduced into a 350 ml tissue homo-genizer (Bellco). 50 mg of TDM were dissolved in 2.5 ml of a 95:5 chloroform - methanol mixture and then added to the homogenizer. The resulting CWS-TDM suspension was mixed for about 15 to 30 seconds and the solvent was evaporated using a sterile air stream. This was followed by the addition of 2 ml of sterile Drakeol 6 VR (a trademark) mineral oil ~Pennreco Company, Butler, Pennsylvania) and the mixture was homogenized for 1 minute using a drill press while an oil paste consistency is obtained. 190 ml of 0.2 percent Tween-80 (a trademark) in saline solution was in-troduced into each homogenizer. Using a drill press, the mixture was homogenized for about ~ to 5 minutes until an emulsion was obtained. Microscopic investigation showed that substantially all of the oil droplets were coated with CWS and TDM.
'J~
Example 2 - Lyophilizing the CWS-TDM Composition 5 ml of the CWS-TDM oil in saline emulsion (a 1~
oil-in-water emulsion) produced in accordance with the proce-dure of Example 1 was introduced into a 10 ml Wheaton serum vial.
The vial was frozen in a Revco (a trademark) freezer at a temperature of -95 C. and lyophilized in a sterile container on a Labconco freeze dryer. The vial was then capped using a sterile technique.
Example 3 - Reconstitution of Lyophilized CWS TDM Composition A vial containing the lyophilized CWS-TDM emulsion produced in accordance with the procedure of Example 2 containing, for example, 3.75 mg CWS and 1.25 mg TDM in 5 ml.
of a 1% oil droplet emulsion was injected with sterile distilled water using a No. 20 gauge sterile syringe. The suspension was then emulsified by repeated aspirations and injections using the syringe for at least two minutes until the resulting emulsion gave a cloudy-milky appearance. ~
Example 4 - CWS-TDM Composition - Comparison Data (Animals) Dose per Animal (~g) Animals (guinea pig)* Treated Regression CWS + TDM 300 + 150 125 76 CWS + TDM 150 -~ 150 56 63 CWS 150 65 ~3 CWS + TDM 50 + 50 16 19 ~20~g~3 Oil-tween-saline controls - 66 0 *Strain 2 The CWS was obtained rom M. bovis.
Example 5 - CWS-TDM Composition - Clinical Test Results 17 adult patients afflicted with metastatic malignant melanoma were treated with doses of 300 to 1050 ug of CWS
(obtained from M. smegmatis) and 150 to 525 ug ~DM for l to 2 weeks for a total of 8 treatments. This therapy was effective in 7 patients; of the 7 patients, 6 had complete regression of at least l injected lesion.
The subject matter of the present invention is directed to a stable, therapeutically efective composition containing cell wall skeleton (CWS~ and purified trehalose dimycolates (TDM). Both substances are isolates of bacteria and when used together as a composition, are effective in obtaining suppres-sion and regression of tumor cells. The present invention also includes a method of preparing the composition as well as use of the composition in treating cancerous tumors and as an ad-juvant.
~ he comoination of CWS and TDM is known in the art (SeeBiologically Active Compounds from Mycobacterial Cell Walls.
II. Suppression and Regression of Strain-2 Guinea Pig Hepatoma;
Meyer et al, Journal of the National Cancer Institute, Volume 52, No. 1, January, 1974; and Mycobacterial Cell Wall Components in Tumor Suppression and Regression; Ribi et al, National Can-cer Institute Monograph No. 39, pgs. 115-120, October, 1972) 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 mucopeptide containing remnants of trehalose mycolates ("P3") and undigested tuberculoproteins. Cell wall skeleton is ob-tained from any mycobacteria including, but not limited to, M.smegmatis, M.phlei, M. avium, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M. bovinis, M. Kansasii, M. Tuberculosis (Strain H 37 RV and Ayoma B), and M. Bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abor-tus and Coxiella Burnettii.
,~
, .
Z9~3 The process of producing cell wall skeleton is time consuming. The bacteria such as M. Bovis Strain BCG (Bacillus Calmette - Guerin) is grown and harvested. The resulting ~-hole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-l)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic impurities. 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.
The second component of the instant composition, tr~halose dimycolates (TDM), may be obtained from any mycobacteria as, for example, _. 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 is 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 ~cobacte~ial Cell 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). As disclosed in A~uma, et al, crude TDM may then be further purified by centrifu~al microparticulate silica gel chromatography to give purified TDM.
CWS and TDM produced as described above have been combined in an oil droplet emulsion. The non-iiving components are ground with a small amount of mineral oil and emulsified in saline to produce an anti-tumor composition suitable for ~.' injection (See Immunotherapy with Non-viable Micro~ial Components, Ribi, et al; Annals of the National Academy o~
Siences, Volume 227, pgs. 228-238, September 20th, 1976).
However, the prior art oil in saline emulsions con-taining CWS and TDM suffer from a major disadvantage. The emul-sion has a relatively short shelf life at room temperature and, therefore, must be used shortly after preparation to produce the desired results.
It is well-known in the art that lyophilizing a pharmaceutical preparation can extend shelf life considerably (see, for example, USP 3,932,943; USP 3,594,471; and USP
4,134,21~). To be successul, the lyophilized product must be able to be reconstituted at a later time without any loss in potency, that is, with the same potency as the pre-lyophilized product. However, the prior art CWS-TDM oil in saline emul-sions have not been effectively lyophilized. Applicants have discovered that if these emulsions are not stabilized without delay, as, for example, by lyophili~ation, they will begin a process of degradation resulting in a significant percent of oil droplets becoming uncoated. The uncoated material is not active in tumor regression. The therapeutically effective emul-sion of the present invention is stabilized by a lyophilization procedure; other stabilization procedures can also be utilized.
It is therefore an object of the invention to provide a stable CWS-TDM composition which may be effectively recon-stituted to produce an effective anti-tumor preparation having a superior shelf life that is, a shelf life o~ a year and even longer. It is another object of the invention to provide a CWS-TDM composition having a large number of coated oil drop lets which are very effective in tumor regression, without side ~2~
effects.
It is still another object of the invention to provide a method of treating various cancer tumors with a stable and potent CWS-TDM composition. It is another object of the invention to employ the CWS-TDM composition as an adjuvant as for example, to increase the immune response to immunogens including, for example, microorganisms r proteins, carbohydrates, allergens, viruses and the like.
THE INVENTION
The present invention is directed to a stable composition comprising CWS and TDM in which the active materials are coated on oil droplets. The composition may be effectively reconstituted in an aqueous solution with the same potency as in the pre-lyophilized state.
The process of producing the instant composition comprises mixing CWS and TDM for a time sufficient to form a uniEorm suspension. If desirable, the TDM may be dissolved in a suitable solvent known to those skilled in the art. For example, such solvents include chloroform, ether, methanol, ethanol, combinations thereof and the like. The weight r~tio of C~S to TDM is in the range of between about 1.0 and 6:1, preferably between about 2.5 and 3.0:1.
A light, non-biodegradable oil is then added and the resulting mixture is homogeni3ed to form a paste-like substance.
The use of a light hydrocarbon non-biodegradable oil is an essential element of the process since biodegradable oils do not achieve the objects of the invention. Furthermore, the oil must be light weight typically having a viscosity of between about 8 and 12 centistokes measured at 100 F. Preferably~ the viscosity is in the range of between about 10 and 10.6 centistokes.
The amount of oil used in the proccss is in the range of between about 0.5 and 3.0 pcrcent by volumc based on the total volume of the composition. It is preferred to use between about 0.75 and 1.5 percent by volume of thè oil. Examples oE such oils include light mineral oil, squalane, 7-n-hexyloctadecane, Conoco (a trademark) superoil and Drakeol 6 VR la trademark) mineral oil (produced by -the Pennreco Company, Butler, Pennsylvania).
The homogeni~ed oil containing mi~t~re i5 then combined with a detergent ~hich may optionally be dissolved in a saline solution prior to oixing. The amount of detexgent is typically between about 0.02 and 0.20 percent by volume and preferably between about 0.10 and 0.20 percent by volume based on the total volume of the composition. ~ny common detergent material may be used including Tween-80 (a trademark), and ~rlacel, ta trademarX) (pxoduced by the Atlas Chemical Company~.
The mixture resultinc3 from the addition of detergent is then homogeni~ed -to form a suspension which has a high percentage of oil drople-ts coated ~ith CWS and TDM as determined by observation under a microscope.
The novel composition of thc present invention is an effective a~cnt in the treatmcnt of cancerous tumors in humans ancl animals and the composi-tion, in thc form of an oil droplet emulsion, is injected directly into thc -tumor tissue. Cancers which can be treated includc bovinc fibrosarcoma, equine sarcoid, equine melanoma, canine melanoMa, bovine ocular sarcoma, etc. The daily dosage of the composition to a typical adult patient weic3h1ng about 70 kiloc3rams is sufficient to provide about 150-300 ~cJ of CWS and 50-100 ~cJ oE TDM. Therapy may be continued for approximately two -to six months providing a ~202~3 total dosage of up to 15 mg of CWS and 15 mg. of TDM.
The instant composi-tion, when used to increase a response to immunogens, is administered at a dosage of between about 10 and 500 micrograms. CWS and TDM can also be used separately as adjuvants; each in amounts of about 10 to about 500 micro-grams.
Description of the Preferred Embodiments The following examples are for illustrative purposes only and are not intended to limit or in any way redefine the invention as claimed i~ the claims appended hereto.
Example 1 - Preparation of CWS-TDM Composition 150 mg of CWS are introduced into a 350 ml tissue homo-genizer (Bellco). 50 mg of TDM were dissolved in 2.5 ml of a 95:5 chloroform - methanol mixture and then added to the homogenizer. The resulting CWS-TDM suspension was mixed for about 15 to 30 seconds and the solvent was evaporated using a sterile air stream. This was followed by the addition of 2 ml of sterile Drakeol 6 VR (a trademark) mineral oil ~Pennreco Company, Butler, Pennsylvania) and the mixture was homogenized for 1 minute using a drill press while an oil paste consistency is obtained. 190 ml of 0.2 percent Tween-80 (a trademark) in saline solution was in-troduced into each homogenizer. Using a drill press, the mixture was homogenized for about ~ to 5 minutes until an emulsion was obtained. Microscopic investigation showed that substantially all of the oil droplets were coated with CWS and TDM.
'J~
Example 2 - Lyophilizing the CWS-TDM Composition 5 ml of the CWS-TDM oil in saline emulsion (a 1~
oil-in-water emulsion) produced in accordance with the proce-dure of Example 1 was introduced into a 10 ml Wheaton serum vial.
The vial was frozen in a Revco (a trademark) freezer at a temperature of -95 C. and lyophilized in a sterile container on a Labconco freeze dryer. The vial was then capped using a sterile technique.
Example 3 - Reconstitution of Lyophilized CWS TDM Composition A vial containing the lyophilized CWS-TDM emulsion produced in accordance with the procedure of Example 2 containing, for example, 3.75 mg CWS and 1.25 mg TDM in 5 ml.
of a 1% oil droplet emulsion was injected with sterile distilled water using a No. 20 gauge sterile syringe. The suspension was then emulsified by repeated aspirations and injections using the syringe for at least two minutes until the resulting emulsion gave a cloudy-milky appearance. ~
Example 4 - CWS-TDM Composition - Comparison Data (Animals) Dose per Animal (~g) Animals (guinea pig)* Treated Regression CWS + TDM 300 + 150 125 76 CWS + TDM 150 -~ 150 56 63 CWS 150 65 ~3 CWS + TDM 50 + 50 16 19 ~20~g~3 Oil-tween-saline controls - 66 0 *Strain 2 The CWS was obtained rom M. bovis.
Example 5 - CWS-TDM Composition - Clinical Test Results 17 adult patients afflicted with metastatic malignant melanoma were treated with doses of 300 to 1050 ug of CWS
(obtained from M. smegmatis) and 150 to 525 ug ~DM for l to 2 weeks for a total of 8 treatments. This therapy was effective in 7 patients; of the 7 patients, 6 had complete regression of at least l injected lesion.
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for producing a stable composition containing cell wall skeleton (CWS) and trehalose dimycolate (TDM) comprising:
(a) mixing CWS and TDM useful for treating tumors selected from the group consisting of bovine fibrosarcoma, equine sarcoid, equine melanoma, canine melanoma and bovine ocular sarcoma in a weight ratio of CWS to TDM of between about 1.0:1 and 6:1;
(b) adding a light hydrocarbon non-biodegradable oil to the resulting mixture; wherein said light hydrocarbon non-biodegrad-able oil has a viscosity of between about 8 and 12 centistokes measured at 100°F. and is present in an amount between about 0.5 and 3.0 percent by volume based on the total volume of the composition;
(c) homogenizing the resulting oil containing mixture;
(d) adding a detergent in an amount of between about 0.02 and 0.20 percent by volume based on the total volume of the composition selected from the group consisting of polyoxyethylene sorbition mono oleates and non-ionic emulsifiers of fatty acid polyols and polyanhydrides to said homogenized oil containing mixture;
(e) homogenizing the resulting mixture to form an emulsion containing oil droplets coated with CWS and TDM;
(f) lyophilizing the resulting emulsion and subsequently reconstituting said emulsion with sterile distilled water.
(a) mixing CWS and TDM useful for treating tumors selected from the group consisting of bovine fibrosarcoma, equine sarcoid, equine melanoma, canine melanoma and bovine ocular sarcoma in a weight ratio of CWS to TDM of between about 1.0:1 and 6:1;
(b) adding a light hydrocarbon non-biodegradable oil to the resulting mixture; wherein said light hydrocarbon non-biodegrad-able oil has a viscosity of between about 8 and 12 centistokes measured at 100°F. and is present in an amount between about 0.5 and 3.0 percent by volume based on the total volume of the composition;
(c) homogenizing the resulting oil containing mixture;
(d) adding a detergent in an amount of between about 0.02 and 0.20 percent by volume based on the total volume of the composition selected from the group consisting of polyoxyethylene sorbition mono oleates and non-ionic emulsifiers of fatty acid polyols and polyanhydrides to said homogenized oil containing mixture;
(e) homogenizing the resulting mixture to form an emulsion containing oil droplets coated with CWS and TDM;
(f) lyophilizing the resulting emulsion and subsequently reconstituting said emulsion with sterile distilled water.
2. The method of claim 1, wherein said ratio is between about 2.5:1 and 3.5:1.
3. The method of claim 1, wherein said light hydrocarbon non-biodegradable oil is present in an amount between about 0.75 and 1.5 percent by volume.
4. The method of claim 1, wherein the amount of said deter-gent is between about 0.10 and 0.20 percent by volume based on the total volume of the composition.
5. A pharmaceutical composition prepared by the method of claim 1.
6. The composition of claim 5 wherein said weight ratio is between about 2.5:1 and 3.5:1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US37284482A | 1982-04-29 | 1982-04-29 | |
| US372,844 | 1982-04-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1202903A true CA1202903A (en) | 1986-04-08 |
Family
ID=23469853
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000426843A Expired CA1202903A (en) | 1982-04-29 | 1983-04-27 | Stable composition and preparation thereof |
Country Status (4)
| Country | Link |
|---|---|
| AU (1) | AU555085B2 (en) |
| CA (1) | CA1202903A (en) |
| GB (1) | GB2120548B (en) |
| NZ (1) | NZ204020A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5059518A (en) * | 1988-10-20 | 1991-10-22 | Coulter Corporation | Stabilized lyophilized mammalian cells and method of making same |
| 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. |
| US6090385A (en) * | 1993-12-10 | 2000-07-18 | Maes; Hubert | Method of treating cancer |
| AUPO359396A0 (en) | 1996-11-13 | 1996-12-05 | Amrad Operations Pty. Limited | A method of treatment and pharmaceutical compositions useful for same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1163470A (en) * | 1967-11-13 | 1969-09-04 | Cecil Arthur Clark | Improvements in or relating to Adjuvant Vaccines |
| US3932943A (en) * | 1970-08-14 | 1976-01-20 | E. I. Du Pont De Nemours And Company | Method of preparation of lyophilized biological products |
| US4134214A (en) * | 1977-08-05 | 1979-01-16 | Merck & Co., Inc. | Freeze-drying process for the preparation of meningococcus vaccine without degradation of potency |
-
1983
- 1983-04-27 NZ NZ20402083A patent/NZ204020A/en unknown
- 1983-04-27 CA CA000426843A patent/CA1202903A/en not_active Expired
- 1983-04-28 AU AU14015/83A patent/AU555085B2/en not_active Ceased
- 1983-04-28 GB GB8311592A patent/GB2120548B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB2120548B (en) | 1986-11-26 |
| GB8311592D0 (en) | 1983-06-02 |
| GB2120548A (en) | 1983-12-07 |
| AU555085B2 (en) | 1986-09-11 |
| AU1401583A (en) | 1983-11-03 |
| NZ204020A (en) | 1987-01-23 |
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