CA1216846A - Purification of trehalose dimycolates and product - Google Patents
Purification of trehalose dimycolates and productInfo
- Publication number
- CA1216846A CA1216846A CA000426844A CA426844A CA1216846A CA 1216846 A CA1216846 A CA 1216846A CA 000426844 A CA000426844 A CA 000426844A CA 426844 A CA426844 A CA 426844A CA 1216846 A CA1216846 A CA 1216846A
- Authority
- CA
- Canada
- Prior art keywords
- fact
- tdm
- process according
- trehalose dimycolates
- solvent
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07H13/06—Fatty acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
Abstract
ABSTRACT OF THE DISCLOSURE
A method of purifying crude trehalose dimycolates (TDM) is disclosed which comprises dissolving crude TDM in a solvent and then subjecting said solution to a low pressure silica gel column operating at a pressure between about 10 and 300 psi wherein the silica gel comprises particles in the range of between about 15 and 63 microns. The product of this process is also disclosed.
A method of purifying crude trehalose dimycolates (TDM) is disclosed which comprises dissolving crude TDM in a solvent and then subjecting said solution to a low pressure silica gel column operating at a pressure between about 10 and 300 psi wherein the silica gel comprises particles in the range of between about 15 and 63 microns. The product of this process is also disclosed.
Description
BACKGROUND OF THE INVENTION
The subject matter of the present invention is directed to a method of purifying crude trehalose dimycolates (TDM). TDM is an isolate of bacteria and when combined with cell wall skeleton (CWS) forms a composition which ~s effective in obtaining suppression and regression of tumor cells.
The combination of cell wall skeleton and TDM is known in the art (See Biologically Active Components from Mycobacterial Cell Walls. II. -Suppression and Re2ression 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 Reqression; Ribi, et al, National Cancer Institute Monograph No. 39, pages 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 obtained from any mycobacteria including, but limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia astero;des, Corynebacterium diphtheriae, Corvnebacterium parvum, M.kansasii, M.tuberculosis (Strain H 37 RV and Ayoma B3, and M.bovis Strain BC~ Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abortus and Coxiella burnettiiO
The process of producing cell wall skeleton is time ~z~
consuming. The bacteria such as M.bovis, Strain BC~ (bacillus Calmette-Guerin) is grown and harvested. The resulting whole 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 ~he protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.gO, trypsin and/or chymotrypsin) to give purified cell wall skeleton.
The second component trehalose dimycolates (TDM) may be obtained from any mycobacteria including, for example, M.avium, M.phlei, M.tuberculosis (Strain ~1 37 RV and Ayoma B), M.bovis BCG, M.sme~matis, 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 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 TD~ (See Biolo~ically Active Components from Mycobacterial Cell Walls. I. Isolation and Composition of Cell Wall Skeleton_and Component P3; Azuma, et al, Journ~l oE the National Cancer Institute, Volume 52, pages 95-101, 1974). As disclosed in Azuma, et al, crude TDM may then be further purified by centri~ugal microparticulate silica gel chromotography to g~ve purified TDM.
CWS and ~DM produced as described above can be used as an oil droplet emulsion to obtain an anti-tumor composition suitable for injection (See Immuno herapy with Non-viable Microbial Components; Ribi, et al; Annals of the New York Academy of Science, Volume 227, pages 228-238, September 2~th, 1976).
The prior art emulsions, however, suffer from a major disadvantage. Impurities remaining in the purified TDM
seriously affect the potency of the composition limiting its effectiveness in treating tumors. Prior art attempt~ at further purifying TDM have generally involved repeated time consuming costly elutions through high pressure silica gel columns.
Applicants have discovered that the use of a low pressure (i.e., between about 10 and 300 psi) column with silica gel particles having a size between about 15 and 63 microns surprisingly and effectively removes virtually all impurities from crude TDM.
It is thereEore an object of the invention to provide a process for purifying crude TDM. It is another object of the invention to provide a purified TDM product which can be combined with CWS to produce an oil in saline emulsion which is effective as an anti-tumor agent.
THE INVENTION
The present invention is directed to a process for purifying TDM to eliminate virtually all impurities normally associated with crude TDM. The process compri~es dissolving the crude TDM in a solvent and then subjecting the solution to a low pressure isilica gel column having a particle size of between about 15 and 63 microns~ The pressure employed in the column is normally betwe~n about 10 and 300 psi, preferably between about 30 and 80 psi.
A wide variety of non-polar solvents may be used to dissolve the crude TDM. The preferred solvents include, for example, chloroform, ether, hexane~ methanol, ethanol, tetrahydrofuran, petroleum ether, heptane, methylene chloride, ligroin, propanol, butanol, ethyl acetate, ben~ene, toluene, acetic acid and the like including combinations thereof.
Fractions oE the purified TDM are combined and the solvent !$~
removed. The resulting product has virtually ns detectable impurities (i.e., purity equal to or greater than 99.9 percent)~
By employing the process described above, a highly pure TDM
product is obtained without the need for repetitious purifying steps. The product can be ef~ectively combined with CWS in a conventional manner to produce a potent anti-tumor composition.
The following examples are for illustrati~e 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 Crude TDM
600 g (moist weight) of whole cells of M.Phlei which had previously been heat killed were stirred overnight on a magnetic stirrer in 4 liters of 95 percent ethanol and then vacuum filtered through a 27 cm Buchner funnel, fitted with a 24.0 cm Whatman No. l filter paper, into a 2 liter filter flask. The ethanol solution was removed. The cell residue was resu~pended in a 2 liter flask with 1500 ml of a 1:1 ethyl ether-ethanol solution and stirred overnight on a magnetic stirrer and then filtered as described above. The ether-ethanol solution was removed and a second ethyl ether-ethanol extraction and filtration was then performed. After removal of the ether-ethanol solution, the cell residue was resuspended in 1500 ml of a 2:1 chloroform-methanol solution and stirred overnight on a magnetic stirrer and filtered through a Buchner funnel or centrifuged at 10,000 rpm for 30 seconds. The chloroform-methanol solution was removed and the extractlon and filtration process repeated twice. The cell residue was air dried and bottled. The three chloroEorm-methanol solutions were combined and evaporated on a Buchi Ro~o-vapor in a tared round bottom flask. The weight of the chloroform-methanol residue was 13.0 g.
The residue was dissolved in 500 ml of a 2.1 chloroform-methanol solution and stirred Eor 1 hour on a magnetic stirrer and then filtered through a sintered glass Buchner ~unnel tcoarse, 300 ml). The solvent was evaporated on a Buchi Roto-vapor in a tared round bottom flask to provide a residue weighing 12.5 grams.
The resulting residue was resuspended in 400 ml oE ethyl ether and stirred on a magnetic stirrer for 1 hour. The suspension was then centrifuged in 2 screw-cap centrifuge bottles in a GSA Rotor at 5000 rpm for 30 minutes. The ether soluble ~raction was then decanted. Both the ether soluble and ether insoluble fractions were preserved.
The ether insoluble material was disssolved in a 200 ml
The subject matter of the present invention is directed to a method of purifying crude trehalose dimycolates (TDM). TDM is an isolate of bacteria and when combined with cell wall skeleton (CWS) forms a composition which ~s effective in obtaining suppression and regression of tumor cells.
The combination of cell wall skeleton and TDM is known in the art (See Biologically Active Components from Mycobacterial Cell Walls. II. -Suppression and Re2ression 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 Reqression; Ribi, et al, National Cancer Institute Monograph No. 39, pages 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 obtained from any mycobacteria including, but limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia astero;des, Corynebacterium diphtheriae, Corvnebacterium parvum, M.kansasii, M.tuberculosis (Strain H 37 RV and Ayoma B3, and M.bovis Strain BC~ Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abortus and Coxiella burnettiiO
The process of producing cell wall skeleton is time ~z~
consuming. The bacteria such as M.bovis, Strain BC~ (bacillus Calmette-Guerin) is grown and harvested. The resulting whole 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 ~he protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.gO, trypsin and/or chymotrypsin) to give purified cell wall skeleton.
The second component trehalose dimycolates (TDM) may be obtained from any mycobacteria including, for example, M.avium, M.phlei, M.tuberculosis (Strain ~1 37 RV and Ayoma B), M.bovis BCG, M.sme~matis, 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 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 TD~ (See Biolo~ically Active Components from Mycobacterial Cell Walls. I. Isolation and Composition of Cell Wall Skeleton_and Component P3; Azuma, et al, Journ~l oE the National Cancer Institute, Volume 52, pages 95-101, 1974). As disclosed in Azuma, et al, crude TDM may then be further purified by centri~ugal microparticulate silica gel chromotography to g~ve purified TDM.
CWS and ~DM produced as described above can be used as an oil droplet emulsion to obtain an anti-tumor composition suitable for injection (See Immuno herapy with Non-viable Microbial Components; Ribi, et al; Annals of the New York Academy of Science, Volume 227, pages 228-238, September 2~th, 1976).
The prior art emulsions, however, suffer from a major disadvantage. Impurities remaining in the purified TDM
seriously affect the potency of the composition limiting its effectiveness in treating tumors. Prior art attempt~ at further purifying TDM have generally involved repeated time consuming costly elutions through high pressure silica gel columns.
Applicants have discovered that the use of a low pressure (i.e., between about 10 and 300 psi) column with silica gel particles having a size between about 15 and 63 microns surprisingly and effectively removes virtually all impurities from crude TDM.
It is thereEore an object of the invention to provide a process for purifying crude TDM. It is another object of the invention to provide a purified TDM product which can be combined with CWS to produce an oil in saline emulsion which is effective as an anti-tumor agent.
THE INVENTION
The present invention is directed to a process for purifying TDM to eliminate virtually all impurities normally associated with crude TDM. The process compri~es dissolving the crude TDM in a solvent and then subjecting the solution to a low pressure isilica gel column having a particle size of between about 15 and 63 microns~ The pressure employed in the column is normally betwe~n about 10 and 300 psi, preferably between about 30 and 80 psi.
A wide variety of non-polar solvents may be used to dissolve the crude TDM. The preferred solvents include, for example, chloroform, ether, hexane~ methanol, ethanol, tetrahydrofuran, petroleum ether, heptane, methylene chloride, ligroin, propanol, butanol, ethyl acetate, ben~ene, toluene, acetic acid and the like including combinations thereof.
Fractions oE the purified TDM are combined and the solvent !$~
removed. The resulting product has virtually ns detectable impurities (i.e., purity equal to or greater than 99.9 percent)~
By employing the process described above, a highly pure TDM
product is obtained without the need for repetitious purifying steps. The product can be ef~ectively combined with CWS in a conventional manner to produce a potent anti-tumor composition.
The following examples are for illustrati~e 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 Crude TDM
600 g (moist weight) of whole cells of M.Phlei which had previously been heat killed were stirred overnight on a magnetic stirrer in 4 liters of 95 percent ethanol and then vacuum filtered through a 27 cm Buchner funnel, fitted with a 24.0 cm Whatman No. l filter paper, into a 2 liter filter flask. The ethanol solution was removed. The cell residue was resu~pended in a 2 liter flask with 1500 ml of a 1:1 ethyl ether-ethanol solution and stirred overnight on a magnetic stirrer and then filtered as described above. The ether-ethanol solution was removed and a second ethyl ether-ethanol extraction and filtration was then performed. After removal of the ether-ethanol solution, the cell residue was resuspended in 1500 ml of a 2:1 chloroform-methanol solution and stirred overnight on a magnetic stirrer and filtered through a Buchner funnel or centrifuged at 10,000 rpm for 30 seconds. The chloroform-methanol solution was removed and the extractlon and filtration process repeated twice. The cell residue was air dried and bottled. The three chloroEorm-methanol solutions were combined and evaporated on a Buchi Ro~o-vapor in a tared round bottom flask. The weight of the chloroform-methanol residue was 13.0 g.
The residue was dissolved in 500 ml of a 2.1 chloroform-methanol solution and stirred Eor 1 hour on a magnetic stirrer and then filtered through a sintered glass Buchner ~unnel tcoarse, 300 ml). The solvent was evaporated on a Buchi Roto-vapor in a tared round bottom flask to provide a residue weighing 12.5 grams.
The resulting residue was resuspended in 400 ml oE ethyl ether and stirred on a magnetic stirrer for 1 hour. The suspension was then centrifuged in 2 screw-cap centrifuge bottles in a GSA Rotor at 5000 rpm for 30 minutes. The ether soluble ~raction was then decanted. Both the ether soluble and ether insoluble fractions were preserved.
The ether insoluble material was disssolved in a 200 ml
2:1 chloroform-methanol solution and filtered through a Buchner funnel.
The ether soluble fraction was evaporated on a Buchi Roto-vapor in a tared round bottom flask and an ether soluble residue was obtained. This residue was dissolved in 300 ml of ether and precipitated into 900 ml of methanol. The precipitate was filtered through a Buchner funnel using Whatman No. 1 filter paper and combined with the ~:1 chloroform-methanol solutlon containing the ether insoluble material described above. The resulting solution was evaporated on a Buchi Roto-vapor in a tared round bottom flask to obtain 6.5 grams of a residue.
The ras~due was dissolved in 200 ml o~ a 2:1 chloroform-methanol solution and poure~ into a 500 ml separatory funnel.
This solution was then added drop-wise into a 2 liter flask, ~n a magnetic stirrer, containing 600 ml of acetone. The resulting precipitate was filtered into a Buchner funnel, air dried and placed in a tared bottle to obtain 4.5 9 of crude TDM.
Exa~ple 2 - Purification of Crude ~DM
2 grams of crude TDM as obtained in Example 1 were dissolved in 2 ml of a 10:1 chloroform-methanol solution and then drawn onto a 5 ml sample loop. The remainder o~ the loop was filled with solvent. The solution was pumped onto a silica gel 60 column (25 X 1000 mm) having a particle size of from about 15 to 63 microns, using an Isco Model 132 pump. The column was eluted with 800 ml of chloroform, followed by 1200 ml of a 98:2 chloroform-methanol solution at a rate of 4 ml/minute.
(The flow rate is critical as it has been found that resolution is less when the flow rate is unduly increased. Consequently~
elutlon is generally affected at a flow rate varying between about 0.1 ml to 20 mls./minute and normally at a rate between about 2 and 5 mIs./minute.) The effluent from the column was then connected to a fraction collector and fractions of 8 ml per tube were collected while the column was being eluted with 3500 ml of a 96:4 chloroform-methanol solution. The tubes containing purified TDM were determined by spotting aliquots of the various fractions on TLC plates (silica gel F-254, 5XlOcm, 0.25mm thick) using a 10:1 chloroform-methanol solution as an eluant and comparing these fractions with pure TDM previously isolated.
Visualization of TLC plates were produced by spraying plates with 10 percent (w/v) of phosphomolybdic acid in ethanol.
Fractions containing purified TDM were combined and solvent evaporated on a Buchi Roto-vapor in a tared round bottom flask~
358 mg of purified TDM were obtained.
The ether soluble fraction was evaporated on a Buchi Roto-vapor in a tared round bottom flask and an ether soluble residue was obtained. This residue was dissolved in 300 ml of ether and precipitated into 900 ml of methanol. The precipitate was filtered through a Buchner funnel using Whatman No. 1 filter paper and combined with the ~:1 chloroform-methanol solutlon containing the ether insoluble material described above. The resulting solution was evaporated on a Buchi Roto-vapor in a tared round bottom flask to obtain 6.5 grams of a residue.
The ras~due was dissolved in 200 ml o~ a 2:1 chloroform-methanol solution and poure~ into a 500 ml separatory funnel.
This solution was then added drop-wise into a 2 liter flask, ~n a magnetic stirrer, containing 600 ml of acetone. The resulting precipitate was filtered into a Buchner funnel, air dried and placed in a tared bottle to obtain 4.5 9 of crude TDM.
Exa~ple 2 - Purification of Crude ~DM
2 grams of crude TDM as obtained in Example 1 were dissolved in 2 ml of a 10:1 chloroform-methanol solution and then drawn onto a 5 ml sample loop. The remainder o~ the loop was filled with solvent. The solution was pumped onto a silica gel 60 column (25 X 1000 mm) having a particle size of from about 15 to 63 microns, using an Isco Model 132 pump. The column was eluted with 800 ml of chloroform, followed by 1200 ml of a 98:2 chloroform-methanol solution at a rate of 4 ml/minute.
(The flow rate is critical as it has been found that resolution is less when the flow rate is unduly increased. Consequently~
elutlon is generally affected at a flow rate varying between about 0.1 ml to 20 mls./minute and normally at a rate between about 2 and 5 mIs./minute.) The effluent from the column was then connected to a fraction collector and fractions of 8 ml per tube were collected while the column was being eluted with 3500 ml of a 96:4 chloroform-methanol solution. The tubes containing purified TDM were determined by spotting aliquots of the various fractions on TLC plates (silica gel F-254, 5XlOcm, 0.25mm thick) using a 10:1 chloroform-methanol solution as an eluant and comparing these fractions with pure TDM previously isolated.
Visualization of TLC plates were produced by spraying plates with 10 percent (w/v) of phosphomolybdic acid in ethanol.
Fractions containing purified TDM were combined and solvent evaporated on a Buchi Roto-vapor in a tared round bottom flask~
358 mg of purified TDM were obtained.
Claims (7)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for purifying crude trehalose dimycolates character-ized by:
(a) dissolving crude trehalose dimycolates in a solvent; and (b) subjecting the solution to a low pressure silica gel col-umn having a particle size of between about 15 and 63 microns, and at a pressure between about 10 and 300 psi.
(a) dissolving crude trehalose dimycolates in a solvent; and (b) subjecting the solution to a low pressure silica gel col-umn having a particle size of between about 15 and 63 microns, and at a pressure between about 10 and 300 psi.
2. The process of Claim 1, characterized by the fact that said pres-sure is between about 30 and 80 psi.
3. The process of Claim 1 or 2, characterized by the fact that the solvent is chloroform, ether, hexane, ethanol, methanol, tetrahydro-furan, petroleum ether, heptane, methylene chloride, ligroin, pro-panol, butanol, ethyl acetate, benzene, toluene, acetic acid or mixtures thereof.
4. The process according to Claim 1 or 2, characterized by the fact that said trehalose dimycolates are obtained from mycobacteria.
5. The process according to Claim 1 or 2, characterized by the fact that said trehalose dimycolates are obtained from the mycobacteria M. avium, M. phlei, M. tuberculosis (Strain H 37 RV and Ayoma B), M. bovis, BCG, M. smegmatis, M. kansasii, Norcardia rubra, M. bov-inis, or Corynebacterium diphtheriae.
6. The process according to Claim 1 or 2, characterized by the fact that the elution is conducted at a rate varying between about 0.1 ml.
to 20 mls./minute.
to 20 mls./minute.
7. The process according to Claim 1 or 2, characterized by the fact that the elution is conducted at a rate varying between about 2 and 5 mls./minute.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US37284382A | 1982-04-29 | 1982-04-29 | |
| US372,843 | 1982-04-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1216846A true CA1216846A (en) | 1987-01-20 |
Family
ID=23469848
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000426844A Expired CA1216846A (en) | 1982-04-29 | 1983-04-27 | Purification of trehalose dimycolates and product |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS58219122A (en) |
| AU (1) | AU565095B2 (en) |
| CA (1) | CA1216846A (en) |
| DE (1) | DE3315264A1 (en) |
| FR (1) | FR2526027B1 (en) |
| GB (1) | GB2119794B (en) |
| IT (1) | IT1164201B (en) |
| NZ (1) | NZ204021A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63200162U (en) * | 1987-06-05 | 1988-12-23 | ||
| WO2012003205A2 (en) * | 2010-07-02 | 2012-01-05 | Intervet International B.V. | Vaccine against ehrlichia canis and associated methods |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2001118A1 (en) * | 1968-02-01 | 1969-09-26 | Kyowa Hakko Kogyo Kk | |
| FR2160326B1 (en) * | 1971-11-19 | 1975-02-07 | Anvar | |
| JPS5129292A (en) * | 1974-09-02 | 1976-03-12 | Kyowa Hakko Kogyo Kk | HATSUKOHONYORUGURUKOOSU OYOBI TOREHAROOSU NO SHIBOSANESUTERU NO SEIZOHO |
| FR2319373A1 (en) * | 1975-07-29 | 1977-02-25 | Anvar | (2,2)-Diesterified diholosides prodn. - from cpd. with all other hydroxy gps. silylated, and a fatty acid or its reactive deriv. |
| US4307229A (en) * | 1979-11-07 | 1981-12-22 | Avraham Liav | 6,6'-Diesters of trehalose and process for the synthesis thereof |
-
1983
- 1983-04-27 NZ NZ204021A patent/NZ204021A/en unknown
- 1983-04-27 DE DE19833315264 patent/DE3315264A1/en active Granted
- 1983-04-27 CA CA000426844A patent/CA1216846A/en not_active Expired
- 1983-04-28 FR FR8307012A patent/FR2526027B1/en not_active Expired
- 1983-04-28 IT IT20835/83A patent/IT1164201B/en active
- 1983-04-28 GB GB08311593A patent/GB2119794B/en not_active Expired
- 1983-04-28 AU AU14016/83A patent/AU565095B2/en not_active Ceased
- 1983-04-28 JP JP58074182A patent/JPS58219122A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| FR2526027A1 (en) | 1983-11-04 |
| FR2526027B1 (en) | 1987-03-27 |
| IT8320835A1 (en) | 1984-10-28 |
| JPS58219122A (en) | 1983-12-20 |
| JPS6257166B2 (en) | 1987-11-30 |
| NZ204021A (en) | 1986-11-12 |
| GB2119794A (en) | 1983-11-23 |
| GB8311593D0 (en) | 1983-06-02 |
| AU565095B2 (en) | 1987-09-03 |
| GB2119794B (en) | 1986-04-03 |
| IT1164201B (en) | 1987-04-08 |
| AU1401683A (en) | 1983-11-03 |
| DE3315264A1 (en) | 1983-12-01 |
| DE3315264C2 (en) | 1988-02-25 |
| IT8320835A0 (en) | 1983-04-28 |
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