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• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
  • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
• • 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
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/63—Vibrio

Definitions

• the present invention relates to a new substance having an adjuvant effect, obtained from
• the inventors have isolated from substances supernatants of Vibrio cholerae a substance which they have called "D.G.Z.” and which is characterized in that:
• its sugar composition essentially comprises: rhamnose approximately 4.1% annose approximately 42% galactose approximately 32% glucose 2/3% approximately
• its amino acid composition essentially comprises asparagma 8.2-8.7% approximately glutamine 8.9-10.5% approximately serine 5.5- 7.2% approximately glycine 4.4- 8.6% approximately histidine 2.3- 3.4% approximately arginine 2.8- 4.1% approximately threonine 5.1- 5.6% approximately alanine 7.2- 8.1% approximately proline, 5.8- 7.6% approximately ammonia 11.0-14 Approximately 7% tyrosine 2.0- 2.9% approximately valine 5.6- 6.3% approximately methionine 0.9- 1.1% approximately cysteine 0.6- 1.1% approximately isoleucine 2.4- 2.8% approximately leucine 6.9- approximately 8.8% phenylalanine 3.4- 4.2% approximately lysine 4.6- 7.0% approximately
• its fatty acid composition essentially comprises C- fatty acids
• Ci4.30H about 0.1% C- j -j about 0.13%
• the present invention also relates to an antimitotic composition characterized in that it comprises the adjuvant substance extracted from the supernatant of Vibri cholerae in accordance with the present invention, associated with at least one antimitotic agent.
• the antimitotic agent is advantageously taken from the group which comprises in particular Cis platinum, 5-fluorouracil, adriamycin, mitomycin.
• the adjuvant and the anti mitotic are present in the composition in an adjuvant / antimitotic ratio of the order of 1 to 10.
• the present invention also relates to methods of obtaining the substance having an adjuvanting effe, defined above, from. cultures of Vibri cholerae.
• a process for obtaining such a substance is characterized in that the supernatant of Vibrio cholerae cultures is precipitated then centrifuged, then the centrifugation pellet is dissolved in distilled water, and the solution heated then centrifuged dialyzed and lyophilized to give rise to the antimitotic effect substance identified above.
• the culture supernatant is obtained from a culture of Vibrio cholerae on alkaline nutritive agar, cultivated for approximately 8 hours at approximately 37 ° C.
• Another process for obtaining the substance having an adjuvant effect is characterized in that the culture supernatant of V. cholerae is treated with chloroform, which gives rise to the formation of a gel. chloroform-protein which is eliminated by decanting, while the adjuvant substance according to the invention is found in the aqueous phase.
• the culture is carried out in a Fernbach flask of 2 liters half-filled with glass beads and sterilized in the autoclave, then made up to 200 ml with trypsinized peptone water of alkaline calf at pH 8.2.
• the Fernbach flask is inoculated with this culture and is carried for 18 to 20 hours in an oven
• the culture supernatant is then precipitated with d polyethylene glycol at 28% final concentration.
• the supernatant is centrifuged at 20,000 G for 20 minutes.
• the supernatant is then rejected and the pellet dried. This is dissolved in distilled water. Thereof is gradually faley added to obtain complete dissolution of the product with the help of a few beads to homogenize the solution.
• the activity was demonstrated on cell cultures of Sarcoma 180 cells, by bringing together in a culture medium constituted by trypsinized peptone water of alkaline calf at pH 8.2, the cells and increasing amounts of the substance according to the invention.
• the activity of said substance was evaluated on the number of living cells by vital staining for 24 hours.
• Sarcoma 180 multiplies until the 6th - 7th day to give 10 ⁇ cells / l, in the presence of DGZ.
• FIG. 1 shows the viability% of a culture of Sarcoma 180 cells (10 6 cells / dish) as a function of time in the presence of DGZ alone (curve Ib) by comparison with a control (culture of sarcoma 180 cells alone) (curve la).
• FIG. 2 shows the viability% of a culture of cells (10 6 cells / dish) of Sarcoma 18 as a function of time, in the presence of a combination of Cisplatin and DGZ; o notes a regular decrease in the rate of viability from 80% on the 1st day to around 15 on the 8th day.
• FIG. 3 shows the viability% of a culture of cells (10 6 cells / dish) of Sarcoma 18 as a function of time, in the presence of
• mice were carried out on Balb / c mice weighing approximately 20 g.
• Acute toxicity an injection of 20 mg intraperitoneally does not cause any problems.
• Chronic toxicity injections of 16 to 20 mg intraperitoneally twice a week for 4 weeks cause diarrhea.
• Tests were carried out on Balb / c mice inoculated with 105 Sarcoma 180 cells and treated from the 24th hour intraperitoneally for 4 days with doses of 2 mg of the substance according to the invention, ie alone or in combination with Cisplatin.
• mice survived without ascites or tumor according to the experimental method. Note that they could not be reinoculated whatever the dose of cells injected.
• FIG. 6 shows the survival rate of mice in which 10 ⁇ Sarcoma 180 cells were inoculated, treated by injection of buffered physiological water, twice a week for 4 weeks: no mouse surviving on the 12th day.
• FIG. 7 shows the rate of mice inoculated by 10 ⁇ Sarcoma 180 cells treated by injections of DGZ (in peptone water) heated for one hour at 100 ° C. and of Cisplatin to raiso of:
• mice 18 were still alive on the 75th day after the start of treatment (2 deaths observed on the 39th day). 2. Tests were also carried out on C57 black mice inoculated with Lewis tumor cells, 10 4 cells in the left hind paw. The progress of the tumor is followed, in controls like che treated mice, by measuring the thickness of the leg. The treatment was carried out intraperitoneally for 4 days, at a rate of 1 mg of the substance according to the invention and 0.0125 mg of Cisplatin in the same syringe per mouse and per day, for 4 days, and under a volume of 0.20 ml
• Tests were also carried out on LTCH mice inoculated with mouse teratoblastoma cells.
• the evolution of the tumor is done in 45 days in 20 female mice and in 60 days in males, for the controls.
• the treatment was carried out as described in connection with the cells inoculated with Lewis tumor cells (1 mg of substance in accordance with the invention and 0.0125 mg of e Cisplatin / mouse / day / for 4 days): on 10 females 25- LTC ' E ,. only one developed tumor ascites within the same time frame as the controls; similarly, only one in ten male mice developed tumor ascites.
• the survival times were extended specacularly since the failure rate was only 5 and the survival times of the recidivist patients treated reached from 13 months to 92 months, with a total disappearance of the ascites and huge belly linked to the presence of. ascites, thus providing the patients not only with an improvement at least temporarily in terms of the disease itself, but also an important improvement on the psychological level resulting from the improvement of their physical aspect and their comfort.
• metastasized cancer pulmonary, digestive
• similar results have been obtained, with reduction of the main tumor and metastases.
• the administration conditions were identical to those used in the treatment of ovarian cancer.

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• 1987
  • 1987-04-06 FR FR8704799A patent/FR2613380B1/fr not_active Expired
• 1988
  • 1988-04-06 JP JP63503341A patent/JPH02500670A/ja active Pending
  • 1988-04-06 EP EP88903273A patent/EP0308475A1/fr not_active Withdrawn
  • 1988-04-06 WO PCT/FR1988/000167 patent/WO1988007864A1/fr not_active Application Discontinuation

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