Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific... – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1995-06-05
2001-02-20
Swartz, Rodney P. (Department: 1645)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
Bacterium or component thereof or substance produced by said...
C424S093400, C514S928000, C514S885000
Reexamination Certificate
active
06190671
ABSTRACT:
The subject of the invention is a new medicinal product containing, as active ingredient, envelopes or fractions of envelopes of non-photosynthetic and non-fruiting filamentous bacteria, as well as its preparation.
It is known that certain constituents of the envelopes of Gram-negative bacteria, and especially the lipopolysaccharide fractions which can be extracted therefrom, exhibit properties of non-specific stimulation of the immune defences of the body; see for example Int. Archs Allergy Appl. Immun. 76 Suppl. 1.119-127, (1985), and Journal of immunopharmacology 3(2), 119-132(1981).
It has now been discovered that the envelopes of certain bacteria called, according to the classification of Bergey's Manual, non-photosynthetic and non-fruiting filamentous bacteria, exhibit particularly advantageous immunomodulatory properties. They stimulate especially, in a non-specific manner, the immune defences, including cellular immunity and in particular the macrophages.
The subject of the invention is therefore a medicinal product having especially an immunomodulatory effect, containing bacteria envelopes or fractions of these envelopes, characterized by the fact that the said bacteria are chosen from the non-photosynthetic and non-fruiting filamentous bacteria as defined according to the classification of Bergey's Manual of Systematic Bacteriology, Volume 3, Section 23, 9th Edition 1989.
“Envelope” is called herein the bacterial wall and optionally the underlying membranes.
Among the bacteria whose envelopes or envelope fractions constitute an active ingredient of the immunomodulatory medicinal product of the invention, there may be mentioned more particularly the bacteria belonging to the order Beggiatoales, and especially the bacteria belonging to the genus Beggiatoa, such as for example various strains of Beggiatoa alba according to the definition given in Arch. Microbiol. (1984) 137,139-144. It should be noted that this definition of B. alba corresponds to the former names Beggiatoa arachnoidea, B. gigantea, B. leptomiformis, B. minima, B. mirabilis of Bergey's Manual, 8th Edition.
There may be mentioned, moreover, the bacteria belonging to the genus Vitreoscilla which is known to be related to and often difficult to differentiate from the genus Beggiatoa.
The bacteria which have just been defined, and of which several have already been described, generally have an aquatic habitat and can be found especially in thermal springs.
Among the bacteria which can be used there may be mentioned for example:
Vitreoscilla beggiatoïdes
(ATCC 43181)
Beggiatoa alba
(ATCC 33555).
It is known that the culture of non-photosynthetic and non-fruiting filamentous bacteria is relatively difficult, like the production of pure cultures. Most others recommend the use of poorly defined media, including various maceration fluids using tap water. The carbon source recommended is an acetate.
It has now been discovered that it is possible to adapt these bacteria, by counter-selection, to the use of a monosaccharide, instead of acetate, as carbon source.
It has been discovered, in addition, that it is possible to culture these bacteria on a perfectly defined culture medium. A culture can in particular be carried out in the following medium:
TABLE 1
COMPOSITION
CONCENTRATION
Autolytic yeast extract
0.5 to 5
g/l
Peptone
0.5 to 5
g/l
Glucose, anhydrous
0.5 to 7
g/l
Heller microelements
0.5 to 5
ml/l
CaCl
2
.10H
2
O
0.010 to 0.200
g/l
The mixture is adjusted to 1000 ml with distilled water. Among the peptones which can be used, may be mentioned for example soya bean papain peptone.
This special medium differs from the media generally used in the absence of catalase and sulphide.
The Heller microelements, of which the composition is given in the experimental section below, have been described by Heller, Ann Sci. Nat. Biol. Veg. 14:1-223 (1953). They are mixtures of various mineral elements which were recommended by Heller, not for the culture of bacteria but for the nutrition of plant tissues cultivated in vitro. It should be noted here that attempts were not made to determine if the Heller micro-elements are all essential or useful in the culture of non-photosynthetic and non-fruiting filamentous bacteria. It has nevertheless been found that the Heller microelements, when used together, for example in combination with the other constituents mentioned in Table 1 above, indeed permit the culture of the bacteria considered.
The culture can be performed at the appropriate temperature suitable for the cultured bacterial species. Generally, this temperature is between 18 and 40° C. according to the strains. The pH of the culture medium is preferably between 5.5 and 8.
By virtue of the process of the invention, it was possible to obtain, from samples of thermal springs, axenic cultures, in spite of the known difficulties of obtaining pure cultures of non-photosynthetic and non-fruiting filamentous bacteria.
The subject of the invention is therefore also a process for the culture, in aqueous medium, of non-photosynthetic and non-fruiting filamentous bacteria, mainly characterized by the fact that the culture is carried out using a monosaccharide, for example glucose, as main carbon source. Strains which have been adapted and selected, as indicated above, for the use of a monosaccharide, instead of acetate, as main carbon source, are therefore thus cultured. The culture can therefore be carried out in the absence of acetate. According to other embodiments, the procedure can, in addition, be carried out in the absence of H
2
S (or of sulphides) and/or in the absence of catalase, although these ingredients were generally considered as being essential up until now. The procedure can also be carried out in the presence of Heller microelements, for example in the proportions indicated in Table 1. The culture medium defined in Table 1 can be used in particular.
The subject of the invention is also a process for the preparation of a medicinal product as defined above, characterized by the fact that the said bacteria are cultured in an appropriate culture medium, and that the strains capable of multiplying using a monosaccharide as main carbon source are then selected and cultured, that the biomass or the bacterial envelopes are separated, and that fractions of the said envelopes, especially lipopolysaccharide fractions, are optionally extracted according to known methods.
When strains are used which have not yet been adapted, for example strains or mixtures of strains from a sample of thermal springs or of sea water, they are first cultured in a conventional culture medium containing acetate (for example sodium acetate) as sole carbon source. The strains capable of multiplying using a monosaccharide, for example glucose, as sole carbon source are then selected, in particular by culturing in the defined medium which was indicated earlier. For the selection and production of pure cultures, methods for the inoculation of solid culture media (agar gel) by means of dilutions of liquid culture media, are used in a known manner in order to isolate colonies derived from the same bacterial cell.
After culturing the bacteria, the biomass can be isolated by various known methods, for example by filtration, by coagulation with an alcohol (ethanol, isopropanol or isobutanol), by drying on a scraped prelayer (starch, diatomes and the like) cylinder, or by freeze-drying. A preliminary concentration, for example at 80° C. under reduced pressure, improves this separation.
An operation for disrupting the envelopes can be carried out, for example by the action of ultrasound. Extracts can, in addition, be prepared having an immunostimulant activity, especially by means of an alcohol such as ethanol or propanol.
Lipopolysaccharide extracts can also be prepared according to known methods; see for example Noris and Ribbons, Methods in Microbiology, Vol. 5B, Academic Press (1971). The method generally used is the well known so-called Westphal method (or a related method), which consists in carrying out the extraction with water-phen
Aubert Lucien
Martin Richard
L'Oreal
Nixon & Vanderhye P.C.
Swartz Rodney P.
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