FR285F - Drug containing antibiotic-resistant E. coli bacteria. - Google Patents

Description

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International Clarification: A 61 k

Do you think that? <<■ -;! anti-magnetism.

Mr. Lucien NOUVEL Resilient in Fiance (Paris).

[Principal patent taken on i March 1963.)

Deistamie I April 16, 1968, at 31 hours, in Paris.

Issued by anêlé of November 12, 1969.

(Official Bulletin of Industrial Property [/J.S..1/.], No. 51 of 22 December 1969.)

In this main patent, a medicament for the treatment of lic diseases in a keystone of the intestinal flora has been described, the medicament being based on non-pathogenic, live, antibiotic-resistant E. coli bacteria; more particularly, the drug in question contains an amino acid, and is buffered to have a pH of 4 to 9.

The present invention provides a perfumeion to this drug, which itself was a significant step forward on the known medical compositions of non-pathogenic live E. coli. It aims at the use of a type of bacterial strains which have unexpectedly proved to be much more active than the strains usually employed. The strains according to the present invention are of such an efficiency that not only do they render the drug of the main patent more active, but they also make it possible to obtain very effective compositions, even if the particular measures according to the main patent are not applied. ; however, the presence of amino acid and a pH buffer in the drug is preferred because without Wunpon substances the colibacilli die, very quickly in aqueous suspension. It makes it possible to improve the old compositions, both of E. coli alone and of these bacilli, accompanied by other non-pathogenic bacteria, lacteal enzymes, milk and other substances.

When one examines the compositions based on living E. coli bacteria, used until now, one finds that their bacteria are, at least in the great majority, in the form "R", that is to say of the type which forms irregularly contoured columns with a rough, dry, matte surface, and is known in enterobacteriology ("Laboratory Diagnosis of Euerobac-tenes", Léon Le Minor; 3rd edition), that the form

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| 't S' of colibacillus, giving on agar I colonies round, convex, smooth, shiny and wet, undergo - in strains in collection - a progressive transformation into the form "R". A distinct characteristic! Ve between the two kinds of strains is the absence of the "0" antigen in the "R" form, while the "3" form alone, an example of "R", has it at maximum.

The present invention is based on the quite unexpected finding that living, non-pathogenic, "S" shaped bacteria exert much more beneficial beneficial effects in the intestine than do bacteria of the "R" form. The effectiveness of a strain of coli bacteria is mainly a function of its enzymatic equipment that induces the "0" antigen.

The novel medicament according to the invention therefore consists of living, preferably antibiotic-resistant, nonpathogenic colibacilli, comprising a high proportion of bacteria of the "S" form, and one or more of the materials generally present in this type of compositions.

The medicament according to the invention may be in any of the usual forms. in particular in that of an aqueous suspension island germs of E. coli "S" or rich in this form, paste, powder, and especially freeze-dried powder.

Preferably, the strain of E. coli is entirely pure bacteria of the form "S"; practically complete absence of the form '<R' being quite difficult to obtain, very effective drugs are nevertheless obtained with stem islands containing about 10 to 95% or better 50 to 98% of "S" form, the rest being in the form "II". In other words, the antigen richness of the strains used may be of the order of 10 to 95% of the maximum which corresponds to the form "S has no" R ".

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As indicated above, the drug may be used in more than one day in May, when it is present in the blood. 'e <l <• •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• In particular, as far as the addition of useful amino acids is concerned, it may have recourse to one or more acids taken in the free or tic state. preferably self: -; the fomu: of an alkaline salt, such as for example glvco-glue, alanine. U'ueine. isoleuciile, aiT.inine. a-pa-ragin, glulamine, glutamic acid, iistidine, ivsine, omithine, phenylalanine, proline, serine, tluvcime, thyroxine, iryntophane, tvro.-ine. valinr, cysineine, cystine, mcihioninr, etc.

Where the new medicinal product contains, besides E. coli, other non-pathogenic Enterobacteriaceae, it is preferable that they also, in whole or in part, be in the form

"S".

It is also advisable to incorporate a mucin into the drug; the role of this kind of mucleopolysaccharides, already reported in BF No. 1,482,817, is confirmed when it comes to the preparation of freeze-dried culture rich in "S" form, according to the present invention.

When in the drug of the main patent, most of the colibacilli is replaced by seeds of the form "S", the clinical results, reported in Examples 2, 3 and 5 dndil patent are well improved: in all similar cases, the patients are relieved and undergo a marked improvement more rapidly; their healing is advanced from several hours to two days depending on the case.

The invention also includes a process for the preparation of the novel enteric bacterial rich "S" form drug. This method is particularly useful when it comes to the preparation of antibiotic resistant culture; the reason is that it is precisely in environments with antibiotics that the evolution towards the "R" form is particularly exalted; this has resulted in the difficulty of obtaining antibiotic-resistant "S" strains in a medium containing antibiotics.

The method according to the invention consists in culturing non-pathogenic E. coli in a nutrient medium containing antibiotics in increasing dose. After isolation of the strain, it is cultured in a nutrient medium containing fresh guinea pig serum which lyses the "R" forms. An antibiotic-resistant form of <S "shape is thus obtained.

According to a particular feature of the invention, the properties of the fresh guinea-pig serum are used for the elimination of "R" forms; for this purpose, the culture broth is supplemented with such a serum.

By way of non-limiting example, given solely by way of illustration of the pre-invention,

the operations applied in a preparation of the medicament according to the invention.

In culture medium, the following composition was analyzed:

Arginiiic 1000 g

Amino extract of yeast 2000 g

Agar 500 g

Di-odium phosphate 4000 g

Potassium dihydrogen phosphate - 400 g

Distilled water - sterile q.s. 500 1.

This broth, whose fold is adjusted to about 7.3, is autoclaved and then centrifuged and sterilized. After cooling, it is inoculated with 2 liters of an aqueous suspension of non-pathogenic bacteriophage living, antibio-resistant, containing a majority of "S" form; this suspension was obtained by culturing in a solid or liquid nutrient medium containing 10 to 25% fresh guinea-pig serum.

In a particularly advantageous embodiment of the invention, the powder, obtained by lyophilization of a culture of non-pathogenic, antibiotic-resistant coliforms, for the most part in the "S" form, is sterilely coated with silicones. Suitable for this purpose is the silicone known in the trade under the name SI 200, preferably used in a proportion of 5 to 25% of the weight of the powder to be treated. The preferred silicones have viscosities of the order of 100 to 1000 cSt, this indication not being however limiting.

The coating of the powder with silicone provides several advantages; it isolates, so to speak, the particles of the powder, protecting them from direct contact with the air, which renders them less alterable by atmospheric oxygen; it makes it possible to add to the powder a substance that is not compatible with E. coli bacteria, a substance that can destroy them, such as for example an antibiotic. Thanks to its hydrophobic properties, the silicone makes it possible to preserve, in the freeze-dried powder, the residual moisture of the latter, necessary for a good conservation. Moreover, the silicones being stable in acid medium, they do not disintegrate in the stomach, but only in the intestine where the medium is basic; thus, the use of E. coli becomes much more rational and efficient, since they are released only at the level of the intestine, that is to say precisely where they must act.

By way of non-limiting example, a powder according to the invention is prepared as follows, 100 g silicone island SI 200, viscosity 350 cSt., Are mixed with 1000 g of lyophilized powder of a culture of non-pathogenic E. coli, antimicrobially resistant, predominantly "S" form. The thus silicone powder is mixed with tetracyoline hydrochloride at the rate of 250 mg of hydrochloride

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piMir (<0 m ;. 'ilt- 11111: •! r •: each <1 island 60 tn {* «1 *»

! i <-11:> i: i • uiiiiri '<"11; <- -; The number of germs *, living * included, and 150 million.

nÉscMi-:

1. Medicine. for the treatment of conditions which are read off from an equilibrium of the plant species, characterized in that it contains living non-pathogenic E. coli, including a high proportion of colibiae of the ton "S".

2. Medicament according to 1, wherein the proportion of coli bacilli of the Ion • S "is 10 to 98%, the jacket being care of the ton" II ".

3. Drug according to 1 or 2, characterized in that it contains one or more materials such as amino or salified acid, pH buffer, yeast extract, thickener or other substances usually present in drugs based on enterobacterial cultures.

4. Drug according to 1, 2 or 3, characterized in that it contains other non-pathogenic enterobacteria, at least a portion of which is preferably in the form "S".

5. Drug according to 1, 2, 3 or 4, characterized in that the colibacillus of the form "S" is anti-bioresi-tant.

C. Medicain according to one or more of

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points 1 to 5, characterized in that the colibacillus is therein in the form of a freeze-dried powder culture, coated with silicone.

7. Powder according to 6, characterized in that it contains. 5 to 25 parts by weight of silicone per 100 parts of culture, the silicone having a pre-set viscosity of 100 to 1000 cSt.

8. Drug following 6 or / and 7, characterized in that it contains a drug substance not compatible with E. coli, especially an antibiotic.

9. A process for the preparation of the medicament according to any one of 1 to 8 which comprises culturing the useful bacteria or bacteria in a medium containing a "R" form lysant of these bacteria.

10. Process according to 6, characterized in that the Ivsant is that of a fresh serum, in particular that of the guinea pig.

11. Process for the preparation of the drug according to 8, characterized in that the powder culture is coated with silicone before the addition of. The substance incompatible with it.

Lucien NEW

Vicarious :

Armand Kohn

DOCUMENTARY NOTICE ON NEW

Documents likely to affect the novelty of the medicine: none.

Documents illustrating the state of the art in the matter:

- French Patent (B.S.M.) No. 2.828 M.

BAD ORIGINAL

on the pages of the papers, address to the National Assembly, 27, iuc of the Convention, Paria (15 *).