Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Patent
1981-08-18
1983-06-21
Hazel, Blondel
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
424180, 424 88, A61K 39108, A61K 3902, C07H 2102, A61K 3170
Patent
active
043893961
DESCRIPTION:
BRIEF SUMMARY
This invention which was developed at the Centre d'Immunologie et de Biologie PIERRE FABRE relates to non-specific immunostimulating preparations containing ribosomal RNA's and to processes for the preparation of these RNA's.
The vaccinating power of ribosomes and RNA's is known but is only developed in the presence of adjuvants, such as membranal proteoglycans or membranal polysaccharides, in addition to which the activity developed is essentially preventative.
Now, the Applicants have found that certain RNA's may be used on their own for the treatment of diseases attributable to immunodeficits, such as leprosy and cancer.
Thus, the Applicants have demonstrated the very considerable non-specific immunostimulating power of the ribosomal RNA's of:
Accordingly, the present invention relates to non-specific immunostimulating preparations for the treatment of immunodeficits such as those encountered in leprosy and cancer, characterised in that they contain as sole active principle one or more bacterial ribosomal RNA's extracted from the following strains:
The immunostimulating preparations according to the invention are preferably made up in injectable form, the concentrations and frequency of the injections being of course variable according to the disease to be treated. In the majority of cases, each dose contains of the order of 10 .mu.g to 50 .mu.g of ribosomal RNA in a support acceptable in human therapeutics which corresponds to the daily doses for an adult.
The present invention also relates to a process for the preparation of RNA suitable for working on a commercial scale. The processes which have hitherto been described for the preparation of ribosomal RNA's are all based on a complex technology which does not lend itself to large-scale production under satisfactory conditions. Thus, in the process in which the RNA's are extracted with phenol, the extraction phases are difficult to separate with existing industrial apparatus.
Accordingly, the process according to the present invention is a process for the preparation of bacterial ribosomal RNA's which is characterised in that: with an aqueous solution of sodium dodecyl sulphate under heat and precipitating the crude RNA's, and heat, followed by precipitation of the purified RNA's.
In one preferred embodiment, this process is characterised in that: with an aqueous solution of sodium dodecyl sulphate having a concentration of from 1 to 5% at a temperature in the range from 30.degree. to 50.degree. C., the crude RNA's are precipitated by adding sodium acetate and ethanol to the mixture and collecting the precipitate of crude RNA's, the range from 20.degree. to 40.degree. C., after which the purified RNA is precipitated by adding ammonium cetyl trimethyl bromide to the solution obtained, the precipitate of purified RNA's is collected and the ammonium cetyl trimethyl bromide is eliminated from the precipitate.
The treatment with sodium dodecyl sulphate enables the majority of proteins attached to the RNA by ionic bonds to be liberated. The treatment with proteolytic enzymes enables the proteins remaining after the preceding treatment to be eliminated by cleavage.
In one preferred embodiment of the process, a 2.5% solution of sodium dodecyl sulphate (SDS) is used for about 30 minutes at a temperature of +40.degree. C.
After precipitation of the crude RNA's, it is of advantage to repeatedly wash the deposit of RNA with aqueous ethanol containing sodium acetate before exposing it to the action of the proteolytic enzyme.
Proteolytic enzymes suitable for use in accordance with the invention include in particular pronase and trypsin.
The proteolysis mixture is treated with a solution containing from 2 to 10% by volume of ammonium cetyl trimethyl bromide (CETAVLON), the precipitate being recovered by centrifuging.
The RNA may be recovered from the precipitate by washing the deposit with an excess of aqueous ethanol containing sodium acetate which enables the CETAVLON to be eliminated.
The processes by which the ribosomes are prepared from ground bacteria are know
Durand Jacques
Dussourd d'Hinterland Lucien
Normier Gerard
Pinel Anne-Marie
Hazel Blondel
Pierre Fabre S.A.
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