FR2546752A1 - PROCESS FOR THE PRODUCTION OF ACETHER PLATELET AGGREGATION FACTOR-BASED PRODUCT USED AS A MEDICINAL PRODUCT AND LABORATORY REAGENT, PRODUCTS AND REAGENTS THUS OBTAINED TO PREVENT HISTAMINE RELEASE IN BLOOD AND LIVING TISSUES - Google Patents

Abstract

THE INVENTION RELATES TO A MEDICINAL PRODUCT AND A REAGENT, PREVENTING THE RELEASE OF HISTAMINE INTO THE BLOOD. WE START FROM A SAMPLE OF PAF-ACETHER THAT WE DILUTE UP TO A HOMEOPATHIC DOSE (BETWEEN 6 CH AND 15 CH), BY THE HAHNNEMANNIAN METHOD, WITH DYNAMIZATION OF INTERMEDIATE DILUTIONS. APPLICATIONS: INVERSION OF THE KNOWN PROPERTIES OF PAF-ACETHER, AT ALLOPATHIC DOSE.

Description

The present invention relates to a

  It has been diluted rapidly, and can be used both as a drug and as a laboratory reagent. It also concerns its manufacturing process.

  Did you know that a number of pa-

  These diseases are accompanied by the release of histamine by the leucocytes and thrombocytes of the blood, and the tissue mast cells. These affections can be divided into two main categories, namely: allergic phenomena; platelet aggregation (the first stage in the formation of blood clots): this phenomenon marks in particular the

  onset of thrombosis, whether of inflammatory origin or

', or others.

  The present invention relates to a pr Dduit

  highly diluted, likely to be used, either as

  dicament to fight against the aforementioned affections, either as laboratory reagent, to carry out tests, not only qualitative, but also quantitative

  for the study of phenomena: degranulation of certain elements

figured blood.

  We know a chemical called "Plate-

  let activating factor acether "or, abbreviated" PAF-Acether.

  We know that when using this product

  at concentrations of the order of nanomolarity, it pro-

  the release of histamine, by certain

  This process may even have consequences

which lead to death.

  The present invention aims to prepare, for

  from this known product, a drug or a laboratory reagent

  of a new type whose effects on the human organism are exactly the opposite of those just recalled.

  The process according to the invention for the manufacture

  This new product consists of a PAFAcether sample and is characterized by the dilution of this sample by the Hahnemannian method as it

  is described in the Pharmacopoeia; French, to bring the

  appears at a dilution less than or equal to 6 CH, each of the successive dilution operations being accompanied by a-dynamisation of shaking the container of each

  intermediate method, or by any other method

  both to achieve a concentration equal to or less than the

molar co.

  According to another characteristic of the invention

  the product obtained by this process is a dilution

  the above described PAF-Acether, which can be used both as a drug and as a laboratory reagent.

  According to another characteristic of the invention

  each intermediate dilution shall be carried out centrally

  tth, the final product being preferably diluted to 11 CH, according to the method-Hahnemannienne Of course, any other method of dilution

  could be used, whose use is known in homeopathy.

  thie, in particular: Korsakovian dilution of emptying each cycle a bottle which is kept only the liquid adhering to

  walls, after which the flask is filled with

  lution, and it is energized; turbulence dilution, which consists in agitating the liquid at

as the dilution progresses.

  The particular cases that will follow, given

  as non-limitative examples, will enable better understanding

  take the invention, and the features it presents.

  ) The basophil degranulation test

  humans (TDBH) or mast cells of tissue origin.

  Experimental model The experiment is based on the use of the human basophilic degranulation test (TDBH) according to the technique of Jacques BENVENISTE This test consists in bringing together the basophils obtained by concentration of a

  blood sample, and sensitizing allergen.

  When basophils carry

  anaphylactic bodies attached to their membrane receptors

  res, they degenerate to 370, that is to say they release

  in the external environment, preformed or neoformed mediators

  contained in the cytoplasmic granules.

  This reaction serves as a definition

  immediate hypersensitivity (type I) according to the classi-

  of GELL and COM 4 BS Basophils Degranulated

  are differentiated from those who have not been degraded by

  te of their metachromatic dye affinity Vis-à-vis

Toluidine blue.

  An application-based statistical calculation

  Poisson's law, allows to define a threshold of signifi-

  on average between 20 and 35%, depending on the

number of basophiles counted -

Technical

  The product to be studied is used for dilution

  mineral buffer, called basophil wash buffer

the, concentrated ten times.

  The basophils contained in the sample

  are centrifuged at a very slow rate, resuspended in this half

  place and preincubated twenty minutes to 37.

  An aliquot of this concentration is then

  used to realize the TDBH To do this, ten micro-

  liters of the suspension of basophils are introduced into

  cups of a microtiter plate at the bottom of which

  the antigen was previously dried after 15 minutes

  incubation at 370, the basophils that have not degranulated are counted in the presence of a solution

Toluidine blue acid.

  Six dilutions of the antigen are systematically

  tested and one thus generally obtains a

  be in "bell" with its two zones of inhibition by excess of

antibodies and excess of antigen.

  The maximum percentage of degranulation is

then calculated.

  An identical method is applicable to mas-

tocytes of tissue origin.

  20) The histamine release test Under the experimental conditions that we

  report, this test has the same goals as the test of

granulation of basophils.

  It allows a quantitative study of the libera-

  of histamine by human basophils of a pedi-

  feeling allergic to a known allergen.

  As in the previous test, histamine release is brought about by placing the basophils of a given subject in contact with the allergen to which this subject is found.

be allergic.

  The degranulation of basophils that is thus caused results in a release of histamine in the

  extracellular medium The test on which we are

  puyons consists of an assay of histamine in the extracellular medium, before and after contact with basophils

  and allergen This assay is done by the method of

  radioactive cells thanks to H 3 histamine.

  In this case, the basophiles

  are divided into two batches, one of which is previously incubated with the product to be tested, and the other with water

  In either case, the solution of

  gene is then added at varying concentrations to

avoid the - zone phenomenon.

  The histamine dosage in both lots

  allows to evaluate the inhibition of the release of this same

me histamine.

  3) Platelet aggregation test Measurement of aggregating activity Aggregating activity of different analogues

  is tested on washed rabbit platelets at 37 under

  tation (1500 revolutions per minute) in an Icare aggregmeter (Marseille France) Fifty> l of the suspension

  platelet concentrates are added to the aggregate tube.

  2 + meter, at three cents> l of TG Ca, in order to obtain a

  percentage of constant light transmission, corresponding to

  At a platelet concentration of about 2.5 X 10 8 platelets / ml, we added CP / CPK (im M / 10 U / ml) in the Ca 2 + aggregation buffer: final concentrations to inhibit the formation of platelets. 67-induced aggregation of ADP) to transform into ATP, the ADP being

secreted during aggregation.

  The cyclooxygenase pathway is blocked by incubating the platelets for fifteen minutes with ASA (100 FIH) just before the second wash (except

  when the aggregation indudt: paar 1 'AA is studied).

  Platelets are stimulated by one to four

  1 of a solution of PAF-Acether or analogues in 60% ethanol

  A standard solution of synthetic PAF-Acether

  tick draws a standard range The activity

  aggregation of products is expressed by the EC 50, ie

  say the molar concentration required to obtain 50% of the macimal aggregation induced by the standard solution of PAF 4 Acether (mean + standard deviation of three to six experiments). Inhibition test

  Washed rabbit platelets are preincub

  0.2 or 3 minutes, without shaking at 22 o C, with the platelet aggregation inhibiting agent, in our case

  homeopathic dilutions of PAF-Acether.

  Synthetic PAF-Acether (0.13 to 1.4 / 4 M)

  AA (0.2 to 4.7) LI 4) or ADP (1.3 to 13 pM) are then added.

  at a concentration which induces a submaximal aggregation

  on control platelets (incubated in the presence of

ethanol solvent).

  In order to separate the three paths of

  we studied the aggregation induced by PAF-

  acetone on ASA-treated platelets (100 kL) in the presence of the CP / CPK complex (1 m M / 10 U / ml). For the study of AA-induced aggregation, or ADP, ASA or

CP / CPK are omitted respectively.

  The results are expressed as percentage inhibition by comparison of the aggregations obtained with

  the aggregation of platelets controlled, incubated

  solvent (average + a standard deviation of three ex-

ences).

LEGEND

  CPLCPK: creatine phosphate / creatine phosphokinase ADP: adenosinediphosphate ATP: adenosine-triphosphate AA: arachinodic acid ASA: acetylsalicylic acid

Claims (5)

  1 Process for the manufacture of a product   for use as a medicament, and as a laboratory reagent.   re, initially using a sample of PAF-   Acether, characterized in that said sample is diluted   successive homeopathic dilutions performed up to   dilute between 10 12 and 10 30 each intermediate dilution   re being accompanied by a boosting operation 2 Homeopathic medicine obtained through the   method according to claim 1, characterized   rised in that it consists of a Hahnemannian dilution   PAF-Acether, at a concentration between 6 CH and 15 CH, while, moreover, it has properties curative anti-histamines.   3 Homeopathic medicine obtained by placing   of the process according to claim 1, characterized   in that it consists of a Korsakovian dilution of PAFL Acether, at a concentration between 100 K and   10,000 K, while, moreover, he owns curative anti-histamines.   4 Homeopathic medicine obtained by placing   of the process according to claim 1, characterized   in that it consists of a turbulent dilution of PAFAcether, while, moreover, it possesses   antihistaminic properties.   Laboratory reagent obtained by carrying out the process according to claim 1, characterized in   what it consists of a Hahnemannian dilution of PAF-   Acether, at a dilution lower than 6 CH, each dilution   The intermediate procedure has been accompanied by a dynamisation operation, which gives it properties that inhibit degranulation of the subject's basophils, so that it can be used in quantitative tests to measure   the degree of degranulation of the basophils of the blood or mas-   tissue, or the degree of aggregation of platelets. 6 Homeopathic product according to any one of   Claims 2 to 5, characterized in that the solvent dilution used is water.   7 Homeopathic product according to any one   of claims 2 to 5, characterized in that the   Dilution solvent used is ethyl alcohol.   8Homeopathic product according to any   of claims 2 to 5, characterized in that the   Solvent used is a mixture of water, and alcohol.