WO1991013611A1

WO1991013611A1 - Process for making highly diluted homeopathic compositions or preparations from an initial solution containing an active substance

Info

Publication number: WO1991013611A1
Application number: PCT/FR1991/000206
Authority: WO
Inventors: Jacques Benveniste
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 1990-03-13
Filing date: 1991-03-13
Publication date: 1991-09-19
Also published as: FR2659553A1; FR2659553B1

Abstract

The object of the invention is a process for making highly diluted homeopathic compositions or preparations from a solution of given dilution, itself originating from an initial solution containing an active substance, in which said initial solution is subject to successive dilutions, each solution going from that of first dilution to that of last dilution, constituting a solution of given dilution, wherein the solution of given dilution is subjected to an agitation stage at least after every tenth dilution and preferably after every third dilution and more preferably after every dilution, where the agitation consists of a stage of creating bubbles in the solution of given dilution (hereinafter referred to as ''bubbling'') by blowing gas in at an adequate rate of agitation to form a vortex in the solution of given dilution to be agitated.

Description

PROCESS FOR THE MANUFACTURE OF HOMEOPATHIC COMPOSITIONS OR HIGH DILUTION PREPARATIONS FROM AN INITIAL SOLUTION CONTAINING AN ACTIVE SUBSTANCE

The subject of the invention is a new process for manufacturing homeopathic compositions or preparations at high dilution from an initial solution containing an active substance.

The invention also relates to an automatic device for implementing the above-mentioned method.

Until today, the manufacturing techniques used for the preparation of homeopathic compositions are inspired by those recommended by Hannemann and it is mainly about trituration, impregnation and dilution (for example, centesimal or decimal) ).

The dilution technique makes it possible to prepare homeopathic dilutions which can be used as such or which are incorporated into a neutral excipient for the galenic shaping which constitutes the finished medicament.

Regarding the manufacturing technique by centesimal dilution, we can proceed as follows:

- there are a series of bottles and caps washed with water and dried, in number corresponding to the number of the centesimal dilution to be obtained;

- one puts in the first bottle a part by weight of the basic substance supplemented to 100 parts by weight by volume by means of the appropriate vehicle;

- shake at least 100 times; the dilution obtained is the first CH; a part by volume of this first CH is taken and poured into the second bottle already containing 99 parts of the vehicle; - we also shake 100 times; the dilution thus obtained is the second CH.

For decimal dilutions, the procedure is identical, but according to the decimal series.

The shaking step previously mentioned constitutes revitalization.

This shaking step is also called agitation or succussion, and it is of fundamental importance, for obtaining active homeopathic compositions.

To date, industrially, the process for preparing homeopathic medicines is based on the use of two devices: one is a conventional dilution device and the second a stirring device. Between each step, the solution to be diluted is extracted from the dilution device and is put in the succussion device, then again in the dilution device for the next step.

The implementation of this process involves manual handling of the tubes, which is a waste of time and a possibility of error.

One aspect of the invention is to provide a process for the preparation of homeopathic compositions in which the succussion step is simplified.

One aspect of the invention is to provide a preparation process which can be automated by reducing wasted time and sources of error.

Another aspect of the invention is to provide a fully automated machine.

The process of the invention for the preparation of homeopathic compositions from determined dilution solutions, themselves originating from an initial solution containing an active substance, in which the initial solution containing the active substance undergoes a series of dilution steps successive, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a solution dilution, to give the second dilution solution and so on, up to the last dilution solution,

each of the solutions going from the solution of first dilution to the solution of last dilution, constituting a solution of determined dilution, is characterized in that one proceeds to a stage of agitation of the dilution solution determined at least after every ten dilutions, preferably after all three dilutions, advantageously after each dilution, the stirring being constituted by a step of creating bubbles in the dilution solution determined using gas blowing at a stirring rate sufficient to create the formation of a vortex in the dilution solution determined to agitate.

By "vortex" is meant a vortex created in an intermediate solution, by gas blown under sufficient pressure.

For simplification, the expression "creation of bubbles" will be designated by the term "bubbling".

By “homeopathic compositions”, it is necessary to understand the compositions obtained from determined dilution solutions containing active substances at very low doses, or even at infinitesimal doses and compositions in which there is no longer any active substance.

Preferably, the first dilution is carried out by taking a part of the initial solution, and each of the determined dilutions is carried out by taking a part of the previous dilution solution.

To fix ideas, the homeopathic compositions coming from determined dilution solutions obtained by the process of the invention are such that the active substance is less than

10 ^-10 moles / l, advantageously less than

10 ^-12 moles / l, and preferably less than

10 ^-14 moles / l, or no longer contain active substance.

In the process of the invention, the stirring step was simplified by a bubbling step using gas blowing at a stirring rate sufficient to create the formation of a vortex in the solution to be shake.

According to an advantageous embodiment of the method of the invention, the bubbling is carried out after each dilution.

According to another embodiment of the invention, some of the stirring steps can be carried out manually, for example by placing the container containing the solution to be stirred on an eccentric stirring device.

One can for example after the first dilution, shake the first dilution solution manually as indicated above, then use bubbling, according to the invention.

The blown gas can be constituted by nitrogen, advantageously by air.

To create the formation of a vortex in the solution to be agitated via bubbling, the gas responsible for stirring can be blown at a pressure of about 5 to about 6 bars.

According to a preferred embodiment of the process of the invention, the appropriate stirring rate is obtained by blowing air with a volume ranging from approximately one third to approximately once the volume of the solution to be stirred, the volume blown air advantageously being a volume of approximately 100 μl to approximately 10 ml, in particular 500 μl, under sufficient pressure, and such that the temperature does not exceed approximately 45ºC.

According to another embodiment of the method of the invention, between two successive dilutions, a step of rinsing the device is carried out which allows the taking of a fraction of the whole of the initial solution and of each intermediate solution and the mixing of this fraction or all in a dilution solution.

The rinsing step between two successive dilutions (respectively designated by "dilution coming first" and "dilution coming second") can be carried out between the dilution coming first and the bubbling of the corresponding determined dilution solution, or may take place after bubbling, or may be concomitant with each dilution.

In the latter case, to do this, and by way of example relating to the rinsing concomitant with the dilution coming secondly, a quantity of the latter is taken from the dilution coming first with the rinsed device (concomitantly at the dilution coming first), quantity which one introduces into the tube intended for the dilution coming second, then one takes with the same device approximately half of the volume necessary for the dilution coming second, and one throw it back in the tube intended for the dilution coming second and one takes again approximately half of the volume necessary for the dilution coming second and one rejects it in the tube intended for the dilution coming second, which has the effect of rinsing twice the device which followed the sampling used to carry out the second dilution, and so on.

In what follows, the method as defined above will be designated by "method comprising the bubbling step".

The invention relates to an automatic apparatus for implementing the method defined above, comprising:

means for carrying out successive dilutions of an initial solution containing an active substance, which dilutions lead to solutions of determined dilution,

bubbling means programmed to carry out bubbling at least after all ten dilutions, advantageously after all three dilutions and preferably after each dilution,

- optionally rinsing means between each dilution.

The dilution step consists of what has been indicated above, but can be carried out in any other way, for example according to the method of Korsakow in a single bottle, or else according to the method of 50 millésimales or also according to the method by flow continues.

The initial solution and the dilution solution can be alcoholic or glycerin solutions and are advantageously aqueous solutions.

The process of the invention for preparing homeopathic compositions can also include control of the dilution and contamination of determined dilution solutions.

The process for the preparation of homeopathic compositions from determined dilution solutions originating from an initial solution containing an active substance, which process comprises controlling the dilution and the contamination of the above-mentioned determined dilution solution, and is such that the initial solution

undergoes successive dilutions, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a dilution solution, this which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a dilution solution, to give the solution of second dilution and so on, until the solution of the last dilution, each of the solutions going from the solution of first dilution to the solution of last dilutior constituting a determined dilution solution ,

the successive dilutions being such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ^-10 moles, advantageously less than 10 ^-12 moles / l and preferably less than 10 ^-14 moles / l, or no longer contains an active substance, said determined dilution solution still having activity at dilutions greater than that at which the active substance has disappeared,

is characterized in that - Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1, and the control substance having the property of disappearing between the dilution n - 1 + m and n + m, m being the number of dilutions in which the control substance is present, and being comprised in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the range from dilution n to dilution n - 1 + m and at least once in the range from dilution n + m at dilution n + m + y, y being the range of dilution free of control substance and advantageously being from 3 to 5,

- and from dilution n to dilution n - 1 + m, the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution are compared, which allows quantitative control dilutions, and

- from the dilution n + m to the dilution n + m + y, it is checked that there is no longer any control substance, which makes it possible to control on the one hand the quality of the dilutions and on the other hand the absence of contamination.

In this embodiment of the method of the invention, a substance which is easily detectable and which is detectable until it disappears is used as a control substance. Advantageously, an enzyme detectable by its chromogenic activity is used.

The presence of the control substance in the first dilutions and its absence in the extreme dilutions, (that is to say dilutions greater than the fourteenth dilution and in particular the twenty-third dilution) which are nevertheless active, affirm the relationship between the observed phenomenon and the mechanism of high dilutions. We start with an initial solution containing an active substance and dilute it a first time using a dilution solution, which leads to a first dilution solution, which in turn is diluted, to give a solution. second dilution, and so on to give successive dilutions, up to the last dilution solution. Each of the dilutions leads to a determined dilution solution.

The method of the invention is such that it makes it possible to check at each dilution (which gives rise to a determined dilution solution), whether the dilution has been correctly made and if there is no contamination. An incorrect dilution could, for example, consist of forgetting a previous dilution or introducing a drop, containing for example the active substance at the last detected dilution, or using an incorrectly rinsed pipette , which at such dilutions may change everything.

The method is such that it makes it possible to control each of the dilutions and when the control substance is still present, to quantify the dilution by comparing the calculated theoretical quantity of control substance and the quantity actually found. When there is no longer any control substance, there is also no longer any active substance since the control substance disappears after the active substance. After a certain number of dilutions one should expect not only the absence of active substance, but also the absence of control substance.

As indicated above, the dilutions will be identified by first dilution, second dilution, third dilution, umpteenth dilution or even dilution 1, 2, 3, 4, ... n. Usually, the last dilutions are dilutions 30 or 40 (decimal).

These dilutions can be made on any scale, in particular decimal or centesimal.

In all of the above and in everything that follows, the figures correspond, unless otherwise indicated, to decimal dilutions.

However, the term "dilution" can apply to centesimal dilutions. As the dilutions progress, the diluted solution will be such that it contains 10 ^-10 moles / l, then 10 ^-12 moles / l, then an amount less than 10 ^-14 moles / l. Generally beyond this molar concentration per liter there are no more molecules in the solution, which does not prevent said dilute solution from still showing, and quite unexpectedly, activity.

In the process of the invention, the control substance can be introduced before any dilution, that is to say in any determined dilution solution.

By "control substance which does not interfere with the dilution solution n - 1" is defined a control substance such that its presence has no effect on the possible activity of the dilution solution n - 1.

When the control substance has been introduced at dilution n - 1 and a first dilution of the dilution solution n - 1 containing the control substance is carried out, the dilution is then continued until it reaches a dilution at which the control substance disappears. The method of the invention involves at least one assay of the control substance after dilution n. This assay preferably takes place at least once in the range from dilution n to dilution n - 1 + m, i.e. at least once from the first dilution of the control substance to the dilution at which the control substance disappears.

Over the interval from dilution n to dilution n - 1 + m of the control substance, it is possible in principle to make a quantitative assay since it is possible after each of the dilutions from n to n - 1 + m to dose the control substance and compare it with the corresponding theoretical calculated value.

When the control substance is such that it is no longer present in any given dilution solution, it is also advantageous to make a determination, for example on the three dilutions which follow that from which the control substance has disappeared, dilution for to which no control substance is added.

This confirms that there has been no contamination compared to the dilution at which it is found that there is no longer any control substance. In this case, it is no longer a quantitative control but a qualitative control allowed by the absence of the control substance, the control substance then behaving like a negative control.

According to another preferred embodiment of the invention, the control substance is introduced at least twice, one of the two introduction of the control substance being carried out in the dilution solution n - 1, the control substance disappearing between the dilution n - 1 + m and n + m, the other introduction is carried out as soon as possible in the dilution solution n + m, and preferably in the dilution solution n + m + y, n being an integer greater than 0 , m being advantageously included from 5 to 8, being advantageously included there from 3 to 5.

This process corresponds to the fact that the control substance is introduced a first time into the dilution solution n - 1, diluted until a dilution solution in which the control substance has disappeared and without adding a control substance again before it has disappeared, otherwise the assay carried out on each dilution of dilution solution n to the dilution in which the control substance disappeared would no longer make sense.

We therefore wait until the control substance has disappeared before adding it again. The control substance can be added again to the dilution immediately following that for which the control substance has disappeared, but advantageously, the control substance is added again from 2 to 10 dilutions which follow the dilution at which the control substance has disappeared for the first time, and advantageously from the third, fourth or fifth dilution following the dilution corresponding to the disappearance of the control substance.

We can thus repeat the process of introducing the control substance whenever it has disappeared or while waiting for 3 dilutions to 5 dilutions after the dilution from which the control substance has disappeared.

In practice, according to the method of the invention, the control substance can be reintroduced at regular intervals, and it suffices for example to be able to detect the control substance over 3 or 4 dilutions, to show that the decrease in the control substance is regular ( therefore that the dilutions are correct) and that outside the areas where the control substance is detectable, there is no contamination.

According to one embodiment of the process of the invention, the control substance is introduced for the first time into the dilution solution n - 1, then the control substance is reintroduced into the dilution solution which follows that which corresponds to the disappearance of the control substance introduced into the dilution n - 1, then the control substance is reintroduced a second time in the dilution solution which follows that which corresponds to the disappearance of the reintroduced control substance and so on.

According to another embodiment of the process of the invention, the control substance is introduced for the first time into the dilution solution n - 1, the control substance disappearing between the dilution n - 1 + m and n + m, m being understood in particular from 5 to 8, the control substance is reintroduced into the dilution solution n + m + y, including 2 to 10, advantageously from 3 to 5, and so on.

According to another embodiment, the control substance is introduced for the first time into the dilution solution n - 1, then all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15 .

According to an advantageous embodiment, the method of the invention is such that

- the initial solution undergoes successive dilutions, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a solution dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the solution of the last dilution. each solution ranging from the first dilution solution to the last dilution solution constituting a determined dilution solution,

- each determined dilution solution undergoes vigorous stirring,

characterized in that:

- Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1,

* the control substance having the property of being detectable at dilutions greater than that from which the active substance is no longer detectable,

and

* the control substance also having the property of disappearing between the dilution n - 1 + m and n + m, m being the number of dilutions in which the control substance is present and being included in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the interval from dilution n to dilution n - 1 + m and at least once in the interval from there dilution n + m at dilution n + m + y, y being the free dilution range of control substance and advantageously being from 3 to 5,

- and from dilution n to dilution n - 1 + m we compare the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution, which makes it possible to quantitatively control the dilutions, and

The control substance is endowed with the property of being detectable at dilutions greater than that from which the active substance is no longer detectable, that is to say if it was introduced into the initial solution before the first dilution of the starting solution it would disappear at a dilution greater than that at which the active substance is no longer detectable.

To fix the ideas, taking into account the technical means available to date, a substance is detectable by the usual biochemical means up to a quantity of approximately 10 ^-6 moles / 1.

In some cases, the active substances can be detectable up to 10 ^-12 moles / 1. However, this implies very sensitive detection systems.

As for the control substance, in general, it is detectable up to approximately 3 × 10 -10 −3 × 10 ^-11 moles / l, which corresponds to dilution 7 when the initial concentration is approximately 0.1 approximately 10 mg / l, in particular approximately 1 mg / ml. The quantity in moles / l, up to which the control substance is detectable corresponds to the limit from which. it is considered that there is no longer any control substance. However, the active substances which can be used in the process of the invention are detectable at concentrations of approximately 1x10 ^-3 to 1x10 ^-6 moles / 1, that is to say up to approximately the third decimal dilution, for a initial concentration of approximately 1x10 ^-3 moles / l.

According to an advantageous embodiment of the method of the invention, the control substance is introduced at the dilution n - 1, and disappearing between the dilution n - 1 + m and n + m, it is reintroduced at the dilution n + m + y the interval m + y + 1 being such that,

- over approximately one of the two halves of this interval the dilutions are such that the corresponding dilution solutions are not active and

- over approximately the other half of this interval, at least one of the corresponding dilution solutions is active.

Represented in Figure 1

- in dotted lines the variation in the activity of the dilution solution as a function of the dilution, in solid lines the variation in the initial concentration of the control substance added at the start as a function of the dilution, and

- in short dotted lines - long dotted lines, the variation in initial concentration of the active substance as a function of the dilution solution.

To fix this, the initial concentration of the control substance is approximately 1 mg / ml, and that of the active substance is approximately 1 mg / ml.

In this FIG. 1, given by way of nonlimiting illustration, the control substance disappears between dilution 7 and 8, the active substance disappears between dilution 4 and 5, the dilution interval over which the control substance is present is 0 at 8 dilutions. Over half of this interval, say from 0 to 4 dilutions, we see that the solution has a certain activity whereas on the other half of the interval, that is to say from the fourth to the eighth dilutions, the solution no longer has activity. Over the interval from 0 to 4 dilutions, the control substance is such that there is activity in the corresponding dilution solution and over the interval from 4 to 8 dilutions the control substance is such that there is no . only no activity of the corresponding dilution solutions.

According to another embodiment, the control substance has the property that if it is introduced into the initial solution before the first dilution of the starting solution and if the active substance disappears (is no longer detectable) between dilution p and the dilution p + 1, the control substance disappears between the dilution p + x and the dilution p + x + 1, x being understood in particular from 2 to 4, p being understood in particular from 3 to 6.

In the above definitions, m corresponds to p + x, when the control substance is introduced into the initial solution before the first dilution of the starting solution.

According to another embodiment of the invention, the method comprises the following steps:

the control substance is introduced into the initial solution containing the active substance before the first dilution of said initial solution,

the successive dilutions of the initial solution containing the active substance and the control substance are carried out, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or of the entire initial solution in a dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first dilution solution in a diluting solution to give the solution of second dilution and so on, until the solution of the last dilution,

the active substance disappears between dilution p and dilution p + 1 and the control substance disappears between dilution p + x and dilution p + x + 1, p being between 3 and 6, x being between 2 and 4, the solution still having activity for at least one dilution greater than or equal to the dilution p + 1,

- After the first dilution, a sufficient quantity of solution is taken from the solution obtained at the end of said dilution for at least a given dilution, and preferably the control substance is dosed at each dilution from dilution 1 to dilution p + x and preferably at least once from dilution p + x + 1 to dilution 1 + p + x + y, being there advantageously from 2 to 10, advantageously from 3 to 5 , and advantageously also at each dilution from dilution p + x + 1 to dilution 1 + p + x + y,

- and from dilution 1 to dilution p + x, the value of the concentration of the control substance obtained and the value of the concentration of the control substance calculated according to the dilution are compared, which makes it possible to quantitatively control the dilutions and from the dilution p + x + 1 to the dilution 1 + p + x + y, it is checked that there is no longer any control substance, which makes it possible to check on the one hand that there is no contamination and on the other hand the quality of the dilutions,

and a stirring step of the dilution solution determined at least after every ten is carried out dilutions, preferably after all three dilutions, advantageously after each dilution, the stirring being constituted by a bubbling step of the dilution solution determined using gas blowing at a stirring rate sufficient to create the formation of a vortex in the dilution solution determined to agitate.

According to another embodiment, the control substance is introduced for the first time before the first dilution, then the control substance is introduced for the second time into the dilution solution which follows that at which the control substance introduced for the second time disappears, then the control substance is introduced a third time into the dilution solution which follows that which corresponds to the disappearance of the control substance introduced the second time, and so on.

Advantageously, each introduction of the control substance takes place from 2 to 10, advantageously 3 to 5 dilutions after that which corresponds to the disappearance of the control substance previously introduced.

According to another embodiment, the control substance is introduced before the first dilution and all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

According to another embodiment, the control substance is introduced for the first time into the initial solution, then every ten dilutions starting from the initial solution.

According to another embodiment, the control substance is assayed advantageously every 10 dilutions, and preferably at each dilution. According to another embodiment, the initial solution contains an active substance at a rate of approximately 1x10 ^-3 to approximately 1x10 ^-6 moles / l.

According to another embodiment, when the control substance consists of an enzyme, this is chosen from peroxidase, is in particular horseradish peroxidase, detectable by its reactivity with the substrate D-phenylene diamine in H ₂ O ₂ medium .

The initial solution and the dilution solution are aqueous solutions, of pure alcohol, or of glycerin, and advantageously aqueous solutions.

The invention also relates to an automatic device for implementing the method defined above, comprising:

- optionally rinsing means between each dilution,

means for controlling the dilution and the contamination of the determined dilution solutions obtained respectively after the successive dilutions.

The determined dilution solutions, obtained in accordance with the process defined above comprising the bubbling step and optionally controlled as to their dilution and their contamination can be used as such as finished medicament, or can be used as active solutions, for the manufacture of solid galenic homeopathic compositions and of any preparation at high dilution (beyond 1 x 10 ^-10 M) for experimental and clinical pharmacological purposes (in animals and humans, but also in plant biology) .

With regard to solid homeopathic pharmaceutical compositions, mention may be made of granules or globules, which are made active by impregnation in a determined dilution solution obtained according to the process defined above comprising the bubbling step and optionally controlled as to its purity and its contamination as indicated above.

The invention also relates to a process for obtaining a dilute solution having an average activity in which successive determined dilution solutions are mixed, obtained in accordance with the process described above, each solution being of different dilution, but corresponding to the same dilution scale.

The determined dilution solutions obtained in accordance with the process defined above comprise the bubbling step and optionally controlled as to their dilution and their contamination, can be used either individually, in particular for fundamental pharmacology studies, or after mixing together . By mixing several specific dilution solutions, for example from 2 to 20 successive dilution solutions (preferred number of mixed dilutions: 10) corresponding to the same dilution scale (i.e. for example only decimal or only centesimal, etc ...), a dilute solution is obtained hereinafter designated by "mixture" which, experimentally, has a medium activity. This process, consisting in using mixtures of determined dilution solutions successive, therefore overcomes the drawback of using determined individual dilution solutions for which one cannot always predict whether they will be active or not, in favor of an average activity, but more constant.

The subject of the invention is also a method of increasing the activity of a determined dilution solution in which a determined dilution solution obtained according to the method described above is used, characterized in that this solution is subjected determined dilution to a single high dilution.

The invention also relates to a method for increasing the activity of a dilute solution (mixture) in which the determined dilution solutions used are obtained according to the method described above, characterized in that said mixture is subjected to a unique high dilution.

A determined dilution solution or a mixture obtained as indicated above can be used as it is in experimental pharmacology or in therapy. However, the activity of this determined dilution solution or of this mixture can be considerably increased by carrying out a very high single dilution, that is to say a small amount of a determined dilution solution or of the above mixture, diluted all at once in a large volume of diluent liquid (water, water-alcohol, alcohol, etc.), for example 10 microliters in 10 ml with vigorous stirring. Maximum biological activity is thus obtained. The single dilution level must be adapted to the activity of the starting substance and to the nature of the diluted substance. The level of dilution is at least 100, but it can be diluted in one go without upper limit, this being indicated only by the material possibility of distribute a very small volume in a very large one. In practice, it can be from 100 to 10 ⁵ . For example, a factor of 1 x 10 ⁸ requires diluting one microliter in one hundred liters. The best dilution zone must therefore be determined in each case according to the sensitivity of the biological system and the nature of the substance to be diluted.

EXAMPLE I:

This example relates to the control of the activation and inhibition of achromasia of human basophils, using the method comprising the steps of bubbling and of checking the dilution and the contamination according to the invention.

More precisely, in this example, the effect of the high dilutions of an anti-IgE antibody on human basophils is verified, the high dilution solutions being prepared and checked as to their dilution and their contamination according to the process of invention.

I- BLOOD SAMPLING:

It is carried out in subjects presenting no recognized allergy, neither seropositive nor hepatitis-positive.

Twenty ml of blood from these donors is collected in two glycerin glass tubes, each containing 250 μl of anticoagulant thus prepared:

- Mix (1: 1) two solutions of EDTA-Na ₂ and EDTA-Na ₄ (Merck, Darmstadt, RFA) 0.2 M, pH 7.40.

* EDTA-NaCl ₂ (MW = 372.24): 3.7 g dissolved in 50 ml of heated distilled water,

* EDTA-Na ₄ (MW = 452.24): 4.5 g dissolved in 50 ml of cold distilled water.

- To 100 ml of the mixture, heparin without phenol (Choay, Paris, France) is added to the concentration final 40 U / ml (ie 4000 U in 100 ml of EDTA solution).

The tubes containing the anticoagulant (250 μl / tube) are prepared in advance and stored at + 4ºC.

II- PREPARATION OF PADS:

There are two buffers:

- a washing buffer, not containing calcium, necessary for the preparation of the cells;

- a dilution buffer, containing calcium, necessary for the preparation of dilutions.

These two buffers are prepared extemporaneously from the following Tyrode stock buffer:

1. Tyrode stamp buffered with HEPES: "Tyrode- HEPES"

The products are dissolved hot by stirring in ultra-pure water (obtained after treatment by a reverse osmosis machine and filtration). The pH is adjusted to 7.40 with 5N NaOH and IN. The buffer is then filtered (2 μm filter, Costar, Cambridge, USA) in a sterile hood and stored at + 4ºC for 10 days maximum. Source of products:

KCl, NaCl, Glucose, EDTA-Na ₄ are reagents for cell culture, Sigma Chemical Company, Saint Louis, Missouri, USA.

HEPES, Seromed ^® , Biochrom KG, Berlin, RDA.

2. The washing buffer:

It is the Tyrode-HEPES buffer brought to room temperature and adjusted to pH 7.40 extemporaneously.

3. The dilution buffer:

It is the preceding Tyrode's buffer, brought to ambient temperature and adjusted extemporaneously to pH 7.40 after addition of calcium.

a) Stock solution of CaCl ₂ 220 mM:

1.62 g of CaCl ₂ 2H ₂ O in 50 ml of Tyrode-HEPES buffer at pH 7.40. Storage takes place at + 4 ° C.

b) Dilution buffer:

The final concentration in the dilutions is 11 mM: 5 ml of CaCl ₂ 220 mM qs 100 ml of Tyrode-HEPES buffer. Adjust to pH 7.40 with 1N or 0.1N NaOH. III- PREPARATION OF THE RANGES OF ANTI-IgE DILUTIONS (TEST), ANTI-IgG AND ULTRA-PURE DISTILLED WATER (CONTROLS):

The dilutions of ultra-pure distilled water are not systematically prepared and tested for all the experiments.

1. Anti-IgE and anti-IgG antisera:

It is a goat anti-human IgG antiserum (specific Fc, GAHu / IgG (Fc)) and a goat anti-human IgE antiserum (specific Fc, GAHu / IgE (Fc)) (Nordic Immunology , Tilburg, The Netherlands) with an antibody concentration of 1 mg / ml.

The lyophilized antisera are taken up in 1 ml of ultra-pure distilled water then aliquoted into eppendorf tubes (15 μl / tube) and stored at -20ºC. The antibody concentration is 1 mg / ml.

2. Dilution of antisera and ultra-pure distilled water:

The dilutions are made under the control of a researcher foreign to the laboratory and responsible for the statistical processing of the results (INSERM U292).

They are performed in a laminar flow hood on a Gilson 222-401E Programmable Logic Controller (Gilson Médical Electronics, France) using new sterile tubes drawn by lot of 5 ml in polypropylene (Greiner). The dilution buffer used is Tyrode-HEPES buffer containing 11 mM calcium and adjusted to pH 7.40.

First dilute the ultra-pure distilled water, then the anti-IgG, then the anti-IgE. A rinse cycle is programmed at the beginning and at the end of each of the dilution ranges. These comprise 29 tubes ranging from the 1 × 10 ² dilution to the 1 × 10 ³⁰ dilution of ultra-pure distilled water or of the starting anti-IgG or anti-IgE antiserum solution.

As an enzymatic tracer making it possible to judge the good practice of the dilutions, peroxidase (Sigma) is added at the same time as the distilled water or the anti-IgG or anti-IgE antiserum. The peroxidase solution was prepared at 1 mg / ml, aliquoted in eppendorf tubes (15 μl / tube) and stored at -20ºC.

Realization of a range of dilutions:

The 29 tubes of the range, empty and bearing the number corresponding to their dilution marked with felt, are placed on the rack of the Gilson automatic device.

In the first tube, corresponding to the dilution 1 x 10 ² , 10 μl of distilled water or anti-IgG or anti-IgE (1 mg / ml) and 10 μl of peroxidase (1 mg / ml) are added to 980 μl of Tyrode buffer containing 11 mM calcium (dilution buffer). The tube is closed and shaken for 30 sec on a Vortex.

The 1 x 10 ² dilution made manually is replaced on the rack and the automatic dilution program is then started. After a rinse cycle with the dilution buffer, a 500 μl syringe (stainless steel plunger) withdraws 100 μl of the 1 x 10 ² dilution and draws 400 μl of the dilution buffer. The whole is rejected in the following tube, corresponding to the dilution 1 x 10 ³ . Five hundred μl of dilution buffer are still aspirated and discharged into the 1 x 10 ³ dilution tube, which rinses the syringe. Stirring of the dilution is ensured by a suction-delivery of 500 μl of air, 5 times in succession, at the maximum delivery speed. The needle then takes 100 μl of the 1 x 10 ³ dilution, aspirates 400 μl then 500 μl of dilution buffer according to the same process as above to obtain the 1 x 10 ⁴ dilution and so on.

At a dilution of 1 x 10 ³⁰ , the device stops automatically and starts a rinsing cycle. A new range can then be implemented.

Diagram of the automatic dilution process of the anti-IgE antiserum:

This diagram is the subject of FIG. 2, in which the automatic dilution method has been shown in which the stirring steps mentioned by "bubbling" are carried out in accordance with the method of the invention and in which the step of shaking following the first dilution is done manually on an eccentric shaking device.

3. Coding of the tubes of the dilutions of distilled water and of antisera:

During an "activation" experiment, we test: - 1 x 10 ² to 1 x 10 ⁴ weight dilutions of anti-IgE and of the anti-IgG and / or distilled water control;

- high dilutions 1 x 10 ²¹ to 1 x 10 ³⁰ (beyond the limit number of molecules calculated thanks to the number of Avogadro) of anti-IgE and of the anti-IgG and / or distilled water control. Any part of the dilution range beyond the limit given by the number of Avogadro can be used;

- the internal controls controlling the calcium sensitivity of basophils and corresponding to Tyrode buffers without calcium and Tyrode with calcium.

a) Realization of the code:

In this "activation" protocol, all the tubes are tested blind.

The researcher foreign to the laboratory and controlling the performance of the experiments assigns to each of the tubes, according to a randomization table:

- A number between 1 and 30 when experience compares the effectiveness of dilutions of anti-IgE and anti-IgG;

- a number between 1 and 43 when experience compares the effectiveness of dilutions of distilled water, anti-IgG and anti-IgE.

To do this, the numbers corresponding to the dilutions and marked with felt on the tubes are erased with alcohol and labels with a code number are stuck on the tubes.

Example: in the case of a code between 1 and 30, the coded tubes will be:

- 1x10 ² to 1x10 ⁴ dilution tubes of anti-IgG (3 tubes) and anti-IgE (3 tubes);

- tubes of 1 x 10 ²¹ to 1 x 10 ³⁰ dilutions of anti-IgG (10 tubes) and anti-IgE (10 tubes); - the internal control tubes: 1) dilution buffer, Tyrode with calcium (2 tubes); 2) washing buffer, Tyrode without calcium (2 tubes).

b) Split the coded range from 1 to 30 or from 1 to 43:

200 μl (Pipetman 200) are taken from each of the coded tubes, which are placed in 1 ml aggregameter tubes. The tubes are blocked and taken away by the person who made the code for a possible, subsequent and independent control assay, immunoglobulins which may be contained in the dilutions (assay by electrophoresis on polyacrylamide gel) or any other appropriate control (mass spectrometry etc ...).

IV- CELLULAR PREPARATION:

Before proceeding to obtain a suspension enriched in basophils from the blood collected on the heparin-EDTA anticoagulant (paragraph I), the number of basophils present per mm ³ of whole blood of the subject is determined.

1. Coloring and counting of basophils on whole blood:

Basophils are counted thanks to their metachromatic coloring property with toluidine blue.

a) Coloring solution: toluidine blue:

One hundred mg of toluidine blue (Toluidine Blue, CI N ° 52040 C ₁₅ H ₁₆ CIN ₃ S, PM = 305.84, Fluka, Mulhouse, France) are dissolved in 100 ml of 25% ethanol and adjusted to pH 3, 20-3.40 with 80-100 μl of glacial acetic acid. The solution is stored at room temperature in a tightly closed bottle and protected from light.

b) Coloring: It is performed by mixing 90 μl of dye and 10 μl of whole blood in the round bottom well of a microtiter plate (Costar). The mixture is immediately and gently stirred by 5 to 6 aspirations and discharges using the pipette (Pipetman 200) used to deposit the dye.

c) Basophil count:

Five to 10 minutes after mixing the blood and the dye, the latter is again gently agitated and immediately deposited in a Fuchs-Rosenthal chamber (3.2 mm ³ ) using a pipette (Pipetman 200) .

The slide is placed in a humid atmosphere (in a closed and humidified box) to prevent it from drying out. After 3 to 5 minutes corresponding to the time necessary for the colored suspension to settle in the hemocytometer chamber, counting is carried out on an Olympus microscope at magnification G x 10 x 20.

Basophils are the only cells with a colored cytoplasm. They appear red and are very easily identified on a pale background. In case of doubt, it is necessary to modify the focusing so as to clearly distinguish the cytoplasms colored in pink-red of the basophils from those of the other cells which remain transparent. The nuclei of other leukocytes are slightly colored blue.

Generally, on whole blood, there are on average 7 to 15 basophils per Fuchs-Rosenthal chamber, their number possibly ranging from 2-3 to 30-35.

2. Obtaining a suspension enriched in basophils:

When the 10 μl necessary for counting basophils on whole blood are taken, Dextran T500 4,% (Pharmacia, Uppsala, Sweden) is added at the rate of one volume per five volumes of blood. The tubes are tilted and as the sedimentation takes place (approximately 20 min at 1 xg at room temperature), plasma rich in leukocytes is collected as well as red blood cells in suspension. The presence of the latter reinforces the coloration of basophils by toluidine blue (empirically observed in the laboratory during experimental tests).

The sedimentation is collected in 2 plastic tubes of 10 ml to which washing buffer is added (Tyrode-HEPES without calcium, pH 7.40). After centrifugation (150 x g, 10 min), the leukocyte pellets (+ red blood cells) are combined in a single tube, suspended in 10 ml of washing buffer and centrifuged again (150 x g, 10 min). The plasma rich in leukocytes is initially separated into 2 tubes in order to better wash the cells.

The pellet is finally taken up in an aliquot of the same buffer, between 400 and 900 μl approximately, depending on the number of wells to be deposited (10 μl of suspension x times the number of wells + 40 to 60 μl additional for losses on the walls of the tube).

FIG. 3 shows the diagram of cell preparation.

V- PROTOCOL FOR THE ACTIVATION OF HUMAN BASOPHILES BY ANTI-IGE (ANTI-IGG OR DISTILLED WATER FOR CONTROL):

After the preparation of the anti-IgG and anti-IgE antiserum dilutions (and, for fifteen experiments, dilutions of distilled water) and their coding, after obtaining the suspension enriched in basophils, the test is carried out properly. says, under laminar flow hood. The process is identical for the 30-tube code and the 43-tube code. 1. The protocol:

- Ten μl of washing buffer (Tyrode without calcium) are deposited at the bottom of 30 (or 43) round bottom wells of a sterile microtiter plate (Costar), avoiding the peripheral wells where the risks of contamination and evaporation are larger,

- twenty μl of each of the coded dilutions (code from 1 to 30 or from 1 to 43) are then deposited at the bottom of these same wells,

- the plate is pre-incubated for 5 min at 37 ° C., under tape and cover to avoid evaporation of the contents of the wells,

- ten μl of enriched suspension are then added,

- The plate is then gently agitated by slow rotational movements in order to homogenize the content of each of the wells; it is important that this agitation is gentle in order to avoid any contamination from one well to another,

- the plate is then incubated for 15 min at 37ºC, under adhesive tape and cover to avoid any evaporation,

- after incubation, 90 μl of toluidine blue are added to each of the wells and immediately agitated by 5 to 6 aspirations and discharges with a multichannel pipette. We change cones between each row of wells,

- after coloring, the plate is adhesive tape and kept overnight at + 4ºC before reading. On cells, washed, the coloration is more homogeneous after several hours. 2. Plate diagram:

Example: in the case of a code from 1 to 30

10 μl washing buffer (Tyrode without Ca ⁺⁺ )

+ 20 μl coded dilutions (1 to 30)

PREINCUBATION 5 min at 37ºC

+ 10 μl suspension rich in basophils (+ red blood cells)

INCUBATION 15 min at 37ºC

+ 90 μl toluidine blue

CONSERVATION one night at + 4ºC then READING

3. Reading under an optical microscope. Basophil count:

Basophils are counted the day after the experiment by the same person who prepared the cells the day before. The technique is identical to that of counting basophils in whole blood (paragraph IV-1-c).

When the number of basophils is large (greater than 100 over an entire Fuchs-Rosenthal chamber), it is possible to read (for all wells) only a half-chamber. If more than 150 basophils appear on a half-chamber of Fuchs-Rosenthal, it is preferable to deposit the contents of the wells in the chamber of a hemocytometer of Malassez (1 mm ³ ).

It takes three to five minutes to count the basophil content of a hemocytometer chamber. A trained experimenter can prepare 3 to 4 hemocytometers at the same time on condition that they are stored in a humidified box to avoid drying out. In case of doubt On an account, it is possible to redeposit the contents of a well in a room of Fuchs-Rosenthal to proceed to a new account. But it is recommended not to repeat this operation more than twice for the same well because it seems that repeated withdrawals with agitation can damage the sample and then lead to erratic results.

The numbers of basophils are given in a table like the diagram on the plate.

4. Quality control of dilutions of anti-IgG and anti-IgE antisera: Peroxidase assay:

The peroxidase is assayed on the same day of the experiment, independently, by the second person taking part in the protocol and who has not experienced it on that day. Its purpose is to control the dilution process and to detect possible contamination of high dilutions by weight concentrations of antiserum, contamination which would then be responsible for the biological activity observed at high dilution.

It is an assay by spectrocolorimetry at 490 nm based on the reactivity of the peroxidase with the substrate O-Phenylene-Diamine in H ₂ O ₂ medium.

a) Preparation of the buffers and solutions:

- Citrate buffer pH 5.0

It is a mixture of 20.5 ml of a 0.1M solution of citric acid (MW = 210.1) and 29.5 ml of a 0.1M solution of sodium citrate (MW = 294, 1).

- O-Phenylene-Diaminé solution (OPD):

Eight mg of OPD (Sigma) are dissolved extemporaneously in 10 ml of citrate buffer pH 5.0. The solution is stored protected from light under aluminum foil.

b) The dosage:

In the wells of a 96-well flat-bottom microtiter plate (Costar), successively:

- 50 μl of each of the coded dilutions (from 1 to 30 or from l to 43) and non-coded dilutions (1 x 10 ⁵ to 1 x 10 ²⁰ ) of the ranges of distilled water, anti-IgG and anti-IgE (Pipetman 200).

- 50 μl of 8 mg / 10 ml OPD (eppendorf dispensing pipette).

- 10 μl of H ₂ O ₂ 30 vol. (eppendorf dispensing pipette).

There is a yellow-orange coloration of the wells corresponding to the most concentrated dilutions.

Leave the reaction to take place for 10 minutes, protected from light, under aluminum.

Add 50 μl (eppendorf dispensing pipette) of H ₂ SO ₄ 9% (H ₂ SO ₄ concentrated, diluted 10 times, 3% final in the well). The previously observed coloration is accentuated (ocher-orange coloration).

Read immediately at 490 nm with an automatic plate reader spectrophotometer (Dynatech Laboratories). The results are automatically saved and printed.

Plate diagram:

Example in the case of a code from 1 to 30:

5) The "activation" results:

The results (numbers of basophils + peroxidase assay) are given every day to the person who made the codes.

The tubes corresponding to each experiment are enclosed in a sealed and dated envelope and kept at + 4ºC, for the purpose of subsequent checks, a) Interpretation of the results:

It will be done after an independent statistical analysis, on the experiences selected according to the following criteria:

1. Number of basophils in the controls greater than 35. The controls correspond to dilutions of distilled water, anti-IgG and to internal controls (Tyrode buffer with and without Ca ⁺⁺ ).

2. Anti-IgE activity at a weight dose greater than 40% achromasia compared to the respective weight dilutions of distilled water or anti-IgG. (Anti-IgG by weight dose can theoretically lead to achromasia of basophils compared to the same dilutions of distilled water or compared to the "Tyrode with calcium" controls. One can invoke recognition of the light chains of IgE by l anti-IgG or an anaphylactic IgG-dependent reaction).

3. In the presence of calcium alone, that is to say without the addition of anti-IgE, the number of basophils should not vary by more than 25%. This is verified by comparing the number of basophils in the presence of Tyrode-calcium buffer with that of the basophils in the presence of Tyrode buffer without calcium.

Achromasia is thus determined:

No. basos of control well - No. basos of test well _x100

No. basos of the control well

basos = basophiles For each experience selected according to the criteria:

1) calculation of the difference between the average number of basophils counted in the wells containing the anti-IgE solution and the average the number of basophils counted in the wells containing the control solution (distilled water or anti-IgG), for all dilutions between 1 x 10 ²¹ and 1 x 10 ³⁰ .

2) also, for high dilutions of anti-IgE, research for the presence of at least one achromasia peak of 3 to 4 significant successive points according to the data in the chart (paragraph VI-2). b) Representation of results:

It is done after opening the codes, after 18 interpretable activation experiences (i.e. meeting the selection criteria).

The number of experiments required was determined after statistical analysis of the results provided by preliminary experiments.

The results are represented as follows:

Logarithmic dilutions (anti-IgE, anti-IgG or distilled water) are plotted on the abscissa while the number of basophils is plotted on the ordinate.

Each graph corresponds to an experience. It comprises :

1) a curve which represents the variations in the number of basophils as a function of the dilutions of anti-IgE;

2) one (or two) control curve (s) showing the variations in the number of basophils as a function of the dilutions of distilled water and / or of anti-IgG.

Depending on the average number of basophils obtained for the control dilutions of distilled water and for the "Tyrode with calcium" control, a significance limit is defined corresponding to the number below which the effect of anti-IgE (or anti-IgG) will be considered significant. This limit most often corresponds to around 20% of achromasia. It is given by an abacus (see Figure 4) and is shown in dotted lines on the graphs.

The lower the number of basophils compared to the average of the controls, the more significant the effect observed.

FIG. 4 shows the abacus to determine the significance of the achromasia of human basophils.

The chart indicates the significance (p <0.05) of the achromasia observed for basophils. Example: when 70 basophils are counted in the control well, at most 56 basophils must be counted in the test well for the achromasia to be significant.

VI- EXPERIMENTAL PROTOCOL OF THE MODULATION OF BASOPHILIC ACHROMASIS BY APIS MELLIFICA:

This protocol is practiced either at the same time as the "activation" protocol when the number of basophils in the whole blood of the donor is sufficient (greater than 15 on a Fuchs-Rosenthal chamber), or independently of the "activation" protocol, on another donor's blood.

1) Principle:

The modulating effect of dilutions of Apis mellifica from 15 to 20CH (Hahnemannian Centesimal) is tested compared to the corresponding control, NaCl 137 mM 20CH on the achromasia of basophils in the presence of anti-IgE weight dilutions. The effect of Apis mellifica and of 137 mM NaCl is tested, as a control, on basophils put in the presence of the only anti-IgE dilution buffer, without anti-IgE. 2) Dilutions of Apis mellifica and 137 mM NaCl:

They are supplied by Boiron-L.H.F. (Lyon, France) in sterile 1 ml ampoules, in 137 mM NaCl. The contents of the ampoules are transferred to new sterile 5 ml polypropylene tubes, under a laminar flow hood. The tubes are closed progressively and agitated for 30 seconds on a Vortex.

Identical bulbs are sent to an outside laboratory in order to control the quality of the products by mass spectrometry.

3) Coding of the dilutions of Apis mellifica and of 137 mM NaCl:

In this protocol, we studied the modulating effect of the 15 to 20CH dilutions of Apis mellifica compared to a control, the 20CH dilution of 137 mM NaCl.

All these dilutions are tested blind. An arbitrary code number between 1 and 8 is randomly assigned to each dilution (6 dilutions 15 to 20CH of Apis mellifica and 2 dilutions 20CH of 137 mM NaCl) by the researcher foreign to the laboratory which controls the process and is responsible statistical interpretation of the results.

To do this, a label with a code number is stuck on each of the tubes containing the corresponding dilution. The code is changed with each new experience, new labels bearing a new number replacing the previous ones.

The coded tubes are kept from one experiment to another at + 4ºC under aluminum foil for 2 weeks. After this time, a new procedure (transfer of the ampoules to the tubes and coding) is carried out throughout the duration of the experimental protocol.

4) The dilutions by weight of anti-IgE:

The dilutions (1 x 10 ² to 1 x 10 ⁴ ) are prepared manually in dilution buffer (Tyrode-HEPES + Ca ⁺⁺ 11 mM final buffer, pH 7.40), under a laminar flow hood, in sterile tubes of 5ml in polypropylene, from anti-human IgE goat antiserum (1 mg / ml of antibody) aliquoted in eppendorf tubes and stored at -20ºC.

Ten μl of anti-IgE (1 mg / ml) are added to 990 μl of dilution buffer. The tube is closed and stirred for 30 seconds on a Vortex: the dilution 1 x 10 ² is obtained.

One hundred μl are taken from it and added to 900 μl of dilution buffer contained in a second tube. This is blocked and agitated in turn for 30 seconds on the Vortex. The 1 x 10 ³ dilution is thus obtained and the same is done for the 1 x 10 ⁴ dilution.

5) The protocol itself:

At first, therefore, were prepared:

- the dilutions of Apis mellifica,

- anti-IgE dilutions,

- the cell suspension enriched in basophils (paragraph IV).

The test :

- Ten μl of each of the 8 coded dilutions are deposited at the bottom of the round bottom wells of a sterile microtiter plate (Costar). Each dilution is deposited as many times as doses of anti-IgE to be inhibited and once for the dilution buffer. In this protocol, the coded dilutions of Apis mellifica and of 137 mM NaCl are therefore deposited 4 times (anti-IgE 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ ; dilution buffer). - Ten μl of suspension rich in basophils are then deposited in each of the wells.

- The plate is gently agitated by very gentle rotation in order to homogenize the content of the wells and left for 30 minutes at room temperature, under adhesive tape and cover to avoid any evaporation.

- After this preincubation time of basophils with the products 20 μl of anti-IgE at dilutions 1x10 ² , 1x10 ³ , 1x10 ⁴ and 20 μl of Tyrode-HEPES buffer containing calcium 11 mM (and without anti-IgE) are added to the wells for each of the coded dilutions of Apis mellifica and 137 mM NaCl.

- The plate is gently shaken to homogenize the contents of the wells and placed for 15 minutes at 37 ° C under adhesive tape and cover to avoid evaporation in the wells.

- After incubation, 90 μl of toluidine blue are added to each of the wells and immediately stirred by suction and delivery, using a multichannel pipette. The cones are changed between each row of wells.

- After staining, the plate is covered with an adhesive tape and stored overnight at + 4 ° C before reading. The coloration of the washed cells is more homogeneous after several hours.

Plate diagram:

1 2 3 4 5 6 7 8 ← code number

with 6 dilutions of Apis mel algE 1 x 10 ² 15CH at 20CH and with 2 dilutions algE 1 x 10 ³ controls of 137 mM NaCl.

alGE 1 X 10 ⁴

'' Tyrode-Ca ⁺⁺

- 10 μl of coded dilutions and

- 10 μl of basophils are placed in the wells of the PREINCUBATION plate 30 min at room temperature

- 20 μl algE or Tyrode-Ca ⁺⁺

INCUBATION 15 min 37 ° C

Witnesses without Ca ⁺⁺ : 10 μl of NaCl - 90 μl blue toluidine

137 mM + 10 μl basopniles are placed CONSERVATION 1 night + 4 ° C in each of the two control wells which READ

are called "calcium-free controls", then

preincubation, + 20 μl of Tyrode without

Ca ⁺⁺ → incubation → staining →

reading

The "Tyrode without Ca ⁺⁺ " controls (*) are added, as in the "activation" protocol, to control the sensitivity of basophils to calcium.

6) Reading under the microscope and counting basophils:

The principle is identical to that described for the activation of basophils (paragraph V-3).

7) The results:

The basophil counts are reported in a table like the diagram on the plate and are given for each experiment to the person who did the code.

a) Interpretation of results:

Experiments, after decoding, are only retained for statistical interpretation if they meet 3 criteria: 1. Number of basophils in the controls greater than 35 (basophils preincubated with 137 mM NaCl or with Apis mellifica and not having been put in the presence of anti-IgE).

2. Spontaneous sensitivity of basophils to calcium alone less than 25% achromasia by comparing on the one hand the basophils which, preincubated with 137 mM NaCl, are, in the absence of any anti-IgE, placed in the presence of buffer of Tyrode-calcium with, on the other hand, those placed in the presence of Tyrode buffer without calcium.

3. Presence of at least one dose of anti-IgE with achromasia of between 40 and 60% of basophils which, preincubated with 137 mM NaCl, are put in the presence of anti-IgE 1x10 ² to 1x10 ⁴ relative to those placed in the presence of Tyrode-Ca ⁺⁺ buffer without anti-IgE. This is based on preliminary studies which have shown that below 40%, the achromasia of basophils is too weak for the study of inhibition to be carried out. Above 60%, it is too strong to be able to be significantly modulated by high dilution agonists.

Basophile achromasia is thus determined: Nb basos of the control well - Nb basos of the test well _x100

No. basos of the control well

basos = basophiles

b) Representation of results:

Three types of results (in number of basophils) are obtained and must be compared:

- the wells corresponding to the basophils placed in the presence of Apis mellifica or of 137 mM NaCl but without anti-IgE give the maximum number of basophils. These are the reference witnesses.

- wells corresponding to basophils in the presence of 137 mM NaCl and anti-IgE without Apis mellifica give the minimum number of basophils (maximum achromasia).

- the wells corresponding to the basophils brought into contact with Apis mellifica and anti-IgE give a number of basophils on which the possible modulating effect of the product is evaluated. The closer this number is to the maximum number of basophils, the greater the inhibitory effect. Conversely, the closer this number is to the minimum number of basophils, the weaker or zero the inhibitory effect. If it becomes less than this minimum number, the effect is activating.

The graphic representation:

For each dose of anti-IgE tested, the dilutions of Apis mellifica and the control dilutions of 137 mM NaCl are plotted on the abscissa while the number of basophils is plotted on the ordinate.

Each graph includes:

1) a curve which represents the variations in the number of basophils in the presence of anti-IgE as a function of the dilutions of Apis mellifica and of 137 mM NaCl;

2) a curve which represents the variations in the number of basophils in the presence of Tyrode-Ca ⁺⁺ buffer without anti-IgE as a function of the dilutions of Apis mellifica and of 137 mM NaCl.

c) Statistical analysis of the results: The modulating effect of the dilutions of APis mellifica is studied statistically by a Whitney-Wilcoxon rank test after a series of fifteen independent experiments. For each dilution of APis mellifica, the number of basophils in the presence of a dose of anti-IgE will be compared with the number of basophils preincubated with 137 mM NaCl and in the presence of the same dose of anti-IgE. IgE. These studies are carried out independently by the persons responsible for monitoring the experiments and for the statistical interpretation of the results.

Claims

1. Process for the manufacture of homeopathic compositions or high-dilution preparations from a determined dilution solution, itself originating from an initial solution containing an active substance, in which the initial solution containing the active substance undergoes a series of successive dilution steps, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or all of the initial solution in a dilution solution, which gives the first dilution solution, the second dilution of the initial solution being obtained by taking a fraction or all of the first dilution solution, and mixing this fraction or all of the first solution dilution in a dilution solution, to give the second dilution solution and so on, up to the last solution re dilution,

each of the solutions ranging from the first dilution solution to the last dilution solution constituting a determined dilution solution, characterized in that a step of stirring the dilution solution determined at least after all ten dilutions is carried out, preferably after all three dilutions and advantageously after each dilution, the stirring being constituted by a step of creating bubbles in the determined dilution solution (hereinafter referred to as "bubbling") using gas blowing at a sufficient agitation rate to create the formation of a vortex in the dilution solution determined to agitate.

2. Method according to claim 1, characterized in that the blown gas is constituted by nitrogen, advantageously by air.

3. Method according to any one of claims 1 or 2, characterized in that the blown gas has a pressure of about 5 to about 6 bars.

4. Method according to any one of claims 1 to 3, in which the stirring rate is obtained by blowing air with a volume ranging from approximately one third to approximately once the volume of the solution to be stirred, the volume of blown air advantageously being a volume of approximately 100 μl to approximately

10 ml, especially 500 μl, under sufficient pressure and such that the temperature does not exceed approximately 45ºC.

5. Method according to any one of claims 1 to 4, characterized in that between two successive dilutions, is performed a step of rinsing the device which allows the removal of a fraction of the entire initial solution and each solution intermediate and mixing this fraction or all in a dilution solution.

6. Method according to claim 5, in which the rinsing step takes place between two successive dilutions (respectively designated by "dilution coming first" and "dilution coming second") can be carried out between the dilution coming first place and the bubbling of the corresponding determined dilution solution, or may take place after bubbling, or may be concomitant with each dilution.

7. Automatic apparatus for implementing the method according to one of claims 1 to 6, comprising: means for carrying out successive dilutions of an initial solution containing an active substance, which dilutions lead to solutions of determined dilution,

- optionally rinsing means between each dilution.

8. A method of manufacturing homeopathic compositions or high dilution preparations according to claim 1, wherein

the successive dilutions are such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ^-10 moles, advantageously less than 10 ^-12 moles / l and preferably less than 10 ^-14 moles / l, or no longer contains an active substance, said determined dilution solution still having activity at dilutions greater than that at which the active substance has disappeared,

and characterized in that

- Introducing, before at least any of the dilutions n, n being an integer greater than 0, in the dilution solution n-1 a control substance soluble in said solution and not interfering with the dilution solution n -1, and the control substance having the property of disappearing between the dilution n - 1 + m and n + m, m being the number of dilutions in which the control substance is present, and being comprised in particular from 5 to 8,

- the control substance is assayed at least once after dilution n, preferably at least once in the interval from dilution n to dilution n - 1 + m and at least once in the interval going from the dilution n + m to the dilution n + m + y, y being the dilution range free of control substance and being advantageously from 3 to 5,

9. A method of manufacturing homeopathic compositions or high dilution preparations according to claim 8, characterized in that the control substance is introduced at least twice, one of the two introductions of the control substance being carried out in the dilution solution n - 1, the control substance disappearing between the dilution n - 1 + m and n + m, the other introduction is carried out as soon as possible in the dilution solution n + m, and preferably in the dilution solution n + m + y, n being an integer greater than 0, m being advantageously between 5 and 8, y being advantageously between 3 and 5.

10. A method of manufacturing homeopathic compositions or high dilution preparations according to claim 8, wherein

the successive dilutions are such that at least one of the determined dilution solutions contains the active substance in an amount less than 10 ^-10 moles, advantageously less than 10 ^-12 moles / l and preferably less than 10 ^-14 moles / l, or no longer contains any active substance, said determined dilution solution still exhibiting activity at dilutions higher than that at which the active substance has disappeared,

and characterized in that

and

- from the dilution n + m to the dilution n + m + y, it is checked that there is no longer any control substance, which makes it possible to control on the one hand the quality of the dilutions and on the other hand 1 ' absence of contamination.

11. Process for the manufacture of homeopathic compositions or high-dilution preparations according to any one of claims 8 to 10, characterized in that the control substance has the property that if it is introduced into the initial solution before the first dilution of the starting solution and if the active substance disappears (is not more detectable) between dilution p and dilution p + 1, the control substance disappears between dilution p + x and dilution p + x + 1, x being understood in particular from 2 to 4, p being understood in particular from 3 to 6 .

12. A method of manufacturing homeopathic compositions or high-dilution preparations according to any one of claims 8 to 11, characterized in that the control substance is introduced for the first time into the dilution solution n-1, then reintroduced the control substance in the dilution solution which follows that which corresponds to the disappearance of the control substance introduced in dilution n - 1, then the control substance is reintroduced a second time in the dilution solution which follows that which corresponds to the disappearance of the reintroduced control substance and so on.

13. A method of manufacturing homeopathic compositions or high-dilution preparations according to any one of claims 8 to 12, characterized in that the control substance is introduced for the first time into the dilution solution n-1, the control substance disappearing between the dilution n - 1 + m and n + m, m being included in particular from 5 to 8, the control substance is reintroduced into the dilution solution n + m + y, including being from 2 to 10, advantageously from 3 to 5, and so on.

14. A method of manufacturing homeopathic compositions or high dilution preparations according to any one of claims 8 to 13, characterized in that the control substance is introduced for the first time in the dilution solution n - 1, then all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

15. A method of manufacturing homeopathic compositions or high-dilution preparations according to any one of claims 8 to 14, characterized in that

the successive dilutions of the initial solution containing the active substance and the control substance are carried out, the first dilution of the initial solution being obtained by taking a fraction or all of the initial solution, and mixing this fraction or of the whole of the initial solution in a dilution solution, which gives the solution of first dilution, the second dilution of the initial solution being obtained by taking a fraction or all of the solution of first dilution, and the mixing this fraction or all of the first dilution solution in a dilution solution, to give the second dilution solution and so on, until the solution of the last dilution,

- After the first dilution, a sufficient quantity of solution is taken from the solution obtained at the end of said dilution for at least a given dilution, to measure the control substance. and preferably the control substance is assayed at each dilution from dilution 1 to dilution p + x and preferably at least once from dilution p + x + 1 to dilution 1 + p + x + y, y being advantageously comprised from 2 to 10, advantageously from 3 to 5, and advantageously still at each dilution from the dilution p + x + 1 to the dilution 1 + p + x + y,

and a step of stirring the dilution solution determined at least after every ten dilutions, preferably after all three dilutions, advantageously after each dilution, is carried out, the stirring being constituted by a step of bubbling the solution of dilution determined using gas blowing at a rate of agitation sufficient to create the formation of a vortex in the dilution solution determined to agitate.

16. A method of manufacturing homeopathic compositions or high-dilution preparations according to any one of claims 8 to 15, characterized in that the control substance is introduced for the first time before the first dilution, then the control substance is introduced for the second time in the dilution solution following that at which the control substance introduced for the second time disappears, then the control substance is introduced a third time into the solution dilution which follows that which corresponds to the disappearance of the control substance introduced the second time, and so on,

17. A method of manufacturing homeopathic compositions or high-dilution preparations according to any one of claims 8 to 16, characterized in that the control substance is introduced before the first dilution and all the m dilutions, m being comprised from 5 to 15, advantageously from 10 to 15, and advantageously still 10 or 15.

18. A method of manufacturing homeopathic compositions or preparations at high dilution according to any one of claims 8 to 17, characterized in that the control substance is metered, advantageously every 10 dilutions, and more preferably at each dilution.

19. A method of manufacturing homeopathic compositions or preparations at high dilution according to any one of claims 8 to 18, characterized in that the initial solution contains an active substance in an amount of about 1x10 ^-3 to about 1x10 ^-6 moles / l.

20. A method of manufacturing homeopathic compositions or high dilution preparations according to any one of claims 8 to 19, characterized in that the control substance consists of an enzyme, the presence of which is advantageously detectable by its chromogenic activity.

21. Process for the manufacture of homeopathic compositions or high-dilution preparations according to any one of claims 8 to 20, characterized in that the enzyme is chosen from peroxidase, is in particular horseradish peroxidase, detectable by its reactivity with the substrate D-phenylene diamine in H ₂ O ₂ medium.

22. Method for obtaining a dilute solution (mixture) having an average activity, characterized in that successive determined dilution solutions obtained according to any one of claims 1 to 6 or 8 to 21 are mixed, each solution being of different dilution, but corresponding to the same dilution scale.

23. A method of increasing the activity of a determined dilution solution in which the determined dilution solution is obtained according to any one of claims 1 to 6 or 8 to 21, characterized in that a dilution solution determined at a single high dilution.

24. Method for increasing the activity of a dilute solution (mixture) in which the dilute solution is obtained according to claim 22 and in which the determined dilution solutions used are obtained according to any one of claims 1 to 6 or 8 to 21, characterized in that said mixture is subjected to a single high dilution.