WO2000001412A1

WO2000001412A1 - Method for activating an inactive solution

Info

Publication number: WO2000001412A1
Application number: PCT/FR1999/000908
Authority: WO
Inventors: Jacques Benveniste; Didier Guillonnet
Original assignee: Digibio
Priority date: 1998-07-01
Filing date: 1999-04-16
Publication date: 2000-01-13
Also published as: AU3335899A; EP1091758A1; FR2780651A1; FR2780651B1

Abstract

The invention concerns a method for activating an inactive solution with very low concentration of a specific biological and/or chemical substance. The method comprises a step which consists in subjecting said solution to a mechanical excitation field, in particular generated by a vortex.

Description

METHOD FOR ACTIVATING AN INACTIVE SOLUTION

The present invention relates to a method for activating an inactive and very low concentration solution of a determined biological and / or chemical substance in a solvent. The present invention also relates to the applications of said activation method. The term "very low concentration" denotes concentrations between 10 ^"6 and zero moles per liter (M). Methods of preparing a" highly diluted "solution are known by successive dilution and stirring. preparation traditionally used in homeopathy (Hannemahn method) consists, starting from a relatively concentrated solution made using a mother tincture (concentration greater than 10 ^"6 M), to dilute one factor 10 or 100, then mechanical agitation

(called "dynamization"). It is noted after each operation that the solution has remained active in the sense that it triggers a reaction within a sensitive biological system. As examples of solutions which have been prepared by the method described above, mention may be made of all homeopathic preparations. By way of examples of sensitive biological systems making it possible to test the active nature of such solutions, there may be mentioned: the isolated guinea pig heart (Langendorff experiment) or the skin test carried out on the skin of a living guinea pig or rabbit . The need to start with an active solution before diluting and then stirring seemed essential to obtain active diluted solutions. Dilutions carried out up to 10 ^"6 M and beyond, without implementing this successive stirring and dilution process, have not so far made it possible to obtain solutions active. π is not without interest either to recall that the stirring and dilution technique implemented by Hannemann has been extrapolated without being able to show the varnishes of such high dilutions beyond factor 5 up to now CH.

However, the inventors, who are known for their work on high dilutions (Nature 1988: "Degranulation of human Basophils triggered by a high dilution solution of anti-IgE antibodies"), have surprisingly found that it was possible to obtain an active solution from an inactive and very low concentration solution of a determined biological and / or chemical substance in a solvent, they have shown that a very low concentration solution whose activity is nonexistent at at the start, can be made active by methods which are particularly simple to implement. Thus, they designed a process which makes it possible to make active solutions at very low concentration without it being necessary to prepare them beforehand by the traditional technique of the dilutions and successive agitations, they thus solved a problem whose industrial implications are considerable . Indeed: - it is now possible to detect specific biological and / or chemical substances in solution at very low concentration in a solvent,

- it is now possible to design and produce drugs using specific biological and / or chemical substances in solution at very low concentration in a solvent,

- it is now possible to control the production of very low concentration products, in particular homeopathic products.

The present invention therefore relates to a method for activating a solution at very low concentration of a determined biological substance and / or chemical in a solvent. Said method comprises the step of placing said solution in a mechanical excitation field. The mechanical excitation field could be created, for example, by a shock wave propagating in the solution, by ultrasound or sound waves diffused in the solution, by vibrations transmitted by the container containing the solution. Preferably, the mechanical excitation field results from violent agitation, in particular obtained by means of a Vortex-type agitator consisting of a disc which rotates rapidly until a vortex is formed in the column of liquid in restlessness course. Advantageously, said solution is subjected to a mechanical excitation field for at least 15 seconds. As examples of substances in solution which have been activated by the method described above, mention may be made of: arnica, ovalbumin, acetylcholine, calcium ionophore. The method according to the invention is of interest only when the concentration of said determined substance in said solution is less than 10 ^"6 moles per liter. Indeed above 10 ^{" 6} moles per liter the solution is already active (pharmacology traditional). Advantageously, the concentration of said substance determined in said solution is within a range of less than 10 ⁻⁶ moles per liter and greater than

10 ^"16 moles per liter.

Preferably also said solvent contains at least 5% water. It has indeed appeared that below a certain proportion of water in the solvent the solution subjected to a mechanical excitation field remains inactive.

It has also been found that a percentage of alcohol of at least 2% in the solvent has the effect that the solution remains active for a longer period.

The method according to the invention further comprises the step of checking the state active of said solution by implementing an experimental protocol identical or similar to that which one would implement to account for the presence of said determined substance in a medium which would contain it. By way of example of a sensitive biological system making it possible to test the active nature of the solutions of the following determined substances: calcium ionophore, acetylcholine, Wstamine, ovalbumin (in animals made sensitive), arnica, bradykinin, anti-IgE antibodies, may cite: the Langendorff experience (isolated guinea pig heart) as well as the skin test carried out on the skin of a living guinea pig or rabbit. Thus, for example, to control the active state of the acetylcholine solution, it is verified that an injection of the latter, in particular under the skin of a guinea pig, causes skin reactions. The present invention also relates to the application of the method to the detection of a determined substance, diluted in very low concentration.

In fact, once the solution is activated, it is possible to carry out identification tests by implementing an experimental protocol identical or similar to that which would be used to account for the presence of said determined substance. in an environment that would contain it. Examples of specific substances which can be detected in solution include the following substances: calcium ionophore, caffeine, nicotine, bacterial toxins and endotoxins; and the following identification tests: the Langendorff experiment (isolated guinea pig heart) as well as the skin test carried out on a living guinea pig or rabbit skin.

The present invention also relates to the application of the process to the production of medicaments using biological and / or chemical substances at very low concentration. Indeed, as soon as it is possible to prepare active solutions with a very low concentration of determined biological and / or chemical substances, new therapeutic applications of these substances become possible. Examples of drugs which can thus be produced include the following drugs: coronary vasodilators (eg trinitroglycerin), beta-blocker (propranolol), caffeine, nicotine.

The present invention also relates to the application of the method to the control of the production of products at very low concentration, in particular homeopathic products. One of the problems to be solved when manufacturing homeopathic products is that of the production control of successive dilutions. The method according to the invention makes it possible to test the activity of homeopathic products during the various phases of their manufacturing process. By way of example of homeopathic products whose production can thus be controlled, the following drugs may be cited: arnica 5 to 30 CH, Wstaminum 5 to 30 CH, acetylcolinum 5 to 30 CH, apis mellifica 5 to 30 CH.

Other characteristics and advantages of the invention will appear on reading the description of variant embodiments of the invention, given by way of non-limiting example, as well as on reading the examples of experiments which have enabled to validate the activation process, object of the present invention, and which refer to the appended drawings in which:

- Figure 1 shows a perspective view of an alternative embodiment of the stirring system.

- Figure 2 shows a perspective view of a system for testing the activity of the solution (Langendorff experiment).

- Figure 3 shows an image of the skin of a guinea pig

We will now describe Figure 1 which shows a perspective view of an alternative embodiment of the stirring system. A rubber roller 2 is pivotally mounted about a vertical axis. The pebble in rubber is rotated around the vertical axis by an electric motor (not shown), located inside the housing 3. The electric motor is powered by a cable 10. The speed of rotation of the roller is controlled by a potentiometer 4. Tube 1 contains solution 6 of acetylcholine at the concentration of lpM, to be shaken violently. The operator 5 maintains the lower end 7 of the tube applied against the roller 2 by pressing on the upper part 8 of the tube 1. The lower end 7 of the tube 1 describes a circle 9. As a result, a vortex occurs within solution 6. This vortex shakes and violently mixes the solution.

By way of example, we will now describe, with reference to FIG. 2, the test carried out using an isolated guinea pig heart perfused, known since 1897 (under the name of the "Langendorff experiment") and described in the books of traditional pharmacology, in particular in “Animal experimentation in cardiology” - Medicine - Science Insertion - Flammarion,

Bernard SWYNGHEDAUW, Chapter 3.1 P. 81 "Isolated organ: isolated heart according to Langendorff". The fraction collector collects the tubes at the rate of one tube per minute and thus measures the flow rate of the guinea pig heart minute by minute. Here are the results from the experiments carried out with the following substances, after they have vortexed (agitated) as described with reference to FIG. 1, in a 15 ml tube containing 10 ml of solution:

- for the first, a mixture of acetate-choline (A-C) 1 μM (sodium acetate 1 μM + choline chloride 1 μM) vortexed for 15 seconds,

- for the second, acetylcholine (ACh) lpM vortexed for 15 seconds,

- for the third, acetylcholine (ACh) lpM vortexed for 5 seconds

- for the third, acetylcholine (ACh) lpM vortexed for 2 seconds

- for the third, acetylcholine (ACh) lpM vortexed for 1 second

The buffer solution had the following composition: Ca ²⁺ 2mM, NaHC03

25 mM.

The solvent used in all five cases was water.

The table below indicates (in ml) the quantity of buffer solution recovered in the collecting tubes over time.

This table highlights several things: a) in the experimental protocol the solutions are tested before being vortexed to verify that they are inactive. The results of Experiment 5 illustrate one of these tests. The solution is inactive before being vortexed. In fact, the variation in flow rate of 0.2 ml / min (min = 3.6; max = 3.8) corresponds to measurement uncertainties and to normal variations in flow rate in the biological system that is the isolated guinea pig heart perfused. b) the comparison of the results of experiment 1 with experiment 2 shows that the action of agitation is not sufficient in itself: still it is necessary that molecules to which the biological system is sensitive are present. In fact, a product very close to acetylcholine, acetate-choline, prepared under the same conditions as acetylcholine, does not trigger a reaction (Exp 1). c) the series of experiments 2, 3, and 4 was carried out on the same guinea pig core and shows the influence of the agitation time using the Vortex on the activity produced in the substance. Thus for a 2 second agitation we obtain a maximum variation of flow rate of 0.3 ml / min (min = 3.5; max = 3.8) while for a 15 second agitation we obtain a maximum variation of flow rate of 0.6 ml / min (min = 2.8; max = 3.4) As further examples, here are the results from the experiments carried out with acetylcholine lpM as substance, vortexed for 15 seconds for different ethanol contents in the solvent. The experiments were carried out 9 days after stirring the solution.

This table shows that the ethanol content promotes the conservation of activity in water.

We will now describe with reference to Figure 3 the skin test in guinea pigs

We use a living guinea pig, to which we inject a venous blue dye (Evans blue) which is fixed on the blood albumin. Albumin does not leave the vessels, unless there is inflammation, therefore vasodilation and permeation of the vessels, the typical example of such a reaction in humans being urticaria.

The test is carried out by injecting 0.1 ml of the solution, the activity of which must be monitored, under the skin of the animal thus prepared. The diameter of the blue spots that appeared around the injection points is then measured. To do this, we scan the skin, then save the bitmap file. Then we measure the pixel size of the blue spots due to the reaction.

The numbers 3, 4, 10, 11 which appear in the first column of the table below correspond to the references of FIG. 3.

- Injection number 3 shows that the very low concentration (lpM) vortexed solution of the neuromediator acetylcholine (ACh) triggers a significant skin reaction (1,949 10 ³ pixels) compared to the same non-vortexed solution which does not trigger a reaction as shown in injection number 4 (43 10 ³ pixels). - The comparison of injection number 3 (1,949 10 ³ pixels) and injection number 11 (1,154 10 ³ pixels) shows that the activity of a solution at very low vortexed concentration is entirely comparable to that of a non-vortexed solution with a higher concentration (1 μM, usual concentration in traditional pharmacology)

- Injection number 10 is carried out with a vortexed picomolar solution

(lpM) of a product close to acetylcholine but inactive: the acetate / choline (AC) mixture. This injection shows that the skin reaction of the guinea pig is very specific to the nature of the substance in solution because this acetate / choline solution vortexed under the same conditions as injection number 1 has no effect (25 10 ³ pixels).

Claims

claims

1. A method for activating an inactive and very low concentration solution of a determined biological and / or chemical substance in a solvent, said method comprising the step: - of placing said solution in a field of mechanical excitation.

2. Method according to claim 1, said method comprising the step:

- subjecting said solution to agitation to create said mechanical excitation field.

3. Method according to any one of claims 1 or 2, such that the concentration of said determined substance in said solution is less than 10 ^"6 moles per liter.

4. Method according to any one of claims 1 to 3, such that the concentration of said substance determined in said solution is within a range less than 10 ^"6 mole per liter and greater than

10 ^"16 mole per liter,

5. Method according to any one of claims 1 to 4, such that said solvent contains at least 5% water.

6. Method according to any one of claims 1 to 5, such that said solvent contains at least 2% alcohol,

(so that the solution remains active for a longer period).

7. Method according to any one of claims 1 to 6, said method comprising the step:

- subjecting said solution to a mechanical excitation field for at least 15 seconds.

8. Method according to any one of claims 1 to 7 further comprising the step:

- to control the active state of said solution by implementing an experimental protocol identical or similar to that which would be implemented works to account for the presence of said determined substance in a medium which would contain it.

9. The method of claim 8 such that to control the active state of said solution it is verified that an injection thereof causes skin reactions

10. Application of the method according to any one of claims 1 to 9 to the detection of a determined substance, diluted in very low concentration.

11. Application of the method according to any one of claims 1 to 9 to the production of medicaments.

12. Application of the method according to any one of claims 1 to 9 to the control of the production of products at very low concentration, in particular homeopathic products.

AMENDED CLAIMS

[received by the International Bureau on September 27, 1999 (September 27, 1999); claims 1-12 as amended; (2 pages)]

1. Method for reactivating a biological and / or chemical substance in dilute solution at very low concentration, in particular at concentrations lower than 10 ^"6 moles per liter, likely to present in solution at least two states:

- an inactive state and

an active state, said states being able to be controlled by the implementation of an experimental protocol comparable or identical to that making it possible to demonstrate the effects of said substance on a sensitive receptor; said method comprising at least the second of the following two steps:

the step of checking the state of said substance in solution,

- The step of subjecting said substance in solution, when it is in an inactive state at very low concentration, to a mechanical excitation field.

2. The method of claim 1, said method comprising the step of subjecting said substance in solution to agitation to create said mechanical excitation field.

3. Method according to any one of claims 1 or 2, such that the concentration of said substance in solution is less than 10 ^"6 moles per liter.

4. Method according to any one of claims 1 to 3, such that the concentration of said substance in solution is within a range of less than 10 ^"6 moles per liter and greater than 10 ^{" 16} moles per liter.

5. Method according to any one of claims 1 to 4, such that said substance is in solution in a solvent containing at least 5% water.

6. Method according to any one of claims 1 to 6, such that said substance is in solution in a solvent containing at least 2% alcohol,

MODIFIED SHEET (ARTICLE 19) (so that the solution remains active for a longer period). 7. Method according to any one of claims 1 to 6, said method comprising the step of subjecting said substance in solution to a mechanical excitation field for at least 15 seconds. 8. Method according to any one of claims 1 to 7 further comprising the step of controlling the active state of said substance in solution by implementing an experimental protocol identical or similar to that which would be used for give an account of the presence of said determined substance in a medium which would contain it. 9. The method of claim 8 such that to control the active state of said substance in solution it is verified that an injection of it causes skin reactions

10. Application of the method according to any one of claims 1 to

9 upon detection of a determined substance diluted in very low concentration.

11. Application of the method according to any one of claims 1 to

10 to drug production.

12. Application of the method according to any one of claims 1 to 10 to the control of the production of products at very low concentration, in particular homeopathic products.