FR2729082A1 - Homeopathic compsn. for restoring immune system function - Google Patents

Abstract

Compsn. for restoration of the immune system contains, as active ingredients, very high dilutions of at least one each of (1) a differentiation factor for B lymphocytes, (2) a cytokine and (3) a thymus hormone.

Description

 The present invention relates to an immunoreparative composition and its homeopathic applications.

 Immunosuppression is a pathological condition that can be either spontaneous (immune deficiencies), or caused by irradiation, by the administration of immunosuppressants, whether chemical such as antimetabolites (anti-cancer drugs) or biological as serums antilymphocyte or by immunodepression viral origin.

 To palliate this pathological state, the administration of molecules, issued from the immune system, has been proposed. Among these, there may be mentioned certain cytokines, and especially interleukin 2 (IL-2) or thymic hormones.

 However, these substances have the disadvantage either of being poorly tolerated (IL-2) or of having an immunostimulatory action that is too weak, for an effective therapeutic application. In no case do these substances exhibit an immunoreparative function.

 It has therefore been the object of the present invention to provide an immunoreparative composition which is more responsive to the needs of the practice, particularly in that it is both well tolerated and actually has sufficient immunoreparative activity, effective therapeutic application.

The subject of the present invention is an immunoreparative composition, characterized in that it comprises, in combination, as active substances, at least one lymphocyte differentiation factor.
B, at least one cytokine and at least one thymic hormone, at very high dilutions.

 For the purposes of the present invention, the term "immunoreparative composition" is intended to mean a composition capable of operating an immune system whose function is interrupted.

 For the purposes of the present invention, the concentrations of active substances are defined by their dilutions and the term "very high dilution" means dilution obtained from an initial solution of active substance, dynamically diluted, a sufficient number of times in a solution of dilution, so that the dilution of active substance is at least equal to about 4 successive dilutions at least 1 / 5th or more and especially 1 / 100th or even 1 / 50000th; in the case where one carries out 4 successive dilutions to 1 / 100th, the solution obtained corresponds, in the system "Centésimale Hahnemannienne", to 4CH; said very high dilutions can also be obtained by any other method of successive dilutions with boosting (prolonged stirring).

 Most of them are expressed in the CH system for convenience only.

 Preferably, each of said active substances is present in said immunoreparative composition according to the invention, in the form of a set of said very high dilutions, for example between 4 successive dilutions with dynamization and 20 successive dilutions with dynamization, or more.

 Preferably, each of said active substances is present in said immunorepairable composition according to the invention, in the form of a set of dilutions, between 15CH and 30CH, preferably between 15CH and 20CH or any other set of several dilutions leading to the same immunoreparative activity.

 A dilution at 15CH corresponds to 15 dilutions per hundredth from the initial solution of active substance and a dilution at 20CH corresponds to 20 dilutions per hundredth, from the initial solution of active substance.

The initial solution of active substance undergoes successive dilutions
the first dilution of the initial solution is obtained by taking a fraction or all of the initial solution and the mixture of the sample taken in a dilution solution, followed by a boosting of this mixture.

 the second dilution of the initial solution is obtained by taking a fraction or all of the solution of the first dilution and the mixture of this sample thus taken in a dilution solution, followed by a boosting of this mixture, to give a second dilution solution and so on until the solution of the last desired dilution is obtained.

According to the invention, the differentiation factor of B lymphocytes is selected from bursin and bursopoietin (including tuftsin (Thr
Lys-Pro-Arg), from bursopoietin) or fragments thereof; the thymic hormones are especially selected from thymulin, thymopoietin and its derivatives, thymosin and its derivatives and THF and its derivatives; the cytokine is preferably interleukin-3 (IL-3).

 Advantageously, said substances can also be obtained from extracts of the corresponding organs (Fabricius bursa, marrow, thymus).

 Preferably, the immunoreparative composition according to the invention comprises a set of dilutions of 15CH to 20CH, equal parts, bursine, interleukin-3 and thymulin (BIT).

 Unexpectedly, the composition according to the invention has, at these dilutions, effectively immunoreactive properties, is particularly well tolerated and is suitable for use in the treatment of immunosuppression (or immunodepression, including immunosuppression). viral origin), spontaneous or induced, as defined above and in particular in immunosuppression induced by irradiations, by chemical radiomimetics and anticancer agents.

 Also unexpectedly, such a composition allows a recolonization of the red marrow after irradiation.

 Preferably, interleukin-3 is an interleukin of the same species as the species to be treated.

 In addition to the foregoing, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention, with reference to the appended drawings, in which which Figures 1 to 9 show the results of experiments.

 It should be understood, however, that these examples are given solely by way of illustration of the object of the invention, of which they in no way constitute a limitation.

 EXAMPLE 1 Method for Preparing a Coating According to the Invention (BIT Mixture)

The composition according to the invention can be produced
or by mixing in a 1: 1: 1 (equal parts) ratio of the three active substances, for example bursine, human interleukin-3 and thymulin, and then carrying out the successive dilutions starting from the initial solution containing the 3 active substances,
or by preparing 3 initial solutions, corresponding respectively to bursin, interleukin-3 and thymulin, then making 3 sets of dilutions and mixing dilution sets obtained from the initial 3 solutions.

* Raw materials
- bursine, tripeptide of Lys-His-Gly structure (Neosystem, France), in the form of an initial solution at 2CH
thymulin, nonapeptide-Zn (Neosystem,
France) in the form of an initial solution at 3ci;
mouse interleukin-3 (Pepro Tech USA, at 1 μg / ml), that is 1CH,
- milliporee treated water quality Mille Q
More (Q + 1 thousand water).

* Preparation process proper
1) The different products are reduced to 4CH dilution
- the bursin undergoes two dilutions 1/100 followed by dynamization (shaking on Agitelec Type L vertical shaking, 540 to 560 shakes / minute (Carteau & OTS, Bagnolet, France), for 2 minutes and 30 seconds)
- the thymulin is diluted once to 1/100 with dynamisation, under the conditions explained above
interleukin-3 is subjected to 3 successive dilutions to 1/100 with dynamization, under the conditions exposed above.

 2) The three 4CH solutions are mixed to equal parts and give a 4CH BIT composition.

 The 1/100 dilutions are then prepared dynamically, under the conditions described above, until dilution to 20CH and give a range of BIT dilutions between 15CH and 20CH (15, 16, 17, 18, 19 and 20CH). , in equal parts, which are packaged in vials covered with aluminum foil.

 = KI1PXa 2: Enhancement of the X unoreparative activity of the composition according to the invention.

 Balb / C mice are subjected to a total body irradiation (cobalt 60, emission y) of 7 gy calculated (ie 6.3 gy real, evaluated by a probe) at a dose rate of 1.68 gy / min.

These mice are treated by different compositions
1) drinking water containing 1/100 of water Mille Q + (lot control, called A),
2) a composition according to Example 1, called
BIT,
3) Q + water-enriched drinking water, diluted on itself under the same conditions as those used for the preparation of the immunoreparative composition according to the invention (negative control lot, called B), according to the same protocol, namely
- start of treatment 2 days before irradiation and
continuous treatment after the irradiation, and under the same conditions - either the drinking water, or the dilutions of the composition according to Example 1 or of B mixed in the drinking water at a rate of 1/100 without particular agitation, are given to said mice in the form of bottles. The bottles are changed every 4 days.

The different lots (control or A, witness or
B and BIT) are stored in different refrigerators.

The evaluation is done by counting the survivors in each batch and is obtained from 5 different experiments, carried out under the following conditions
For each experiment, the dilutions are raised from the 14CH of the BIT range and prepared, as specified in Example 1.

 The administration is carried out in accordance with the protocol set out above.

The results obtained are illustrated in the
Tables below and in Figures 1 to 4.

TABLE I
Percent mortality of mice at 30 days

<tb><SEP> A <SEP> B <SEP> BIT <SEP> GM <SEP> Bursine
<tb><SEP> CSF *
<tb> 1st <SEP> experiment <SEP> 50 <SEP>% <SEP> 20 <SEP>% <SEP> 9 <SEP>%
<tb> lots <SEP> of <SEP> 11 <SEP> mice
<tb> 2nd <SEP> experiment <SEP> 90 <SEP>% <SEP> 82 <SEP>% <SEP> 10 <SEP>%
<tb> lots <SEP> of <SEP> 11 <SEP> mice
<tb> 3rd <SEP> experiment <SEP> 50 <SEP>% <SEP> 43 <SEP>% <SEP> 22 <SEP>% <SEP> 65 <SEP>%
<tb> lots <SEP> of <SEP> 14 <SEP> mice
<tb> 4th <SEP> experiment <SEP> 100 <SEP>% <SEP> 86 <SEP>% <SEP> 58 <SEP>% <SEP> 100 <SEP>% <SEP> 84 <SEP>%
<tb> lots <SEP> of <SEP> 14 <SEP> mice
<Tb>
TABLE II
Compared reports of 30-day mouse mortality

<tb><SEP> BIT / A <SEP> BIT / B <SEP> B / A <SEP> GM <SEP> Burs / B
<tb><SEP> CSF / B
<tb> 1st <SEP> experiment <SEP> 0.18 <SEP> 0.45 <SEP> 0.4
<tb> 2nd <SEP> experience <SEP> 0.11 <SEP> 0.12 <SEP> 0.91
<tb> 3rd <SEP> experiment <SEP> 0.44 <SEP> 0.51 <SEP> 0.86 <SEP> 1.51
<tb> 4th <SEP> experiment <SEP> 0.58 <SEP> 0.62 <SEP> 0.86 <SEP> 1.16 <SEP> 0.97
<tb> GM CSF = Granulocyte Macrophage - Colony Stimulating
Mouse factor.

TABLE III
Compared results of the blood formulas of the surviving mice (average of two samples) expressed as a percentage compared to unirradiated control mice of the same age and of the same batch.

<Tb>

<SEP> 2nd <SEP> experiment <SEP> J <SEP> 6 <SEP> J <SEP> 15 <SEP> J <SEP> 39
<tb> A <SEP>% <SEP> neutrophils <SEP> 0.67 <SEP> 0 <SEP> 0.93
<tb><SEP>%<SEP> lymphocytes <SEP> 0.93 <SEP> 0 <SEP> 0.96
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 80 <SEP>% <SEP> 90 <SEP> t <SEP>
<tb> B <SEP>% <SEP> Neutrophils <SEP> 1.1 <SEP> O <SEP> 1.07
<tb><SEP>%<SEP> lymphocytes <SEP> 1.08 <SEP> 0 <SEP> 1.02
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 35 <SEP> t <SEP> 82 <SEP> t <SEP>
<tb> BIT <SEP>% <SEP> Neutrophils <SEP> 1.21 <SEP> 0 <SEP> 0.95
<tb><SEP>%<SEP> lymphocytes <SEP> 0.99 <SEP> 0 <SEP> 0.84
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 10 <SEP> t <SEP> 10 <SEP> t <SEP>
<tb><SEP> 3rd <SEP> experiment <SEP> J <SEP> 6 <SEP> J <SEP> 13 <SEP> J <SEP> 19 <SEP> J <SEP> 35
<tb> A <SEP>% <SEP> neutrophils <SEP> 1.41 <SEP> 0 <SEP> 1.19 <SEP> 1.34
<tb><SEP>%<SEP> lymphocytes <SEP> 0.44 <SEP> 0 <SEP> 0.49 <SEP> 0.60
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 22 <SEP> t <SEP> 50 <SEP> t <SEP> 50 <SEP>%
<tb> B <SEP>% <SEP> neutrophils <SEP> 1.51 <SEP> 0 <SEP> 1.78 <SEP> 2.0
<tb><SEP>%<SEP> lymphocytes <SEP> 0.5 <SEP> 0 <SEP> 0.35 <SEP> 0.25
<tb><SEP> Mortality <SEP> o <SEP> t <SEP> 15 <SEP> t <SEP> 43 <SEP> t <SEP> 43 <SEP> t <SEP>
<tb> BIT <SEP>% <SEP> neutrophils <SEP> 1.61 <SEP> 0 <SEP> 1.0 <SEP> 0.67
<tb><SEP>%<SEP> lymphocytes <SEP> 0.40 <SEP> 0 <SEP> 0.74 <SEP> 1.02
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 7 <SEP> t <SEP> 22 <SEP> t <SEP> 22 <SEP> t <SEP>
<tb> GM <SEP> CSF <SEP>% <SEP> Neutrophils <SEP> 1.68 <SEP> 0 <SEP> 1.27 <SEP> 1.41
<tb><SEP>%<SEP> lymphocytes <SEP> 0.49 <SEP> 0 <SEP> 0.54 <SEP> 0.62
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 15 <SEP> t <SEP> 65 <SEP> t <SEP> 65 <SEP>%
<Tb>

<tb><SEP> 4th <SEP> experiment <SEP> J <SEP> 0 * <SEP> J <SEP> 6 <SEP> J <SEP> 13 <SEP> J <SEP> 22
<tb> A <SEP>% <SEP> neutrophils <SEP> 1.61 <SEP> 0 <SEP> 0
<tb><SEP>%<SEP> lymphocytes <SEP> 0.52 <SEP> 0 <SEP> 0
<tb><SEP> Mortality <SEP> o <SEP> t <SEP> 0 <SEP> t <SEP> 86 <SEP>% <SEP> 100 <SEP>%
<tb> B <SEP>% <SEP> Neutrophils <SEP> 1.77 <SEP> 0 <SEP> 0 <SEP> 1.55
<tb><SEP>%<SEP> lymphocytes <SEP> 0.52 <SEP> 0 <SEP> 0 <SEP> 0.63
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 0 <SEP> t <SEP> 79 <SEP> t <SEP> 86 <SEP>%
<tb> BIT <SEP>% <SEP> Neutrophils <SEP> 1.19 <SEP> 0 <SEP> 0 <SEP> 0.94
<tb><SEP>%<SEP> lymphocytes <SEP> 0.56 <SEP> 0 <SEP> 0 <SEP> 1.06
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> o <SEP> t <SEP> 50 <SEP> t <SEP> 57 <SEP> t <SEP>
<tb> GM <SEP> CSF <SEP>% <SEP> Neutrophils <SEP> 1.67 <SEP> 0 <SEP> 0
<tb><SEP>%<SEP> lymphocytes <SEP> 0.61 <SEP> 0 <SEP> 0
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 0 <SEP> t <SEP> 79 <SEP>% <SEP> 100 <SEP> t <SEP>
<tb> Bursin <SEP>% <SEP> Neutrophils <SEP> 1.67 <SEP> 0 <SEP> 0 <SEP> 1.42
<tb><SEP>%<SEP> lymphocytes <SEP> 0.60 <SEP> 0 <SEP> 0 <SEP> 0.75
<tb><SEP> Mortality <SEP> 0 <SEP> t <SEP> 0 <SEP> t <SEP> 69 <SEP> t <SEP> 84 <SEP> t <SEP>
<tb> 4 h post irradiation.

 Hematological monitoring of treated mice shows a recovery of neutrophilic granulocytes and lymphocytes comparable to non-irradiated controls.

 This is obtained after 35 days in experiment 3 and after 22 days in experiment 4.

 FIG. 1, which comprises the number of days on the abscissa and the percentage of survival of the irradiated mice, corresponds to the first experiment carried out on a batch of 11 animals and shows that the surviving mice have a survival time of at least 90 days.

 FIG. 2 illustrates the results obtained with a second experiment and also comprises, on the abscissa, the number of days and on the ordinate the percentage of survival of irradiated mice; the curve -O- corresponds to (A) (water) (MilliQ, n = 11), the curve -A- corresponds to (B) (water dyn; n = 11), the curve --- corresponds to the administration of the composition according to the invention, after irradiation (BIT post-y) (n = 10), the curve -A- corresponds to the administration of the composition according to the invention during irradiation (pre-post BIT) -y) (n = 11).

 The third experiment is illustrated in Figure 3; it was performed on a batch of 14 animals and includes the number of days on the x-axis and the number of surviving animals on the y-axis.

 FIG. 4 illustrates the results obtained with a fourth experiment and also comprises, on the abscissa, the number of days after irradiation and on the ordinate the percentage of survival of mice irradiated the curve -s- corresponds to (A) (water), the curve - - corresponds to (B), the curve -O- corresponds to GM-CSF, the curve - corresponds to the administration of the composition according to the invention, the curve -s- corresponds to the administration of bursin.

The results obtained show the repeatability of the tests on different batches of mice (11 to 14 mice per batch) and show that
in control lots (A), 50 to 100% mortality is observed,
in negative control batches (B), 50 to 90% mortality is observed,
in batches comprising the composition according to the invention at high dilution (BIT), only 10 to 57% mortality is observed.

 In addition, the mitogen proliferative response curves of the treated (BIT) mouse lymphocytes with B and A of experiments 1 and 3 show a significantly higher proliferative response of T and B lymphocytes in BIT-treated mice are illustrated in FIGS. to 9.

 In these Figures 5 to 9, the curves (-s-) illustrate the results obtained with a composition according to the invention (BIT), the curves (---) illustrate the results obtained with (B) and (-d-) illustrate the results obtained with (A).

 These different figures comprise, on the ordinate, the number of cpm, and on the abscissa, the concentration expressed in Rg / ml in PHA (FIGS. 5 and 8), in LPS (FIGS. 6 and 9) and in ConA (FIG. 7), FIGS. and 6 corresponding to the first experiment, Figures 7, 8 and 9 corresponding to the 3rd experiment.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (10)

 1 ') immunoreparative composition, characterized in that it comprises in combination, as active substances, at least one differentiating factor of B lymphocytes, at least one cytokine and at least one thymic hormone, at very high dilutions.  2 ') immunoreactive composition according to claim 1, characterized in that the dilution in each active substance is at least equal to about 4 successive dilutions at least 1 / 5th.  3 ') immunoreactive composition according to claim 1 or claim 2, characterized in that the dilution in each active substance is at least equal to about 4 successive dilutions, between 1 / 5th and 1 / 100th.  4 ') Composition according to any one of claims 1 to 3, characterized in that each of said active substances is present in the form of a set of said very high dilutions.  5 ') Composition according to any one of claims 1 to 4, characterized in that each of said active substances is present in the form of a set of said very high dilutions between 4 successive dilutions and 20 successive dilutions.  6 ') Composition according to any one of claims 1 to 5, characterized in that each of said active substances is present in the form of a set of dilutions, between 15 and 30 dilutions 1 / 100th, that is, that is to say between 15 and 30CH (Hahnemannian Centésimal), preferably between 15CH and 20cl.  7 ') Composition according to any one of claims 1 to 6, characterized in that the differentiation factor of B lymphocytes is selected from bursine, bursopoietin, a fragment of these substances or Fabricius purse extracts; the thymic hormones are selected from thymulin, thyopoietin and its derivatives, thymosin and its derivatives and THF and its derivatives or thymus extracts and the cytokine is selected from interleukin-3 (IL-3) or extracts of marrow.  8 ') Composition according to any one of claims 1 to 7, characterized in that it comprises a set of dilutions of 15CH to 20CH, equal parts of bursine, interleukin-3 and thymulin.  9) Use of a composition according to any one of claims 1 to 8, for obtaining a medicament for the treatment of immunosuppression, including viral origin.  10 ') Use of a composition according to any one of claims 1 to 8, for obtaining a cancer adjuvant drug.