RU2643934C2 - Method for determining expression of the modifying activity associated with carrier - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to pharmacology and can be used to determine the extent of modifying activity, associated with the carrier acquired in during processing of the initial substance in form of repeated successive reduction of its concentration using a carrier, which manifests itself in the ability to directly change physico-chemical and/or biological properties of initial substance when exposed to it by said carrier. For this purpose presence or absence of molecules of the initial substance in said carrier is determined, and in presence of molecules of the initial substance before measurement of the characteristic parameter molecules of the initial substance are removed from the carrier, and one or more characteristic parameters of the initial substance are measured by analytical methods before and after interaction with said carrier, wherein, degree of modifying activity expression is determined by magnitude of the change in the characteristic parameter in relative units of activity calculated by formula (1): X=C|A-A_M|:A, (1), where X is the number of units of activity (UA); C is the dimensionless coefficient of proportionality; A is value of the characteristic parameter of the initial substance prior to interaction of said activated carrier with it; A_M is the value of the same characteristic parameter of the initial substance after interaction of the above activated carrier with it.

EFFECT: invention allows to determine the extent of modifying activity associated with the carrier, acquired during processing of the initial substance in form of repeated successive reduction of the concentration of the latter using a carrier.

4 cl, 1 dwg, 4 tbl, 7 ex

Description

The invention relates to medicine, namely to pharmacy, and can be used for reliable and reliable determination of the pharmacological activity of drugs, including those prepared according to homeopathic technology.

The prior art method for detecting biological activity (RU 2181890 C1, G01N 33/68, 2002), in which in vitro the ratio of the rate of the enzymatic reaction on the test object after adding the substance and the speed of the enzymatic reaction to adding the substance at "optimal concentrations of the substance in sample. " However, this method is not applicable for determining the activity of drugs prepared, for example, using a homeopathic potentiation technology by repeatedly sequentially decreasing the concentration — diluting the starting material in a carrier (aqueous or hydroalcoholic solvent) in combination with multiple shaking of each dilution (RU 2191601 C1 , A61K 39/395, 2002; RU 2192888 C1, A61K 39/395, 2002; RU 2332236 C1, A61K 39/395, 2008; RU 2438707 C2, A61K 39/395, 2012), which, when diluted with close and higher Avogadro numbers (N _A = 6.02252 × 10 ²³ mol ^-1 ) contain small and ultra-small th doses of the starting drug substance.

It is also known from the prior art that in the process of activation (potentiation) during processing of a starting substance by repeatedly sequentially decreasing the concentration (dilution or grinding) in a carrier using homeopathic technology, the carrier acquires modifying activity, which is manifested in the ability to change physicochemical and / or biological properties of the starting substance when exposed to it by the specified activated (potentiated) carrier (RU 2161955 C1, A61K 9/00, 2001).

A known method for determining the quality of homeopathic medicines by illuminating coherent linearly polarized optical radiation with a homeopathic multiple dilution of a drug substance that does not contain its molecules, which is placed in a constant magnetic field and records the scattered transmitted radiation by temporarily accumulating the intensity values of its polarized component in the optical bias mode different points of the studied environment, and the analysis is carried out with techniques, are infraslow frequency spectrum of the intensity fluctuations of the transmitted radiation and its comparison to the reference sample (RU 2112976 C1, G01N 33/15, 1998).

In addition, there is a method for the qualitative determination of a homeopathic medicinal product or a potentiated form of a substance, including exposure to a test reagent on a test medium and recording changes in physicochemical parameters, in which a set of known substances that are similar or similar in structure and / or composition are used as a reference reagent to a defined homeopathic medicinal product or substance in potentiated form, and antibodies to these known substances, an opatic drug or a potentiated form of a substance is produced according to a known substance, the reaction of which with an appropriate antibody when a homeopathic medicinal product or a potentiated form of a substance is introduced into the reaction medium proceeds with changes registered by immunochemical analysis methods based on the antigen-antibody reaction (RU 2195648 C2, G01N 33/15, 2002).

However, the known above methods do not provide reliable and reliable qualitative and quantitative determination of the authenticity of the drug and the activity associated with the drug carrier, including those prepared mainly by homeopathic technology.

The technical result to which the invention is directed is to increase the reliability and reliability of determining “in vitro” (outside the body) the authenticity and pharmacological modifying activity associated with a drug carrier, including those prepared mainly using homeopathic technology and practically do not containing molecules of the source (active) substance.

The solution of this problem with the achievement of the claimed technical result is ensured by the fact that in the method for determining the severity of modifying activity associated with the carrier, acquired during activation during technological processing of the starting substance in the form of a multiple successive decrease in the concentration of the latter using the carrier, which manifests itself in the ability to directly change (or influence) the physicochemical and / or biological properties of the starting material during the action on it of the specified activated carrier, according to the invention, by analytical methods determine the presence or absence of the molecules of the starting substance in the specified activated carrier and measured by analytical methods one or more characteristic parameters of the starting substance before and after the interaction of the specified activated carrier with it, while the degree of expression of the modifying activity determined by the magnitude of the change in the characteristic parameter in relative units.

In addition, if there is a molecule of the starting material in the modified solvent, the molecules of the starting material are removed from the latter before measuring the characteristic parameter of the starting material by reacting the indicated activated carrier.

In this case, a liquid solvent is used as a carrier, or a solid carrier during trituration.

In addition, additionally, by analytical methods, the effect on the physicochemical properties of the starting material of an inactive starting medium and / or other substance, which is processed by repeated successive dilutions or triturations with intermediate shaking of each dilution using homeopathic technology, and / or the starting substance is recorded.

The severity of modifying activity is determined by the magnitude of the change in a characteristic parameter in relative units of activity (release activity):

where X is the number of units of activity;

C is a dimensionless coefficient of proportionality, which depends on the analytical methodology used to measure a characteristic parameter that displays the initial physicochemical and / or biological properties and values of the characteristic parameter. In the particular case, for example, C = 10 ^k , where k is an integer from the series 1, 2, 3, ... etc .;

A is the value of the characteristic parameter of the starting material prior to the interaction of the specified technologically processed carrier with it;

Am is the value of the same characteristic parameter of the starting material after the interaction of the specified technologically processed carrier with it.

When implementing the inventive method, various methods of qualitative and quantitative analysis can be used that provide high sensitivity and reliability at ultralow concentrations of the substance, including spectrometry methods, including mass spectrometry, chromatography-mass spectrometry (gas-liquid (GLC) and high-performance liquid (HPLC) ) chromatography), nuclear magnetic resonance spectroscopy (NMR); enzyme immunoassay methods (ELISA); biological methods and other methods suitable for implementing the claimed method (see, for example, Zolotov Yu.A. (ed.). Fundamentals of analytical chemistry in two books. A textbook for universities. 3rd ed., revised and additional M .: Higher school, 2004; Vasiliev VP Analytical chemistry. 1989; Otto M. Modern methods of analytical chemistry. Volume 2003).

In addition, if there are molecules of the starting material in the modified solvent, they are removed by known methods, for example, when a protein is used as the starting material, its molecules are removed by heating to denaturation followed by filtration, or by ultrafiltration (molecular filtration) with preliminary filtration through larger pores and etc. (see, for example, "The Production of High-Purity Water" by V.M. Stewart The production of high-purity water in the clinical laboratory // Laboratory Medicine. - 2000. - V.31 (11) - P.605 -611; "Review of Electro-assisted methods for water purification" J. Grimm, D. Bessarabov, R. Sanderson Review of electro-assisted methods for water purification // Desalination. - 1998. - VI 15 (3) - P. 285-294; "reverse flow filtration" IA Koznacheev et al. Water purification of organic inclusions in a reverse flow filtration combustion reactor // International Journal of Heat and Mass Transfer - 1998.54 - P.932-937).

Due to the combination of the determination by analytical methods of the presence or absence of molecules of the starting material in the specified activated carrier with the measurement by analytical methods of at least one characteristic parameter of the starting material before and after the interaction of the specified activated carrier, it is confirmed (justified), firstly, that the associated with the carrier modifying activity is due not to the presence of molecules of the starting substance, but to the physicochemical and / or biological properties indicated of the carrier differ from the physicochemical and / or biological properties of the starting material, and secondly, that the activated support was obtained using the starting material, and its modifying activity is due precisely to the process of processing the starting material in the form of a multiple sequential decrease in the concentration of the latter using the specified carrier, and, accordingly, in vitro (outside the body), the reliability and authenticity of the drug prepared on the basis of the decree is proved This activated carrier using the starting substance, mainly according to homeopathic technology that does not contain the molecules of the starting (active) substance. In addition, the claimed measurement by analytical methods of at least one characteristic parameter of the starting material before and after the interaction of the specified activated carrier provides the ability to quantify the degree of severity of modifying activity associated with the carrier in relative dimensionless units of activity (release - activity). The claimed method is implemented as follows.

To determine the severity of the modifying activity associated with the carrier, the following operations are performed sequentially:

a. the representation of a carrier with modifying activity, activated in the process of processing the starting material by repeatedly sequentially reducing the concentration using the specified carrier, in which the specified carrier does not contain a molecular form of the specified starting material,

b. Verification of step a. solution for the specificity of the substance, while the check includes

i. impact specified in step a. carrier to the molecular form of the starting material

ii. preferably, the exposure indicated in step a. carrier to the molecular form of another substance and / or solvent

iii. measuring by analytical methods at least one physicochemical and / or biological characteristic parameter of the indicated molecular form of the starting material (A) and the specified combination according to b.i. (Am), in which the specified carrier specifically modifies the effect-ability to change the physicochemical and / or biological properties of the starting substance, it is considered specific to the substance if the specified characteristic parameter is statistically significantly changed during the implementation of section b.i. (and does not change statistically significantly in the implementation of paragraph b.ii.)

c. determination of modifying activity associated with the carrier in relative units of activity according to the formula:

where C is preferably equal to 100 or 1000.

A carrier associated with modifying activity is prepared, mainly by homeopathic technology, by uniformly decreasing the concentration as a result of successive dilution of 1 part of the matrix solution of the starting material in 9 parts (for decimal dilution) or in 99 parts (for hundredth dilution) or 999 parts (for thousandth dilution) of a neutral solvent with multiple intermediate vertical shaking ("dynamization or activation") of each dilution obtained and use separate containers for each subsequent dilution until the required dilution of multiplicity, for example, by homeopathic method (see., e.g., V. Schwabe "Homeopathic medicines", M., 1967, p. 14-29).

For example, to prepare the 12th hundredth dilution of C 12, in accordance with homeopathic technology, one part of the aforementioned matrix solution of the starting material, for example, with a concentration of 2.5 mg / ml, is diluted in 99 parts of a neutral aqueous or aqueous-alcoholic solvent (mainly 70% ethyl alcohol) and repeatedly (10 or more times) vertically shake - potentiate the obtained 1st hundredth C1 dilution. From the 1st hundredth dilution C1, the second hundredth dilution C2 is prepared. This operation is repeated 11 times, obtaining the 12th hundredth dilution of C12. Thus, the 12th hundredth dilution of C12 is a solution obtained by diluting successively in different containers 12 times of the first part of a matrix solution of the starting material with a concentration of 2.5 mg / ml in 99 parts of a neutral solvent, i.e. a solution prepared from a matrix solution diluted 100 to ¹² times. Similar operations with an appropriate dilution ratio are carried out to obtain dilutions of C30, C50 or C200.

When using as a biologically active liquid component a mixture of various homeopathic, mainly hundred, dilutions, starting materials, each component of the composition (for example, C12, C30, C50 or C200) is prepared separately according to the technology described above to the penultimate dilution (respectively, to obtain C11, C29, C49 or C199) and then, in accordance with the composition of the mixture, make one part of each component in one container and mix with the required amount of solvent (respectively, with 97 parts for hundred percent dilution I am). An activated carrier is prepared from a matrix solution diluted in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ or 100 ²⁰⁰ , which is equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C50 or C200.

If a solid substance poorly soluble in a neutral carrier is used as the starting material, the activated carrier associated with the modifying activity is prepared in the form of powder grinding (trituration), and the starting material is triturated to obtain a powder, one part of which is triturated over an hour from 99 parts of a solid neutral carrier (for example, lactose or isomalt) to obtain 1 hundredth grinding. Then one part of the resulting mixture is similarly triturated with 99 parts of lactose, etc. until a predetermined multiplicity is obtained, or, for example, after trituration of the third hundredth grinding (equivalent to C3) is obtained, and dissolved in 79 parts of a neutral aqueous or aqueous-alcoholic solvent (mainly 70% ethyl alcohol) and repeatedly (10 times or more) vertically shake, potentiate, getting the 4th hundredth dilution of C4. Further dilutions are made according to the above homeopathic technology.

Example 1

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material - a matrix solution of rabbit antibodies to human gamma interferon (AT to IFN-γ) in the process of multiple sequential decrease in the concentration of the latter with multiple intermediate shaking and using the original carrier in the form of water-alcohol solution, by dilution in 100 ¹² , in 100 ³⁰ and in 100 ⁵⁰ , which is equivalent to a mixture of hundreds of homeopathic dilutions C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material of rabbit antibodies to human gamma interferon (AT to IFN-γ)) was determined by high performance liquid chromatography (HPLC), and the amount of the substance was measured as a characteristic parameter.

List of abbreviations:

AT - antibodies

IFN gamma - interferon gamma,

HPLC - high performance liquid chromatography,

AN - activated carrier,

FSB - phosphate-buffered saline,

AFSB - phosphate-buffered saline activated by homeopathic technology,

IgG is a human class G immunoglobulin including IFN gamma-interferon gamma.

And water - activated by homeopathic technology water

The determination of the amount of the substance was carried out by estimating the peak area by HPLC. A mixture of IgG + AN was selected as the test sample. The following mixtures were taken as control samples: IgG + A water, IgG + water, IgG + APSB.

Test samples were prepared by mixing IgG (50 mg / ml) and AN (or A water, or APSB, or water) with a ratio of 1: 2 by volume. The resulting mixtures were filtered using cellulose acetate filters with a pore size of 0.45 μm.

The analysis was carried out on a system of high performance liquid chromatographic separation in a gradient mode. An anion exchange column was used as the stationary phase, and a mixture of two phases was used as the mobile phase (1 phase — acetonitrile, 2 phase — solutions of potassium hydrogen phosphate and potassium chloride). Detection was performed using a UV-Vis detector; the scanning wavelength was 280 nm.

Calibration and zeroing of the baseline of the UV-Vis detector was carried out before and after each analysis.

The prepared mixtures were placed in vials and a chromatographic system was introduced using an autosampler. The analysis time for each test sample was approximately 23 minutes.

At the end of the analysis, the chromatographic column was balanced under the initial gradient conditions of the mobile phase at a constant flow rate.

The signal from the test samples was recorded in the form of a chromatogram of peaks corresponding, presumably, to the light and heavy chains of IgG. The spectrophotometric peak areas of the first maximum (Max-1 - corresponds to the heavy chains of immunoglobulins) and the second maximum (Max-2 - correspond to the light chains of immunoglobulins) were calculated. The results of the study are presented in table 1.

Table 1 The peak areas of the chromatographic peak No. Test sample The peak areas of the chromatographic peak The modifying activity of a substance in EA units at C = 100 Max-1 Max-2 Max-1 Max-2 one IgG + AN 17207.9 ± 434.7 45860.6 ± 9427.3 2 IgG + water 30270.2 ± 980.6 5577.4 ± 467.5 43,2 722.3 3 IgG + A water 28704.0 ± 4265.3 5626 ± 686.6 40.1 715.2 four IgG + AFSB 25888.7 ± 1514D 7135.7 ± 746.0 33.5 542.7

It was experimentally shown that AN to IFN gamma reduce the peak area of the IgG of the first maximum (Max-1 - corresponds to the heavy chains of immunoglobulins) and increase the peak area of the IgG peak of the second maximum (Max-2 - corresponds to the light chains of immunoglobulins) compared to the controls.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that said activated carrier is prepared on the basis of the starting material IFN gamma-interferon gamma.

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of the manifestation of modifying activity associated with the carrier, and makes it possible to express the specified modified activity determined using the method (HPLC) in dimensionless units of activity according to the formula (1 ) (see Table 1).

Example 2

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material — a matrix solution of rabbit antibodies to human gamma interferon (AT to IFN-γ) in the course of multiple successive decrease in the concentration of the latter with multiple intermediate shaking and using the original in the carrier in the form of water-alcohol solution, by dilutions in 100 ¹² , in 100 ³⁰ and in 100 ⁵⁰ , which is equivalent to a mixture of hundreds of homeopathic dilutions C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material of rabbit antibodies to human gamma-interferon (AT to IFN-γ)) was determined by high performance liquid chromatography (HPLC), and the amount of the substance was measured as a characteristic parameter.

List of abbreviations:

AT - antibodies

IFN gamma - interferon gamma

HPLC - High Performance Liquid Chromatography

AN - activated carrier

And water is water activated by homeopathic technology.

The determination of the amount of the substance was carried out by estimating the peak area by HPLC. A mixture of AT to IFN gamma + AN was chosen as the test sample. The following mixtures were taken as control samples: AT to IFN gamma + A water, AT to IFN gamma + water.

Test samples were prepared by mixing AT to IFN gamma and AN (or A water or water) with a ratio of 1: 2 by volume. The resulting mixtures were mixed on Vortex for 15 s, incubated at room temperature for 18 hours, and then AN (or A water or water) was added.

The analysis was carried out on a system of high performance liquid chromatographic separation in a gradient mode. A reverse phase octadecylsilane column was used as a stationary phase, a mixture of two phases was used as the mobile phase (phase 1 was acetonitrile with methyl and trifluoroacetic acids, phase 2 was deionized water with methyl and trifluoroacetic acids). Detection was performed using a UV-Vis detector; the scanning wavelength was 280 nm.

Calibration and zeroing of the baseline of the UV-Vis detector was carried out before and after each analysis.

The prepared mixtures were placed in vials and a chromatographic system was introduced using an autosampler. The analysis time for each test sample was approximately 15 minutes.

At the end of the analysis, the chromatographic column was balanced under the initial gradient conditions of the mobile phase at a constant flow rate.

The signal from the test samples was recorded as a peak chromatogram corresponding to a protein. The area of the spectrophotometric peak was calculated. The results of the study are presented in table 2.

table 2 The peak areas of the chromatographic peak No. Test sample Chromatographic Peak Area The modifying activity of a substance in EA units at C = 100 one AT to IFN gamma + PAP AT to IFN gamma 113.1 ± 3.6 2 AT to IFN gamma + RAP water 123.2 ± 3.6 8.2 3 AT to IFN gamma + water 128.3 ± 0.3 11.8

It was shown that PAP AT to IFN gamma reduce the peak areas of AT to IFN gamma compared to controls.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that said activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma;

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of the manifestation of modifying activity associated with the carrier, and makes it possible to express the specified modified activity determined using the method (HPLC) in dimensionless units of activity according to the formula (1 ) (see Table 2).

Example 3

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material, diclofenac sodium, in the process of repeatedly sequentially decreasing the concentration of the latter with repeated intermediate shaking and using the starting medium in the form of an aqueous-alcoholic solution by dilution in 10012, 10030 and 100200, which equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C200. The change in the physicochemical and / or biological properties of the starting material of diclofenac was determined by high performance liquid chromatography (HPLC), and the amount of the substance was measured as a characteristic parameter.

List of abbreviations

HPLC - High Performance Liquid Chromatography

AN - activated carrier

FSB - phosphate buffered saline

AFSB - phosphate-buffered saline activated by homeopathic technology

Quantitative determination of diclofenac was carried out by estimating the peak area by HPLC. A mixture of diclofenac + lactose saturated with AN was chosen as the test sample. The following mixtures were taken as control samples: diclofenac + lactose, saturated APSB, diclofenac + lactose, unsaturated.

The test samples were presented in the form of diclofenac sodium powder, unsaturated lactose and saturated AN (or APSB) lactose. The powders were dissolved in distilled water (the ratio of the weight of the sample of diclofenac to the weight of the sample of lactose was 1: 3, the volume of water for dissolution was the same). The prepared solutions were mixed with a solution of diclofenac sodium 1: 3 by volume. The solutions were mixed by vertically shaking the bottles manually for 15 seconds. Mixtures of solutions were diluted with double distillate to a final diclofenac concentration of 0.3 μg / ml. The solutions were mixed by hand for 15 seconds, shaking flasks vertically. The resulting mixtures were incubated in the dark at room temperature for 18 hours.

The analysis was carried out on a system of high performance liquid chromatographic separation in a gradient mode. An octadecyl modified silica gel column was used as the stationary phase, a mixture of two phases was used as the mobile phase (phase 1 was distilled water with formic and trifluoroacetic acid additives, phase 2 was acetonitrile with formic and trifluoroacetic acid additives). Detection was performed using a diode array detector; the scanning wavelength was 276 nm.

Zeroing the baseline of the UV-Vis detector was carried out before each analysis.

The prepared mixtures were placed in vials and a chromatographic system was introduced using an autosampler. The analysis time for each test sample was approximately 15 minutes.

At the end of the analysis, the chromatographic column was balanced under the initial gradient conditions of the mobile phase at a constant flow rate.

The signal from the test samples was recorded as a chromatogram of the peak corresponding to diclofenac. The area of the spectrophotometric peak was calculated. The results of the study are presented in table 3 (detection at 276 nm).

Table 3 The peak areas of the chromatographic peak No. Test sample Chromatographic Peak Area The modifying activity of a substance in EA units at C = 100 one Diclofenac + Lactose Saturated with AN 66039.3 ± 549.1 2 Diclofenac + lactose, saturated APSB 42652.0 ± 484.2 54.8 3 Diclofenac + Lactose Unsaturated 32004.3 ± 1113.7 106.3

It was shown that the peak area of diclofenac mixed with AN exceeds the peak area of diclofenac mixed with controls: unsaturated lactose and lactose saturated with APSB.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that the specified activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of the manifestation of modifying activity associated with the carrier and provides the possibility of expressing the specified modified activity, determined using the HPLC method, in dimensionless units of activity according to the formula (1) (see . Table 3).

Example 4

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material — a matrix solution of rabbit antibodies to human gamma interferon (AT to IFN-γ) in the course of multiple successive decrease in the concentration of the latter with multiple intermediate shaking and using the original in the carrier in the form of water-alcohol solution by diluting 100 ^12, 100 ³⁰ and 100 ^50, which is equivalent to a mixture of centesimal homeopathic dilutions C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material of rabbit antibodies to human gamma interferon (AT to IFN-γ)) was determined by enzyme-linked immunosorbent assay (ELISA), and the change in the number of antigen-antibody complexes was measured as a characteristic parameter.

List of abbreviations

AT - antibodies

ELISA - enzyme-linked immunosorbent assay,

IFN gamma - interferon gamma,

AN - activated carrier,

OP - optical density,

FSB - phosphate-buffered saline,

AFSB - phosphate-buffered saline activated by homeopathic technology.

The authenticity of preparations based on an activated carrier associated with modifying activity can be confirmed by ELISA by determining the change in the physicochemical properties of the starting material IFN gamma-interferon gamma after exposure to it with the indicated activated carrier, which is expressed in a change in the number of antigen-antibody complexes.

Before analysis, AT for IFN gamma and AN (or AFSB as a placebo) was preincubated for 24 hours at 4 ° C, during which AT for IFN gamma was bound to IFN gamma solution. After incubation, the amount of AT to IFN gamma binding to IFN gamma adsorbed on the solid phase of the ELISA plate and remaining after binding to IFN gamma in solution was determined. The analysis was performed using a tablet on the surface of which IFN gamma antigen was absorbed.

After pre-incubation, the obtained samples were tested in accordance with the standard ELISA scheme. The amount of antigen-antibody complexes formed adsorbed on the solid phase was determined by measuring the optical density of the solutions in the wells of the plate, taking into account the extinction reaction of the chromogen solution, which changes color when the substrate decomposes by the enzyme. The extinction of the solution was determined spectrophotometrically at a wavelength of 490 nm in the single-wave mode. The more antigen-antibody complexes formed on the plate, the less AT to IFN gamma bound to IFN gamma in solution.

The average OD of samples for two similar experiments incubated with AN was 0.603 ± 0.075 (in the case when AN or placebo was immediately mixed with antigen and AT to IFN gamma and incubated for a day) or 0.251 ± 0.027 (if AN or placebo was mixed with AT to IFN gamma, and after 40 minutes of incubation the antigen was reduced and incubated for 24 hours), while for IFN gamma incubated with AFSB, the optical densities were 0.812 ± 0.084 and 0.391 ± 0.023, respectively.

As a result of the experiment, it was found that aqueous solutions of AN reduce the number of antigen-antibody complexes in solution compared to the control, which confirms the authenticity of the drug, prepared on the basis of rabbit antibodies to human gamma-interferon (AT to IFN-γ).

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that said activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma;

3. A change in the physicochemical properties of the initial substance after exposure to it with the indicated activated carrier confirms the presence of the manifestation of modifying activity associated with the carrier, and provides the possibility of expressing the specified modified activity, determined using the method of enzyme-linked immunosorbent assay, in dimensionless units of activity according to the formula (1 ) The severity of modifying activity, calculated (compared with placebo) by the formula (1), was 25.7-35.8 EA.

Example 5

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material — a matrix solution of rabbit antibodies to human gamma-interferon (AT to IFN-γ) in the process of multiple sequential decrease in the concentration of the latter with repeated intermediate shaking and using the original in the carrier as an aqueous alcohol solution by dilution in 100 ¹² , in 100 ³⁰ and in 100 ⁵⁰ , which is equivalent to a mixture of hundreds of homeopathic dilutions C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material of rabbit antibodies to human gamma-interferon (AT to IFN-γ) was determined by the biological method of a piezoelectric quartz immunosensor with a measurement of the change in the binding of antibodies adsorbed on the surface of the immunosensor to the antigen under the action of the above activated carrier .

List of abbreviations

Δf is the change in the oscillation frequency of the piezoelectric crystal,

APTS - gamma-aminopropyltriethoxysilane,

Sr - standard deviation,

AT - antibodies

IFN gamma - interferon gamma,

AN - activated carrier,

FSB - phosphate-buffered saline,

AFSB - phosphate-buffered saline activated by homeopathic technology.

An analytical signal of the piezoelectric crystal immunosensor is a change in the frequency of oscillations of the piezoelectric crystal resonator (Δf) with an increase or decrease in the mass of the bioreceptor coating due to the formation or destruction of the immune complex on its surface. In this study, we studied the effect of the composition of the analyzed samples on the analytical signal of the sensor. A mixture of IFN gamma and AN was selected as the test sample. A mixture of IFN gamma and AFSB was taken as a control sample. Samples were tested as aqueous solutions.

To create an immunosensor, a bio-recognizing receptor layer was formed based on APTS. Using a microsyringe, 0.8 μl of APTS (sequentially dried at 80 ° C in an oven for 20 min), 5 μl of glutaraldehyde (2.5% solution in bidistilled water) were sequentially applied to the surface of the sensor’s gold electrode with a diameter of 8 mm and then 5 μl of a solution of antibodies to IFN gamma (9.6 ng / ml). A new bioreceptor layer was formed for each measurement.

Preliminary sample preparation included mixing 50 μl IFN gamma (30 mg / ml) with 50 μl test sample. Then the sample was heated at 37 ° C for 45 minutes and stirred for 10 minutes using a centrifuge (1000 rpm). Then, 2.5 μl of the resulting solution was applied onto the surface of the sensor with immobilized AT to IFN gamma, kept for 30 minutes, washed with distilled water, dried to constant weight, and the sensor signal was measured in static mode. The change in the binding of antigen and antibodies is fixed by the change in the mass of the interferon molecule.

The sensors used were AT-cut piezoelectric quartz resonators with 8 mm gold electrodes (ZAO Etna, Moscow). The analytical signal was recorded using a computer and a Diskop transducer (Bafika, Moscow).

The results of the experiment are presented in table 4.

Table 4 The influence of the composition of the analyzed samples on the analytical signal of the sensor Sample Composition Δf (M ± SD) The modifying activity of a substance in EA units at C = 100 IFN gamma + AN 89 ± 4 IFN gamma + AFSB 123 ± 5 2764.2

As a result of the experiment, it was found that aqueous solutions of AN have an effect on the oscillation frequency of the piezoelectric crystal, causing it to decrease compared to controls.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that said activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma;

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of the manifestation of modifying activity associated with the carrier and makes it possible to express the specified modified activity in dimensionless units of activity according to formula (1), which, when measured using the piezoelectric method the immunosensor was 2764.2 EA.

Example 6

The study of changes in the neutralizing activity of antibodies to interferon gamma under the action of release-active antibodies to interferon gamma (Anaferon children), the method is the measurement of neutralizing activity.

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material — a matrix solution of rabbit antibodies to human gamma-interferon (AT to IFN-γ) in the process of multiple sequential decrease in the concentration of the latter with repeated intermediate shaking and using the original carrier in the form of water alcohol solution by diluting 100 ^12, 100 ³⁰ and 100 ^50, which is equivalent to a mixture of centesimal homeopathic dilutions C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material of rabbit antibodies to human gamma interferon (AT to IFN-γ) was determined by changing the neutralizing activity of antibodies to gamma interferon under the action of the indicated activated carrier associated with modifying activity, and was determined as a characteristic parameter by the number of surviving cells after infection with the virus.

List of abbreviations

R (X) - RPMI-1640 culture medium containing X% fetal calf serum,

AT - antibodies

IFN gamma - interferon gamma,

NOT - neutralizing activity,

AN - activated carrier,

CPE - cytopathic effect,

FSB - phosphate-buffered saline.

The neutralizing activity (HE / ml) of antibody-based preparations is based on the inhibition of the binding of IFN-gamma to its receptor expressed on the cell membrane.

The neutralizing activity of samples of rabbit polyclonal antibodies to IFN gamma was determined in the culture medium [R (1)] in the presence of AN or a control sample (distilled water).

The grown lung fibroblast cells of the HEP-2 human embryo were trypsinized, suspended in 10 ml of R (10) and applied onto a plate at a concentration of 2.3 × 105 cells / well (100 μl / well). Cells were incubated 24 hours at 37 ° C in a humid chamber containing 7% CO ₂ . IFN gamma was stepwise diluted in R (l), starting from 500 pg / ml to 3.9 pg / ml, and placed in wells. Rabbit polyclonal antibodies to IFN gamma (sequentially 2 times diluted in R (l), starting with 1000 times dilution) were mixed with IFN gamma at a fixed concentration (250 pg / ml) and AN (10% (v / v) in R (l)) or distilled water (10% (v / v) in R (l)). The plates were incubated for 1 h at 37 ° C.

After incubation, the wells were empty and the vesicular stomatitis virus in R (l) was added in an amount of 100 μl / well. After that, the cells were incubated until the CPE in the control line reaches> 90% for 20-24 hours at 37 ° C.

After removing the culture medium, the remaining cells were washed with PBS (200 μl / well) and treated with crystalline violet in formalin (50 μl / well) for 15 min at RT. 100% staining of the monolayer occurred if all cells were alive, but if the cells were dead (CPE), staining of the monolayer was not observed.

The tablet was washed with water. Dilutions of wells that showed 50% CPE were determined visually and then checked spectrophotometrically.

The effect is estimated by the number of surviving cells (the proportion of surviving cells of the total number of cells). Assessment of the effect of AN is carried out to reduce the neutralizing activity of AT to IFN gamma. The neutralizing activity of the preparations was determined by the formula F × A × 10 / C, in which F is the reverse dilution of antibodies, A is EE / ml of the standard preparation at 50% CPE, and C is the concentration of antibodies in mg / ml.

The experiment established:

- upon incubation of human gamma IFN (500 pg per 1 ml of culture medium) with the cell line of lung fibroblasts of the human embryo HEP-2 (2.3⋅105 cells / well) infected with the vesicular stomatitis virus (1.6⋅105 PFU / ml) , complete prevention of the cytopathic effect of the virus was observed (100% of infected cells survived);

- upon incubation of IFN human gamma (500 pg per 1 ml of culture medium) and rabbit polyclonal AT to IFN gamma (0.525 μg per 1 ml of culture medium) with the cell line of lung fibroblasts of the human embryo HEP-2 (2.3 × 105 cells / well ) infected with the virus of vesicular stomatitis (1.6-105 PFU / ml), 50% prevention of the cytopathic effect of the virus was observed (50% of infected cells survived);

- upon incubation of human gamma IFN (500 pg per 1 ml of culture medium) and rabbit polyclonal AT to IFN gamma (0.525 μg per 1 ml of culture medium), as well as AN (10% (v / v) culture medium) with a fibroblast cell line human lung embryo HEP-2 (2.3 × 105 cells / well) infected with the vesicular stomatitis virus (1.6 × 105 PFU / ml), a 75% prevention of the cytopathic effect of the virus was observed (75% of infected cells survived).

It was revealed that AN has a modulating effect on the neutralizing activity of polyclonal antibodies.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that said activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma;

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of manifestation of modifying activity associated with the carrier and makes it possible to express the specified modified activity in dimensionless units of activity according to formula (1), which, when measured using the piezoelectric method the immunosensor was 50 EA.

Example 7

To determine the severity of the modifying activity, we used an activated carrier prepared from the starting material - a matrix solution of rabbit antibodies to human gamma interferon (AT to IFN-γ) in the process of multiple sequential decrease in the concentration of the latter with multiple intermediate shaking and using the original carrier in the form of water-alcohol solution, by dilution in 100 ¹² , in 100 ³⁰ and in 100 ⁵⁰ , which is equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50. The change in the physicochemical and / or biological properties of the starting material - rabbit antibodies to human gamma-interferon (AT to IFN-γ) was determined by spectroscopy and nuclear magnetic resonance (NMR), and as a characteristic parameter on the molecular structure of the substance.

List of abbreviations

AT - antibodies

IFN gamma - interferon gamma

NMR - nuclear magnetic resonance

AN is an activated carrier.

The change in the conformation of antibodies to interferon gamma (AT to IFN gamma) under the influence of AN was determined by NMR spectroscopy. Homeopathic dilutions of purified water were used as a placebo.

To prepare the tested samples, AN or placebo was mixed with a solution of AT to IFN gamma in a ratio of 2: 1, while the final concentration of AT to IFN gamma in each sample was 0.8 mg / ml.

The experiments were performed on a Brucker Avance 700 NMR spectrometer with an operating frequency of 700 MHz. Test samples were placed in the magnetic field of the device. In this case, magnetically active hydrogen isotopes 1H were excited. The signal from the test samples was recorded in the form of NMR spectra (signal accumulation over 12 hours) characterizing the structure and conformational state of AT to IFN gamma.

The conformational state of AT to IFN gamma was evaluated as part of a comparative analysis of the spectra obtained from a sample containing AT to IFN gamma + AN or AT to IFN gamma + placebo. The comparison was carried out by superposing the spectra on top of each other after appropriate scaling.

As a result of the study, it was shown that when AN is added to AT to IFN gamma, there are changes in the AT spectrum for IFN gamma in the ranges of 8-8.5 ppm, 7.6-7.8 ppm, 6-6.6 ppm in comparison with the AT spectrum for IFN gamma + placebo ( figure 1).

As a result of the experiment, it was found that ANs affect the conformation of AT to IFN gamma in solution.

Thus, we can state that:

1. a priori activated carrier due to the technology used in the prepared homeopathic dilutions C12, C30, C50 does not contain molecules of the starting substance;

2. The change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier reliably confirms that the specified activated carrier is prepared on the basis of the starting material IFN gamma - interferon gamma

3. A change in the physicochemical properties of the starting material after being exposed to it by the indicated activated carrier confirms the presence of a manifestation of modifying activity associated with the carrier.

Claims (9)

1. A method for determining the severity of modifying activity associated with a carrier acquired in the process of processing a starting material in the form of a multiple successive decrease in the concentration of the starting material using the carrier, which is manifested in the ability to directly change the physicochemical and / or biological properties of the starting material when exposed to it the specified carrier, characterized in that analytical methods determine the presence or absence of molecules of the original of the substance in the specified carrier and in the presence of molecules of the starting substance, the molecules of the starting substance are removed from the carrier before measuring the characteristic parameter, and one or more characteristic parameters of the starting substance are measured by the analytical method before and after the specified carrier interacts with it, while the degree of expression of the modifying activity is determined by the magnitude of the change in the characteristic parameter in relative units of activity, calculated by the formula (1):

where X is the number of units of activity (EA); C is the dimensionless coefficient of proportionality; A is the value of the characteristic parameter of the starting substance before the interaction of the specified activated carrier; Am - the value of the same characteristic parameter of the starting substance after the interaction of the specified activated carrier. 2. The method according to p. 1, characterized in that a liquid solvent is used as a carrier. 3. The method according to p. 1, characterized in that a solid carrier is used as a carrier. 4. The method according to p. 1, characterized in that it further register by analytical methods the influence on the physicochemical properties of the starting material of an inactive carrier and / or other substance that is processed by repeated serial dilution or trituration with intermediate shaking of each dilution using homeopathic technology, and / or starting material.

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