Method of comparative estimation of physiological activity of pharmaceutical substances and preparations on their basis

FIELD: medicine.

SUBSTANCE: invention refers to pharmacy, namely identification, estimation of quality and safety of original and reproduced medical products. Described comparative estimation of physiological activity of original medical products and reproduced medical products or related preparations implies that by means of EPR spectroscopy and spin probes influence of these products on erythrocyte, lymphocyte and thrombocyte membrane structures released of the same blood sample depending on active component concentration in solution and contact time are examined by comparison of corresponding data obtained within concentration corresponding to maximum therapeutic dose.

EFFECT: method enables comparing of pharmaceutical preparations - originals and generics, or pharmaceutical substances, with various impurity compounds, both within quality assessment and from the point of safety for patients.

3 ex, 3 tbl

 

The invention relates to the field of medicine and relates to the field of pharmacy, namely the identification, evaluation, quality and safety of medicines.

Now the actual problem is a reliable comparison of the properties of the original pharmaceutical substances and preparations on their basis and reproduced copies of generics (generic), which are becoming increasingly popular as a cheaper medicines. Their release is very attractive for companies because it provides the ability to quickly obtain high profits.

Problem generic pharmaceuticals (generics or generics) is complex and controversial. It is relevant for all countries, including the Russian pharmaceutical market. Until recently, most industry experts had no idea of the differences between the original (innovative medicines) and reproduced drugs. The RF Federal law "On medicines" (1998) introduced the definition of the term "generic drug".

Original products manufactured by well-known pharmaceutical companies, are manufactured in accordance with GMP requirements; they have undergone extensive clinical trials. For generic clinical trials of these drugs are very rare and in limited about the EME.

An important role in the safe use of medicines play an excipient. When creating generic drugs it is reasonable to require the preservation of the original excipients. However, it is not always known. The use of AIDS drugs-generic drugs are regulated on the basis of the recommendations of the who [WHO. Drug information, 1998. V.12].

The forms reproduced medicines, exactly the same as the original products, should be applied the same requirements:

Quality

Efficiency

Security

At the same time in the world practice generic drug (generic drugs) in the vast majority of cases are not tested in the clinic. When registering reproduced medicines today are only required to establish bioequivalence compared with the original, which does not reflect therapeutic effect (RUSSIAN BIOMEDICAL JOURNAL, Vol.2, St (SS-216) // October, 2001, DETERMINATION of BIOEQUIVALENCE: a COMPARATIVE APPROACH, Kgurevich, Apostolski).

A generic contains the same active ingredient as the original, therefore it is considered that there is no need for expensive trials on therapeutic efficacy, side effects and possible consequences of its use.

Tracosopoulou therapeutic effectiveness of the original products and the corresponding reproduced funds which is sometimes on a small sample of healthy volunteers, showed that in some cases they can give the original in therapeutic efficacy in 3-4 times, and sometimes more than once. Even with approximately the same therapeutic efficacy of the original and generic in the case of the latter, often there are multiple side effects that are absent in the case of the original. The observed difference can probably be explained by the different composition of impurities present in the original medicinal product and reproduced medicinal product, which includes the difference in the composition of impurities included in pharmaceutical substances.

Although, according to common belief, original and generic are considered identical if the content of the active component in them about the same, in principle it's wrong, because even pharmaceutical substance, obtained by different technologies, in which the content of active substance of about the same, are a different matter due to the fact that they contain a different number of impurities, characterized by qualitative and quantitative composition.

Currently there is no clear system that would protect the market and consumers from poor-quality copies of the original pharmaceutical products.

According to current practice ID is tificate and analysis of physiologically active substances is carried out by comparing the physico-chemical characteristics and activity of the investigated sample and sample - standard. For these purposes use a variety of analysis techniques: spectroscopy, spectrometry, chromatography, electrochemistry, NMR, polarimetry and other

There is a method of identifying a large number of essential drugs and fillers using capillary electrophoresis (Capillary electrophoresis without method development - the use of generic operating methods, Kevin Altria. 2000).

For example, a known method for the identification of waxes by matching their molecular structure in electronic spectra of para-magnetic resonance absorption (SU 920484, publ. 1982.04.15).

There is a method of establishing the identity of pharmaceutical products, cosmetic, perfume, etc. and identify impurities in them, including in trace quantities (RF patent 2249811, publ. 2005.04.10). The method consists in comparing the set of absorption and spectral-luminescent properties of the analyzed sample fluid with the same set of properties obtained for the control sample of known liquid.

There is a method of study of pharmacological activity of drugs by the time the sorption of dye by the organs of animals treated with pharmaceuticals and not receiving it (SU 1221599, publ. 1986.03.30).

There is also known a method for the identification of both compounds and their properties by genomic screening (RF patent 2244751, publ. 2005.01.20). The method of identification pharmaco is oricheskogo agent in the plant extract was carried out by treatment of cells (corresponding biological target - cancer cell, the cell vessel, etc.) of plant extracts, isolation of a protein or RNA of these cells; the identification of those selected protein or RNA, the concentration of which is different from that in untreated cells, detection of compounds (compounds) in the plant extract. Next, process the detected connection (connections) cells secrete a protein or RNA from cells treated with this compound (compounds) and identify (te) connection (connection)that is (are) also causes the stimulation or suppression of expression of a protein or RNA, the concentration of which is different from that in untreated cells.

Given as examples of known methods, as well as most others, allow you to either compare the structure and physico-chemical properties of substances, or physiological activity and most of them do not solve the problem of the safety of medicines.

In determining the physiological activity and the comparative evaluation of the quality of reproduced and original drugs currently almost no development.

As the closest analogue may be specified approved August 10, 2004 by the MINISTRY of HEALTH AND SOCIAL development of the Russian Federation HOWTO "CONDUCTING QUALITATIVE STUDIES IS of BIOEQUIVALENCE STUDIES of DRUGS". According to the recommendations of two medicinal drugs are bioequivalent if they provide the same bioavailability of the drug. Bioequivalence studies suggest that a pharmacokinetic equivalent (biosimilar) to the original generic drug provide the same efficacy and safety of pharmacotherapy, i.e. that they are therapeutic equivalents. As the comparison drug, it is recommended to use the corresponding original drug, registered in the Russian Federation. The content of active substance in the investigational medicinal product and the reference product should not differ by more than 5%. Trials of medicinal products is carried out in an open, randomized and cross-balanced scheme for the volunteers: each subject sequentially receives the study drug (T) and the reference product (R)or Vice versa (scheme RT/TR"). Assessment of the bioavailability of the medicinal product or its main biologically active metabolite (if the study drugs are prodrugs) is based on the comparison of the values of the pharmacokinetic parameters estimated directly according to the concentration (C) - time (t)for the investigated what about the drug and the comparator drug. To determine the concentration of active substances in plasma, serum or whole blood can be used a variety of methods (physical, chemical, immunological, microbiological and other).

However, the current approach to the testing of pharmaceutical products does not take into account external manifestations of a number of vital side effects associated with changes in the structure of the membranes of blood cells, which leads in some cases to serious consequences for patients.

The present invention is to develop a reliable method of analysis that allows to map pharmaceuticals - originals and generics, or pharmaceutical substance, as in quality ratio, and from a position of safety for patients.

The problem is solved developed a rapid method of mapping the physiological activity of original drugs and reproduced medicines and pharmaceutical substances with different composition of impurities.

The proposed method is based on studying the influence of these drugs and substances on the structure of the membranes of blood cells (red cells, platelets and lymphocytes) using EPR spectroscopy and spin probes, namely: using EPR spectroscopy and spin probes to study the impact of these funds on the structure of membranes e is atrocitas, lymphocytes and platelets isolated from the same sample of human blood, depending on the concentration of the active component in the solution and contact time followed by comparison of the corresponding data obtained at a concentration corresponding to the maximum therapeutic dose.

EPR spectroscopy of the spin probes used in the study of structural reorganization of cell membranes under the influence of various substances, including pharmaceutical substances, in order to clarify the possible mechanism of action on the body. As test objects used for biological cell membrane of red blood cells, liver cells, bacteria. Drugs and other substances have different physical-chemical properties and different alter the structural characteristics of cell membranes and, as a consequence, the activity of biological processes in cells, because the cell membrane is most sensitive to changes in internal and external environment.

Various compounds, including pharmaceutically active compounds, can contact the membrane of red blood cells, causing changes in the structure of the membrane (increase or decrease the stiffness of the membrane). These studies were conducted only with cells of erythrocytes for individual chemicals depending on the concentration of substances which in aqueous solution (Luneva, and others, CHANGES in the MEMBRANE STRUCTURE AND SHAPE of ERYTHROCYTES INDUCED by SYNTHETIC ANTIOXIDANTS, II Congress of biophysicists Russia. The abstracts. M, 1999).

Studies comparing the impact on the structure of the membrane pharmaceuticals (originals and generics) was carried out. No research has been done and the effect on the structure of the membranes of blood cells of pharmaceutical substances of different degrees of purity obtained in the same way and in different ways.

Review of literature showed a lack of work on mapping quality of pharmaceuticals, based on the study of the effect of various pharmaceutical preparations containing the same active substance, on the structure of cell membranes 3 of blood cells - red cells, platelets and lymphocytes using EPR spectroscopy of spin probes.

The aim of our research was to study the influence on the structure of the membranes of human blood cells - red cells, platelets and lymphocytes - the original drug and the corresponding reproduced medicines and drugs and their mapping on the basis of obtained using EPR with spin probes data on changing patterns of the membranes of the respective cells. Compared to the penetration depth of the spin labels in membranes corresponding ele the patients blood.

Choosing us as test objects 3 blood corpuscles (erythrocytes), platelets and lymphocytes is based on the fact that these three types of cells in control of the most important processes in the body: breathing, blood clotting and immunity.

In addition, we match the appropriate substances or pharmaceuticals on a multidimensional basis (the change in stiffness of the membrane, the duration of this change in time for the membranes of the studied cells). Differences in pharmaceuticals, due to the presence of unknown impurities in drug substance and the filler used for the manufacture of tabloids compared pharmaceuticals, can manifest itself in different cells in different ways.

Analysis procedure and its features

Obtaining blood fractions to study the effects of substances on the cell membrane of blood cells

Obtaining plasma enriched with platelets

The portion of the blood of a healthy donor with the addition of heparin is subjected to centrifugation at 1800 rpm for 12 minutes After the deposition of the selected layer plasma (it is about half the volume of blood samples). Platelets - not less than 5000 cells/ál. Above the layer of red blood cells leave the plasma layer height 3-4 mm

Obtaining lymphocytes

Is taken over the remaining layer of eritr is Titov layer (3-4 mm) together with the upper part of the layer of red blood cells and diluted with phosphate buffer (pH=7.4) and mixed thoroughly by pipetting.

4 tubes 10-12 ml is added 1 ml of ficoll-urografin (density of 1.024 g/ml at 30°). Tube layer ficoll-urografin neatly on the wall of the tube is overlapped with a portion of the diluted buffer corresponding fractions of blood.

Tubes centrifugeuse at 2000 rpm for 10 minutes Lymphocytes accumulate in the form of a thin white layer or ring above the layer of ficoll-urografin. Their carefully collected with a pipette and washed with phosphate buffer in a centrifuge at 2000 rpm for 10 minutes and Then the liquid above the sediment lymphocytes collected by pipette and discarded. The residue is diluted with buffer and mixed thoroughly by pipetting. The content of lymphocytes is not less than 4×104cells/μl in the absence of platelets.

Obtaining red blood cells

Remaining after extraction of plasma enriched with platelets, and lymphocytes precipitate is washed erythrocytes phosphate buffer or saline solution in a centrifuge at 1700 rpm for 8 minutes If the selection of the supernatant liquid on the layer of red blood cells visible precipitate of other cells, they are selected together with a small layer of red blood cells and discarded. The content of the erythrocytes is not less than 5×103cells/µl.

Procedure EPR analysis

For EPR analysis in the selected aliquots of the cell suspensions add solutions of the studied drug is of rest (or pharmaceutical substances or preparations on their basis, then the samples are incubated for one hour at 37°and mixed with a solution of the spin probe (16-doxylstearic acid). Then carry out incubation at 37°C for 10 minutes Prepared samples collect in glass capillaries, which are placed in the EPR spectrometer and record the corresponding spectra. From the spectra calculated structural parameter of the membrane "S" - rigidity index (degree of order) of the membrane. The concentration of funds or pharmaceuticals in the solution are selected in accordance with the recommended dose or below it, which study the dependence of changes in the structural characteristics of the membranes studied blood cells depending on the drug concentration and time of exposure. The study of this dependence increases the reliability of the mapping physiological properties of the original pharmaceuticals and generics and related substances.

The invention can be illustrated by the following specific examples of implementation.

Examples illustrating the ability of the proposed method

Example 1

Experiments were conducted to identify differences in the effects of original pharmaceuticals and generics (generic drug, where the active substance - indapamide) on the structure of the membranes of blood cells: erythrocytes, lymphocytes and t is ambiatol. Changing the structure of the membrane were fixed using ESR method and 16-doxylstearic acid - EPR probe (nitroxyl derivative of stearic acid).

On the basis of the obtained spectra was calculated main parameter of membrane S by the formula:

,

where S is a measure of the orderliness of the membrane (stiffness), characterizing the mobility of the fatty acid chains of the molecules of the probe;

Andzz, Axx, Ayyconstants introduced in the calculation formulae for the spectral characteristics of nitroxyl EPR probes. They take into account the hyperfine interaction of the unpaired spin with the nucleus of the nitrogen atom in the molecule of the probe, the chemical structure of the probe, its orientation to the applied magnetic field. Get the constant values of the EPR spectra of single crystals of probes, pre-determining the direction of the principal axes of the single crystal x-ray structural analysis method, and consists of a table of such constants. When calculating S use-valued constants AndzzAndxxand Ayy.

for probe DK this value is 27.55;

2Amax- the distance between the first maximum and the last minimum in the spectrum of the first derivative of the absorption signal of the spin probe;

2Amin- the distance from the first minimum to the maximum of the spectrum of the first derivative of the absorption signal of the spin probe.

A measure of the stiffness of the membranes calculated for the control sample corresponding blood cells, to which were added the studied pharmaceuticals, for sample, to which these drugs were added, and the change in the stiffness of the cell membranes (ΔS)associated with the addition of pharmaceuticals, calculated relative to the control sample.

Table 1 shows the changes of the rigidity index of the cell membranes, depending on the concentration of the substance and time of exposure.

Table 1
The change of the rigidity index (ΔS %) of cell membranes relative to control, depending on the concentration of the active substance (indapamide) in solution and time of exposure to the original drug and the generic
MedicationThe concentration of the active substance in solutionErythrocytes, ΔS%Lymphocytes, ΔS% (1 hour)Platelets, ΔS% (1 hour)
Incubation of 1 hourIncubation 24 hours
Original drug5×10-6M-6.0±0.60+6.9±0.7+17.1±1.8
7.5×10-6M-8.4±0.8---
Generic5×10-6M-8.0±0.8-6,8±0.6+16.9±0.7+17.8±1.8
7.5×10-6M+16.8±1.5+16.7±1.5
* the table shows the average data from 5 experiments; a dash indicates the absence of experimental data

As can be seen from Table 1, the effects of original pharmaceuticals and generics at the same concentrations (5×10-6M) for 1 hour change in the stiffness of the membranes of lymphocytes and platelets is almost the same, however, in the case of erythrocytes, it varies approximately in 1.5 times. When increasing the incubation time to 24 hours erythrocyte membranes treated original pharmaceuticals, fully restored, and in the case of a generic change is irreversible.

Probably, this data is associated with the influence of impurities, the composition and content of which in both drugs is different. By increasing the concentration of active substance in 1.5 times in the case of the original drug, the change in the stiffness of the membrane occurred in 1.5 times, and in the case of generic - 2 times. Thus, the resulting Dan is haunted testify, the original pharmaceuticals has significantly less impact on the cell membrane of erythrocytes compared to generic.

Example 2

The influence of the original drug (active ingredient is simvastatin) and his two generics on the structural condition of the membranes of red blood cells when using the EPR and EPR probe as in example 1.

The concentration of the active substance, which conducted mapping the effects of drugs on the cell membrane, consistent with the maximum recommended daily dose of the drug.

Table 2
The change of the rigidity index (ΔS %) of cell membranes relative to control, depending on the time of exposure at the same concentration of the active substance (simvastatin) of the original drug and two generics
MedicationThe concentration of the active substance in solutionErythrocytes, ΔS%Lymphocytes, ΔS% (1 hour)Platelets, ΔS% (1 hour)
Incubation of 1 hourIncubation 24 hours
Original drug 2-01×10-5M+4.8±0.5 0-10.3±1.0+8.4±0.9
Generic-11×10-5M+11.3±1.2-6.5±0.7+6.0±0.6+12.2±1.3
Generic 2-21×10-5M+1.2±0.10-12.2±1.20

The data show that the impact of generic 2-1 increases the stiffness of the membranes of all investigated cells. Original drug acts on the membrane of lymphocytes in the opposite way, razzhizhaya them (ΔS negative) and a much weaker effect on erythrocyte membranes and platelets compared with drug 2-1, and the effects on erythrocyte membrane, in contrast to drug 2-1, reversible within 24 hours. Close to the original drug and to some extent superior to its influence on the structure of the membranes was studied cells is generic 2-2. When its impact on the membrane of the erythrocyte membrane structure changes less than when exposed to the original drug, and at the same time, after 24 hours of incubation is returned, as in the case of the original product, in its original state. The rigidity of the membrane of lymphocytes, as well as the effects of the original drug, decreases (ΔS negative), and the structure of the membrane tro is boltov remains unchanged.

Example 3

The influence of the original drug (active ingredient is amlodipine) and his two generics on the structural condition of the membranes of red blood cells when using the EPR and EPR probe, as in examples 1 and 2.

The concentration of the active substance, which conducted mapping the effects of drugs on the cell membrane, consistent with the maximum recommended daily dose of the drug.

The data obtained are given in Table 3.

Table 3
The change of the rigidity index (ΔS %) of cell membranes relative to control, depending on the time of exposure at the same concentration of the active substance (enalapril) of the original drug and two generics
MedicationThe concentration of the active substance in solutionErythrocytes, ΔS%Lymphocytes, ΔS% (1 hour)Platelets, ΔS% (1 hour)
Incubation of 1 hourIncubation 24 hours
Original drug 3-01×10-5M+7.0±0.7000
Generic 3-11×10-5the +11.7±1.5+7.0±0.7+8.6±0.9+8.4±0.9
Generic 3-21×10-5M+11.3±1.5+6.0±0.6+15.0±1.54.7±0.5

From the Table 3 data shows that the original drug has less than generics, the effect on structural state of erythrocyte membranes, and it is reversible within 24 hours and has no impact on the structural condition of the membranes of platelets and lymphocytes.

At the same time, the rigidity of the membranes of all investigated cells due to the impact of generics increases significantly compared with the impact of the original, and the effect on erythrocyte membrane irreversibly after 24 hours of incubation. Generics 3-1 and 3-2 differ in its effect on the membranes of platelets and lymphocytes.

Thus, the above examples indicate that the proposed method allows a reliable comparison of original pharmaceuticals and generics in their effects on the structural condition of the membranes of erythrocytes, platelets and lymphocytes.

Special studies conducted with inducers of blood clots (adenosine triphosphate (ADP), platelet activating factor (PHAGE), epinephrine, thrombin and other) when studying the x effect on structural state of the membranes of platelets, showed that the impact of all inductors cause a decrease in the stiffness of the membranes (ΔS negative) platelets, and therefore creates conditions that increase the sensitivity of spontaneous platelet aggregation and thrombus formation.

Increasing the stiffness of the membranes of platelets leads to a decrease in blood clotting and increases the likelihood of bleeding.

In the case of lymphocytes increase the rigidity of the membrane leads to a reduction of the protective functions of the body and its immune system.

Our findings not only confirm the possibility of accurate mapping of original drugs and reproduced medicines (generics), but also identify undesirable side effects, dangerous for patients who cannot be identified under normal narrowly focused clinical trials.

Method for comparative evaluation of physiological activity of original drugs and reproduced medicines or drugs based on them, characterized in that by using EPR spectroscopy and spin probes to study the impact of these devices or drugs on the structure of the membranes of erythrocytes, lymphocytes and platelets isolated from the same sample of human blood, depending on the concentration of the active component in the solution and contact time will follow them by comparing the respective data such as the change in stiffness of the membrane, the duration of this change in time for membranes obtained at a concentration corresponding to the maximum therapeutic dose.



 

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