RESEARCH AND DEVELOPMENT OF NEW DRUG PRODUCTS
Introduction. In recent years, there has been a steady increase in gastroenterological diseases in many countries due to adverse environmental conditions and unsustainable nutrition. Diseases of digestive organs are not only chronic, but also combined, and varieties of synthetic drugs are widely used in therapy. The range of official multicomponent plant collections recommended in the therapy of diseases of the digestive system is limited, and their disadvantage is especially felt in the treatment of chronic hepatitis, gastric ulcer, cholecystitis.
Aim. Search and creation of new multicomponent agents for prevention and complex treatment of digestive system diseases.
Materials and methods. The objects of the study were developed multi-component means: «Pentafit», «Octafit», «Hexafit». To assess the pharmacotherapeutic effect of the developed agents «Pentafit» hepatoprotective activity studied in experimental models tetrachloromethane and D-galactosamine hepatitis, «Octafit» gastroprotective activity studied in models of acute aspirins, neurogenic and chronic butadione ulcer, сholeretic activity of «Hexafit» as studied in models of experimental cholecystitis and toxic liver damage using laboratory animals. The results of the studies processed statistically using the Statistica 10 program.
Results and discussion. Based on the obtained data, the general patterns in molecular biological action of the studied multicomponent agents revealed, which consist in their ability to inhibit the free radical oxidation of lipids and stabilize the biological membranes of gastric cells and organs of the hepatobiliary system. «Pentafit» profitably distinguished by its multilateral nature of influence on the functional state and structure of the organs of the liver. «Octafit» provides accelerated healing and restoration of damaged structures of functional systems of the stomach. «Hexafit» secreted by the polyvalent spectrum of action in case of damage to the hepatobiliary system.
Conclusion. Developed multicomponent drugs will expand the register of medicines of plant origin of domestic production for the prevention and treatment of digestive diseases.
PHARMACEUTICAL TECHNOLOGY
Introduction. An original N-glycoside derivative of indolocarbazole LHS-1269 with a carbohydrate xylose residue was synthesized at the N.N. Blokhin Russian Cancer Research Center Ministry of Health of Russia. It is characterized by high antitumor activity against a number of transferable ascitic and solid tumors in vivo experiments. Due to the hydrophobic properties of the substance LHS-1269, a method of solubilization by encapsulation in phospholipid vesicles - liposomes is proposed for creating an injectable dosage form.
Aim. Development of the composition of a model of a liposomal dosage form for injection of an indolocarbazole derivative LHS-1269.
Materials and methods. Substance LHS-1269 (N.N. Blokhin Russian Cancer Research Center Ministry of Health of Russia), egg phosphatidylcholine (PC, E PC S, Lipoid, Germany), cholesterol >99 % (Sigma-Aldrich, Japan), polyethylene glycol-2000-distearoylphosphatidylethanolamine (PEG-PE, Lipoid, Germany). To prepare phospholipid vesicles, the method of lipid film hydration with subsequent filtration and extrusion was used. The obtained liposomes were analyzed using the method of spectrophotometry, laser scattering spectroscopy, the method of determining the electrophoretic mobility of particles, and viscometry.
Results and discussion. On the basis of lipid components and active substance in various molar ratios, compositions were made and experimental models of liposomal dispersion were obtained. The compositions were evaluated based on the effectiveness of LHS-1269 incorporation into liposomes and physical and chemical parameters - dispersion viscosity, vesicle size distribution, and zeta-potential. As a result of the analysis of 7 studied compositions, the optimal molar ratios of the drug form components - LHS-1269/PC 1:160 and PC/cholesterol/PEG-PE 1:0.33:0.003 were determined. The use of this composition allows us to obtain relatively stable (zeta-potential -33 mV) homogeneous liposomes with a diameter of 190 nm with a maximum level of encapsulation of the active substance of 98 %.
Conclusions. As a result of technological and chemical-pharmaceutical research, the composition of a model of a liposomal dosage form for injection of the indolocarbazole derivative LHS-1269 has been developed.
Introduction. Saturated polymer microspheres represent one of the leading developments in the field of chemoembolization. Due to the complicated multi-stage production in the Russian market, mainly foreign manufacturers of these products are represented, which may indicate the relevance of studying this topic.
Aim. The aim of the work was to develop a technological scheme for the production of polymer microspheres and a description of critical control points of production.
Materials and methods. The study of the intermediate product was carried out using a Levenhuk D320L microscope (Levenhuk Inc, USA). Analysis of production control points was carried out by the HACCP (Hazard analysis and critical control points) method.
Results and discussion. As a result of the study, the main technological stages of the production of microspheres, which are a copolymer of vinyl alcohol and sodium acrylate, have been identified and described. For each technological stage, control critical points are determined with an indication of the controlled parameters and control methods. The photographs of the intermediate product at intermediate stages are shown.
Conclusion. A technological scheme for the production of microspheres has been developed. The analysis of critical control points of the stages of the main production process is carried out. Identified irreversible critical points of production.
Aim. Study of interpolymer complexes based on a pair of Eudragit® EPO and Eudragit® S100 copolymers obtained in a medium of various organic solvents (ethyl alcohol and a mixture of isopropanol-acetone (in a ratio of 60:40, v/v) from the point of view of their use in oral matrix systems for the delivery of active pharmaceutical ingredients.
Materials and methods. The synthesis of interpolymer complexes based on Eudragit® copolymers was carried out in organic solvents [ethyl alcohol or isopropanol-acetone mixture (in a ratio of 60:40, v/v)]. The structural features of the obtained IPCs were assessed by FTIR spectroscopy using a Nicolet iS5 instrument with a Smart iTR single ATR attachment (Thermo Scientific, USA) and differential scanning calorimetry with modulated temperature (mDSC) using a Discovery™ DSC instrument (TA Instruments, USA). The composition was determined using the method of elemental analysis on a Thermo Flash 2000 CHNS/O device (Thermo Fisher Scientific, UK). The swelling capacity of the obtained IPCs was evaluated in buffer media with pH values that mimic the pH of different parts of the gastrointestinal tract (GIT): 1.2; 5.8; 6.8 and 7.4. The release of theophylline (TEO) and diclofenac sodium (DS) was assessed on a ERWEKA DFZ II (ERWEKA GmbH, Germany) flow through cell apparatus in buffer media simulating the pH of various parts of the gastrointestinal tract for 7 hours.
Results and discussion. All obtained IPCs are characterized by an excess of Eudragit® S100 in their composition. The obtained IPCs are stabilized by a cooperative system of ionic and hydrogen bonds. DSC thermograms of IPC samples are characterized by the presence of a single glass transition temperature, which confirms the absence of impurities of free copolymers in their composition, while with an increase in the content of Eudragit® S100 in the IPC composition, the glass transition temperature increases. The matrix based on IPC-1 disintegrated during the first 15 minutes of the experiment in a medium with pH 1.2, while matrices based on the rest of the IPC samples retain their shape for 7 hours and are characterized by pH-independent behavior. The release of TEO from polycomplex matrices is diffusion-controlled and occurs in accordance with Fick's law (IPC-2, IPC-4, and IPC-6) or can be characterized as «abnormal transport» (IPC-1, IPC-3 and IPC- 5), and also depends on the swelling capacity of the matrix. In contrast, the release of DS is dependent on surface erosion of the matrix, which is consistent with the Super Case II transport mechanism.
Conclusion. The resulting interpolymer complexes Eudragit® EPO/Eudragit® S100 are promising carriers in matrix oral systems for the controlled delivery of active pharmaceutical ingredients, providing a prolonged release of BCS class I substances, depending on the diffusion rate from the swollen matrix, and a delayed release of BCS class II substances, controlled by surface processes erosion of the matrix.
Introduction. The development of polymer carriers for micro- and nanoscale drug delivery systems is an emerging area of modern pharmaceutical technology. One of the urgent needs in this area is the development of effective methods to study the drug release from these systems.
Aim. This work aimed to study the release of a model drug (MD) haloperidol from polycomplex nanoparticles prepared based on interpolyelectrolyte complexes (IPEC) using various methods.
Materials and methods. IPECs were prepared in the form of nanoparticles based on pharmaceutical polymers (Eudragit® EPO and Eudragit® L100-55). Size distribution of these nanoparticles were determined using dynamic light scattering on Zetasizer Nano-ZS equipment (Malvern Instruments, UK). The release of haloperidol was studied in a medium simulating an artificial nasal fluid using a vertical Franz diffusion cell (PermeGear, USA) as well as a modified USP IV method in a flow-through cell apparatus (DFZ II, Erweka, Germany).
Results and discussion. Statistically significant increase in the release of haloperidol from polycomplex nanoparticles in contrast with the control (haloperidol solution) is observed when using a vertical diffusion or Franz cell, after almost 2.5 hours. At the same time, it was not possible to study the release of drug using the flow-through cell method (USP IV), due, apparently, to the effect of crystallization of haloperidol on the surface of dialysis membranes in the Float-A-Lyzer® G2 nanoadapters. The attempts to eliminate this effect and to improve the membrane permeability to haloperidol by adding surfactants (tween-80) and penetration enhancers (DMSO) were not successful.
Conclusion. Both methods are promising for studying the release of drugs from nanosized carriers; however, in the case of using poorly-soluble drugs, including haloperidol, the diffusion method using a vertical Franz cell is effective.
Introduction. Quality, along with efficacy and safety, are the key characteristics of a drug. Therefore, it is important in frame of pharmaceutical development to lay the foundation for obtaining a quality product and achieving the desired product characteristics. One of the tools used for this is the «Quality by design» approach (QbD) - quality through development. The ICH Q8 «Pharmaceutical development» manual defines it as a systematic approach to development that starts with pre-defined goals and focuses on understanding the product and process, as well as managing the process based on reliable scientific data and quality risk management.
Aim. The aim of the research is to develop the composition of tablets based on clover meadow grass phytosubstance using the QbD tool with application of mathematical model that links the composition of tablets (CMA) with its quality attributes (CQA).
Materials and methods. The research was based on the concept of quality by design/quality through development. The main method for conducting the development process was design of experiments method/experiment planning with creating of individual design. The experiment planning was performed in the software package JMP Pro 14 (ver. 14. 3. 0), SAS Institute Inc., USA. The method of risk analysis-failure mode and effects analysis (FMEA)/analysis of the types and consequences of failures was used for risk assessment in research. As methods of analysis of the tablet mixture and the tablets were used the following the tests: disintegration of tablets, crushing strength of tablets, determination of hygroscopicity, determination of Carr's and Hausner index.
Results and discussion. The composition of tablets based on the clover meadow grass phytosubstance obtained by direct pressing was developed. In order to develop the composition of tablets in accordance with the ICH Q8 guidelines, the first step was to create a quality target product profile. To ensure the properties specified in the quality target product profile, the component composition of the developed tablets was selected based on the selected production technology and key characteristics of both the tablet mixture and the finished product. The risk assessment for the product composition determined that the quantitative content of 4 excipients are considered as critical quality attributes of materials (CMA). CMAs affect the critical quality attributes (CQA) of the dosage form, which determine the effectiveness of the composition.
Based on the initial data analysis, optimal content boundaries for each component were established. Potentially critical qualitative characteristics of the drug under development (CQA) were identified. A mathematical model linking critical quality parameters and tablet composition was obtained and analyzed.
Conclusion. Based on a mathematical model, the optimal composition of tablet mixture and tablets obtained from it was determined. The tablets quality indicators corresponding to the selected composition were calculated, and the adequacy of the obtained model was evaluated by comparing calculated and real indicators. It is shown that the calculation error does not exceed 10 %, and the proposed optimization algorithm and the model derived from it can be successfully used to optimize the composition of tablets.
ANALYTICAL METHODS
Introduction. Recently, much attention has been paid in the literature to the primary assessment of the pharmacological effect of various drugs using in vivo and in vitro tests. Great interest in determining the antioxidant activity (АА) of drugs, including herbal remedies, medicinal plant materials and their biologically active substances (BAS). It is known that such an officinal medicinal plant, like dioica nettle, is rich in natural antioxidants (АО) in its phytochemical composition: flavonoids, carotenoids, ascorbic acid, etc. In some publications, there is information about the АА of nettle leaves and preparations based on it. However, information on the comparative characteristics of the use of various methods for determining the АА of this type of medicinal plant material and the results obtained are not found in the scientific literature.
Aim. The aim of this work was a comparative determination of the АА of medicinal plant raw materials of nettle dioica by various methods.
Materials and methods. The object of the study was the finished crushed raw material of dioica nettle leaves (Folia Urticae) in filter bags produced by a domestic manufacturer. AA of water and water-alcohol extracts was determined titrimetrically according to the method developed by T. V. Maksimova with co-authors; the ability to inhibit the autooxidation of adrenaline in vitro. The Paramecium caudatum cell culture was also used as a biological model to determine the АА of aqueous extracts from the studied raw material on a living cell. The determination of the qualitative and quantitative composition of phenolic AO was determined by HPLC-DMD-MS.
Results and discussion. The total АА of water and water-alcohol extracts from dioica nettle leaves was determined using various techniques recommended in the literature. The effect of the extractant polarity on the value of АА was investigated and an inversely proportional relationship was revealed. It was found that the highest content of АО in the extraction is observed when using 96% ethanol as an extractant. Evaluation of the АА of the test object by the biological method was carried out in accordance with the values of the index of biological activity. HPLC-DMD-MS analysis of water-methanol extraction from nettle leaves showed the presence of 17 substances - representatives of the group of phenolic compounds.
Conclusion. Using four independent methods, the prospects of using dioica nettle leaves and preparations based on it as a source of AO are shown. The data obtained undoubtedly open up new possibilities for the use of a long-known plant and confirm the feasibility and prospects of its use for obtaining new dosage forms.
Introduction. Mycotoxins (MT) are secondary metabolites of microscopic mold fungi with pronounced toxic properties.
Text. Currently, the development of express methods for determining the content of MT with mutagenic, teratogenic and carcinogenic properties is of particular importance in the food and pharmaceutical industries. The creation of methods for their analytical control continues to be the focus of attention of the largest international organizations (IUPAC, AOAC International and IFJU), specialized national organizations of the EU and the USA, as well as academies and ministries of many countries of the world. By the Decree of the President of the Russian Federation (RF), the Doctrine of Food Security of the RF № 120 dated January 30, 2010 was approved. The Ministry of Agriculture of the RF issued an order «On the implementation of the state veterinary laboratory monitoring plan» № 780 dated May 30, 2003. In our country, MT studies, according to the plan Monitoring and Recommendations of the EU Commission are limited to determining the content of six tested MTs - the sum of aflatoxins (AFL), ochratoxins (OT), zearalenone (ZEA), trichothecenes (TT), patulin (P), T-2 toxin in agricultural raw materials, food products and feed. MTs are natural pollutants not only of grains of cereals, legumes, sunflower seeds, as well as vegetables and fruits, but also of medicinal plant materials (MPM), as well as pharmaceutical substances of plant origin, in particular, such as fatty vegetable oils widely used in pharmacy in as independent drugs, as well as auxiliary components for the production of drugs, dietary supplements, solvents for injection drugs, etc. In the RF, the MT content in MPM is not yet standardized. It is almost impossible to prevent contamination of the MPM MT, therefore, strict control of these substances is necessary. However, this problem and the need to introduce such a study into the list of indicators for evaluating the quality of MPM requires an in-depth study and an assessment of the degree of its infection. In addition, standard methods for determining normalized MTs are usually not designed to detect transformed forms, and their content remains unaccounted for. Therefore, MT metabolites modified by a medicinal plant are called masked (conjugated, transformed) MT plants, the content of which also needs to be evaluated. MTs can also form during storage of MPM under the action of microscopic fungi developing in them. Since the basis for obtaining herbal medicines are medicinal plants, which are raw materials that are most contaminated with various microorganisms, viruses, and molds, not only MPM, but also almost all substances and herbal medicines (HM) can be infected. It has been shown that MTs remain in dried MPM for more than 100 years. It was found that a lot of MPM densely seeded with Deuteromycetes class molds, which can be detected by microscopic analysis. The aim of this work was to systematize the methods for determining the main MT based on the analysis of domestic literature for their possible further use in assessing the quality of MPM and HM based on it.
Conclusion. MTs adversely affect the body of animals and humans and are difficult to decontaminate. MTs may be present in MPM without visible mold growth. MT does not lose toxicity for many years. It is not possible to completely eliminate the formation of MT in raw materials, therefore, the task of controlling its quality of services is to identify the presence of MT and compare the detected amount with the norms of maximum content. The content of MT in MPM is currently not determined in the RF by the RF GF XIV ed. However, by the decision of the Council of the Eurasian Economic Commission dated January 26, 2018 №15, the Rules of Good Practice for the cultivation, collection, processing and storage of raw materials of plant origin were approved, which stipulate the inadmissibility of mold formation during the primary processing, storage and transportation of cattle. Therefore, the accumulation of experimental material, as well as the development of pharmacopoeial methods for the analysis of MPM, constantly tightening quality requirements, can lead to the inclusion of this indicator in pharmacopoeial articles (PA). In this connection, it was interesting to conduct a review of existing methods of MT analysis, with their possible subsequent inclusion in the GPA and PA. The traditional methods of MT analysis are physicochemical methods. The most widely used chromatographic methods: HPLC, GLC using various detectors (mass spectrometric, fluorescence, amperometric, etc.). TLC occupies a special place among the separation methods. These methods, of course, are able to provide sufficient specificity and the necessary sensitivity of the determination, however, they are expensive, designed primarily for specialized and well-equipped stationary laboratories, and also require highly qualified personnel. None of them can also be attributed to express methods. The complexity of determining MT in MPM is associated with long and laborious sample preparation, as well as with the use of expensive highly sensitive methods (HPLC, GLC), which cannot be applied directly at the place of production and processing of MPM. The most promising are immunochemical test methods for determining MT, based on the immunochemical reaction between the antibody and antigen. It was shown that the high specificity and the possibility of detecting MT in low concentrations in combination with the existing diverse instrumentation make it possible to consider immunochemical methods of analysis as the most promising for wide practical use.
Introduction. The quality control of a medicinal product needs to be systematically improved in connection with a continuous increase in the requirements for the quality of medicines. When assessing the quality of substances and drugs, the most important indicators are authenticity, the content of the main substance and impurities. This makes it relevant to introduce modern physical and chemical methods of analysis into industrial practice in determining parameters such as authenticity, the content of the main substance and impurities. The choice and applicability of a particular method is possible only when conducting comparative studies on the application of a particular method to solve this problem. The method should preferably be recommended by the Pharmacopoeia, give unambiguous results, and the analysis should not be expensive and affordable. The methods chosen for comparative analysis are well known, but it should be noted that in recent years, instrument-making companies have significantly improved their instrument base and expanded their capabilities. Therefore, one of the objectives of this article is to demonstrate the capabilities of the selected methods for solving the problems of drug quality control using dalargin as an example.
Aim. To develop methods for determining the authenticity of the active substance in a substance - Dalargin powder using NMR spectroscopy, IR spectrometry, and Mass spectroscopy. Conduct a comparative analysis of the applicability of the selected methods to solve the problem.
Materials and methods. To determine the authenticity of the substance - the powder «Dalargin» used the methods of mass spectrometry, 13C NMR and 1H NMR spectrometry and IR spectrometry.
Results and discussions. Methods have been developed for determining the authenticity of the test substance by NMR, IR, and mass spectrometry. A comparative study of the possibilities of the selected methods in solving the problem. Conclusions were drawn about the applicability of the NMR method not only in determining the authenticity of dalargin, but also in solving the problem of determining the content of the main substance and impurities.
Conclusion. Authenticity methods have been developed in the substance - Dalargin powder by Mass, IR, and NMR spectrometry. It has been established that 1H NMR spectrometry is a more informative method for authenticating.
Introduction. Creating a new herbal remedy with a mild generalized effect is an urgent task of modern pharmacology and pharmaceutical technology. The dosage form of an innovative medicinal product should take into account its properties, ensure the effectiveness of the drug and ease of intake.
Aim. Creation of hard gelatin capsules with a plant composition-dry extract and validation of methods for quantifying the amount of flavonoids for the preparation of draft regulatory documentation.
Materials and methods. The physicochemical and technological properties of the developed dry extract, as well as experimental capsule masses, such as mass loss during drying, bulk volume and granulometric composition, flowability, angle of natural slope, compaction, and parameters of the obtained capsules (solubility, disintegration, etc.) were studied according to the methods described in the State Pharmacopoeia of the XIV edition. Methods were developed for quantitative determination of flavonoid content in terms of rutin in dry extract and capsules using differential spectrophotometry, which were validated.
Results and discussion. During the study of the experimental dry extract, its high hygroscopicity was found, which worsens its technological properties and complicates the encapsulation process. As a result of the addition of auxiliary substances, such as anhydrous lactose and talc, it was possible to obtain an optimal mass for encapsulation, which served as the basis for the creation of the drug «Sedoflav».
Conclusion. On the basis of a dry extract of plant composition capsules «Sedoflavt» were developed, its standardization was produced. Methods for quantifying the amount of flavonoids in terms of rutin in experimental dry extract and capsules have been developed and validated.
Introduction. When developing and validating methods of impurity determination, it is important to test the spectral peak purity (SPP) of the main substances using diode array detectors (DAD).
Text. It is emphasized that the task of testing SPP is to detect invisible coeluting impurities under the peak of the main substance, and not the control the absolute purity of the peak. SPP testing reduces the risk of getting non enough specific methods. In this regard, we considered the main criteria and key points for assessing the spectral purity of peaks when using computer programs of Agilent's, Shimadzu's, and Waters's chromatographs.
Conclusion. Recommendations are given, which should be taken into account for the correct assessment of spectral purity of peaks during development of chromatographic methods and validation of their specificity.
Introduction. Seeds of Nigella sativa L. are widely known as a source of fatty oil with a very rare component-thymoquinone. The therapeutic potential of biologically active compounds of plant seeds covers a positive effect on the gastrointestinal tract, cardiovascular and immune systems. In addition, hypoglycemic, antioxidant, antitumor, oncoprotective, immunomodulating effects were found in thymoquinone. In addition to fatty oil and thymoquinone, a number of other significant compounds are present in the seeds, which also make up their own pharmacological asset. Aim. Study was to develop a processing flow chart and analyze the lipophilic and hydrophilic components of the seeds of N. sativa L.
Materials and methods. The following methods were used as analytical methods: gas-liquid chromatography with a flame ionization detector - for the analysis of sterols and triterpenes; chromatography-mass spectrometry (gas-liquid chromatography with mass spectrometric detection) - for the study of essential oil; chromatography-mass spectrometry (high performance liquid chromatography with mass spectrometric detection) - to study flavonoids. To obtain individual fractions of biologically active compounds of seeds of N. sativa L., fractional extraction was used.
Results and discussion. The principle of this approach in this case is to extract the entire lipid complex of seeds with n-hexane, followed by removal of the extractant. The thickened hexane extract is treated with ethyl alcohol, which extracts unsaponifiable compounds - terpenes, quinones, sterols and does not dissolve triacylglycerides. The seed meal remaining after hexane extraction is treated with ethyl alcohol 70 %, into which hydrophilic molecules, in particular flavonoids, pass.
Conclusion. After transesterification, 5 compounds were identified in the lipophilic fraction of saponifiable lipids, the linoleic and oleic acids being dominant. A characteristic feature of this fraction is the presence of cis-11,14-eicosadiene acid, which can act as a marker element of the fatty oil of the seeds of N. sativa L. Sterols and triterpenes were found in the unsaponifiable fraction. The major component of this fraction is β-sitosterol. In addition to the indicated sterol, campesterol and stigmasteri are present. Triterpenes are represented by cycloartenol and its derivatives. Simple phenols, quinones and monoterpenes were found in the essential oil complex, p-cymol, thymoquinone and α-thuyen predominate. The hydrophilic fraction of the seeds of N. sativa L. includes flavonoids of the methoxylated series of flavones and kempferol glycosides.
Introduction. Vitex agnus-castus is an important plant in medicine. Extract from its fruits is used as part of drugs for the treatment of premenstrual syndrome.
Aim. To study the chemical composition of water-ethanol extract from Vitex fruits, to establish the dopaminergic activity of various extract fractions separated by lipophilicity, as that of its dominant iridoid-agnuside
Materials and methods. The composition of water-ethanol extraction was studied using HPLC-UV and HPLC-MS/MS methods. Identification of the extract components was performed by comparing the retention times on the chromatograms of the test and standard samples. The extract and its fractions were analyzed in vitro using the tyrosine hydroxylase biotest system. Cyclodinone® (oral drops, Vitex agni casti fructuum extract) was used as a reference drug.
Results and discussion. Protocatechuic acid, chlorogenic acid, p-hydroxybenzoic acid, agnuside, mizodendron, 6'-O-p-hydroxybenzoylmusaenoside acid, luteolin-7-glucoside, 5-O-caffeoylquinic acid, vitexin, luteolin, apigenin, maslinic acid, corosolic acid and linolenic acid were identified. The liquid extract obtained in the laboratory showed the same activity as Cyclodinone®, ethyl acetate fraction, and agnuside.
Conclusion. This work is the most extensive study of the vitex fruit in relation to the nomenclature of identified compounds. The total extract was shown to be more active than any of its fractions proving the synergistic effect of the extract components on the dopamine neurotransmitter system. This observation justifies the feasibility of using the total extract of the vitex fruit. In vitro agnuside demonstrates high dopaminergic activity. This makes it possible to standardize the extract and medicinal plant materials according to the agnuside content.
Introduction. Modern pharmacognostic research is aimed at searching for plant biologically active individual compounds (RBAIS) isolated from plant extracts.
Aim. Study of the content of the steroid sapogenin diosgenin in fenugreek seeds in plant extracts by HPLC-UV method.
Materials and methods. The object of study was raw materials-fenugreek seeds produced as medicinal plant raw materials by LLC «Sage» (Irkutsk). Series of extraction and chromatographic separation by HPLC-UV method were performed.
Results and discussion. In this work, the method of diosgenin extraction was successfully optimized (after acid hydrolysis of dioscin with a 5 % aqueous solution of hydrochloric acid when heated for at least 4 hours in a sand bath at a power of 150 W) from raw fenugreek seeds using a 50 % aqueous solution of isopropanol, h.h. A simplified technique for chromatographic separation of diosgenin from fenugreek seed extracts using an isocratic mode of 99.9 % acetonitrile elution (h.h.) was proposed.
Conclusion. The average content of diosgenin (after acid hydrolysis of dioscin) in fenugreek seeds, taking into account the humidity of raw materials, is (M ± σ) 5,3 ± 0,05 mg/g (95 % CI: 5,1-5,4 mg/g; n = 6).
PRECLINICAL AND CLINICAL STUDIES
Introduction. The pharmacokinetic profiling of active compounds is necessary for drug development and application. Comlex extract of biologically active compounds was isolated from gonads of green sea urchin Strongylocentrotus droebachiensis from Barents Sea. It contains fatty acids, carotenoids and tocopherols and have inhibition activity to ensyme dypeptydylpeptidase IV (DPP-4). Traditional approaches to pharmacokinetic study based on chromatographic methods are not effective for such complex mixtures because they had not enough selectivity and sensitivity. Methods of immunoassay for special enzyme, bioassay, etc. may be used as an alternative way.
Aim. The aim: to find approach to pharmacokinetic study of sea urchin gonads extract in rabbits after a three doses oral administration.
Materials and methods. Various spectroscopic and chromatographic (HPLC and TLC) methods were used for chemical characteristic of sea urchin gonads extract ant different target analytes quantification in biosamples. The pharmacokinetic of sea urchin gonads extract was studied after single dose oral administration to male rabbits. The correlation between concentration of sea urchin gonads extract in the blood plasma and its biological activity marked by DPP-4 activity was established. The activity of DPP-4 was determined by the chromogenic optical method.
Results and discussion. Main groups of sea urchin gonads extract chemical compounds were characterized. Total peptides content was 15-22 %; a-tocopherol content - 0.05-0.15 %, total tocopherols content - 0.23-0.38 %; total carotenoid content - 0.005-0.07 %; total fatty acids content calculated to linolenic acid - 11,03 to 12,74 %. After sea urchin gonads extract chemical composition results it was established that concentrations of identified individual biologically active compound are low, there is no dominant compound or group of compounds responsible for pharmacological activity of sea urchin gonads extract which may be unambiguously chosen as target analyte for its pharmacokinetic study. Approach based on correlation of special marker activity (DPP-4) and extract concentration appears to be the best for pharmacokinetic study of complex extract from green sea urchin gonads. The method for the quantitative determination of sea urchin gonads extract in the blood plasma of rabbits by its effect on DPP-4 activity was developed. The method was validated by parameters: selectivity, lower limit of quantification, linearity range, accuracy, and precision. The pharmacokinetics was linear in the dose range of 5-25 mg/kg after oral administration. The mean maximal concentration in plasma (Cmax) was dose dependent and it was 37.12-114.71 pg/mL for various doses, area under the curve (AUC0-24) was 192.92-597.14 h • pg/mL, mean time to reach maximum plasma concentration (Tmax) was 3-3.5 h, half-life (T1/2) was 7.88-9.89 h, and the mean retention time (MRT) was 1.73-14.31.
Conclusion. After nontraditional approach based on correlation of special marker activity (DPP-4) and active substance concentration the plasma pharmacokinetics of sea urchin gonads extract after oral administration to the rabbits in three doses was characterized. Similar approaches may be effective for compounds and complex mixtures when it is difficult or impossible to analyze them traditionally by chromatographic (HPLC-UV/FL/MS, GC-MS, etc.) methods.
Introduction. The early detection of tumor growth remains one of the most important tasks of diagnostic nuclear medicine. The vascular growth being created malignant neoplasms is a good target for the purpose. The molecular participants in the process are gallium-68 labeled vector to the delivery of radionuclides and positron-emission tomography (PET) imaging.
Aim. Possibility researching of using the complex gallium-68 labeled peptide sequence RGD for PET imaging of tumor with different grade of neovascularization.
Materials and methods. Complex compound of Ga-68 with the peptide NODAGA-cRGD2 (the proposed name used below is «Vascular, 68Ga») was used for studying of the distribution in mice with transplanted heterotopic xenografts of glioblastoma U-87 MG and breast adenocarcinoma Ca-755 after intravenous injection, as well as the possibility of visualization the tumor.
Results and discussion. The «Vascular, 68Ga» biodistribution in the mice body is usual of laboratory animals is usual of the behavior of peptides in vivo: rapid clearance from the blood, intense urinary excretion. The accumulation of the drug in the area of glioblastoma in mice is on average 2 times higher than that in adenocarcinoma. The results of in vivo PET imaging of experimental tumor lesions correlate well with ex vivo radiometry data.
Conclusion. The «Vascular, 68Ga» actively accumulated in well-vascularized tumor foci after intravenous injection and rapidly excreted from the body through the kidneys without noticeable non-specific accumulation in other organs and tissues. The PET imaging possibility of experimental tumor foci was shown.
REGULATORY ISSUES
Introduction. Qualification of equipment is one of the crucial aspects of quality assurance of medicinal products that ensures consistency of metrological characteristics. Elaboration of local guidances on qualification of measuring instruments, and harmonisation of the qualification procedure with the requirements of the European and American regulations will help to improve the quality of medicinal products manufactured in Russia and to facilitate their competitiveness on the global pharmaceutical market.
Text. The aim of this review is to analyse the Russian regulatory framework regarding the consistency of metrological characteristics of measuring equipment used in the pharmaceutical industry. The article contains information on the special aspects of confirmation of compliance of equipment to the metrological requirements stated in various regulatory documents in force in the Russian Federation.
Conclusion. The article demonstrates the ambiguity of interpretation of the Russian regulatory framework and European quality recommendations regarding metrological requirements to measuring equipment. Elaboration of the local guidance harmonizing metrological requirements to equipment will help to focus on the result and the quality of medicinal products, and to eliminate the ambiguity of interpretation of the Russian regulatory framework.
Introduction. This article reviews main steps of the transfer of analytical procedures following the recommendations of the Russian and foreign regulatory bodies. Each step provides thorough evaluation of an analytical procedure, checks its current validation status and readiness of the receiving laboratory. Each step of the process is crucial for a reliable transfer. The article contains examples of formatting of analytical procedures to be revalidated, characteristics of test samples, reference standards of impurities. The authors described procedures to be transferred depending on the dosage form.
Text. The aim of this article is to review possible types of the transfer, including a transfer waiver, and main components of a transfer protocol following the information from the Russian and foreign literature sources.
Conclusion. Reliability and reproducibility of analytical method data are crucial as they form the basis for a decision-making process. Therefore a procedure should be capable of producing consistent data.
Introduction. This paper is devoted to analysis of a new risk-based regulatory approach to the classification of deficiencies found by inspections of medicines manufacturers, which is undoubtedly relevant.
Text. The article describes the new definitions for the deficiencies found in the medicinal products manufacturing, the proposed gradation of non-conformances distinguishes from the one that is currently used. The proposed innovation in regulatory activity is that a critical deficiency can consist of several related nonconformities, none of which can be critical on their own, but which together can constitute a critical nonconformity or system failure. The proposed approach to the classification of deficiencies, which is based on a risk assessment for the patient, is described. The main stages of the risk-based algorithm are characterized: a detailed assessment of the deficiency for its preliminary classification; assessment of risk increase or decrease factors, regardless of the initial classification; making a decision on the final classification, taking into account the factors of increasing, reducing risk or their absence. Block diagrams and illustrative examples are described, on the basis of which preliminary assignment of a revealed deficiency of one or another level of criticality is carried out. The factors of increasing or decreasing risk are considered. These factors are extrapolated to the category of deficiency and they can both increase the preliminary assigned category or decrease it, and can leave the preliminary classification unchanged.
Conclusion. A new risk-oriented regulatory approach to the classification of non-conformances with the requirements of Good Manufacturing Practice, proposed by the Pharmaceutical Inspection Co-operation Scheme PIC / S, is described. An analysis of this approach and its characterization are made. The new approach is based on the systematic and methodological application of risk management tools, aimed at justifiably assigning a certain criticality category to a revealed deficiency. The implementation of the new approach allows to increase the objectivity and transparency of inspection procedures. Further detailed study and possible step-by-step implementation of this new approach is very important for regulators so for medicines manufacturers.
ISSN 2658-5049 (Online)
