МЕТОДОЛОГИЧЕСКИЙ ПОДХОД К ПЛАНИРОВАНИЮ ЭКСПЕРИМЕНТА ПРИ ВЫБОРЕ КАЧЕСТВЕННЫХ РЕАКЦИЙ ДЛЯ ПОДТВЕРЖДЕНИЯ ПОДЛИННОСТИ КОМПОНЕНТОВ ЛЕКАРСТВЕННОГО СРЕДСТВА (НА ПРИМЕРЕ АСКОРБИНОВОЙ КИСЛОТЫ)

The need for identification  testing of active substances or excipients in multi-component medicinal products,  including the use of qualitative tests, calls for research substantiating the choice of tests and test conditions  with due regard to interference  effects caused by other components  of medicinal products and the amount of the sample used. The aim of the study was to develop a methodological approach to designing experiments while selecting qualitative reactions for identification testing of a medicinal product component based on the results of studies investigating the possibility of using known qualitative tests (as illustrated by ascorbic acid in a multi-component product — 0.4 mg of ascorbic acid per 100 mg of the vial contents)  with due regard to interference on the part of other medicinal product components and the amount of the sample used. Material and methods: the study focused on a multi-component medicinal product — lyophilisate for solution for intravenous and intramuscular  injections containing an antiinflammatory active substance and ascorbic acid as a stabilizing agent (antioxidant). The analysis of literature sources helped to determine qualitative tests that were assessed for potential use for identification testing of ascorbic acid as a component of the analysed medicinal product. The study involved experimental testing of the qualitative reactions based on acidic and reducing properties of ascorbic acid. Results: it was demonstrated that several well-known qualitative tests could be used for identification  testing of ascorbic acid as a component of the analysed medicinal product,  namely, the reaction of ferrous ascorbate formation  and the reaction of silver nitrate reduction to metallic silver after preliminary separation of ascorbic acid from the other medicinal product components, as well as the reaction of Prussian blue formation,  iodine test and reaction with a potassium permanganate solution, which do not require additional sample preparation.  It is not practicable to use the reaction with a methylene blue solution and the Fehling’s reagent reaction for this particular medicinal product,  since their results are feeble. Conclusions: the analysis of the multi-component medicinal product helped to develop a methodological  approach to choosing qualitative reactions for identification testing of one of the medicinal product’s components  (e.g., ascorbic acid). The suggested algorithm includes the choice of reactions, determination of their sensitivity and applicability for a particular medicinal product, analysis of the other components’ effects on the results of the chemical reaction,  and the need for additional sample preparation.  The whole complex of the studies performed helped to determine qualitative reactions and optimal conditions for identification testing of the analysed substance.Необходимость подтверждения подлинности  действующих  или  вспомогательных веществ  многокомпонентных лекарственных средств,  в том числе с использованием качественных  реакций, влечет за собой  необходимость  проведения исследований по выбору реакций  и условий их проведения с учетом мешающего  влияния других компонентов лекарственного средства  и количества  используемого образца.  Цель работы: разработка  методологического подхода к планированию эксперимента при выборе  качественных  реакций  для подтверждения подлинности определяемого компонента лекарственного средства на основании результатов исследований возможности использования известных качественных  реакций  (на примере  аскорбиновой кислоты  в многокомпонентном лекарственном средстве — 0,4 мг аскорбиновой кислоты / 100 мг содержимого  флакона) с учетом мешающего влияния других компонентов лекарственного средства и количества  используемого образца.  Материалы и методы: в качестве  объекта исследования было выбрано  многокомпонентное лекарственное средство  — лиофилизат для приготовления раствора  для внутривенного и внутримышечного введения  с лекарственным веществом,  обладающим  противовоспалительным действием,  в состав которого  входит аскорбиновая кислота  в качестве  стабилизатора (антиоксиданта). В результате анализа  данных литературы  выбраны  качественные реакции  для проведения исследований возможности их использования для подтверждения  подлинности аскорбиновой кислоты  в изучаемом  лекарственном средстве.  Проведена  экспериментальная проверка  реакций, основанных на кислотных  и восстановительных свойствах аскорбиновой кислоты.  Результаты: установлено, что в изучаемом многокомпонентном лекарственном средстве для подтверждения подлинности аскорбиновой кислоты могут быть применимы несколько известных качественных реакций: реакции образования аскорбината железа и восстановления нитрата серебра до металлического серебра после предварительного отделения аскорбиновой кислоты  от других компонентов лекарственного средства,  а также реакция  образования берлинской лазури,  йодная проба и реакция  с раствором  перманганата калия,  не требующие  дополнительной пробоподготовки. Использование реакций  с раствором  метиленового  синего и реактивом  Фелинга  применительно к данному лекарственному средству нецелесообразно, так как результат указанных реакций  слабо выражен. Выводы: на примере многокомпонентного лекарственного средства разработан  методологический подход к выбору качественных  реакций  для подтверждения подлинности одного  из компонентов лекарственного средства  (например, аскорбиновой кислоты).  Алгоритм действий включает в себя выбор реакций, определение их чувствительности и целесообразности применения для конкретного лекарственного средства,  изучение  влияния других его компонентов на результат химической реакции, а также необходимость  или отсутствие дополнительной пробоподготовки. Совокупность проведенных  исследований позволяет сделать выбор качественных  реакций  и оптимальных  условий  их проведения для достижения поставленной цели — подтверждения подлинности определяемого вещества

Unprocessed Viral DNA Could Be the Primary Target of the HIV-1 Integrase Inhibitor Raltegravir

Integration of HIV DNA into host chromosome requires a 3′-processing (3′-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 3′-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 5′C4pA33′ step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance

A METHODOLOGICAL APPROACH TO DESIGNING EXPERIMENTS WHEN DEALING WITH IDENTIFICATION TESTS FOR MEDICINAL PRODUCT COMPONENTS (AS ILLUSTRATED BY ASCORBIC ACID)

The need for identification  testing of active substances or excipients in multi-component medicinal products,  including the use of qualitative tests, calls for research substantiating the choice of tests and test conditions  with due regard to interference  effects caused by other components  of medicinal products and the amount of the sample used. The aim of the study was to develop a methodological approach to designing experiments while selecting qualitative reactions for identification testing of a medicinal product component based on the results of studies investigating the possibility of using known qualitative tests (as illustrated by ascorbic acid in a multi-component product — 0.4 mg of ascorbic acid per 100 mg of the vial contents)  with due regard to interference on the part of other medicinal product components and the amount of the sample used. Material and methods: the study focused on a multi-component medicinal product — lyophilisate for solution for intravenous and intramuscular  injections containing an antiinflammatory active substance and ascorbic acid as a stabilizing agent (antioxidant). The analysis of literature sources helped to determine qualitative tests that were assessed for potential use for identification testing of ascorbic acid as a component of the analysed medicinal product. The study involved experimental testing of the qualitative reactions based on acidic and reducing properties of ascorbic acid. Results: it was demonstrated that several well-known qualitative tests could be used for identification  testing of ascorbic acid as a component of the analysed medicinal product,  namely, the reaction of ferrous ascorbate formation  and the reaction of silver nitrate reduction to metallic silver after preliminary separation of ascorbic acid from the other medicinal product components, as well as the reaction of Prussian blue formation,  iodine test and reaction with a potassium permanganate solution, which do not require additional sample preparation.  It is not practicable to use the reaction with a methylene blue solution and the Fehling’s reagent reaction for this particular medicinal product,  since their results are feeble. Conclusions: the analysis of the multi-component medicinal product helped to develop a methodological  approach to choosing qualitative reactions for identification testing of one of the medicinal product’s components  (e.g., ascorbic acid). The suggested algorithm includes the choice of reactions, determination of their sensitivity and applicability for a particular medicinal product, analysis of the other components’ effects on the results of the chemical reaction,  and the need for additional sample preparation.  The whole complex of the studies performed helped to determine qualitative reactions and optimal conditions for identification testing of the analysed substance

Methodological approaches to the choice of identification test methods for medicines

The article summarises the main selective and non-selective methods of physical, chemical and physico-chemical analysis which are used in medicines identification testing and which differ in selectivity, sensitivity, informative value, sample preparation, and availability. The article demonstrates that the choice of methods is governed by chemical, physical and physico-chemical properties of medicines and the type of medicine (whether it is a substance or a finished dosage form). The article describes a complex approach based on the use of several analytical methods, the cumulative results of which are used to support medicines identification

Major challenges in evaluating the quality of pharmaceutical substances (the assay)

Common topical issues of the quality assessment of pharmaceutical substances in terms of «Assay», are related to the compliance of actual test results to assay requirements as well as to the proper elaboration of normative documents (completeness, accuracy of the descriptions of analytical methods) and the appropriate test conditions validation with the materials in the scope of the mentioned regulatory documents. It was shown that an objective assessment of the quantitative content of an active ingredient in pharmaceutical substance of synthetic or mineral origin depends largely on the choice of the analytical method, use of the appropriate techniques considering the materials included in the monographs of the State Pharmacopoeia the Russian Federation and leading foreign pharmacopoeias

Основные проблемы экспертизы качества фармацевтических субстанций (оценка количественного содержания)

Common topical issues of the quality assessment of pharmaceutical substances in terms of «Assay», are related to the compliance of actual test results to assay requirements as well as to the proper elaboration of normative documents (completeness, accuracy of the descriptions of analytical methods) and the appropriate test conditions validation with the materials in the scope of the mentioned regulatory documents. It was shown that an objective assessment of the quantitative content of an active ingredient in pharmaceutical substance of synthetic or mineral origin depends largely on the choice of the analytical method, use of the appropriate techniques considering the materials included in the monographs of the State Pharmacopoeia the Russian Federation and leading foreign pharmacopoeias.Общие проблемные вопросы, возникающие при экспертизе качества фармацевтических субстанций по показателю «Количественное определение», связаны с соответствием фактически полученных величин предусмотренным нормам количественного содержания, а также уровнем подготовки представленных нормативных документов (полнота, правильность изложения методик анализа) и соответствием условий проведения испытаний с валидационными материалами по данному разделу нормативной документации. Показано, что объективность оценки количественного содержания действующего вещества в фармацевтической субстанции синтетического или минерального происхождения во многом определяется выбором метода анализа, приведением соответствующих методик с учетом материалов, включенных в фармакопейные статьи Государственной фармакопеи Российской Федерации и монографии ведущих зарубежных фармакопей

Методологические подходы к выбору методов установления подлинности лекарственных средств

The article summarises the main selective and non-selective methods of physical, chemical and physico-chemical analysis which are used in medicines identification testing and which differ in selectivity, sensitivity, informative value, sample preparation, and availability. The article demonstrates that the choice of methods is governed by chemical, physical and physico-chemical properties of medicines and the type of medicine (whether it is a substance or a finished dosage form). The article describes a complex approach based on the use of several analytical methods, the cumulative results of which are used to support medicines identification.Представлен обзор основных селективных и неселективных методов физического, химического и физико-химического анализа, используемых при подтверждении подлинности лекарственных средств и различающихся по селективности, чувствительности, информативности, пробоподготовке, доступности. Показано, что выбор методов исследования зависит от химических, физических и физико-химических свойств определяемых веществ, типа лекарственного средства (фармацевтическая субстанция или лекарственная форма). Рассмотрен комплексный подход, основанный на использовании нескольких методов анализа, по совокупности результатов которых подтверждается идентичность лекарственного средства