Determination of biological activity of gonadotrophins in inbred and outbred animals. Part 1: Determination of biological activity of follicle-stimulating hormone

Scientific relevance. The biological activity of medicinal products may vary depending on the method of production (i.e. biological or recombinant products). The widening variety of gonadotrophin preparations, the diversity of their production methods, and the irreplaceability of biological activity bioassays with physicochemical tests require improvement of animal testing conditions.Aim. This study aimed to determine the biological activity of follicle-stimulating hormone (FSH) in several rat lines, analyse the findings, and select the most optimal testing conditions.Materials and methods. The biological activity was determined using in vivo methods. The comparative analysis used test results obtained over several years in inbred and outbred rats treated with FSH. In all cases, the authors used a three-dose randomised method based on the determination of the biological activity of test samples by comparison with that of the WHO international standard (IS) containing 183 IU of FSH bioactivity and 177 IU of LH bioactivity per ampoule (NIBSC code: 10/286). The study included immature female rats, inbred (Wistar-Kyoto or Sprague Dawley) and outbred. Testing conditions depended on the selected rat line, with the main variables being the test dose and the number of animals per group.Results. The authors compared responses of inbred and outbred rats to various doses of the FSH IS and test samples. Given the narrow range of the analytical procedure, Wistar-Kyoto rats showed a relatively weak dose–response relationship. The study demonstrated that the doses and testing duration depended on the sensitivity of the animals. Test results were less variable in inbred rats than in outbred ones. The statistical analysis of the results of FSH bioactivity testing in inbred and outbred rats showed that, with inbred rats, the number of animals could be halved without compromising the validity of the test.Conclusions. The approach proposed in this study provides for testing the biological activity of FSH with fewer experimental animals, improved cost-effectiveness, and the same reliability of results

Determination of the Biological Activity of Insulin and Its Analogues in Animals

In recent years, there has been a tendency to equate the results of insulin testing by physicochemical methods with its biological activity. However, it should be emphasised that the effectiveness of insulin preparations can be judged only by the reaction of the whole organism, i.e. by the hypoglycaemic action, which is a composite indicator. The determination of the biological activity of insulin has become especially relevant with the pharmaceutical market entry of insulin biosimilars, which are practically not analogous to the original medicinal products. The effectiveness of insulin products cannot be adequately evaluated using only physicochemical testing. At present, the biological activity of insulin is tested in rabbits. Additionally, the State Pharmacopoeia of the Russian Federation includes an alternative method to determine this parameter using mice. Owing to the physiological characteristics of mice, an adequate test would require special attention to the selection of test concentrations of insulin. The aim of the study was to offer recommendations for choosing the range of insulin concentrations for determining its biological activity in mice with due consideration of changes in their sensitivity depending on the season. Materials and methods: the authors analysed the results of testing the biological activity of insulin in female mice, bearing in mind the analytical range of the method specified in the State Pharmacopoeia of the Russian Federation. Test concentrations of insulin were selected taking into account seasonal shifts in insulin sensitivity of the animals. Results: the study demonstrated significant dose dependence and linearity of the responses of female mice to insulin in the concentration range of 0.03–0.3 IU/mL. Therefore, this range can be recommended as a guideline for determining the biological activity of insulin in female mice. The selection of test concentrations is illustrated with specific examples. Conclusions: the authors offer a methodological approach to the selection of insulin concentrations and the assessment of the validity of test results during biological activity determination using twin-crossover tests in female mice

Современные подходы к определению биологической активности инсулина и его аналогов

The paper considers insulin’s specific action on the patient’s body, types of insulin preparations and insulin analogues which are used for the treatment of diabetes, as well as applicable requirements for these products. It was demonstrated that determination of biological activity is one of the key quality parameters of this type of medicines. The paper summarises the methods used for evaluation of insulin and its analogues, which are based both on the hormone’s general action on the body (in vivo: double crossing, euglycemic clamp, etc.), and on certain aspects of the hormone’s interaction with the body systems (in vitro: receptor-binding assay, phosphorylation, metabolic methods). Due to the appearance of insulin biosimilars on the pharmaceutical market, the article raises the issue that the «Biological potency» parameter tested in animals should be kept as part of the product specification. The analysis of the in vivo and in vitro methods of biological activity determination convincingly demonstrates that animal models can not be replaced with the modern analytical methods based on cell cultures. Consequently, animal models are still necessary, as they allow for an adequate assessment of the quality of insulins in terms of «Biological potency». Taking into account the global trend towards reduction of animal testing, the authors point out the need to develop modern methods, the results of which will be comparable to the results of in vivo determination of the biological activity.Рассмотрены особенности действия инсулина на организм пациента, виды препаратов инсулина и его аналогов, применяемые для лечения сахарного диабета, и предъявляемые к ним требования. Показано, что определение биологической активности является одним из главных показателей качества данной группы препаратов. Приведено краткое описание методов анализа инсулина и его аналогов, основанных как на действии гормона на организм в целом (in vivo: двойной перекрест, эугликемический клэмп), так и на отдельных этапах его взаимодействия с системами организма (in vitro: рецепторно-связывающий анализ, фосфорилирование, метаболические методы). В связи с появлением на фармацевтическом рынке биоаналогов инсулина, в статье поднимается вопрос о необходимости сохранения в нормативных документах показателя «Биологическая активность», выполняемого на животных. Проведенный анализ методов определения биологической активности in vivo и in vitro убедительно показывает невозможность замены моделей с использованием животных на существующие в настоящее время методы анализа, выполняемые на клеточных культурах. Следовательно, модели на животных по-прежнему необходимы, так как только они позволяют дать адекватную оценку качества инсулинов по показателю «Биологическая активность». Принимая во внимание мировые тенденции к сокращению использования животных в испытаниях, авторы указывают на необходимость разработки методов, результаты которых будут сопоставимы с результатами определения биологической активности in vivo

Методические подходы к определению депрессорных веществ в лекарственных средствах

In order to harmonise national and foreign requirements for quality control of medicinal products, a second variant of the test method, which complies with the requirements of the European Pharmacopoeia, was included into the general monograph OFS.1.2.4.0008.18 “Test for depressor substances”. The aim of the study was to compare the two variants of the in vivo test method that uses cats for determination of depressor substances in medicinal products and to develop recommendations for the use of these two variants of the test method. The comparative analysis of the two ways of determining impurities that lower blood pressure revealed different approaches to performing the test and to testing an animal's sensitivity to the reference solution (histamine dihydrochloride). It was demonstrated that different concentrations, volumes, and doses of medicinal products are used to assess the results of testing performed by variants 1 and 2 of the test method according to the pharmacopoeial requirements. Based on the established differences in the methodological approach to determination of depressor substances in medicinal products, the authors developed recommendations for each of the test variants. The inclusion of two variants of the test method in the general monograph provides an opportunity to evaluate the quality of medicinal products under the most appropriate conditions and, consequently, to improve the validity of test results.Для гармонизации отечественных и зарубежных требований к оценке качества лекарственных средств в ОФС.1.2.4.0008.18 «Испытание на депрессорные вещества» включен второй вариант метода проведения испытания, который соответствует требованиям Европейской фармакопеи. Цель работы — сравнительный анализ двух вариантов метода определения депрессорных веществ в лекарственных средствах в условиях in vivo на кошках и подготовка рекомендаций по применению каждого из этих вариантов. В результате проведенного сравнительного анализа двух вариантов определения в лекарственных средствах примесей, снижающих артериальное давление, показаны различные подходы к проведению испытаний, проверке чувствительности животного к раствору сравнения (гистамина дигидрохлориду). Показано, что в соответствии с фармакопейными требованиями в вариантах 1 и 2 результаты оцениваются с использованием разных концентраций, а также объемов и доз лекарственных средств. На основании установленных различий методического подхода к определению депрессорных веществ в лекарственных средствах разработаны рекомендации для использования каждого из рассмотренных вариантов проведения испытания. Благодаря включению в общую фармакопейную статью двух вариантов метода появляется возможность оценивать качество лекарственных средств в более подходящих для каждого конкретного препарата условиях и тем самым повышать достоверность результатов

Определение биологической активности инсулина и его аналогов на животных

In recent years, there has been a tendency to equate the results of insulin testing by physicochemical methods with its biological activity. However, it should be emphasised that the effectiveness of insulin preparations can be judged only by the reaction of the whole organism, i.e. by the hypoglycaemic action, which is a composite indicator. The determination of the biological activity of insulin has become especially relevant with the pharmaceutical market entry of insulin biosimilars, which are practically not analogous to the original medicinal products. The effectiveness of insulin products cannot be adequately evaluated using only physicochemical testing. At present, the biological activity of insulin is tested in rabbits. Additionally, the State Pharmacopoeia of the Russian Federation includes an alternative method to determine this parameter using mice. Owing to the physiological characteristics of mice, an adequate test would require special attention to the selection of test concentrations of insulin. The aim of the study was to offer recommendations for choosing the range of insulin concentrations for determining its biological activity in mice with due consideration of changes in their sensitivity depending on the season. Materials and methods: the authors analysed the results of testing the biological activity of insulin in female mice, bearing in mind the analytical range of the method specified in the State Pharmacopoeia of the Russian Federation. Test concentrations of insulin were selected taking into account seasonal shifts in insulin sensitivity of the animals. Results: the study demonstrated significant dose dependence and linearity of the responses of female mice to insulin in the concentration range of 0.03–0.3 IU/mL. Therefore, this range can be recommended as a guideline for determining the biological activity of insulin in female mice. The selection of test concentrations is illustrated with specific examples. Conclusions: the authors offer a methodological approach to the selection of insulin concentrations and the assessment of the validity of test results during biological activity determination using twin-crossover tests in female mice.В последние годы появилась тенденция приравнивать результаты испытаний инсулина физико-химическими методами к его биологической активности. Однако следует подчеркнуть, что об эффективности препаратов инсулина можно судить только по реакции целостного организма, т.е. по гипогликемическому действию, которое является интегральным показателем. Определение биологической активности инсулина особенно актуально в связи с появлением на фармацевтическом рынке биосимиляров, которые практически не являются аналогами оригинальных препаратов. Испытаний только физико-химическими методами для оценки эффективности препаратов инсулина недостаточно. В настоящее время биологическую активность определяют путем испытаний на кроликах. При этом согласно требованиям Государственной фармакопеи Российской Федерации (ГФ РФ) для определения данного показателя также предложена альтернативная методика испытаний на мышах. В связи с физиологическими особенностями мышей для проведения адекватного испытания особое внимание следует уделять подбору исследуемых концентраций инсулина. Цель работы: предложить рекомендации по выбору диапазона концентраций инсулина для определения его биологической активности на мышах с учетом изменения чувствительности животных в зависимости от сезона. Материалы и методы: проведен анализ результатов испытаний биологической активности инсулина на мышах-самках с учетом аналитической области методики, указанной в ГФ РФ. При подборе концентраций вводимых проб учитывали чувствительность животных в конкретный временной период. Результаты: продемонстрировано, что реакция мышей-самок на инсулин в диапазоне концентраций 0,03–0,3 МЕ/мл характеризуется статистически значимой дозозависимостью и линейностью, что позволяет рекомендовать данный диапазон в качестве ориентировочного для определения биологической активности инсулина на мышах-самках. Приведены конкретные примеры, иллюстрирующие подбор концентраций испытуемых растворов. Выводы: предложен методический подход к выбору концентраций инсулина и оценке правильности результатов испытания при определении биологической активности двойным перекрестом на мышах-самках

Этапы стандартизации препаратов эритропоэтинов

Preparations of recombinant human erythropoietin (rhEPO) are included in the list of vital and essential drugs for medical use. When evaluating the quality of EPO preparations during the manufacturing process, under the state marketing authorization and certification procedures, in order to confirm their quality in terms of assay (specific activity), identification, dimer and high-molecular related substances content, sialic acids, it is required to use erythropoietin reference standard. The present article describes various stages of the development of erythropoietin reference standards. It provides the comparative description of the existing methods for evaluating the quality of erythropoietin preparations using reference standards. The necessity of the development and validation of national erythropoietin reference standard is justified.Препараты рекомбинантных эритропоэтинов человека (рчЭПО) входят в перечень жизненно необходимых и важнейших лекарственных препаратов для медицинского применения. При оценке качества препаратов ЭПО в процессе производства, при государственной регистрации и сертификации для подтверждения их качества по таким показателям, как количественное определение (специфическая активность), подлинность, димеры и высокомолекулярные родственные вещества, сиаловые кислоты, необходимы стандартные образцы эритропоэтина. В работе приведены сведения об этапах разработки различных стандартных образцов эритропоэтина. Дано сравнительное описание существующих методов оценки качества препаратов эритропоэтина с использованием стандартных образцов. Показана необходимость разработки и аттестации отечественного стандартного образца эритропоэтина

Изучение принципов стандартизации фармакологической активности препаратов рекомбинантных эритропоэтинов

Analysis of the publications devoted to the structure, functions, mechanism of action of erythropoietin is given in the article. Erythropoietin preparations derived from recombinant DNA technology are a mixture of isoforms with different biological activity, which determine the biological properties pharmacological activity, pharmacokinetics, efficacy and safety of medicinal product. Erythropoietin preparations derived by using recombinant DNA technology are a mixture of isoforms with different biological activity, that determine the biological properties, pharmacological activity, pharmacokinetics, safety and therapeutic efficacy of the drug. However, at production of erythropoietin, its pharmacological activity is controlled only by the ability to stimulate erythropoiesis, despite the multiplicity of different directions of action of drugs erythropoietin. The drug is dispensed and applied on this indicator. The international reference standard, a European reference biologic drug or calibrated by him enterprise standard samples are used by manufacturers to assess the quality of erythropoietin in the Russian Federation. The urgency of developing domestic standard samples for the evaluation of biological activity and physico-chemical characteristics of erythropoietin preparations produced by recombinant DNA technology.В статье проведен анализ публикаций, посвященных структуре, функциям, механизму действия эритро-поэтина. Препараты эритропоэтина на основе технологии рекомбинантной ДНК представляют собой смесь изоформ с различной биологической активностью, которые определяют биологические свойства, фармакологическую активность, фармакокинетику, терапевтическую эффективность и безопасность лекарственного средства. Несмотря на множественность и разнонаправленность действия лекарственных препаратов эритропоэтина, при их выпуске фармакологическая активность контролируется только по способности стимулировать эритропоэз, по которой и дозируется, и применяется препарат. Для оценки качества эритропоэтина в РФ производители применяют международный стандартный образец, европейский биологический референс-препарат или откалиброванные по ним стандартные образцы предприятия. Показана актуальность разработки отечественных стандартных образцов для определения биологической активности и оценки физико-химических показателей препаратов эритропоэтина на основе технологии рекомбинантной ДНК

Methodological Approaches to Determination of Depressor Substances

In order to harmonise national and foreign requirements for quality control of medicinal products, a second variant of the test method, which complies with the requirements of the European Pharmacopoeia, was included into the general monograph OFS.1.2.4.0008.18 “Test for depressor substances”. The aim of the study was to compare the two variants of the in vivo test method that uses cats for determination of depressor substances in medicinal products and to develop recommendations for the use of these two variants of the test method. The comparative analysis of the two ways of determining impurities that lower blood pressure revealed different approaches to performing the test and to testing an animal's sensitivity to the reference solution (histamine dihydrochloride). It was demonstrated that different concentrations, volumes, and doses of medicinal products are used to assess the results of testing performed by variants 1 and 2 of the test method according to the pharmacopoeial requirements. Based on the established differences in the methodological approach to determination of depressor substances in medicinal products, the authors developed recommendations for each of the test variants. The inclusion of two variants of the test method in the general monograph provides an opportunity to evaluate the quality of medicinal products under the most appropriate conditions and, consequently, to improve the validity of test results

Calculation of the Median Lethal Dose and Low Lethal Dose Using the CombiStats Biometric Software

The median lethal dose (LD50) and low lethal dose (LD10) are calculated in acute toxicity studies, as well as during specific activity assessment of some medicines. The aim of the study was to develop a procedure for using CombiStats to calculate LD50 and LD10. The authors proposed a step-by-step algorithm for processing bioassay results using the CombiStats biometric software (median effective dose determination model, probit analysis) with conversion of doses to simple fractions (fractions of the maximum dose) to calculate LD50 and LD10. They compared LD50 and LD10 calculation results obtained using CombiStats with those obtained using electronic spreadsheets according to the Bliss–Miller–Tainter–Prozorovsky method described in the State Pharmacopoeia of the Russian Federation (General Monograph 1.1.0014.15). It has been demonstrated that the use of CombiStats sometimes has advantages over the use of the pharmacoepoeial method

Modern Approaches to Determination of the Biological Activity of Insulin and its

The paper considers insulin’s specific action on the patient’s body, types of insulin preparations and insulin analogues which are used for the treatment of diabetes, as well as applicable requirements for these products. It was demonstrated that determination of biological activity is one of the key quality parameters of this type of medicines. The paper summarises the methods used for evaluation of insulin and its analogues, which are based both on the hormone’s general action on the body (in vivo: double crossing, euglycemic clamp, etc.), and on certain aspects of the hormone’s interaction with the body systems (in vitro: receptor-binding assay, phosphorylation, metabolic methods). Due to the appearance of insulin biosimilars on the pharmaceutical market, the article raises the issue that the «Biological potency» parameter tested in animals should be kept as part of the product specification. The analysis of the in vivo and in vitro methods of biological activity determination convincingly demonstrates that animal models can not be replaced with the modern analytical methods based on cell cultures. Consequently, animal models are still necessary, as they allow for an adequate assessment of the quality of insulins in terms of «Biological potency». Taking into account the global trend towards reduction of animal testing, the authors point out the need to develop modern methods, the results of which will be comparable to the results of in vivo determination of the biological activity