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

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