Generation of mouse lines with conditionally or constitutively inactivated Snca gene and Rosa26-stop-lacZ reporter located in cis on the mouse chromosome 6

α-Synuclein is involved in many important molecular processes in neuronal cells and their synapses, and its malfunction has been linked to the development of Parkinson’s and certain other neurodegenerative diseases. Animal models allowing tightly monitored conditional inactivation of the encoding gene, Snca, are indispensible for studies aimed at understanding normal function of α-synuclein in various neuronal populations and its role in pathogenesis of neurodegenerative diseases. We have recently reported the production of several novel mouse lines for manipulating expression of the endogenous Snca gene, including a line for Cre-recombinase-driven conditional inactivation of the gene (mice with floxed Snca) and a new line with a constitutive knockout of α-synuclein. Rosa26-stop-lacZ reporter cassette is commonly used for monitoring efficiency of Cre-recombination but in mouse genome Snca and Rosa26 loci are located on the same chromosome. Here we describe production of lines with a modified Snca locus, either floxed or constitutively inactivated and the Rosa26-stop-lacZ reporter cassette located in cis on the mouse chromosome 6. These new mouse lines are invaluable for fast identification of cells with inactivation of Snca by Cre-recombination and represent useful tools for in vivo studies of α-synuclein function and dysfunctio

Transcriptomic changes in glomeruli in response to a high salt challenge in the Dahl SS rat

Salt sensitivity impacts a significant portion of the population and is an important contributor to the development of chronic kidneydisease. One of the significant early predictors of salt-induced damage is albuminuria, which reflects the deterioration of the renal filtration barrier: the glomerulus. Despite significant research efforts, there is still a gap in knowledge regarding the molecularme chanisms and signaling networks contributing to instigating and/or perpetuating salt-induced glomerular injury. To address this gap, we used 8-wk-old male Dahl salt-sensitive rats fed a normal-salt diet (0.4% NaCl) or challenged with a high-salt diet (4%NaCl) for 3 wk. At the end of the protocol, a pure fraction of renal glomeruli obtained by differential sieving was used for nextgeneration RNA sequencing and comprehensive semi-automatic transcriptomic data analyses, which revealed 149 differentially expressed genes (107 and 42 genes were down regulated and up regulated, respectively). Furthermore, a combination of predictivegene correlation networks and computational bio informatic analyses revealed pathways impacted by a high salt dietary challenge, including renal metabolism, mitochondrial function, apoptotic signaling and fibrosis, cell cycle, inflammatory and immune responses, circadian clock, cytoskeletal organization, G protein-coupled receptor signaling, and calcium transport. In conclusion, we report here novel transcriptomic interactions and corresponding predicted pathways affecting glomeruli under salt-inducedstress. NEW & NOTEWORTHY Our study demonstrated novel pathways affecting glomeruli under stress induced by dietary salt. Predictive gene correlation networks and bioinformatic semi-automatic analysis revealed changes in the pathways relevant to mitochondrialfunction, inflammatory, apoptotic/fibrotic processes, and cell calcium transport

Specific Aspects and Significance of Subgroup Assessment in Confirmatory Clinical Trials

Patient subgroup analysis plays an important role in interpretation of confirmatory clinical trial results and is mandatory in most cases. The purpose of subgroup analysis is to assess the consistency (heterogeneity) of the treatment effect in subgroups of patients identified based on such characteristics as demographics, stage and severity of the underlying disease, presence of a certain genetic mutation, etc. However, existing methodological issues (the problem of multiple comparisons, detection of differences between subgroups by chance alone, etc.) make it difficult to carry out the analysis and often lead to controversial conclusions. The aim of  the study was to analyse and summarise foreign regulatory approaches to subgroup analysis in confirmatory clinical trials, and to elaborate science-based requirements for subgroup analysis and interpretation of the results by clinical trial sponsors and experts when assessing the risk-benefit ratio of medicinal products for the purpose of their authorisation in Russia and the  Eurasian Economic Union. This paper discusses the objectives of subgroup  analysis and statistical approaches to its performance, provides relevant examples of such analysis from regulatory practice. It describes approaches to interpretation of subgroup analysis depending on the presence/absence of evidence supporting the primary hypothesis of the study, the nature of the experimental medicinal product’s heterogeneous effects in the  subgroups, and selection of the subgroups. The paper highlights areas of concern in subgroup analysis, potential controversies in interpretation of the  obtained results, and regulatory expectations. The recommendations  presented in the paper can be used by experts in the assessment of the risk-benefit ratio, as well as by medicine developers in the preparation of clinical trial protocols and reports

Регуляторные подходы к программе разработки лекарственных препаратов, применяемых для лечения инфекционных заболеваний

In February 2017 the World Health Organization first published the list of antibiotic-resistant «priority pathogens» — a catalogue of 12 species of bacteria that pose the greatest threat to human health. The list highlights the danger posed by Gramnegative bacteria that are resistant to multiple antibiotics. Thus, the development of new antimicrobial medicines is becoming a pressing issue. The list is an important reference point and incentive to secure and guide research and development related to new antibiotics that will help solve the issue of growing global resistance to antimicrobial medicines. The aim of the study was to determine the main regulatory approaches to planning preclinical and clinical development programmes for new antimicrobial medicines. On the basis of current requirements and recommendations in force in the Russian Federation and guidelines of the European Medicines Agency, the issues of planning antimicrobial drug development programs were considered. The autors analysed the main stages and aspects of preclinical studies of medicines for infectious diseases (specific activity in vitro and in vivo, PK-PD modeling), as well as requirements for the clinical trial stage, including the rationale for the choice of clinically relevant efficacy and safety endpoints, study design, and statistical methods.Всемирная организация здравоохранения в феврале 2017 г. впервые опубликовала документ, содержащий перечень устойчивых к действию антибиотиков «приоритетных патогенов» — 12 видов бактерий, которые представляют наибольшую угрозу для здоровья человека. В документе подчеркивается опасность, которую представляют грамотрицательные бактерии, устойчивые к действию сразу нескольких антибиотиков. В связи с этим очевидна актуальность разработки новых антибактериальных препаратов, а данный документ призван стать ориентиром и стимулом для научных исследований и разработок в области создания новых антибиотиков, которые помогут в решении масштабной проблемы устойчивости к противомикробным препаратам. Цель работы — определение основных регуляторных подходов к планированию программы доклинических и клинических исследований новых антимикробных препаратов. На основании актуальных требований и рекомендаций, действующих в Российской Федерации, и руководств Европейского агентства по лекарственным средствам, рассмстрены вопросы планирования программ разработки антибактериальных препаратов. Проанализированы основные этапы и особенности проведения доклинических исследований лекарственных препаратов, применяемых для лечения инфекционных заболеваний (специфическая активность in vitro и in vivo, фармакокинетическое-фармакодинамическое моделирование). Рассмотрены требования, предъявляемые к этапу клинических исследований данной группы препаратов, включая обоснование выбора клинически значимых конечных точек оценки безопасности и эффективности, дизайна исследования, статистического расчета

Kinetics of alpha-synuclein depletion in three brain regions following conditional pan-neuronal inactivation of the encoding gene (Snca) by tamoxifen-induced Cre-recombination in adult mice

Conditional pan-neuronal inactivation of the Snca gene in 2-month old male and female mice causes dramatic decrease in the level of the encoded protein, alpha-synuclein, in three studied brain regions, namely cerebral cortex, midbrain and striatum, 12 weeks after the last injection of tamoxifen. Kinetics of alpha-synuclein depletion is different in these brain regions with a longer lag period in the cerebral cortex where this protein is normally most abundant. Our results suggest that efficient post-developmental pan-neuronal knockout of alpha-synuclein in adult, i.e. 5- to 6-month old, animals, could be achieved by tamoxifen treatment of 2-month old mice carrying loxP-flanked Snca gene and expressing inducible Cre-ERT2 recombinase under control of the promoter of neuron-specific enolase (NSE) gene

Potency evaluation of erythropoietin products: current status

INTRODUCTION. Given the widespread clinical use of recombinant human erythropoietin (rhEPO) products from different manufacturers, potency assays should ensure that patients receive comparable doses of rhEPO across medicinal products. The harmonisation of approaches to potency testing requires the use of pharmacopoeial bioassays and appropriate international/pharmacopoeial reference standards (RSs).AIM. This study aimed to summarise information on pharmacopoeial requirements, relevant RSs, and bioassays (in vivo and in vitro) for the assessment of rhEPO potency, as well as to analyse the pharmacopoeial compliance of manufacturers’ specifications for rhEPO products authorised in Russia.DISCUSSION. This article presents information on the molecular structure of erythropoietin. The glycosylation profile of erythropoietin not only accounts for most of the differences in the half-life and biodegradation rate but also significantly influences the potency of rhEPO products. The authors outlined the pharmacopoeial requirements for potency assays in vivo, summarised the information on RSs for potency determination, characterised the development of potency assays in vitro, and studied the possibility of including in vitro assays in pharmacopoeias. The analysis showed that some potency assays used for rhEPO products manufactured in Russia did not comply with the requirements of the State Pharmacopoeia of the Russian Federation.CONCLUSIONS. The study identified the need to develop and certify a national pharmacopoeial RS for the potency of rhEPO in order to satisfy the demands of Russian manufacturers in the context of import substitution. To implement adequate 3R-compliant methods for rhEPO quality assessment, it is necessary to harmonise approaches to potency testing of rhEPO products and develop a consolidated document governing such testing

International standards for monoclonal antibodies for assessing the biological activity of medicines: A status update

Scientific relevance. The clinical effects and the expiration of patents for original (reference) biotechnological medicines based on monoclonal antibodies (mAbs) stimulated the development of biosimilar mAbs. The quality profile of a biosimilar mAb should correspond to the quality of the reference medicinal product. When demonstrating biosimilarity and determining the activity of medicines as part of batch quality control, analysts should study the biological properties of mAbs using suitable reference standards. The lack of international standards (ISs) makes mAb manufacturers use in-house reference standards. There is a risk of obtaining non-uniform quality and efficacy data because of the use of in-house reference standards, the heterogeneity and structural complexity of mAbs, and the relationship between the biological activity and efficacy of mAbs.Aim. This study aimed to analyse the relevance of and need for ISs for the biological activity of biotherapeutic mAbs and to define the role of reference medicinal products and ISs in assessing biosimilarity and testing medicines throughout their lifecycle.Discussion. This review covers the issues arising from the lack of ISs for assessing the biological activity of mAbs and the role and significance of reference products and ISs for biosimilars. The authors describe the specifics of studying the biological properties of mAbs and summarise the data on the need to develop and use ISs for the standardisation of biological tests. This review presents the results of studies on the first ISs established by the World Health Organisation to assess the biological activity of mAbs; these results suggest the need to standardise mAbs using ISs to ensure the quality, safety, and efficacy of mAb therapy.Conclusions. The use of ISs for mAbs plays a key role in harmonising biological activity assessments. Publicly available ISs serve as primary standards for the calibration of secondary reference materials. Moreover, ISs are required for the harmonisation of activity evaluation (in IU) between laboratories and for the consistency of the activity of various medicinal products from different manufacturers that share the same INN. The use of ISs by mAb manufacturers will contribute to ensuring the quality of mAbs and clinical monitoring of the effectiveness of their use

Особенности и значимость оценки подгрупп в подтверждающих клинических исследованиях

 Patient subgroup analysis plays an important role in interpretation of confirmatory clinical trial results and is mandatory in most cases. The purpose of subgroup analysis is to assess the consistency (heterogeneity) of the treatment effect in subgroups of patients identified based on such characteristics as demographics, stage and severity of the underlying disease, presence of a certain genetic mutation, etc. However, existing methodological issues (the problem of multiple comparisons, detection of differences between subgroups by chance alone, etc.) make it difficult to carry out the analysis and often lead to controversial conclusions. The aim of  the study was to analyse and summarise foreign regulatory approaches to subgroup analysis in confirmatory clinical trials, and to elaborate science-based requirements for subgroup analysis and interpretation of the results by clinical trial sponsors and experts when assessing the risk-benefit ratio of medicinal products for the purpose of their authorisation in Russia and the  Eurasian Economic Union. This paper discusses the objectives of subgroup  analysis and statistical approaches to its performance, provides relevant examples of such analysis from regulatory practice. It describes approaches to interpretation of subgroup analysis depending on the presence/absence of evidence supporting the primary hypothesis of the study, the nature of the experimental medicinal product’s heterogeneous effects in the  subgroups, and selection of the subgroups. The paper highlights areas of concern in subgroup analysis, potential controversies in interpretation of the  obtained results, and regulatory expectations. The recommendations  presented in the paper can be used by experts in the assessment of the risk-benefit ratio, as well as by medicine developers in the preparation of clinical trial protocols and reports.  Анализ подгрупп пациентов играет важную роль в интерпретации результатов подтверждающих клинических исследований и в большинстве случаев обязателен к выполнению. Целью анализа является оценка согласованности (гетерогенности) терапевтического эффекта в подгруппах пациентов, выделенных на основании  демографических характеристик, стадии и степени тяжести основного  заболевания, наличия определенной генетической мутации и др. Однако существующие методологические проблемы (проблема  множественных сравнений, случайные различия между подгруппами и т.д.) затрудняют его проведение и часто приводят к спорным выводам.  Цель работы — анализ и систематизация подходов зарубежных  регуляторных органов к оценке подгрупп в подтверждающих  клинических исследованиях, формирование научно обоснованных  требований к ее выполнению и интерпретации результатов спонсорами клинических исследований и экспертами при проведении экспертизы  отношения ожидаемой пользы к возможному риску применения лекарственных препаратов с целью их регистрации по национальным  правилам и по правилам Евразийского экономического союза. В работе  рассмотрены цели проведения анализа подгрупп и статистические  подходы к его выполнению, приведены примеры такого анализа из регуляторной практики. Описаны подходы к интерпретации анализа подгрупп в зависимости от наличия или отсутствия доказательства  основной гипотезы исследования, характера обнаруженных  гетерогенных эффектов исследуемого препарата в подгруппах и выбора  самих подгрупп. Освещены проблемные аспекты анализа подгрупп, возможные противоречия интерпретации полученных результатов,  регуляторные ожидания. Представленные рекомендации могут быть  использованы экспертами при проведении экспертизы  отношения ожидаемой пользы к возможному риску применения лекарственного  препарата, а также разработчиками лекарственных средств при подготовке протоколов и отчетов клинических исследований.

Stem cells in human breast milk

Recent studies have demonstrated that breast milk contains a population of cells displaying many of the properties typical of stem cells. This review outlines progress made in this newly emerging field of stem cell biology and provides an analysis of the available data on purification, propagation and differentiation of certain types of progenitor cells from breast milk. The possible fates of breast milk cells, including microchimerism caused by their transmission to the distant organs of the infant, are also discussed. Unique properties of breast milk-derived stem cells, such as their unusually low tumorigenic potential and their negligible ability to form teratomas, are highlighted as obvious advantages for using these cells in regenerative therapy

Сверхвысокомолекулярный полиэтилен (СВМПЭ) как основа клеточного матрикса для создания 3D клеточной культуры

The study is devoted to the development of an artificial material based on the ultrahigh-molecular weight polyethylene (UHMWPE) with a porous or cellular 3D structure as a cellular matrix – a framework for growing cell cultures. The development of such matrix provides support for neuronal cell culture under conditions that mimick those that exist in the living body. Typically, in vitro cellular studies are conducted in a 2D format, which limits intercellular interactions, morphology, differentiation, survival, signaling responses, gene expression and proliferation that are found in vivo. Here, we propose to use UHMWPE as a material of the cellular matrix, the ultra-high molecular weight polyethylene. UHMWP is a bioinert substance, wich allows forming a system of open connected pores needed to provide cellular life conditions with supply of nutrients and oxygen as well as the removal of waste products, the possibility of intercellular communication, etc. As a result, the use of UHMWPE as a cellular matrix will allow to study the processes occurring in cells in the 3D environment.Работа посвящена анализу свойств искусственного материала на основе сверхвысокомолекулярного полиэтилена (СВМПЭ) с пористой или ячеистой 3D-структурой, который используется в качестве клеточного матрикса – каркаса для выращивания культуры клеток. Разработка такого каркаса обеспечивает культивирование клеточной культуры в условиях, приближенных к тем, которые существуют в живом организме. Как правило, клеточные исследования in vitro проводят в 2D-формате, который по своей природе ограничивает межклеточные взаимодействия, морфологию, дифференцировку, выживаемость, сигнальные ответы, экспрессию генов и пролиферацию, наблюдаемые in vivo. В качестве материала клеточного матрикса предлагается использовать биоинертный сверхвысокомолекулярный полиэтилен (СВМПЭ), который позволяет сформировать систему открытых связанных пор с целью обеспечения клеточной жизнедеятельности – “подвод” питания и кислорода, удаление продуктов жизнедеятельности, возможность осуществления межклеточных связей и т.д. В результате использование СВМПЭ в качестве клеточного матрикса позволит изучить процессы, протекающие в клетках в условиях 3D-среды