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Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Transcriptoma , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Humanos , Transcriptoma/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células CultivadasRESUMO
Biotherapeutics exhibit high efficacy in targeted therapy, but their oral delivery is impeded by the harsh conditions of the gastrointestinal (GI) tract and limited intestinal absorption. This article presents a strategy to overcome the challenges of poor intestinal permeability by using a protein shuttle that specifically binds to an intestinal target, the leptin receptor (LepR), and exploiting its capacity to perform a receptor-mediated transport. Our proof-of-concept study focuses on the characterization and transport of robust affinity proteins, known as Nanofitins, across an ex vivo porcine intestinal model. We describe the potential to deliver biologically active molecules across the mucosa by fusing them with the Nanofitin 1-F08 targeting the LepR. This particular Nanofitin was selected for its absence of competition with leptin, its cross-reactivity with LepR from human, mouse, and pig hosts, and its shuttle capability associated with its ability to induce a receptor-mediated transport. This study paves the way for future in vivo demonstration of a safe and efficient oral-to-systemic delivery of targeted therapies.
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A novel series of potent agonists of the bile acid receptor TGR5 bearing a dihydropyridone scaffold was developed from a high-throughput screen. Starting from a micromolar hit compound, we implemented an extensive structure-activity-relationship (SAR) study with the synthesis and biological evaluation of 83 analogues. The project culminated with the identification of the potent nanomolar TGR5 agonist 77A. We report the GLP-1 secretagogue effect of our lead compound ex vivo in mouse colonoids and in vivo. In addition, to identify specific features favorable for TGR5 activation, we generated and optimized a three-dimensional quantitative SAR model that contributed to our understanding of our activity profile and could guide further development of this dihydropyridone series.
Assuntos
Relação Quantitativa Estrutura-Atividade , Fatores de Transcrição , Animais , Camundongos , Peptídeo 1 Semelhante ao Glucagon , Ácidos e Sais BiliaresRESUMO
Recombinant biological molecules are at the cutting-edge of biomedical research thanks to the significant progress made in biotechnology and a better understanding of subcellular processes implicated in several diseases. Given their ability to induce a potent response, these molecules are becoming the drugs of choice for multiple pathologies. However, unlike conventional drugs which are mostly ingested, the majority of biologics are currently administered parenterally. Therefore, to improve their limited bioavailability when delivered orally, the scientific community has devoted tremendous efforts to develop accurate cell- and tissue-based models that allow for the determination of their capacity to cross the intestinal mucosa. Furthermore, several promising approaches have been imagined to enhance the intestinal permeability and stability of recombinant biological molecules. This review summarizes the main physiological barriers to the oral delivery of biologics. Several preclinical in vitro and ex vivo models currently used to assess permeability are also presented. Finally, the multiple strategies explored to address the challenges of administering biotherapeutics orally are described.
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The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, Kp,uu,brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.
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The blood-brain barrier (BBB) is localized at the brain microvascular endothelial cells. These cells form a tight barrier, limiting the access of cells, pathogens, chemicals, and toxins to the brain due to tight junctions and efflux transporters. As the BBB plays a role in the assessment of neurotoxicity and brain uptake of drugs, human in vitro BBB models are highly needed. They allow to evaluate if compounds could reach the central nervous system across the BBB or can compromise its barrier function. Past decade, multiple induced pluripotent stem cell (iPSC)-derived BBB differentiation protocols emerged. These protocols can be divided in two groups, the one-step protocols, direct differentiation from iPSC to BBB cells, or the two-step protocols, differentiation for iPSC to endothelial (progenitor) cells and further induction of BBB characteristics. While the one-step differentiation protocols display good barrier properties, reports question their endothelial nature and maturation status. Therefore protocol characterization remains important. With transcriptomics becoming cheaper, this may support iPSC-derived model characterization. Because of the constraints in obtaining human brain tissue, good human reference data is scarce and would bear inter-individual variability. Additionally, comparison across studies might be challenging due to variations in sample preparation and analysis. Hopefully, increasing use of transcriptomics will allow in-depth characterization of the current iPSC-BBB models and guide researchers to generate more relevant human BBB models.
Assuntos
Células-Tronco Pluripotentes Induzidas , Barreira Hematoencefálica , Diferenciação Celular , Células Endoteliais/fisiologia , Humanos , TranscriptomaRESUMO
Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.
Assuntos
Herbicidas , Células-Tronco Pluripotentes Induzidas , Animais , Herbicidas/metabolismo , Herbicidas/toxicidade , Humanos , Fígado/metabolismo , Estresse Oxidativo , Paraquat/toxicidadeRESUMO
Permeation is one of the most evaluated parameters using preclinical in vitro blood-brain barrier models, as it has long been considered to be one of the major factors influencing central nervous system drug delivery. Blood-brain barrier permeability can be defined as the speed at which a compound crosses the brain endothelial cell barrier and is employed to assess barrier tightness, which is a crucial feature of brain capillaries in vivo. In addition, it is used to assess brain drug penetration. We review traditionally used methods to assess blood-brain barrier permeability in vitro and summarize often neglected in vivo (e.g., plasma protein and brain tissue binding) or in vitro (e.g., culture insert materials or methodology) factors that influence this property. These factors are crucial to consider when performing BBB permeability assessments, and especially when comparing permeability data obtained from different models, since model diversification significantly complicates inter-study comparisons. Finally, measuring transendothelial electrical resistance can be used to describe blood-brain barrier tightness; however, several parameters should be considered while comparing these measurements to the blood-brain barrier permeability to paracellular markers.
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Barreira Hematoencefálica , Células Endoteliais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , PermeabilidadeRESUMO
The proinflammatory cytokine Interleukin-1 (IL-1), with its two isoforms α and ß, has important roles in multiple pathogenic processes in the central nervous system. The present study aimed to evaluate and compare the blood-to-brain distribution of anakinra (IL-1 receptor antagonist), bermekimab (IL-1α antagonist) and canakinumab (IL-1ß antagonist). A human in vitro model of the blood-brain barrier derived from human umbilical cord blood stem cells was used, where isolated CD34+ cells co-cultured with bovine pericytes were matured into polarized brain-like endothelial cells. Transport rates of the three test items were evaluated after 180 âmin incubation at concentrations 50, 250 and 1250 ânM in a transwell system. We report herein that anakinra passes the human brain-like endothelial monolayer at a 4-7-fold higher rate than the monoclonal antibodies tested. Both antibodies had similar transport rates at all concentrations. No dose-dependent effects in transport rates were observed, nor any saturation effects at supraphysiological concentrations. The larger propensity of anakinra to pass this model of the human blood-brain barrier supports existing data and confirms that anakinra can reach the brain compartment at clinically relevant concentrations. As anakinra inhibits the actions of both IL-1α and IL-1ß, it blocks all effects of IL-1 downstream signaling. The results herein further add to the growing body of evidence of the potential utility of anakinra to treat neuroinflammatory disorders.
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Physiologically-based kinetic (PBK) models can simulate concentrations of chemicals in tissues over time without animal experiments. Nevertheless, in vivo data are often used to parameterise PBK models. This study aims to illustrate that a combination of kinetic and dynamic readouts from in vitro assays can be used to parameterise PBK models simulating neurologically-active concentrations of xenobiotics. Baclofen, an intrathecally administered drug to treat spasticity, was used as a proof-of-principle xenobiotic. An in vitro blood-brain barrier (BBB) model was used to determine the BBB permeability of baclofen needed to simulate plasma and cerebrospinal concentrations. Simulated baclofen concentrations in individuals and populations of adults and children generally fall within 2-fold of measured clinical study concentrations. Further, in vitro micro-electrode array recordings were used to determine the effect of baclofen on neuronal activity (cell signalling). Using quantitative in vitro-in vivo extrapolations (QIVIVE) corresponding doses of baclofen were estimated. QIVIVE showed that up to 4600 times lower intrathecal doses than oral and intravenous doses induce comparable neurological effects. Most simulated doses were in the range of administered doses. This show that PBK models predict concentrations in the central nervous system for various routes of administration accurately without the need for additional in vivo data.
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Baclofeno/administração & dosagem , Agonistas dos Receptores de GABA-B/administração & dosagem , Modelos Biológicos , Relaxantes Musculares Centrais/administração & dosagem , Adulto , Animais , Baclofeno/líquido cefalorraquidiano , Baclofeno/farmacocinética , Bioensaio , Barreira Hematoencefálica/metabolismo , Bovinos , Criança , Técnicas de Cocultura , Simulação por Computador , Eletrodos , Células Endoteliais/metabolismo , Feminino , Agonistas dos Receptores de GABA-B/líquido cefalorraquidiano , Agonistas dos Receptores de GABA-B/farmacocinética , Humanos , Cinética , Masculino , Relaxantes Musculares Centrais/líquido cefalorraquidiano , Relaxantes Musculares Centrais/farmacocinética , Pericitos/metabolismoRESUMO
Microfluidic lab-on-a-chip (LOC) devices allow the study of blood-brain barrier (BBB) properties in dynamic conditions. We studied a BBB model, consisting of human endothelial cells derived from hematopoietic stem cells in co-culture with brain pericytes, in an LOC device to study fluid flow in the regulation of endothelial, BBB and glycocalyx-related genes and surface charge. The highly negatively charged endothelial surface glycocalyx functions as mechano-sensor detecting shear forces generated by blood flow on the luminal side of brain endothelial cells and contributes to the physical barrier of the BBB. Despite the importance of glycocalyx in the regulation of BBB permeability in physiological conditions and in diseases, the underlying mechanisms remained unclear. The MACE-seq gene expression profiling analysis showed differentially expressed endothelial, BBB and glycocalyx core protein genes after fluid flow, as well as enriched pathways for the extracellular matrix molecules. We observed increased barrier properties, a higher intensity glycocalyx staining and a more negative surface charge of human brain-like endothelial cells (BLECs) in dynamic conditions. Our work is the first study to provide data on BBB properties and glycocalyx of BLECs in an LOC device under dynamic conditions and confirms the importance of fluid flow for BBB culture models.
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Barreira Hematoencefálica/metabolismo , Glicocálix/metabolismo , Dispositivos Lab-On-A-Chip/normas , Animais , Bovinos , Modelos Animais de Doenças , HumanosRESUMO
The blood-brain barrier (BBB) is a highly restrictive barrier that preserves central nervous system homeostasis and ensures optimal brain functioning. Using BBB cell assays makes it possible to investigate whether a compound is likely to compromise BBBs functionality, thereby probably resulting in neurotoxicity. Recently, several protocols to obtain human brain-like endothelial cells (BLECs) from induced pluripotent stem cells (iPSCs) have been reported. Within the framework of the European MSCA-ITN in3 project, we explored the possibility to use an iPSC-derived BBB model to assess the effects of repeated dose treatment with chemicals, using Cyclosporine A (CsA) as a model compound. The BLECs were found to exhibit important BBB characteristics up to 15 days after the end of the differentiation and could be used to assess the effects of repeated dose treatment. Although BLECs were still undergoing transcriptional changes over time, a targeted transcriptome analysis (TempO-Seq) indicated a time and concentration dependent activation of ATF4, XBP1, Nrf2 and p53 stress response pathways under CsA treatment. Taken together, these results demonstrate that this iPSC-derived BBB model and iPSC-derived models in general hold great potential to study the effects of repeated dose exposure with chemicals, allowing personalized and patient-specific studies in the future.
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Barreira Hematoencefálica , Ciclosporina/toxicidade , Células Endoteliais/efeitos dos fármacos , Imunossupressores/toxicidade , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Testes de Toxicidade/métodos , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Transcriptoma/efeitos dos fármacosRESUMO
PEGylation of therapeutic agents is known to improve the pharmacokinetic behavior of macromolecular drugs and nanoparticles. In this work, we performed the conjugation of polyethylene glycols (220-5000 Da) to a series of non-steroidal small agonists of the bile acids receptor TGR5. A suitable anchoring position on the agonist was identified to retain full agonistic potency with the conjugates. We describe herein an extensive structure-properties relationships study allowing us to finely describe the non-linear effects of the PEG length on the physicochemical as well as the in vitro and in vivo pharmacokinetic properties of these compounds. When appending a PEG of suitable length to the TGR5 pharmacophore, we were able to identify either systemic or gut lumen-restricted TGR5 agonists.
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Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Barreira Hematoencefálica/metabolismo , Células CACO-2 , Células HEK293 , Humanos , Hipoglicemiantes/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Polietilenoglicóis/química , Receptores Acoplados a Proteínas G/química , Relação Estrutura-AtividadeRESUMO
Therapies targeting neurological conditions such as Alzheimer's or Parkinson's diseases are hampered by the presence of the blood-brain barrier (BBB). During the last decades, several approaches have been developed to overcome the BBB, such as the use of nanoparticles (NPs) based on biomaterials, or alternative methods to open the BBB. In this review, we briefly highlight these strategies and the most recent advances in this field. Limitations and advantages of each approach are discussed. Combination of several methods such as functionalized NPs targeting the receptor-mediated transcytosis system with the use of magnetic resonance imaging-guided focused ultrasound (FUS) might be a promising strategy to develop theranostic tools as well as to safely deliver therapeutic molecules, such as drugs, neurotrophic factors or antibodies within the brain parenchyma.
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Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Doenças do Sistema Nervoso Central/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Humanos , Nanopartículas/metabolismoRESUMO
Formation, maintenance, and repair of the blood-brain barrier (BBB) are critical for central nervous system homeostasis. The interaction of endothelial cells (ECs) with brain pericytes is known to induce BBB characteristics in brain ECs during embryogenesis and can be used to differentiate human ECs from stem cell source in in vitro BBB models. However, the molecular events involved in BBB maturation are not fully understood. To this end, human ECs derived from hematopoietic stem cells were cultivated with either primary bovine or cell line-derived human brain pericytes to induce BBB formation. Subsequently, the transcriptomic profiles of solocultured vs. cocultured ECs were analysed over time by Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3'-end targeted, tag-based, reduced representation transcriptome profiling technique, that can reliably quantify all polyadenylated transcripts including those with low expression. By analysing the generated transcriptomic profiles, we can explore the molecular processes responsible for the functional changes observed in ECs in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identified several up- and downregulated genes and signaling pathways that provide a valuable data source to further delineate complex molecular processes that are involved in BBB formation and BBB maintenance. In addition, this data provides a source to identify novel targets for central nervous system drug delivery strategies.
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Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/citologia , Pericitos/metabolismo , Transcriptoma , Técnicas de Cocultura , Perfilação da Expressão Gênica , Humanos , Transdução de SinaisRESUMO
The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand-P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand-P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (hP-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π-sigma, π-alkyl, and π-π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand-protein complex in the binding site. It was also observed that some interacting residues in hP-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Descoberta de Drogas , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Teoria da Densidade Funcional , Descoberta de Drogas/métodos , Ligação de Hidrogênio , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
Characterizing interaction of newly synthetized molecules with efflux pumps remains essential to improve their efficacy and safety. Caco-2 cell line cultivated on inserts is widely used for measuring apparent permeability of drugs across biological barriers, and for estimating their interaction with efflux pumps such as P-gp, BCRP and MRPs. However, this method remains time consuming and expensive. In addition, detection method is required for measuring molecule passage across cell monolayer and false results can be generated if drugs concentrations used are too high as demonstrated with quinidine. For this reason, we developed a new protocol based on the use of Caco-2 cell directly seeded on 96- or 384-well plates and the use of fluorescent substrates for efflux pumps. We clearly observed that the new method reduces costs for molecule screening and leads to higher throughput compared to traditional use of Caco-2 cell model. This accelerated model could provide quick feedback regarding the molecule design during the early stage of drug discovery and therefore reduce the number of compounds to be further evaluated using the traditional Caco-2 insert method.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Miniaturização , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fluoresceínas/metabolismo , Humanos , Quinidina/farmacologia , Rodamina 123/metabolismoRESUMO
Assessing the rate of drug delivery to the central nervous system (CNS) in vitro has been used for decades to predict whether CNS drug candidates are likely to attain their pharmacological targets, located within the brain parenchyma, at an effective dose. The predictive value of in vitro blood-brain barrier (BBB) models is therefore frequently assessed by comparing in vitro BBB permeability, usually quoted as the endothelial permeability coefficient (Pe) or apparent permeability (Papp), to their rate of BBB permeation measured in vivo, the latter being commonly assessed in rodents. In collaboration with AstraZeneca (DMPK department, Södertälje, Sweden), the in vitro BBB permeability (Papp and Pe) of 27 marketed CNS drugs has been determined using a bovine in vitro BBB model and compared to their in vivo permeability (Pvivo), obtained by rat in-situ brain perfusion. The latter was taken from published data from Summerfield et al. (2007). This comparison confirmed previous reports, showing a strong in vitro/in vivo correlation for hydrophilic compounds, characterized by low brain tissue binding and a weak correlation for lipophilic compounds, characterized by high brain tissue binding. This observation can be explained by the influence of brain tissue binding on the uptake of drugs into the CNS in vivo and the absence of possible brain tissue binding in vitro. The use of glial cells (GC) in the in vitro BBB model to mimic brain tissue binding and the introduction of a new calculation method for in vitro BBB permeability (Pvitro) resulted in a strong correlation between the in vitro and in vivo rate of BBB permeation for the whole set of compounds. These findings might facilitate further in vitro to in vivo extrapolation for CNS drug candidates.