RESUMO
A growing number and diversity of complex medicines is in development and reaching the market, with many of these medicines utilising innovative delivery technology to achieve appropriate biodistribution and exposure. Accurate assessment of biodistribution, cell penetration, internalised form, cargo release and efficacy are essential for the development of these medicines. Advanced imaging technologies, deploying different labelling techniques that allow the assessment of both carrier and cargo, are enabling in-depth analysis and providing a mechanistic understanding of each step in the drug delivery pathway. Translation across cell, tissue and whole-body settings using multiple imaging methods can provide decision-making information that is critical for clinical phase selection and for the development of complex medicines.
Assuntos
Imagem Molecular , Preparações Farmacêuticas , Distribuição TecidualRESUMO
Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Microbolhas/uso terapêutico , Ondas Ultrassônicas , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/patologia , Terapia Combinada/métodos , Feminino , Humanos , Irinotecano , Técnicas Analíticas Microfluídicas , Tomografia por Emissão de Pósitrons , Distribuição Tecidual/efeitos da radiação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Organs-on-a-chip (OOAC) are research platforms containing cellular models designed to recapitulate relevant biological cues and, in some cases, enable communication between 'on-chip' connected organs. With enhanced physiological relevance, improvements in predictivity of the efficacy and toxicity of test compounds are anticipated. However, there are challenges to demonstrate the 'gain of confidence' of this technology for patient benefit. Translational challenges, the opportunities and deficiencies of the organ models, their intercommunication and the platform technology are all issues to be resolved. Sensitive, real-time detection technologies and data-rich readouts are needed to understand OOAC biology. Thus, the validation of normal and disease biology on chip, and modelling to translate these data to patients, will help position this technology in mainstream drug discovery.
Assuntos
Descoberta de Drogas , Dispositivos Lab-On-A-Chip , Engenharia Tecidual , Animais , Humanos , Modelos Biológicos , Pesquisa Translacional BiomédicaRESUMO
The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. Optimisation of inhibitory potency against multiple CDK's (1, 2 and 9) resulted in imidazole pyrimidine amides with potent in vitro anti-proliferative effects against a range of cancer cell lines. Excellent physiochemical properties and large margins against inhibition of CYP isoforms and the hERG ion channel were achieved by modification of lipophilicity and amine basicity. A candidate with disease model activity in human cancer cell line xenografts and with suitable physiochemical and pharmacokinetic profiles for intravenous (i.v.) dosing was selected for further development as AZD5597.
Assuntos
Amidas/química , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Imidazóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/química , Linhagem Celular Tumoral , Físico-Química/métodos , Cristalografia por Raios X , Proteínas Inibidoras de Quinase Dependente de Ciclina/farmacologia , Desenho de Fármacos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Imidazóis/farmacologia , Infusões Intravenosas , Modelos Químicos , Conformação Molecular , Transplante de Neoplasias , Isoformas de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologiaRESUMO
High-content screening (HCS), a process that combines fluorescence microscopic imaging and automated image analysis, has had a significant impact on drug discovery since its introduction in the mid 1990s. The application of HCS within pharmaceutical drug discovery has become widespread, notably within oncology drug discovery. The trends, challenges and considerations for HCS that affect the successful and pragmatic implementation of this process in drug discovery will be outlined.
Assuntos
Antineoplásicos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , FenótipoRESUMO
Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Humanos , Immunoblotting , Neurônios/citologia , Neurônios/metabolismo , Fatores de TempoRESUMO
Robust and reliable methods for the manipulation of neural cell lines, by passaging, plating, dye labeling, imaging, fixation, and immunocytochemistry, are required to enable consistent, reproducible screens to be performed. We describe herein procedures and processes we have established to maximize the level of consistency of cell plating, fixation, and dye or antibody labeling, to ensure that assays which we are running on a routine basis remain consistent across long periods of time. These procedures involve a variety of fully or semiautomated steps, using high-quality commercially available liquid handling and dispensing technology.
Assuntos
Análise Serial de Tecidos/métodos , Autoanálise/métodos , Humanos , Fixação de TecidosRESUMO
High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.
Assuntos
Sinalização do Cálcio , Técnicas de Cultura de Células/métodos , Células Cultivadas/química , Algoritmos , Animais , Encéfalo/embriologia , Córtex Cerebral/metabolismo , Química Farmacêutica/métodos , Desenho de Fármacos , Análise de Fourier , Cinética , Neurônios/metabolismo , Ratos , Transdução de Sinais , Fatores de TempoRESUMO
The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional high-throughput screening approaches.
Assuntos
Bioensaio/métodos , Neurônios/citologia , Células-Tronco/citologia , Animais , Cálcio/metabolismo , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Cinética , Neuritos/fisiologia , Neuritos/ultraestrutura , Neurônios/fisiologia , Ratos , Transdução de Sinais/fisiologia , Células-Tronco/fisiologiaRESUMO
Open innovation in pharmaceutical R&D evolved from a triple helix of convergent paradigm shifts in academic, industrial and government research sectors. The birth of the biotechnology sector catalyzed shifts in location dynamics that led to the first wave of open innovation in pharmaceutical R&D between big pharma and startup companies. The National Institutes of Health (NIH) Roadmap was a crucial inflection point that set the stage for a new wave of open innovation models between pharmaceutical companies and universities that have the potential to transform the pharmaceutical R&D landscape. We highlight the attributes of leading protected open innovation models that foster the sharing of proprietary small molecule collections by lowering the risk of premature escape of intellectual property, particularly structure-activity data.
Assuntos
Descoberta de Drogas , Pesquisa Biomédica , Indústria Farmacêutica , Humanos , Invenções , National Institutes of Health (U.S.) , Prostaglandina-Endoperóxido Sintases , Estados UnidosRESUMO
Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.
Assuntos
Piridazinas/síntese química , Receptores de GABA-A/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Técnicas In Vitro , Ligantes , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Técnicas de Patch-Clamp , Subunidades Proteicas/fisiologia , Piridazinas/farmacocinética , Piridazinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/fisiologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
A great deal of information can be gained from kinetic fluorescence-based measurement of cellular responses; however, until recently the use of such approaches has been limited by the manual nature of the instrumentation available. Higher-throughput kinetic studies of signaling pathways are greatly facilitated by new confocal, liquid handling-enabled, high content screening (HCS) platforms. In the present work, we have implemented one such instrument, the BD(TM) Pathway HT bioimager (BD Biosciences, Rockville, MD), for studying regulation of neuronal signaling pathways. We have established a neuronal calcium oscillation model, whereby rate of oscillation, amplitude of oscillation, and level of synchronicity across the culture can be measured. We have implemented membrane potential measurement using fluorescence resonance energy transfer-based dyes, for single cell characterization on this platform, showing the benefits of a truly flexible excitation and recording system; this dye combination cannot be readily implemented on all HCS platforms because of constraints of excitation wavelengths. We have validated long-term intracellular calcium imaging experiments, using innovative dyes and BD Pathway HT's spinning disk-based confocal excitation. To maximize both throughput and reproducibility, walk-away automation integration of this bioimaging technology has been implemented, producing an affordable, compact platform for fully automated kinetic HCS.
Assuntos
Sinalização do Cálcio , Avaliação Pré-Clínica de Medicamentos/métodos , Neurônios/fisiologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Automação , Bicuculina/farmacologia , Células Cultivadas , Córtex Cerebral , Avaliação Pré-Clínica de Medicamentos/instrumentação , Embrião de Mamíferos , Corantes Fluorescentes , Antagonistas GABAérgicos/farmacologia , Humanos , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Reprodutibilidade dos Testes , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Invertebrate glutamate-gated chloride channels (GluCls) are important targets for anthelmintics and insecticides such as ivermectin. To facilitate screening for novel GluCl modulators, the Caenorhabditis elegans GluCl alpha2beta channel was chosen as a surrogate for parasite channels not yet cloned, and an inducible stable human embryonic kidney cell line was generated. Functional expression of the alpha2 and beta subunits was confirmed by whole-cell voltage clamp assays. Using this cell line, a high-throughput assay was developed that detects membrane potential changes associated with the activation of GluCls. In this assay, membrane depolarization was quantified via changes in fluorescence resonance energy transfer between two membrane-associated dyes. Robust and reproducible signals were detected in response to addition of glutamate or ivermectin. This assay was used for the screening of over 180,000 samples from natural and synthetic sources.
Assuntos
Bioensaio/métodos , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , Ácido Glutâmico/farmacologia , Ativação do Canal Iônico/fisiologia , Rim/fisiologia , Técnicas de Patch-Clamp/métodos , Espectrometria de Fluorescência/métodos , Animais , Caenorhabditis elegans , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação do Canal Iônico/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Robótica/métodosRESUMO
Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.
Assuntos
Subunidades Proteicas/metabolismo , Receptores de GABA-A/metabolismo , Esteroides/farmacologia , Animais , Células Cultivadas , Humanos , Cinética , Camundongos , Conformação Molecular , Subunidades Proteicas/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Esteroides/química , Relação Estrutura-Atividade , TransfecçãoRESUMO
Cell migration is vital for many physiological processes, and its modulation is likely to be of therapeutic benefit. In this study we have developed fluorescence image-based chemokinesis and chemotaxis assays on the Cellomics (Pittsburgh, PA) ArrayScan platform, which would be suitable for industrial drug discovery in a variety of fields. Studying the migratory characteristics of neural stem cells is of interest for understanding the therapeutic potential of these cells, in terms of both cellular transplantation and the activation of endogenous populations of stem cells. Growth conditions were identified whereby human neural precursors could be maintained as neurospheres and plated out into microtitre plates for high-content assays. Chemokinesis was assessed using fluorescent bead-coated 96-well microtitre plates, whilst chemotaxis was assessed using BD Biosciences (Oxford, UK) Fluoroblok 24-well plates. Assays for both chemokinesis and chemotaxis were developed that were quantified automatically using the ArrayScan and appropriate algorithms. Using the two complementary techniques, foetal bovine serum was observed to have chemokinetic effects on the cells, whilst platelet-derived growth factor isoform AB was chemotactic. The two assays described here are suitable for screening for novel modulators of cell migration, or for performing more detailed mechanistic follow-up studies. These assays enable us to perform cell motility studies with minimal laboratory handling, in an automated manner, thereby allowing quantitative studies of cell behaviour to be incorporated in a routine drug discovery screening cascade.
Assuntos
Quimiocinas/fisiologia , Quimiotaxia/fisiologia , Citometria de Fluxo , Neurônios/citologia , Células-Tronco/citologia , Contagem de Células/métodos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Separação Celular , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo/métodos , Humanos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptor's downstream effects.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Corantes , Retículo Endoplasmático/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Inositol 1,4,5-Trifosfato/farmacologia , Neoplasias Hepáticas/metabolismo , Lisofosfolipídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
We have studied the pharmacological regulation of mitochondrial activity in a human neuroblastoma cell line. Cyclosporin A was found to directly alter mitochondrial membrane potential and to decrease mitochondrial permeability as measured using calcein. The estrogen receptor ligands tamoxifen, nafoxidine and clomiphene were identified as agents which affect mitochondrial membrane potential in a cyclosporin A-like manner. Also when mitochondrial permeability was measured using calcein, tamoxifen, nafoxidine and clomiphene were effective in inhibiting dye loss from mitochondria. Nafoxidine and cyclosporin A inhibit effects of mastoparan on SH-SY5Y mitochondria. These studies indicate that estrogen receptor ligands appear to affect mitochondria in a cyclosporin A-like manner in human neuroblastoma cells.