RESUMO
The iminosugar UV-4 is a broad-spectrum antiviral drug candidate with activity in vitro and in vivo against multiple, diverse viruses. The toxicological profile of UV-4, dosed as the hydrochloride salt, was evaluated in single-dose and repeat-dose oral toxicity studies in mice, rats, dogs, and non-human primates (NHP). No moribundity or deaths were associated with the drug up to the maximum tolerated dose. No treatment-related adverse effects were observed following single oral doses in dogs, rats, and mice up to 250, 400, 1000 mg/kg, respectively, and in NHP up to 180 mg/kg administered three times daily for 10 days. UV-4-related findings were generally seen at higher doses after 7- or 14-day exposure. The most common clinical pathology findings (increase in aspartate aminotransferase and decreased platelet count) were consistently found across species and each appeared dose related. The kidney, mesenteric lymph nodes, stomach including gastrointestinal tract, and thymus were identified as target organs in mice, rats, and dogs. In 14-day repeat-dose toxicology studies in mice and dogs conducted in compliance with Good Laboratory Practice regulations, the dog was considered to be the most sensitive species to UV-4 exposure based on the treatment-related adverse effects noted in the identified target organs. The results of these studies demonstrate the safety profile of UV-4 hydrochloride and supported the selection of starting and maximal doses for a single ascending dose first-in-human clinical study.
Assuntos
Antivirais , Drogas em Investigação , Administração Oral , Animais , Antivirais/uso terapêutico , Antivirais/toxicidade , Cães , Drogas em Investigação/toxicidade , Dose Máxima Tolerável , Camundongos , RatosRESUMO
The development of novel therapeutics is associated with high rates of attrition, with unexpected adverse events being a major cause of failure. Serious adverse events have led to organ failure, cancer development and deaths that were not expected outcomes in clinical trials. These life-threatening events were not identified during therapeutic development due to the lack of preclinical safety tests that faithfully represented human physiology. We highlight the successful application of several novel technologies, including high-throughput screening, organs-on-chips, microbiome-containing drug-testing platforms and humanised mouse models, for mechanistic studies and prediction of toxicity. We propose the incorporation of similar preclinical tests into future drug development to reduce the likelihood of hazardous therapeutics entering later-stage clinical trials.
Assuntos
Desenvolvimento de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Drogas em Investigação/farmacologia , Drogas em Investigação/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Humanos , Tecnologia Farmacêutica/métodos , Tecnologia Farmacêutica/tendênciasRESUMO
Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.
Assuntos
Desenvolvimento de Medicamentos/métodos , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Bibliotecas de Moléculas Pequenas/toxicidade , Animais , Medula Óssea/efeitos dos fármacos , Carcinógenos/toxicidade , Aberrações Cromossômicas/induzido quimicamente , Dano ao DNA/efeitos dos fármacos , Drogas em Investigação/toxicidade , Feminino , Humanos , Técnicas In Vitro/métodos , Linfoma/induzido quimicamente , Masculino , Camundongos , Mutação/efeitos dos fármacos , Ratos , RoedoresRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) is a target for cancer therapy as it is overexpressed in a wide variety of cancers. Therapeutic antibodies that bind EGFR are being evaluated in clinical trials as imaging agents for positron emission tomography and image-guided surgery. However, some of these antibodies have safety concerns such as infusion reactions, limiting their use in imaging applications. Nimotuzumab is a therapeutic monoclonal antibody that is specific for EGFR and has been used as a therapy in a number of countries. METHODS: Formulation of IRDye800CW-nimotuzumab for a clinical trial application was prepared. The physical, chemical, and pharmaceutical properties were tested to develop the specifications to determine stability of the product. The acute and delayed toxicities were tested and IRDye800CW-nimotuzumab was determined to be non-toxic. Non-compartmental pharmacokinetics analysis was used to determine the half-life of IRDye800CW-nimotuzumab. RESULTS: IRDye800CW-nimotuzumab was determined to be non-toxic from the acute and delayed toxicity study. The half-life of IRDye800CW-nimotuzumab was determined to be 38 ± 1.5 h. A bi-exponential analysis was also used which gave a t1/2 alpha of 1.5 h and t1/2 beta of 40.8 h. CONCLUSIONS: Here, we show preclinical studies demonstrating that nimotuzumab conjugated to IRDye800CW is safe and does not exhibit toxicities commonly associated with EGFR targeting antibodies.
Assuntos
Drogas em Investigação/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/toxicidade , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/farmacocinética , Benzenossulfonatos/toxicidade , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Estabilidade de Medicamentos , Drogas em Investigação/farmacologia , Drogas em Investigação/toxicidade , Receptores ErbB/antagonistas & inibidores , Feminino , Meia-Vida , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/toxicidade , Indóis/administração & dosagem , Indóis/farmacocinética , Indóis/toxicidade , Aplicação de Novas Drogas em Teste , Masculino , Camundongos , Neoplasias/patologia , Neoplasias/cirurgia , Cirurgia Assistida por Computador/métodos , Testes de Toxicidade Aguda , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present study entails the toxicity evaluation of 7-methyl xanthine (7-MX), first of its kind molecule found effective in phase II clinical trials for the treatment of myopia, in comparison to other clinically used xanthines i.e., caffeine and theobromine. For acute toxicity evaluation, 7-MX was administered orally in two rodent species (rat and mice) at the doses of 300 mg/kg and 2000 mg/kg and for repeated dose 28-d oral toxicity, at 250, 500, and 1000 mg/kg in rats. Further, cellular toxicity was evaluated in normal breast epithelial (fR2), rat brain C6 glioma (C6 glioma) and human colorectal (Caco-2) cell lines. Also, the cell uptake assay to determine the intestinal permeability of drug was performed in Caco-2 cells. In acute toxicity, 7-MX treatment showed no mortality and toxicity, whereas 66.6% (mice) and 33.3% (rat) mortality was observed in both caffeine and theobromine treatment groups. In repeated dose 28-d oral toxicity, 7-MX treatment was found to have no-observed-adverse-effect level up to the dose of 1000 mg/kg in the present study conducted as per OECD guidelines 407. Also, very high IC50 value of 305.5 and 721 µg/mL was observed for 7-MX in fR2 and C6 glioma cells, respectively. In Caco-2 cells, linear bioavailability and high % cell viability was observed. Thus, 7-MX may be classified as Globally Harmonized System (GHS) category 5 drug with LD50 >2000-5000 mg/kg. Also, the repeated dose 28-d oral toxicity study demonstrated 7-MX to be nontoxic in nature, with cell line toxicity results further endorsing its nontoxic nature.
Assuntos
Drogas em Investigação , Miopia , Xantinas , Animais , Células CACO-2 , Drogas em Investigação/toxicidade , Humanos , Camundongos , Miopia/tratamento farmacológico , Ratos , Xantinas/toxicidadeRESUMO
An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15 % of compounds not cytotoxic to HepG2 cells (below 50 µM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.
Assuntos
Células Epiteliais/efeitos dos fármacos , Rim/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/toxicidade , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Hep G2 , Humanos , Concentração Inibidora 50 , Rim/metabolismo , Rim/patologia , NADPH Oxidase 4/genética , Nifedipino/toxicidadeRESUMO
BACKGROUND: The programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in suppressing antitumor immunity; axis dysregulation can be used by cancer cells to evade the immune system. Tislelizumab, an investigational monoclonal antibody with high affinity and binding specificity for PD-1, was engineered to minimize binding to FcγR on macrophages to limit antibody-dependent phagocytosis, a potential mechanism of resistance to anti-PD-1 therapy. The aim of this phase IA/IB study was to investigate the safety/tolerability, antitumor effects and optimal dose and schedule of tislelizumab in patients with advanced solid tumors. METHODS: Patients (aged ≥18 years) enrolled in phase IA received intravenous tislelizumab 0.5, 2, 5 or 10 mg/kg every 2 weeks; 2 or 5 mg/kg administered every 2 weeks or every 3 weeks; or 200 mg every 3 weeks; patients in phase IB received 5 mg/kg every 3 weeks. Primary objectives were to assess tislelizumab's safety/tolerability profile by adverse event (AE) monitoring and antitumor activity using RECIST V.1.1. PD-L1 expression was assessed retrospectively with the VENTANA PD-L1 (SP263) Assay. RESULTS: Between May 2015 and October 2017, 451 patients (n=116, IA; n=335, IB) were enrolled. Fatigue (28%), nausea (25%) and decreased appetite (20%) were the most commonly reported AEs. Most AEs were grade 1-2 severity; anemia (4.9%) was the most common grade 3-4 AE. Treatment-related AEs led to discontinuation in 5.3% of patients. Grade 5 AEs were reported in 14 patients; 2 were considered related to tislelizumab. Pneumonitis (2%) and colitis (1%) were the most common serious tislelizumab-related AEs. As of May 2019, 18% of patients achieved a confirmed objective response in phase IA and 12% in phase IB; median follow-up duration was 13.6 and 7.6 months, respectively. Pharmacokinetics, safety and antitumor activity obtained from both phase IA and IB determined the tislelizumab recommended dose; ultimately, tislelizumab 200 mg intravenous every 3 weeks was the dose and schedule recommended to be taken into subsequent clinical trials. CONCLUSIONS: Tislelizumab monotherapy demonstrated an acceptable safety/tolerability profile. Durable responses were observed in heavily pretreated patients with advanced solid tumors, supporting the evaluation of tislelizumab 200 mg every 3 weeks, as monotherapy and in combination therapy, for the treatment of solid tumors and hematological malignancies. TRIAL REGISTRATION NUMBER: NCT02407990.
Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Drogas em Investigação/toxicidade , Inibidores de Checkpoint Imunológico/toxicidade , Neoplasias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacocinética , Área Sob a Curva , Colite/induzido quimicamente , Colite/epidemiologia , Colite/imunologia , Relação Dose-Resposta a Droga , Drogas em Investigação/administração & dosagem , Drogas em Investigação/farmacocinética , Feminino , Seguimentos , Meia-Vida , Humanos , Inibidores de Checkpoint Imunológico/administração & dosagem , Inibidores de Checkpoint Imunológico/farmacocinética , Infusões Intravenosas , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/imunologia , Pneumonia/induzido quimicamente , Pneumonia/epidemiologia , Pneumonia/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Critérios de Avaliação de Resposta em Tumores Sólidos , Estudos Retrospectivos , Adulto JovemRESUMO
The importance of speed to clinic for medicines that may address unmet medical needs puts pressure on product development timelines. Historically, both toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming, and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, we have used pools of up to six clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and then the final single clone was selected for production of clinical materials. This approach was enabled by using platform processes across all stages of early development, including expression vectors, host cell lines, media, and production processes. Through comprehensive cell culture and product quality analysis, we demonstrated that the toxicology material was representative of the clinical material for all six monoclonal antibody programs evaluated. Our extensive development experience further confirmed that using a pool of clones for toxicology material generation is a reliable approach to shorten the early development timeline.
Assuntos
Anticorpos Monoclonais/imunologia , Células Clonais/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação/metabolismo , Proteínas Recombinantes/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/toxicidade , Células CHO , Células Clonais/efeitos dos fármacos , Cricetinae , Cricetulus , Drogas em Investigação/uso terapêutico , Drogas em Investigação/toxicidade , Humanos , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/toxicidade , Testes de Toxicidade/métodosRESUMO
Growing evidence suggests that Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) can be used as a new human cell-based platform to assess cardiac toxicity/safety during drug development. Cardiotoxicity assessment is highly challenging due to species differences and various toxicities, such as electrophysiological and contractile toxicities, which can result in proarrhythmia and heart failure. To explore proarrhythmic risk, the Multi-Electrode Array (MEA) platform is widely used to assess QT-interval prolongation and the proarrhythmic potential of drug candidates using hiPSC-CMs. Several consortiums, including the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japanese iPS Cardiac Safety Assessment (JiCSA), have demonstrated the applicability of hiPSC-CMs/MEA for assessing the torsadogenic potential of drug candidates. Additionally, contractility is a key safety issue in drug development, and efforts have been undertaken to measure contractility by a variety of imaging-based methods using iPS-CMs. Therefore, hiPSC-CMs might represent a standard testing tool for evaluating the proarrhythmic and contractile potentials. This review provides new insights into the practical application of hiPSC-CMs in early or late-stage nonclinical testing during drug development.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Desenvolvimento de Medicamentos , Drogas em Investigação/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Neoplasias , Medição de RiscoRESUMO
JD5037 is a novel peripherally restricted CB1 receptor (CB1R) inverse agonist being developed for the treatment of visceral obesity and its metabolic complications, including nonalcoholic fatty liver disease and dyslipidemia. JD5037 was administered by oral gavage at 10, 40, and 150â¯mg/kg/day dose levels for up to 34 days to Sprague Dawley rats, and at 5, 20, and 75â¯mg/kg/day dose levels for 28 consecutive days to Beagle dogs. In rats, higher incidences of stereotypic behaviors were observed in 10â¯mg/kg females and 40â¯mg/kg males, and slower responses for reflex and sensory tests were observed only in males at 10 and 40â¯mg/kg during neurobehavioral testing. Sporadic minimal incidences of decreased activity (males) and seizures (both sexes) were observed in rats during daily clinical observations, without any clear dose-relationship. Male dogs at 75â¯mg/kg during treatment period, but not recovery period, had an increased incidence of gut associated lymphoid tissue hyperplasia and inflammation in the intestine. In both species, highest dose resulted in lower AUCs indicative of non-linear kinetics. Free access to food increased the plasma AUC∞ by ~4.5-fold at 20â¯mg/kg in dogs, suggesting presence of food may help in systemic absorption of JD5037 in dogs. Based on the study results, 150â¯mg/kg/day in rats, and 20 and 75â¯mg/kg/day doses in male and female dogs, respectively, were determined to be the no-observed-adverse-effect-levels (NOAELs).
Assuntos
Drogas em Investigação/toxicidade , Pirazóis/toxicidade , Receptor CB1 de Canabinoide/agonistas , Convulsões/induzido quimicamente , Comportamento Estereotipado/efeitos dos fármacos , Sulfonamidas/toxicidade , Animais , Área Sob a Curva , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/uso terapêutico , Feminino , Humanos , Aplicação de Novas Drogas em Teste , Masculino , Nível de Efeito Adverso não Observado , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêuticoRESUMO
History has established that many drugs, such as the antibiotics, chemotherapies, and loop diuretics, are capable of inducing both nephrotoxicity and ototoxicity. The exact mechanisms by which cellular damage occurs remain to be fully elucidated. Monitoring the indices of renal function conducted in the Food and Drug Administration's prescribed set of early investigational new drug (IND)-enabling studies may be the first signs of ototoxicity properties of the new drug candidate. In developing improved and efficacious new molecular entities, it is critically necessary to understand the cellular and molecular mechanisms underlying the potential ototoxic effects as early in the drug development program as possible. Elucidation of these mechanisms will facilitate the development of safe and effective clinical approaches for the prevention and amelioration of drug-induced ototoxicity prior to the first dose in man. Biomarkers for nephrotoxicity in early tier I or tier II nonclinical IND-enabling studies should raise an inquiry as to the need to conduct a full auditory function assay early in the game to clear the pipeline with a safer candidate that has a higher probability of continued therapeutic compliance once approved for distribution.
Assuntos
Drogas em Investigação/toxicidade , Rim/patologia , Ototoxicidade , Animais , Orelha , Humanos , Rim/efeitos dos fármacosRESUMO
Interest has been expressed in using a joint test procedure that requires that the results of both a trend test and a pairwise comparison test between the control and the high groups be statistically significant simultaneously at the levels of significance recommended in the FDA 2001 draft guidance for industry document for the separate tests in order for the drug effect on the development of an individual tumor type to be considered as statistically significant. Results of our simulation studies show that there is a serious consequence of large inflations of the false negative rate through large decreases of false positive rate in the use of the above joint test procedure in the final interpretation of the carcinogenicity potential of a new drug if the levels of significance recommended for separate tests are used. The inflation can be as high as 204.5% of the false negative rate when the trend test alone is required to test if the effect is statistically significant. To correct the problem, new sets of levels of significance have also been developed for those who want to use the joint test in reviews of carcinogenicity studies.
Assuntos
Bioestatística/métodos , Testes de Carcinogenicidade/estatística & dados numéricos , Drogas em Investigação/toxicidade , Neoplasias/induzido quimicamente , Animais , Simulação por Computador , Interpretação Estatística de Dados , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Modelos Estatísticos , Reprodutibilidade dos Testes , Medição de Risco , Fatores de TempoRESUMO
High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Drogas em Investigação/toxicidade , Hepatócitos/efeitos dos fármacos , Cultura Primária de Células/métodos , Esferoides Celulares/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Hepatócitos/patologia , Humanos , Modelos Biológicos , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Esferoides Celulares/patologia , Fatores de TempoRESUMO
Drug-induced liver injury (DILI) is a leading cause of drug attrition during drug development and a common reason for drug withdrawal from the market. The poor predictability of conventional animal-based approaches necessitates the development of alternative testing approaches. A body of evidence associates DILI with the induction of stress-response genes in liver cells. Here, we set out to identify signal transduction pathways predominantly involved in the regulation of gene transcription by DILI drugs. To this end, we employed ATTAGENE's cell-based multiplexed reporter assay, the FACTORIAL transcription factor (TF), that enables quantitative assessment of the activity of multiple stress-responsive TFs in a single well of cells. Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nat. Methods 5, 253-260). Using this assay, we assessed TF responses of the human hepatoma cell line HepG2 to a panel of 64 drug candidates, including 23 preclinical DILI and 11 clinical DILI compounds and 30 nonhepatotoxic compounds from a diverse physicochemical property space. We have identified 16 TF families that specifically responded to DILI drugs, including nuclear factor (erythroid-derived 2)-like 2 antioxidant response element, octamer, hypoxia inducible factor 1 alpha, farnesoid-X receptor, TCF/beta-catenin, aryl hydrocarbon receptor, activator protein-1, E2F, early growth response-1, metal-response transcription factor 1, sterol regulatory element-binding protein, paired box protein, peroxisome proliferator-activated receptor, liver X receptor, interferone regulating factor, and P53, and 2 promoters that responded to multiple TFs (cytomegalovirus and direct repeat 3/vitamin D receptor). Some of TFs identified here also have previously defined role in pathogenesis of liver diseases. These data demonstrate the utility of cost-effective, animal-free, TF profiling assay for detecting DILI potential of drug candidates at early stages of drug development.
Assuntos
Alternativas ao Uso de Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação/química , Drogas em Investigação/toxicidade , Fatores de Transcrição/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Células Hep G2 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Respiratory tract toxicity represents a significant cause of attrition of inhaled drug candidates targeting respiratory diseases. One of the key issues to allow early detection of respiratory toxicities is the lack of reliable and predictive in vitro systems. Here, the relevance and value of a physiologically relevant 3D human airway in vitro model (MucilAir) were explored by repeated administration of a set of compounds with (n = 8) or without (n = 7) respiratory toxicity following inhalation in vivo. Predictability for respiratory toxicity was evaluated by readout of cytotoxicity, barrier integrity, viability, morphology, ciliary beating frequency, mucociliary clearance and cytokine release. Interestingly, the data show that in vivo toxicity can be predicted in vitro by studying cell barrier integrity by transepithelial electrical resistance (TEER), and cell viability determined by the Resazurin method. Both read-outs had 88% sensitivity and 100% specificity, respectively, while the former was more accurate with receiver operating characteristic (ROC) AUC of 0.98 (p = .0018) compared with ROC AUC of 0.90 (p = .0092). The loss of cell barrier integrity could mainly, but not fully, be attributed to a loss of cell coverage in 6 out of 7 compounds with reduced TEER. Notably, these effects occurred only at 400 µM, at concentration levels significantly above primary target cell potency, suggesting that greater attention to high local lung concentrations should be taken into account in safety assessment of inhaled drugs. Thus, prediction of respiratory toxicity in 3D human airway in vitro models may result in improved animal welfare and reduced attrition in inhaled drug discovery projects.
Assuntos
Drogas em Investigação/toxicidade , Células Epiteliais/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Modelos Biológicos , Sistema Respiratório/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Impedância Elétrica , Células Epiteliais/patologia , Humanos , Valor Preditivo dos Testes , Sistema Respiratório/patologiaRESUMO
During the last decades, imaging mass spectrometry has gained significant relevance in biomedical research. Recent advances in imaging mass spectrometry have paved the way for in situ studies on drug development, metabolism and toxicology. In contrast to whole-body autoradiography that images the localization of radiolabeled compounds, imaging mass spectrometry provides the possibility to simultaneously determine the discrete tissue distribution of the parent compound and its metabolites. In addition, imaging mass spectrometry features high molecular specificity and allows comprehensive, multiplexed detection and localization of hundreds of proteins, peptides and lipids directly in tissues. Toxicologists traditionally screen for adverse findings by histopathological examination. However, studies of the molecular and cellular processes underpinning toxicological and pathologic findings induced by candidate drugs or toxins are important to reach a mechanistic understanding and an effective risk assessment strategy. One of IMS strengths is the ability to directly overlay the molecular information from the mass spectrometric analysis with the tissue section and allow correlative comparisons of molecular and histologic information. Imaging mass spectrometry could therefore be a powerful tool for omics profiling of pharmacological/toxicological effects of drug candidates and toxicants in discrete tissue regions. The aim of the present review is to provide an overview of imaging mass spectrometry, with particular focus on MALDI imaging mass spectrometry, and its use in drug development and toxicology in general.
Assuntos
Descoberta de Drogas/instrumentação , Drogas em Investigação , Substâncias Perigosas , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Toxicologia/instrumentação , Animais , Biomarcadores/análise , Descoberta de Drogas/métodos , Drogas em Investigação/farmacocinética , Drogas em Investigação/toxicidade , Substâncias Perigosas/farmacocinética , Substâncias Perigosas/toxicidade , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massa de Íon Secundário/métodos , Distribuição Tecidual , Toxicologia/métodosRESUMO
INTRODUCTION: This report summarizes and comments key talks on the five traditional senses (ear, vestibular system, vision, taste, olfaction, and touch) which were delivered during the 2015 Annual Meeting of the Safety Pharmacology (SP) Society. AREAS COVERED: The functional observational battery (FOB) can detect major candidate drug liabilities only on ear, touch and vision. Anatomy, physiology, pharmacology, and pathology notions on each sensory system introduce speaker talks. Techniques for evaluating drug effects on hearing functions are reviewed. Nonclinical approaches to assess vestibular toxicity leading to balance deficits are presented. Retinal explants studied with multielectrode arrays allow the identification of drug liability sites on the retina. Routinely performed Safety Pharmacology assays are not powered to address candidate drug-induced disturbances on taste and smell. This weakness needs correction since unintended pharmacological impairment of these sensorial functions may have serious health consequences. Neuropathy produced by chemotherapeutic agents may cause multiple sensorial perception distortions. CONCLUSIONS: Safety Pharmacology studies should ensure the safety of any candidate drug on the five sensorial systems.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Drogas em Investigação/toxicidade , Farmacologia/métodos , Córtex Sensório-Motor/efeitos dos fármacos , Animais , Qualidade de Produtos para o Consumidor , HumanosRESUMO
In this work, for the first time, a method has been developed for the determination of AZD6118, a candidate drug, in dog plasma samples. The method is based on microextraction by packed sorbent (MEPS) of the drug prior to liquid chromatography-electrospray ionization tandem mass spectrometry assay. Various important factors affecting MEPS performance were optimized, and under the optimized condition, a linear calibration curve in the concentration range of 20-25,000nmolL-1 with a coefficient of determination over 0.99 was obtained. The back-calculated values of the calibration points showed good agreement with the theoretical concentrations (coefficients of variation percent between 0.3-3.8). The lower limit of quantification and limit of detection were 20.0 and 2.9nmolL-1, respectively. The repeatability and accuracy of the method was evaluated by determination of quality control samples at three concentration levels (low, medium and high) using the developed method, and the results (coefficients of variation values were between 1.9% and 3.2%, relative recoveries ranged between 93.5-102.1%) confirm that a powerful method has been developed for the extraction and determination of the investigated drug in dog plasma.
Assuntos
Cromatografia Líquida/métodos , Drogas em Investigação/análise , Microextração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Drogas em Investigação/química , Drogas em Investigação/farmacocinética , Drogas em Investigação/toxicidade , Feminino , Limite de Detecção , Modelos Lineares , Masculino , Reprodutibilidade dos TestesRESUMO
This study was conducted to investigate the use of a nanosuspension for intravenous injection into dogs to increase exposure without toxic additives for preclinical studies in the discovery stage. Nanosuspensions were prepared with a mixer mill and zirconia beads with a vehicle of 2% (w/v) poloxamer 338, which was confirmed to lead to no histamine release in dogs. Sterilized nanosuspensions of poorly water-soluble compounds, cilostazol (Cil), spironolactone (Spi) and probucol (Pro), at 10 mg ml(-1) were obtained by milling for 30 min, followed by autoclaving for 20 min at 121 °C and milling for 30 min (mill-autoclave-mill method). The particle sizes (d50) of Cil, Spi and Pro were 0.554, 0.484 and 0.377 µm, respectively, and the percentages of the nominal concentration were 79.1%, 99.6% and 75.4%, respectively. In chromatographic data, no extra peaks were observed. The particle size of Cil was 0.564 µm after storage for 16 days at 2-8 °C. Cil in nanosuspension, but not in microsuspension, rapidly dissolved in dog plasma. Cil nanosuspension at 0.4 mg kg(-1) and Cil saline solution at 0.03 mg kg(-1) , around the saturation solubility, were intravenously administered to dogs. Nanosuspension increased exposure. The versatility of the mill-autoclave-mill method was checked for 15 compounds, and the particle size of 12 compounds was in the nano range. The nanosuspension optimized in this study may be useful for intravenous toxicological and pharmacological studies in the early stage of drug development. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Descoberta de Drogas , Drogas em Investigação/administração & dosagem , Drogas em Investigação/química , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Testes de Toxicidade , Animais , Disponibilidade Biológica , Cães , Composição de Medicamentos , Drogas em Investigação/farmacocinética , Drogas em Investigação/toxicidade , Injeções Intravenosas , Masculino , Nanoestruturas/toxicidade , Nanotecnologia , Tamanho da Partícula , Veículos Farmacêuticos/administração & dosagem , Veículos Farmacêuticos/química , Veículos Farmacêuticos/farmacocinética , Veículos Farmacêuticos/toxicidade , Solubilidade , Tensoativos/administração & dosagem , Tensoativos/química , Tensoativos/farmacocinética , Tensoativos/toxicidade , Tecnologia FarmacêuticaRESUMO
A new more efficient preclinical study design (referred to as a compact design) is proposed that removes the need for satellite animals for the collection of toxicokinetic (TK) data by sampling from the main study animals, taking no more than one sample in 24 h to build up a full profile over the course of the study. The compact design's performance was tested with a simulation study, using an example of chemotherapy-induced myelosupression in rats. Data sets were simulated from a model based on available data, following both the compact design and a traditional design using satellite animals, with 100 studies being simulated for each. The effect of the compact design on parameter and variance estimates for the TK and neutrophil models were investigated, as well as the potential effect of interoccasion variability (IOV). The compact design performed equally as well as the traditional design, and had little impact on parameter or variation estimates, indicating that it would be a suitable alternative to traditional satellite designs while reducing the number of animals required. When IOV was present but not accounted for during the TK analysis some parameter estimates were biased and interindividual variation and residual errors inflated; this was reduced by allowing for IOV in the analysis. Using the compact design removes the need for a satellite group, reducing the number of animals required, without affecting the ability to model the data. If large IOV is suspected, caution should be exercised to avoid parameter estimation bias, and inflation of variability and residual error.