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1.
Regul Toxicol Pharmacol ; 150: 105632, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679316

RESUMO

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.


Assuntos
Testes de Toxicidade , Toxicologia , Animais , Toxicologia/métodos , Testes de Toxicidade/métodos , Humanos , Bases de Dados Factuais , Medição de Risco
2.
Genes Dev ; 30(19): 2152-2157, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798842

RESUMO

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.


Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Mutações Sintéticas Letais/genética , Trissacarídeos/genética , Animais , Apoptose/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Instabilidade Genômica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Tolerância a Radiação/genética , Trissacarídeos/metabolismo
3.
Toxicol Pathol ; 51(6): 390-396, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-38293937

RESUMO

In the last decade, numerous initiatives have emerged worldwide to reduce the use of animals in drug development, including more recently the introduction of Virtual Control Groups (VCGs) concept for nonclinical toxicity studies. Although replacement of concurrent controls (CCs) by virtual controls (VCs) represents an exciting opportunity, there are associated challenges that will be discussed in this paper with a more specific focus on anatomic pathology. Coordinated efforts will be needed from toxicologists, clinical and anatomic pathologists, and regulators to support approaches that will facilitate a staggered implementation of VCGs in nonclinical toxicity studies. Notably, the authors believe that a validated database for VC animals will need to include histopathology (digital) slides for microscopic assessment. Ultimately, the most important step lies in the validation of the concept by performing VCG and the full control group in parallel for studies of varying duration over a reasonable timespan to confirm there are no differences in outcomes (dual study design). The authors also discuss a hybrid approach, whereby control groups comprised both concurrent and VCs to demonstrate proof-of-concept. Once confidence is established by sponsors and regulators, VCs have the potential to replace some or all CC animals.


Assuntos
Desenvolvimento de Medicamentos , Patologia , Animais , Grupos Controle , Projetos de Pesquisa
4.
Toxicol Pathol ; 49(6): 1164-1173, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34060353

RESUMO

The approach undertaken to deliver a Good Laboratory Practice (GLP) validation of whole slide images (WSIs) and the associated workflow for the digital primary evaluation and peer review of a GLP-compliant rodent inhalation toxicity study is described. The contract research organization (CRO) undertook validation of the slide scanner, scanner software, and associated database software. This provided a GLP validated environment within the database software for the primary histopathologic evaluation using WSI and viewed with the database software web viewer. The CRO also validated a cloud-based digital pathology platform that supported the upload and transfer of WSI and metadata to a cache within the sponsor's local area network. The sponsor undertook a separate GLP validation of the same cloud-based digital pathology platform to cover the download and review of the WSI. The establishment of a fit-for-purpose GLP-compliant workflow for WSI and successful deployment for the digital primary evaluation and peer review of a large GLP toxicology study enabled flexibility in accelerated global working and potential future reuse of digitized data for advanced artificial intelligence and machine learning image analysis.


Assuntos
Inteligência Artificial , Roedores , Animais , Processamento de Imagem Assistida por Computador , Revisão por Pares , Software
5.
Toxicol Pathol ; 47(2): 100-107, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30636543

RESUMO

Whole slide imaging (WSI) technology has advanced to a point where it has replaced the glass slide as the primary means of pathology evaluation within many areas of medical pathology. The deployment of WSI in the field of toxicologic pathology has been delayed by a lack of clarity around the degree of validation required for its use on Good Laboratory Practice (GLP) studies. The current opinion piece attempts to provide a high-level overview of WSI technology to include basic methodology, advantages and disadvantages over a conventional microscope, validation status of WSI scanners, and perceived concerns over regulatory acceptance for the use of WSI for (GLP) peer review in the field of toxicologic pathology. Observations are based on the extensive use by AstraZeneca of WSI for the peer review of non-GLP studies conducted at Charles River facilities and represent the experiences of the authors. Note: This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the author(s). It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.


Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Patologia/métodos , Revisão dos Cuidados de Saúde por Pares/métodos , Toxicologia/métodos , Humanos , Interpretação de Imagem Assistida por Computador/normas , Patologia/normas , Revisão dos Cuidados de Saúde por Pares/normas , Toxicologia/normas
6.
Regul Toxicol Pharmacol ; 73(3): 737-46, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26517939

RESUMO

We have previously reported the profile of target organs (defined as organs showing histopathological changes) in rodent and non-rodent toxicity studies conducted prior to first-time-in-man (FTiM) for 77 AstraZeneca candidate drugs (CDs). Here, we test the assumption that toxicity is exacerbated by dosing duration by comparing the incidence and severity of target organ toxicities in these ≤ 6 week FTiM studies with those observed in subsequent subchronic/chronic (≥ 3 month) studies. Looking at the effect of dosing duration on severity (pathological score) and incidence (percentage of animals within the group) for the 39 CDs that met the criteria for inclusion (comparable doses between FTiM and subchronic/chronic studies), new toxicities appeared for 31 target organs but existing ones resolved for 29 target organs. Increased severity was more frequent for rodent (16 target organs) than for non-rodent (4 target organs). Most notable changes were a large increase in severity/incidence in liver and in non-rodent lung in contrast to a large decrease in severity and incidence for kidneys/ureter and for the non-rodent thymus. Overall this analysis shows that, even with continued exposure, target organ toxicities of CDs are as likely to show partial or complete recovery as they are to progress in severity.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação/toxicidade , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Drogas em Investigação/administração & dosagem , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Medição de Risco , Especificidade da Espécie , Timo/efeitos dos fármacos , Timo/patologia , Fatores de Tempo , Ureter/efeitos dos fármacos , Ureter/patologia
7.
Xenobiotica ; 44(2): 186-95, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24417752

RESUMO

1. The biotransformation, hepatic transporter and blood chemistry effects of troglitazone were investigated following 7 days of dosing at 600 mg/kg/day to chimeric murinized or humanized FRG mice, Mo-FRG and Hu-FRG mice, respectively. 2. Clinical chemistry and histopathology analysis revealed a significant drop in humanization over the time course of the study for the Hu-FRG mice but no significant changes associated with troglitazone treatment in either the Mo-FRG or the Hu-FRG models. No changes in transporter expression in livers of these mice were observed. Oxidative and conjugative metabolic pathways were identified with a 15- to 18-fold increase in formation of troglitazone sulfate in the Hu-FRG mice compared with the Mo-FRG mice in blood and bile, respectively. This resembles the troglitazone metabolism in human and these data are comparable with the formation of this metabolite in the chimeric uPA(+/+)/SCID mice. 3. However, larger amounts of troglitazone glucuronide were also observed in the Hu-FRG mouse compared with the Mo-FRG mouse which may be an effect of the drop in humanization of the Hu-FRG mouse during the study. 4. Highly humanized mice have a considerable potential in providing a useful first insight into circulating human metabolites of candidate drugs metabolized in the liver.


Assuntos
Cromanos/metabolismo , Cromanos/farmacocinética , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacocinética , Animais , Bile/metabolismo , Análise Química do Sangue , Hepatócitos/transplante , Humanos , Hidrolases/genética , Inativação Metabólica , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Ésteres do Ácido Sulfúrico/metabolismo , Quimeras de Transplante , Troglitazona
8.
Artigo em Inglês | MEDLINE | ID: mdl-23349040

RESUMO

BACKGROUND: A cross-laboratory analytic evaluation of a commercially available human inhibin B ELISA for measuring inhibin B in rat serum and plasma has been undertaken. METHODS: Dilution linearity, spiked recovery, intra- and inter-assay precision, functional sensitivity, matrix effects, and frozen stability were assessed across five laboratories. Reference ranges were generated for male Sprague Dawley and Han Wistar rats. RESULTS: Acceptable performance was defined as an overall assay coefficient of variation ≤ 20% with an intraday LLOQ ≤ 20 pg/ml. Intra- and inter-assay precision and functional sensitivity (≤6.4 pg/ml) generally met these criteria, but with occasional evidence of greater variability, particularly at lower concentrations. Dilution linearity was acceptable with occasional low recovery. Acceptable recovery of kit calibrators from rat serum confirmed the absence of matrix effects. Matched serum and plasma samples gave comparable results. The signal increased on freezing, remained constant for ≥3 freeze-thaw cycles and was generally stable for at least 8 weeks. Mean inhibin B ranged from 33.5 to 140.6 pg/ml in adult rats across laboratories, with some evidence for a decline from 6 to 9 weeks of age. Power calculations using preliminary reference range data indicated 10 animals/group would generally detect a 40% decrease in inhibin B at AstraZeneca, but laboratories with lower control values would require larger groups. CONCLUSIONS: The assay meets the analytical performance criteria; however, precision at the low end of the standard curve, biological variability, and low control values observed in some laboratories indicate that the utility of the assay may be limited in some laboratories.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Inibinas/sangue , Animais , Bioensaio , Congelamento , Humanos , Masculino , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Valores de Referência , Soro/metabolismo
10.
Toxicol Pathol ; 40(8): 1106-16, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22673116

RESUMO

The aims of this study were to assess the utility of the PXB mouse model of a chimeric human/mouse liver in studying human-specific effects of an important human hepatotoxic drug, the PPARγ agonist, troglitazone. When given orally by gavage for 7 days, at dose levels of 300 and 600 ppm, troglitazone induced specific changes in the human hepatocytes of the chimeric liver without an effect on the murine hepatic portions. The human hepatocytes, in the vehicle-treated PXB mouse, showed an accumulation of electron-dense lipid droplets that appeared as clear vacuoles under the light microscope in H&E-stained sections. Following dosing with troglitazone, there was a loss of the large lipid droplets in the human hepatocytes, a decrease in the amount of lipid as observed in frozen sections of liver stained by Oil-red-O, and a decrease in the expression of two bile acid transporters, BSEP and MRP2. None of these changes were observed in the murine remnants of the chimeric liver. No changes were observed in the expression of three CYPs, CYP 3A2, CYP 1A1, and CYP 2B1, in either the human or murine hepatocytes, even though the baseline expression of the enzymes differed significantly between the two hepatocyte species with the mouse hepatocytes consistently showing increased expression of the protein of all three enzymes. This study has shown that the human hepatocytes, in the PXB chimeric mouse liver, retain an essentially normal phenotype in the mouse liver and, the albeit limited CYP enzymes studied show a more human, rather than a murine, expression pattern. In line with this conclusion, the study has shown a differential response of the human versus the mouse hepatocytes, and the effects observed are highly suggestive of a differential handling of the compound by the two hepatocyte species although the exact reasons are not as yet clear. The PXB chimeric mouse system therefore holds the clear potential to explore human hepatic-specific features, such as metabolism, prior to dosing human subjects, and as such should have considerable utility in drug discovery and development.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cromanos/toxicidade , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tiazolidinedionas/toxicidade , Quimeras de Transplante , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Pré-Escolar , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Hepatócitos/metabolismo , Hepatócitos/transplante , Hepatócitos/ultraestrutura , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteína 2 Associada à Farmacorresistência Múltipla , Tamanho do Órgão/efeitos dos fármacos , PPAR gama/agonistas , Especificidade da Espécie , Troglitazona
11.
Xenobiotica ; 42(6): 503-17, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22201515

RESUMO

The pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone were investigated following daily oral dosing, at 300 and 600 mg/kg, for 7 days to control (SCID) and chimeric (PXB) mice with humanized livers. Clinical chemistry revealed no consistent pattern of changes associated with troglitazone treatment in the PXB mouse. Human MRP2 but not mouse mrp2 was down-regulated following troglitazone treatment. Pharmacokinetic analysis revealed similar T(max) values for troglitazone in both mouse groups, a mono- and bi-phasic elimination phase in PXB and SCID mice, respectively, but a 3- to 5- and 2- to 5-fold higher C(max) and AUC, respectively, in PXB mice. Oxidative and conjugative metabolic pathways were identified, with the sulfate being the predominant metabolite in PXB compared to SCID mice (4- to 13-fold increase in liver and blood, respectively). The glucuronide conjugate was predominant in SCID mice. There was no evidence of glutathione conjugation. The primary oxidative pathways were mono- and di-oxidations which may also be attributed to quinone or hydroquinone derivatives. Several metabolites were observed in PXB mice only. As the troglitazone metabolic profiles in the PXB mouse were similar to reported human data the PXB mouse model can provide a useful first insight into circulating human metabolites of xenobiotics metabolized in the liver.


Assuntos
Cromanos/farmacologia , Cromanos/farmacocinética , Hepatócitos/transplante , Hipoglicemiantes , Fígado/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Tiazolidinedionas/farmacocinética , Transplante Heterólogo , Animais , Cromanos/metabolismo , Feminino , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteína 2 Associada à Farmacorresistência Múltipla , Oxirredução , Sulfatos/metabolismo , Tiazolidinedionas/metabolismo , Quimeras de Transplante , Troglitazona
12.
Nat Commun ; 9(1): 1700, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703891

RESUMO

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease characterized by segmental premature aging, with cardiovascular disease being the main cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated, toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation or aiming to reduce progerin levels have provided only partial health improvements. Recently, we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or genetic depletion, significantly enhances the healthspan in a Lmna G609G HGPS mouse model. Collectively, the data provided here highlights NAT10 as a potential therapeutic target for HGPS.


Assuntos
Senilidade Prematura/tratamento farmacológico , Instabilidade Genômica/efeitos dos fármacos , Hidrazonas/farmacologia , Acetiltransferase N-Terminal A/antagonistas & inibidores , Progéria/tratamento farmacológico , Tiazóis/farmacologia , Senilidade Prematura/genética , Senilidade Prematura/mortalidade , Senilidade Prematura/patologia , Animais , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Modelos Animais de Doenças , Feminino , Instabilidade Genômica/genética , Humanos , Hidrazonas/uso terapêutico , Estimativa de Kaplan-Meier , Lamina Tipo A/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferases N-Terminal , Progéria/genética , Progéria/mortalidade , Progéria/patologia , Tiazóis/uso terapêutico
13.
Cancer Res ; 70(1): 288-98, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20028854

RESUMO

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (approximately 1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 micromol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.


Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Proteínas Quinases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Serina-Treonina Quinases TOR , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Arch Toxicol ; 78(7): 410-7, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15141287

RESUMO

Male F344 rats exposed to bromodichloromethane (BDCM) by gavage at 50 or 100 mg/kg/day for 5 days a week for 28 days excreted large amounts of formic acid in their urine, which was accompanied by a change in urinary pH. Male B6C3F1 mice exposed to BDCM at 25 or 50 mg/kg/day for 5 days a week for 28 days also excreted increased amounts of formic acid in their urine. In rats, formate excretion was dose and time dependant, being markedly elevated after four doses and remaining at that level after 3 weeks of dosing at 100 mg/kg/day BDCM, while at 50 mg/kg/day there was some suggestion of a decline after 3 weeks. In contrast, in mice formate excretion did not start to a major extent until 3 weeks of dosing, with the biggest response at 4 weeks. There was no increase in clinical chemistry markers of liver or kidney injury in either rats or mice following 28-day exposure to BDCM. However, morphological examination of the kidneys showed some mild renal tubule injury in two out of five rats exposed to 100 mg/kg/day BDCM. This was associated with a marked increase in cell proliferation in the renal cortex of all rats exposed to 100 mg/kg/day. No increase in cell proliferation was seen in the renal cortex of rats exposed to BDCM at 50 mg/kg/day, or in mice exposed to 25 or 50 mg/kg/day BDCM for 28 days. Long-term exposure to formic acid is known to cause kidney damage, suggesting that excretion of this acid may be a contributory factor to the increase in cell proliferation and kidney damage seen in the longer-term studies with BDCM.


Assuntos
Formiatos/urina , Túbulos Renais/efeitos dos fármacos , Trialometanos/toxicidade , Poluentes Químicos da Água/toxicidade , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Creatinina/metabolismo , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Córtex Renal/efeitos dos fármacos , Córtex Renal/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , Fatores de Tempo , Trialometanos/administração & dosagem , Poluentes Químicos da Água/administração & dosagem
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