Assessing how in vitro assay types predict in vivo toxicology data.

In vivo animal bioassays are increasingly being supplemented with in vitro assays to serve as the new standard for chemical toxicity tests. Despite this shift, investigators face challenges related to increased reliance on in vitro data. The aim of this study was to deploy a streamlined method to assess the ability of in vitro data to predict similar results as in vivo data by correlating chemical toxicity rankings obtained using Benchmark Doses and Benchmark Dose Lower Limits (BMD(L)s) derived from in vivo and in vitro assays. In vitro and in vivo assay characteristics were assessed for their impact on the predictive ability of in vitro data. Minimum best-fit BMD(L)s were calculated for chemicals using Environmental Protection Agency's (EPA's) Benchmark Dose Software (BMDS). Forty-one chemicals met the inclusion criteria of this study. Relative chemical toxicity rankings were assessed through Kappa statistics, Pearson correlations, and/or Ordinary Least Squares (OLS) regressions. Results illustrated likely ability of in vitro data to predict similar results as short-term in vivo data. Further, rankings derived from in vitro cytotoxicity assays, unlike stress response assays, significantly correlated with rankings derived from short-term in vivo assays. These results support the use of in vitro data as a prioritization tool within toxicity testing.

Usage of In Vitro Metabolism Data for Drug-Drug Interaction in Physiologically Based Pharmacokinetic Analysis Submissions to the US Food and Drug Administration.

The key parameters necessary to predict drug-drug interactions (DDIs) are intrinsic clearance (CLint ) and fractional contribution of the metabolizing enzyme toward total metabolism (fm ). Herein, we summarize the accumulated knowledge from 53 approved new drug applications submitted to the Office of Clinical Pharmacology, US Food and Drug Administration, from 2016 to 2018 that contained physiologically based pharmacokinetic (PBPK) models to understand how in vitro data are used in PBPK models to assess drug metabolism and predict DDIs. For evaluation of CLint and fm , 29 and 20 new drug applications were included for evaluation, respectively. For CLint , 86.2% of the PBPK models used modified values based on in vivo data with modifications ranging from -82.5% to 2752.5%. For fm , 45.0% of the models used modified values with modifications ranging from -28% to 178.6%. When values for CLint were used from in vitro testing without modification, the model resulted in up to a 14.3-fold overprediction of the area under the concentration-time curve of the substrate. When values for fm from in vitro testing were used directly, the model resulted in up to a 2.9-fold underprediction of its DDI magnitude with an inducer, and up to a 1.7-fold overprediction of its DDI magnitude with an inhibitor. Our analyses suggested that the in vitro system usually provides a reasonable estimation of fm when the drug metabolism by a given CYP pathway is more than 70% of the total clearance. In vitro experiments provide important information about basic PK properties of new drugs and can serve as a starting point for building a PBPK model. However, key PBPK parameters such as CLint and fm still need to be optimized based on in vivo data.

The use of in vivo and in vitro tests in children with beta lactam allergy.

BACKGROUND: Drug allergies are reactions within the context of drug hypersensitivity reactions, which are caused by immunological mechanisms due to a previously sensitising drug. Beta-lactam antibiotics (BLA) are the leading agents causing drug hypersensitivity reactions in children. The aim of this study is to evaluate the diagnostic importance of in vivo and in vitro diagnostic tests in children with suspected immediate-type BLA hypersensitivity and to investigate the frequency of their use for the final diagnosis. METHODS: Patients admitted to the Outpatient Clinic of Division of Paediatric Allergy and Immunology with suspicion of immediate-type BLA hypersensitivity between December 2014 and December 2018 were investigated. Patients with a history of immediate reactions to BLA were examined by performing drug specific IgE, skin prick tests, intradermal tests and drug provocation tests (DPT). RESULTS: During the study period, 148 patients were admitted to our clinic with suspected immediate-type BLA hypersensitivity. Forty-eight patients completed all assessment steps and were enrolled in the study. It has been shown that 27 patients did not have drug allergy. BLA hypersensitivity was proven in 21 patients by using in vivo test algorithm. More than half of the patients were diagnosed via skin tests with culprit drug. CONCLUSION: Allergy work-up should be performed in patients with immediate reactions to BLA. A skin test can demonstrate BLA hypersensitivity in most patients. Thus, skin tests should be performed prior to the drug provocation test.

Método químico simplificado de desmineralización del esmalte dental humano / Simplified chemical method of demineralization in human dental enamel

Introducción: Se requieren métodos experimentales abreviados para simular las lesiones de desmineralización temprana de forma controlada y reproducible. Objetivo: Realizar una evaluación in vitro de un método simple de desmineralización incipiente del esmalte. Métodos: Estudio experimental aleatorizado con doble diseño factorial de réplicas. Se seleccionaron 12 terceros molares de sujetos humanos saludables para su desmineralización en solución de ácido láctico racémico. Las muestras se distribuyeron aleatoriamente: Grupo 1 (G1) (n= 6) ácido láctico a pH 2,4 y Grupo 2 (G2) (n= 6) ácido láctico a pH 5,4. A continuación, cada grupo se subdividió (n = 2) para evaluar el efecto de las soluciones a tres tiempos de exposición (7, 15 y 30 días) a 37 °C. La evaluación se llevó a cabo con estereomicroscopios, equipo de radiografía digital con un software de análisis digital de imágenes y microscopía de polarización. Se formuló una integración de los índices de respuesta y se realizó un ANOVA. Resultados: Los hallazgos visuales, radiográficos e histológicos mostraron que en el G1 en los tiempos 1 a 3, la desmineralización se caracterizó por una gran pérdida de la integridad del esmalte (80 por ciento a 100 por ciento). Visualmente, el G2 a los 7 días mostró opacidad y pérdida de brillo (16 por ciento) con preservación de la estructura superficial del esmalte. Conclusiones: Se demuestra que el empleo de ácido láctico durante 7 días a pH 5,4 produce una lesión clínica, radiográfica e histológica similar a una lesión temprana del esmalte(AU)

Introduction: Abridged experimental methods are required to simulate early demineralizing lesions in a controlled and reproducible way. Objective: Perform an in vitro evaluation of a simple method of incipient enamel demineralization. Methods: Randomized experimental study with a double factorial replication design. Twelve third molars from healthy human subjects were selected for demineralization in a racemic lactic acid solution. Samples were then distributed randomly: Group 1 (G1) (n= 6) lactic acid at pH 2.4 and Group 2 (G2) (n= 6) lactic acid at pH 5.4. Each group was then subdivided (n = 2) to evaluate the effect of the solutions at three exposure times (7, 15 and 30 days) at 37°C. The evaluation used stereomicroscopes, a digital x-rays apparatus with software for the digital analysis of images, and polarization microscopy. An integration of the response indices was formulated and ANOVA was performed. Results: Visual, radiographic and histological findings showed that G1 at time 1 through 3 displayed demineralization characterized by extensive loss (80 percent to 100 percent) of enamel integrity. Visually, G2 at 7 days exhibited opacity and loss of brightness (16 percent), with preservation of the surface structure of the enamel. Conclusions: It was shown that employing lactic acid for 7 days at pH 5.4 develops a clinical, radiographic and histological injury similar to an early enamel lesion(AU)

The Overt and Hidden Use of Animal-Derived Products in Alternative Methods for Skin Sensitisation: A Systematic Review.

In vitro methods that can replace animal testing in the identification of skin sensitisers are now a reality. However, as cell culture and related techniques usually rely on animal-derived products, these methods may be failing to address the complete replacement of animals in safety assessment. The objective of this study was to identify the animal-derived products that are used as part of in vitro methods for skin sensitisation testing. Thus, a systematic review of 156 articles featuring 83 different in vitro methods was carried out and, from this review, the use of several animal-derived products from different species was identified, with the use of fetal bovine serum being cited in most of the methods (78%). The use of sera from other animals, monoclonal antibodies and animal proteins were also variously mentioned. While non-animal alternatives are available and methods free of animal-derived products are emerging, most of the current methods reported used at least one animal-derived product, which raises ethical and technical concerns. Therefore, to deliver technically and ethically better in vitro methods for the safety assessment of chemicals, more effort should be made to replace products of animal origin in existing methods and to avoid their use in the development of new method protocols.

Monoamine Transporter and Receptor Interaction Profiles in Vitro Predict Reported Human Doses of Novel Psychoactive Stimulants and Psychedelics.

Background: Pharmacological profiles of new psychoactive substances can be established rapidly in vitro and provide information on potential psychoactive effects in humans. The present study investigated whether specific in vitro monoamine transporter and receptor interactions can predict effective psychoactive doses in humans. Methods: We correlated previously assessed in vitro data of stimulants and psychedelics with human doses that are reported on the Internet and in books. Results: For stimulants, dopamine and norepinephrine transporter inhibition potency was positively correlated with human doses, whereas serotonin transporter inhibition potency was inversely correlated with human doses. Serotonin 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2C receptor affinity was significantly correlated with psychedelic doses, but 5-HT1A receptor affinity and 5-HT2A and 5-HT2B receptor activation potency were not. Conclusions: The rapid assessment of in vitro pharmacological profiles of new psychoactive substances can help to predict psychoactive doses and effects in humans and facilitate the appropriate scheduling of new psychoactive substances.

Ortho-nitrobenzyl derivatives as potential anti-schistosomal agents

ABSTRACT In the search for new anti-schistosomal agents, a series of fifteen ortho-nitrobenzyl derivatives was assayed in vitro against both the schistosomulum (somule) and adult forms of Schistosoma mansoni. Compounds 8 and 12 showed significant activity against somules at low micromolar concentrations, but none was active against adults. The SAR demonstrated that the compounds most active against the parasite were mutagenic to the human cell line RKO-AS45-1 only at concentrations 10- to 40-fold higher than the worm-killing dose. Given their electrophilicity, compounds were also screened as inhibitors of the S. mansoni cysteine protease (cathepsin B1) in vitro. Amides 5 and 15 exhibited a modest inhibition activity with values of 55.7 and 50.6 % at 100 µM, respectively. The nitrobenzyl compounds evaluated in this work can be regarded as hits in the search for more active and safe anti-schistosomal agents.

Can a pharmacokinetic/pharmacodynamic (PKPD) model be predictive across bacterial densities and strains? External evaluation of a PKPD model describing longitudinal in vitro data.

BACKGROUND: Pharmacokinetic/pharmacodynamic (PKPD) models developed based on data from in vitro time-kill experiments have been suggested to contribute to more efficient drug development programmes and better dosing strategies for antibiotics. However, for satisfactory predictions such models would have to show good extrapolation properties. OBJECTIVES: To evaluate if a previously described mechanism-based PKPD model was able also to predict drug efficacy for higher bacterial densities and across bacterial strains. METHODS: A PKPD model describing the efficacy of ciprofloxacin on Escherichia coli was evaluated. The predictive performance of the model was evaluated across several experimental conditions with respect to: (i) bacterial start inoculum ranging from the standard of ∼106 cfu/mL up to late stationary-phase cultures; and (ii) efficacy for seven additional strains (three laboratory and four clinical strains), not included during the model development process, based only on information regarding their MIC. Model predictions were performed according to the intended experimental protocol and later compared with observed bacterial counts. RESULTS: The mechanism-based PKPD model structure developed based on data from standard start inoculum experiments was able to accurately describe the inoculum effect. The model successfully predicted the time course of drug efficacy for additional laboratory and clinical strains based on only the MIC values. The model structure was further developed to better describe the stationary phase data. CONCLUSIONS: This study supports the use of mechanism-based PKPD models based on preclinical data for predictions of untested scenarios.

In vitro evaluation of the effect of antioxidants on the bond strength of bleached teeth / Avaliação in vitro do efeito de antioxidantes sobre a resistência de união de dentes clareados

Objective: To evaluate the in vitro effect of antioxidants on the bond strength between composite resin and enamel subjected to bleaching agents. Methods: Nineteen sound human molars had their roots sectioned, while their surfaces were flattened and filled with composite resin to produce the specimens, which were then divided into seven groups: G1 ­ unbleached and without antioxidant (control); G2 ­ 36% carbamide peroxide; G3 ­ 36% carbamide peroxide and 10% ascorbic acid solution; G4 ­ 36% carbamide peroxide and 10% ascorbic acid gel; G5 ­ 16% carbamide peroxide; G6 ­ 16% carbamide peroxide and 10% ascorbic acid solution; and G7 ­ 16% carbamide peroxide and 10% ascorbic acid gel. The results of microtensile strength were submitted to an analysis of variance (ANOVA) and Tukey's test, with a significance level of 5%. Results: The bond strength between composite resin and enamel was not affected after the use of bleaching agents. In addition, ascorbic acid did not appear to neutralize the oxidative effects of these agents, given that group 3 showed the lowest mean of bond strength (13.85 MPa). In other groups, the mean of bond strength ranged from 24.06 MPa to 32.02 MPa, with no significant differences when compared to the control. Conclusion: The presence of ascorbic acid immediately after bleaching did not increase the bond strength between the resin and the enamel surface.(AU)

Objetivo: Avaliar in vitro o efeito de antioxidantes sobre a resistência de união entre resina composta e o esmalte sujeito a agentes clareadores. Métodos: Dezenove molares humanos hígidos tiveram suas raízes seccionadas e suas superfícies planificadas e restauradas para obtenção dos corposde-prova, os quais foram divididos em sete grupos: G1 - não clareado e sem agente antioxidante (controle); G2 - Peróxido de carbamida a 36%; G3 - Peróxido de carbamida a 36% e Ácido ascórbico 10% solução; G4 - Peróxido de carbamida a 36% e Ácido ascórbico 10% gel; G5 - Peróxido de carbamida a 16%; G6 - Peróxido de carbamida a 16% e Ácido ascórbico 10% solução; G7 - Peróxido de carbamida a 16% e Ácido ascórbico 10% gel. Os resultados da resistência a microtração foram submetidos à análise de variância (ANOVA) e ao teste de Tukey em nível de significância 5%. Resultados: A resistência adesiva entre resina e o esmalte não foi afetada após a utilização dos agentes clareadores, além disto, o ácido ascórbico não se apresentou capaz de neutralizar os efeitos oxidantes destes agentes, tendo em vista que o grupo 3 mostrou menor média quanto a resistência de união (13,85 Mpa). Nos demais grupos, as médias variaram de 24,06 Mpa a 32,02 Mpa, não havendo diferença significante entre essas médias e o controle. Conclusão: A presença do ácido ascórbico logo após o clareamento dentário não aumento a resistência de união entre a resina e a superfície do esmalte.(AU)

Efeito da ciclagem térmica nas propriedades mecânicas de materiais restauradores indiretos: isolamento do fator água / Effect of thermal cycling on mechanical properties of indirect restorative materials: water effect exception

O sucesso a longo prazo das restaurações dentárias é limitada pela sua durabilidade no meio bucal. Testes in vitro continuam sendo ferramentas indispensáveis para avaliação inicial dos materiais dentários. Ciclagem térmica é um dos procedimentos mais utilizados para simular o envelhecimento fisiológico no qual os biomateriais são submetidos na prática clínica. Os objetivos desse estudo foram determinar o efeito da variação da temperatura, isolando o efeito hidrolítico, na degradação de uma cerâmica feldspática e de um cimento resinoso por meio de comparação entre os métodos artificiais de envelhecimento: armazenamento em água destilada, termociclagem em água e termociclagem em óleo mineral seguido pelo ensaio de flexão de 3 pontos e mini-flexão. Blocos de CAD-CAM de cerâmica feldspática foram cortados e lixados para obter-se 100 barras, com dimensões de 14 x 4 x 1,2 mm. Foram confeccionadas 100 barras de cimento resinoso autoadesivo, com dimensões de 12 x 2 x 2 mm. As barras foram aleatoriamente divididas (n = 10) e envelhecidos por meio de termociclagem em água e óleo mineral por 500 (norma ISO 11405), 5000 e 10000 ciclos com banhos de 30 s a 5 oC e 55 oC e armazenados em água destilada em estufa a 37 oC pelo mesmo tempo que foi realizado a ciclagem (9 h, 4 dias e 8 dias), além de dois grupos controles, um de cerâmica feldspática e outro de cimento resinoso ambos sem envelhecimento, totalizando 20 grupos. Após o envelhecimento, as barras foram fraturadas por meio do ensaio de flexão de 3 pontos e mini-flexão e os dados foram submetidos à análise estatística. Amostras representativas de cada grupo foram submetidas ao teste de dureza Knoop (cimento resinoso) e Vickers (cerâmica feldspática). Algumas superfícies fraturadas foram analisadas em Microscópio eletrônico de varredura. Para cerâmica feldspática não houve diferença significativa entre os grupos, apontando que os envelhecimentos propostos pelo trabalho não degradaram as amostras, já para o cimento resinoso houve diminuição da resistência à flexão após 8 dias ou 10000 ciclos térmicos, sendo que a termociclagem degradou ainda mais a amostra quando comparada ao armazenamento em água por 8 dias. Para dureza Vickers na cerâmica houve decréscimo após 10000 ciclos térmicos em óleo mineral. Para dureza Knoop no cimento resinoso, os valores diminuíram conforme aumentou o número de ciclos para a termociclagem em óleo e aumentou no armazenamento em água e não houve diferença para a termociclagem em água. Dentre as limitações desse estudo concluiu-se que é necessário mais ciclos térmicos/tempo para degradar a cerâmica feldspática e que para o cimento resinoso 10000 ciclos térmicos são suficientes para apontar degradação desse material(AU)

The long-term success of dental restorations is limited by their durability in oral environments. In vitro tests remain essential to evaluate dental materials. Thermocycling is one of the most commonly used test to simulate physiological aging, to which biomaterials are exposed in clinical practice. The aim of this study was to determine the effect of thermocycling, in non-aqueous surroundings, on feldspathic ceramic and resin cement degradation comparing 3-point bending flexural strength test and mini-flexural strength test respectively. CAD-CAM feldspathic ceramic blocks were cut and polished to obtain 100 bars (14 x 4 x1.2 mm). One hundred resin cement bars were fabricated (12 x 2 x 2 mm). The bars were randomly divided into groups (n = 10) and submitted to aging using thermocycling in water (ISO 11405) and in mineral oil for 500, 5000 and 100000 cycles from 5 °C to 55 °C, with 30 s dwell time and stored in distilled water in a dry oven at 37°C for the same time as cycling occurred (9 h, 4 days and 8 days) besides the two control groups, one feldspathic ceramic and one resin cement both without aging, totaling 20 groups. After aging, the bars were fractured using 3 point bending test and mini-flexural test and the data was subjected to statistical analysis. Representative samples of each group were subjected to Knoop (resin cement) and Vickers (feldspathic ceramic) hardness test. Some of the fractured surfaces were analyzed through scanning electron microscope. No significant difference was observed between the feldspathic ceramic groups, showing that aging processes suggested in the study did not degrade the samples. As for the resin cement, a decrease in flexural strength after 8 days or 10000 thermal cycles was observed, in which thermocycling degraded the sample further when compared to the water storage for 8 days. For Vickers hardness in the ceramic there was decrease after 10000 thermal cycles in mineral oil. For Knoop hardness in the resin cement, values decreased as the number of thermal cycles for increased in oil and values increased in water storage and there was no difference for thermocycling in water. Within the limitations of the study, it is concluded that more cycles/days are necessary to degrade feldspathic ceramic and that for resin cement degradation to become evident 10000 thermal cycles are sufficient(AU)

Espectros luminosos no desenvolvimento de plântulas de Curcuma longa cultivadas in vitro / Luminous spectrum in the development of in-vitro Curcuma longa seedlings / Espectros luminosos en el desarrollo de plántulas de Curcuma longa cultivadas in vitro

Curcuma longa é uma espécie asiática perene e rizomatosa com grandes propriedades medicinais, alimentícias e ornamentais. No processo micropropagativo carece de muitas informações dentre a faixa espectral mais adequada ao seu desenvolvimento. Sendo assim, o objetivo desse trabalho foi avaliar diferentes filmes espectrais no desenvolvimento de plântulas de C. longa cultivadas in vitro. Para tanto, brotações de C. longa foram inoculadas em meio Murashig e Skoog e suplementado com 30 g/L de sacarose, 6,5 g/L de ágar 4, 44 µM/L de Benzil aminopurina (BAP) e 1,08 µM/L ácido naftaleno acético (ANA). O pH do meio foi ajustado para 5,8. Os brotos foram submetidos a diferentes intensidades e qualidade espectral de luz: branco, vermelho, amarelo, azul e verde. As plântulas foram mantidas em sala de crescimento em luz constante de 25C° e fotoperíodo de 24 horas por 145 dias. Foram avaliadas características morfológicas e anatômicas. Os resultados obtidos demonstraram que as diferenças espectraris propiciaram diferenças no desenvolvimento das plântulas de C. longa e também na contaminação in vitro. O diâmetro da base das plântulas, a massa seca e fresca da parte área e raiz foram influenciadas pelas diferentes faixas espectrais, sendo que de modo geral os filmes, amarelo e branco foram os que propiciaram maiores valores para essas características. Já o filme verde foi o que menos favoreceu o ganho de massa das plântulas, além disso, alta taxa de contaminação foi observada na presença desse filme.

Curcuma longa is a perennial Asiatic rhizomelic species with important medicinal, food and ornamental properties. However, its micro-propagation process lacks information regarding the most adequate spectral range for its development. Thus, this work aimed to evaluate different spectrum films in the development of in vitro C. longa seedlings. In order to do this, C. longa sprouts were inoculated in Murashig and Skoog media supplemented with 30 g/L of sucrose, 6.5 g/L of agar 4, 44 µM/L of benzylaminopurine (BAP) and 1.08 µM/L naphthalene acetic acid (ANA). The pH was adjusted to 5.8. Sprouts were put under different light spectrum intensity and quality: white, red, yellow, blue and green. The seedlings were maintained in a growth room with constant light at 25 ºC and a 24-hours photoperiod for 145 days. Morphological and anatomical characteristics were analyzed. The results demonstrated that spectral differences propitiated differences in the development of C. longa seedlings, and also in the in vitro contamination. The seedling base diameter, aerial and root dry and fresh mass were influenced by the different spectral ranges, with the yellow and white ranges being those that resulting the highest values for each characteristic. The green spectrum was the least favorable for the seedling regarding mass gain, as well as presenting the highest contamination rate.

Curcuma longa es una especie rizomélica asiática perenne con grandes propiedades medicinales, alimenticias y ornamentales. En el proceso de micropropagación no se dispone de informaciones sobre el rango espectral más adecuado para su desarrollo. Así, el objetivo de ese trabajo fue evaluar distintos espectros en el desarrollo de plántulas de C. longa cultivadas in vitro. Para esto, se inocularon los brotes de C. longa en medio Murashig y Skoog suplementados con 30 g/L de sacarosa, 6,5 g/L de agar 4, 44 µM/L de Benzilaminopurina (BAP) y 1,08 µM/L de ácido naftaleno acético (ANA). El pH de lo medio fue ajustado para 5.8. Los brotes fueron sometidos a diferentes intensidades y calidad de espectro luz: blanco, rojo, amarillo, azul y verde. Las plántulas se mantuvieron en un salón de crecimiento con luz constante a 25ºC y fotoperiodo de 24 horas durante 145 días. Se evaluaron características morfológicas y anatómicas. Los resultados obtenidos demostraron que las diferencias espectrales propiciaron diferencias en el desarrollo de plántulas de C. longa y sobre la contaminación in vitro. El diámetro de la base de las plántulas, la masa seca y fresca de la parte aérea y raíces fueron influenciadas por los diferentes rangos espectrales, siendo que el amarillo y el blanco fueron los que propiciaron valores más altos para esas características. La película verde fue la menos favorable a la plántula en gano de masa, además, alta tasa de contaminación se ha observado en la presencia de esa película.