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While previously the dose selection in early maximum tolerated (MTD) or dose range finding (DRF) studies was mainly based on findings in body weight, clinical observation, food consumption, clinical pathology, and feces evaluation, it can now also comprise evaluation of CNS, respiratory and cardiovascular safety assessment data. This study suggests a feasible study designs and discuss potential advantages and disadvantages considering different pharmacological activities of compounds (small vs large molecule). While classical examinations are still essential for such studies, more comprehensive physical and neurological examinations and spinal reflexes testing as well as neurobehavioral observation (modified Irwin) can be included. Furthermore, quantitative assessment of locomotor activity videos from conscious, freely moving and group housed animals can be taken by video-tracking EthoVision™ XT system and evaluated for distance moved, velocity, duration of movement, time in seconds spent per zone, duration movement in seconds per zone as necessary. Respiratory measurements include blood gas saturation and respiratory rate. For cardiovascular assessment, blood pressure as well as electrocardiogram can be monitored by minimally invasive jacketed external telemetry. Conclusion: In these study designs 4 to 5 days will leave sufficient time to decide about the next dose level of the DRF/MTD phase not only based on “classical” endpoints (e.g. food consumption, clinical observation, feces), but also on CNS, cardiovascular, and respiratory safety pharmacology endpoints. With appropriate bioanalytical methods established, the exposure can also be measured in parallel.

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NASH (Non-Alcoholic Steatohepatitis) is emerging as a real health concerning the progression from simple steatosis to more severe liver pathologies like steatohepatitis and cirrhosis. The characterization of this pivotal step using predictive biomarkers can improve the knowledge on the pathogenesis of the disease in various aspects such as modifications of lipid metabolism, inflammatory processes and development of fibrosis. As the hallmarks required for the diagnosis of NASH still rely on liver biopsy, the identification of biomarkers using a Mass Spectrometry Imaging (MSI) approach associated with histopathological evaluation has a very interesting role to play. Here, we investigated the targeted metabolite profiling in two translational mouse models of metabolism disorders (APOE*3Leiden.CETP and LDLR-/-.Leiden) exposed to various diets to assess liver histological specificities. A multimodal approach focused on various molecular classes (small metabolites & lipids) was used to describe alterations in the liver lobules in these NASH models. Potential disease or histology related biomarkers were observed especially at the level of lipids, phospholipids or lysophospholipids classes. Smaller molecules were detected such as bile acids in the portal vein area or the GSH/GSSG couple. Potential association with fibrotic and inflammatory processes has been advanced at a biological point of view. Molecular distribution was correlated with H&E (for classical histology) staining on adjacent tissue section to highlight histological specificities related to metabolites levels. In conclusion, MSI was used to achieve a better understanding of the underlying process in development of NASH by linking changes in metabolite profiles to histopathological alterations.

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Mass spectrometry Imaging (MSI) applications to ophthalmic drug discovery have recently gained growing interest especially for pharmacological or toxicological studies. MSI was applied to assess the distribution of benzalkonium chloride (BAK); a common eye drops preservative, in specific areas of the eye after instillations in animal model tissues. Preservatives have been reported to cause ocular surface disorders with tear film alteration, eye irritation and to promote dry eye. Therefore, BAK distribution was investigated in small specific histological regions of the eyes in New Zealand rabbits in order to estimate the possible localizations of adverse effects of the treatment. Local drug concentration differences were observed according to the histological area and positions on eye cryosections, at anterior, posterior, temporal or nasal sides. MSI and immunohistochemistry results were put side-by-side to correlate inflammatory areas, degradation of corneal epithelium or apoptosis phenomena, within cornea/conjunctiva regions, with BAK localizations. Moreover, an important accumulation of BAKs was observed at the sclerocorneal junction where is located the trabecular meshwork, a crucial structure involved in aqueous humor outflow. Moreover, differential analysis was carried out to find disease state biomarkers in each ocular structure. Thus, MSI offers new insight in ocular therapeutic/pharmaceutical research, especially to improve the eye distribution understanding for any new drug candidate in order to assist studies on drug efficiency or toxicity in specific tissues targeted by eye diseases.

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The author has established an organotypic culture method using adipose tissue slices (Cell Biol Int. 2015;39:1288-98). For further evaluation of its usefulness as a platform for adipose tissue research including cell responses against the drug treatment, lithium chloride, which is a GSK-3β inhibitor, was added to the culture medium along with the adipogenic stimulation by insulin, dexamethasone, and 3-isobutyl-1-methylxanthine and the histomorphological evaluation of adipose tissue slices was conducted.
In the histological evaluation of H-E-stained adipose tissue slices, small-sized multilocular adipocytes appeared between the unilocular-mature adipocytes and/or perivascular spaces following the adipogenic stimulation. With supplementation of lithium chloride, the cytoplasmic area of newly formed multilocular cells were smaller than those for cells found in the other condition and the number of multilocular adipocytes was remarkably decreased. The 3-D observation of adipose tissue slices using confocal microscopy revealed adipose differentiation of mesenchymal cells and the inhibiting effects of lithium chloride on the adipogenesis induced by adipogenic stimulation.
Based on the above, the organotypic culture method developed by the author was confirmed to be a useful in vitro research tool for the adipose tissue biology and the investigation of histomorphological changes observed in in vivo toxicology studies.

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Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Candidate Reduced Risk Products (RRPs) are being developed to reduce smoking-related health risks. We investigated over an 8-month period the effects of exposure to cigarette smoke (CS) or to the aerosol of a candidate RRP, Tobacco Heating System (THS) 2.2, on DNA methylation in the lung of apolipoprotein E-deficient mice. Cessation or switching to THS2.2 after 2 months of CS exposure were also assessed. High-throughput sequencing of bisulfite treated DNA revealed a gradual increase in the number of hypermethylated CpG loci in DNA extracted from lungs of mice exposed to CS. The number of hypermethylated CpG loci after 2 month exposure to THS2.2 (nicotine concentration matched to CS) also increased, however, hypermethylation was limited from 3 month on. Cessation or switching to THS2.2 resulted in a decrease in the number of hypermethylated CpG loci. In this mouse model initial exposure to CS or THS2.2 resulted in hypermethylation of CpG loci in lung. Continuous exposure to CS further increased the number of hypermethylated CpG loci while continuous exposure to THS2.2 reverted the CpG methylation level to the level observed in the lung of mice exposed to fresh air. Likewise, cessation or switching to THS2.2 reverted the hypermethylation of CpG loci.

Reduced Risk Products (“RRPs”) is the term we use to refer to products with the potential to reduce individual risk and population harm in comparison to smoking cigarettes.

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Gender-specific effect is widely existed, but the shortage of appropriate model restricts the study of gender-specific effect developmentally. Embryonic stem cell test (EST) has been utilized as an alternative test for developmental toxicity; despite numerous improvements to the EST, female mouse embryonic stem cells (ESC) has not been used in this test previously.
Therefore, in current study, a SP3 ESC with XX karyotype was applied to establish a “female” EST, while R1 ESC with XY karyotype was used as a “male” control, which allows testing of teratogenicity potential following chemicals exposure and their effect on each gender. Pluripotency determination and karyotype analysis were performed to assure the cellular quality of ESCs, and cardiac specific morphological and molecular endpoints were detected to monitor cardiac differentiation. A panel of test chemicals was selected for EST establishment, in which chemicals with different teratogenicity potential in vivo and without known gender-specific effect were chosen; classical microscopy observation, together with molecular endpoints Myh6 and cTnT were used to determine chemically induced embryotoxicity. The “female” EST could predict the embryotoxicity of this panel of chemicals correctly, as the “male” EST did. In addition, another three test chemicals with known gender-specific effect were assessed; an index was constructed for comparison between “male” and “female” EST, which would suggest if any gender-specific effect existed preliminarily. Endocrine disruptors with known gender-specific effect were predicted applying the “two genders” mouse EST, and their gender-specific effect could be distinguished.

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Multiple blood sampling times over a short period are often required in preclinical studies. Bioanalytical methods typically use blood volumes that are too large for repeated sampling in mice. The objective of our study was to improve blood sampling and bioanalytical techniques, allowing multiple sampling of each mouse over the full duration of the study. Sixteen untreated CD1 mice were sampled for T4 levels (6 timepoints over 24 hr) on Days 9 and 50 and on Days 22 and 38. The blood collection site was the saphenous vein. Fifty microliters of blood/timepoint was collected using a heparinised capillary. The capillary was placed in a tube for centrifugation, and spun to separate the plasma. The sample processing was by solid phase extraction followed by mass spectroscopy. Overall, T4 plasma levels ranged from 9 to 69 ng/ml. The results indicate that a slight decrease in T4 concentrations appears at the beginning of the nocturnal cycle. Levels of T4 showed individual variation throughout the day, and inter-individual variations during the study. In conclusion, our laboratory has developed a successful approach to sampling blood in mice on multiple occasions over a 24-hour period combined with a bioanalytical method with volumes as low as 10 uL of plasma. The results indicated circadian variation in T4 levels in mice between age 6-13 weeks. This approach is in line with 3R principles and could reduce the number of animals utilized for bioanalysis in toxicology studies.

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The Bovine Corneal Opacity and Permeability Assay (BCOP) is an ex vivo assay, which may be used to assess the eye irritation potential of new chemicals and finished products. The BCOP assay has been accepted by several regulatory agencies for the identification of severe and corrosive ocular irritants, replacing the rabbit eye test. As per the TG OECD 437 for eye irritation (BCOP assay), non-surfactant solid materials are typically tested as 20% dilutions prepared in 0.9% sodium chloride solution or other solvent that has been demonstrated to have no adverse effects on the test system. However, the limited solubility of some chemicals adds technical challenges in finding a suitable vehicle that would ensure the material’s availability to the excised corneas without affect the test system. In this study, we evaluated three solvents in the BCOP assay: normal saline, olive oil and propylene glycol. Based on the available classification systems, our preliminary data showed that water and olive oil were predicted as non-irritants, while propylene glycol was predicted as a mild irritant. To demonstrate the influence of the type of solvent on the outcome/prediction of the BCOP assay for solid materials, we tested a 20% suspension of dicamba prepared in these solvents. Previous animal tests have reported corrosive effect of dicamba. Our results demonstrated that when mixed in normal saline and corn oil dicamba was predicted to be a corrosive, while it was predicted to be a moderate irritant when mixed in propylene glycol. These results support the need for further investigation of the solvent’s influence in the BCOP assay to allow the correct prediction of the irritation potential of solid materials.

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