The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected for further development.

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Alveolar macrophage (AM) responses are commonly induced in inhalation toxicology studies, typically being observed as an increase in number or a vacuolated 'foamy' morphology. Discriminating between adaptive AM responses and adverse events during nonclinical and clinical development is a major scientific challenge. When measuring and interpreting induced AM responses, an understanding of macrophage biology is essential; this includes 'sub-types' of AMs with different roles in health and disease and mechanisms of induction/resolution of AM responses to inhalation of pharmaceutical aerosols. In this context, emerging assay techniques, the utility of toxicokinetics and the requirement for new biomarkers are considered. Risk assessment for nonclinical toxicology findings and their translation to effects in humans is discussed from a scientific and regulatory perspective. At present, when apparently adaptive macrophage-only responses to inhaled investigational products are observed in nonclinical studies, this poses a challenge for risk assessment and an improved understanding of induced AM responses to inhaled pharmaceuticals is required.

SummaryPrior to controlled clinical trial s in human volunteers or patients it is required that novel pharmaceuticals are evaluat ed for pre-clinical safet y in a rodent and a non-rodent (`second') species. In most cases the rodent species used has been the rat and the second species has been the dog or macaque (usually cynomolgus or rhesus) monkey. However, there is an increasing trend within the United Kingdom (UK) pharm aceutical industry to use the common marm oset (C a llith rix ja cc h us) for pre-clinical toxicology programmes.T his paper examines the practicality of using the common marm oset (henceforth referred to as`the marm oset') in toxicological testing and reviews metabolic and pharmacodynam ic similarities between this species and humans. It then discusses some of the advan tages and disadvantages of the use of this species when compared with two other alternat ives to the dog and macaque, nam ely the ferret and minipig. In particular, the marm oset has clear advantages over the macaque in terms of animal welfare and practicality. T here is regulatory acceptance of this species for Investigational New Drug (IND ), Clinical Trial Exemption (CT X), New Drug Application (NDA) and Mark eting Authorization Application (MAA) registrat ions. Whilst the dog is likely to be maintained as the primary non-rodent species in toxicology, the marm oset has been, and will likely continue to be, adopted as an additional non-rodent species in pre-clinical toxicology program mes where appropriat e.

Contrast-enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T 1 -weigthed 3D pulse sequences were acquired before and after administration of Gd-DTPA-albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I-II) or in the core (zones III-IV) of the tumor itself. Significant differences (p<0.001) were found in the values of Kps and fpv of zones I-II with respect to zones III-IV. In the peripheral rim, permeability was significantly higher (p<0.01) in the muscle-peripheral region (zone I) with respect to the skin-peripheral region (zone II). In areas with high Kps, histological and ultrastructural examination revealed clusters of newly formed vessels and signs of intense permeability. Numerous vascular vesicular organs were visible in these areas. In the tumoral core, analysis of the microcirculatory parameters revealed regions with mild permeability (zone III) and regions with negligible permeability (zone IV). These 2 zones were discriminated by the average value of Kps (p<0.05), while their fpv was not significantly different. Upon histological examination, the tumoral core exhibited necrotic areas; CD31 immunocytochemistry exhibited that it was diffusely hypovascularized with large avascular areas. Upon ultrastructural examination, capillaries were rarely visible and exhibited signs of endothelial cell damage. The results suggest that segmentation based on microvascular parameters detects in vivo zones characterized by immunocytochemical and ultrastructural aspects of intense angiogenesis. The finding that a certain amount of contrast agent penetrates in the tumoral core suggests that high oncotic and hydrostatic pressure only partially hinders the penetration of macromolecules. Tumor-induced angiogenesis plays an important role in tumor growth and there is a significant interest in attacking the tumor vasculature using anti-angiogenesis or vascular targeting therapies. 1,2 The study of the vascular function in tumors is hampered by the lack of an in vivo model of the complex microenvironment present in the tumor stroma. New magnetic resonance imaging (MRI)-based methods allow morphological and functional evaluation of the microvascular bed in living animals at high spatial/ temporal resolution. An interesting approach is dynamic contrastenhanced MRI (DCE-MRI), which detects peculiar properties of vascular beds by virtue of the differential distribution of contrast media in normal and pathological regions. 3,4 MRI enhanced with small molecular contrast media (Ͻ1 kDa) is currently being used to define tumor microvasculature...

PurposeProgress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.MethodsHuman (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation.ResultsCell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.ConclusionsA high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Within drug development and pre-clinical trials, a common, significant and poorly understood event is the development of drug-induced lipidosis in tissues and cells. In this manuscript, we describe a mass spectrometry imaging strategy, involving repeated analysis of tissue sections by DESI MS, in positive and negative polarities, using MS and MS/MS modes. We present results of the detected distributions of the administered drug, drug metabolites, lipid molecules and a putative marker of lipidosis, di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate (di-22:6-BMP). A range of strategies have previously been reported for detection, isolation and identification of this compound, which is an isomer of di-docosahexaenoic (22:6 n-3) phosphatidylglycerol (di-22:6 PG), a commonly found lipid that acts as a surfactant in lung tissues. We show that MS imaging using MS/MS can be used to differentiate these compounds of identical mass, based upon the different distributions of abundant fragment ions. Registration of images of these fragments, and detected drugs and metabolites, is presented as a new method for studying drug-induced lipidosis in tissues. Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00216-019-02151-z) contains supplementary material, which is available to authorized users.

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