Ultrastructural analysis in preclinical safety evaluation.

The first electron microscopic images of biological specimens were made in the 1940s, and the next 30 years comprised an era of descriptive ultrastructure during which transmission electron microscopy (TEM) was integral to an explosion in cellular and molecular biology. However, when questions could no longer be answered by ultrastructural information alone, the use of TEM in biological research declined. Innovative molecular techniques and newer imaging technologies such as confocal fluorescence microscopy filled the gap, providing faster answers with less rigorous training as a prerequisite to data collection. The use of TEM in toxicologic pathology has paralleled the rise and fall of its popularity in other disciplines. However, TEM remains an essential resource that provides direct and unequivocal data to explain and address safety concerns in preclinical toxicity studies. There is still an important place for TEM in preclinical safety evaluation and mechanistic studies, particularly when visualization of subcellular structures provides a link to other endpoints. This review reinforces the value of TEM in preclinical safety testing and model development and encourages best practices for ultrastructural evaluation.

Asymptomatic macrothrombocytopenia in a young pure-bred beagle dog: a case report.

During baseline evaluation prior to a preclinical safety study, a 10-month-old male pure-bred Beagle dog was found to have marked thrombocytopenia (6 × 10(3) platelets [PLT]/µL) associated with a mean platelet volume (MPV) of 17.9 fL. Tests for Rickettsia rickettsii, Ehrlichia canis, and Borrelia burgdorferi were negative. Buccal bleeding time was normal. Over 3 months, PLT were 4 to 141 × 10(3) PLT/µL, and MPV was 11.4 to 25.1 fL; however, PLT were <50 × 10(3) PLT/µL and MPV was >16 fL during most of this period. Antinuclear antibody (ANA) and anti-PLT antibody tests were negative. Genotyping for the presence of a beta 1-tubulin mutation demonstrated the normal wild-type gene. Treatment with prednisone resulted in normal values after only 3 days. Ultrastructure of enlarged PLT was consistent with that of immature PLT, characterized by reduced numbers of peripheral microtubules and the presence of rough endoplasmic reticulum, free ribosomes, Golgi apparatus, and a prominent canalicular system. PLT ultrastructure and glucocorticoid responsiveness supported a diagnosis of immune-mediated thrombocytopenia that was masked by the cyclic nature of PLT decreases and lack of clinical signs. Inclusion of such a dog in a preclinical safety study could result in misinterpretation of clinical pathology findings.

ABT-751, a novel tubulin-binding agent, decreases tumor perfusion and disrupts tumor vasculature.

ABT-751 is an orally bioavailable tubulin-binding agent that is currently under clinical development for cancer treatment. In preclinical studies, ABT-751 showed antitumor activity against a broad spectrum of tumor lines including those resistant to conventional chemotherapies. In this study, we investigated the antivascular properties of ABT-751 in a rat subcutaneous tumor model using dynamic contrast-enhanced magnetic resonance imaging. A single dose of ABT-751 (30 mg/kg, intravenously) induced a rapid, transient reduction in tumor perfusion. After 1 h, tumor perfusion decreased by 57% before recovering to near pretreatment levels within 6 h. In contrast, ABT-751 produced little change in muscle perfusion at either time point. To further elucidate mechanisms of drug action at the cellular level, we examined the effects of ABT-751 on endothelial cells using an in-vitro assay. ABT-751, at concentrations corresponding to plasma levels achieved in vivo, caused endothelial cell retraction and significant loss of microtubules within 1 h. The severity of these morphological changes was dose-dependent but reversible within 6 h after the discontinuation of the drug. Taken together, these results show that ABT-751 is a tubulin-binding agent with antivascular properties. Microtubule disruption and morphological changes in vascular endothelial cells may be responsible, at least in part, for the dysfunction of tumor blood vessels after ABT-751 treatment.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

Computer-assisted imaging algorithms facilitate histomorphometric quantification of kidney damage in rodent renal failure models.

INTRODUCTION: Surgical 5/6 nephrectomy and adenine-induced kidney failure in rats are frequently used models of progressive renal failure. In both models, rats develop significant morphological changes in the kidneys and quantification of these changes can be used to measure the efficacy of prophylactic or therapeutic approaches. In this study, the Aperio Genie Pattern Recognition technology, along with the Positive Pixel Count, Nuclear and Rare Event algorithms were used to quantify histological changes in both rat renal failure models. METHODS: Analysis was performed on digitized slides of whole kidney sagittal sections stained with either hematoxylin and eosin or immunohistochemistry with an anti-nestin antibody to identify glomeruli, regenerating tubular epithelium, and tubulointerstitial myofibroblasts. An anti-polymorphonuclear neutrophil (PMN) antibody was also used to investigate neutrophil tissue infiltration. RESULTS: Image analysis allowed for rapid and accurate quantification of relevant histopathologic changes such as increased cellularity and expansion of glomeruli, renal tubular dilatation, and degeneration, tissue inflammation, and mineral aggregation. The algorithms provided reliable and consistent results in both control and experimental groups and presented a quantifiable degree of damage associated with each model. CONCLUSION: These algorithms represent useful tools for the uniform and reproducible characterization of common histomorphologic features of renal injury in rats.

AhR activation underlies the CYP1A autoinduction by A-998679 in rats.

Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 [3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile], was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity.

Comparison of the pharmacological effects of paricalcitol and doxercalciferol on the factors involved in mineral homeostasis.

Vitamin D receptor agonists (VDRAs) directly suppress parathyroid hormone (PTH) mRNA expression. Different VDRAs are known to have differential effects on serum calcium (Ca), which may also affect serum PTH levels since serum Ca regulates PTH secretion mediated by the Ca-sensing receptor (CaSR). In this study, we compared the effects of paricalcitol and doxercalciferol on regulating serum Ca and PTH, and also the expression of PTH, VDR, and CaSR mRNA. The 5/6 nephrectomized (NX) Sprague-Dawley rats on a normal or hyperphosphatemia-inducing diet were treated with vehicle, paricalcitol, or doxercalciferol for two weeks. Both drugs at the tested doses (0.042-0.33 mug/kg) suppressed PTH mRNA expression and serum PTH effectively in the 5/6 NX rats, but paricalcitol was less potent in raising serum Ca than doxercalciferol. In pig parathyroid cells, paricalcitol and the active form of doxercalciferol induced VDR translocation from the cytoplasm into the nucleus, suppressed PTH mRNA expression and inhibited cell proliferation in a similar manner, although paricalcitol induced the expression of CaSR mRNA more effectively. The multiple effects of VDRAs on modulating serum Ca, parathyroid cell proliferation, and the expression of CaSR and PTH mRNA reflect the complex involvement of the vitamin D axis in regulating the mineral homeostasis system.