Barbara Lenz

Neonatal lethality in mice deficient in XCE, a novel member of the endothelin-converting enzyme and neutral endopeptidase family

XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in specific areas of the central nervous system including spinal chord and medulla. To elucidate the importance and function of XCE, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in XCE. The resulting phenotype is characterized by neonatal lethality. All XCE −/− homozygous mice died of respiratory failure shortly after birth, and in most cases their lungs were never ventilated. Apart from the atelectasis, anatomical and histological examinations of embryonic day 18.5 XCE −/− embryos and newborn homozygotes did not reveal any obvious abnormalities in organs and tissues. Malformations that are related to the knock-out were also not found in the skeletons of XCE −/− mice. In addition, XCE knock-out animals showed no deficiency of pulmonary surfactant proteins and had normal heart beat frequencies. Taken together, our results demonstrate that XCE is an essential gene. The phenotype of the XCE-deficient mice together with the central nervous system-specific expression further suggest that XCE may play a vital role in the control of respiration.

In silico assay for assessing phospholipidosis potential of small druglike molecules: training, validation, and refinement using several data sets.

Phospholipidosis (PLD) is a lysosomal storage disorder induced by compounds, notably cationic amphiphilic drugs, which although reversible interferes with cellular phospholipids.The in silico method described utilizes the amphiphilic moment ΔΔG(AM) (kJ/mol) together with basic pK(a) values to assign PLD inducing potential to a compound. The new model was accurate and sensitive (85% and 82%, respectively) when compared to other data sets. Therefore, the parallel in vitro assay for PLD was discontinued. The data reinforce our view that the amphiphilic moment is far more informative for determining a compounds potential to induce PLD than the combined use of basic pK(a) and ClogP values.

Characterizing “Adversity” of Pathology Findings in Nonclinical Toxicity Studies Results from the 4th ESTP International Expert Workshop

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

β-Carotene-Induced Changes in RARβ Isoform mRNA Expression Patterns Do Not Influence Lung Adenoma Multiplicity in the NNK-Initiated A/J Mouse Model

Abstract: A number of epidemiological studies have reported associations of β-carotene plasma levels or intake with decreased lung cancer risk. However, intervention studies in smokers reported increased lung tumor rates after high long-term β-carotene supplementation. For insight into these conflicting results, we studied the influence of β-carotene on tobacco smoke carcinogen-induced lung cancer development in the A/J-mouse using 4-(N-Methyl-N-nitro samino)-1-(3-pyridyl)-1-butanone (NNK) as the initiator and lung adenoma multiplicity as the functional endpoint. Gene regulation of the putative tumor suppressor RARβ in mouse lung was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for its relevance in predicting the endpoint of lung cancer. A/J-mice achieved plasma β-carotene levels of up to 3 μmol/L within 4 wk and up to 6 μmol/L after 6 mo of supplementation on a diet modified to enhance β-carotene absorption. Despite high lung β-carotene concentrations of up to 6 μmol/kg, tumor multiplicity was not significantly affected by the β-carotene treatment, either in carcinogen-initiated or non-initiated mice, and was unrelated to β-carotene dose and the time point of treatment during cancer formation. Tumor multiplicity did not correlate with β-carotene plasma levels in NNK-treated animals. All RARβ isoforms were significantly suppressed in the lungs of NNK- and NNK plus high dose β-carotene-treated animals. However, the number of tumors per mouse did not correlate with the RARβ-isoform expression levels. β-carotene alone after 3 mo of supplementation mildly but significantly increased levels of RARβ1, β2, and β4. This increase persisted for 6 mo for RARβ2 and β4. In summary, we found no effect of β-carotene on tumor formation in the NNK-initiated A/J-mouse lung cancer model with respect to dose or time point of treatment. β-Carotene-induced changes in RARβ isoform gene expression levels were not predictive for the number of lung tumors but were indicative of intact β-carotene metabolism and persistent sensitivity to retinoic acid in the mice. Down-regulation of RARβ in NNK-induced adenoma-bearing lungs was similar to that observed in human lung cancer and further confirms the A/J-mouse as a valuable model for lung carcinogenesis.

Increased levels of urinary phenylacetylglycine associated with mitochondrial toxicity in a model of drug-induced phospholipidosis

Background: Phospholipidosis (PLD) is a lysosomal storage disorder induced by a class of cationic amphiphilic drugs. However, drug-induced PLD is reversible. Evidence of PLD from animal studies with some compounds has led to discontinuation of development. Regulatory authorities are likely to request additional studies when PLD is linked to toxicity. Objective: We conducted a trial to investigate urinary phenylacetylglycine (uPAG) as a biomarker for PLD. Materials and methods: Five groups of 12 male Wistar rats were dosed once with vehicle, 300 mg/kg or 1500 mg/kg of compound A (known to induce PLD), or 300 mg/kg or 1000 mg/kg of compound B (similar structure, but does not induce PLD) to achieve similar plasma exposures. Following dosing, urine and blood samples underwent nuclear magnetic resonance (NMR), proteomic, and biochemical analyses. Necropsies were performed at 48 and 168 h, organ histopathology evaluated, and gene expression in liver analyzed by microarray. Electron microscopic examination of peripheral lymphocytes was performed. Results: For compound A, uPAG increased with dose, correlating with lamellar inclusion bodies formation in peripheral lymphocytes. NMR analysis showed decreased tricarboxylic acid cycle intermediates, inferring mitochondrial toxicity. Mitochondrial dysfunction was suggested by uPAG increase, resulting from a switch to anaerobic metabolism or disruption of the urea cycle. Discussion and conclusion: uPAG shows utility as a noninvasive biomarker for mitochondrial toxicity associated with drug-induced PLD, providing a mechanistic hypothesis for toxicity associated with PLD likely resulting from combined direct and indirect mitochondrial toxicity via impairment of the proton motor force and alteration of fatty acid catabolism.

Modulation of Oral Squamous Cell Carcinoma Incidence in Rats Via Diet and a Novel Calcium Channel Antagonist

An unexpected dose related increase in oral squamous cell carcinomas was observed in a standard 2-year carcinogenicity study with a novel calcium channel blocker, in which Wistar rats received daily doses of 0, 1.5, 7, 20, or 40 mg/kg of the compound mixed with a standard diet containing fibers from barley. This finding was associated with an increased incidence of severe (destructive) periodontitis and the formation of oro-nasal fistulae at the 2 highest doses. Five assays of the compound for genotoxicity were negative indicating that a genotoxic effect was highly improbable. To investigate the underlying pathogenic mechanisms a second 2-year study in the same strain of rats was initiated and the influence of the diet and/or a possible local irritancy by the drug was assessed. In this second study the compound was administered by oral gavage at daily doses of 0, 7, or 40 mg/kg (later reduced to 20 mg/kg due to systemic intolerance) to rats maintained either on the standard diet or on a low fiber diet assumed to be less aggressive in terms of inducing periodontal lesions. Dose dependent gingival overgrowth (a class-related effect) was observed in the incisor and molar teeth area of all treated groups but was independent of the diet used. No oral tumors were found in the standard diet or low fiber diet controls and all treatment groups fed the low fiber diet, whereas in the high-dose group fed the standard diet a total of 8 oral squamous cell carcinomas were detected in association with an increased incidence of severe periodontitis. These results indicate that the increased incidence of squamous cell carcinomas observed upon chronic administration of the compound is not due to a direct tumorigenic effect of the drug. Tumor formation is attributable to severe periodontal disease favored by the diet and class related gingival overgrowth.

The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

&NA; Non‐human primates (NHPs) are currently considered to be the non‐rodent species of choice for the preclinical safety assessment of single‐stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid‐based SSOs (3 antisense and 1 anti‐miR), all with known safety profiles, were administered to minipigs using similar study designs and read‐outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in‐life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose‐dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high‐dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off‐target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non‐rodent species in SSO candidate non‐clinical safety packages.

Application of Imaging Techniques to Cases of Drug-Induced Crystal Nephropathy in Preclinical Studies

A number of drugs can cause precipitates within renal tubules leading to crystal nephropathy. Crystal nephropathy is usually an exposure-related finding and is not uncommon in preclinical studies, where high doses are tested. An understanding of the nature of precipitates is important for human risk assessment and further development. Our aim was to investigate the ability of various imaging techniques to detect the presence of drugs or metabolites in renal crystals. We applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) imaging, Raman and infrared microspectroscopy, scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and standard histopathology to cases of drug-induced crystal nephropathy, induced in rodents and primates by 4 compounds. MALDI-FTICR MS imaging enabled the identification of the drug-related crystal content in all 4 cases of nephropathy, without reference material and with high accuracy. Crystals were composed of unchanged parent drug and/or metabolites. Similar results were obtained using Raman and infrared microspectroscopy for 2 compounds. In the absence of reference standards of metabolites, Raman and infrared microspectroscopy showed that the crystals consisted of components similar, but not identical, to the administered drug for the other compounds, a limitation for these techniques. SEM/EDX showed which counter ions were colocalized with the identified drug-related material, complementing the MALDI-FTICR MS findings. Therefore, we recommend MALDI-FTICR MS as a first-line methodology to characterize crystal nephropathies. Raman and infrared microspectroscopy may be useful when MALDI-FTICR MS imaging cannot be applied. SEM/EDX could be considered as a complementary technology.

Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop:

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.