Integration of the rat liver micronucleus assay into a 28-day treatment protocol: testing the genotoxicity of four small-molecule nitrosamines with different carcinogenic potencies and tumor target specificities.

Several potent carcinogenic nitrosamines, including N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), induce micronuclei in the micronucleated hepatocyte (MNHEP) assay but not in the micronucleated reticulocyte (MNRET) assay. However, the MNHEP assay is not as frequently used as the MNRET assay for evaluating in vivo genotoxicity. The present study evaluated MN formation in the liver of Big Blue transgenic rats exposed to four small-molecule nitrosamines, NDMA, N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisoporpylamine (NEIPA), and N-nitrosomethylphenylamine (NMPA), using a repeat-dose protocol typically used for in vivo mutagenicity studies.

Mutagenicity and genotoxicity evaluation of 15 nitrosamine drug substance-related impurities in human TK6 cells.

Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT).

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test.

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used β-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems.

Appropriate in vivo follow-up assays to an in vitro bacterial reverse mutation (Ames) test positive investigational drug candidate (active pharmaceutical ingredient), drug-related metabolite, or drug-related impurity.

Kirkland et al. [Mutation Research/Genetic Toxicology and Environmental Mutagenesis 847 (2019) 403035, https://doi.org/10.

The US Food and Drug Administration's Perspective on the New Antipsychotic Pimavanserin.

Objective: To summarize the US Food and Drug Administration's (FDA's) review of the safety and effectiveness for pimavanserin, an atypical antipsychotic, for the treatment of hallucinations and delusions associated with Parkinson's disease psychosis. We describe the regulatory and clinical issues important to the FDA's approval of this New Drug Application, with special focus on the risk-benefit balance. We also describe a new labeling feature that presents additional efficacy data to clinicians.

Interpretation and considerations on the safety evaluation of human drug metabolites.

The final Food and Drug Administration guidance on the safety testing of drug metabolites was published in February 2008. Discussions of the role and applications of this guidance were addressed at several public scientific meetings over the past year. One of the main differences between the draft and the finalized guidance is that in the latter, the human metabolite level was correlated to the parent drug level in plasma, whereas this parameter was considered in relationship to administered dose or total exposure in the draft guidance.

Current and future application of genetic toxicity assays: the role and value of in vitro mammalian assays.

With the advent of new technologies (e.g., genomics, automated analyses, and in vivo monitoring), new regulations (e.

A regulatory perspective on issues and approaches in characterizing human metabolites.

This document captures the current thinking within FDA/CDER on the non-clinical safety assessment of human drug metabolites in new drug products. Examples are provided, which define a scientific based approach to the safety evaluation of human metabolites in new drug candidates. A discussion of the need for, and the adequacy of, the assessment of human drug metabolites with specific regard to their potential as mediators of toxicity is presented from a regulatory perspective.

Challenges and lessons learned since implementation of the safety pharmacology guidance ICH S7A.

The International Conference on Harmonization, Topic S7A guidance (ICH S7A) on safety pharmacology for human pharmaceuticals has been in effect for 3 years in Europe, the United States and Japan. Surveys of the pharmaceutical industry, regulatory agencies and the audience attending the 4th Annual Meeting of the Safety Pharmacology Society have helped identify and address areas of controversy, as well as those challenges that have emerged since implementation of the guidance worldwide. Overall, ICH S7A has been successfully implemented.

Regulatory science: a special update from the United States Food and Drug Administration: Preclinical issues and status of investigation of botanical drug products in the United States.

A recent survey was conducted across the therapeutic divisions within the CDER, U.S. FDA regarding the number of submissions related to botanical drug products over the past ten years.

Increased Na(+)-K+ pump number and decreased pump activity in soleus muscles in SHR.

We have previously demonstrated electrophysiological and contractile abnormalities in soleus muscles of the spontaneously hypertensive rat (SHR). The age-related decrease in force and fatigue resistance observed in SHR muscles may be produced by alterations in sarcolemmal ion conductance and/or Na+ pump function. The experiments reported in the present paper were designed to assess the functional capacity of the Na+ pump in 6- to 8- and 24- to 28-wk-old SHR and Wistar-Kyoto (WKY) soleus muscles and to correlate pump activity with Na+ pump number and binding affinity ([3H]ouabain binding).

Development of soleus muscles in SHR: relationship of muscle deficits to rise in blood pressure.

Skeletal muscles from 24- to 28-wk-old spontaneously hypertensive rats (SHR) exhibit decreased contractile capacity and resistance to fatigue. The present study was designed to determine the age at which these deficits first appear and their relationship to the development and progression of the rise in blood pressure. SHR soleus was significantly weaker than age-matched Wistar-Kyoto (WKY) soleus at all ages studied, but resistance to fatigue varied with age.

Hypomagnesemia and isoproterenol cardiomyopathies: Protection by probucol.

Chronic magnesium deficiency is associated with injury of heart muscle, blood vessels, and neuronal tissue. Despite its clinical significance, the mechanism of magnesium deficiency-induced damage remains unclear. The myocardial necrosis induced by injecting catecholamines, which is augmented by magnesium deficiency, is thought to involve a free radical component through catecholamine autoxidation.

Antioxidants and the cardiomyopathy of Mg-deficiency.

For several decades the animal models of Mg-deficiency have been studied with particular attention to the cardiomyopathy that develops due to dietary deficiency. In recent years we have studied the effects of nutrients and drugs with antioxidant properties on the development of the cardiomyopathy. We have found that treatment of the Mg-deficient animals with alpha-tocopherol, a naturally-occurring antioxidant, significantly diminishes the number and size of lesions.

Apparent upregulation of Na+,K+ pump sites in SHR skeletal muscle with reduced transport capacity.

Slow-twitch, oxidative skeletal muscles in SHR exhibit several physiological defects, including a reduced ability to maintain force during high frequency repetitive stimulation (1). Muscle fatigue may be produced by one of a variety of factors acting at different levels of the neuromuscular system. Several lines of evidence, however, suggest that SHR soleus fatigues more rapidly than WKY soleus because SHR muscles allow more K+ to accumulate in the extracellular space during repetitive muscle activity.

Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine.

The influx of Ca ions into the presynaptic nerve terminal through ion channels is a key link between the action potential and the release of chemical transmitters. It is not clear, however, which types of Ca channel are involved in neurosecretion at vertebrate synapses. In particular, there is disagreement as to whether these channels are sensitive to dihydropyridine blockers, characteristic of L-type Ca channels.

Magnesium deficiency-induced cardiomyopathy: protection by vitamin E.

Syrian male hamsters weighing 80-100g were placed on either a magnesium deficient diet (MgD) or an identical diet supplemented with 100 mmols/Kg MgCl. Animals from each group received vitamin E 10, 15, and 25mg three-week slow release pellets, as subcutaneous implants. The animals were sacrificed after 14 days and their hearts isolated for morphological analysis.

Beta-receptor properties in soleus muscles from spontaneously hypertensive rats.

We have compared the properties of beta-adrenergic receptors in slow-twitch, oxidative skeletal muscles (soleus) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at three different ages. The investigation was based on the hypothesis that the increase in Na+ content and decrease in fatigue resistance observed previously in the soleus of SHR might be the result of a down regulation of muscle beta-receptors. Activation of beta-adrenergic receptors in skeletal muscle stimulates sarcolemmal sodium-potassium adenosine triphosphatase, which produces an efflux of Na+ and an influx of K+.