Toxicology Species Research Articles

The Current State of Biotransformation Science - Industry Survey of In Vitro and In Vivo Practices, Clinical Translation, and Future Trends.

Embedded within the field of drug metabolism and pharmacokinetics (DMPK), biotransformation is a discipline that studies the origins, disposition, and structural identity of metabolites to provide a comprehensive safety assessment, including the assessment of exposure coverage in toxicological species. Spanning discovery and development, metabolite identification (metID) scientists employ various strategies and tools to address stage-specific questions aimed at guiding the maturation of early chemical matter into drug candidates. During this process, the identity of major (and minor) circulating human metabolites is ascertained to comply with the regulatory requirements such as the Metabolites in Safety Testing (MIST) guidance. Through the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the "Translatability of MetID In Vitro Systems Working Group" was created within the Translational and ADME Sciences Leadership Group. The remit of this group was to objectively determine how accurate commonly employed in vitro systems have been with respect to prediction of circulating human metabolites, both qualitatively and quantitatively. A survey composed of 34 questions was conducted across 26 pharmaceutical companies to obtain a foundational understanding of current metID practices, preclinically and clinically, as well as to provide perspective on how successful these practices have been at predicting circulating human metabolites.The results of this survey are presented as an initial snapshot of current industry-based metID practices, including our perspective on how a harmonized framework for the conduct of in vitro metID studies could be established. Future perspectives from current practices to emerging advances with greater translational capability are also provided.

Utility of common in vitro systems for predicting circulating metabolites.

In vitro systems such as cultured hepatocytes are used early in drug development as a proxy for in vivo data to predict metabolites in human and the potential pre-clinical species. These data support preclinical species selection for toxicology studies as well as provide early evidence for potential active and reactive metabolites that can be generated in human. While in vivo data would be best to select preclinical species for a given compound, only in vitro systems are available when selecting tox species. However, as with any in vitro system, the correlation to actual in vivo results can be variable. Understanding the predictivity of a given in vitro assay for in vivo metabolism would help drug development teams appreciate the significance of early cross-species metabolite profiles relative to the eventual clinical outcomes. In a retrospective analysis of historic metabolite profiling data from Abbott/AbbVie, in vitro systems predicted ~50% of circulating metabolites present in vivo, across preclinical species and human, with no correlation between apparent exposures in vitro vs in vivo A direct comparison of five common in vitro systems using commercial compounds with known metabolism resulted in suspension hepatocytes and co-cultured hepatocytes slightly outperforming the other systems in successfully generating major human circulating metabolites. Current in vitro systems have value early in development when in vivo studies are not feasible and are required for regulatory filings to support pre-clinical toxicology species selection but should not be treated as wholly representative of a given drug's in vivo metabolism. Significance Statement This is a comprehensive assessment of historic metabolism data quantitating the success rate of in vitro to in vivo predictivity. Reliability of in vitro systems for metabolite profiling is important for early drug development, and understanding predictivity will help give appropriate context to the data. New data were also generated to compare common in vitro liver models to determine whether any could be definitively identified as more predictive of human circulating metabolites than others.

Determination of toxicological relevant arsenic species in urine by hydride generation microwave-induced plasma optical emission spectrometry

An analytical method for the determination of toxicological relevant species of arsenic in urine was developed and validated using hydride generation microwave-induced emission spectrometry (HG-MP-AES). This strategy can be used as an alternative to HG-HPLC-ICP-MS considered as a reference technique for arsenic speciation. This procedure is notably less expensive than other techniques and sample preparation and requires only a few steps.•Hydride generation with MP-AES detection has proven to be an effective technique for measuring arsenic metabolites in urine, which is relevant for occupational monitoring and health risk assessment purposes.•This method offers simplicity and cost-effectiveness, serving as an alternative to classical analytical procedures typically used for arsenic analysis in urine.•The methodology has been successfully applied for the purpose of workers' health surveillance.

Using a Novel Multiplexed Algal Cytological Imaging (MACI) Assay and Machine Learning as a Way to Characterize Complex Phenotypes in Plant-Type Organisms.

High-throughput phenotypic profiling assays, popular for their ability to characterize alternations in single-cell morphological feature data, have been useful in recent years for predicting cellular targets and mechanisms of action (MoAs) for different chemicals and novel drugs. However, this approach has not been extensively used in environmental toxicology due to the lack of studies and established methods for performing this kind of assay in environmentally relevant species. Here, we developed a multiplexed algal cytological imaging (MACI) assay, based on the subcellular structures of the unicellular microalgae, Raphidocelis subcapitata, a toxicology and ecological model species. Several different herbicides and antibiotics with unique MoAs were exposed to R. subcapitata cells, and MACI was used to characterize cellular impacts by measuring subtle changes in their morphological features, including metrics of area, shape, quantity, fluorescence intensity, and granularity of individual subcellular components. This study demonstrates that MACI offers a quick and effective framework for characterizing complex phenotypic responses to environmental chemicals that can be used for determining their MoAs and identifying their cellular targets in plant-type organisms.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

Metabolism and Disposition of [14C]Pevonedistat, a First-in-Class NEDD8-Activating Enzyme Inhibitor, after Intravenous Infusion to Patients with Advanced Solid Tumors.

Metabolism and disposition of pevonedistat, an investigational, first-in-class inhibitor of the NEDD8-activating enzyme (NAE), were characterized in patients with advanced solid tumors after intravenous infusion of [14C]pevonedistat at 25 mg/m2 (∼60-85 μCi radioactive dose). More than 94% of the administered dose was recovered, with ∼41% and ∼53% of drug-related material eliminated in urine and feces, respectively. The metabolite profiles of [14C]pevonedistat were established in plasma using an accelerator mass spectrometer and excreta with traditional radiometric analysis. In plasma, unchanged parent drug accounted for approximately 49% of the total drug-related material. Metabolites M1 and M2 were major (>10% of the total drug-related material) circulating metabolites and accounted for approximately 15% and 22% of the drug-related material, respectively. Unchanged [14C]pevonedistat accounted for approximately 4% and 17% of the dose in urine and feces, respectively. Oxidative metabolites M1, M2, and M3 appeared as the most abundant drug-related components in the excreta and represented approximately 27%, 26%, and 15% of the administered dose, respectively. Based on the unbound plasma exposure in cancer patients and in vitro NAE inhibition, the contribution of metabolites M1 and M2 to overall in vivo pharmacological activity is anticipated to be minimal. The exposure to these metabolites was higher at safe and well tolerated doses in rat and dog (the two preclinical species used in toxicology evaluation) plasma than that observed in human plasma. Reaction phenotyping studies revealed that CYP3A4/5 are primary enzymes responsible for the metabolic clearance of pevonedistat. SIGNIFICANCE STATEMENT: This study details the metabolism and clearance mechanisms of pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, after intravenous administration to patients with cancer. Pevonedistat is biotransformed to two major circulating metabolites with higher exposure in nonclinical toxicological species than in humans. The pharmacological activity contribution of these metabolites is minimal compared to the overall target pharmacological effect of pevonedistat. Renal clearance was not an important route of excretion of unchanged pevonedistat (∼4% of the dose).

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 1013 vg/kg) and day 14 in monkeys (≥3.3 × 1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Abstract 1148: SynOV1.1: A synthetic gene circuit controlled oncolytic adenovirus demonstrating high tumor specificity, potent antitumor efficacy and high synergy with an anti-PD-L1 monoclonal antibody in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is yet the fourth most common cause of cancer-related death although its treatment has been significantly advanced in recent years. Oncolytic viruses are among a novel class of immunotherapies that have shown promise to HCC patients with convincing abilities to selectively replicate in and kill cancer cells as well as to boost tumor immunogenicity. In order to further enhance the safety and efficacy of oncolytic viruses, SynOV1.1, a recombinant replication-competent serotype 5-based adenovirus, was constructed by deleting E1B and partial E3 genes, and incorporating a synthetic sensory switch circuit. The gene circuit was designed to enhance tumor-specific expression of E1A gene and an immune effector, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by sensing cancer-specific alpha-fetoprotein (AFP) promoter and two microRNA inputs. The specificity of SynOV1.1 to selectively kill target tumor cells and the ability to specifically express hGM-CSF in the target cells were confirmed in numerous human and mouse HCC cell lines with an IC50 approximately 3 to 35-fold less than that of normal cell lines. In vivo, intratumoral injections of SynOV1.1 in immune-deficient mice bearing human HCC cells resulted in a significant reduction of tumor volume (approximately 80%) as compared with the controls (approximately 30% for sorafenib). To further evaluate the effects of antitumor and immune stimulation, SynOV1.1m (SynOV1.1 murine surrogate) was intratumorally injected to the immune-competent mice bearing mouse HCC cells (ie., mHepa1-6), which showed SynOV1.1m induced a greater antitumor effect as well as a more robust tumor-specific immune response as compared to the controls. In addition, SynOV1.1m could significantly inhibit the growth of non-injected distal tumors after it was injected into a mHepa1-6 tumor on the other side of the mouse body, indicating that a systemic immune response was induced by SynOV1.1m. The combination of SynOV1.1m and an anti-mPD-L1 monoclonal antibody (mAb) was further explored and showed a synergistic anti-tumor effect as compared to SynOV1.1m or anti-mPD-L1 mAb alone. Preclinical safety of SynOV1.1 was examined in various toxicology species including golden hamsters, tumor-bearing mice and cynomolgus monkey, in all of which SynOV1.1 was found safe. Overall, preclinical pharmacology and toxicology data support clinical investigation of SynOV1.1 alone or in combination with atezolizumab in advanced HCC patients with positive serum AFP levels. Citation Format: Qiang Liu, Yubing Cao, Man Zhang, Shuguang Peng, Yiqi Liu, Huiya Huang, Bin Chen. SynOV1.1: A synthetic gene circuit controlled oncolytic adenovirus demonstrating high tumor specificity, potent antitumor efficacy and high synergy with an anti-PD-L1 monoclonal antibody in preclinical hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1148.

Transcriptomic analysis of lead-induced hepatoxicology in female Japanese quails (Coturnix japonica): Implications of triglyceride synthesis, degradation and transport disruption

Lead (Pb) pollution poses great threats to mammals including human and it is also hazardous to bird life. In this study, RNA sequencing analysis was employed to examine the molecular responses to lead exposure in the liver of a toxicological model species Japanese quails (Coturnix japonica). Female birds were exposed to 0, 50, 500 and 1000 ppm waterborne Pb for 49 days. The results showed that hepatic microstructure was damaged under lead exposure featured by sinusoids dilation and irregularity as well as cell necrosis. Moreover, ultrastructural injury in the liver including mitochondrial swelling and vacuolization as well as nuclear deformation was induced by lead exposure. Lead exposure also caused the decrease of lipid droplets in the liver by oil red O staining. In addition, liver transcriptomic analysis revealed that molecular signaling and functional pathways were disrupted by lead exposure. Meanwhile, the expression of genes involved with hepatic glycerophospholipids metabolism of triglyceride synthesis and lipid transport of triglyceride transfer was significantly down-regulated by lead exposure. Moreover, the up-regulation of genes associated with fatty acid oxidation and the down-regulation of genes related with fatty acid synthesis were caused by lead exposure. The present study implied that lead induced liver malfunction and bird health risks through histopathological damages, molecular signaling disruption, genetic expression alteration and triglyceride metabolism disturbance.

Justification for species selection for pharmaceutical toxicity studies.

Toxicity studies using mammalian species are generally required to provide safety data to support clinical development and licencing registration for potential new pharmaceuticals. International regulatory guidelines outline recommendations for the order (rodent and/or non-rodent) and number of species, retaining flexibility for development of a diverse range of drug modalities in a manner relevant for each specific new medicine. Selection of the appropriate toxicology species involves consideration of scientific, ethical and practical factors, with individual companies likely having different perspectives and preferences regarding weighting of various aspects dependent upon molecule characteristics and previous experience of specific targets or molecule classes. This article summarizes presentations from a symposium at the 2019 Annual Congress of the British Toxicology Society on the topic of species selection for pharmaceutical toxicity studies. This symposium included an overview of results from a National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and Association of British Pharmaceutical Industry (ABPI) international collaboration that reviewed the use of one or two species in regulatory toxicology studies and justification for the species selected within each programme. Perspectives from two pharmaceutical companies described their processes for species selection for evaluation of biologics, and justification for selection of the minipig as a toxicological species for small molecules. This article summarizes discussions on the scientific justification and other considerations taken into account to ensure the most appropriate animal species are used for toxicity studies to meet regulatory requirements and to provide the most value for informing project decisions.

Disposition and Metabolism of [14C]Lemborexant in Healthy Human Subjects and Characterization of Its Circulating Metabolites.

Lemborexant is a novel dual orexin receptor antagonist recently approved for the treatment of insomnia in the United States and Japan. Here, disposition and metabolic profiles were investigated in healthy human subjects. After single oral administration of 10 mg [14C]lemborexant (100 µCi), plasma concentrations of lemborexant and radioactivity peaked at 1 hour postdose and decreased biphasically. Cumulative recovery of the administered radioactivity within 480 hours was 86.5% of the dose, with 29.1% in urine and 57.4% in feces. Unchanged lemborexant was not detected in urine but accounted for 13.0% of the dose in feces, suggesting that the main elimination pathway of lemborexant was metabolism. Metabolite analyses revealed that the major metabolic pathways of lemborexant are oxidation of the dimethylpyrimidine moiety and subsequent further oxidation and/or glucuronidation. In plasma, lemborexant was the dominant component, accounting for 26.5% of total drug-related exposure. M4, M9, M10, and M18 were detected as the major radioactive components; M10 was the only metabolite exceeding 10% of total drug-related exposure. Although M4, M9, and M10 showed binding affinity for orexin receptors comparable to that of lemborexant, their contributions to the sleep-promoting effects of lemborexant are likely low because of the limited brain penetration by P-glycoprotein. Exposure comparison between humans and nonclinical toxicology species confirmed that plasma exposure of M10 was higher in at least one animal species compared with that in humans, indicating that there is no disproportionate metabolite in humans, as defined by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M3(R2) and U.S. Food and Drug Administration Metabolite in Safety Testing guidance; therefore, no additional toxicology studies are needed. SIGNIFICANCE STATEMENT: This study provides detailed data of the disposition and metabolism of lemborexant, a novel therapeutic drug for insomnia, in humans, as well as a characterization of the circulating metabolites and assessment of their contributions to efficacy and safety. The information presented herein furthers our understanding of the pharmacokinetic profiles of lemborexant and its metabolites and will promote the safe and effective use of lemborexant in the clinic.

Outcomes of the European Federation of Pharmaceutical Industries and Associations Oligonucleotide Working Group Survey on Nonclinical Practices and Regulatory Expectations for Therapeutic Oligonucleotide Safety Assessment

The Oligonucleotide Working Group of the European Federation of Pharmaceutical Industries and Associations (EFPIA) conducted a survey of companies to understand the trends in nonclinical practices and regulatory expectations for oligonucleotide drug safety assessment. Twenty-two companies of different types, with varying oligonucleotide experience levels in the field, participated. The survey identified key regulatory challenges and areas of perceived health authority (HA) concern regarding nonclinical safety strategies for oligonucleotides, such as the choice of toxicology species, approaches to dose setting in toxicity studies, dose scaling from animals to humans, the implementation (and regulatory acceptability) of lean packages, and methods for dealing with impurities and human-specific off-targets. The perceived oligonucleotide experience of HAs and the relevance of guidance to oligonucleotide development were also assessed. The results showed a general lack of consensus on nonclinical safety assessment approaches being used for this growing class of medicines and highlight the need for continuing collaboration between sponsors and HAs to better define best practices.

Long-Term Nonclinical Pulmonary Safety Assessment of Afrezza, a Novel Insulin Inhalation Powder.

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

Development of peptide therapeutics: A nonclinical safety assessment perspective

Novel peptide drugs continue to gain interest as effective modalities against previously undruggable targets. As with any other technology, development and safety assessment of peptides presents with various complex challenges. Additionally, there is a lack of specific regulatory guidance for peptide development, with the industry relying mainly on associating existing small molecule [ICH M3(R2)] and biologic [ICH S6(R1)] guidance. To gain insights into regulatory requirements for therapeutic peptides, we developed a dataset of peptides approved in the United States from 1998 through 2019 for which the summary basis of approval (SBA) packages are publicly available. The dataset comprises a total of 47 peptides (22 chemically synthesized, 6 semi-synthetic, 18 recombinant, and 1 natural). This article summarizes our learnings from the dataset in regards to the development paradigm, guidances followed, strategies for selection of toxicology species; requirements and/or value of genotoxicity and immunogenicity assessment; impurity, metabolite, and safety pharmacology assessment; and safety assessment of peptides containing non-proteogenic amino acids. In the context of the learnings from the dataset, the authors provide their recommendations for improvement of strategies to develop peptide drugs.

Prodiamesa olivacea: de novo biomarker genes in a potential sentinel organism for ecotoxicity studies in natural scenarios

Along with traditional ecotoxicological approaches in model organisms, toxicological studies in non-model organisms are being taken into consideration in order to complement them and contribute to more robust approaches. This allows us to figure out the complexity of the exposures involved in natural ecosystems. In this context, in the present research we have used the model species Chironomus riparius (Chironomidae, Diptera) and the non-model species Prodiamesa olivacea (Chironomidae, Diptera) to assess the aquatic toxic effects of acute 4-h and 24-h exposures to 1 μgL−1 of three common environmental pollutants: butyl benzyl phthalate (BBP), bisphenol A (BPA), and benzophenone 3 (BP3). Individuals of both species were collected from a contaminated river (Sar) in Galicia (Spain).Regarding Chironomus, there are four OECD standardized tests for the evaluation of water and sediment toxicity, in which different species in this genus can be used to assess classical toxicity parameters such as survival, immobilization, reproduction, and development. In contrast, Prodiamesa is rarely used in toxicity studies, even though it is an interesting toxicological species because it shares habitats with Chironomus but requires less extreme conditions (e.g., contamination) and higher oxygen levels. These different requirements are particularly interesting in assessing the different responses of both species to pollutant exposure.Quantitative real-time PCR was used to evaluate the transcriptional changes caused by xenobiotics in different genes of interest. Since information about P. olivacea in genomic databases is scarce, its transcriptome was obtained using de novo RNAseq. Genes involved in biotransformation pathways and the oxidative stress response (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a14-like and Cyp6a2-like) were de novo identified in this species. Our results show differential toxic responses depending on the species and the xenobiotic, being P. olivacea the dipteran that showed the most severe effects in most of the studied biomarker genes.This work represents a multi-species approach that allows us to deepen in the toxicity of BBP, BPA, and BP3 at the molecular level. Besides, it provides an assessment of the tolerance/sensitivity of natural populations of model and non-model insect species chronically exposed to complex mixtures of pollutants in natural scenarios. These findings may have important implications for understanding the adverse biological effects of xenobiotics on P. olivacea, providing new sensitive biomarkers of exposure to BBP, BPA, and BP3. It also highlights the suitability of Prodiamesa for ecotoxicological risk assessment, especially in aquatic ecosystems.

Comparative toxicokinetic profiles of multiple-components of Gelsemium elegans in pigs and rats after a single oral administration

Gelsemium elegans Benth (G. elegans) is highly toxic to humans and rats, but has insecticides and growth promoting effects on pigs and goats. G. elegans is widely used in livestock, but its in vivo dynamics are entirely unknown. Hence, we investigated the toxicokinetic profiles of G. elegans alkaloids after a single oral dose of G. elegans to pigs (1.0 g/kg) and rats (0.1 g/kg). The results indicated that rats were more susceptible to the toxicity of G. elegans than pigs. The toxicokinetic parameters of 22 and 6 components were obtained in pigs and rats, respectively. The components included 9 and 5 gelsedine-type alkaloids in pigs and rats, respectively. The Tmax results of the 5 gelsedine-type alkaloids indicated that these alkaloids were rapidly absorbed in pigs and rats. The T1/2 values of the 5 gelsedine-type alkaloids indicated that the elimination rates of these alkaloids in pigs were slower than those in rats. In addition, the Cmax and AUC results indicated that the degrees of absorption and exposure of most alkaloids in pigs were higher than those in rats except GS-2. However, the Cmax value of GS-2 (11-methoxy-14-hydroxygelsenicine) in rats was greater than that of pigs, and the Cmax value of 14-hydroxygelsenicine in pigs was merely greater than 3 times that of rats. The present results suggested that the cause of the toxicological differences species of G. elegans might be related to the degrees of absorption and exposure of gelsedine-type alkaloids, especially for the 14-hydroxygelsenicine and GS-2 in different animals.

Comparative toxicokinetics of Trans-resveratrol and its major metabolites in Harlan Sprague Dawley rats and B6C3F1/N mice following oral and intravenous administration

Trans-resveratrol (RES) is a naturally occurring stilbene found in numerous plants and foods. Due to its widespread human exposure and lack of toxicity and carcinogenicity data, RES was nominated to the National Toxicology Program for testing. To aid the toxicology studies, the dose, sex, and species differences in RES toxicokinetics was investigated in Harlan Sprague Dawley rats and B6C3F1/N mice following single intravenous (IV) (10 mg/kg) or oral gavage administration (312.5, 625, and 1250 mg/kg and 625, 1250, and 2500 mg/kg in rats and mice, respectively).Following IV and gavage administration, systemic exposure of RES based on AUC was trans-resveratrol-3-O-β-D-glucuronide (R3G)> > trans-resveratrol-3-sulfate (R3S) > RES in both species. Following gavage administration Tmax_predicted values were ≤ 263 min for both species and sexes. RES elimination half-life was longer in rats than mice, and shortest in male mice. Clearance was slower in mice with no apparent sex difference in both species. In both rats and mice, following gavage administration AUC increased proportionally to the dose. After gavage administration, enterohepatic recirculation of RES was observed in both rats and mice with secondary peaks occurring around 640 min in the concentration-time profiles. RES was rapidly metabolized to R3S and R3G in both species. Extensive first pass conjugation and metabolism resulted in low levels of the parent compound RES which was confirmed by the low estimates for bioavailability. The bioavailability of RES was low, ~12–31% and ~2–6% for rats and mice, respectively, with no apparent difference between sexes.

A Potent Pan-TGFβ Neutralizing Monoclonal Antibody Elicits Cardiovascular Toxicity in Mice and Cynomolgus Monkeys.

Transforming growth factor β (TGFβ) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFβ therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFβ signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFβ receptor and not considered to be a liability associated with pan-TGFβ neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFβ neutralizing mAb (pan-TGFβ mAb; neutralizes TGFβ1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFβ signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFβ inhibiting mAb.