Role of NaV1.7 in postganglionic sympathetic nerve function in human and guinea-pig arteries.

NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF 05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P < 0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA 121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF 05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.

Nonclinical pharmacokinetics, pharmacodynamics and safety assessment of a FLT3L-Fc molecule for cancer immunotherapy.

FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.

Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

INVESTIGATION OF THE USE OF SERUM BIOMARKERS FOR THE DETECTION OF CARDIAC DISEASE IN MARINE MAMMALS.

Cardiac disease has been extensively documented in marine mammals; however, it remains difficult to diagnose antemortem. Assays measuring cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are used as sensitive and specific biomarkers of cardiac disease in many species, but have not been widely investigated in marine mammals. This study aimed to provide a set of reference values for cTnI and NT-proBNP in belugas (BW) (Delphinapterus leucas), sea otters (SO) (Enhydra lutris), Steller sea lions (SSL) (Eumetopias jubatus), and California sea lions (CSL) (Zalophus californianus) with and without cardiac disease, and to determine if these biomarkers are useful indicators of cardiac disease in these species. First, existing immunoassays for cTnI and NT-proBNP were successfully validated utilizing species-specific heart lysate spiked serum. Cohorts were defined by histopathology as animals with no evidence of cardiac disease ("control"), with confirmed cardiac disease ("disease"), and with concurrent renal and cardiac disease ("renal") for which serum samples were then analyzed. Serum concentration ranges for cTnI (ng/ml) and NT-proBNP (pmol/L) were determined for control and disease cohorts. There was significantly higher cTnI (P= 0.003) and NT-proBNP (P= 0.004) concentrations in the CSL disease cohort, as well as positive trends in BW, SO, and SSL disease cohorts that did not reach statistical significance. NT-proBNP concentrations were significantly higher in the CSL renal cohort compared with the control (P < 0.001) and disease (P= 0.007) cohorts. These results suggest that cTnI and NT-proBNP may be clinically useful in the antemortem diagnosis of cardiac disease in CSL, and warrant further investigation in BW, SO, and SL.

Nonclinical Pharmacokinetics and Pharmacodynamics Characterization of Anti-CD79b/CD3 T Cell-Dependent Bispecific Antibody Using a Surrogate Molecule: A Potential Therapeutic Agent for B Cell Malignancies.

The T cell-dependent bispecific (TDB) antibody, anti-CD79b/CD3, targets CD79b and CD3 cell-surface receptors expressed on B cells and T cells, respectively. Since the anti-CD79b arm of this TDB binds only to human CD79b, a surrogate TDB that binds to cynomolgus monkey CD79b (cyCD79b) was used for preclinical characterization. To evaluate the impact of CD3 binding affinity on the TDB pharmacokinetics (PK), we utilized non-tumor-targeting bispecific anti-gD/CD3 antibodies composed of a low/high CD3 affinity arm along with a monospecific anti-gD arm as controls in monkeys and mice. An integrated PKPD model was developed to characterize PK and pharmacodynamics (PD). This study revealed the impact of CD3 binding affinity on anti-cyCD79b/CD3 PK. The surrogate anti-cyCD79b/CD3 TDB was highly effective in killing CD79b-expressing B cells and exhibited nonlinear PK in monkeys, consistent with target-mediated clearance. A dose-dependent decrease in B cell counts in peripheral blood was observed, as expected. Modeling indicated that anti-cyCD79b/CD3 TDB's rapid and target-mediated clearance may be attributed to faster internalization of CD79b, in addition to enhanced CD3 binding. The model yielded unbiased and precise curve fits. These findings highlight the complex interaction between TDBs and their targets and may be applicable to the development of other biotherapeutics.

Establishment of baseline profiles of 50 bile acids in preclinical toxicity species: A comprehensive assessment of translational differences and study design considerations for biomarker development.

The use of bile acids as functional biomarkers for hepatobiliary injury and disease has been proposed for decades, but the utility has been generally limited due to lack of sensitivity in diagnosis and assay availability. However, recent advances in liquid chromatography and mass spectrometry have allowed for highly sensitive profiling of individual bile acids across several different matrices. In the current work, a panel of 54 bile acids were quantified in plasma by high resolution mass spectrometry in the common species used for preclinical toxicity studies, including rat (both Wistar and Sprague-Dawley strains), Beagle dog, Cynomolgus macaque monkey, and New Zealand White rabbit. In each species, blood draws were collected across three days in such a way to derive overall interpretations of: 1) biological variability across species, 2) sex differences, 3) diurnal fluctuations in the bile acid pool (including over light/dark cycles), and 4) changes due to fed or fasting state. Various methods of normalization were applied to the dataset to overcome notable inter-individual variability in bile acid concentrations to allow for better data derivations and interpretation. As such, the current work elucidates not only key differences in the bile acid pool across species, but also informs best practices in protocol design and analytical methods for interpreting large sets of bile acid data. When taken together, these data facilitate better species translation and application of bile acids as biomarkers for hepatobiliary injury and disease.

Polysorbate 80-Induced Anaphylactoid Reaction and the Effects on Cardiovascular Function: Dose Threshold and Species Comparison.

Polysorbate 80 (PS80) is commonly used in pre-clinical formulations. The dose threshold for cardiovascular (CV) changes and hypersensitivity reaction in the dog was assessed and compared to other species. PS80 was administered by intravenous (IV) bolus (.5, 1 mg/kg), IV infusion (.3, .5, 1, 3 mg/kg), subcutaneous (SC) injection (5, 10, 15 mg/kg) and oral gavage (10 mg/kg) to dogs with CV monitoring. Monkeys and minipigs received PS80 by IV infusion at 3 mg/kg. Plasma histamine concentration was measured following PS80 IV infusion and with diphenhydramine pre-treatment in dogs only. In dogs, PS80 was not associated with CV changes at doses up to 15 mg/kg SC and 10 mg/kg oral, but decreased blood pressure and increased heart rate with IV bolus at ≥ .5 mg/kg and IV infusion at ≥ 1.0 mg/kg and decreased body temperature with IV infusion at 3 mg/kg was observed. Transient edema and erythema were noted with all administration routes, in all three species including doses that were devoid of CV effects. In monkeys and minipigs, PS80 did not induce CV, cutaneous or histamine concentration changes. These results suggest that mild, transient skin changes occur following PS80 administration at doses that are not associated with CV effects in the dogs. In dogs, the cardiovascular effect threshold was <.5 mg/kg for IV bolus, .3 mg/kg for IV infusion, 15 mg/kg for SC injection, and 10 mg/kg for oral administration. Monkey and minipig were refractory to PS80-induced histamine release at 3 mg/kg by IV infusion over 15 minutes.

Phase I/II Trial of Vemurafenib in Dogs with Naturally Occurring, BRAF-mutated Urothelial Carcinoma.

BRAF-targeted therapies including vemurafenib (Zelboraf) induce dramatic cancer remission; however, drug resistance commonly emerges. The purpose was to characterize a naturally occurring canine cancer model harboring complex features of human cancer, to complement experimental models to improve BRAF-targeted therapy. A phase I/II clinical trial of vemurafenib was performed in pet dogs with naturally occurring invasive urothelial carcinoma (InvUC) harboring the canine homologue of human BRAF V600E The safety, MTD, pharmacokinetics, and antitumor activity were determined. Changes in signaling and immune gene expression were assessed by RNA sequencing and phosphoproteomic analyses of cystoscopic biopsies obtained before and during treatment, and at progression. The vemurafenib MTD was 37.5 mg/kg twice daily. Anorexia was the most common adverse event. At the MTD, partial remission occurred in 9 of 24 dogs (38%), with a median progression-free interval of 181 days (range, 53-608 days). In 18% of the dogs, new cutaneous squamous cell carcinoma and papillomas occurred, a known pharmacodynamic effect of vemurafenib in humans. Upregulation of genes in the classical and alternative MAPK-related pathways occurred in subsets of dogs at cancer progression. The most consistent transcriptomic changes were the increase in patterns of T lymphocyte infiltration during the first month of vemurafenib, and of immune failure accompanying cancer progression. In conclusion, the safety, antitumor activity, and cutaneous pharmacodynamic effects of vemurafenib, and the development of drug resistance in dogs closely mimic those reported in humans. This suggests BRAF-mutated canine InvUC offers an important complementary animal model to improve BRAF-targeted therapies in humans.

Gaining Efficiency in Clinical Trials With Cardiac Biomarkers: JACC Review Topic of the Week.

The momentum of cardiovascular drug development has slowed dramatically. Use of validated cardiac biomarkers in clinical trials could accelerate development of much-needed therapies, but biomarkers have been used less for cardiovascular drug development than in therapeutic areas such as oncology. Moreover, there are inconsistences in biomarker use in clinical trials, such as sample type, collection times, analytical methods, and storage for future research. With these needs in mind, participants in a Cardiac Safety Research Consortium Think Tank proposed the development of international guidance in this area, together with improved quality assurance and analytical methods, to determine what biomarkers can reliably show. Participants recommended the development of systematic methods for sample collection, and the archiving of samples in all cardiovascular clinical trials (including creation of a biobank or repository). The academic and regulatory communities also agreed to work together to ensure that published information is fully and clearly expressed.

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

The use of emerging safety biomarkers in nonclinical and clinical safety assessment - The current and future state: An IQ DruSafe industry survey.

Pharmaceutical and biotechnology companies rarely disclose their use of translational emerging safety biomarkers (ESBs) during drug development, and the impact of ESB use on the speed of drug development remains unclear. A cross-industry survey of 20 companies of varying size was conducted to understand current trends in ESB use and future use prospects. The objectives were to: (1) determine current ESB use in nonclinical and clinical drug development and impact on asset advancement; (2) identify opportunities, gaps, and challenges to greater ESB implementation; and (3) benchmark perspectives on regulatory acceptance. Although ESBs were employed in only 5-50% of studies/programs, most companies used ESBs to some extent, with larger companies demonstrating greater nonclinical use. Inclusion of ESBs in investigational new drug applications (INDs) was similar across all companies; however, differences in clinical trial usage could vary among the prevailing health authority (HA). Broader implementation of ESBs requires resource support, cross-industry partnerships, and collaboration with HAs. This includes generating sufficient foundational data, demonstrating nonclinical to clinical translatability and practical utility, and clearly written criteria by HAs to enable qualification. If achieved, ESBs will play a critical role in the development of next-generation, translationally-tailored standard laboratory tests for drug development.

An Industry Survey With Focus on Cardiovascular Safety Pharmacology Study Design and Data Interpretation.

INTRODUCTION: The Safety Pharmacology Society (SPS) conducted a membership survey to examine industry practices related mainly to cardiovascular (CV) safety pharmacology (SP). METHODS: Questions addressed nonclinical study design, data analysis methods, drug-induced effects, and conventional and novel CV assays. RESULTS: The most frequent therapeutic area targeted by drugs developed by the companies/institutions that employ survey responders was oncology. The most frequently observed drug-mediated effects included an increased heart rate, increased arterial blood pressure, hERG (IKr) block, decreased arterial blood pressure, decreased heart rate, QTc prolongation, and changes in body temperature. Broadly implemented study practices included Latin square crossover study design with n = 4 for nonrodent CV studies, statistical analysis of data (eg, analysis of variance), use of arrhythmia detection software, and the inclusion of data from all study animals when integrating SP studies into toxicology studies. Most responders frequently used individual animal housing conditions. Responders commonly evaluated drug effects on multiple ion channels, but in silico modeling methods were used much less frequently. Most responders rarely measured the J-Tpeak interval in CV studies. Uncertainties relative to Standard for Exchange of Nonclinical Data applications for data derived from CV SP studies were common. Although available, the use of human induced pluripotent stem cell cardiomyocytes remains rare. The respiratory SP study was rarely involved with identifying drug-induced functional issues. Responders indicated that the study-derived no observed effect level was more frequently determined than the no observed adverse effect level in CV SP studies; however, a large proportion of survey responders used neither.

Discovery of two highly divergent negative-sense RNA viruses associated with the parasitic nematode, Capillaria hepatica, in wild Mus musculus from New York City.

Recent advances in high-throughput sequencing technology have led to a rapid expansion in the number of viral sequences associated with samples from vertebrates, invertebrates and environmental samples. Accurate host identification can be difficult in assays of complex samples that contain more than one potential host. Using unbiased metagenomic sequencing, we investigated wild house mice (Mus musculus) and brown rats (Rattus norvegicus) from New York City to determine the aetiology of liver disease. Light microscopy was used to characterize liver disease, and fluorescent microscopy with in situ hybridization was employed to identify viral cell tropism. Sequences representing two novel negative-sense RNA viruses were identified in homogenates of wild house mouse liver tissue: Amsterdam virus and Fulton virus. In situ hybridization localized viral RNA to Capillaria hepatica, a parasitic nematode that had infected the mouse liver. RNA from either virus was found within nematode adults and unembryonated eggs. Expanded PCR screening identified brown rats as a second rodent host for C. hepatica as well as both nematode-associated viruses. Our findings indicate that the current diversity of nematode-associated viruses may be underappreciated and that anatomical imaging offers an alternative to computational host assignment approaches.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Adverse Drug-Induced Inotropic Effects in Early Drug Development. Part 1: General Considerations for Development of Novel Testing Platforms.

Drug-induced effects on cardiac contractility can be assessed through the measurement of the maximal rate of pressure increase in the left ventricle (LVdP/dtmax) in conscious animals, and such studies are often conducted at the late stage of preclinical drug development. Detection of such effects earlier in drug research using simpler, in vitro test systems would be a valuable addition to our strategies for identifying the best possible drug development candidates. Thus, testing platforms with reasonably high throughput, and affordable costs would be helpful for early screening purposes. There may also be utility for testing platforms that provide mechanistic information about how a given drug affects cardiac contractility. Finally, there could be in vitro testing platforms that could ultimately contribute to the regulatory safety package of a new drug. The characteristics needed for a successful cell or tissue-based testing platform for cardiac contractility will be dictated by its intended use. In this article, general considerations are presented with the intent of guiding the development of new testing platforms that will find utility in drug research and development. In the following article (part 2), specific aspects of using human-induced stem cell-derived cardiomyocytes for this purpose are addressed.

What evidence do we need for biomarker qualification?

Biomarkers can facilitate all aspects of the drug development process. However, biomarker qualification-the use of a biomarker that is accepted by the U.S. Food and Drug Administration-needs a clear, predictable process. We describe a multistakeholder effort including government, industry, and academia that proposes a framework for defining the amount of evidence needed for biomarker qualification. This framework is intended for broad applications across multiple biomarker categories and uses.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.