A systems approach reveals species differences in hepatic stress response capacity.

To minimize the occurrence of unexpected toxicities in early phase preclinical studies of new drugs, it is vital to understand fundamental similarities and differences between preclinical species and humans. Species differences in sensitivity to acetaminophen (APAP) liver injury have been related to differences in the fraction of the drug that is bioactivated to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI). We have used physiologically based pharmacokinetic modeling to identify oral doses of APAP (300 and 1000 mg/kg in mice and rats, respectively) yielding similar hepatic burdens of NAPQI to enable the comparison of temporal liver tissue responses under conditions of equivalent chemical insult. Despite pharmacokinetic and biochemical verification of the equivalent NAPQI insult, serum biomarker and tissue histopathology analyses revealed that mice still exhibited a greater degree of liver injury than rats. Transcriptomic and proteomic analyses highlighted the stronger activation of stress response pathways (including the Nrf2 oxidative stress response and autophagy) in the livers of rats, indicative of a more robust transcriptional adaptation to the equivalent insult. Components of these pathways were also found to be expressed at a higher basal level in the livers of rats compared with both mice and humans. Our findings exemplify a systems approach to understanding differential species sensitivity to hepatotoxicity. Multiomics analysis indicated that rats possess a greater basal and adaptive capacity for hepatic stress responses than mice and humans, with important implications for species selection and human translation in the safety testing of new drug candidates associated with reactive metabolite formation.

Slow dynamics in a single bead with mechanical conditioning and transient heating.

The contact stiffness of an aluminum bead confined between two slabs diminishes upon mechanical conditioning, and then recovers as log(t) after the conditioning ceases. Here that structure is evaluated for its response to transient heating and cooling, with and without accompanying conditioning vibrations. It is found that, under heating or cooling alone, stiffness changes are mostly consistent with temperature-dependent material moduli; there is little or no slow dynamics. Hybrid tests in which vibration conditioning is followed by heating or cooling lead to recoveries that begin as log(t) and then become more complex. On subtracting the known response to heating or cooling alone we discern the influence of higher or lower temperatures on slow dynamic recovery from vibrations. It is found that heating accelerates the initial log(t) recovery, but by an amount more than predicted by an Arrhenius model of thermally activated barrier penetrations. Transient cooling has no discernible effect, in contrast to the Arrhenius prediction that it slows recovery.

Acceptable intakes (AIs) for 11 small molecule N-nitrosamines (NAs).

Low levels of N-nitrosamines (NAs) were detected in pharmaceuticals and, as a result, health authorities (HAs) have published acceptable intakes (AIs) in pharmaceuticals to limit potential carcinogenic risk. The rationales behind the AIs have not been provided to understand the process for selecting a TD50 or read-across analog. In this manuscript we evaluated the toxicity data for eleven common NAs in a comprehensive and transparent process consistent with ICH M7. This evaluation included substances which had datasets that were robust, limited but sufficient, and substances with insufficient experimental animal carcinogenicity data. In the case of robust or limited but sufficient carcinogenicity information, AIs were calculated based on published or derived TD50s from the most sensitive organ site. In the case of insufficient carcinogenicity information, available carcinogenicity data and structure activity relationships (SARs) were applied to categorical-based AIs of 1500 ng/day, 150 ng/day or 18 ng/day; however additional data (such as biological or additional computational modelling) could inform an alternative AI. This approach advances the methodology used to derive AIs for NAs.

Pharmacokinetics of N,N-dimethyltryptamine in Humans.

BACKGROUND AND OBJECTIVE: N,N-dimethyltryptamine (DMT) is a psychedelic compound under development for the treatment of major depressive disorder (MDD). This study evaluated the preclinical and clinical pharmacokinetics and metabolism of DMT in healthy subjects. METHODS: The physiochemical properties of DMT were determined using a series of in vitro experiments and its metabolic profile was assessed using monoamine oxidase (MAO) and cytochrome P450 (CYP) inhibitors in hepatocyte and mitochondrial fractions. Clinical pharmacokinetics results are from the phase I component of a phase I/IIa randomised, double-blind, placebo-controlled, parallel-group, dose-escalation trial (NCT04673383). Healthy adults received single escalating doses of DMT fumarate (SPL026) via a two-phase intravenous (IV) infusion. Dosing regimens were calculated based on pharmacokinetic modelling and predictions with progression to each subsequent dose level contingent upon safety and tolerability. RESULTS: In vitro clearance of DMT was reduced through the inhibition of MAO-A, CYP2D6 and to a lesser extent CYP2C19. Determination of lipophilicity and plasma protein binding was low, indicating that a high proportion of DMT is available for distribution and metabolism, consistent with the very rapid clinical pharmacokinetics. Twenty-four healthy subjects received escalating doses of DMT administered as a 10-min infusion over the dose range of 9-21.5 mg (DMT freebase). DMT was rapidly cleared for all doses: mean elimination half-life was 9-12 min. All doses were safe and well tolerated and there was no relationship between peak DMT plasma concentrations and body mass index (BMI) or weight. CONCLUSION: This is the first study to determine, in detail, the full pharmacokinetics profile of DMT following a slow IV infusion in humans, confirming rapid attainment of peak plasma concentrations followed by rapid clearance. These findings provide evidence which supports the development of novel DMT infusion regimens for the treatment of MDD. CLINICAL TRIAL REGISTRATION: Registered on ClinicalTrials.gov (NCT04673383).

The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

Design, Synthesis, and Evaluation of Lung-Retentive Prodrugs for Extending the Lung Tissue Retention of Inhaled Drugs.

A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 µM. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.

Diffuse energy transport and coda-wave interferometry for resonant transmission between reverberant structures.

Approximate analytic and numerical theories are developed with which to model, and compare with laboratory measurements, the diffuse ultrasonic field transmitted from one elastic body to another through a resonant coupling. Particular focus is on the sensitivity of the transmitted field to perturbations in the natural frequency of the coupling and the manifestation of such perturbations as time delays in the second body. The immediate application is to measurements of slow dynamic elastic nonlinearity. It is found that the developed approximate theories do a good job of predicting the time dependence of the mean square transmitted signal and signal spectra. They also predict the time delays, both their erratic character and means. Analytic dependence of these time shifts on the fractional frequency perturbation df/f is derived.

Evaluation of Lotions of Botanical-Based Repellents Against Aedes aegypti (Diptera: Culicidae).

Thirteen botanical product repellent compounds such as 2-undecanone, capric, lauric, coconut fatty acids (and their methyl ester derivatives), and catnip oil were formulated in either Coppertone or Aroma Land lotions and evaluated against laboratory-reared Aedes aegypti L. (Diptera: Culicidae) mosquitoes. These formulations contained 7-15 wt/wt of the botanical repellent as the major active ingredient either pure or as mixtures. USDA standard repellent test cages were used to determine the complete protection time (CPT) of the different formulated repellents. Two of the evaluated formulations, a 7% capric acid in Coppertone (CPT 2.7 ± 0.6 h) and 7% coconut fatty acids containing carrylic acid, capric acid, and lauric acid in Coppertone (CPT 2.3 ± 2.0 h), provided strong repellency against mosquitoes up to 3 h, which was equivalent to the (N,N-diethyl-m-toluamide) DEET control (CPT 2.7 ± 0.6 h). This work suggests future potential for these botanical product-based repellents as alternatives to commercial DEET-containing products.

Slow dynamic elastic recovery in unconsolidated metal structures.

Slow dynamic nonlinearity is widely observed in brittle materials with complex heterogeneous or cracked microstructures. It is seen in rocks, concrete, and cracked glass blocks. Unconsolidated structures show the behavior as well: aggregates of glass beads under pressure and a single glass bead confined between two glass plates. A defining feature is the loss of stiffness after a mechanical conditioning, followed by a logarithmic-in-time recovery. Materials observed to exhibit slow dynamics are sufficiently different in microstructure, chemical composition, and scale (ranging from the laboratory to the seismological) to suggest some kind of universality. There lacks a full theoretical understanding of the universality in general and the log(time) recovery in particular. One suspicion has been that the phenomenon is associated with glassy grain boundaries and microcracking. Seminal studies were focused on sandstones and other natural rocks, but in recent years other experimental venues have been introduced with which to inform theory. Here, we present measurements on some simple metallic systems: an unconsolidated aggregate of aluminum beads under a confining pressure, an aluminum bead confined between two aluminum plates, and a steel bead confined between steel plates. Ultrasonic waves are used as probes of the systems, and changes are assessed with coda wave interferometry. Three different methods of low-frequency conditioning are applied; all reveal slow dynamic nonlinearities. Results imply that glassy microstructures and cracking do not play essential roles, as they would appear to be absent in our systems.

Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation.

An international expert working group representing 37 organisations (pharmaceutical/biotechnology companies, contract research organisations, academic institutions and regulatory bodies) collaborated in a data sharing exercise to evaluate the utility of two species within regulatory general toxicology studies. Anonymised data on 172 drug candidates (92 small molecules, 46 monoclonal antibodies, 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates) were submitted by 18 organisations. The use of one or two species across molecule types, the frequency for reduction to a single species within the package of general toxicology studies, and a comparison of target organ toxicities identified in each species in both short and longer-term studies were determined. Reduction to a single species for longer-term toxicity studies, as used for the development of biologicals (ICHS6(R1) guideline) was only applied for 8/133 drug candidates, but might have been possible for more, regardless of drug modality, as similar target organ toxicity profiles were identified in the short-term studies. However, definition and harmonisation around the criteria for similarity of toxicity profiles is needed to enable wider consideration of these principles. Analysis of a more robust dataset would be required to provide clear, evidence-based recommendations for expansion of these principles to small molecules or other modalities where two species toxicity testing is currently recommended.

Slow dynamic nonlinearity in unconsolidated glass bead packs.

Slow dynamic nonlinearity describes a poorly understood, creeplike phenomena that occurs in brittle composite materials such as rocks and cement. It is characterized by a drop in stiffness induced by a mechanical conditioning, followed by a log(time) recovery. A consensus theoretical understanding of the behavior has not been developed. Here we introduce an alternative experimental venue with which to inform theory. Unconsolidated glass bead packs are studied rather than rocks or cement because the structure and internal contacts of bead packs are less complex and better understood. Slow dynamics has been observed in such systems previously. However, the measurements to date tend to be irregular. Particular care is used here in the experimental design to overcome the difficulties inherent in bead pack studies. This includes the design of the bead pack support, the use of low-frequency conditioning, and the use of ultrasonic waves as a probe with coda wave interferometry to assess changes. Slow dynamics is observed in our system after three different methods for low-frequency conditioning, one of which has not been reported in the literature previously.

Slow dynamics in a single glass bead.

Slow dynamic nonlinearity is ubiquitous amongst brittle materials, such as rocks and concrete, with cracked microstructures. A defining feature of the behavior is the logarithmic-in-time recovery of stiffness after a mechanical conditioning. Materials observed to exhibit slow dynamics are sufficiently different in microstructure, chemical composition, and scale (ranging from the laboratory to the seismological) to suggest some kind of universality. A consensus of theoretical understanding of the universality in general and the log(time) recovery in particular is lacking. Seminal studies were focused on sandstones and other natural rocks, but in recent years other experimental venues have been introduced with which to inform theory. One such system is unconsolidated glass bead packs. However, bead packs still contain many contact points. The force distribution amongst the contacts is unknown. Here, we present slow dynamics measurements on a yet simpler system-a single glass bead confined between two large glass plates. The system is designed with a view towards rapid control of the contact zone environment. Ultrasonic waves are used as a probe of the system, and changes are assessed with coda wave interferometry. Three different methods of low-frequency conditioning are applied; all lead to slow dynamic recoveries. Results imply that force chains do not play an essential role in granular media slow dynamics, as they are absent in our system.

The hepatotoxic fluoroquinolone trovafloxacin disturbs TNF- and LPS-induced p65 nuclear translocation in vivo and in vitro.

Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB. We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.

Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2-trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

Evaluation of the effects of CBD hemp extract on opioid use and quality of life indicators in chronic pain patients: a prospective cohort study.

Context: Chronic pain is highly prevalent in most of the industrialized nations around the world. Despite the documented adverse effects, opioids are widely used for pain management. Cannabinoids, and specifically Cannabidiol, is proposed as an opioid alternative, having comparable efficacy with better safety profile.Objectives: We aim to investigate the impact of full hemp extract cannabidiol (CBD) on opioid use and quality of life indicators among chronic pain patients.Methods: An initial sample of 131 patients was recruited from a private pain management center's investigative population. Ninety-seven patients completed the 8-week study. The primary inclusion criteria included patients between 30 and 65 years old with chronic pain who have been on opioids for at least 1 year. Data were collected at three different time points: baseline, 4, and 8 weeks. Opioid and other medication use were evaluated via the medication and psychiatric treatment receipt. Improvement was evaluated using four indices: Pain Disability Index (PDI-4); Pittsburgh Sleep Quality Index (PSQI), Pain Intensity and Interference (PEG); and Patient Health Questionnaire (PHQ-4).Results: Over half of chronic pain patients (53%) reduced or eliminated their opioids within 8 weeks after adding CBD-rich hemp extract to their regimens. Almost all CBD users (94%) reported quality of life improvements. The results indicated a significant relationship between CBD and PSQI (p = 0.003), and PEG (p = 0.006). There was a trend toward improvement but no significant relationship between CBD use and PHQ and PDI.Conclusion: CBD could significantly reduce opioid use and improve chronic pain and sleep quality among patients who are currently using opioids for pain management.Key Message: This is a prospective, single-arm cohort study for the potential role of cannabinoids as an alternative for opioids. The results indicate that using the CBD-rich extract enabled our patients to reduce or eliminate opioids with significant improvement in their quality of life indices.

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models.

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI.

Differential toxic effects of bile acid mixtures in isolated mitochondria and physiologically relevant HepaRG cells.

Bile acids (BAs) are recognised as the causative agents of toxicity in drug-induced cholestasis (DIC). Research in isolated mitochondria and HepG2 cells have demonstrated BA-mediated mitochondrial dysfunction as a key mechanism of toxicity in DIC. However, HepG2 cells are of limited suitability for DIC studies as they do not express the necessary physiological characteristics. In this study, the mitotoxic potentials of BA mixtures were assessed in isolated mitochondria and a better-suited hepatic model, HepaRG cells. BAs induced structural alterations and a loss of mitochondrial membrane potential (MMP) in isolated mitochondria however, this toxicity did not translate to HepaRG cells. There were no changes in oxygen consumption rate, MMP or ATP levels in glucose and galactose media, indicating that there was no direct mitochondrial toxicity mediated via electron transport chain dysfunction in HepaRG cells. Assessment of key biliary transporters revealed that there was a time-dependent reduction in the expression and activity of multi-drug resistance protein 2 (MRP2), which was consistent with the induction of cytotoxicity in HepaRG cells. Overall, the findings from this study have demonstrated that mitochondrial dysfunction is not a mechanism of BA-induced toxicity in HepaRG cells.

A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists.

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

Identification and characterization of a naïve CD8+ T cell repertoire for benzylpenicillin.

BACKGROUND: Beta-lactams allergy is the most commonly reported drug allergy and constitutes an important health problem. We previously showed the pre-existence of a naïve CD4+ T cell repertoire for benzylpenicillin (BP) coupled to human serum albumin (HSA) but little is known about the naïve CD8+ T cell repertoire specific for BP. OBJECTIVE: The purpose of this work was to identify naïve CD8+ T cells specific for BP and to explore mechanisms dictating their activation. METHODS: Co-cultures were established with naïve CD8+ T cells and autologous dendritic cells (DCs) loaded with HSA-BP or free BP. T cells were restimulated once a week with autologous DCs loaded with HSA-BP or BP. The specific CD8+ T cell response was measured using an IFN-γ ELISpot assay. RESULTS: When using free BP, we were able to detect a naïve CD8+ T cell repertoire for BP in the 6 out of 7 tested healthy donors. However, our results showed that HSA-BP was recognized by naïve CD8+ T cells in only one donor out of five tested healthy donors. Using free BP, we evidenced its binding to cellular proteins in DCs that was concentration dependent and was correlated with BP-specific CD8+ T cell activation. Moreover, the BP-specific CD8+ cell response was MHC class I-dependent and required intracellular processing and proteasome activity. CONCLUSION AND CLINICAL RELEVANCE: This work showed the existence of a naïve CD8+ T cell repertoire for BP when DCs were treated with free BP suggesting that patients could be immunized by haptenated peptides from cellular proteins generated in antigen-presenting cells.

Today's Challenges to De-Risk and Predict Drug Safety in Human "Mind-the-Gap".

Current gaps in drug safety sciences can result from the inability (1) to identify hazard across multiple target organs, (2) to predict and risk assess with certainty against drug safety liabilities for the major target organs, (3) to optimally manage and mitigate against drug safety liabilities, and (4) to apply principles of governance on the generation, integration, and use of experimental data. Translational safety assessment to evaluate several target-organ drug toxicities can only be partially achieved by use of current in vitro and in vivo test systems. What remains to be tackled necessitates the deployment of in vitro-human-relevant test systems to address human specific or selective forms of toxicities. Nevertheless, such models may only address in part some of the requirements in today's armament of biomedical tools essential for improving the discovery of drug candidates. Refinement of in silico tools, Target Safety Assessment and a greater understanding of mechanistic insights of toxicities might provide future opportunities to better identify drug safety liabilities. The increasing diversity of drug modalities present further challenges for nonclinical and clinical development requiring further research to develop suitable test systems and technologies. Our ability to optimally manage and mitigate safety risk will come from the greater refinement of safety margin estimates, provision and use of human-relevant safety biomarkers, and understanding of the translation from in silico, in vitro, and in vivo studies to human. An improvement of governance frameworks and standards at all levels within organizations, national, and international, can only help facilitate drug discovery and development programs.