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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.

Overexpression of the antiapoptotic protein B-cell lymphoma-extra large (BCL-X L ) is associated with drug resistance and disease progression in numerous cancers. The compelling nature of this protein as a therapeutic target prompted efforts to develop selective small-molecule BCL-X L inhibitors. Although efficacious in preclinical models, we report herein that selective BCL-X L inhibitors cause severe mechanism-based cardiovascular toxicity in higher preclinical species. To overcome this liability, antibody-drug conjugates were constructed using altered BCL-X L -targeting warheads, unique linker technologies, and therapeutic antibodies. The epidermal growth factor receptor-targeting antibody-drug conjugate AM1-15 inhibited growth of tumor xenografts and did not cause cardiovascular toxicity nor dose-limiting thrombocytopenia in monkeys. While an unprecedented BCL-X L -mediated toxicity was uncovered in monkey kidneys upon repeat dosing of AM1-15, this toxicity was mitigated via further drug-linker modification to afford AM1-AAA (AM1-25). The AAA drug-linker has since been incorporated into mirzotamab clezutoclax, the first selective BCL-X L -targeting agent to enter human clinical trials.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.

Rationale: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation., Methods: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach., Results: A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement., Conclusions: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo., (© 2024 John Wiley & Sons Ltd.)

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Warner van Dijk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cadmium chloride (CdCl2) is a known genotoxic carcinogen, with a mechanism of action thought to partly involve the generation of reactive oxygen species (ROS). We applied here a multi-endpoint approach in vitro to explore the impact of CdCl2 on both the genome and on wider cell biology pathways relevant to cancer. Multi-endpoint approaches are believed to offer greater promise in terms of understanding the holistic effects of carcinogens in vitro. This richer understanding may help better classification of carcinogens as well as allowing detailed mechanisms of action to be identified. We found that CdCl2 caused DNA damage [micronuclei (MN)] in both TK6 and NH32 cells in a dose-dependent manner after 4 h exposure (plus 23 h recovery), with lowest observable effect levels (LOELs) for MN induction of 1 μM (TK6) and 1.6 μM (NH32). This DNA damage induction in TK6 cells was ROS dependent as pretreatment with the antioxidant N-Acetyl Cysteine (1 mM), abrogated this effect. However, 2',7'-dichlorofluorescin diacetate was not capable of detecting the ROS induced by CdCl2. The use of NH32 cells allowed an investigation of the role of p53 as they are a p53 null cell line derived from TK6. NH32 showed a 10-fold increase in MN in untreated cells and a similar dose-dependent effect after CdCl2 treatment. In TK6 cells, CdCl2 also caused activation of p53 (accumulation of total and phosphorylated p53), imposition of cell cycle checkpoints (G2/M) and intriguingly the production of smaller and more eccentric (elongated) cells. Overall, this multi-endpoint study suggests a carcinogenic mechanism of CdCl2 involving ROS generation, oxidative DNA damage and p53 activation, leading to cell cycle abnormalities and impacts of cell size and shape. This study shows how the integration of multiple cell biology endpoints studied in parallel in vitro can help mechanistic understanding of how carcinogens disrupt normal cell biology., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Rheumatoid arthritis (RA) is a systemic autoimmune and inflammatory disease. Plasma biomarkers are critical for understanding disease mechanisms, treatment effects, and diagnosis. Mass spectrometry-based proteomics is a powerful tool for unbiased biomarker discovery. However, plasma proteomics is significantly hampered by signal interference from high-abundance proteins, low overall protein coverage, and high levels of missing data from data-dependent acquisition (DDA). To achieve quantitative proteomics analysis for plasma samples with a balance of throughput, performance, and cost, we developed a workflow incorporating plate-based high abundance protein depletion and sample preparation, comprehensive peptide spectral library building, and data-independent acquisition (DIA) SWATH mass spectrometry-based methodology. In this study, we analyzed plasma samples from both RA patients and healthy donors. The results showed that the new workflow performance exceeded that of the current state-of-the-art depletion-based plasma proteomic platforms in terms of both data quality and proteome coverage. Proteins from biological processes related to the activation of systemic inflammation, suppression of platelet function, and loss of muscle mass were enriched and differentially expressed in RA. Some plasma proteins, particularly acute-phase reactant proteins, showed great power to distinguish between RA patients and healthy donors. Moreover, protein isoforms in the plasma were also analyzed, providing even deeper proteome coverage. This workflow can serve as a basis for further application in discovering plasma biomarkers of other diseases.

The expression of six transmembrane epithelial antigen of the prostate (STEAP2) is increased in prostate cancer when compared to normal tissue, suggesting a role for STEAP2 in disease progression. This study aimed to determine whether targeting STEAP2 with an anti-STEAP2 polyclonal antibody (pAb) or CRISPR/Cas9 knockout influenced aggressive prostate cancer traits. Gene expression analysis of the STEAP gene family was performed in a panel of prostate cancer cell lines; C4-2B, DU145, LNCaP and PC3. The highest increases in STEAP2 gene expression were observed in C4-2B and LNCaP cells (p<0.001 and p<0.0001 respectively) when compared to normal prostate epithelial PNT2 cells. These cell lines were treated with an anti-STEAP2 pAb and their viability assessed. CRISPR/Cas9 technology was used to knockout STEAP2 from C4-2B and LNCaP cells and viability, proliferation, migration and invasion assessed. When exposed to an anti-STEAP2 pAb, cell viability significantly decreased (p<0.05). When STEAP2 was knocked out, cell viability and proliferation was significantly decreased when compared to wild-type cells (p<0.001). The migratory and invasive potential of knockout cells were also decreased. These data suggest that STEAP2 has a functional role in driving aggressive prostate cancer traits and could provide a novel therapeutic target for the treatment of prostate cancer.

In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.

Over the past two decades, we have seen an increase in the complexity and diversity of biotherapeutic modalities pursued by biopharmaceutical companies. These biologics are multifaceted and susceptible to post-translational modifications and in vivo biotransformation that could impose challenges for bioanalysis. It is vital to characterize the functionality, stability and biotransformation products of these molecules to enable screening, identify potential liabilities at an early stage and devise a bioanalytical strategy. This article highlights our perspective on characterization and bioanalysis of biologics using hybrid LC-MS in our global nonregulated bioanalytical laboratories. AbbVie's suite of versatile, stage-appropriate characterization assays and quantitative bioanalytical approaches are discussed, along with guidance on their utility in answering project-specific questions to aid in decision-making.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Objective: This work describes a simplified, 96-well plate method for determining the blood-to-plasma concentration ratio (BP ratio) for small molecules., Methods: The need for calibration curves was eliminated using a matrix-matching approach in which blood samples were mixed with blank plasma and plasma samples were mixed with blank blood. As a result, both blood- and plasma-origin samples shared an equivalent matrix ahead of bioanalysis. In the in vitro assay, identical sample matrices were achieved by using the same source of blank plasma and blood., Results: In humans, a good correlation (R2 = 0.84) was observed between the data obtained in this matrix-matching method and literature values for 11 commercial compounds possessing a wide range of logD values across multiple chemical classes. In addition, this method showed good agreement with in vitro BP ratios for 10 proprietary compounds determined radiometrically (R2 = 0.72) in human and preclinical species. Finally, the in vitro matrix matching method compared favorably to BP ratios determined ex vivo for 13 proprietary and literature compounds (R2 = 0.87) in rat., Conclusion: This method, suitable for in vitro and ex vivo BP ratio determinations, is operationally efficient, robust, and a useful improvement upon previously published methods., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Rationale: The in-sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked-in stable isotope labeled internal standard (SIL-IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high-resolution mass spectrometers, quantitation on high-resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high-resolution mass spectrometer equipped with an Orbitrap detector., Methods: Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL-IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves., Results: A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product-ion-based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments., Conclusions: ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non-reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high-resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High-resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high-resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high-resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies., (© 2022 John Wiley & Sons Ltd.)

Due to the expansive application of TiO 2 and its variance in physico-chemical characteristics, the toxicological profile of TiO 2 , in all its various forms, requires evaluation. This study aimed to assess the hazard of five TiO 2 particle-types in relation to their cytotoxic profile correlated to their cellular interaction, specifically in human lymphoblast (TK6) and type-II alveolar epithelial (A549) cells. Treatment with the test materials was undertaken at a concentration range of 1-100 μg/cm 2 over 24 and 72 h exposure. TiO 2 interaction with both cell types was visualised by transmission electron microscopy, supported by energy-dispersive X-ray. None of the TiO 2 materials tested promoted cytotoxicity in either cell type over the concentration and time range studied. All materials were observed to interact with the A549 cells and were further noted to be internalised following 24 h exposure. In contrast, only the pigmentary rutile was internalised by TK6 lymphoblasts after 24 h exposure. Where uptake was observed there was no evidence, as determined by 2D microscopy techniques, of particle localisation within the nucleus of either cell type. This study indicates that industrially relevant TiO 2 particles demonstrate cell interactions that are cell-type dependent and do not induce cytotoxicity at the applied dose range., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Coalitions are increasingly utilized to promote positive community health outcomes. Typically, coalitions produce more desirable outcomes when coalition functioning is strong and the coalition model is implemented with fidelity. Although theory indicates that coalitions proceed through predictable stages of development, minimal research explicitly examines functioning and fidelity at these different stages., Method: Within a larger evaluation of Washington State Community Prevention and Wellness Initiative, this cross-sectional study employs qualitative comparative analysis to illuminate the coalition functioning conditions necessary and sufficient to produce high model fidelity at different stages of development in 43 substance misuse prevention coalitions in one state., Results: In the formation stage, only the presence of high levels of coalition leadership was sufficient to produce high model fidelity. In the maintenance stage, three combinations of conditions were sufficient: (1) sustainability planning if, and only if, accompanied by the absence of coalition participation costs, (2) coordinator leadership, and (3) a combination of coalition leadership and team cohesion. In the institutionalization stage, two solutions were sufficient: (1) coalition leadership if, and only if, accompanied by the absence of sustainability planning, and (2) sustainability planning if, and only if, accompanied by the absence of coordinator leadership., Conclusions: This study illustrates several tangible steps technical assistance providers may take to increase the likelihood of achieving model fidelity. In the formation stage, skillful and inclusive coalition leadership is important. In the maintenance stage, technical assistance should focus on reducing participant-perceived costs; increasing sustainability planning; enhancing coordinator-specific leadership; and developing team cohesion and coalition leadership. For coalitions in the institutionalization stage, coalition leadership and sustainability planning may be prime targets for technical assistance. Plain Language Summary: Community coalition approaches to addressing social problems are common and have some evidence of producing positive community outcomes. Research shows that coalitions produce more desirable outcomes when coalition functioning is strong and the coalition model is implemented as it was designed. Although theory suggests that coalitions proceed through predictable stages of development, few studies look at which supports are most needed, at each stage, to improve coalition functioning and implementation. This study aims to help answer that question by determining which aspects of coalition functioning, in which combinations, are key to strong, well-functioning coalitions at different developmental stages in a sample of prevention coalitions in Washington State focused on youth substance misuse. The results suggest that having a strong coalition leadership team is key for all coalitions, regardless of development stage. For coalitions earlier in their formation, this was especially true. For coalitions further into their development, the combination of strong coordinator leadership and strong team cohesion was particularly important. Finally, for the most well-established coalitions, if sustainability planning was weak, strong coalition team leadership was key; but if coordinator leadership was weak, strong sustainability planning was critical to offset the lack of strong leadership. The results can be used to inform the types of technical assistance provided to support coalitions at different developmental stages., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Background: In order to achieve wide-scale impact in community settings, programs must be sustained. Theory and empirical evidence suggest that intervention characteristics, organizational context, capacity for program implementation, and processes related to implementation are associated with continued program delivery. However, few studies examine how combinations of these factors work together in different settings to influence program sustainment., Methods: Using scales specified in the Program Sustainability Assessment Tool (PSAT), the current cross-sectional study aims to identify the necessary and sufficient conditions for the sustainment of the Strengthening Families Program for Parents and Youth 10-14 (SFP 10-14). Staff (n = 59) at SFP 10-14 implementation sites across Washington State completed an online survey reporting on their current level of SFP 10-14 sustainment. They also completed PSAT, with eight scales designed to assess conditions that consistently produce sustainment. Data were analyzed using qualitative comparative analysis., Results: Environmental support was the only necessary condition for sustainment success. Four solutions sufficient to achieve sustainment were also identified. These included the combined presence of (1) environmental support, organizational capacity, and funding stability; (2) environmental support, organizational capacity, communication, and program evaluation, in the absence of strategic planning; (3) environmental support, organizational capacity, program evaluation, and partnerships, in the absence of strategic planning; and (4) environmental support, communication, partnerships, and funding stability, in the absence of program evaluation., Conclusions: Environmental support in combination with organizational capacity appeared to most consistently produce sustainment of SFP 10-14 programs in Washington State. Program providers will benefit from a focusing on enhancing those conditions to increase program sustainment., (© 2022. The Author(s).)

This study aims to develop a 4-limb canine gait analysis system using wireless inertial measurement units (IMUs). 3D printed sensor holders were designed to ensure quick and consistent sensor mounting. Signal analysis algorithms were developed to automatically determine the timing of swing start and end in a stride. To evaluate the accuracy of the new system, a synchronized study was conducted in which stride parameters in four dogs were measured simultaneously using the 4-limb IMU system and a pressure-sensor based walkway gait system. The results showed that stride parameters measured in both systems were highly correlated. Bland-Altman analyses revealed a nominal mean measurement bias between the two systems in both forelimbs and hindlimbs. Overall, the disagreement between the two systems was less than 10% of the mean value in over 92% of the data points acquired from forelimbs. The same performance was observed in hindlimbs except for one parameter due to small mean values. We demonstrated that this 4-limb system could successfully visualize the overall gait types and identify rapid gait changes in dogs. This method provides an effective, low-cost tool for gait studies in veterinary applications or in translational studies using dog models of neuromuscular diseases., (© 2022. The Author(s).)

Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Aged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests D.D. is a member of the scientific advisory board for Solid Biosciences and an equity holder in Solid Biosciences. The Duan lab received research support unrelated to this project from Solid Biosciences and Edgewise Therapeutics., (© 2021. Published by The Company of Biologists Ltd.)

During September 1, 2020-April 30, 2021, the California Department of Public Health, Richmond, California, USA, received 255 positive influenza molecular test results that matched with severe acute respiratory syndrome coronavirus 2 molecular test results; 58 (23%) persons were co-infected. Influenza activity was minimal in California, and co-infections were sporadic.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na + /K + -ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na + /K + -ATPase and γ -glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug ( 3 , ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing., (©2021 American Association for Cancer Research.)

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14 + CD1c + monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

Few-layer graphene (FLG) has garnered much interest owing to applications in hydrogen storage and reinforced nanocomposites. Consequently, these engineered nanomaterials (ENMs) are in high demand, increasing occupational exposure. This investigation seeks to assess the inhalation hazard of industrially relevant FLG engineered with: (i) no surface functional groups (neutral), (ii) amine, and (iii) carboxyl group functionalization. A monoculture of human lung epithelial (16HBE14o - ) cells is exposed to each material for 24-h, followed by cytotoxicity and genotoxicity evaluation using relative population doubling (RPD) and the cytokinesis-blocked micronucleus (CBMN) assay, respectively. Neutral-FLG induces the greatest (two-fold) significant increase (p < 0.05) in micronuclei, whereas carboxyl-FLG does not induce significant (p < 0.05) genotoxicity. These findings correlate to significant (p < 0.05) concentration-dependent increases in interleukin (IL)-8, depletion of intracellular glutathione (rGSH) and a depletion in mitochondrial ATP production. Uptake of FLG is evaluated by transmission electron microscopy, whereby FLG particles are observed within membrane-bound vesicles in the form of large agglomerates (>1 µm diameter). The findings of the present study have demonstrated the capability of neutral-FLG and amine-FLG to induce genotoxicity in 16HBE14o - cells through primary indirect mechanisms, suggesting a possible role for carboxyl groups in scavenging radicals produced via oxidative stress., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Opioid use disorders are chronic and relapse is common. Both negative affect and craving have been suggested antecedents of relapse and have been shown to demonstrate within- and between-person variability, as well as association with each other. The present study extends previous research by examining the covariation of negative affect and craving both within-day and at the person-level during 12 days of treatment among opioid-dependent patients. Ecological momentary assessment (EMA) data were collected from 73 participants starting between 10 and 14 days after admission to an inpatient treatment facility. These data were analyzed using multivariate multilevel models and time-varying effect models. Results demonstrated strong association between negative affect and craving. Within-day, negative affect and craving were most associated in the early afternoon. At the person-level, association between negative affect and craving declined during the first week of data collection. Following this initial decline in association, negative affect and craving increasingly covaried during days 8-12 of data collection. To our knowledge, this is the first study to report a lagged increase in the association between negative affect and craving among patients during inpatient treatment for opioid dependence. Implications for research and treatment providers are discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages))., Results: In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress., Conclusions: Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89-85% RPD. Furthermore, an exposure of 20-50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent., (© The Author(s) 2020. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amorphous solid dispersion (ASD) is a widely employed formulation technique for drugs with poor aqueous solubility. Polymers are integral components of ASDs, but mechanisms by which polymers lead to the generation and maintenance of supersaturated solutions, which enhance oral absorption in vivo, are poorly understood. Herein, a diverse group of newly synthesized cellulose derivatives was evaluated for their ability to inhibit crystallization of enzalutamide, a poorly soluble compound used to treat prostate cancer. ASDs were prepared from selected polymers, specifically a somewhat hydrophobic polymer that was extremely effective at inhibiting drug crystallization, and a less effective, but more hydrophilic, crystallization inhibitor, that might afford better release. Drug membrane transport rate was evaluated in vitro and compared to in vivo performance, following oral dosing in rats. Good correlation was noted between the in vitro diffusion cell studies and the in vivo data. The ASD formulated with the less effective crystallization inhibitor outperformed the ASD prepared with the highly effective crystallization inhibitor in terms of the amount and rate of drug absorbed in vivo. This study provides valuable insight into key factors impacting oral absorption from enabling ASD formulations, and how best to evaluate such formulations using in vitro approaches.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure., (© The Author(s) 2020. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo 'features' such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.

Crystallization of drug from an amorphous formulation is expected to negatively impact its bioperformance following oral delivery. In evaluating this in vivo, neat crystalline drug is typically mixed with the amorphous formulation. However, this approach may not adequately mimic the effect of drug crystals that form within the amorphous matrix, because crystal properties are highly dependent on the crystallization environment. The aim of this study was to evaluate the in vivo impact of crystals formed in a generic tacrolimus amorphous formulation, relative to noncrystallized formulations and a reference suspension containing neat crystalline drug. Crystallization of tacrolimus was induced in the generic product by exposing it to moderate temperatures and high relative humidity. Controlled levels of crystallinity in the formulations were achieved by mixing maximally crystallized and fresh formulations at the desired ratios. These formulations were then characterized in vitro and used for oral dosing to beagle dogs. Analysis of blood concentrations versus time revealed that formulations containing 50 and 100% crystalline tacrolimus resulted in lower area under the curve (AUC) and maximum concentration ( C max ) values as compared to the fresh amorphous formulation. However, the AUC and the C max values for these formulations were significantly higher than those observed after dosing the pure crystalline tacrolimus suspension. The innovator formulation, Prograf, showed comparable pharmacokinetics before and after exposure to accelerated stability conditions, confirming the robustness of the innovator product to drug crystallization. This study provides insight into the impact of endogenously crystallized material on the oral absorption of a poorly water-soluble compound and highlights the importance of using representative crystalline material when undertaking risk assessment of amorphous formulations.

The pro-inflammatory cytokine interleukin (IL)-23 is a key modulator of the immune response, making it an attractive target for the treatment of autoimmune disease. Correspondingly, several monoclonal antibodies against IL-23 are either in development or approved for autoimmune indications such as psoriasis. Despite being a clinical validated target, IL-23 pharmacokinetics (e.g., IL-23 synthesis and elimination rates) and the degree of target suppression (i.e., decrease in free "active" IL-23) associated with clinical efficacy are not well understood, primarily due to its ultra-low circulating levels and the lack of sensitive and accurate measurement methods. In the current work, this issue was overcome by using accelerator mass spectrometry (AMS) to measure the concentration and pharmacokinetics of human recombinant [ 14 C]-IL-23 following an intravenous trace-dose in cynomolgus monkeys. IL-23 pharmacokinetic parameters along with clinical drug exposure and IL-23 binding affinities from four different anti-IL-23 antibodies (ustekinumab, tildrakizumab, guselkumab, and risankizumab) were used to build a pharmacokinetics/pharmacodynamics (PK/PD) model to assess the time course of free IL-23 over one year in psoriasis patients following different dosing regimens. The predicted rank order of reduction of free IL-23 was consistent with their reported rank order of Psoriasis Area and Severity Index (PASI) 100 scores in clinical efficacy trials (ustekinumab < tildrakizumab < guselkumab < risankizumab), thus demonstrating the utility of highly sensitive AMS for determining target pharmacokinetics to inform PK/PD modeling and assessing target suppression associated with clinical efficacy.