The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration.

Next-generation risk assessment (NGRA) for environmental chemicals involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Among existing NAMs, the omic-based technologies are of particular importance based on the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omics level, such as transcriptome, proteome, metabolome, epigenome and genome. Transcriptomic assay plays a leading role in providing relatively conservative PoDs compared with apical endpoints. However, it is unclear whether and how parameters measured with other omics techniques predict the cellular response to chemical perturbations, especially at exposure levels below the transcriptomically defined PoD. Multi-omics coverage may provide additional sensitive or confirmative biomarkers to complement and reduce the uncertainty in safety decisions made using targeted and transcriptomics assays. In the present study, we conducted multi-omics studies of transcriptomics, proteomics and phosphoproteomics on two prototype compounds, coumarin and 2,4-dichlorophenoxyacetic acid (2,4-D), with multiple chemical concentrations and time points, to understand the sensitivity of the three omics techniques in response to chemically-induced changes in HepG2. We demonstrated that, phosphoproteomics alterations occur not only earlier in time, but also more sensitive to lower concentrations than proteomics and transcriptomics when the HepG2 cells were exposed to various chemical treatments. The phosphoproteomics changes appear to approach maximum when the transcriptomics alterations begin to initiate. Therefore, it is proximal to the very early effects induced by chemical exposure. We concluded that phosphoproteomics can be utilized to provide a more complete coverage of chemical-induced cellular alteration and supplement transcriptomics-based health safety decision making.

Particle Image Velocimetry (PIV) measurements of USP Apparatus 1 hydrodynamics with 500 mL fill volume.

Changes to hydrodynamics arising from changes within dissolution testing systems, such as the fill volume level, can potentially cause variability in dissolution results. However, the literature on hydrodynamics in Apparatus 1 is quite limited and little information is available for vessels with different liquid volumes. Here, velocities in a USP Apparatus 1 vessel with a liquid fill volume of 500 mL, a common alternative to 900 mL, were experimentally measured using 2D-2C Particle Image Velocimetry (PIV) for different basket rotational speeds. Tangential velocities dominated the flow field, while axial and radial velocities were much lower and varied with location. The velocities distribution increased proportionately with the basket rotational speed almost everywhere in the vessel excepting for underneath the basket. A nearly horizontal radial liquid jet was found to originate close to the basket upper edge. Comparison of these results with those previously reported with 900-mL liquid volume (Sirasitthichoke et al., Intern. J. Pharmaceutics:X; 3 (2021) 100078) showed that the flow rate through the baskets was similar in both systems, implying that, at least initially, the amount of drug in solution would increase linearly with time. In other words, the flow rate through the baskets would be independent of the liquid volume. Velocity profiles were also found to be similar, except in the region above the basket, which was affected by the radial jet with an orientation significantly different between the 500-mL and the 900-mL systems.

Using transcriptomics, proteomics and phosphoproteomics as new approach methodology (NAM) to define biological responses for chemical safety assessment.

Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments. We showed that the methodology presented here was able to capture the complete chain of events from the first chemical-induced changes at the phosphoproteome level, to changes in gene expression, and lastly to changes in protein abundance, each with vastly different points of departure (PODs). In HepG2 cells we found that the metabolism of lipids and general cellular stress response to be the dominant biological processes in response to caffeine and coumarin exposure, respectively. The phosphoproteomic changes were detected early in time, at very low doses and provided a fast, adaptive cellular response to chemical exposure with 7-37-fold lower points of departure comparing to the transcriptomics. Changes in protein abundance were found much less frequently than transcriptomic changes. While challenges remain, our study provides strong and novel evidence supporting the notion that these three omic technologies can be used in an integrated manner to facilitate a more complete understanding of pathway perturbations and POD determinations for risk assessment of chemical exposures.

Are Non-animal Systemic Safety Assessments Protective? A Toolbox and Workflow.

An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions. The toolbox includes physiologically based kinetic (PBK) models to estimate systemic Cmax levels in humans, and 3 bioactivity platforms, comprising high-throughput transcriptomics, a cell stress panel, and in vitro pharmacological profiling, from which points of departure are estimated. A Bayesian model was developed to quantify the uncertainty in the Cmax estimates depending on how the PBK models were parameterized. The feasibility of the evaluation approach was tested using 24 exposure scenarios from 10 chemicals, some of which would be considered high risk from a consumer goods perspective (eg, drugs that are systemically bioactive) and some low risk (eg, existing food or cosmetic ingredients). Using novel protectiveness and utility metrics, it was shown that up to 69% (9/13) of the low risk scenarios could be identified as such using the toolbox, whilst being protective against all (5/5) the high-risk ones. The results demonstrated how robust safety decisions could be made without using animal data. This work will enable a full evaluation to assess how protective and useful the toolbox and workflow are across a broader range of chemical-exposure scenarios.

Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells.

Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.

Impact of Select Geometric and Operational Parameters on Hydrodynamics in Dissolution Apparatus 2 (Paddle Apparatus): A Design of Experiments Analysis Based on Computational Fluid Dynamics Simulations.

PURPOSE: A Design of Experiments (DOE) analysis driven by Computational Fluid Dynamics (CFD) simulations was used to evaluate individual and two-factor interaction effects of varying select geometric and operational parameters on the hydrodynamics in dissolution apparatus 2 (paddle apparatus). METHODS: Simulations were run with meshing controls and solution strategies retained from a mesh-independent validated baseline model. Distance between vessel and impeller bottom surfaces, impeller offset, vessel radius and impeller rotation speed were considered as input parameters. The velocity magnitudes at four locations near the vessel bottom surface were considered as output parameters. Response surfaces and Pareto charts were generated to understand individual and two-factor interaction effects of input parameters on the output parameters. RESULTS: Impeller offset has a dominating influence of a linear and quadratic nature on the output parameters and affects overall hydrodynamics. Changes to other input parameters have limited influence on velocity magnitudes at locations closest to the vessel axis and on overall hydrodynamics. However, these parameters have important influences of varying degrees on velocity magnitudes at locations away from the vessel axis. CONCLUSIONS: The hydrodynamics in Apparatus 2 is influenced differently by different parameters and their combinations. Impeller offset has a stronger influence when compared to parameters that do not alter apparatus symmetry.

Latent Variables Capture Pathway-Level Points of Departure in High-Throughput Toxicogenomic Data.

Estimation of points of departure (PoDs) from high-throughput transcriptomic data (HTTr) represents a key step in the development of next-generation risk assessment (NGRA). Current approaches mainly rely on single key gene targets, which are constrained by the information currently available in the knowledge base and make interpretation challenging as scientists need to interpret PoDs for thousands of genes or hundreds of pathways. In this work, we aimed to address these issues by developing a computational workflow to investigate the pathway concentration-response relationships in a way that is not fully constrained by known biology and also facilitates interpretation. We employed the Pathway-Level Information ExtractoR (PLIER) to identify latent variables (LVs) describing biological activity and then investigated in vitro LVs' concentration-response relationships using the ToxCast pipeline. We applied this methodology to a published transcriptomic concentration-response data set for 44 chemicals in MCF-7 cells and showed that our workflow can capture known biological activity and discriminate between estrogenic and antiestrogenic compounds as well as activity not aligning with the existing knowledge base, which may be relevant in a risk assessment scenario. Moreover, we were able to identify the known estrogen activity in compounds that are not well-established ER agonists/antagonists supporting the use of the workflow in read-across. Next, we transferred its application to chemical compounds tested in HepG2, HepaRG, and MCF-7 cells and showed that PoD estimates are in strong agreement with those estimated using a recently developed Bayesian approach (cor = 0.89) and in weak agreement with those estimated using a well-established approach such as BMDExpress2 (cor = 0.57). These results demonstrate the effectiveness of using PLIER in a concentration-response scenario to investigate pathway activity in a way that is not fully constrained by the knowledge base and to ease the biological interpretation and support the development of an NGRA framework with the ability to improve current risk assessment strategies for chemicals using new approach methodologies.

Experimental determination of the velocity distribution in USP Apparatus 1 (basket apparatus) using Particle Image Velocimetry (PIV).

The USP Apparatus 1 (basket apparatus) is commonly used to evaluate the dissolution performance of oral solid dosage forms. The hydrodynamics generated by the basket contributes, in general, to the dissolution rate and hence the dissolution results. Here, the hydrodynamics of Apparatus 1 was quantified in a vessel filled with 900-mL de-ionized water at room temperature by determining, via Particle Image Velocimetry (PIV), the velocity profiles on a vertical central plane and on 11 horizontal planes at different elevations at three different basket agitation speeds. The flow field was dominated by the tangential velocity component and was approximately symmetrical in all cases. Despite all precautions taken, small flow asymmetries were observed in the axial and radial directions. This appears to be an unavoidable characteristic of the flow in Apparatus 1. The magnitudes of the axial and radial velocity components varied with location but were always low. A small jet was seen emanating radially near the top edge of the basket. Velocities typically scaled well with increasing agitation speed in most regions of the vessel except for a region directly below the basket. The results of this work provide a major insight into the flow field inside the USP Apparatus 1.

Evaluation of intratympanic formulations for inner ear delivery: methodology and sustained release formulation testing.

A convenient and efficient in vitro diffusion cell method to evaluate formulations for inner ear delivery via the intratympanic route is currently not available. The existing in vitro diffusion cell systems commonly used to evaluate drug formulations do not resemble the physical dimensions of the middle ear and round window membrane. The objectives of this study were to examine a modified in vitro diffusion cell system of a small diffusion area for studying sustained release formulations in inner ear drug delivery and to identify a formulation for sustained drug delivery to the inner ear. Four formulations and a control were examined in this study using cidofovir as the model drug. Drug release from the formulations in the modified diffusion cell system was slower than that in the conventional diffusion cell system due to the decrease in the diffusion surface area of the modified diffusion cell system. The modified diffusion cell system was able to show different drug release behaviors among the formulations and allowed formulation evaluation better than the conventional diffusion cell system. Among the formulations investigated, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer systems provided the longest sustained drug delivery, probably due to their rigid gel structures and/or polymer-to-cidofovir interactions.

Transport behavior of hairless mouse skin during constant current DC iontophoresis, part 2: iontophoresis of nonionic molecules with cotransport of polystyrene sulfonate oligomers.

The purpose of this study was to characterize changes that occur in the iontophoretic transport of nonionic probe permeants in hairless mouse skin epidermal membrane from the anode to cathode when polystyrene sulfonate (PSS) oligomers are cotransported from the cathode to anode. The experiments were conducted with trace levels of the nonionic probe permeants: urea, mannitol, and raffinose. In order to systematically assess changes that occur as a result of having PSS in the cathodal chamber, the steady-state transport parameters of the membrane and the experimental permeability coefficients of the probe permeants were determined and compared with results obtained from earlier baseline experiments where both the cathodal and anodal chamber media were phosphate buffered saline. In addition, the physicochemical properties of the PSS solutions were determined including the solution viscosity and conductance as well as the mobilities of individual PSS oligomers. The effective pore radii of the transport pathways were calculated using a theoretical expression based on simultaneous diffusion and electroosmosis. Compared with the baseline results, the calculated radii were found to have increased up to around twofold and the iontophoretic fluxes of the probe permeants increased by as much sixfold.

Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis.

Macromolecules such as therapeutic proteins currently serve an important role in the treatment of eye diseases such as wet age-related macular degeneration and diabetic retinopathy. Particularly, bevacizumab and ranibizumab have been shown to be effective in the treatment of these diseases. Iontophoresis can be employed to enhance ocular delivery of these macromolecules, but the lack of information on the properties of these macromolecules has hindered its development. The objectives of the present study were to determine the effective electrophoretic mobilities and charges of bevacizumab, ranibizumab, and model compound polystyrene sulfonate (PSS) using capillary zone electrophoresis. Salicylate, lidocaine, and bovine serum albumin (BSA), which have known electrophoretic mobilities in the literature, were also studied to validate the present technique. The hydrodynamic radii and diffusion coefficients of BSA, bevacizumab, ranibizumab, and PSS were measured by dynamic light scattering. The effective charges were calculated using the Einstein relation between diffusion coefficient and electrophoretic mobility and the Henry equation. The results show that bevacizumab and ranibizumab have low electrophoretic mobilities and are net negatively charged in phosphate buffered saline (PBS) of pH 7.4 and 0.16M ionic strength. PSS has high negative charge but the electrophoretic mobility in PBS is lower than that expected from the polymer structure. The present study demonstrated that capillary electrophoresis could be used to characterize the mobility and charge properties of drug candidates in the development of iontophoretic drug delivery.

Transport behavior of hairless mouse skin during constant current DC iontophoresis I: baseline studies.

The fluxes of charged and nonionic molecules across hairless mouse skin (HMS) were induced by direct current iontophoresis and used to characterize the transport pathways of the epidermal membrane. Experimental data were used to determine permeability coefficients from which the effective pore radii (Rp) of the transport pathways were calculated. Permeants used in these experiments were nonionic permeants (urea, mannitol, and raffinose), monovalent cationic permeants (sodium, tetraethylammonium, and tetraphenylphosphonium ions), and monovalent anionic permeants (chloride, salicylate, and taurocholate ions). The Rp estimates obtained by the anionic permeant pairs were 49, 22, and 20 Å for the chloride/salicylate (Cl:SA), chloride/taurocholate (Cl:TC), and salicylate/taurocholate (SA:TC) pairs, respectively; with the cationic permeant pairs, the Rp values obtained were 19, 30, and 24 Å for the sodium/tetraethylammonium (Na:TEA), sodium/tetraphenylphosphonium (Na:TPP), and the tetraethylammonium/tetraphenylphosphonium (TEA:TPP) pairs, respectively. Rp estimates for HMS obtained from nonionic permeant experiments ranged from 6.7 to 13.4 Å. When plotted versus their respective diffusion coefficients, all of the permeability coefficients for the cationic permeants were greater than those of the anionic permeants. Additionally, the magnitudes of permeability coefficients determined in the current study with HMS were of the same order of magnitude as those previously determined in our laboratory using human epidermal membrane under similar iontophoresis conditions.

The USP Performance Verification Test, Part I: USP Lot P Prednisone Tablets: quality attributes and experimental variables contributing to dissolution variance.

PURPOSE: Beyond instrumental qualification, proficiency testing is not usually a prerequisite for many analytical procedures, given reliance on a manufacturer's assay validation coupled with regulatory review and inspection. Given the special features of the dissolution procedure, proficiency testing was put in place initially by pharmaceutical manufacturers and carried on by USP. Proficiency testing is designed to help ensure that execution of a dissolution procedure for solid oral dosage forms adequately supports administrative and legal decisions so that measurements made at different times, by different analysts, or with different methods can be confidently compared. USP has applied metrological principles to aid practitioners in carrying out the dissolution procedure alone and in collaborative studies to facilitate understanding potential sources of variability. MATERIALS AND METHODS: The present study aimed to identify key dissolution variables associated with USP Lot P Prednisone Tablets in conjunction with the USP Performance Verification Test (PVT). Using five dissolution test assemblies from different manufacturers, at least four of six analysts determined percents prednisone dissolved on dissolution Apparatus 1 (basket) and Apparatus 2 (paddle) on each assembly. Six replicate experiments were performed on each analyst-assembly combination with a set of six to eight tablets in each experiment. RESULTS AND CONCLUSIONS: Statistical analysis demonstrated that dissolution test assemblies were the largest factor contributing to dissolution variability. Inherent tablet variability was low, and USP Lot P Prednisone Tablets did not contribute importantly to dissolution variability. Contributions from analyst and analytical procedure also were estimated to be low.

Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis.

Previous studies in our laboratory have shown that enhanced, constant permeant fluxes across human skin can be achieved by applying an alternating current (AC) to maintain skin electrical conductance at a constant level. Relative to conventional direct current (DC) iontophoresis, for which current is maintained at a constant level, this newly developed constant conductance alternating current (CCAC) method achieves constant fluxes with less inter- and intra-sample variability. The present study focused upon further investigating the permeability properties of human skin during CCAC iontophoresis at a variety of target resistance/conductance values. A three-stage experimental protocol was used with flux measurements determined on 3 consecutive days. Stage I was an AC only protocol (symmetrical AC square-wave signal), stage II was an AC plus DC protocol (AC square-wave with DC offset voltage), and stage III was a repeat of stage I. During this three-stage protocol, the skin electrical resistance was maintained at a constant target value by manually adjusting the applied AC voltage. Radiolabeled mannitol and urea were model permeants in all experiments. Their fluxes were determined and used to characterize the permeability properties of human skin. The results from the present study established that: (i) the CCAC protocol made it possible to reduce HEM electrical resistance to different target levels as low as 0.8 kOmega cm(2) and maintain the specific resistance level throughout the flux experiment, (ii) permeant fluxes are proportional to skin electrical conductance, (iii) under the studied CCAC passive conditions, membrane pore size tends to increase as skin resistance decreases, and (iv) as the membrane breaks down, its pore sizes become larger.