Simplifying the Extended Clearance Concept Classification System (EC3S) to Guide Clearance Prediction in Drug Discovery.

PURPOSE: The Extended Clearance Concept Classification System was established as a development-stage tool to provide a framework for identifying fundamental mechanism(s) governing drug disposition in humans. In the present study, the applicability of the EC3S in drug discovery has been investigated. In its current format, the EC3S relies on low-throughput hepatocyte uptake data, which are not frequently generated in a discovery setting. METHODS: A relationship between hepatocyte uptake clearance and MDCK permeability was first established along with intrinsic clearance from human liver microsomes. The performance of this approach was examined by categorizing 64 drugs into EC3S classes and comparing the predicted major elimination pathway(s) to that observed in humans. As an extension of the work, the ability of the simplified EC3S to predict human systemic clearance based on intrinsic clearance generated using in-vitro metabolic systems was evaluated. RESULTS: The assessment enabled the use of MDCK permeability and unscaled unbound intrinsic clearance to generate cut-off criteria to categorize compounds into four EC3S classes: Class 12ab, 2cd, 34ab, and 34cd, with major elimination mechanism(s) assigned to each class. The predictivity analysis suggested that systemic clearance could generally be predicted within threefold for EC3S class 12ab and 34ab compounds. For classes 2cd and 34cd, systemic clearance was poorly predicted using in-vitro systems explored in this study. CONCLUSION: Collectively, our simplified classification approach is expected to facilitate the identification of mechanism(s) involved in drug elimination, faster resolution of in-vitro to in-vivo disconnects, and better design of mechanistic pharmacokinetic studies in drug discovery.

Effects of geometric individualisation of a human spine model on load sharing: neuro-musculoskeletal simulation reveals significant differences in ligament and muscle contribution.

In spine research, two possibilities to generate models exist: generic (population-based) models representing the average human and subject-specific representations of individuals. Despite the increasing interest in subject specificity, individualisation of spine models remains challenging. Neuro-musculoskeletal (NMS) models enable the analysis and prediction of dynamic motions by incorporating active muscles attaching to bones that are connected using articulating joints under the assumption of rigid body dynamics. In this study, we used forward-dynamic simulations to compare a generic NMS multibody model of the thoracolumbar spine including fully articulated vertebrae, detailed musculature, passive ligaments and linear intervertebral disc (IVD) models with an individualised model to assess the contribution of individual biological structures. Individualisation was achieved by integrating skeletal geometry from computed tomography and custom-selected muscle and ligament paths. Both models underwent a gravitational settling process and a forward flexion-to-extension movement. The model-specific load distribution in an equilibrated upright position and local stiffness in the L4/5 functional spinal unit (FSU) is compared. Load sharing between occurring internal forces generated by individual biological structures and their contribution to the FSU stiffness was computed. The main finding of our simulations is an apparent shift in load sharing with individualisation from an equally distributed element contribution of IVD, ligaments and muscles in the generic spine model to a predominant muscle contribution in the individualised model depending on the analysed spine level.

The control effort to steer self-propelled microswimmers depends on their morphology: comparing symmetric spherical versus asymmetric L-shaped particles.

Active goal-directed motion requires real-time adjustment of control signals depending on the system's status, also known as control. The amount of information that needs to be processed depends on the desired motion and control, and on the system's morphology. The morphology of the system may directly effectuate or support the desired motion. This morphology-based reduction to the neuronal 'control effort' can be quantified by a novel information-entropy-based approach. Here, we apply this novel measure of 'control effort' to active microswimmers of different morphology. Their motion is a combination of directed deterministic and stochastic motion. In spherical microswimmers, the active propulsion leads to linear velocities. Active propulsion of asymmetric L-shaped particles leads to circular or-on tilted substrates-directed motion. Thus, the difference in shape, i.e. the morphology of the particles, directly influence the motion. Here, we quantify how this morphology can be exploited by control schemes for the purpose of steering the particles towards targets. Using computer simulations, we found in both cases a significantly lower control effort for L-shaped particles. However, certain movements can only be achieved by spherical particles. This demonstrates that a suitably designed microswimmer's morphology might be exploited to perform specific tasks.

Primary human hepatocyte spheroids as an in vitro tool for investigating drug compounds with low clearance.

Characterizing the pharmacokinetic properties of drug candidates represents an essential task during drug development. In the past, liver microsomes and primary suspended hepatocytes have been extensively used for this purpose, but their relatively short stability limits the applicability of such in vitro systems for drug compounds with low metabolic turnover. In the present study, we used 3D primary human hepatocyte spheroids to predict the hepatic clearance of seven drugs with low to intermediate clearance in humans. Our results indicate that hepatocyte spheroids maintain their in vivo like phenotype during prolonged incubations allowing to monitor the depletion of parent drug for seven days. In contrast, attempts to increase the relative metabolic capacity by pooling hepatocyte spheroids resulted in an immediate fusion of the spheroids followed by hepatocellular de-differentiation processes, demonstrating limited applicability of the pooling approach for quantitative pharmacokinetic studies. The hepatic clearance values obtained from incubations with individual spheroids were in close correlation with the clinical reference data with six out of seven drug compounds being predicted within a three-fold deviation and average fold and absolute average fold errors of 0.57 and 1.74, respectively. In conclusion, the hepatocyte spheroid model enables accurate hepatic clearance predictions for slowly metabolized drug compounds and represents a valuable tool for determining the pharmacokinetic properties of new drug candidates as well as for mechanistic pharmacokinetic studies. Significance Statement Traditional in vitro systems often fail to predict the hepatic clearance of slowly metabolized drug compounds. The current study demonstrates the ability of primary human hepatocyte spheroids to provide accurate projections on the hepatic clearance of drug compounds with low and intermediate clearance.

Intuitive assessment of modeled lumbar spinal motion by clustering and visualization of finite helical axes.

A variety of medical imaging procedures, cadaver experiments, and computer models have been utilized to capture, depict, and understand the motion of the human lumbar spine. Particular interest lies in assessing the relative movement between two adjacent vertebrae, which can be represented by a temporal evolution of finite helical axes (FHA). Mathematically, this FHA evolution constitutes a seven-dimensional quantity: one dimension for the time, two for the (normalized) direction vector, another two for the (unique) position vector, as well as one for each the angle of rotation around and the amount of translation along the axis. Predominantly in the literature, however, movements are assumed to take place in certain physiological planes on which FHA are projected. The resulting three-dimensional quantity - the so-called centrode - is easily presentable but leaves out substantial pieces of available data. Here, we investigate and assess several possibilities to visualize subsets of FHA data of increasing dimensionality. Finally, we utilize an agglomerative hierarchical clustering algorithm and propose a novel visualization technique, namely the quiver principal axis plot (QPAP), to depict the entirety of information inherent to hundreds or thousands of FHA. The QPAP method is applied to flexion-extension, lateral bending, and axial rotation movements of a lumbar spine within both a reduced model as well as a complex upper body system.

Loads distributed in vivo among vertebrae, muscles, spinal ligaments, and intervertebral discs in a passively flexed lumbar spine.

The load distribution among lumbar spinal structures-still an unanswered question-has been in the focus of this hybrid experimental and simulation study. First, the overall passive resistive torque-angle characteristics of healthy subjects' lumbar spines during flexion-extension cycles in the sagittal plane were determined experimentally by use of a custom-made trunk-bending machine. Second, a forward dynamic computer model of the human body that incorporates a detailed lumbar spine was used to (1) simulate the human-machine interaction in accordance with the experiments and (2) validate the modeled properties of the load-bearing structures. Third, the computer model was used to predict the load distribution in the experimental situation among the implemented lumbar spine structures: muscle-tendon units, ligaments, intervertebral discs, and facet joints. Nine female and 10 male volunteers were investigated. Lumbar kinematics were measured with a marker-based infrared device. The lumbar flexion resistance was measured by the trunk-bending machine through strain gauges on the axes of the machine's torque motors. Any lumbar muscle activity was excluded by simultaneous sEMG monitoring. A mathematical model was used to describe the nonlinear flexion characteristics. The subsequent extension branch of a flexion-extension torque-angle characteristic could be significantly distinguished from its flexion branch by the zero-torque lordosis angle shifted to lower values. A side finding was that the model values of ligament and passive muscle stiffnesses, extracted from well-established literature sources, had to be distinctly reduced in order to approach our measured overall lumbar stiffness values. Even after such parameter adjustment, the computer model still predicts too stiff lumbar spines in most cases in comparison with experimental data. A review of literature data reveals a deficient documentation of anatomical and mechanical parameters of spinal ligaments. For instance, rest lengths of ligaments-a very sensitive parameter for simulations-and cross-sectional areas turned out to be documented at best incompletely. Yet by now, our model well reproduces the literature data of measured pressure values within the lumbar disc at level L4/5. Stretch of the lumbar dorsal (passive) muscle and ligament structures as an inescapable response to flexion can fully explain the pressure values in the lumbar disc. Any further external forces like gravity, or any muscle activities, further increase the compressive load on a vertebral disc. The impact of daily or sportive movements on the loads of the spinal structures other than the disc cannot be predicted ad hoc, because, for example, the load distribution itself crucially determines the structures' current lever arms. In summary, compressive loads on the vertebral discs are not the major determinants, and very likely also not the key indicators, of the load scenario in the lumbar spine. All other structures should be considered at least equally relevant in the future. Likewise, load indicators other than disc compression are advisable to turn attention to. Further, lumbar flexion is a self-contained factor of lumbar load. It may be worthwhile, to take more consciously care of trunk flexion during daily activities, for instance, regarding long-term effects like lasting repetitive flexions or sedentary postures.

Mechanisms of Chronic Fialuridine Hepatotoxicity as Revealed in Primary Human Hepatocyte Spheroids.

Drug hepatotoxicity is often delayed in onset. An exemplar case is the chronic nature of fialuridine hepatotoxicity, which resulted in the deaths of several patients in clinical trials as preclinical studies failed to identify this human-specific hepatotoxicity. Conventional preclinical in vitro models are mainly designed to evaluate the risk of acute drug toxicity. Here, we evaluated the utility of 3D spheroid cultures of primary human hepatocytes (PHHs) to assess chronic drug hepatotoxicity events using fialuridine as an example. Fialuridine toxicity was only detectable after 7 days of repeated exposure. Clinical manifestations, including reactive oxygen species formation, lipid accumulation, and induction of apoptosis, were readily identified. Silencing the expression or activity of the human equilibrative nucleoside transporter 1 (ENT1), implicated in the mitochondrial transport of fialuridine, modestly protected PHH spheroids from fialuridine toxicity. Interference with the phosphorylation of fialuridine into the active triphosphate metabolites by silencing of thymidine kinase 2 (TK2) provided substantial protection, whereas simultaneous silencing of ENT1 and TK2 provided near-complete protection. Fialuridine-induced mitochondrial dysfunction was suggested by a decrease in the expression of mtDNA-encoded genes, which correlated with the onset of toxicity and was prevented under the simultaneous silencing of ENT1 and TK2. Furthermore, interference with the expression or activity of ribonucleotide reductase (RNR), which is critical to deoxyribonucleoside triphosphate (dNTP) pool homeostasis, resulted in selective potentiation of fialuridine toxicity. Our findings demonstrate the translational applicability of the PHH 3D spheroid model for assessing drug hepatotoxicity events which manifest only under chronic exposure conditions.

Examining P-gp efflux kinetics guided by the BDDCS - Rational selection of in vitro assay designs and mathematical models.

The generation of reliable kinetic parameters to describe P-glycoprotein (P-gp) activity is essential for predicting the impact of efflux transport on gastrointestinal drug absorption. The compound-specific selection of in vitro assay designs and ensuing data analysis methods is explored in this manuscript. We measured transcellular permeability and cellular uptake of five P-gp substrates in Caco-2 and LLC-PK1 MDR1 cells. Kinetic parameters of P-gp-mediated efflux transport (Km, Vmax) were derived from conventional and mechanistic compartmental models. The estimated apparent Km values based on medium concentrations in the conventional permeability model indicated significant differences between the cell lines. The respective intrinsic Km values based on unbound intracellular concentrations in the mechanistic compartmental models were significantly lower and comparable between cell lines and assay formats. Non-specific binding or lysosomal trapping were shown to cause discrepancies in the kinetic parameters obtained from different assay formats. A guidance for the selection of in vitro assays and kinetic assessment methods is proposed in line with the Biopharmaceutics Drug Disposition Classification System (BDDCS). The recommendations are expected to aid the acquisition of robust and reproducible kinetic parameters of P-gp-mediated efflux transport.

Current In Vitro Methods to Determine Hepatic Kpuu: A Comparison of Their Usefulness and Limitations.

Unbound intrahepatic drug concentrations determine the interaction potential with intracellular targets related to toxicity, pharmacokinetics, or pharmacodynamics. Recently, the unbound liver-to-blood partition coefficient (Kpuu) based on the Extended Clearance Model (ECM) has been developed providing indirect estimates of unbound intrahepatic drug concentrations. This study aimed to determine Kpuu for 18 diverse drug compounds by 3 alternative in vitro methods and to compare the outcome with the ECM approach. Kpuu was calculated from independent measurements of hepatocellular drug accumulation (Kp) and unbound fraction in hepatocytes (fuhep) either assessed from steady-state accumulation at 4°C (temperature method), using equilibrium dialysis (homogenization method), or empirically from logD7.4 (logD7.4 method). Deviations to ECM-based Kpuu data were closely linked to the absence of intrinsic clearance processes (metabolism, biliary secretion) in the investigated methods. Differences in fuhep additionally contributed to deviations in Kpuu. The homogenization method generally provided lowest fuhep values, especially for compounds with high molecular weight or low logD7.4. Kpuu values of compounds with low intrinsic clearance correlated well between the ECM and temperature methods independent of physicochemical properties. Therefore, only the ECM provides an integrated quantitative determination of hepatic Kpuu. Temperature and homogenization methods, however, represent useful alternatives if compound properties are appropriately considered.

Assessing the Risk of Drug-Induced Cholestasis Using Unbound Intrahepatic Concentrations.

Inhibition of the bile salt export pump (BSEP) has been recognized as a key factor in the development of drug-induced cholestasis (DIC). The risk of DIC in humans has been previously assessed using in vitro BSEP inhibition data (IC50) and unbound systemic drug exposure under assumption of the "free drug hypothesis." This concept, however, is unlikely valid, as unbound intrahepatic drug concentrations are affected by active transport and metabolism. To investigate this hypothesis, we experimentally determined the in vitro liver-to-blood partition coefficients (Kpuu) for 18 drug compounds using the hepatic extended clearance model (ECM). In vitro-in vivo translatability of Kpuu values was verified for a subset of compounds in rat. Consequently, unbound intrahepatic concentrations were calculated from clinical exposure (systemic and hepatic inlet) and measured Kpuu data. Using these values, corresponding safety margins against BSEP IC50 values were determined and compared with the clinical incidence of DIC. Depending on the ECM class of a drug, in vitro Kpuu values deviated up to 14-fold from unity, and unbound intrahepatic concentrations were affected accordingly. The use of in vitro Kpuu-based safety margins allowed separation of clinical cholestasis frequency into three classes (no cholestasis, cholestasis in ≤2%, and cholestasis in >2% of subjects) for 17 out of 18 compounds. This assessment was significantly superior compared with using unbound extracellular concentrations as a surrogate for intrahepatic concentrations. Furthermore, the assessment of Kpuu according to ECM provides useful guidance for the quantitative evaluation of genetic and physiologic risk factors for the development of cholestasis.

New IVIVE method for the prediction of total human clearance and relative elimination pathway contributions from in vitro hepatocyte and microsome data.

Total human clearance is a key determinant for the pharmacokinetic behavior of drug candidates. Our group recently introduced the Extended Clearance Model (ECM) as an accurate in vitro-in vivo extrapolation (IVIVE) method for the prediction of hepatic clearance. Yet, knowledge about relative elimination pathway contributions is needed in order to predict the total human clearance of drug candidates. In the present work, a training set of 18 drug compounds was used to describe the affiliations between in vitro sinusoidal uptake clearance and the fractional contributions of hepatic (metabolic and biliary) or renal clearance to overall in vivo elimination. By means of these quantitative relationships and using a validation set of 10 diverse drug molecules covering different (sub)classes of the Extended Clearance Concept Classification System (ECCCS), the relative contributions of elimination pathways were calculated and demonstrated to well correlate with human reference data. Likewise, ECM- and pathway-based predictions of total clearances from both data sets demonstrated a strong correlation with the observed clinical values with 26 out of 28 compounds within a three-fold deviation. Hence, total human clearance and relative contributions of elimination pathways were successfully predicted by the presented method using solely hepatocyte and microsome in vitro data.

The quantitative nuclear matrix proteome as a biochemical snapshot of nuclear organization.

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining bona fide NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology.