Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model.

Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole. SIGNIFICANCE STATEMENT: In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.

Can Bile Salt Export Pump Inhibition Testing in Drug Discovery and Development Reduce Liver Injury Risk? An International Transporter Consortium Perspective.

Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma ). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow-up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case-by-case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

In Vitro Drug-Induced Liver Injury Prediction: Criteria Optimization of Efflux Transporter IC50 and Physicochemical Properties.

Drug-induced liver injury (DILI) is a severe drug adverse response, which cannot always be reliably predicted in preclinical or clinical studies. Lack of observation of DILI during preclinical and clinical drug development has led to DILI being a leading cause of drug withdrawal from the market. As DILI is potentially fatal, pharmaceutical companies have been developing in vitro tools to screen for potential liver injury. Screens for physicochemical properties, mitochondrial function, and transport protein inhibition have all been employed to varying degrees of success. In vitro inhibition of the bile salt export pump (BSEP) has become a major risk factor for in vivo DILI predictions, yet discrepancies exist in which methods to use and the extent to which BSEP inhibition predicts clinical DILI. The presented work focuses on optimizing DILI predictions by comparing BSEP inhibition via the membrane vesicle assay and the hepatocyte-based BSEPcyte assay, as well as dual and triple liabilities. BSEP transport inhibition of taurcholic acids and glycocholic acids were similar for up to 29 drugs tested, in both the vesicle and hepatocyte-based assays. Positive and negative DILI predictions were optimized at a 50-µM cutoff value for 50 drugs using both NIH Livertox and PharmaPendium databases. Additionally, dual inhibition of BSEP and other efflux transporters (multidrug resistance-associated protein [MRP]2, MRP3, or MRP4) provided no observable predictive benefit compared with BSEP inhibition alone. Eighty-five percent of drugs with high molecular weight (>600 Da), high cLogP (>3), or a daily dose >100 mg and BSEP inhibition were associated with DILI. Triple liability of BSEP inhibition, high molecular weight, and high cLogP attained a 100% positive prediction rate.

Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans.

Aging and degeneration of human tissue come with the loss of tissue water retention and associated changes in physical properties partially due to degradation and subsequent loss of proteoglycans. We demonstrated a novel method of fabrication of biomimetic proteoglycans, which mimic the three-dimensional bottlebrush architecture and physical behavior of natural proteoglycans responsible for tissue hydration and structural integrity. Biomimetic proteoglycans are synthesized by an end-on attachment of natural chondroitin sulfate bristles to a synthetic poly(acryloyl chloride) backbone. Atomic force microscopy imaging suggested three-dimensional core-bristle architecture, and hydrodynamic size of biomimetic proteoglycans was estimated at 61.3 ± 12.3 nm using dynamic light scattering. Water uptake results indicated that biomimetic proteoglycans had a ∼50% increased water uptake compared to native aggrecan and chondroitin sulfate alone. The biomimetic proteoglycans are cytocompatible in the physiological ranges of concentrations and could be potentially used to repair damaged or diseased tissue with depleted proteoglycan content.

Hierarchically ordered polymer nanofiber shish kebabs as a bone scaffold material.

The ideal bone scaffold harnesses the body's ability to regenerate bone in critical size defects. A potential strategy to design the ideal bone scaffold is to mimic the natural structure of bone at the molecular level. The orientation and spatial distribution of nanocrystals in the organic matrix are two important and distinctive structural characteristics associated with natural bone. There has yet to be a synthetic system or scaffold that is able to control both the spatial distribution and orientation of the hydroxyapatite crystalline structure. To mimic the unique hybrid structure of natural bone using synthetic materials, there have been a number of reported approaches to control the mineral nanocrystal orientation on synthetic scaffolds. However, the spatial distribution of minerals is challenging to reproduce in a biomimetic manner. Herein we report using block copolymer-decorated polymer nanofibers to achieve biomineralized fibrils with precise control of both mineral crystal orientation and spatial distribution. We show that the crystallization of poly(caprolactone)-b-poly(acrylic acid) block copolymer on poly(caprolactone) nanofibers resulted in a unique shish kebab structure that significantly enhances the mechanical properties of the nanofibers, and is cytocompatible to L-929 mouse fibroblast cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1786-1798, 2017.

Quantifying the biomechanics of conception: L-selectin-mediated blastocyst implantation mechanics with engineered "trophospheres".

An estimated 12% of women in the United States suffer from some form of infertility. In vitro fertilization (IVF) is the most common treatment for infertility encompassing over 99% of all assisted reproductive technologies. However, IVF has a low success rate. Live birth rates using IVF can range from 40% in women younger than 35 years to 4% in women older than 42 years. Costs for a successful IVF outcome can be upward of $61,000. The low success rate of IVF has been attributed to the inability of the blastocyst to implant to the uterus. Blastocyst implantation is initiated by L-selectin expressing cells, trophoblasts, binding to L-selectin ligands, primarily sialyl Lewis X (sLeX), on the uterine surface endometrium. Legal and ethical considerations have limited the research on human subjects and tissues, whereas animal models are costly or do not properly mimic human implantation biochemistry. In this work, we describe a cellular model system for quantifying L-selectin adhesion mechanics. L-selectin expression was confirmed in Jeg-3, JAR, and BeWo cell lines, with only Jeg-3 cells exhibiting surface expression. Jeg-3 cells were cultured into three-dimensional spheres, termed "trophospheres," as a mimic to human blastocysts. Detachment assays using a custom-built parallel plate flow chamber show that trophospheres detach from sLeX functionalized slides with 2.75 × 10(-3) dyn of force and 7.5 × 10(-5) dyn-cm of torque. This work marks the first time a three-dimensional cell model has been utilized for quantifying L-selectin binding mechanics related to blastocyst implantation.