3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Friend or Foe: Xenobiotic Activation of Nrf2 in Disease Control and Cardioprotection.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that governs a highly conserved pathway central to the protection of cells against various oxidative stresses. However, the biological impact of xenobiotic intervention of Nrf2 in physiological and pathophysiological conditions remains debatable. Activation of Nrf2 in cancer cells has been shown to elevate drug resistance and increase cell survival and proliferation, while inhibition of Nrf2 sensitizes cancer cells to drug treatment. On the other hand, activation of Nrf2 in normal healthy cells has been explored as a rather successful strategy for cancer chemoprevention. Selective activation of Nrf2 in off-target cells has recently been investigated as an approach for protecting off-target tissues from untoward drug toxicity. Specifically, induction of antioxidant response element genes via Nrf2 activation in cardiac cells is being explored as a means to limit the well-documented cardiotoxicity accompanied by cancer treatment with commonly prescribed anthracycline drugs. In addition to cancers, Nrf2 has been implicated in many other diseases including Alzheimer's and Parkinson's Diseases, diabetes, and cardiovascular disease. In this review, we discuss the roles of Nrf2 and its downstream target genes in the treatment of various diseases, and its recently explored potential for increasing the benefit: risk ratio of commonly utilized cancer treatments.

Development and characterization of rat duodenal organoids for ADME and toxicology applications.

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

CITCO as an Adjuvant Facilitates CHOP-Based Lymphoma Treatment in hCAR-Transgenic Mice.

Non-Hodgkin's lymphoma (NHL) is a malignant cancer originating in the lymphatic system with a 25-30% mortality rate. CHOP, consisting of cyclophosphamide (CPA), doxorubicin, vincristine, and prednisone, is a first-generation chemotherapy extensively used to treat NHL. However, poor survival rates among patients in advanced stages of NHL shows a need to improve this standard of care treatment. CPA, an integral component of CHOP, is a prodrug that requires CYP2B6-mediated bioactivation to 4-hydroxy-CPA (4-OH-CPA). The expression of CYP2B6 is transcriptionally regulated by the constitutive androstane receptor (CAR, NRi13). We have previously demonstrated that the induction of hepatic CYP2B6 by CITCO, a selective human CAR (hCAR) agonist, results in CHOP's enhanced antineoplastic effects in vitro. Here, we investigate the in vivo potential of CITCO as an adjuvant of CPA-based NHL treatment in a hCAR-transgenic mouse line. Our results demonstrate that the addition of CITCO to the CHOP regimen leads to significant suppression of the growth of EL-4 xenografts in hCAR-transgenic mice accompanied by reduced expression of cyclin-D1, ki67, Pcna, and increased caspase 3 fragmentation in tumor tissues. CITCO robustly induced the expression of cyp2b10 (murine ortholog of CYP2B6) through hCAR activation and increased plasma concentrations of 4-OH-CPA. Comparing to intraperitoneal injection, oral gavage of CITCO results in optimal hepatic cyp2b10 induction. Our in vivo studies have collectively uncovered CITCO as an effective facilitator for CPA-based NHL treatment with a pharmacokinetic profile favoring oral administration, promoting CITCO as a promising adjuvant candidate for CPA-based regimens.

Human constitutive androstane receptor agonist DL5016: A novel sensitizer for cyclophosphamide-based chemotherapies.

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC50 value of 0.66 µM and EMAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.

Antibody-Drug Conjugates: Pharmacokinetic/Pharmacodynamic Modeling, Preclinical Characterization, Clinical Studies, and Lessons Learned.

Antibody-drug conjugates are an emerging class of biopharmaceuticals changing the landscape of targeted chemotherapy. These conjugates combine the target specificity of monoclonal antibodies with the anti-cancer activity of small-molecule therapeutics. Several antibody-drug conjugates have received approval for the treatment of various types of cancer including gemtuzumab ozogamicin (Mylotarg®), brentuximab vedotin (Adcetris®), trastuzumab emtansine (Kadcyla®), and inotuzumab ozogamicin, which recently received approval (Besponsa®). In addition to these approved therapies, there are many antibody-drug conjugates in the drug development pipeline and in clinical trials, although these fall outside the scope of this article. Understanding the pharmacokinetics and pharmacodynamics of antibody-drug conjugates and the development of pharmacokinetic/pharmacodynamic models is indispensable, albeit challenging as there are many parameters to incorporate including the disposition of the intact antibody-drug conjugate complex, the antibody, and the drug agents following their dissociation in the body. In this review, we discuss how antibody-drug conjugates progressed over time, the challenges in their development, and how our understanding of their pharmacokinetics/pharmacodynamics led to greater strides towards successful targeted therapy programs.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be nontoxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs.

Insights into CYP2B6-mediated drug-drug interactions.

Mounting evidence demonstrates that CYP2B6 plays a much larger role in human drug metabolism than was previously believed. The discovery of multiple important substrates of CYP2B6 as well as polymorphic differences has sparked increasing interest in the genetic and xenobiotic factors contributing to the expression and function of the enzyme. The expression of CYP2B6 is regulated primarily by the xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in the liver. In addition to CYP2B6, these receptors also mediate the inductive expression of CYP3A4, and a number of important phase II enzymes and drug transporters. CYP2B6 has been demonstrated to play a role in the metabolism of 2%-10% of clinically used drugs including widely used antineoplastic agents cyclophosphamide and ifosfamide, anesthetics propofol and ketamine, synthetic opioids pethidine and methadone, and the antiretrovirals nevirapine and efavirenz, among others. Significant inter-individual variability in the expression and function of the human CYP2B6 gene exists and can result in altered clinical outcomes in patients receiving treatment with CYP2B6-substrate drugs. These variances arise from a number of sources including genetic polymorphism, and xenobiotic intervention. In this review, we will provide an overview of the key players in CYP2B6 expression and function and highlight recent advances made in assessing clinical ramifications of important CYP2B6-mediated drug-drug interactions.

Activation of the Constitutive Androstane Receptor Increases the Therapeutic Index of CHOP in Lymphoma Treatment.

The constitutive androstane receptor (CAR and NR1i3) is a key regulator of CYP2B6, the enzyme predominantly responsible for the biotransformation of cyclophosphamide (CPA) to its pharmacologically active metabolite, 4-hydroxycyclophosphamide (4-OH-CPA). Previous studies from our laboratory illustrated that CAR activation increases the formation of 4-OH-CPA; however, CPA is rarely used clinically outside of combination therapies. Here, we hypothesize that including a selective human CAR activator with the CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen can improve the efficacy without exacerbating off-target toxicity of this regimen in non-Hodgkin lymphoma treatment. In this study, we have developed a novel multiorgan coculture system containing human primary hepatocytes for hepatic metabolism, lymphoma cells as a model target for CHOP, and cardiomyocytes as a major site of off-target toxicity associated with this regimen. We found that a selective human CAR activator, CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime), altered expression of key drug-metabolizing enzymes and transporters in human hepatocytes, which positively affects the metabolic profile of CHOP. Coadministration of CITCO and CHOP in the coculture model led to significantly enhanced cytotoxicity in lymphoma cells but not in cardiomyocytes. Moreover, the beneficial effects of CITCO were abrogated when CAR knockout HepaRG cells were used in the coculture model. Importantly, synergistic anticancer effects were observed between CITCO and CHOP, in that inclusion of CITCO alongside the CHOP regimen offers comparable antineoplastic activity toward lymphoma cells at significantly reduced drug concentrations, and the decreased CHOP load attenuates cardiotoxicity. Overall, these findings provide a potentially promising novel strategy for facilitating CHOP-based chemotherapy.

Transporter-Mediated Disposition of Opioids: Implications for Clinical Drug Interactions.

Opioid-related deaths, abuse, and drug interactions are growing epidemic problems that have medical, social, and economic implications. Drug transporters play a major role in the disposition of many drugs, including opioids; hence they can modulate their pharmacokinetics, pharmacodynamics and their associated drug-drug interactions (DDIs). Our understanding of the interaction of transporters with many therapeutic agents is improving; however, investigating such interactions with opioids is progressing relatively slowly despite the alarming number of opioids-mediated DDIs that may be related to transporters. This review presents a comprehensive report of the current literature relating to opioids and their drug transporter interactions. Additionally, it highlights the emergence of transporters that are yet to be fully identified but may play prominent roles in the disposition of opioids, the growing interest in transporter genomics for opioids, and the potential implications of opioid-drug transporter interactions for cancer treatments. A better understanding of drug transporters interactions with opioids will provide greater insight into potential clinical DDIs and could help improve opioids safety and efficacy.