Pharmacodynamic Modeling Identifies Synergistic Interaction Between Chloroquine and Trastuzumab in Refractory HER2- positive Breast Cancer Cells.

BACKGROUND/AIM: Despite improvements in HER2-positive breast cancer (BC) patients' outcomes with trastuzumab, the occurrence of intrinsic or acquired resistance presents a clinical challenge. Here, we quantitatively assess the combinatorial effects of chloroquine, an autophagy inhibitor, with trastuzumab on JIMT-1 cells, a HER2-positive BC cell-line primarily resistant to trastuzumab. MATERIALS AND METHODS: The temporal changes in JIMT-1 cellular viability were assessed using the CCK-8 kit, where JIMT-1 cells were exposed for 72-h to trastuzumab (0.007-17.19 µM) or chloroquine (5-50 µM) as single-agents, in combination (trastuzumab: 0.007-0.688 µM; chloroquine: 5-15 µM), or control (no drug). Concentration-response relationships were built for each treatment arm to determine drugs' concentrations inducing 50% of cell-killing (IC50). Cellular pharmacodynamic models were built to characterize the time-trajectory of JIMT-1 cellular viability under each treatment arm. The nature of trastuzumab and chloroquine interaction was quantified by estimating the interaction parameter (Ψ). RESULTS: The IC50 were estimated at 19.7 and 24.4 µM for trastuzumab and chloroquine. The maximum killing effect was about thrice higher for chloroquine than trastuzumab (0.0405 vs. 0.0125 h-1), validating chloroquine's superior anti-cancer effect on JIMT-1 cells compared to trastuzumab. The time-delay for chloroquine cell-killing was twice longer than that for trastuzumab (17.7 vs. 7 h), suggesting a chloroquine time-dependent anti-cancer effect. The Ψ was determined at 0.529 (Ψ<1), indicating a synergistic interaction. CONCLUSION: This proof-of-concept study on JIMT-1 cells identified chloroquine and trastuzumab synergistic interaction, warranting further in vivo investigations.

Integrating Forward and Reverse Translation in PBPK Modeling to Predict Food Effect on Oral Absorption of Weakly Basic Drugs.

INTRODUCTION: Ketoconazole and posaconazole are two weakly basic broad-spectrum antifungals classified as Biopharmaceutics Classification System class II drugs, indicating that they are highly permeable, but exhibit poor solubility. As a result, oral bioavailability and clinical efficacy can be impacted by the formulation performance in the gastrointestinal system. In this work, we have leveraged in vitro biopharmaceutics and clinical data available in the literature to build physiologically based pharmacokinetic (PBPK) models for ketoconazole and posaconazole, to determine the suitability of forward in vitro-in vivo translation for characterization of in vivo drug precipitation, and to predict food effect. METHODS: A stepwise modeling approach was utilized to derive key parameters related to absorption, such as drug solubility, dissolution, and precipitation kinetics from in vitro data. These parameters were then integrated into PBPK models for the simulation of ketoconazole and posaconazole plasma concentrations in the fasted and fed states. RESULTS: Forward in vitro-in vivo translation of intestinal precipitation kinetics for both model drugs resulted in poor predictions of PK profiles. Therefore, a reverse translation approach was applied, based on limited fitting of precipitation-related parameters to clinical data. Subsequent simulations for ketoconazole and posaconazole demonstrated that fasted and fed state PK profiles for both drugs were adequately recapitulated. CONCLUSION: The two examples presented in this paper show how middle-out modeling approaches can be used to predict the magnitude and direction of food effects provided the model is verified on fasted state PK data.

Navigating Through Cell-Based In vitro Models Available for Prediction of Intestinal Permeability and Metabolism: Are We Ready for 3D?

Traditionally, in vitro studies to quantify the intestinal permeability of drugs have relied on two-dimensional cell culture models using human colorectal carcinoma cell lines, namely Caco-2, HT 29 and T84 cells. Although these models have been commonly used for high-throughput screening of xenobiotics in preclinical studies, they do not fully recapitulate the morphology and functionality of enterocytes found in the human intestine in vivo. Efforts to improve the physiological and functional relevance of in vitro intestinal models have led to the development of enteroids/intestinal organoids and microphysiological systems. These models leverage advances in three-dimensional cell culture techniques and stem cell technology (in addition to microfluidics for microphysiological systems), to mimic the architecture and microenvironment of the in vivo intestine more accurately. In this commentary, we will discuss the advantages and limitations of these established and emerging intestinal models, as well as their current and potential future applications for the pre-clinical assessment of oral therapies.

Multiscale and Translational Quantitative Systems Toxicology, Pharmacokinetic-Toxicodynamic Modeling Analysis for Assessment of Doxorubicin-Induced Cardiotoxicity.

Dose-dependent life-threatening doxorubicin-induced cardiotoxicity (DIC) is a major clinical challenge that needs to be addressed. Here, we developed an integrated multiscale and translational quantitative systems toxicology and pharmacokinetic-toxicodynamic (QST-PK/TD) model for optimization of doxorubicin dosing regimens for early monitoring and minimization of DIC. A QST model was established by exposing human cardiomyocytes, AC16 cells, to doxorubicin over a time course, and measuring the dynamics of intracellular signaling proteins, AC16 cell viability and released biomarkers of cardiomyocyte injury such as the B-type natriuretic peptide (BNP). Experiments were scaled up to a three-dimensional and dynamic (3DD) cell culture system to evaluate DIC under various dosing regimens. The PK determinants of doxorubicin influencing DIC were identified in vitro and then translated to the in vivo setting through hybrid physiologically based PK (PBPK)/TD models using preclinical- and clinical-level data extracted from literature. The developed cellular-level QST model captured well the observed dynamics of intracellular proteins, AC16 cell viability and BNP kinetics. In the 3DD setting, dose fractionation of doxorubicin displayed a significant reduction in cardiotoxicity compared to single intravenous doses with equal exposure, implying doxorubicin peak concentrations as the PK determinant for DIC. The in vivo hybrid PBPK/TD models captured well doxorubicin PK and DIC. Peak doxorubicin concentrations correlated well with acute DIC for dose-fractionated regimens, while maximum 48-h moving average concentrations correlated with DIC for dose-fractionated and long-term infusion regimens in vivo. The developed multiscale and translational QST-PK/TD modeling platform may serve as an in silico tool for assessment of early toxicity and/or efficacy of developmental drugs in vitro.

A quantitative systems pharmacological approach identified activation of JNK signaling pathway as a promising treatment strategy for refractory HER2 positive breast cancer.

HER2-positive breast cancer (BC) is a rapidly growing and aggressive BC subtype that predominantly affects younger women. Despite improvements in patient outcomes with anti-HER2 therapy, primary and/or acquired resistance remain a major clinical challenge. Here, we sought to use a quantitative systems pharmacological (QSP) approach to evaluate the efficacy of lapatinib (LAP), abemaciclib (ABE) and 5-fluorouracil (5-FU) mono- and combination therapies in JIMT-1 cells, a HER2+ BC cell line exhibiting intrinsic resistance to trastuzumab. Concentration-response relationships and temporal profiles of cellular viability were assessed upon exposure to single agents and their combinations. To quantify the nature and intensity of drug-drug interactions, pharmacodynamic cellular response models were generated, to characterize single agent and combination time course data. Temporal changes in cell-cycle phase distributions, intracellular protein signaling, and JIMT-1 cellular viability were quantified, and a systems-based protein signaling network model was developed, integrating  protein dynamics to drive the observed changes in cell viability. Global sensitivity analyses for each treatment arm were performed, to identify the most influential parameters governing cellular responses. Our QSP model was able to adequately characterize protein dynamic and cellular viability trends following single and combination drug exposure. Moreover, the model and subsequent sensitivity analyses suggest that the  activation of the stress pathway, through pJNK, has the greatest impact over the observed declines of JIMT-1 cell viability in vitro. These findings suggest that dual HER2 and CDK 4/6 inhibition may be a promising novel treatment strategy for refractory HER2+ BC, however, proof-of-concept in vivo studies are needed to further evaluate the combined use of these therapies.

Anticancer and cardio-protective effects of liposomal doxorubicin in the treatment of breast cancer.

Breast cancer (BC) is a highly prevalent disease, accounting for the second highest number of cancer-related mortalities worldwide. The anthracycline doxorubicin (DOX), isolated from Streptomyces peucetius var. caesius, is a potent chemotherapeutic drug that is successfully used to treat various forms of liquid and solid tumors and is currently approved to treat BC. DOX exerts its effects by intercalation into DNA and inhibition of topoisomerases I and II, causing damage to DNA and the formation of reactive oxygen species (ROS), resulting in the activation of caspases, which ultimately leads to apoptosis. Unfortunately, DOX also can cause cardiotoxicity, with patients only allowed a cumulative lifetime dose of 550 mg/m2. Efforts to decrease cardiotoxicity and to increase the blood circulation time of DOX led to the US Food and Drug Administration (FDA) approval of a PEGylated liposomal formulation (L-DOX), Doxil® (known internationally as Caelyx®). Both exhibit better cardiovascular safety profiles; however, they are not currently FDA approved for the treatment of metastatic BC. Here, we provide detailed insights into the mechanism of action of L-DOX and its most common side effects and highlight results of its use in clinical trials for the treatment of BC as single agent and in combination with other commonly used chemotherapeutics.

Nanotechnology in retinal drug delivery.

Retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the leading causes of blindness in adults over the age of 50 years in the US. While most of those conditions do not have a cure, currently available treatment options attempt to prevent further vision loss. For many ophthalmic drugs, an efficient delivery system to provide maximum therapeutic efficacy and promote patient compliance remains an unmet medical need. An exploration of literature via PubMed spanning from 2007 to 2017 was conducted to identify studies that have evaluated nanotechnology as platforms for delivering therapeutic agents to the posterior segment of the eye where the retina is located. Until now, four routes that have been utilized for retinal drug delivery are the intravitreal, periocular, subretinal, and systemic routes. Intravitreal injections are now widely used in clinical practice due to their ability to directly target the back of the eye but are highly invasive procedures that may cause several complications, particularly with repeated uses over a short timespan. Nanotechnology shows great promise to revolutionize retinal drug delivery, offering many advantages such as a targeted delivery system towards the specific site of the retina as well as sustained delivery of therapeutic agents. In this review, specific eye anatomy and constraints on ocular drug administration are illustrated. Further, we list and highlight several examples of nanosystems, such as hydrogels, liposomes, dendrimers, and micelles, used via different drug delivery routes to treat various retinal diseases.