MatTek Corporation is presenting posters at the AAPS 2016 annual meeting in Denver, Colorado. Dr. Seyoum Ayheunie is presenting on MatTek’s latest in vitro innovations including the new EpiIntestinal and EpiAlveolar models for drug toxicity, metabolism, and drug-drug interactions. To schedule a meeting with Dr. Ayehunie at AAPS, contact him directly: sayehunie@mattek.com.
35M0830 – Human Alveolar Tissue Model for Inhaled Drug Delivery Applications
Anna Maione, MatTek Corporation (Main Author); Robert Jackson, MatTek Corporation; Olivia O’Connell, MatTek Corporation; Amy Hunter, MatTek Corporation; Steven Coughlin, MatTek Corporation; Seyoum Ayehunie, MatTek Corporation (Poster Presenting Author); Patrick Hayden, MatTek Corporation
Purpose: Reliable in vitro human models for investigating airway toxicity and drug delivery are needed to provide accurate tools to inhaled drug formulators. In vitro airway models based on primary human cells have been described. However, models utilizing animal cells or human cell lines are most commonly employed. The purpose of the present work was to develop an in vitro air-blood barrier model derived from primary human alveolar epithelial cells, pulmonary endothelial cells and monocyte-derived macrophages.
Methods: Reliable in vitro human models for investigating airway toxicity and drug delivery are needed to provide accurate tools to inhaled drug formulators. In vitro airway models based on primary human cells have been described. However, models utilizing animal cells or human cell lines are most commonly employed. The purpose of the present work was to develop an in vitro air-blood barrier model derived from primary human alveolar epithelial cells, pulmonary endothelial cells and monocyte-derived macrophages.
Results: Confocal imaging demonstrated epithelial staining for cytokeratin 19 as well as tight junction proteins ZO-1 and occludin, while the endothelial cell layer stained positive for von Willebrand factor and e-cadherin. Celltracker dye was used to visualize macrophages on the luminal side of the alveolar cultures. The model developed peak TEER of > 1,000 Ω*cm2 within 9-12 days, and maintained TEER > 400 Ω*cm2 for up to 30 days. Macrophage purity by flow cytometry was > 95% (CD14+). Expression of drug transporter genes including ABC family efflux transporters BCRP, MRP1 and MRP2, and organic cation uptake transporters OCTN1, OCTN2 and OCT3 was demonstrated by RT-PCR.
Conclusion: This new human alveolar model shows promise as a useful tool for in vitro airway toxicity and inhaled drug delivery investigations.
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35T0830 – Validation of Organotypic Small Intestine Tissue Model for Drug Permeability, Drug-Drug Interaction, and Metabolism Studies
Seyoum Ayehunie, Mattek Corporation (Main Author, Poster Presenting Author); Zachary Stevens, Mattek Corporation; Timothy Landry, Mattek Corporation; Barry Press, Cyprotex; Alex Armento, Mattek Corporation; Mitchell Klausner, Mattek Corporation; Patrick Hayden, Mattek Corporation
Purpose: The goal of this study is to validate a biologically relevant organotypic small intestinal (SMI) tissue model to predict intestinal drug absorption/bioavailability of orally administered drugs. Primary human cell-based small intestinal (SMI) 3D tissue models that recapitulate in vivo counterpart phenotypically, structurally and functionally will be a relevant testing model for therapeutic drug screening, drug metabolism, and drug-drug interaction studies. Currently used cell-line based assays are not physiological and do not mimic the in vivo microenvironment.
Methods: Human primary SMI epithelial cells and fibroblasts were used to reconstruct SMI tissues. Outcome measurements include transepithelial electrical resistance (TEER), PCR, and histology. bioavailability of drugs or efflux transport was analyzed by LC-MS/MS (N = 16 drugs from different Biopharmaceutics Classification System (BCS) classification). The sensitivity and accuracy of the in vitro method compared to historical absorption data was calculated. Test drug with human absorption of >80% and in vitro Papp of >2 x106 cms-1 was considered as high permeable and drugs that showed <80% and in human absorption and an in vitro Papp value of
Results: In Concordance with the historical % human absorption data, the apparent permeability coefficient (Papp) values differentiate the test articles as high and low permeability drugs. The SMI tissue model categorizes test drugs as high permeable and low permeable with a sensitivity of 100%, specificity of 89%, and accuracy of 94% compared to the % human absorption data available from the literature. Atenolol (paracellular) transport and propranolol (intercellular) transport with known human absorption of 50% and 90% respectively, were used as markers for ranking the test compounds. The FDA recommended P-glycoprotein substrate (Quinidine) was used as control for efflux transport. Drug-drug interactions were examined using efflux transporter inhibitors and the inhibitors increased drug bioavailability while decreasing the efflux ratio. Efflux ratios of Pgp substrates (talinolol, digoxin, and loperamide) were reduced by 45%, 40%, and 60%, respectively, in the presence of the Pgp inhibitor (verapamil). Efflux ratio of nitrofurantoin (BCRP substrate) was reduced by 63% in the presence of its inhibitor novobiocin. Results from drug metabolism studies also showed Midazolam (CPY3A substrate) was metabolized (6.5% conversion) by the intestinal tissue model.
Conclusion: The newly developed SMI tissue models appear to be promising new tool for evaluation of drug safety, permeability, and metabolism. This novel in vitro intestinal model can serve as a platform for testing bioavailability and effectiveness of drug candidates prior to clinical studies Availability of primary cell-based human 3D intestinal tissue model will also reduce the need for unreliable animal studies.
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17W1130 – 3D-Human Small Intestinal Tissue Model System to Screen Drug Induced Gastrointestinal Toxicity and Wound Healing
Timothy Landry, Mattek Corporation (Main Author); Seyoum Ayehunie, Mattek Corporation (Poster Presenting Author); Zachary Stevens, Mattek Corporation; Matt Wagoner, AstraZeneca; Mitchell Klausner, Mattek Corporation
Purpose: The mechanisms of off-target dose-limiting gastrointestinal toxicities for therapeutic compounds are often poorly understood, in large part due to the lack of physiologically relevant in vitro models. The inherent differences in intestinal physiology between patients and preclinical species also add another layer of complexity in gastrointestinal (GI) toxicity studies. The purpose of this work is to evaluate the utility of an in vitro primary human cell-based small intestinal 3D tissue (SMI) model as an investigational tool for drug-induced GI toxicity and wound healing studies.
Methods: To achieve this goal, a blinded study was performed using N=5 therapeutic compounds for which dog and rat GLP toxicity studies were not predictive of human toxicities. These compounds were selected from stopped AstraZeneca clinical development programs representing a diversity of target classes and chemistries and tested at five concentrations. As negative controls, N=3 drugs that are well tolerated in humans were included. To model GI toxicity, we examined cytotoxicity by MTT viability and LDH release assays and tissue barrier integrity using transepithelial electrical resistance (TEER) measurements, following two repeat exposures over 96 hours. We also examine the utility of the tissue model for wound healing studies by inducing a 2 mm wound area using 2 mm biopsy punch. The wounded tissues were cultured for up to 10 days in medium with or without human serum. To examine the rate of wound healing, confocal imaging of tissues was performed at days 2, 4, 7, and 10.using anti-CK19 and anti-vimentin antibodies for epithelial cell migration and fibroblast spreading, respectively.
Results: The results showed that the SMI system detected drug-induced disruption of intestinal barrier function (TEER) in 5/5 problematic drugs with human GI toxicity at concentrations within or below 30x clinical exposure levels. Importantly, the SMI system showed no effect within 1,000x clinical exposure levels for the three negative controls. In terms of wound healing, tissues cultured in human serum completed the wound healing process at day 6 of the culture period compared to > 10 days for control tissues cultured in the absence of serum.
Conclusion: Overall, the use of TEER as an endpoint makes the SMI tissue a sensitive predictive tool to assess clinically relevant exposures of drugs that induce intestinal toxicity in patients but incorrectly predicted in preclinical GLP toxicology studies, suggesting that the in vitro system may serve as a promising model for both investigational and traditional GI drug safety studies. Furthermore, the small intestinal tissue model can be used for evaluating therapeutics for their wound healing potential.
