A Mammary Epithelium on a Chip for Assessing Lofexidine Transport into Breast Milk

Introduction: Opioid addiction is a major health concern in the United States. Detoxification and management of withdrawal symptoms are the first steps in treating opioid dependency. Lofexidine (LUCEMYRA®) is the only FDA-approved, non-opioid, non-addictive treatment for relief of the multiple symptoms of opioid withdrawal [1]. Lactating mothers are often required to terminate their medication or nursing due to the unknown risk of drug exposure in their infants. Here, we have developed a breast epithelium on a chip that will enable the study of the transport characteristics of drugs such as Lofexidine through the breast epithelium and into breast milk and help predict potential adverse effects on infants. Furthermore, the in vitro transport data has been used in an in vitro in vitro extrapolation (IVIVE) approach to estimate milk-to-plasma (M/P) ratios of lofexidine.

Materials and

Methods: The microfluidic device was fabricated by joining various polycarbonate parts. A thin polycarbonate membrane with 0.4 µm pores was used to separate the apical and basolateral chambers in the device. The mammary epithelial cell line MCF10A was used to generate the mammary epithelial barrier. The cells were allowed to adhere to the membrane surface in static conditions for 2 days after which flow was initiated at a shear stress rate of 1.4 mPa. The in vitro mammary epithelium integrity was analyzed with tight junction staining and lucifer yellow transport assay after 3 days of flow. Lofexidine bidirectional permeability was assessed in the device keeping the donor side concentration at 10 µM. For drug transport assays, the apical chamber was circulated with donor human milk and the basolateral chamber with donor human plasma to mimic milk and blood flow respectively. The fraction of unbound lofexidine in the breast milk was determined by a Rapid Equilibrium Dialysis (RED) assay. A previously published IVIVE approach was used to estimate milk-to-plasma (M/P) ratios of lofexidine. Using the in vitro transport data a whole-body lactation model was built in Simcyp® simulator v22.

Results, Conclusions, and Discussions: The device showed uniform fluid shear stress and pressure distribution throughout the cell culture area. MCF10A cells showed cell attachment and viability after 5 days of culture in dynamic conditions in the device. The epithelium differentiated and formed tight junctions within 3 days of flow as indicated through anti-occludin staining. Tight junction formation was also demonstrated by transepithelial electrical resistance values greater than 1000 ohm*cm2. The microfluidic models showed a slightly higher lofexidine permeability than the static transwell models. Using these permeability values, the IVIVE and physiologically based pharmacokinetic (PBPK) modeling approaches predicted M/P ratios with a majority in the range of 1.60-5.36.
The microfluidic device-derived permeability data can be used to develop a whole-body lactation PBPK model to predict the concentration-time profile of lofexidine in breast milk. Simultaneously, the predicted plasma PK profiles will be validated with single and multiple lofexidine dose clinical data. In conclusion, an in vitro cell-based model was developed and used to predict the breast milk secretion of lofexidine, which provides a novel tool for estimating drug exposure and potential adverse effects in nursing infants.

Acknowledgements (Optional): This work was funded by the Food and Drug Administration (FDA) Office of Women’s Health (OWH).
References
[1] I. Urits et al., “A Comprehensive Update of Lofexidine for the Management of Opioid Withdrawal Symptoms,” Psychopharmacol Bull, vol. 50, no. 3, pp. 76–96, Jul. 2020.