Transport of Chlorpromazine in the Caco-2 Cell Permeability Assay: A Kinetic Study

Abstract

The intestinal transport of compounds can be measured in vitro with Caco-2 cell monolayers. We took a closer look at the exposure and fate of a chemical in the Caco-2 cell assay, including the effect of protein binding. Transport of chlorpromazine (CPZ) was measured in the absorptive and secretory direction, with and without albumin basolaterally. Samples were taken from medium, cells, and well plastic. For the secretory transport experiments with albumin, the free CPZ concentration at the start of the experiment was measured by negligible depletion-solid phase microextraction (nd-SPME). Recovery of CPZ from the medium was low, especially in the absorptive transport direction. CPZ was found in the cells (≤20%) and bound to the well plastic (≤25%), and 94% of CPZ was bound to albumin. An initial lag phase was observed, which was likely caused by partitioning of CPZ between the donor concentration and the Caco-2 cells; after 20 min, transport of CPZ to the receiver compartment was linear. The low recovery and the test compound found both inside the Caco-2 cells and bound to the well plastic complicate the calculation of the fraction transported and render reliable estimates of permeability constants impossible. For a chemical like chlorpromazine, which is hydrophobic in its neutral form, but in general also for more lipophilic compounds, the Caco-2 cell assay might not be straightforward, and a more detailed study into the fate and exposure of the test compound might be needed to arrive at meaningful data for transport and permeability.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…Additional efforts are therefore needed to change the conditions of permeability assays to allow for analysis of lipophilic compounds in such application areas. A suitable strategy to measure compound-specific biokinetics (including chemical and metabolic stability, lipophilicity, adsorption to plastics, binding to protein) should also be considered as this information would prove helpful in data interpretation (Broeders et al, 2012). Moreover, there is an urgent need to optimize biobarrier models with respect to chemical applicability domains (not only druglike chemicals) as well as with respect to the biological response domain in the sense that they should be able to quantify extremely low, low and medium membrane permeability and not only high permeability as holds for the Caco-2 cell line.…”

Exploration of the Protection of Riboflavin Laurate on Oral Mucositis Induced by Chemotherapy or Radiotherapy at the Cellular Level: What Is the Leading Contributor?

“…In addition, the Caco-2 cell monolayer is a well-established model for screening the transport, intestinal absorption and metabolism properties of new drugs [18]. Therefore, using the model, we first investigate whether riboflavin laurate could transport across Caco-2 cell monolayer as the prototype and illuminate the transport characteristics of riboflavin laurate, helping us clearly understand what the active ingredient ameliorating or relieving the pain of oral or gastrointestinal mucositis is.…”

EURL ECVAM strategy for achieving 3Rs impact in the assessment of toxicokinetics and systemic toxicity

“…This pertains to in vitro ADME testing (Broeders et al, 2012) and to the collection of human in vivo data on TK (biomonitoring and human volunteer studies). This also concerns in vitro toxicity testing where the in vitro kinetics should be measured, i.e.…”