Cell permeability measures how easily a substance passes through the cell membrane, the thin, complex barrier surrounding every cell. This membrane is composed of a double layer of lipids, creating a highly selective filter between the cell’s internal environment and the external world. A cell permeability assay is an experiment designed to quantify the rate at which molecules move across this selectively permeable boundary. Measuring permeability is a fundamental step in understanding how a compound will behave in the body, as the membrane’s hydrophobic interior restricts the movement of most substances.
Why Permeability Must Be Measured
Measuring a compound’s permeability directly predicts the likelihood of a drug reaching its target in the body. For orally administered medication, the active substance must move from the gastrointestinal tract into the bloodstream, a process known as absorption. If a molecule cannot efficiently cross the cellular barrier of the intestinal wall, it will be poorly absorbed and ineffective as a medicine.
This measurement is part of the ADME framework (Absorption, Distribution, Metabolism, and Excretion) used in drug development. Permeability determines Absorption and influences Distribution, controlling whether a compound can reach organs like the brain or liver. A compound with poor permeability, even if potent, will have low bioavailability, meaning only a small fraction reaches the systemic circulation to exert a therapeutic effect.
Predicting absorption early allows scientists to filter out poor candidates before investing in costly later phases of research. Permeability data helps optimize the chemical structure of a compound, steering development toward molecules that can be effectively delivered. Quantifying this characteristic informs decisions about dosage form, route of administration, and potential drug-drug interactions.
How Molecules Cross the Cell Membrane
Movement across the cell membrane is divided into passive and active transport, differing based on energy expenditure and interaction with the lipid bilayer. Passive transport, such as simple diffusion, does not require cellular energy and relies on the concentration gradient, moving molecules from high to low concentration. Small, uncharged, and highly lipophilic compounds can pass directly through the hydrophobic core of the lipid bilayer this way.
Passive diffusion efficiency is influenced by a molecule’s size and its partition coefficient, which reflects its solubility in lipids versus water. Large, polar, or electrically charged molecules, such as ions and glucose, are repelled by the membrane’s interior and cannot cross via simple diffusion. These molecules often require active transport, which involves specialized transmembrane carrier proteins or channels.
Active transport requires metabolic energy, often supplied by ATP, because it allows molecules to move against their concentration gradient. This process is performed by specific transporter proteins embedded in the membrane. These proteins can facilitate nutrient uptake or, in the case of efflux pumps like P-glycoprotein, actively push compounds back out of the cell.
Standard Assays for Permeability Screening
Permeability screening relies on in vitro models that mimic the body’s biological barriers, such as the intestinal lining. Two widely used methods are the Caco-2 cell monolayer assay and the Parallel Artificial Membrane Permeability Assay (PAMPA). The Caco-2 assay is considered the standard for predicting human intestinal absorption because it uses human colon cancer cells that differentiate into a monolayer resembling the small intestine epithelium.
In the Caco-2 setup, cells are grown on a porous filter separating the apical side (mimicking the gut lumen) from the basolateral side (mimicking the blood). The assay measures the rate a compound moves from the apical to the basolateral side, corresponding to absorption. Since Caco-2 cells express many human gut transporter proteins, the assay measures both passive and active transport. Measuring transport in both directions allows researchers to calculate an efflux ratio, which indicates if a compound is a substrate for efflux pumps that actively prevent absorption.
In contrast, the PAMPA assay is a high-throughput, non-biological method using a synthetic, lipid-infused filter to simulate the cell membrane. PAMPA is faster and less expensive than Caco-2 because it eliminates complex cell culture maintenance. The PAMPA system measures only passive diffusion, as it lacks the living cell components, such as transporter proteins, that mediate active transport. This makes PAMPA valuable for rapidly screening thousands of compounds to assess their intrinsic ability to diffuse through a lipid barrier.
Translating Permeability Data into Drug Success
The quantitative result from these assays is the apparent permeability coefficient, or \(P_{app}\), which represents the velocity at which the compound crosses the membrane barrier. The \(P_{app}\) value is calculated by normalizing the rate of compound movement by the membrane surface area and the initial concentration. This standardized metric allows for the direct comparison of different compounds, providing a consistent measure of intrinsic permeability.
Researchers use established \(P_{app}\) thresholds to classify compounds and predict human absorption. For example, a compound is classified as having high permeability if its \(P_{app}\) value is greater than \(10 times 10^{-6}\) centimeters per second (\(text{cm/s}\)) in the Caco-2 assay, correlating with high absorption. Compounds below a low-permeability threshold, often around \(1 times 10^{-6}\) \(text{cm/s}\), are predicted to be poorly absorbed, signaling a hurdle for development.
These classifications, combined with solubility data, predict the overall in vivo performance of an oral drug. A high permeability prediction suggests that absorption is not limited by membrane crossing, allowing the focus to shift to factors like solubility or formulation. While in vitro models like Caco-2 and PAMPA are predictive, they are simplified systems that cannot fully replicate the complexity of the gastrointestinal tract, such as blood flow or the mucus layer.