Transcript
0:00In vitro ADME assays
Lesson 7.1 of our prediction module: in vitro ADME assays. ADME is the absorption, distribution, metabolism and excretion of a drug. In vitro means in the laboratory, outside the body. An assay is a laboratory test that measures one property of a drug. In vitro ADME assays help to compare new compounds and to plan clinical studies.
0:31Microsomes, hepatocytes and Caco-2 cells
Liver microsomes are fragments of the endoplasmic reticulum, a membrane network inside liver cells. A laboratory separates them from broken-up liver by centrifugation, a fast spin that sorts particles by size and density. They contain the cytochrome P450 enzymes, or CYPs, which metabolize many drugs, and the assay adds NADPH, a cofactor that the CYPs need. Hepatocytes are intact liver cells with all the enzymes that metabolize drugs, and their cofactors. Caco-2 cells come from a human colon tumor. On a porous filter, they grow in about 21 days into a single layer like the lining of the small intestine. The filter separates a gut-side chamber from a blood-side chamber.
1:32CLint, Papp, efflux ratio and fu
In vitro intrinsic clearance, CLint, is the rate of metabolism in the assay divided by the drug concentration. The apparent permeability, Papp, is the rate at which drug appears on the far side, per unit of layer area and of initial concentration. Efflux is the transport of drug out of the cells, back toward the gut. The efflux ratio is Papp from the blood side to the gut side, divided by Papp in the reverse direction. The fraction unbound, fu, is the unbound drug concentration in plasma, the liquid part of the blood, divided by the total concentration. This lesson covers five assays. The first three measure drug properties. The last two test for drug interactions, in which one drug changes the plasma concentrations of another.
2:37Metabolic stability
The first is metabolic stability, the resistance of a drug to metabolism. The assay mixes the drug with microsomes and NADPH, or with hepatocytes. It measures the drug left in samples over 60 to 90 minutes. The natural logarithm of the amount left usually lies on a straight line against time. For microsomes, CLint equals the steepness of this line, k, times the mixture volume per milligram of microsomal protein. A high CLint means fast metabolism, so less of an oral dose probably reaches the blood unchanged.
3:26Permeability
The second is permeability. The assay measures Papp in both directions. Papp from the gut side to the blood side helps to predict oral absorption. Efflux transporters, such as P-glycoprotein, or P-gp, are membrane proteins that pump drugs out and can lower absorption. An efflux ratio of 2 or more suggests efflux by a transporter. If a known inhibitor of the transporter lowers the ratio by more than half, the transporter probably carries the drug.
4:06Plasma protein binding
The third is plasma protein binding. In equilibrium dialysis, a membrane separates plasma with drug from a buffer, a salt solution without protein. Unbound drug crosses the membrane, and protein-bound drug stays in the plasma. After several hours, the unbound concentrations on the two sides are equal. fu is then the buffer concentration divided by the plasma concentration. fu matters because, by a common assumption, only unbound drug can bind to the target.
4:47CYP inhibition and the transporter assay
The fourth is CYP inhibition. A probe drug is a drug that mainly one CYP metabolizes. The IC50 is the concentration of the new drug that halves the probe drug metabolism in microsomes. The fifth is the transporter assay: cells with one added transporter show whether it carries the drug and whether the drug inhibits it.
5:15Apixaban in vitro
Apixaban, a drug that reduces blood clotting, shows the results of all five assays. In human liver microsomes, its metabolism was slow, mainly by CYP3A4 and CYP3A5, two CYP enzymes. In human plasma in vitro, proteins bound about 87 percent, so fu was about 0.13. In one Caco-2 study, the Papp of apixaban was 0.9 times 10 to the minus 6 centimeters per second. The efflux ratio was more than 10, in Caco-2 cells and in cells with added P-gp or BCRP, a second efflux transporter. Inhibitors of the two transporters lowered this efflux, and the authors concluded that both carry apixaban. Apixaban did not inhibit seven CYPs, and the IC50 values were above 20 micromolar, a unit of concentration. It also did not inhibit the P-gp transport of digoxin in Caco-2 cells.
6:33Apixaban in clinical studies
In 18 healthy subjects, ketoconazole, a strong inhibitor of CYP3A4 and P-gp, doubled the apixaban AUC, the area under the plasma concentration-time curve. One later analysis concluded that CYP3A4 inhibition in the gut wall can explain this increase. In a second clinical study, apixaban did not change digoxin concentrations.
7:06Summary
In summary, metabolic stability assays give CLint from the loss of drug in microsomes or hepatocytes. Caco-2 assays give Papp, and an efflux ratio of 2 or more suggests efflux by a transporter. Equilibrium dialysis gives fu. CYP inhibition and transporter assays test for possible drug interactions. For apixaban, the assays showed slow metabolism mainly by CYP3A4 and CYP3A5, efflux by P-gp and BCRP, and no CYP inhibition. In vitro results show possible mechanisms, and clinical studies test which of them matter. This concludes lesson 7.1 on in vitro ADME assays.
Sources
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Illustrations adapted from Servier Medical Art (smart.servier.com), CC BY 4.0. Structures drawn with RDKit.
