Transcript
0:00In vitro, in vivo and IVIVE
Lesson 7.2 of our prediction module: from test tube to human. In vitro means outside a live organism, for example in a test tube. In vivo means inside a live organism. IVIVE, in vitro to in vivo extrapolation, predicts a value in the body from a measurement in vitro. Plasma is the liquid part of the blood. Clearance is the volume of blood or plasma from which the body removes all of the drug per unit of time. Hepatic clearance is the clearance by the liver.
0:41Unbound fraction and intrinsic clearance
Unbound drug is free in solution, not attached to proteins or membranes. The unbound fraction is the unbound concentration divided by the total concentration. Intrinsic clearance is the rate of metabolism divided by the unbound drug concentration at the enzymes. It measures the removal by the enzymes alone, apart from any limit by blood flow or protein binding. It is measured in hepatocytes, the main liver cells, or in microsomes. Microsomes are small vesicles made from the endoplasmic reticulum, a membrane system inside liver cells.
1:30The in vitro assay and scaling factors
In the assay, the drug is incubated with microsomes or hepatocytes at a low concentration. The concentration then falls exponentially, and the rate constant of this fall is the rate of loss divided by the amount of drug that remains. The rate constant, times the incubation volume, divided by the amount of microsomal protein, is the in vitro intrinsic clearance of total drug. For hepatocytes, the number of cells replaces the amount of protein. Scaling factors convert this value to the whole liver. Published data from human livers give about 32 milligrams of microsomal protein and 99 million hepatocytes per gram of liver. IVIVE studies typically use a liver weight of 21 to 26 grams per kilogram of body weight.
2:33Scaling to the whole liver
After the assay, IVIVE has three steps: scaling, binding corrections and a liver model. A fourth part, the comparison with the hepatic clearance observed in people, tests the result. The first is scaling to the whole liver. The in vitro value is multiplied by the protein or cells per gram of liver and by the liver weight per kilogram of body weight. The result is the whole-liver intrinsic clearance.
3:06Correction for binding
The second is the correction for binding, in the incubation and in blood. The assay value uses the total drug concentration, but the microsomes or hepatocytes bind part of the drug. Division by the unbound fraction in the incubation gives the intrinsic clearance of unbound drug. The unbound fraction in blood is the unbound fraction in plasma divided by the blood-to-plasma concentration ratio. For 29 drugs in human liver microsomes, the unbound fraction in the incubation ranged from 0.11 to 1.0. Both corrections together gave the best overall agreement with the observed clearance.
3:57The well-stirred model and observed clearance
The third is the well-stirred model, which combines this intrinsic clearance with the unbound fraction in blood and hepatic blood flow to give hepatic clearance. The fourth is the comparison with observed hepatic clearance. The fold error is the predicted clearance divided by the observed clearance. A prediction is within twofold when the fold error lies between 0.5 and 2, and the fraction within twofold measures accuracy. The geometric mean of the fold errors, an average on a log scale, shows whether the predictions are too low or too high. IVIVE tends to underpredict clearance, with fold errors below 1. The underprediction is larger for drugs with a high hepatic clearance.
4:54A published analysis of IVIVE
A published analysis of a large database shows the size of this error. The database held published predictions for 101 drugs in human hepatocytes and 83 in human microsomes. The authors calculated hepatic clearance for every drug with the same three steps. With hepatocytes, 31 percent of the predictions were within twofold, 62 percent were below this range and 7 percent above it. With microsomes, the values were 42, 48 and 10 percent. On average, the predictions were 2.7 times too low with hepatocytes and 2.0 times too low with microsomes. Hepatic blood flow is the upper limit of hepatic clearance. For drugs with an observed hepatic clearance above 70 percent of this limit, the hepatocyte predictions were on average 4.8 times too low.
6:02Promethazine
Promethazine shows that a prediction within twofold can still put a drug in the wrong clearance range. In the microsome set, promethazine, an antihistamine, had a predicted hepatic clearance of 9.4 milliliters per minute per kilogram and an observed one of 16. This prediction was within twofold and 1.7 times too low. The observed value is above 70 percent of hepatic blood flow, but the predicted value falls in the intermediate range, 30 to 70 percent.
6:42Summary
In summary, IVIVE predicts hepatic clearance in people from intrinsic clearance in vitro. Scaling factors convert the in vitro value to the whole liver. For 29 drugs, both binding corrections together gave the best overall agreement. The well-stirred model then combines blood flow, binding and intrinsic clearance into hepatic clearance. The predictions tend to be too low, more so at high hepatic clearance. In the database, 31 percent of hepatocyte predictions and 42 percent of microsome predictions were within twofold. This concludes lesson 7.2 on in vitro to in vivo extrapolation.
Sources
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Illustrations adapted from Servier Medical Art (smart.servier.com), CC BY 4.0. Structures drawn with RDKit.
