TRANSIL Microsomal Binding Kit
TRANSIL Microsomal Binding Kit
When estimating intrinsic clearance in metabolic stability incubations with microsomes or hepatocytes, the disappearance of the parent compound is measured over a time course. The measured clearance rate is only reflecting the intrinsic clearance rate, when the test compound is available to interact freely with the CYP enzymes at the assumed target concentration.
However, this is frequently not the case, because compounds tend to bind quantitatively to microsomal or hepatocyte membranes. This can significantly reduce the available concentration – even to 1/100 or less of the total concentration in the incubation (Obach, 1999; McLure et al., 2000; Austin et al., 2002; Hallifax and Houston, 2006). Consequently, it is important to estimate microsomal binding in these in vitro incubations.
The TRANSIL Microsomal Binding kit measures the affinity of drugs to human microsomal membranes and determines microsomal binding in stability incubation experiments. This allows the accurate estimation of intrinsic clearance from stability incubations by correcting the experimental clearance with the fraction of drug unbound in the incubation. Internal quality controls provide easy assessment of recovery, experiment and data quality.
The kit consists of ready-to-use 96 well microtiter plates. One plate can be used for measuring HSA binding of up to 12 compounds. The assay requires only 5 steps: (i) addition of drug candidate, (ii) mixing and incubation for 12 minutes, (iii) removal of beads by centrifugation, (iv) sampling of supernatant, and (v) quantification of drug candidate.
Choosing a diverse compound set of 22 drugs we observed a strong correlation (r2=0.97) between the results from the ready-to-use TRANSIL kits and dialysis with microsomal homogenates. This indicates that the methods are highly comparable and validates the assumption that microsomal binding is attributable to membrane binding alone.
How comparable are the results of the TRANSIL Microsomal Binding kit to literature data and QSAR predictions?
Comparing a set of 6 drugs with reported literature values for fu mic that were obtained by dialysis with microsomal homogenates and results from the ready-to-use TRANSIL kits we observed strong correlation with an r2 of 0.88, however, when comparing the experimental data to quantitative structure-activity relationship model predictions the correlation deteriorates down to an r2 of 0.66. In fact, the Austin model consistently underpredicts fu mic while the Turner as well as Halifax & Houston models have less systematic bias but still low precision.
fu mic experimental fu mic QSAR Drug TRANSIL Literature Cp [mg/ml] Poulin Halifax & Houston Turner et al. Austin et al. Diazepam 86% 75% 0.5 91% 81% 75% 55% Alprazolam 66% 66% 5 90% 51% 45% 29% Midazolam 74% 88% 1 34% 37% 34% 14% Propranolol 41% 50% 1 48% 42% 41% 17% Triazolam 85% 78% 1 93% 76% 69% 50% Warfarin 100% 100% 0.1 100% 99% 99% 99%
Subcellular fractions such as liver microsomes are useful in vitro models for investigation of hepatic clearance and drug-drug interactions. However, it is the unbound substrate or inhibitor concentration which is important in the prediction of in vivo pharmacokinetics as only the free drug can interact with drug metabolizing enzymes in the microsomal incubations. Knowledge of the extent of microsomal binding leads to a better understanding of the relationship between in vitro metabolism and in vivo pharmacokinetics.
- Clearance may be underpredicted if fu mic is ignored
- QSAR models predict fu mic poorly
- Interpretation of drug-drug interaction data can be affected by fu mic as inhibitor potency can be underestimated
Why is microsomal binding important in prediction of clearance from in vitro microsomal clearance incubations?
Only the free unbound compound is available to be metabolized by the enzymes present in microsomal incubations. Therefore, it is important to consider the extent of binding when performing microsomal clearance studies. Several studies show that correcting for non-specific microsomal binding improves the prediction of in vivo clearance (Carlile et al. 1999, Obach 1997, 1999).
Can I use the results from the TRANSIL Microsomal Binding kit also for estimating hepatocyte binding?
Yes, the TRANSIL Microsomal Binding Kit can also be used to estimate hepatocyte binding because it determines the affinity of test items to liver microsomal membranes and they differ only marginally in their composition to hepatocyte membranes.
The TRANSIL Microsomal Binding Kit measures the affinity of a test item to immobilized microsomal membranes with natural membrane fluidity. This membrane affinity is a partitioning coefficient of drug between membrane and buffer. It is defined as the concentration of drug in membrane (cl) over the concentration of drug in buffer (cb):
The membrane affinity is calculated from the assay data using the mass balance equation:
which is rearranged such that the membrane affinity can be determined from the slope of plotting the ratio of total amount of drug (nt) over remaining concentration in supernatant (cb) against the lipid membrane volume present in each well:
We recommend fluoxetine as positive control and warfarin as negative control.
The TRANSIL Quality Index (TQI) is based on independent measures derived from the data analysis.
- Model fit (see equation 3 of the section “how the assay works”
- Recovery: does the model derived compound concentration equal the true concentration?
- Data consistency: does membrane binding increase proportionally with the increasing TRANSIL bead content in each well?
- Data consistency: are the estimated reference concentrations in alignment with the compound concentration used?
- Missing data and outliers.
One assay plate can be used for 12 compounds. Thus, you’ll need to pipette 15 µl of test item to each of 8 wells and repeat this for all 12 compounds. This takes less than 10 minutes even with manual pipetting. After compound addition, the plate is ready for incubation. You can do this with an electronic 8 or 12 channel pipette by aspirating and dispensing a volume of 120 µl for 15 times. That takes just over a minute for each column or row. In total, that makes 8 to 15 minutes depending on your pipette. When using a pipetting robot with a 96 well head this time decreases to 2 minutes. After incubation, the plate needs to be spun in a plate centrifuge for 10 minutes. The supernatants are then ready for quantification by LC/MS/MS, UV, fluorescence or any other method of your choice.
Thus, the total time the start and end of the experiment varies between 7 and 25 minutes depending upon your equipment.
One assay plate can be used for 12 compounds. A special feature of the 96 well plates used for these kits is that each of the 12 columns can be separated from the plate. Thus, it is possible to use the plate for one compound at a time.
The TRANSIL assay kits utilize Micronic 96 well plates with ultra-low-binding tubes. Standard polypropylene tubes have 41x higher non-specific binding and low-binding tubes from other vendors have 2.6x higher non-specific binding.