Benefits of TRANSIL Technology
• Matrix free method:
– Enables rapid separation of the drug from the biological phase = fast quantification
• Requires 15 minutes equilibration time
– in comparison dialysis requires 4-6 hours
• Requires only 5 minutes manual handling time
– In comparison dialysis requires 100 minutes
• Fully automatable
• No need for analytical techniques with very high sensitivity
• Overcomes analytical limitations for highly bound drugs
• Reduces laboratory animal consumption
Quantification relative to references
= no calibration curve needed
= fast quantification
= minimal labor requirement
For protein binding application our chemists prime the bead surface so that it can covalently bind proteins. The resulting protein-coated beads have very little exposed silica surface which practically eliminates any contact of the silica with the solution and keeps unspecific binding of drug candidates or other test substances minimal.
For membrane permeability applications the beads have lipid bilayer membranes attached via non-covalent interactions. This mimics the fluidity of native membranes, and allows the complete range of complex interactions that occur between a drug candidate, or other test substance, and the membrane molecules.
TRANSIL Membrane affinity (MA) Assay Principle
Consists of silica beads covered with porcine brain lipid or other bilayers suspended in
Assess the drugs brain membrane affinity by
incubating fixed concentration of drug with varying concentrations (six) of lipids
immobilized on the silica beads and analyzing the drug free fraction
Prediction of tissue absorption via specific statistical models using MA as key parameter
Early rational drug candidate selection is important, since later stage preclinical and clinical development is much more costly than early stage development. The soft drug development paradigm calls for inclusion of activity, specificity and ADME/PK data in the candidate selection process. During the secondary screening and lead optimization phase it is useful to obtain such data from in vitro studies as it is nearly impossible to identify causes of unsatisfactory ADME/Tox or pharmacokinetic data from in vivo animal studies. However, this calls for relevant in vitro models, since confidence in pharmacokinetic tools decreases as their relevance to the clinical endpoint decreases.
Therefore, it is not only important to obtain any estimate of how well lead compounds permeate epithelial cell’s membranes such as the intestine’s, but to obtain meaningful and reliable estimates of membrane permeation. Our TRANSIL membrane permeability in vitro models are based on biological membranes with true phospholipid bilayers purified from in vivo sources, which are reconstituted in a highly controlled process. Moreover, all TRANSIL assay kits have an affinity measure as endpoint, which is much more robust to variations in experimental conditions and unspecific binding than rate or concentration endpoints alone. Thus, TRANSIL membrane permeability assay kits are an ideal combination of a highly meaningful in vitro model through truly biological materials, a highly reproducible test system through their production process, and a very robust endpoint. Furthermore, it is possible to determine if the permeability is pH-dependent.
In our standard product line we offer two TRANSIL membrane permeation assays. TRANSIL Intestinal Absorption is based on silica beads covered with phosphatidyl choline as a classical natural membrane bilayer model for the small intestine, while TRANSIL Brain Absorption is based on silica beads covered with porcine brain phospholipids to assess drug candidates’ ability to cross the blood brain barrier.
TRANSIL Protein binding
Consists of silica beads covered with human plasma albumin
PBS buffer(HSA) and human α1-acid glycoprotein (AGP) suspended in PBS buffer.
Assess the drugs proteins affinity by incubating fixed concentration of drug with varying concentrations (six) of HSA or AGP immobilized on silica beads and analyzing the drug free fraction.
Drugs plasma fu is measured in terms of dissociation constant to HSA (KD HSA ) and to AGP (KD AGP ) assuming HSA and AGP concentration corresponding to the mean abundance of both proteins in healthy individuals
Many drugs bind to plasma proteins so that only a fraction of the drug is free in the plasma and capable of producing pharmacological effects. Most drugs bind to albumin, but some - especially basic drugs - bind to α1-acid glycoprotein. Acid drugs (e.g., warfarin or non-steroidal anti-inflammatory drugs) have high affinity for the binding sites of plasma albumin, but some basic drugs (e.g., antidepressants) are bound as well. Most drugs are pharmacologically active at concentrations that do not saturate plasma protein binding sites and thus the fraction of drug bound is independent of the drug concentration. However, the binding sites of a few drugs (e.g., tolbutamide and some sulphonamides) are almost saturated at the therapeutic concentrations. Therefore the application of a higher dose increases the concentration of the free drug by a much greater amount than might be expected.
Our TRANSIL technology provides simple assay formats that closely resemble typical physiological protein binding conditions and that are highly reproducible. TRANSIL ready-to-use 96-well microplates are implemented in three fast, reliable and easy assays generating affinity values to the major plasma proteins human serum albumin (HSA), α1-acid glycoprotein (AGP) and rat serum albumin (RSA).
– Declining concentration
– Signal < reference
– Predicted vs measured
Intercept of nt/cb vs
[lipid] plot: Vb
Outliers based on regression model
– Robust regression