TRANSIL Intestinal Absorption Kit

TRANSIL Intestinal Absorption Kit

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The TRANSIL Intestinal Absorption kit measures the affinity of drugs to phosphatidylcholine membranes. It is not only a screening tool to predict intestinal permeability coefficients, but also to predict compounds’ tissue binding. As drug-membrane interactions are key to both the process of membrane permeability and to binding to and permeating into the cell membranes of tissues the assay kit is an ideal tool to predict intestinal permeability rates as well as the volume of distribution early in drug discovery phase. 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.

Figure 1: Human intestinal permeability rates obtained by caco-2 experiments and predicted by a model based on the membrane affinity and polar surface area. Figure 2: Comparison of TRANSIL predicted volume of distribution (Vss) with literature data. Vss is predicted based on the measured on the membrane affinity measurement and plasma protein binding (fraction unbound). Figure 3: Illustration of the impact of a membrane’s ionization state on its membrane affinity comparing TRANSIL beads with dialysis with erythrocytes. TRANSIL beads exhibit the same pH dependence as erythrocyte membranes.
  • Is membrane affinity more meaningful than measuring lipophilicity as LogP or LogD?

    The octanol-water partition model has several limitations. First off, it is not very 'biological' as octanol is a poor representation of membranes. In the past, the alternative use of liposomes (which are vesicles with walls made of a phospholipid bilayer) has become more widespread. Liposomes are made of the main ingredients found in all biological membranes and model the structure-structure interaction of a drug passing through a membrane very well.

    Unfortunately, liposomes are difficult to handle and last only about 15 minutes before they begin to fuse into lager multilammellar structure. While they are a much better model for membrane permeability and membrane binding, they are cumbersome to use, which is why LogP or LogD is still frequently used in the physchem description of drugs.

    TRANSIL Intestinal Absorption beads are coated with a floating phosphatidylcholine membrane bilayer. This stabilized the liposomes while retaining their natural fluidity. Thus, the TRANSIL intestinal absorption test kits easier and faster to use than octanol-water partitioning experiments, yet they are as informative as liposome partitioning.

  • How do we know that the immobilized membranes retain their natural fluidity?

    An important goal for designing the TRANSIL kits was to model a compound’s interaction with membranes as close as possible. That requires that the membranes retain their natural fluidity. To achieve that we immobilize single membrane bilayers on porous silica beads such that the membranes float on a thin water layer. The immobilization conditions have been optimized such that both differential scanning calorimetry (c.f. figure 3) and NMR spectrometry (c.f. figure 4) show very similar fluidity patterns.

    Figure 3: Differential scanning calorimetry comparing free floating liposomes (phosphatidylcholine vesicles) with immobilized TRANSIL beads. Both vesicles and beads melt at the same temperature, which indicates that they have the same structure and their fluidity is comparable.

    Figure 4: Comparison of H2-NMR spectrums of free floating membrane vesicles (blue) and TRANSIL bead supported membranes (red). Energy peaks of rotation and flipping of phospholipids occurs at the same frequencies (kHz) in both supported and unsupported membranes. This indicates that the TRANSIL membrane support beads stabilized the phosphatidylcholine membranes such that they retain their natural fluidity.
  • How does the TRANSIL Intestinal Absorption Kit work?

    The TRANSIL Intestinal Absorption Kit measures the affinity of a test item to immobilized phosphatidylcholine 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:

  • What are the main quality control measures applied in TRANSIL assays?

    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.
  • How long does it take to run the assay?

    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.

  • How many compounds can be analyzed with one plate?

    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.

  • Are TRANSIL assay kits supplied in low-binding plates?

    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.