Brain Tissue Binding
Depending on their lipophilicity and solubility, drug-effector molecules can bind to different kinds of tissues, such as fat, brain, liver, and kidney, resulting in drug accumulation in tissues other than the blood circulation. The binding of drugs to brain tissue affects the pharmacokinetic (PK) profile of the compound. Experts from BOC Sciences utilize classical rapid equilibrium dialysis (RED) or ultrafiltration in tissue homogenates to measure the percent binding of test drugs to brain tissue. In addition, we also provide the percentage of unbound (free) compounds.
The composition of the brain is very different from that of plasma. Plasma contains twice as much protein as the brain and the brain contains 20 times more lipids than plasma. Evaluating the in vivo brain to plasma ratio of compounds has long been one of the common methods for establishing brain penetration. However, reliance on brain-to-plasma ratio data alone does not guide the improvement of drug efficacy in vivo, as these data only provide measurements of total brain concentrations, rather than pharmacologically relevant concentrations in the brain. Drug efficacy depends on a combination of factors, including plasma protein binding, brain tissue binding and blood-brain barrier permeability, as well as total brain levels. Therefore, understanding the transport and distribution of drugs in the brain is crucial for the development of new drugs.
Brief Protocol
BOC Sciences has introduced a high-throughput equilibrium dialysis strategy to provide fractional values for compounds not bound to brain tissue. The basic principle of equilibrium dialysis is to add target compound-spiked tissue homogenate to the tissue chamber and dialysis buffer to the buffer chamber. A semipermeable membrane separates the compound-containing chamber in the brain homogenate from the compound-containing chamber in the buffer. Free compounds diffuse from the tissue chamber to the buffer chamber until equilibrium is reached. Then, the compound concentrations in homogenates and buffers were determined by LC-MS and unbound fractions were calculated. All incubations were performed in duplicate. BOC Sciences' brain tissue binding assay has been fully developed to allow our scientists to quickly and accurately determine the unbound fraction of drugs in brain tissue using a 96-well balanced dialysis device.
Data Analysis
The drug-unbound fraction (fu, brain) of the diluted brain homogenate was then calculated according to the following formula:
- fu, brain: The unbound fraction in brain tissue
- fu, homogenate: The unbound fraction in brain homogenate
- Dilution is the dilution factor for the brain homogenate
Deliverable: Fraction unbound (fu) and recovery are calculated based on the LC/MS measurements of the compound concentrations in homogenate and buffer solutions.
Sample submission: Minimum weight of dry compound (~1 mg) or 100~150 μL of 10 mM DMSO stock solution required for this assay.
In addition to routine in vitro ADME assays, BOC Sciences also offers CNS penetration and distribution assays, namely bidirectional MDR1-MDCK cell permeability, plasma protein binding, and brain homogenate assays. Additionally, if the compound proves useful by in vitro assays, we are also able to provide in vivo assessments to determine the compound's pharmacokinetics and brain-to-plasma ratio. If you are interested in our brain tissue binding testing services, please contact us for more information.