Brain Tissue Binding Assay

Creative Bioarray provides Brain Tissue Binding Assay to help customers accurately detect the drug transport and distribution in the brain.

Brain Tissue Binding Assay

Why do we need Brain Tissue Binding Assay?

For drugs targeting the central nervous system (CNS), intracerebral concentrations directly affect the efficacy. For non-brain targeting drugs, excessive intracerebral distribution may cause adverse reactions in the CNS. Therefore, understanding the drug transport and distribution in the brain is essential in developing new drugs.

Assessing the in vivo brain to plasma ratio of compounds has long been one of the most common methods for establishing brain penetration. However, relying on brain to plasma ratio data in isolation does not guide the increase in drug efficacy in vivo, as the data only provide a measure of total brain concentration, not the pharmacologically relevant concentration in the brain.

The brain and plasma are very different in composition, with plasma having twice as much protein as the brain and the brain having 20 times more lipids than plasma, so the free fraction in plasma is not a suitable substitute for the concentration of unbound brain

Creative Bioarray uses a hierarchical assay proposed by Reichel et al. to comprehensively characterize the distribution of compounds in regions of the brain, accurately detecting the relationship between the pharmacologically relevant concentration of a drug in the brain and its efficacy.

Brief Protocol

According to the tiered assay proposed by Reichel et al., compounds are subjected to the following assays.

  • First tier: in vitro assays

A series of in vitro ADME analyses, such as metabolic stability of the hepatic microsomes, Caco-2 permeability, and CYP interactions (induction, inhibition), were performed following the usual procedures of the lead compound characterization and optimization phase. Three additional assays were required regarding CNS permeation: ① MDCK-MDR1 cell permeability (bidirectional form), ② equilibrium permeation of plasma, and ③ brain homogenate (from pharmacological animal species).

Brain Tissue Binding AssayFigure 1. Schematic representation of the first tier of in vitro analysis and parameters characterizing compounds' permeation rate and binding properties to plasma and brain tissue (homogenates).


A: filter surface area;

D: brain homogenate dilution;

fu, Dh: buffer to diluent concentration ratio;

fu,brain: fraction unbound in brain;

fu,plasma: fraction unbound in plasma

  • Second tier: in vivo studies

Once a compound shows favorable in vitro data, in vivo studies will follow to examine the behavior of the compound in the whole animal. For CNS, this means determining the pharmacokinetics and brain to plasma ratio of the compound.

Creative Biarray uses equilibrium dialysis to determine the extent to which compounds bind to brain tissue. A semi-permeable membrane separates the compartment containing the compound in the brain homogenate from the compartment containing the compound in the buffer. The assay was performed at 37°C. LC-MS/MS quantified the test compounds present in each compartment.

Brain Tissue Binding AssayFigure 2. Schematic diagram of the second tier of in vivo PK and rodent brain exposure studies to identify compounds and their main PK parameters for exposure in the brain.


CL: clearance;

Vss: volume of distribution at steady state;

t1/2: elimination half-life;

AUC: area under the plasma concentration-time curve;

F%: oral bioavailability;

Kp: total brain to total plasma concentration ratio;

Kp,uu: unbound brain to unbound plasma concentration ratio;

Cu,brain: unbound concenration in brain, surrogate for brain ISF levels [ng/ml];

Vu,brain: unbound volume of distribution in brain [ml/g brain]

Data Interpretation

Kp, uu: Kp, uu determines the degree of equilibrium in the distribution between the brain and the unbound fraction in the plasma.

Cu, brain: Cu, brain is often a measure of the pharmacologically relevant exposure within the brain and hence gives a critical piece of information for the PD effect compartment. It can be used to interpret dose-response results from efficacy studies and establish dose/exposure/effect.

Vu, brain: Vu, brain is an indicator for the distribution behavior of a compound within the brain and is irrespective of the extent of equilibrium between brain and plasma. It indicates whether a compound is distributed merely in interstitial fluid (ISF).

Brain Tissue Binding AssayFigure 3. Interpretation and use of the brain PK parameters derived from both the first and second tier of in vitro assays and in vivo studies

Quotation and Ordering

If you have any special needs or questions regarding our services, please feel free to contact us. We look forward to cooperating with you in the future.


  1. Reichel, Andreas. "Addressing central nervous system (CNS) penetration in drug discovery: basics and implications of the evolving new concept." Chemistry & biodiversity 6.11 (2009): 2030-2049.
  2. Fridén, Markus, et al. "Measurement of unbound drug exposure in brain: modeling of pH partitioning explains diverging results between the brain slice and brain homogenate methods." Drug metabolism and disposition 39.3 (2011): 353-362.

For research use only. Not for any other purpose.