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Brain tissue binding

Understand the extent of brain partitioning using our brain tissue binding assay.

Brain tissue binding is one of Cyprotex's in vitro experimental ADME services. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

The significance and measurement of brain tissue binding

  • The extent of partitioning into brain tissue influences CNS penetration which in turn influences the efficacy and / or toxicological effects of a drug.
  • The composition of brain and plasma are very different, with plasma having twice as much protein and brain having 20 fold more lipids, therefore free fraction in plasma is not a suitable surrogate for unbound brain concentrations1.
  • Assuming passive equilibrium, it is expected that brain to plasma drug exposure levels for any species will be predicted by the relative ratio of free fractions in these matrices2.
  • For compounds which undergo drug transport, differences between the unbound plasma-to-brain fraction ratios and brain-to-plasma exposure can be used to examine the net influence of active efflux processes on CNS exposure independent of the exact cellular mechanism2.
  • Cyprotex's Brain Tissue Binding assay is performed using equilibrium dialysis, one of the most widely accepted methods for assessing protein and tissue binding.
  • Cyprotex's Brain Tissue Binding assay delivers a value of fraction of compound unbound to brain tissue (fubrain)
Neither total brain levels nor BBB permeability can be taken without considering the binding capacity of the brain tissue, when a link between exposure and efficacy is needed.

1Reichel A (2009) Chemistry and Biodiversity 6; 2030-2049


Brain Tissue Binding assay protocol

Method Equilibrium dialysis using brain homogenate
Typical Test Article Concentration 5 μM (different concentrations available)
Number of Replicates 2
Test Article Requirements 150 μL of a 10 mM DMSO solution
Analysis Method LC-MS/MS quantification (both brain homogenate and buffer standards prepared)
Data Delivery Fraction unbound in brain


Data from Cyprotex's Brain Tissue Binding assay

For the validation, eight compounds were screened in Cyprotex's Brain Tissue Binding assay (rat and mouse) on three separate occasions. Data were compared with literature data (Figure 2).

Figure 1
Graph showing Cyprotex's Rat Brain Tissue Binding data for a set of eight compounds over three separate assays.

These data illustrate good consistency is achieved over a number of different days for compounds with a range of binding values.
Comparison of IC50 values
Figure 2
Graph showing a comparison of fraction unbound in mouse brain between Cyprotex's Brain Binding data (mean ± standard deviation; n = 3) and literature3 data for a set of eight compounds.

Cyprotex's data correlate well with literature data for compounds with a range of different binding values.


Questions and answers on brain tissue binding

Please provide an overview of Cyprotex's Brain Tissue Binding assay

Equilibrium dialysis is used to determine the extent of binding of a compound to brain tissue. A semi-permeable membrane separates a compartment containing compound in brain homogenate from a compartment containing compound in buffer. The system is allowed to equilibrate at 37°C. The test compound present in each compartment is quantified by LC-MS/MS.

A fraction unbound (fu) value in diluted brain tissue is calculated as detailed below;

fumeas = fraction unbound using diluted brain tissue
PC = test compound concentration in protein-containing compartment
PF = test compound concentration in protein-free compartment

As the brain is diluted during homogenization and prior to use, it is necessary to correct the diluted fu value (fumeas) to generate the undiluted fu value (fubrain) using the following equation;

fubrain = fraction unbound in brain
D = dilution factor of brain tissue

Why is brain tissue binding important?

Traditionally, assessing the in vivo brain to plasma ratio of a compound has been one of the most common methods for establishing brain penetration. It is now recognized that relying on the brain to plasma ratio data in isolation does not necessarily lead to an increase in efficacy in vivo as the data only provides a measure of total brain concentration rather than pharmacologically relevant concentrations in the brain.

The link between exposure and efficacy is dependent on a number of factors in combination including the plasma protein binding, brain tissue binding and blood brain barrier permeability in addition to the total brain levels. Reichel (2009)1 has recommended a tiered approach to decision making in CNS drug discovery which utilizes all these parameters. This is described below:

Tier 1: In vitro studies: In addition to regular in vitro ADME assays, a panel of CNS relevant assays should be included to establish CNS penetration and distribution, namely permeability in bidirectional MDR1-MDCK cells, plasma protein binding and brain tissue binding using equilibrium dialysis.

Tier 2: In vivo studies: If the compound demonstrates favorable in vitro data in Tier 1, the compound is then assessed in vivo to determine the pharmacokinetics and the brain to plasma ratio of the compound. The in vivo data can be used in conjunction with Tier 1 binding data to generate three valuable parameters, Kp,uu, Cu,brain and Vu,brain. These parameters assist in the interpretation and understanding of CNS distribution.

How do I interpret the data from the brain tissue binding assay alongside other key CNS parameters?

Based on the approach by Reichel (2009)1, it has been proposed that Tier 1 is used as a pass/fail criteria for progression to Tier 2. For example, acceptance criteria in the MDR1-MDCK permeability assay of Papp > 15 x 10-6 cm/s and efflux ratio < 3 have been proposed, although it is acknowledged that these thresholds are often project specific.

If a compound passes the criteria required for Tier 1, then Tier 2 studies are initiated. The in vitro fraction unbound data from Tier 1 can then be used in conjunction with in vivo data from Tier 2 as described below:

The brain to plasma ratio (Kp) can be converted to the unbound Kp (Kp,uu) using the fraction unbound in plasma and brain tissue as described below:

Kp,uu is more useful than Kp as it determines the extent of distribution equilibrium between the unbound fraction in brain and plasma. If the Kp,uu is close to unity then this indicates passive diffusion across the blood brain barrier (or equal rates of efflux and influx). Kp,uu < 1 indicates efflux at the blood brain barrier and Kp,uu >1 indicates uptake at the blood brain barrier.

The total concentration in the brain (Ctotal,brain) at each time point can be corrected using the unbound fraction in the brain (fubrain) in order to calculate the unbound concentration in the brain (Cu,brain) as detailed in the equation below.

Cu,brain provides a measure of pharmacologically relevant exposure in the brain.

The unbound volume of distribution (Vu,brain) is calculated by dividing the total amount of drug in the brain (corrected for the amount in the cerebral vasculature) (Abrain) by the unbound concentration of drug in the brain (Cu,brain) as detailed in the equation below:

The Vu,brain indicates if a compound is distributed solely in interstitial fluid (i.e., Vu,brain ~0.2 mL/g), throughout brain water space (interstitial and intracellular fluid, i.e., Vu,brain ~ 0.8 mL/g) or binds non specifically to brain tissue (Vu,brain > 0.8 mL/g).

Vu,brain can be estimated from 1/fu,brain from in vitro brain homogenate binding measurements, however significant error in prediction should be expected unless a pH-partitioning model such as the one suggested by Fridén et al., (2011)4 is used. Such models can be used to determine Kp, uu, cell (the unbound drug partitioning coefficient of the cell) which is the used to convert fu, brain to Vu, brain. Kp, uu, cell determination requires knowledge of the pKa and charge type of the compound in question.

What are the effects of poor solubility on the brain tissue binding data?

Compounds are screened for in vitro brain tissue binding at a concentration of 5 μM which could potentially lead to a concentration close to 10 μM if the compound is very highly protein bound. Therefore, if a compound has a solubility value of less than 10 µM at 37°C, it is not recommended that the compound is screened in this assay as the insoluble compound will not be able to freely cross the membrane. In this instance, performing the assay at a lower concentration may be one option depending on the solubility of the compound.

How and why is % recovery calculated?

BufferF = Buffer compartment concentration after dialysis
Brain homogenateF = Brain homogenate compartment concentration after dialysis
BufferI = Initial concentration in buffer
Brain homogenateI = Initial concentration in brain homogenate

In theory, the recovery should be 100%. If the recovery deviates from 100%, it may indicate binding to the dialysis membrane or solubility issues.

What positive control is used in the assay?

The positive control compound used for the assay is diazepam. Diazepam is highly bound to brain tissue in rat and mice.


1 Reichel A (2009) Chem Biodiv 6; 2030-2049
2 Kalvass JC and Maurer TS (2002). Biopharm Drug Dispos 23; 327-338
3 Maurer TS et al. (2005) Drug Metab Dispos 33; 175-181
4 Fridén M et al. (2011) Drug Metab Dispos 39(3); 353-362

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