Brain tissue binding

Brain tissue binding assay protocol

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

High specific binding at the pharmacological target in the CNS and greater free fractions in brain can counterbalance poor BBB permeation and/or extensive plasma protein binding1.

Data from the Cloe Select Brain Tissue Binding assay

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

Figure 1

Graph showing Cloe Select 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.

 

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Questions and answers on brain tissue binding

Please provide an overview of the Cloe Select 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;

fu_meas = 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 homogenisation and prior to use, it is necessary to correct the diluted fu value (fu_meas) to generate the undiluted fu value (fu_brain) using the following equation;

fu_brain = 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 recognised 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 utilises 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 favourable 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, K_p,uu, C_u,brain and V_u,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 P_app > 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 (K_p) can be converted to the unbound K_p (K_p,uu) using the fraction unbound in plasma and brain tissue as described below:

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

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

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

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

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

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.

where
Buffer_F = Buffer compartment concentration after dialysis
Brain homogenate_F = Brain homogenate compartment concentration after dialysis
Buffer_I = Initial concentration in buffer
Brain homogenate_I = 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.

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