In vitro Binding

in vitro Binding

Plasma Protein Binding (PPB)

Most drugs are, at least to some extent, bound to plasma proteins, especially albumin and α1-acid glycoprotein. PPB is mainly dependent on physicochemical properties such as charge and lipophilicity, but an accurate in silico prediction of PPB is often challenging.

The importance of an accurate PPB determination especially for highly bound compounds becomes most evident in the following example.

Whether a compound is 99.0% bound to plasma protein or 99.9% does not appear significantly different. However, when taking the free (unbound) fraction into consideration it becomes obvious that the concentration available to bind to the drug target and elimination decreases 10-fold from 1% to 0.1%.

XenoGesis offers PPB assays for a wide range of species, strains and genders. Using rapid equilibrium dialysis (RED) we can offer a fast, reliable and cost-effective way to assess PPB for your drug candidates. Please speak to us now.

Plasma protein binding is calculated according to equation 1.

gray_reaction111(1)

Where Cb is the compound concentration in buffer and Cp is the compound concentration in plasma.

Typically, PPB is determined over ≥ 4 hours at 37°C. Under these conditions chemical and/or enzymatic degradation of compounds can occur. The determination of compound recovery is, therefore, of pivotal importance. A recovery significantly lower than 100% can flag a variety of potential problems:

• The compound is hydrolytically or metabolically unstable

• The compound is highly bound to the rapid equilibrium dialysis device

• The compound is poorly soluble

Recovery is calculated according to equation 2.

gray_reaction211(2)

Where Cp and Vp are the compound concentration and the volume in the plasma compartment Cb and Vb are the compound concentration and the volume in the buffer compartment and (init.) and (end) indicate concentration before and after the incubation, respectively.

A range of PPB-values determined at XenoGesis for a variety of drugs is illustrated in the figure below.

gray_binding1

Human plasma protein binding. Values are the mean of triplicate determinations ± one standard deviation.

Method Rapid Equilibrium Dialysis
Species Human; rat; dog; cynomolgus monkey; guinea pig; mouse (others upon request)
Plasma concentration 100% (less for very highly bound compounds)
Buffer Phosphate-buffered saline (PBS) pH 7.4
Test compound concentration 5 µM
Incubation time 4 hours
Incubation temperature 37°C
Analytical technique UPLC-MS/MS
Recommended number of replicates ≥ 2
Typical turnaround time 2 weeks

Note: Assay conditions can be tailored to client requirements.

Ian Dearman Media-467Tissue Binding (Fraction Unbound, FUT)

Like plasma protein binding, binding to tissue can significantly reduce a drug’s free fraction, i.e. the fraction of drug available for target binding or elimination. XenoGesis offers equilibrium dialysis using tissue homogenates as a fast, reliable and cost effective way to quantitatively assess tissue binding In vitro.


Blood:Plasma Partitioning

Certain drugs are highly bound to red blood cells (RBC) whilst others are highly bound to plasma proteins. As pharmacokinetics are mostly measured from plasma samples, binding to RBC can lead to dramatically miscalculated blood-derived pharmacokinetic parameters, e.g. clearance, AUC, Cmax and Vss.

Like plasma protein binding, binding to red blood cells (RBC) can significantly reduce a drug’s free plasma concentration, i.e. the fraction of drug that is available for its pharmacological action and its elimination. As a consequence, a compound’s pharmacodynamic and pharmacokinetic profile can be severely affected by its blood partitioning. Likewise, accumulation in RBC can lead to toxicity in these cells. It is, therefore, important to determine blood partitioning early during the drug discovery process.

Method Parallel incubation of test compound in fresh blood and matched plasma
Species Human; rat; dog; cynomolgus monkey; guinea pig; mouse (others upon request)
Test compound concentration 1 µM
Incubation time 30 minutes
Incubation temperature 37°C
pH 7.4
Analytical technique UPLC-MS/MS
Recommended number of replicates ≥ 2
Typical turnaround time 2 weeks

Note: Assay conditions can be tailored to client requirements.

At XenoGesis we offer a quick and cost-effective way to determine blood:plasma (B:P) ratio without the need to measure time-consuming concentrations in RBC preparations. By using blood and plasma from the same donor we avoid extra variability. In parallel incubations test compounds are mixed with fresh blood and plasma freshly prepared from the same batch of blood. After the incubation time RBC are separated from the blood incubations by centrifugation and aliquots from the supernatant are analysed alongside the plasma samples to which test compounds were added directly. B:P ratio is then calculated using equation 1:

gray_plasmapartitioning11

(1)

Note: The directly spiked plasma is a surrogate for directly spiked blood. Therefore, matrix effects during LC-MS/MS analysis are completely abolished.