Ex vivo Receptor Occupancy and In Vitro Radioligand Binding Services

RenaSci is a leading provider of ex vivo receptor occupancy studies in rats and mice. Our dedicated team of experts have been conducting in vitro receptor binding assays and ex vivo occupancy studies for more than 20 years. We have the expertise to design bespoke studies to meet clients’ precise needs by developing novel assays using custom-synthesised radioligands. Alternatively, we can provide off the shelf assays using commercially available radioligands ([³H] and [¹²⁵I]) for well-characterised neurotransmitter receptors and uptake sites.

 

Ex vivo Receptor Occupancy Services

Ex vivo receptor occupancy is a valuable technique in the drug discovery pathway for demonstrating target engagement of centrally penetrant compounds. Alternatively it can be used to obtain a pharmacological profile of binding sites associated with adverse side-effects such as drug abuse liability. This technique provides a link between in vitro activity and in vivo efficacy at considerably lower costs than those associated with in vivo imaging techniques.

At RenaSci ex vivo receptor occupancy studies can be undertaken either as stand-alone or subsequent to in vivo studies eg binge eating  or IVSA  and can be used to determine:

 

• Central receptor occupancy for a range of drug doses. Example data- ex vivo receptor occupancy at kappa opioid receptor…
• Occupancy in different brain regions in the same animal. Example data- ex vivo receptor occupancy at mu opioid receptor…
• Occupancy at a range of molecular targets in the same animal. Example data- ex vivo receptor occupancy at monoamine reuptake transporters…
• Time-course of receptor occupancy. Example data- ex vivo receptor occupancy at MCH receptor…

 

 

 Key Features:

In ex vivo occupancy studies, the test compound or vehicle is administered to the animal peripherally. The level of in vivo receptor occupancy by test compound is subsequently measured ex vivo in a radioligand binding assay:

• Drug administration by a variety of routes (po, ip, sc or iv) in rats and mice.
• Tissue harvested at the time of peak effect with optional blood, CSF and tissue sampling for PK/PD determination with the option for analysis carried out on-site at Sygnature Discovery or shipped to clients at their convenience.
• Ex vivo binding assays performed in brains tissue using homogenate preparations or brains slices for quantitative autoradiography.

• High-throughput quantitative autoradiography is performed using a Biospace Beta-Imager which gives rapid measurements of specific binding along with the ability to detect very low levels of tritium (0.007 cpm/mm²)

• Read-only autoradiography service for brains from external studies

 

 

In vitro receptor binding assays

Radioligand binding assays are used to characterize the binding of a drug to its target receptor providing an important contribution to drug screening and development programmes.

There are three types of in vitro radioligand binding assays: competition, saturation and kinetic, all of which can be performed at RenaSci either in recombinant cell lines or using native tissue (from rat and mouse brains).

Competition radioligand binding assays are used for measuring the affinity (Ki value) of test compounds for binding to a receptor in membrane preparations or using autoradiography.

 

In vitro Competition Binding Assay to determine the affinity of rimonabant for CB1 receptors using inhibition of [3H]SR141716A and [3H]CP55,940 binding to rat frontal cortical membrane preparations

 

Ki = Inhibition constants, (n=3).

 

 

 

Affinity of Rimonabant for CB1 Receptors

 

Representative individual displacement curve for rimonabant for inhibition of [³H]SR141716A.

 

Saturation binding assays are performed to determine the affinity (Kd) of a radioligand for a receptor and the density (Bmax) of a specific receptor in membrane preparations or using autoradiography.

 

 

Saturation Analysis: Kd and Bmax

Binge-eating is associated with decreased D₁ receptor density and increased μ-opioid receptor density in the striatum.

 

Striatal membranes were prepared from rats trained to binge-eat and incubated with [³H]raclopride (D₁) or [³H]DAMGO (μ-opioid).
Results are adjusted means + SEM (n=10). *p<0.05 and **p<0.01 vs. control.

 

Functional Studies

RenaSci also has the expertise to undertake functional pharmacological activity assays; by assessing receptor function using [³⁵S] GTPgammaS binding or by measuring uptake into rat and mouse synaptosomes.

Dipyridamole is a moderately potent inhibitor of adenosine uptake into rat cortical synaptosomes.

Representative individual displacement curve for dipyridamole for inhibition of [3H] adenosine uptake. n=(3)

 

If you are interested in any of our receptor binding services, please get in touch to find out more.