Compound Characterization

Study Goal

Determine ex vivo receptor occupancy of CMPDx at Orexin-1 (OX1) receptors using radiolabeled Orexin antagonist

Prepare stock solutions

Dilutions from stock solutions

Serial dilutions of stock solutions

Phase 1: Radioligand binding assays

Fresh brain homogenates were derived from OX1 receptor rich regions of the rat brain.

Radiolabeled Orexin-A, Almorexant, and SB-674042 (known OX1 receptor ligands) were all assayed.

Assays were performed using conventional filtration-type assays with unlabeled OX-A as a reference compound.

Unfortunately, there was no significant binding in any of the rat brain homogenates prepared.

A different approach was necessary

Brains from adult male rats were sectioned and thaw mounted; sections collected contained OX1 receptor rich regions.

Slides were exposed onto phosphorimage screens and analyzed.

Saturation binding curves were generated for all 3 of the radiolabeled OX1 ligands in rat brain sections.

in vivo autoradiography was now needed to characterize the regions of the rat brain that are dense in OX1 receptors.

All slides were pre-incubated and subjected to binding buffer that contained radiolabeled Orexin-A antagonist for total binding or radiolabeled Orexin-A antagonist and cold CMPDx to determine non-specific binding.

BMAX was determined using GraphPad.

Why?

To confirm the specific binding of OX1 in rat brain homogenates, as this data is lacking in current literature.

Phase 2: in vitro autoradiography of OX1-rich brain regions

Brains from adult male rats were sectioned and thaw mounted; sections collected contained OX1 receptor rich regions.

The following conditions were tested in autoradiography experiments in order to optimize assay conditions:

Slides used

Radiotracer concentration

Buffer composition

Incubation time

Wash time

Optimization was assessed using phosphorimage analysis for calculation of specific binding in OX1 receptor rich regions of the brain.

Why?

Demonstration of specific OX1 receptor binding with CMPDx in rat brain regions using the in vitro autoradiography provided proof of concept to move on to the ex vivo study.

Optimize experimental conditions for ex vivo ARG and demonstrate receptor occupancy at the highest dose and maximum expose to CMPDx.

Receptor occupancy studies and their linkage to in vitro efficacy is important for dose prediction and is required for CD investment decision.

Phase 3: Optimize ex vivo autoradiography conditions

SD rats were injected with a single high dose of CMPDx along with the unlabeled antagonist compound and euthanized 1 hour post-dose.

The following conditions were tested in order to optimize assay conditions:

Slides used

Radiotracer concentration

Buffer composition

Incubation time

Wash time

Receptor occupancy in the brain regions and maximum dose of CMPDx were determined.

Why?

Optimization allows for the minimization of potential washout of test compound from the brain sections.

Testing varying incubation times allows identification of the minimum time needed to obtain a high binding signal.

The results will indicate if the methodology used to optimize the in vitro autoradiography is applicable for this ex vivo receptor occupancy study and future studies.

Phase 4: Final ex vivo autoradiography

Using the previously optimized conditions, SD rats were injected with varying concentrations of CMPDx and the same dose of the OX1 antagonist compound.

Blood samples were collected, inverted, and centrifuged to allow collection of plasma for PK determination.

ex vivo autoradiography was performed under the previously optimized incubation conditions and the radiolabeled OX antagonist.

Levels of bound radioactivity was determined from manual ROI selection.

Animals were euthanized at various time points.

Brains were harvested, preserved, and thaw mounted onto glass slides at the areas of interest with 3 adjacent sections; 2 to observe total binding and 1 to observe non-specific binding.

Slides were exposed to a phosphorscreen screen and then imaged.