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

Phase 1: Radioligand binding assays
  • Fresh brain homogenates were derived from OX1 receptor rich regions of the rat brain.
  • Assays were performed using conventional filtration-type assays with unlabeled OX-A as a reference compound.
  • Radiolabeled Orexin-A, Almorexant, and SB-674042 (known OX1 receptor ligands) were all assayed.
  • 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.
  • 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.
  • 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.
  • BMAX was determined using GraphPad.
  • in vivo autoradiography was now needed to characterize the regions of the rat brain that are dense in OX1 receptors.

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.
  • Animals were euthanized at various time points.
  • Blood samples were collected, inverted, and centrifuged to allow collection of plasma for PK determination.
  • 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.
  • ex vivo autoradiography¬†was performed under the previously optimized incubation conditions and the radiolabeled OX antagonist.
  • Slides were exposed to a phosphorscreen screen and then imaged.
  • Levels of bound radioactivity was determined from manual ROI selection.