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Compound Characterization 1

Compound Characterization

Study Goal

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

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Prepare stock solutions
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Dilutions from stock solutions
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Serial dilutions of stock solutions

Phase 1: Radioligand binding assays

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Fresh brain homogenates were derived from OX1 receptor rich regions of the rat brain.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Radiolabeled Orexin-A, Almorexant, and SB-674042 (known OX1 receptor ligands) were all assayed.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Assays were performed using conventional filtration-type assays with unlabeled OX-A as a reference compound.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Unfortunately, there was no significant binding in any of the rat brain homogenates prepared.

A different approach was necessary

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Brains from adult male rats were sectioned and thaw mounted; sections collected contained OX1 receptor rich regions.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Slides were exposed onto phosphorimage screens and analyzed.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Saturation binding curves were generated for all 3 of the radiolabeled OX1 ligands in rat brain sections.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 in vivo autoradiography was now needed to characterize the regions of the rat brain that are dense in OX1 receptors.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 BMAX was determined using GraphPad.

Why?

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Brains from adult male rats were sectioned and thaw mounted; sections collected contained OX1 receptor rich regions.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 The following conditions were tested in autoradiography experiments in order to optimize assay conditions:
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Optimization was assessed using phosphorimage analysis for calculation of specific binding in OX1 receptor rich regions of the brain.
  • Slides used
  • Radiotracer concentration
  • Buffer composition
  • Incubation time
  • Wash time

Why?

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Optimize experimental conditions for ex vivo ARG and demonstrate receptor occupancy at the highest dose and maximum expose to CMPDx.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 SD rats were injected with a single high dose of CMPDx along with the unlabeled antagonist compound and euthanized 1 hour post-dose.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 The following conditions were tested in order to optimize assay conditions:
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Receptor occupancy in the brain regions and maximum dose of CMPDx were determined.
  • Slides used
  • Radiotracer concentration
  • Buffer composition
  • Incubation time
  • Wash time

Why?

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Optimization allows for the minimization of potential washout of test compound from the brain sections.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Testing varying incubation times allows identification of the minimum time needed to obtain a high binding signal.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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

  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Using the previously optimized conditions, SD rats were injected with varying concentrations of CMPDx and the same dose of the OX1 antagonist compound.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Blood samples were collected, inverted, and centrifuged to allow collection of plasma for PK determination.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 ex vivo autoradiography was performed under the previously optimized incubation conditions and the radiolabeled OX antagonist.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Levels of bound radioactivity was determined from manual ROI selection.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Animals were euthanized at various time points.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 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.
  • Created by potrace 1.10, written by Peter Selinger 2001-2011 Slides were exposed to a phosphorscreen screen and then imaged.

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