Synthesis and Evaluation of Novel Radioligands for Positron Emission Tomography Imaging of the Orexin‑2 Receptor

Orexin receptors (OXRs) in the brain have been implicated in diverse physiological and neuropsychiatric conditions. Here we describe the design, synthesis, and evaluation of OXR ligands related to (1<i>R,</i>2<i>S</i>)-2-(((2-methyl-4-methoxymethylpyrimidin-5-yl)­oxy)­methyl)-<i>N</i>-(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)­cyclopropanecarboxamide (<b>9a</b>) applicable to positron emission tomography (PET) imaging. Structural features were incorporated to increase binding affinity for OXRs, to enable carbon-11 radiolabeling, and to adjust lipophilicity considered optimal for brain penetration and low nonspecific binding. <b>9a</b> displayed nanomolar affinity for OXRs, and autoradiography using rat brain sections showed that specific binding of [¹¹C]<b>9a</b> was distributed primarily to neocortical layer VI and hypothalamus, consistent with reported localizations of orexin-2 receptors (OX₂Rs). In vivo PET study of [¹¹C]<b>9a</b> demonstrated moderate uptake of radioactivity into rat and monkey brains under deficiency or blockade of P-glycoprotein, and distribution of PET signals in the brain was in agreement with autoradiographic data. Our approach and findings have provided significant information for development of OX₂R PET tracers

Representative PET images illustrating distribution of [¹¹C] WAY-100635 in rat brains at baseline and after oral administration of E2110.

<p>PET images were generated by averaging dynamic data at 60-90 min after intravenous radiotracer injection, and were overlaid on the MRI template shown in the far left column. Coronal brain sections shown here were obtained at 1.0 mm (top row), -7.8 mm (middle row) and -12.5 mm (bottom row) from the bregma. ROIs (dotted lines) were defined on the MPFC (top row), DRN (middle row) and cerebellum (CER; bottom row). The radiotracer retention was presented as a percentage of the injected dose per unit tissue volume (%ID/mL).</p

Development of Novel PET Probes for Central 2‑Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic Acid Receptors

We document the development of PET probes for central AMPA receptors and their application to in vivo animal imaging. An initial screening of perampanel derivatives was performed to identify probe candidates. Despite the high autoradiographic contrast yielded by several radioligands, rat PET scans did not support their in vivo suitability. Further focused derivatization and a second screening by ex vivo LC-MS measurements led to the selection of 2-[1-(3-methylaminophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl]­benzonitrile, <b>21a</b>, and its analogues as candidates. [¹¹C]<b>21a</b> was shown by autoradiography to specifically bind to the neocortex and hippocampus, consistent with AMPA receptor localization. PET imaging with [¹¹C]<b>21a</b> demonstrated moderate uptake of radioactivity in rat and monkey brains, with the retention of radiosignals being consistent with that from the autoradiogram data, and the uptake was blocked by pretreatment with unlabeled <b>21a</b> in a dose-dependent manner. The current approach has facilitated the discovery of a PET probe potentially suitable for translational research and development focused on AMPA receptors

Time course data of rat 5-HT_1A RO in MPFC (●) and DRN (◇), and plasma E2110 concentration (thick dashed line) (A), and plot of RO against plasma E2110 concentration at individual time points (B).

<p>5-HT_1A RO was determined at assigned time points after oral administration of E2110 at a dose of 1 mg/kg. Symbols represent mean ± S.E.M at indicated time points (n = 4/time point). Lines indicate the predicted occupancy versus plasma concentration in MPFC (solid line) and DRN (dashed line).</p

Relationship between rat 5-HT_1A RO (MPFC: ●, DRN: ◇) and E2110 plasma concentration.

<p>5-HT_1A RO was determined at 4 hours after oral administration of E2110 at a dose ranging from 0.3 to 10 mg/kg. Symbols represent individual data from all dose levels (n = 4/dose level).</p

Effect compartment model estimation of 5-HT_1A RO in DRN after oral administration of E2110 at doses of 0.03, 0.1 and 0.3 mg/kg to female rats.

<p>Solid circle and error bars represent mean RO ± S.E.M measured by PET scans in female rats (n = 3).</p

Effects of E2110 on micturition interval in 8-OH-DPAT-infused (A) and SCL (B) rats.

<p>Values are expressed as mean ± S.E.M. of eight rats in 8-OH-DPAT-infused and SCL models; * <i>P</i> < 0.05 versus vehicle (Dunnett’s multiple test).</p

Schematic illustration of a model for description of E2110 PK/PD.

<p>C_p, E2110 concentration in the central compartment; C_e, E2110 concentration in the effect compartment; k_a, absorption rate constant; k_e0, equilibrium rate constant; V₁, central volume of distribution; V₂, peripheral volume of distribution.</p

<i>In vitro</i> inhibition profiles of E2110 and WAY-100635 on 5-HT_1A receptors.

<p>Inhibition of specific [³H] MPPF binding in rat hippocampal membrane homogenates was measured at various concentrations of E2110 (●) and WAY-100635 (○).</p

Chemical structure of E2110.

<p>Chemical structure of E2110.</p