Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug–drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2′,7′-dichlorofluorescein (DCF), 4′,5′-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with <i>K</i>_m values of 5.29, 4.16, and 54.1 μM and <i>V</i>_max values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The <i>K</i>_i values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [³H]­estradiol-17β-glucuronide (E₂G) as a substrate, whereas [³H]­estrone-3-sulfate and [³H]­sulfobromophthalein yielded higher <i>K</i>_i values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E₂G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E₂G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent <i>K</i>_i variations

Discovery of 2‑(2-Oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile (Perampanel): A Novel, Noncompetitive α‑Amino-3-hydroxy-5-methyl-4-isoxazolepropanoic Acid (AMPA) Receptor Antagonist

Dysfunction of glutamatergic neurotransmission has been implicated in the pathogenesis of epilepsy and numerous other neurological diseases. Here we describe the discovery of a series of 1,3,5-triaryl-1<i>H</i>-pyridin-2-one derivatives as noncompetitive antagonists of AMPA-type ionotropic glutamate receptors. The structure–activity relationships for this series of compounds were investigated by manipulating individual aromatic rings located at positions 1, 3, and 5 of the pyridone ring. This culminated in the discovery of 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)­benzonitrile (perampanel, <b>6</b>), a novel, noncompetitive AMPA receptor antagonist that showed potent activity in an in vitro AMPA-induced Ca²⁺ influx assay (IC₅₀ = 60 nM) and in an in vivo AMPA-induced seizure model (minimum effective dose of 2 mg/kg po). Perampanel is currently in regulatory submission for partial-onset seizures associated with epilepsy

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

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

<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

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