Presynaptic selectivity of a ligand for serotonin 1A receptors revealed by in vivo PET assays of rat brain

Abstract

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT(1A)) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT(1A) receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT(1A) receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT(1A) receptors. In addition, [(35)S]guanosine 5'-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT(1A) receptors. This finding has lent support to reports that diverse partial agonists for 5-HT(1A) receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.

Figure 1. Binding affinities of Wf-516 and pindolol for central 5-HT_1A receptors quantified by in vitro ARG.

(A) Representative autoradiograms showing distribution and intensity of [¹¹C]WAY-100635 radiosignals in rat brain sections containing the hippocampus (arrows) and raphe nucleus (arrowhead) and its attenuation by different concentrations of Wf-516 or pindolol. (B) Inhibition curves of [¹¹C]WAY-100635 binding to the hippocampus (closed symbols) and raphe nucleus (open symbols) by Wf-516 (left) and pindolol (right). Bars indicate S.E. (n = 3).

Figure 2. Occupancies of 5-HT_1A receptors by Wf-516 and pindolol assessed by ex vivo ARG.

(A) Representative autoradiograms showing distribution and intensity of [¹¹C]WAY-100635 radiosignals in rat brain sections containing the hippocampus (arrows) and raphe nucleus (arrowhead) after oral administration of Wf-516 and intraperitoneal administration of pindolol. (B) Relationships between dose of Wf-516 (left) or pindolol (right) and 5-HT_1A receptor occupancy in the hippocampus (closed symbols) and raphe nucleus (open symbols). Regression curves in the hippocampus and raphe nucleus are denoted by dashed lines and solid lines, respectively, and were generated by the following equation: Occ  = 100×D/(D+ED₅₀), where Occ is 5-HT_1A receptor occupancy and D is the dose of the drug. Bars indicate S.E. (n = 3).

Figure 3. Representative PET images showing distribution of [¹¹C]WAY-100635 in rat brains after oral administration of Wf-516 and intraperitoneal administration of pindolol.

Each pretreatment drug was repeatedly administered to the same rat at different doses. PET images were generated by summation of dynamic data 60–90 min after intravenous injection of [¹¹C]WAY-100635, and were overlaid on the MRI template displayed in the far left column. Coronal brain sections shown here were obtained at −5.2 mm (top row), −7.8 mm (middle row) and −12.5 mm (bottom row) from the bregma. ROIs were placed on the hippocampus (dotted lines), raphe nucleus (solid lines) and cerebellum (dashed lines).

Figure 4. Time-radioactivity curves for [¹¹C]WAY-100635 in the hippocampus (top panels), raphe nucleus (middle panels) and cerebellum (bottom panels) after pretreatments with Wf-516 (A) and pindolol (B) at representative doses.

Data were generated by placing ROIs on different brain structures on the PET images illustrated in Figure 3. Radiotracer uptake into each region was expressed as a percentage of the injected dose per unit tissue volume (%dose/mL). Bars indicate S.E. (n = 4 and 3 in Wf-516 and pindolol treatment groups, respectively).

Figure 5. Time course of specific [¹¹C]WAY-100635 binding to 5-HT_1A receptors in the hippocampus (top panels) and raphe nucleus (bottom panels) after pretreatments with Wf-516 (A) and pindolol (B) at representative doses.

Data were generated by placing ROIs on different brain structures on the PET images illustrated in Figure 3. Binding was estimated as the difference in radiosignals between target and cerebellar regions, and expressed as the percentage of the injected dose per unit tissue volume (%dose/mL). Bars indicate S.E. (n = 4 and 3 in Wf-516 and pindolol treatment groups, respectively).

Figure 6. Relationships between dose or plasma concentration of test drugs and 5-HT_1A receptor occupancies analyzed with [¹¹C]WAY-100635-PET data.

(A) Receptor occupancies in the hippocampus (closed circles) and raphe nucleus (open circles) plotted against oral dose of Wf-516. Regression curves were generated by the following equation: Occ  = Occ_max×D/(D+ED₅₀), where Occ, Occ_max, D and ED₅₀ are 5-HT_1A receptor occupancy, maximal occupancy, dose of Wf-516, and dose of Wf-516 required for 50% of maximal occupancy, respectively. Bars indicate S.E. (n = 4). (B) Receptor occupancies in the hippocampus (closed squares) and raphe nucleus (open squares) plotted against intraperitoneal dose of pindolol. Regression curves were generated by the following equation: Occ  = 100×D/(D+ED₅₀). Bars indicate S.E. (n = 3). (C) Receptor occupancies in the hippocampus (closed squares) and raphe nucleus (open squares) plotted against plasma concentration of pindolol. Regression curves were generated by the following equation: Occ  = 100×C/(C+EC₅₀), where C is plasma concentration of pindolol. Dashed and solid lines represent regressions in the hippocampus and raphe nucleus, respectively.

Figure 7. Alteration of BP_ND for [¹¹C]WAY-100635 in rats treated with a toxicant for 5-HT neurons, 5,7-DHT.

Four rats each underwent three [¹¹C]WAY-100635-PET scans, at baseline, after 5,7-DHT treatment, and after oral administration of 30 mg/kg Wf-516 (5,7-DHT + Wf-516), in the indicated chronological order. Each symbol represents individual BP_ND in the hippocampus (left) and raphe nucleus (right). These changes of BP_ND in each region were statistically examined by one-way repeated-measures ANOVA followed by least significant difference test. *p<0.05 compared with each baseline.

Figure 8. Effects of Wf-516 and pindolol on autoradiographic [³⁵S]GTPγS binding in rat brains.

(A) Representative autoradiograms showing radiolabeling with [³⁵S]GTPγS in the hippocampus and raphe nucleus at baseline (control) or in the presence of 1 µM 8-OH-DPAT (full agonist for 5-HT_1A receptors), 10 µM Wf-516, 10 µM pindolol and 1 µM WAY-100635 (full antagonist for 5-HT_1A receptors). (B) Ratio of [³⁵S]GTPgγS binding to the control level in the hippocampus (left) and raphe nucleus (right). Changes in radiotracer binding were statistically examined using one-way repeated-measures ANOVA followed by least significant difference test. *p<0.01 compared with 1 µM WAY-100635. In the raphe nucleus, a significant interaction between [³⁵S]GTPγS binding and three concentrations each of Wf-516, pindolol and WAY-100635 was demonstrated by two-way repeated-measures ANOVA (p<0.05, F(4,20) = 3.58).