The High Affinity Dopamine D2 Receptor Agonist MCL-536: A New Tool for Studying Dopaminergic Contribution to Neurological Disorders

Abstract

The dopamine D₂ receptor exists in two different states, D₂^high and D_2low; the former is the functional form of the D₂ receptor and associates with intracellular G-proteins. The D₂ agonist [³H]MCL-536 has high affinity for the D₂ receptor (K_d 0.8 nM) and potently displaces the binding of (R-(-)-N-n-propylnorapomorphine (NPA; K_i 0.16 nM) and raclopride (K_i 0.9 nM) in competition binding assays. Here, we further characterize [³H]MCL-536. [³H]MCL-536 was metabolically stable, with about 75% of the compound remaining intact after 1 h incubation with human liver microsomes. Blood-brain barrier penetration in rats was good, attaining at 15 min a % injected dose per gram of wet tissue (%ID/g) of 0.28 in males versus 0.42 in females in the striatum. Specific uptake ratios ([%ID/g striatum]/[%ID/g cerebellum]) were stable in males during the first 60 min and in females up to 15-30 min. The D₂-rich striatum exhibited the highest uptake and slowest washout compared to D₂-poor cortex or cerebellum. In peripheral organs, uptake peaked at 15 min but declined to baseline at 60 min, indicating good clearance from the body. In vitro autoradiography on transaxial and coronal brain sections showed specific binding of [³H]MCL-536, which was abolished by preincubation with D₂/D₃ ligands sulpiride, NPA, and raclopride and in the presence of the stable GTP analogue guanylylimidodiphosphate. In amphetamine-sensitized animals, striatal binding was higher than in controls, indicating specificity for the D₂^high receptor state. [³H]MCL-536's unique properties make it a valuable tool for research on neurological disorders involving the dopaminergic system like Parkinson's disease or schizophrenia.

Keywords: Parkinson’s disease; aporphine; dopamine D2high receptor; schizophrenia; tritiated radioligand.

Figure 1.

Human liver microsome stability assay of MCL-536. Briefly, MCL-536 was preincubated with pooled human microsomes in 100 mM phosphate buffer; then NAPDH (final concentration 1 mM) was added. After 30 or 60 min incubation, the reaction was stopped, and samples were analyzed with a Varian Prostar HPLC system on Agilent Microsorb-MV 100 C18 columns fitted with a Microsorb 100-5 C18 MetaGuard column and a detector set at 265 nm. Dextromethorphan (DXM) was used as positive control (detection at 278 nm); negative controls included DXM incubated with heat-inactivated microsomes (HDXM) and one incubation mix without NADPH. The experiment was performed in triplicate. (A) Structure of MCL 536. (B) Microsome stability assay.

Figure 2.

Biodistribution of [³H]MCL-536 in the rat brain. A total amount of 6 μCi of [³H]MCL-536 was injected into the tail vein of male or female Sprague–Dawley rats, and 15, 30, 60, and 240 min after injection, brain regions were dissected, and the tissue was weighed and dissolved in 1 mL of 0.8 N NaOH. After addition of 5 mL of scintillant, samples were counted in a β-counter and the %ID/g wet tissue was calculated. Data are presented as mean ± SEM of 4 animals per time point and ligand. (A, B) Biodistribution of [³H]MCL-536 in the striatum and cerebellum of female and male rats. (C) Biodistribution of [³H]MCL-536 presented as the brain region/cerebellum ratio of female and male rats. Uptake in brain regions was calculated as the ratio of percent dose per gram of the region versus the cerebellum, where D₂ receptors are scarce.

Figure 3.

Biodistribution of [³H]MCL-536 in peripheral organs in male and female rats. [³H]MCL-536 (6 μCi/300 g body weight) was injected into the tail vein; 15, 30, and 60 min after injection, peripheral organs were dissected, and the tissue weighed and dissolved in 1 mL of 0.8 N NaOH. After addition of 5 mL of scintillant, samples were counted in a β-counter and the %ID/g wet tissue was calculated. Data are presented as mean ± SEM of 4 animals per time point and ligand. (A) Male rats. (B) Female rats.

Figure 4.

In vitro autoradiography showing the binding of [³H]MCL-536 in the rat brain. Transaxial brain sections were preincubated with buffer to eliminate nonspecific binding. Afterward, sections were incubated with [³H]MCL-536 alone or in combination with D₂ receptor antagonists or cold MCL-536 to show specificity. Dried sections were exposed to autoradiography film for visualization. (A) Schematic of a transaxial brain section; CPU, caudate putamen; Ctx, cortex; Cer, cerebellum. (B) Conversion of gray scale values to relative optical densities (ROD). (C) ³H microscales were coexposed on the same films as the experimental samples to allow quantification. (D) In vitro binding of radioligand to 10 μm thick rat brain transaxial slices in the presence or absence of 10 M sulpiride, 100 nM cold ligand, 200 μM Gpp(NH)p, or 100 nM raclopride. (E) Quantification of receptor density for D₂ (fmol/mg). (F) D₂ receptor density ratio in striatum versus cerebellum and cortex versus cerebellum in the presence or absence of other radioligands or cold MCL 536.

Figure 5.

In vitro and ex vivo autoradiography of [³H]MCL-536 binding on transverse brain sections of untreated and amphetamine-sensitized rats. Transverse brain sections of control or amphetamine-sensitized rats were preincubated with buffer to eliminate nonspecific binding. Afterward, sections were incubated with [³H]MCL-536 alone or in combination with D₂ receptor antagonists or cold MCL-536 to show specificity. Dried sections were exposed to autoradiography film for visualization. (A) Schematic of a transverse brain section at the level of the anterior commissure (CPU, caudate putamen; AcbC, nucleus accumbens, core; Acbsh, nucleus accumbens, shell) and in vitro binding of [³H]MCL-536 to transverse rat brain slices at the level of the nucleus accumbens in the absence or presence of D₂ receptor antagonists sulpiride and raclopride, cold ligand, or Gpp(NH)p. (B) Ex vivo binding of [³H]MCL-536 to transverse rat brain sections of amphetamine-sensitized rats in comparison with control rats. Note that panels A and B are separate experiments and that exposure times of the autoradiographs on the film differ between panels A and B for better visualization.