Scopolamine hydrobromide

Scopolamine hydrobromide (Tocris), PHA-543613 hydrochloride (Tocris), and memantine hydrochloride (Tocris) were dissolved in physiological saline to create a final injection volume of 1 ml/kg. Scopolamine was injected intraperitoneally (i.p.) 10 min before experimental sessions, and PHA-543613 and memantine were injected subcutaneously (s.c.) 40 min before experiments (30 min before scopolamine administration). In treatments, where scopolamine, memantine and/or PHA-543613 were not administered, compounds were replaced with saline (vehicle, VEH) injected through the corresponding route of administration. In co-administration treatments, memantine and PHA-543613 were administered consecutively in two separate subcutaneous injections performed on the opposite sides of the body.

T‐495 (Figure 1A: 8‐chloro‐6‐((6‐chloropyridin‐3‐yl)methyl)‐3‐((1S,2S)‐2‐hydroxycyclopentyl)‐7‐methyl‐2,3‐dihydro‐4H‐1,3‐benzoxazin‐4‐one) and MK‐7622 (Figure 1B: 3‐((1S,2S)‐2‐hydroxycyclohexyl)‐6‐((6‐methylpyridin‐3‐yl)methyl)benzo[h]quinazolin‐4(3H)‐one) were synthesized by Takeda Pharmaceutical Company Limited. T‐495 was synthesized according to the procedure described in the patent (WO2016208775, example 24).34 Donepezil hydrochloride was synthesized by Megafine Pharma Limited. Scopolamine hydrobromide and lithium chloride (LiCl) were purchased from Tocris Bioscience and Wako Pure Chemical Industries Limited, respectively. For in vitro experiments, T‐495 and MK‐7622 were dissolved in dimethyl sulfoxide (final concentration: 0.3% for Ca²⁺ mobilization assay and [³H]‐pirenzepine binding assay and 0.1% for spontaneous ileum contraction assay). For in vivo experiments, T‐495 and MK‐7622 were suspended in 0.5% methylcellulose in distilled water and administered orally (p.o.). Donepezil hydrochloride was dissolved in distilled water and administered orally. Scopolamine hydrobromide and LiCl were dissolved in saline and injected subcutaneously (s.c.). All compounds were dosed in a volume of 10 mL/kg body weight in mice and 2 mL/kg body weight in rats.