T-495, a novel low cooperative M1 receptor positive allosteric modulator, improves memory deficits associated with cholinergic dysfunction and is characterized by low gastrointestinal side effect risk

Abstract

M₁ muscarinic acetylcholine receptor (M₁ R) activation can be a new therapeutic approach for the treatment of cognitive deficits associated with cholinergic hypofunction. However, M₁ R activation causes gastrointestinal (GI) side effects in animals. We previously found that an M₁ R positive allosteric modulator (PAM) with lower cooperativity (α-value) has a limited impact on ileum contraction and can produce a wider margin between cognitive improvement and GI side effects. In fact, TAK-071, a novel M₁ R PAM with low cooperativity (α-value of 199), improved scopolamine-induced cognitive deficits with a wider margin against GI side effects than a high cooperative M₁ R PAM, T-662 (α-value of 1786), in rats. Here, we describe the pharmacological characteristics of a novel low cooperative M₁ R PAM T-495 (α-value of 170), using the clinically tested higher cooperative M₁ R PAM MK-7622 (α-value of 511) as a control. In rats, T-495 caused diarrhea at a 100-fold higher dose than that required for the improvement of scopolamine-induced memory deficits. Contrastingly, MK-7622 showed memory improvement and induction of diarrhea at an equal dose. Combination of T-495, but not of MK-7622, and donepezil at each sub-effective dose improved scopolamine-induced memory deficits. Additionally, in mice with reduced acetylcholine levels in the forebrain via overexpression of A53T α-synuclein (ie, a mouse model of dementia with Lewy bodies and Parkinson's disease with dementia), T-495, like donepezil, reversed the memory deficits in the contextual fear conditioning test and Y-maze task. Thus, low cooperative M₁ R PAMs are promising agents for the treatment of memory deficits associated with cholinergic dysfunction.

Keywords: M1 muscarinic acetylcholine receptor; cooperativity; positive allosteric modulator; α-synuclein.

Figure 1

T‐495 and MK‐7622 selectively potentiate M₁R with low and high cooperativity, respectively. (A and B) Chemical structures of T‐495 (A) and MK‐7622 (B). (C and D) Potentiation of ACh‐mediated Ca²⁺ mobilization by T‐495 (C) or MK‐7622 (D) in CHO‐K1 cells expressing human M₁R‐M₅R. The response to an EC₂₀ concentration and 10 μmol/L of ACh was set as the 0% and 100% response, respectively. Data are presented as the mean ± SD (n = 3). (E and F) Effect of T‐495 (E) or MK‐7622 (F) on Ca²⁺ mobilization in the absence of ACh in CHO‐K1 cells expressing human M₁R. The response to solvent and 10 μmol/L of ACh was set as the 0% and 100% response, respectively. Data are presented as the mean ± SD (n = 4). (G and H) Effect of T‐495 (G) or MK‐7622 (H) on [³H]‐pirenzepine binding in cell membranes from human M₁R‐expressing cells (n = 2). Nonspecific binding was defined in the presence of 10 μmol/L atropine

Figure 2

T‐495 and MK‐7622 increase IP1 production mediated by M₁R activation in the rodent brain. (A and B) Effects of T‐495 (A) or MK‐7622 (B) on IP1 production in the rat hippocampus. One hour after oral administration of vehicle (Veh) or a test compound (T‐495:3, 10, and 30 mg/kg; MK‐7622:1, 3, and 10 mg/kg), animals were injected with LiCl (10 mmol/kg, s.c.). One hour after the LiCl injection, animals were sacrificed to collect the hippocampus. Concentrations of T‐495 and MK‐7622 in rat plasma and hippocampus are presented in Tables S2 and S3, respectively. Data are presented as the mean + SEM (n = 6). (C and D) Effects of T‐495 (C) or MK‐7622 (D) on IP1 production in the mouse hippocampus. One hour after oral administration of vehicle or a test compound (3, 10, and 30 mg/kg), animals were injected with LiCl (10 mmol/kg, s.c.). One hour after LiCl injection, animals were sacrificed to collect the hippocampus. Data are presented as the mean + SEM (n = 6). (E and F) Effects of T‐495 (E) or MK‐7622 (F) on IP1 production in the hippocampus, prefrontal cortex, striatum, and brainstem of wild‐type and M₁R KO mice. One hour after oral administration of vehicle or a test compound (T‐495:10 mg/kg; MK‐7622 (MK): 20 mg/kg), mice were injected with LiCl (10 mmol/kg, s.c.). One hour after the LiCl injection, brain tissues were collected. Concentrations of T‐495 and MK‐7622 in the plasma and hippocampus of wild‐type and M₁R KO mice are shown in Table S4. Data are presented as the mean + SEM (n = 10). (G and H) Effects of repeated treatment with T‐495 (G) or MK‐7622 (H) for 13 days on IP1 production in the mouse hippocampus. Vehicle or a test compound (10 mg/kg, p.o.) was administered to mice once daily for 13 days. On the 14th day, 1 hour after the administration of vehicle or a test compound (10 and 30 mg/kg, p.o.), mice were injected with LiCl (10 mmol/kg, s.c.). One hour after the LiCl injection, mice were sacrificed to collect the hippocampus. Basal IP1 levels in the mouse hippocampus after repeated treatment with vehicle or a test compound for 13 days are shown in Tables S5 (T‐495) and S6 (MK‐7622). Concentrations of T‐495 and MK‐7622 in the plasma and hippocampus of mice pretreated with vehicle or a test compound are shown in Tables S7 and S8, respectively. Data are presented as the mean + SEM (n = 10). ^# P ≤ .05 vs vehicle‐treated group by two‐tailed Shirley‐Williams’ test. **P ≤ .01 vs vehicle‐treated group by Student's t‐test or Aspin‐Welch t‐test

Figure 3

T‐495 and MK‐7622 improve scopolamine‐induced memory impairment in a rat NOR test. A test compound (T‐495 (A): 0.3, 1, and 3 mg/kg, p.o.; MK‐7622 (B): 1, 3, and 10 mg/kg, p.o.) and scopolamine (0.1 mg/kg, s.c.) were administered to rats 1 hour and 30 minutes prior to the acquisition trial, respectively. Data are presented as the mean + SEM (n = 6‐8). **P ≤ .01 vs vehicle‐vehicle‒treated group by Student's t‐test. ^# P ≤ .05 vs vehicle‐scopolamine‒treated group by two‐tailed Williams’ test

Figure 4

Effects of T‐495 and MK‐7622 on spontaneous ileum contraction and diarrhea score. (A) Effects of T‐495 and MK‐7622 on spontaneous ileum contraction. The mean amplitude of spontaneous contractions at each concentration was normalized to that at pretreatment. Data are presented as the mean ± SEM (n = 7‐10). (B and C) Diarrhea score. T‐495 (B; 1, 3, 10, 30, and 100 mg/kg, p.o.) or MK‐7622 (C; 3, 10, and 30 mg/kg, p.o.) was administered to rats, and the severity of diarrhea was scored. The highest score during the observation period was used for analysis. Data are presented as the mean + SEM (n = 6). ^# P ≤ .05 vs vehicle‐treated group by two‐tailed Williams’ test

Figure 5

Effects of T‐495 or MK‐7622 in combination with donepezil on scopolamine‐induced memory impairment in a rat NOR test. A test compound (T‐495 at 0.3 mg/kg (A) or MK‐7622 at 1 mg/kg (B)), donepezil (0.1 mg/kg, p.o.), and scopolamine (0.1 mg/kg, s.c.) were administered 1, 0.5, and 0.5 hours, respectively, prior to the acquisition trial. Data are presented as the mean + SEM (n = 7‐8). **P ≤ .01 vs vehicle‐vehicle‐vehicle‒treated group by Student's t‐test. ^# P ≤ .05 vs vehicle‐vehicle‐scopolamine‒treated group by Dunnett's test

Figure 6

ACh content and synaptic protein and M₁R levels in the frontal cortex and hippocampus of the CaMKIIα‐tTA/A53T α‐syn dTg mice. The frontal cortex and hippocampus were dissected from 12‐month‐old mice. (A) The ACh content in the tissues was measured by LC‐MS/MS. Data are presented as the mean + SEM (n = 21‐24), and statistical significance of the differences between the CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T α‐syn dTg mice was determined using Student's t‐test (**P ≤ .01). (B and C) Presynaptic and postsynaptic proteins in the frontal cortical (B) and hippocampal (C) lysates from the CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T α‐syn dTg mice were determined by an automated capillary western blot system. Synaptic protein levels were normalized to the levels of GAPDH, which was a loading control. Results are expressed as percentages of the values obtained from age‐matched CaMKIIα‐tTA sTg mice and are presented as the mean + SEM (n = 10). The statistical significance of the difference between the CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T α‐syn dTg mice was determined using Student's t‐test or Aspin‐Welch test (**P ≤ .01). Representative images of capillary western blot are shown below the quantified results in panels B and C

Figure 7

Effects of donepezil on the memory deficits in the CaMKIIα‐tTA/A53T α‐syn dTg mice. (A) The CFC test was performed to evaluate associative learning of 12‐month‐old CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T dTg mice. The percentage of freezing behavior was analyzed during the conditioning and retention phases. Vehicle or donepezil (1 mg/kg) was orally administered 2 hours prior to both the conditioning and retention phases. (B) The Y‐maze task was performed to evaluate spatial working memory of 12‐month‐old CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T dTg mice. The percentage of alternations was measured. Vehicle or donepezil (1 mg/kg) was orally administered 2 hours prior to the test. Data are presented as the mean + SEM (n = 10); statistical significance between the vehicle‐treated CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T α‐syn dTg mice was determined using Student's t‐test or Aspin‐Welch test (*P ≤ .05; **P ≤ .01). Significant differences between vehicle‐ and donepezil‐treated CaMKIIα‐tTA/A53T α‐syn dTg mice were determined by Student's t‐test or Aspin‐Welch test (^# P ≤ .05)

Figure 8

Effects of T‐495 on the memory deficits in the CaMKIIα‐tTA/A53T α‐syn dTg mice. (A) The CFC test was performed at 12 months of age. The percentage of freezing behavior was analyzed during the conditioning and retention phases. Vehicle or T‐495 (3 mg/kg) was administered orally 1 hour prior to both the conditioning and retention phases. (B) The Y‐maze task was performed at 12 months of age. The percentage of alternations was measured. Vehicle or T‐495 (3 mg/kg) was orally administered 1 hour prior to the test. Data are presented as the mean + SEM (n = 10‐15), and statistical significance between the vehicle‐treated CaMKIIα‐tTA sTg and CaMKIIα‐tTA/A53T α‐syn dTg mice was determined using Student's t‐test or Aspin‐Welch test (*P ≤ .05; **P ≤ .01). Significant differences between vehicle‐ and T‐495‐treated CaMKIIα‐tTA/A53T α‐syn dTg mice were determined by Student's t‐test or Aspin‐Welch test (^# P ≤ .05)