The Dual-Active Histamine H3 Receptor Antagonist and Acetylcholine Esterase Inhibitor E100 Alleviates Autistic-Like Behaviors and Oxidative Stress in Valproic Acid Induced Autism in Mice

Abstract

The histamine H3 receptor (H3R) functions as auto- and hetero-receptors, regulating the release of brain histamine (HA) and acetylcholine (ACh), respectively. The enzyme acetylcholine esterase (AChE) is involved in the metabolism of brain ACh. Both brain HA and ACh are implicated in several cognitive disorders like Alzheimer's disease, schizophrenia, anxiety, and narcolepsy, all of which are comorbid with autistic spectrum disorder (ASD). Therefore, the novel dual-active ligand E100 with high H3R antagonist affinity (hH3R: K_i = 203 nM) and balanced AChE inhibitory effect (EeAChE: IC₅₀ = 2 µM and EqBuChE: IC₅₀ = 2 µM) was investigated on autistic-like sociability, repetitive/compulsive behaviour, anxiety, and oxidative stress in male C57BL/6 mice model of ASD induced by prenatal exposure to valproic acid (VPA, 500 mg/kg, intraperitoneal (i.p.)). Subchronic systemic administration with E100 (5, 10, and 15 mg/kg, i.p.) significantly and dose-dependently attenuated sociability deficits of autistic (VPA) mice in three-chamber behaviour (TCB) test (all p < 0.05). Moreover, E100 significantly improved repetitive and compulsive behaviors by reducing the increased percentage of marbles buried in marble-burying behaviour (MBB) (all p < 0.05). Furthermore, pre-treatment with E100 (10 and 15 mg/kg, i.p.) corrected decreased anxiety levels (p < 0.05), however, failed to restore hyperactivity observed in elevated plus maze (EPM) test. In addition, E100 (10 mg/kg, i.p.) mitigated oxidative stress status by increasing the levels of decreased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), and decreasing the elevated levels of malondialdehyde (MDA) in the cerebellar tissues (all p < 0.05). Additionally, E100 (10 mg/kg, i.p.) significantly reduced the elevated levels of AChE activity in VPA mice (p < 0.05). These results demonstrate the promising effects of E100 on in-vivo VPA-induced ASD-like features in mice, and provide evidence that a potent dual-active H3R antagonist and AChE inhibitor (AChEI) is a potential drug candidate for future therapeutic management of autistic-like behaviours.

Keywords: E100; VPA-induced autism-like behaviors; acetylcholine esterase inhibitor; antagonist; anxiety histamine H3R; cerebellum; mice; oxidative stress; repetitive behaviors; sociability.

Figure 1

Chemical structure of the dual-acting human H3R (hH3R) antagonist and AChE inhibitor E100 and in vitro data with regard to hH1-, hH3-, and hH4R, EeAChE, and EqBuChE. ^a,b,c Binding assays to determine affinity to H1-, H3-, and H4Rs were performed in differently expressed cells as previously described n = 3 [59]. ^d AChE: Acetylcholine esterase; Ee; electric eel; ^e BuChE: Butyrylcholinesterase; Eq: equine [63,64,65].

Figure 2

E100 improved sociability in three-chamber behaviour (TCB) and repetitive behavior in marble-burying behaviour (MBB) paradigms. (A,B) Following acclimatization for a duration of 10 min, male mice were allowed to explore all three chambers for 10 min. The obtained results were expressed in form of Sociability index (SI). Control (CNT) mice received systemic injections of saline (group 1), E100 (10 mg/kg) (group 7), and donepezil (DOZ) (1 mg/kg) (group 8), whereas VPA mice were injected with saline (group 2), E100 (5, 10, and 15 mg/kg) (groups 3–5), or DOZ (1 mg/kg, i.p.) (group 6) subchronically for 21 days (A). Abrogative studies of subchronic (21 days) systemic co-injection of RAM (10 mg/kg, i.p. for group 9), mepyramine (MPA) (10 mg/kg, i.p. group 10), zolantidine (ZLT) (10 mg/kg, i.p., for group 11), or scopolamine (SCO) (0.3 mg/kg, i.p., for group 12) on the E100 (10 mg)-provided improvement of sociability of VPA mice were assessed (B). Marble-burying behavior (MBB) was measured after a 30-min testing session applying the same treatments. VPA mice treated with saline (group 2) displayed significantly increased repetitive behaviors when compared to CNT mice (group 1). E100 (5, 10, or 15 mg/kg, i.p) or DOZ (1 mg/kg, i.p.) were injected systemically and subchronically for 21 days in VPA mice (C). Effects of subchronic (21 days) systemic co-administration of RAM (10 mg/kg, i.p., group 9), MPA (10 mg/kg, i.p., group 10), ZLT (10 mg/kg, i.p., group 11), or SCO (0.3 mg/kg, i.p., group 12) on the E100(10 mg)-provided attenuation of stereotyped repetitive behavior of VPA mice were assessed MBB (D). CNT mice were injected with saline, E100 (10 mg/kg, i.p.), DOZ (1 mg/kg, i.p.). RAM (10 mg/kg, i.p.), MPA (10 mg/kg, i.p.), ZLT (10 mg/kg, i.p.), or SCO (0.3 mg/kg, i.p.) (D). Data are expressed as the mean ± standard errors of the means (SEM) (n = 7 for TCB and n = 5 for MBB). 8 groups of 7 mice per group in TCB (A,B) and 8 groups of 5 mice per group in MBB (C,D) were used. The effects of E100 were analyzed using two-way analysis of variance (ANOVA) with dose of drugs and animals (either VPA or CNT mice) as the between-subjects factor, and post hoc comparisons were performed with Tukey’s test in case of a significant main effect. ^# p < 0.05 vs. CNT mice. ^** p < 0.01 vs. saline-treated VPA mice. ^$ p < 0.05 vs. E100 (10mg)-treated VPA mice.

Figure 3

E100 ameliorated fear-related behavior without affecting locomotor activity in elevated plus maze (EPM). VPA mice injected with saline (group 2) displayed significantly increased deficits in cognitive behaviors compared to CNT mice (group 1). Test compound E100 (5, 10, or 15 mg/kg, i.p.) or DOZ (1 mg/kg) were injected for 21 days to VPA mice (subchronic). E100 (10 and 15 mg/kg, groups 4 and 5) and DOZ (1 mg/kg, group 6) attenuated the decreased time spent on the open arms, however, failed to modify the increased number of entries into the closed arms of the EPM (A–C). Abrogative effects of subchronic (21 days) systemic co-administration of RAM (10 mg/kg, group 9), MPA (10 mg/kg, group 10), ZLT (10 mg/kg, group 11), or SCO (0.3 mg/kg, group 12) on the E100 (10 mg)-provided improvement in number and time spent for open arms of VPA mice were measured (D,E). Number of entries into closed arms was elevated in saline-treated VPA mice (group 2) when compared to saline-treated CNT mice (group 1). E100 (5, 10, and 15 mg/kg) and DOZ failed to modulate the increased number of entries into closed arms (C,F). Additionally, CNT mice treated with E100 (10 mg/kg, group 7) did not show significant difference in number of entries into closed arms when compared with saline-treated CNT mice (group 1). Data are expressed as the mean ± SEM (n = 6). ^# p < 0.05 vs. CNT mice. * p < 0.05 vs. Saline-treated VPA mice. ** p < 0.01 vs. saline-treated VPA mice. ^$ p < 0.05 vs. E100 (10mg)-treated VPA mice. In the EPM test, 8 groups of 6 mice per group were used. The effects of E100 were analyzed using two-way analysis of variance (ANOVA) with dose of drugs and animals (either VPA or CNT mice) as the between-subjects factor, and post hoc comparisons were performed with Tukey’s test in case of a significant main effect (A–D).

Figure 4

E100 restored levels of oxidative stress markers in the cerebellum. Modulated malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) were assessed. VPA mice showed a significant increase in MDA (A) and significant decrease in GSH (B), SOD (C), and CAT (D) compared to CNT mice. Subchronic systemic administration of E100 (10 mg/kg) or DOZ (1 mg/kg) were assessed in VPA mice. E100 (10 mg/kg) or DOZ (1 mg/kg) significantly reduced the increased levels of MDA (A) and significantly increased the reduced levels of GSH, SOD and CAT (A–D). Abrogative effects of subchronic (21 days) systemic co-administration with RAM (10 mg/kg) on modulation of oxidative stress levels provided by E100 (10 mg) were assessed (A–D). Data are expressed as the mean ± SEM (n = 5). ^# p < 0.05 vs. VPA mice. * p < 0.05 vs. VPA mice. ** p < 0.01 vs. VPA mice. *** p < 0.001 vs. VPA mice. ^$ p < 0.01 vs. E100 (10 mg)-treated VPA mice. In biochemical assessments, 5 groups of 5 mice per group were used. The effects of E100 were analyzed using two-way analysis of variance (ANOVA) with dose of drugs and animals (either VPA or CNT mice) as the between-subjects factor, and post hoc comparisons were performed with Tukey’s test in case of a significant main effect (A–D).

Figure 5

Effects of E100 on acetylcholine esterase activity in cerebellum tissues of valproic acid (VPA)-exposed mice. Inhibitory effects of E100 (10 mg/kg, i.p.) on acetylcholine esterase enzyme in the cerebellum of VPA mice. Quantitative analysis revealed a significant increase (^# p < 0.05) in the acetylcholine esterase enzyme activity in cerebellum of VPA mice compared to the CNT mice. However, subchronic treatment with E100 (10 mg/kg, i.p.) or DOZ (1 mg/kg, i.p.) to the VPA mice significantly reduced (* p < 0.05) this activity compared to the VPA mice. Values are expressed as the percent mean ± SEM. For assessment of AChE activity 4 groups were used. 4 CNT mice were used for saline group and 5 VPA mice were used for each treatment group. The effects of E100 were analyzed using two-way analysis of variance (ANOVA) with dose of drugs and animals (either VPA or CNT mice) as the between-subjects factor, and post hoc comparisons were performed with Tukey’s test in case of a significant main effect.

Figure 6

Schematic illustration of systemic treatments, behavioral experiments, and biochemical measurements with VPA and CNT mice. At embryonic day 12.5 (E12.5), pregnant mice were administered intraperitonially with VPA (500 mg/kg) or Saline. After delivery of pups and starting from postnatal day (P44), treatments of VPA mice and CNT mice were carried out. The systemic administrations continued for 21 days until VPA and CNT mice reached P64. Starting from P51, behavioral assessments were conducted. Following behavioral assessments, all mice were sacrificed at P64 for biochemical and immunofluorescence analyses. VPA and CNT treatment groups (8–12 mice/group) were subdivided into 16 subgroups and received intraperitoneally (i.p.) the following treatments and as shown in experimental design: VPA offspring (mice with autistic features; VPA) Group 1: CNT mice injected with saline, group 2: VPA mice injected with saline, group 3: VPA mice injected with E100 (5 mg/kg, i.p.), group 4: VPA mice injected with E100 (10 mg/kg, i.p.), group 5: VPA mice injected with E100 (15 mg/kg, i.p.), group 6: VPA mice injected with DOZ (1 mg/kg, i.p.), group 7: CNT mice injected with E100 (10 mg/kg, i.p.), group 8: CNT mice injected with DOZ (1 mg/kg, i.p.), group 9: E100 (10 mg/kg, i.p.) was co-administered with RAM (10 mg/kg, i.p.), group 10: E100 (10 mg/kg, i.p.) was co-administered with MPA (10 mg/kg, i.p.), group 11: E100 (10 mg/kg, i.p.) was co-administered with ZLT (10 mg/kg, i.p.), group 12: E100 (10 mg/kg, i.p.) was co-administered with SCO (0.3 mg/kg, i.p.), group 13: CNT mice injected with RAM (10 mg/kg), group 14: CNT mice injected with MPA (10 mg/kg, i.p.), group 15: CNT mice injected with ZLT (10 mg/kg, i.p.), and group 16: CNT mice injected with SCO (0.3 mg/kg, i.p.). All co-administrations were carried out in separate injections with 5-min interval following the test compound (E100) administration. CNT; control mice delivered from saline-exposed mice. VPA; autistic mice delivered from VPA-exposed mice.

Figure 7

Putative mode of action of E100 by blocking the acting auto- and hetero histamine H3 receptors (H3Rs) and inhibition of the acetylcholine esterase enzyme (AChE). Regulating the release of brain histamine (HA) and acetylcholine (ACh), respectively and inhibiting the metabolism of ACh.