T Liu et al/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 14 days. Scop-treated mice displayed significantly longer mean latency from 83. 27 s to 63.76 s over training days(p<0.01), indicating that cognitive impairment had been induced by Scop. Subcutaneous RP279 administration of Bis-Mep to Scop-treated mice resulted in signifi cant reduction of mean latencies at doses ranging from 10 ng/kg to 1 ug/kg with no obvious cholinergic adverse responses. The maximal reduction in mean latency was reached at the 100 ng/kg, and the per formance of the mice approached nearly that of the control mice (p>0.05), indicating the complete reversal of Scop-induced cognitive deficits In the probe trial, the percentage of the total distance and spent in the target quadrant was used to evaluate the spatial reten- tion One-way ANOVA analysis showed that the percentage of dis- tance and time was significantly different among the experimental groups(F(5)=7.755.p<0001,Fg5 b and f(1.5)=7465,p<0001 Fig 5c). Compared with the control group, Scop caused 52.5% and HS440 48.7% decrease in the percentage of the total distance and time, re- spectively. Bis-Mep at the dose of 100 ng/kg significantly ameliorated the cognitive deficits as shown by increased percentage of distance (55.6%)and time( 48. 2%)compared to Scop-treated mice As expected, Hup A, a positive control, significantly reversed cognitive impairment by shortening latencies(p<0.05)and increasing the percentage of the total distance(60.6%)and time(48.6%)(p<0.05) compared to Scop-treated mice Furthermore, the cognitive amelioration was obvi- ously observed in the swimming traces on the 5th day after the admin- tration of Bis-Mep( Fig 5d). Taken together, these results demonstrate that Bis-Mep ameliorates Scop-induced cognitive deficits in mice. 3.5. Bis-Mep inhibits Ache activity in mice hippocampus Compared with the control mice, Scop exposure had no obvious effect on AChE activity in hippocampus (p>0.05, Table 1). However, AChE activity in hippocampus was significantly inhibited by 22.0% (p<0.05) and 230%(p<0.05)after the administration of Bis-Mep (100 ng/kg) and Hup A(1 mg/kg), respectively. These data suggested that Bis-Mep would be capable of crossing the blood brain barrier BB)and inhibiting AChE activity in brain. 4. Discussion The neuropathology in AD patients is characterized by a progres ive loss of memory and cognitive abilities(Walsh and Selkoe, 2004). AChEls which can simultaneously bind to the CAs and pas have been shown to be an effective therapeutic approach for mild to moderate Ad by enhancing cholinergic activity as well as preventing .TRP 279 ChE induced aB aggregation( Castro and Martinez, 2006). Therefore, dual-binding AChEls becomes one of the most widely adopted ap- proaches to hit multiple AD biological targets for AD therapy Another challenge for AD therapy, similar to diabetes and psychi- atric illnesses, is treatment compliance. It is known that the non- compliant behavior has been often observed in older AD patients due to the misunderstanding of complicated multiple medication or the confusion with the dosing regimen or forgetfulness. New means of drug administration should be developed and aimed to solve this difficulty. For example, the rivastigmine patch has been approved for Fig. 4. Water bridges"in the dual-binding to AChE. Bis-Mep was docked into AChE at 10E. Bis-Mep was shown dual-site binding to AChE at CAS, the catalytic triad of which of Bis-Mep to stabilize the binding to CAS and PAS, where Hydrogen bond (green dotted arrow )and n-n interaction(green dotted line) were also observed(b). The lual-binding of Bis-Mep to AchE induced the conformational changes of key residu in CAS and PAS (Green residues: before docking: Red residues: after docking), wheredays. Scop-treated mice displayed significantly longer mean latency from 83.27 s to 63.76 s over training days (pb0.01), indicating that cognitive impairment had been induced by Scop. Subcutaneous administration of Bis-Mep to Scop-treated mice resulted in signifi- cant reduction of mean latencies at doses ranging from 10 ng/kg to 1 μg/kg with no obvious cholinergic adverse responses. The maximal reduction in mean latency was reached at the 100 ng/kg, and the per￾formance of the mice approached nearly that of the control mice (p> 0.05), indicating the complete reversal of Scop-induced cognitive deficits. In the probe trial, the percentage of the total distance and time spent in the target quadrant was used to evaluate the spatial reten￾tion. One-way ANOVA analysis showed that the percentage of dis￾tance and time was significantly different among the experimental groups (F(71,5)= 7.755, pb0.001, Fig. 5b and F(71,5)= 7.465, pb0.001, Fig. 5c). Compared with the control group, Scop caused 52.5% and 48.7% decrease in the percentage of the total distance and time, re￾spectively. Bis-Mep at the dose of 100 ng/kg significantly ameliorated the cognitive deficits as shown by increased percentage of distance (55.6%) and time (48.2%) compared to Scop-treated mice. As expected, Hup A, a positive control, significantly reversed cognitive impairment by shortening latencies (pb0.05) and increasing the percentage of the total distance (60.6%) and time (48.6%) (pb0.05) compared to Scop-treated mice. Furthermore, the cognitive amelioration was obvi￾ously observed in the swimming traces on the 5th day after the admin￾istration of Bis-Mep (Fig. 5d). Taken together, these results demonstrate that Bis-Mep ameliorates Scop-induced cognitive deficits in mice. 3.5. Bis-Mep inhibits AChE activity in mice hippocampus Compared with the control mice, Scop exposure had no obvious effect on AChE activity in hippocampus (p>0.05, Table 1). However, AChE activity in hippocampus was significantly inhibited by 22.0% (pb0.05) and 23.0% (pb0.05) after the administration of Bis-Mep (100 ng/kg) and Hup A (1 mg/kg), respectively. These data suggested that Bis-Mep would be capable of crossing the blood brain barrier (BBB) and inhibiting AChE activity in brain. 4. Discussion The neuropathology in AD patients is characterized by a progres￾sive loss of memory and cognitive abilities (Walsh and Selkoe, 2004). AChEIs which can simultaneously bind to the CAS and PAS have been shown to be an effective therapeutic approach for mild to moderate AD by enhancing cholinergic activity as well as preventing AChE induced Aβ aggregation (Castro and Martinez, 2006). Therefore, dual-binding AChEIs becomes one of the most widely adopted ap￾proaches to hit multiple AD biological targets for AD therapy. Another challenge for AD therapy, similar to diabetes and psychi￾atric illnesses, is treatment compliance. It is known that the non￾compliant behavior has been often observed in older AD patients due to the misunderstanding of complicated multiple medication or the confusion with the dosing regimen or forgetfulness. New means of drug administration should be developed and aimed to solve this difficulty. For example, the rivastigmine patch has been approved for Fig. 4. “Water bridges” in the dual-binding to AChE. Bis-Mep was docked into AChE at MOE. Bis-Mep was shown dual-site binding to AChE at CAS, the catalytic triad of which is formed by Ser200, Glu327 and His440; and PAS, the conserved residue of which is Trp279 (a). “Water bridges” (brown dotted line) were revealed in two wings of Bis-Mep to stabilize the binding to CAS and PAS, where Hydrogen bond (green dotted arrow) and π–π interaction (green dotted line) were also observed (b). The dual-binding of Bis-Mep to AChE induced the conformational changes of key residues in CAS and PAS (Green residues: before docking; Red residues: after docking), where the hydrogen bond between Ser200 and His440 (dotted line) was disrupted (c). T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143 141