T Liu et aL/ Pharmacology, Biochemistry and Behavior 104(2013)138-143 Table Ex vivo inhibition of Bis-Mep on AchE activity. Bis-Mep(sc )inhibited AChE activity in hippocampus. AChE inhibitory activity in hippocampus was assayed by a modified Ellman's method Data are expressed as mean+ SD for 10 mice each group AChE activity (U/mg protein) Control Scop(1 mg/kg) Hup A (l mg/kg)+ Sco 0831±0205 p<0.05, one way ANOVA followed by Dunnett's multiple comparison test vs the (b) up to 85.6%. Bis-Mep reached peak plasma concentration rapidly after subcutaneous administration (Tmax=6.6 min), and the t1/2 of Bis-Mep was 18.6 h due to its extensive distribution and slow clearance (Ge et al. 2012). The results provided us advantageous in formation to develop Bis-Mep to be a subcutaneous implant for long time therapy. Therefore, in the current study, Bis-Mep was designed to be administrated by subcutaneous injection in mice, then its effects on the cognitive amelioration and ache activity in hippocampus were evaluated In the present study, the concentration-effect relationship for AChE inhibition and the kinetic features of Bis-Mep were firstly determined.(-)-Meptazinol which is split from race-meptazinol (c) hasshownsomeAcheinhibitoryactivity(shietal.,2005).com- pared with (-)-meptazinol, Bis-Mep was shifted leftward in the concentration-effect curve of ache inhibition the results indicated hat Bis-Mep enhanced its effectiveness greatly, possibly due to its binding to the pas which could promote the inhibition of AChE tivity(Du and Carlier, 2004). bino tt always shows mixed-type inhibition when the inhibitors can to either the free enzyme or the enzyme-substrate complex. As for AChE, those mixed-type inhibitors are in favor of the interac tion with CAS and PAS Donepezil is a typical mixed-type inhibitor with high affinity to AChE in the nanomolar range. Structural biolog ical studies has revealed that donepezil could bind simultaneously to the CAs and PAs (Kryger et al, 1999), indicating its potential benefits for severe Ad patients(Farlow et al, 2010: Tsuno, 2009). In this study, Lineweaver-Burk analysis showed that Bis-Mep inhibited AChE in a linear mixed fashion. These results indicate that Bis-Mep another mixed-type inhibitor that inhibits AChE either by binding on free AChE or on AChE-ASCh complex, which may explain its potent inhibition of AChE. According to others and our reports, the potency of Bis-Mep is higher than denepezil in inhibiting AChE-induced AB aggregation. Inhibition of denepezil-induced AB aggregation at 100 HM was 22%, and those of Bis-Mep was 90.8%(Bartolini et al, 2003: Xie et al., 2008). Compared with those of donepezil, Bis-Mep may exert preferential binding to PAS which is mainly responsible for AB aggregation. To explore the mode of dual-binding of Bis-Mep to AChe,we F performed molecular docking which revealed that the methylene groups of Bis-Mep played a key role in the mixed type inhibition Fig. 5. Bis-Mep ameliorated Scop-induced cognitive deficits in mice. (a)The effects of and its dual-binding to CAS and PAS on AChE. The distance between Bis-Mep(sc)on the latency to the platform during the 4 days of acquisition testing. CAS and PAS is about 14 A. Therefore, the length of the methylene (b)The effects of Bis-Mep(sc)on the distance in target quadrant. (c) The effects of groups should be long enough to cover the distance. The flexible Bis-Mep(sc)on the time spent in target quadrant(d) Swimming traces for ditterent methylene groups are beneficial for the dual site binding of Bis-Mep (10 ng/kg)+Scop: C: Bis-Mep(100 ng/kg)+Scop: D: Bis-Mep(1000 ng/g)+Scop: to AChE. Unfortunately, this flexibility also facilitates the transforma- E:Hup A(1 mg/kg)+Scop: and F: Scop(1 mg/kg)alone. Data were expressed as tion of linear conformation of the molecule to spherical conformation, mean+ SD for 12 mice in each group. p<0.05. p<0.01 vs. Scop-treated group. which could be stabilized by the intramolecular hydrogen bonds between two phenolic hydroxyl groups of the two(-)-meptazinol the long-term treatment of AD patients with severe dementia(Frolich, moieties To compare it with the linear conformation, large volume 2008). Similar to the patch, implants are advantageous in long-time of the spherical conformation makes it more difficult to dock into treatment since they not only avoid liver and gastrointestinal the binding pocket of AChE and then to interact with CAS and PAs. first-pass effects but also relieve the frequent taking of pills. The plas- especially the former one. Therefore, the inhibition against AChE ma pharmacokinetic study of Bis-Mep in rat after subcutaneous ad- would decrease, when the proportion of spherical conformation ministration showed that the absolute bioavailability of Bis-Mep was increases. For example, the inhibition of Bis(6)-(-)-meptazinol wathe long-term treatment of AD patients with severe dementia (Frölich, 2008). Similar to the patch, implants are advantageous in long-time treatment since they not only avoid liver and gastrointestinal first-pass effects but also relieve the frequent taking of pills. The plas￾ma pharmacokinetic study of Bis-Mep in rat after subcutaneous ad￾ministration showed that the absolute bioavailability of Bis-Mep was up to 85.6%. Bis-Mep reached peak plasma concentration rapidly after subcutaneous administration (Tmax= 6.6 min), and the T1/2 of Bis-Mep was 18.6 h due to its extensive distribution and slow clearance (Ge et al., 2012). The results provided us advantageous in￾formation to develop Bis-Mep to be a subcutaneous implant for long time therapy. Therefore, in the current study, Bis-Mep was designed to be administrated by subcutaneous injection in mice, then its effects on the cognitive amelioration and AChE activity in hippocampus were evaluated. In the present study, the concentration–effect relationship for AChE inhibition and the kinetic features of Bis-Mep were firstly determined. (−)-Meptazinol which is split from race-meptazinol has shown some AChE inhibitory activity (Shi et al., 2005). Com￾pared with (−)-meptazinol, Bis-Mep was shifted leftward in the concentration–effect curve of AChE inhibition. The results indicated that Bis-Mep enhanced its effectiveness greatly, possibly due to its binding to the PAS which could promote the inhibition of AChE ac￾tivity (Du and Carlier, 2004). It always shows mixed-type inhibition when the inhibitors can bind to either the free enzyme or the enzyme–substrate complex. As for AChE, those mixed-type inhibitors are in favor of the interac￾tion with CAS and PAS. Donepezil is a typical mixed-type inhibitor with high affinity to AChE in the nanomolar range. Structural biolog￾ical studies has revealed that donepezil could bind simultaneously to the CAS and PAS (Kryger et al., 1999), indicating its potential benefits for severe AD patients (Farlow et al., 2010; Tsuno, 2009). In this study, Lineweaver–Burk analysis showed that Bis-Mep inhibited AChE in a linear mixed fashion. These results indicate that Bis-Mep is another mixed-type inhibitor that inhibits AChE either by binding on free AChE or on AChE-ASCh complex, which may explain its potent inhibition of AChE. According to other's and our reports, the potency of Bis-Mep is higher than denepezil in inhibiting AChE-induced Aβ aggregation. Inhibition of denepezil-induced Aβ aggregation at 100 μM was 22%, and those of Bis-Mep was 90.8% (Bartolini et al., 2003; Xie et al., 2008). Compared with those of donepezil, Bis-Mep may exert preferential binding to PAS which is mainly responsible for Aβ aggregation. To explore the mode of dual-binding of Bis-Mep to AChE, we performed molecular docking which revealed that the methylene groups of Bis-Mep played a key role in the mixed type inhibition and its dual-binding to CAS and PAS on AChE. The distance between CAS and PAS is about 14 Å. Therefore, the length of the methylene groups should be long enough to cover the distance. The flexible methylene groups are beneficial for the dual site binding of Bis-Mep to AChE. Unfortunately, this flexibility also facilitates the transforma￾tion of linear conformation of the molecule to spherical conformation, which could be stabilized by the intramolecular hydrogen bonds between two phenolic hydroxyl groups of the two (–)-meptazinol moieties. To compare it with the linear conformation, large volume of the spherical conformation makes it more difficult to dock into the binding pocket of AChE and then to interact with CAS and PAS, especially the former one. Therefore, the inhibition against AChE would decrease, when the proportion of spherical conformation increases. For example, the inhibition of Bis(6)-(−)-meptazinol was Fig. 5. Bis-Mep ameliorated Scop-induced cognitive deficits in mice. (a) The effects of Bis-Mep (s.c.) on the latency to the platform during the 4 days of acquisition testing. (b) The effects of Bis-Mep (s.c.) on the distance in target quadrant. (c) The effects of Bis-Mep (s.c.) on the time spent in target quadrant. (d) Swimming traces for different groups were presented on the 5th day of Morris Water Maze test. A: Control; B: Bis-Mep (10 ng/kg)+Scop; C: Bis-Mep (100 ng/kg)+Scop; D: Bis-Mep (1000 ng/kg)+ Scop; E: Hup A (1 mg/kg)+ Scop; and F: Scop (1 mg/kg) alone. Data were expressed as mean± SD for 12 mice in each group. ⁎pb0.05, ⁎⁎pb0.01 vs. Scop-treated group. Table 1 Ex vivo inhibition of Bis-Mep on AChE activity. Bis-Mep (s.c.) inhibited AChE activity in hippocampus. AChE inhibitory activity in hippocampus was assayed by a modified Ellman's method. Data are expressed as mean± SD for 10 mice each group. Group AChE activity (U/mg protein) % Scop Control 0.996±0.151 92% Scop (1 mg/kg) 1.079±0.234 100% Bis-Mep (100 ng/kg)+Scop 0.842±0.203⁎ 78% Hup A (1 mg/kg)+ Scop 0.831±0.205⁎ 77% ⁎ pb0.05, one way ANOVA followed by Dunnett's multiple comparison test vs the Scop. 142 T. Liu et al. / Pharmacology, Biochemistry and Behavior 104 (2013) 138–143