| OBJECTIVE:During the last30years, studies have shown that PDE4inhibitors possess promising antidepressant activity and cognitive enhancement effects. However, there are no PDE4inhibitors have been approved as the antidepressants in clinic because of their devastating side effects including nausea and vomiting. According to the published data, we modified the molecular structure of PDE4inhibitors and synthesized a series of novel PDE4inhibitors successfully. Based on their enzyme activities, we selected two representative compounds from synthesized PDE4inhibitors, roflupram (selective PDE4D subtype inhibitor) and chlorbipram (PDE4D allosteric modulator). In the present project, we investigated the antidepressant effect and cognitive enhancement role of these two different classes of PDE4inhibitors. Furthermore, we evaluated their emetic potential in beagle dogs.METHODS:(1) The PDEs used in our experiments were partially purified human recombinant PDEs which were cloned from cDNA library and expressed in S. frugiperda insect cells using a baculovirus expression system. We tested the compounds roflupram and chlorbipram at5concentrations in duplicate against human PDEs to generate dose response curve and determine IC50value.(2) Male C57BL/6mice were randomly divided into eight groups (9-20mice each group):control (5%DMSO), roflupram (0.015,0.03,0.06,0.12,0.25,0.5mg·kg-1) and0.5mg·kg-1rolipram. Weigh the animals and administer the appropriate treatment immediately in a volume of10ml·kg-1. The researchers wait for30min after acute intraperitoneal injection to begin the tail suspension test. The duration of immobility was recorded for6min. Mice were considered immobile only when they hung motionless. Male C57BL/6mice were randomly divided into five groups (10-12mice each group): control (5%DMSO), roflupram (0.03,0.06,0.12mg·kg-1), and0.5mg·kg-1rolipram. Wait for30min after acute intraperitoneal injection to begin the forced swimming test and locomotor activity test.(3) Male Kunming mice were randomly divided into five groups (11-12mice each group):control (5%DMSO), chlorbipram (0.15,0.3,0.6mg·kg-1) and0.5mg·kg-1rolipram. Weigh the animals and immediate administer the appropriate treatment in a volume of10mg·kg-1.The tail suspension test, forced swimming test and locomotor activity test are conducted60min after acute oral administration each time.(4) Male adult SD rats are randomly divided into five groups (10mice each group):control (5%DMSO),1.0mg·kg-1scopolamine,1.0mg·kg-1scopolamine+0.25mg·kg-1roflupram,1.0mg·kg-1scopolamine+0.5mg·kg-1roflupram,1.0mg·kg-1scopolamine+1.0mg·kg-1roflupram. We evaluated whether PDE4inhibitor roflupram can reverse scopolamine-induced cognitive dysfunction of rats in Morris water maze test.(5) Male adult SD rats are randomly divided into five groups (10mice each group):control (5%DMSO),1.0mg·kg-1scopolamine,1.0mg·kg-1scopolamine+0.5mg·kg-1chlorbipram,1.0mg·kg-1scopolamine+1.0mg·kg-1chlorbipram,1.0mg·kg-1scopolamine+1.5mg·kg-1chlorbipram. We evaluated whether PDE4inhibitor chlorbipram can reverse scopolamine-induced cognitive dysfunction of rats in Morris water maze test.(6) Six beagle dogs (with mixed gender) were randomly divided into2groups each time (every week) before the experiment. Rolipram, roflupram and chlorbipram were dissolved in5%DMSO. The animals were administrated by oral route with the volume of1ml·kg-1.After oral dosing, the animals were monitored continuously for120min, the latency (as determined by the interval time from oral administration of drugs to vomit its gastric content) and the incidence of emesis were recorded.RESULTS:(1) The novel PDE4inhibitor, roflupram and chlorbipram, and the classical PDE4inhibitor rolipam all dose-dependently inhibited PDE4activity. roflupram exhibited about44times inhibitory potent for PDE4CAT than rolipram. However, chlorbipram exhibited almost the equal inhibitory potent for PDE4CAT with rolipram. Roflupram and chlorbipram both exhibited lower selectivity for PDE4B2than rolipram. Roflupram exhibited at least3times lower selectivity and16times inhibitory potent for PDE4B2than rolipram. Chlorbipram exhibited at least117times lower selectivity and77times lower inhibitory potent for PDE4B2than rolipram. Also, with regard to selectivity and inhibitory potent for PDE4D4and PDE4D5, chlorbipram were significantly lower than rolipram. However, roflupram exhibited lower selectivity and higher inhibitory potent for PDE4D4and PDE4D5.(2) One-way ANOVA showed significant difference [F=6.426, P=0.000] in TST on the C57BL/6mice among experimental groups. Roflupram at0.03mg·kg-1(P<0.01),0.06mg·kg-1(P<0.01),0.12mg·kg-1(P<0.01),0.25mg·kg-1(P<0.01),0.5mg·kg-1(P<0.05) treated mice significantly decreased immobility time compared with vehicle-treated mice in the TST. However, roflupram at0.015mg·kg-1treated mice marginally decreased immobility time compared with vehicle-treated mice in the TST (P=0.069).The acute antidepressant-like effect of roflupram was similar to that of rolipram, a classical PDE4inhibitor served as positive control (P<0.01). Immobility time in TST decreased from112.02±4.580s in vehicle-treated group to95.20±8.110s (0.015mg·kg-1),76.12±5.994s (0.03mg·kg-1),69.59±4.232s (0.06mg·kg-1),76.87±7.901s (0.12mg·kg-1),76.96±5.664s (0.25mg·kg-1) and90.65±9.478s (0.5mg·kg-1) after roflupram acute intraperitoneal injection, and72.38±6.302s in rolipram-treated mice. Similarly, one-way ANOVA showed significant difference[F=5.799, P=0.01] in FST on the C57BL/6mice among experimental groups. After acute intraperitoneal injection, roflupram at0.03mg·kg-1(P<0.01),0.06mg·kg-1(P<0.01),0.12mg·kg-1(P<0.01), as well as rolipram at0.5mg·kg-1(P<0.01) treated C57BL/6mice had significantly decreased immobility time compared with vehicle-treated mice in the FST. Immobility time decreased from119.13±9.941s in vehicle-treated mice to77.40±8.056s (0.03mg·kg-1),75.25±5.500s (0.06mg·kg-1) and78.32±6.058s (0.12mg·kg-1) after roflupram acute intraperitoneal injection, and80.87±8.814s in rolipram-treated mice. Statistical analysis by one-way ANOVA showed no significant difference[F=0.194, P=0.941]among the groups locomotor activity test. Roflupram produced no significant difference in the total activity number (P>0.05) compared with vehicle treated group.(3) One-way ANOVA showed significant difference [F=3.064, P=0.024] in TST on the Kunming mice among experimental groups. Chlorbipram at0.15mg·kg-1(P<0.05),0.3mg·kg-1(P<0.01),0.6mg·kg-1(P<0.01) treated mice significantly deceased immobility time compared with vehicle-treated mice in the TST. The acute antidepressant-like effect of chlorbipram was similar to that of rolipram. Immobility time decreased from171.75±12.521s in vehicle-treated group to132.98±9.693s (0.15mg·kg-1),126.80±12.161s (0.3mg·kg-1) and122.40±11.611s (0.6mg·kg-1) after chlorbipram oral administration, and132.32±9.109s in rolipram-treated mice. Similarly, one-way ANOVA showed significant difference [F=10.887, P=0.000] in FST on the Kunming mice among experimental groups. After acute oral administration, chlorbipram at0.15mg·kg-1(P<0.01),0.3mg·kg-1(P<0.01),0.6mg·kg-1(P<0.01), as well as rolipram at0.5mg·kg-1(P<0.01) treated Kunming mice had significantly decreased immobility time compared with vehicle-treated mice in the TST. Immobility time decreased from195.56±9.263s in vehicle-treated mice to142.05±8.128s (0.15mg·kg-1),124.37±11.684s (0.3mg·kg-1) and121.34±9.837s (0.6mg·kg-1) after chlorbipram oral administration, and121.77±25.17s in rolipram-treated mice. Statistical analysis by one-way ANOVA showed no significant difference[F=0.067, P=0.992]among the groups in locomotor activity test. Chlorbipram produced no significant difference in the total activity number (P>0.05) compared with vehicle treated group.(4) Intraperitoneal injection of scopolamine hydrobromide (1.0mg·kg-1) significantly extended the latency to reach the platform, and oral administration of roflupram (0.25～1.0mg·kg-1) attenuated the increased latency. During the probe trial on the day following the final day of training trial sessions, a marginal significant effect was observed on percentage of time spent in target quadrant [F=2.399, P=0.065]. In the test, scopolamine hydrobromide significantly reduced the percentage of time spent in the target quadrant where the platform had been located originally, and roflupram attenuated the reduced percentage of time spent (P<0.05). The crossing times to the probe quadrant was significantly different among the groups [F=3.615, P=0.013]. Roflupram attenuated the decrease of crossing times induced by scopolamine (P<0.05). However, no significant differences in the swimming speeds of probe test were observed among the groups. These results indicated that scopolamine-induced learning deficits could be reversed by the novel PDE4inhibitor roflupram.(5) Intraperitoneal injection of scopolamine hydrobromide(1.0mg·kg-1) significantly extended the latency to reach the platform, and oral administration of chlorbipram (0.5～1.5mg·kg-1) attenuated the increased latency. ANOVA indicated significant differences [day1:F=5.732, P=0.001; day2:F=2.345, P=0.072; day3:F=2.793, P=0.039; day4:F=10.341, P=0.000; day5:F=7.080, P=0.000]. During the probe trial on day following the final day of training trial sessions, a significant effect was observed on percentage of time spent in target quadrant [F=2.930, P=0.033]. In the test, scopolamine hydrobromide (1mg·kg-1) significantly reduced the percentage of time spent in the target quadrant where the platform had been located originally, and chlorbipram attenuated the reduced percentage of time spent (P<0.05). The crossing times to the probe quadrant was marginally significantly different among the groups [F=0.353, P=0.071]. Chlorbipram (0.5～1.5mg·kg-1) attenuated the decrease of crossing times induced by scopolamine (P<0.05). However, no significant differences in the swimming speeds of probe test were observed among the groups. These results indicated that scopolamine-induced learning deficits could be reversed by the novel PDE4inhibitor chlorbipram.(6) After oral administration, rolipram expressed emetic profiles in all the dogs in20minutes at0.5mg·kg-1; however, roflupram and chlorbipram did not express emesis profiles in120-min observation period even at1.0mg·kg-1. The hydrochloride of chlorbipram at1.07mg·kg-1also did not express emetic activity in observation period, suggesting roflupram, chlorbipram and its hydrochloride (better solubility) have litter or no emetic activity compared with rolipram. Furthermore, the incidence of emesis was significantly different among the groups. All the dogs vomited following administration of0.5mg·kg-1rolipram (100%), and no dog vomited in vehicle (0%), roflupram and chlorbipram treated group (0%).CONCLUSION:These two compounds have antidepressant-like effects and cognitive enhancement with little or without emetic potential compared with classical PDE4inhibitor rolipram. |
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