Effects of Rolipram and Roflumilast, Phosphodiesterase-4 Inhibitors, On

European Journal of Pharmacology 746 (2015) 138–147

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European Journal of Pharmacology

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Behavioural pharmacology Effects of rolipram and roﬂumilast, phosphodiesterase-4 inhibitors, on hypertension-induced defects in memory function in rats

Sobhana George Sugin Lal Jabaris a, Haridass Sumathy a, Ramadass Satiesh Kumar b, Shridhar Narayanan c, Sadagopan Thanikachalam a, Chidambaram Saravana Babu a,n a Centre for Toxicology and Developmental Research (CEFT), Sri Ramachandra University, Chennai 600116, Tamil Nadu, India b Central Leather Research Institute, Council of Scientiﬁc and Industrial Research, Chennai 600020, Tamil Nadu, India c AstraZeneca India Pvt. Ltd., Off Bellary Road, Hebbal, Bangalore 560024, India article info abstract

Article history: Hypertension (HT) is a prevailing risk factor for cognitive impairment, the most common cause of Received 22 June 2014 vascular dementia; yet, no possible mechanism underlying the cognitive impairment induced by Received in revised form hypertension has been identified so far. Inhibition of PDE-4 has been shown to increase phosphorylation 16 October 2014 of cAMP-response element binding protein in the hippocampus and enhance the memory performance. Accepted 18 October 2014 Here, we examined the effects of PDE-4 inhibitors, rolipram and roflumilast, on the impairment of Available online 6 November 2014 learning and memory observed in hypertensive rats. We used 2k-1c hypertensive model to induce Keywords: learning and memory defects. In addition, mRNA expression of PDE-4 sub-types A-D was also assessed in Hypertension the hippocampus tissue. Systolic blood pressure (SBP) was measured by tail-cuff method was Rolipram significantly increased in 2k-1c rats when compared to sham operated rats; this effect was reversed Roflumilast by clonidine, whereas, PDE-4 inhibitors did not. PDE-4 inhibitors significantly reversed time induced Roflumilast N-oxide fi Cognition memory de cit in novel object recognition task (NORT). Further, the retention latency on the second day in the elevated plus maze model was significantly shortened after repeated administration of rolipram and roflumilast. Plasma and brain concentrations of rolipram, roflumilast and roflumilast N-oxide were also measured after the NORT and showed linear increase in plasma and brain concentrations. The PDE4B and PDE4D gene expression was significantly enhanced in hypertensive rats compared with sham operated however PDE4A and PDE4C remained unaltered. Repeated treatment with PDE-4 inhibitors caused down regulation of PDE4B and PDE4D in hypertensive rats. These results suggest that inhibition of PDE-4 ameliorates HT-induced impairment of learning and memory functions. & 2014 Elsevier B.V. All rights reserved.

1. Introduction et al., 2003; Vermeer et al., 2003b). People with controlled hypertension seem to have a lesser prevalence of white matter Hypertension is a prevailing vascular risk factor for cognitive lesions than people with uncontrolled hypertension (Liao et al., decline and dementia, leading to a distressing loss of indepen- 1996; de Leeuw et al., 2002). dence of the individual (Duron and Hanon, 2008). Several studies Several clinical studies suggest that high blood pressure con- have focused on the possible mechanisms underlying the cognitive tributes to cognitive deficits in aging individuals (Elias et al., 1997; impairment induced by hypertension but a pathophysiological Skoog, 1997). Numerous pre-clinical models have shown positive mechanistic link is still to be ascertained (Carnevale et al., 2012). (Wyss et al., 1992; Meneses et al., 1996; Meneses and Hong, 1998; The existence of memory deficits correlates with the presence of Hirawa et al., 1999; Wyss et al., 2000; Hacioglu et al., 2003) but hypertension, and the subsequent pathological changes are gen- also negative (Kadish et al., 2001) correlations between hyperten- erally foci of ischemic damage in deep cerebral white matter sion and cognitive impairment in rats. (Fazekas et al., 1993; Vermeer et al., 2003a). White matter damage Phosphodiesterase-4 (PDE-4) is a critical regulator of intracel- may contribute to cognitive impairment and its most serious lular level of cAMP that is expressed throughout the brain (Perez- manifestation being dementia (Pantoni and Garcia, 1995; O'Brien Torres et al., 2000). Activation of cyclic AMP (cAMP) signaling enhances memory function and synaptic plasticity (Wang et al., 2012). Therefore, targeting PDE-4 with a selective inhibitor may n Corresponding author. Tel.: þ91 44 2386 0464. offer novel strategies in the treatment of memory impairment E-mail address: [email protected] (C.S. Babu). (Ghavami et al., 2006; Kodimuthali et al., 2008). A number of http://dx.doi.org/10.1016/j.ejphar.2014.10.039 0014-2999/& 2014 Elsevier B.V. All rights reserved. S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147 139 studies have identified that PDE-4 inhibition leads to elevation of In sham animals, incision was made, to expose the left renal artery cAMP levels (Barad et al., 1998), increased protein kinase A (PKA)/ and incision was sutured. Post-operative care was given to the rats phosphorylation of cAMP response element binding protein for one week. The rats were maintained on drinking water (pCREB) in the hippocampus signaling. This signaling cascade is containing 0.9% NaCl except sham group. SBP was measured essential for mediating memory, in particular hippocampus starting three weeks after the renal artery constriction for 11 dependent long-term memory (Monti et al., 2006; Nagakura et weeks. Animals were selected based on the SBP (cut off score al., 2002; Bourtchouladze et al., 2003). Rolipram, a specific PDE-4 Z170 mm Hg) at the 11th week post-surgery; selected animals inhibitor, has been shown to improve the working memory deficits were then stratified into groups based on their mean SBP, so that caused by administration of scopolamine and MK-801in preclinical the mean baseline did not differ between the groups. models (Zhang and O'Donnell, 2000; Zhang et al., 2000). Also, For testing the effects of repeated drug treatments on beha- rolipram treatment reverses cognitive impairment and neuroin- vioral tasks such as novel object recognition task and transfer flammation induced by β-amyloid in rats (Wang et al., 2012). Very latency in elevated plus maze, vehicle (0.5% CMC) or clonidine recently, brain penetrant selective PDE-4 inhibitor-GSK356278 (25 mg/kg., p.o), rolipram (0.03, 0.1, 0.3 mg/kg., i.p, dissolved in demonstrated anxiolytic and improvement of cognition effects in normal saline containing 5% DMSO), roflumilast (0.1, 0.3, 1 mg/kg., preclinical models (Rutter et al., 2014). p.o) were given once per day for ten consecutive days. Here we Roflumilast is a second generation, highly selective PDE-4 inhi- selected clonidine, a α2 receptor agonist, centrally acting anti bitor, and has recently been approved in several countries for severe hypertensive drug to evaluate whether anti-hypertensive treat- COPD (Rabe, 2011). However, the role of PDE-4 inhibitors in improv- ment would attenuate the cognitive impairments associated with ing learning and memory dysfunction induced by hypertension has HT in therapeutic model. For both behavioral experiments, sepa- not been studied so far. To evaluate the possibility of PDE-4 inhibitor rate set of animals were used including sham operated rats (68 for as a therapeutic agent for cognitive dysfunction by hypertension, we NORT and 66 for transfer latency; total no¼134). Both behavioral examined the effects of rolipram and roflumilast on impairment of tests were performed 1 h following the treatment except rolipram learning and memory function in the hypertensive rats using a two- group (45 min after dosing) as per the schedule (Fig. 1). kidney one-clip (2k-1c) hypertensive model to induce deficits in learning and memory function. 2.4. Novel object recognition task paradigm

TheeffectsofrepeatedadministrationofPDE-4inhibitorson 2. Materials and methods recognition memory in hypertensive rats were assessed using NORT similar to that described previously (Prickaerts et al., 2002). Rats 2.1. Animals were acclimated to the open arena (40 60 60) without objects for 15 min. NORT consisted of two trial periods, acquisition trial (T1) Adult male Wistar rats were obtained from Orchid Chemicals and discrimination trial (T2) separated by a 24 h inter trial period. A Pharmaceuticals Ltd, Chennai, India. Animals were housed in groups rat was taken from its home cage and placed into the apparatus, on soft bedding with food and water available ad libitum, in a central from the two objects, facing the wall in front of the observer, temperature controlled environment with a light dark cycle of and the time spent actively exploring (exploration was defined as 12:12 h. All animals were allowed to habituate to the housing the animal sniffing, or touching it) the objects during a 3 min test “ facilities for at least 1 week prior to surgery. Guidelines of Guide period (T1) was recorded and returned to its home cage. The arena ” for the Care and Use of Laboratory Animals (Institute of Laboratory and test objects were cleaned with alcohol in order to remove any Animal Resources, National Academic Press 1996; NIH publication olfactory/taste cues. After 24 h, each rat was again placed in the test number #85-23, revised 1996) were strictly followed throughout the arena for recording T2 in the presence of one of the familiar object study. All experimental procedures were approved by the Institu- and a novel object, and the time spent exploring both objects were tional Animal Ethical Committee (IAEC), Sri Ramachandra University, again recorded and discrimination index (DI) was calculated accord- constitute as per the directions of the Committee for the Purpose of ing to the following formula. Control and Supervision of Experiments on Animals (CPCSEA), India, ¼ =ð (IAEC NO: IAEC/XXX111/SRU/258/2013). DI RI Time spent in exploring novel object þTime spent in exploring familiar objectÞ 2.2. Drugs Recognition IndexðRIÞ¼Time spent in exploring novel object Time spent in exploring familiar object Rolipram and clonidine hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd, Japan. Roflumilast was kindly gifted by Matrix Laboratories, Hyderabad, India. 2.5. Transfer latency in elevated plus maze

2.3. Two-kidney one-clip induced hypertension in rats The procedure and technique for testing learning and memory were followed as per the parameters described earlier (Reddy and Prior to surgical procedure, systolic blood pressure was mea- Kulkarni, 1998; Hlinak and Krejci, 2002). The elevated plus maze sured by tail-cuff method (model MC 4000; Hatteras Instruments, for rats consisted of two open arms (50 cm 10 cm) and two Cary, NC, USA). Two-kidney one-clip induced Reno Vascular closed arms (50 cm 10 cm 40 cm) extended from a central Hypertension (RVHT) was performed as per the method described platform (10 cm 10 cm) and the maze was elevated to a height previously with minor modifications (Zeng et al., 1998; Kalaivani of 50 cm from the floor. On the 10th day of treatment, each rat was et al., 2013). In brief, rats weighing 140–180 g at the time of placed at the end of an open arm, facing away from the central surgery were anaesthetized by intra peritoneal injection of keta- platform. Transfer latency (TL) was defined as the time taken by mine (75 mg/kg) and midazolam (1 mg/kg) mixture. A small the animal to move from the open arm into one of the closed arms incision was made and the left renal artery was exposed and with all its four legs was recorded. If the animal did not enter into cleared. Then a U-shaped silver clip with a gauge of 0.25 mm was one of the closed arm within 180 s, it was gently pushed into one placed around the renal artery and secured in place and the of the two closed arms and TL was assigned as 180 s and rat was incision was sutured and the animals were returned to their cages. allowed to explore the maze for another 60 s. Then, the rat was 140 S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147

Fig. 1. Schedule of drug treatment and experiment orders. Vehicle (0.5% CMC; 10 ml/kg., p.o), clonidine (25 mg/kg., p.o), rolipram (0.03, 0.1, 0.3 mg/kg., i.p) and roﬂumilast (0.1, 0.3, 1 mg/kg., p.o). A, on day 8, habituation (H) of the NORT was performed, followed the next day by the acquisition (A) trial, 24 h after interval discrimination (D) trial. B, on day 10, transfer latency (TL) was performed in elevated plus maze (EPM), next day by retention latency (RL). taken out of the maze and returned to its home cage. The maze Table 1 was cleaned thoroughly with 70% ethanol solution after each rat to Primer sequences for reverse transcriptase—PCR. remove any confounding olfactory cues. Retention latency (RL) was examined 24 h (11th day) after the transfer latency. Name Primer Sequence

PDE4A Upstream 50-gcttgaacaccaacgtcccacggt-30 2.6. Pharmacokinetics of roﬂumilast, roﬂumilast N-oxide and Downstream 50-gctgaggttctggaagatgtcgcag-30 rolipram after oral/i.p administration PDE4B Upstream 50-atgacccagataagtggagtgaag-30 Downstream 50-tcaggctgaaccaggtctgcccag-30

Five min after the NORT experiment, rats were anaesthetized 0 0 fl PDE4C Upstream 5 -atggcccagatcactgggctgcgg-3 using iso urane, and blood was collected retro-orbitally. After Downstream 50-gctgaggttctggaagatgtcgcag-30 blood sampling, rats were euthanized by over dose of isoflurane PDE4D Upstream 50-atggcctccaacaagttcaagagg-30 and the brain was removed. Blood was centrifuged at 3000g for Downstream 50-gctggctttcctcttctgctacagcc-30 1 1 0 0 10 min at 4 C. Blood plasma and brain were frozen at 80 C until β Actin Upstream 5 -tcatgaagtgtgacgttgacatccgt-3 compound level determination took place. The concentration of Downstream 50-cctagaagcatttgcggtgcacgatg-30 roflumilast and roflumilast N-oxide in plasma and brain were measured using a gradient LC/MS/MS procedure as described previously (Thappali et al., 2012). For rolipram, the brain samples following the incubation period of 10 min. The supernatant was were homogenized in 80:20 ratios of acetonitrile (ACN) and water decanted carefully and the pellet was washed three times with with a tissue homogenizer. Two hundred micro liter plasma/brain 75% ethanol and centrifuged at 12,000g for 15 min and the pellet homogenate were vortexed with 200 ml of ACN for 60 s. Then the was dried and resuspended in 20 μl of RNase free water. The samples were centrifuged at 10,000g for 15 min, 50 ml of super- concentration of RNA was determined using nanodrop spectro- natant was injected into the chromatographic system. The HPLC photometer and RNA samples were stored at 80 1Cuntiluse. (Shimadzu Ltd., Japan) system consisted of a LC-20 AD binary Two hundred nanogram of RNA were used for RT-PCR according pump, auto injector (SIL-20 AC HT), column oven (CTO-10 AS VP) to the manufacturer's instructions (Genet Bio, Korea). Isolated and PDA detector (SPD-M20A). The wavelength of the detector RNA was allowed to undergo reverse transcription and polymer- was set at 230 nm. Detector output was quantified on Lab Solu- ization reaction to synthesize cDNA using PCR master cycler tions platform. Separation was carried out on a Enable C18G gradient. The following steps were performed for each PCR column 250 4.6 mm) using mixture of 0.5% formic acid and reaction: 42 1C for 30 s, 94 1Cfor5min(1cycle);941Cfor ACN (60:40) as a mobile phase, at a flow rate of 1 ml/min. Total 1min,β-actin (55.4 1C), PDE 4A (71.0 1C), PDE 4B (59.7 1C), PDE analysis time was 15 min. 4C (58.1 1C) and PDE 4D (64.0 1C) and 72 1C for 1 min (with 35 cycles); and a final extension phase at 72 1Cfor5min.The 2.7. Corticosterone analysis concentration of cDNA was analyzed using nanodrop spectro- photometer. The RT-PCR products were loaded in 1% agarose gel The serum concentration of corticosterone from the NORT and electrophoresed at 40 V for 30 min and bands obtained were experiment was quantified using enzyme-linked immunosorbent quantified using gel documentation (Vilber, France). The primers assay (Cayman Chemical, Ann Arbor, MI, USA). are listed in Table 1.

2.8. Reverse transcriptase PCR 2.9. Statistical analysis

To determine the mRNA expression of PDE4 A, B, C and D in Data are presented as mean7S.E.M. Statistical analysis was rat hippocampus was quantified using reverse transcriptase PCR performed using GraphPad Prism Software (Version 5; Inc). The (RT-PCR). Briefly, total RNA was extracted from hippocampus difference between time spent exploring the novel object versus using TRIzol Reagent (Sigma). After homogenization, the tubes familiar object during discrimination trial was calculated for each were incubated for 10 min and centrifuged at 100g for 5 min. group and the level of significance was analyzed using two-sided 200 μl of chloroform was added to the supernatant, allowed to student's t test. For other parameters, the effect of sham vs vehicle incubate for 5min at room temperature and centrifuged at was analyzed using a two-sided student's t test and each treat- 12,000g for 20 min. Supernatant was transferred to new centri- ment group vs vehicle group was analyzed using one-way analysis fuge tube to which 500 μl of isopropyl alcohol was added to of variance (ANOVA) followed by Dunnet's post hoc test. The level precipitate the total RNA and centrifuged at 12,000g for 15 min of significance was set at Po0.05. S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147 141

Fig. 2. Effect of PDE4 inhibitors on SBP, kidneys, heart and heart index in hypertensive rats. Data represents mean7S.E.M. *Po0.05, **Po0.01, ***Po0.001 (One way ANOVA followed by Dunnett's post hoc test) in comparison with vehicle treated group; ### Po0.001 vs. sham group.

3. Results Fig. 2(B)–(D) gives the mean kidney, heart weights, and heart index (heart weight normalized for body weight). The contralateral 3.1. Effect of PDE-4 inhibitors on Blood pressure non-clipped kidneys had significantly higher weight when compared to clipped kidneys within the same group. As anticipated, The mean systolic blood pressure in male Wistar rats weighing hypertensive control showed significantly increased heart weights 140–180 g which was approximately 120.879.8 mm Hg before (1.4770.05 g vs 1.0370.01 g; Po0.001) and heart indexes renal artery occlusion, rose to 156.178.4 mm Hg at the end of (4.3070.14 vs 2.5070.12; Po0.001) compared with those in three weeks, then exceeded 185.971.6 mm Hg at 11th weeks, normotensive group. Both PDE-4 inhibitors caused no significant respectively, after renal artery occlusion. To determine whether differences in heart weight and heart indexes compared with inhibition of the PDE-4 affects blood pressure in rats, SBP and hypertensive control rats. Clonidine, however, caused a significant responses to i.p/oral administration of rolipram and roflumilast, reduction of heart weight as well as heart indexes (Po0.05). selective PDE4 inhibitors, were examined under the fully conscious state. As shown in Fig. 2(A), PDE-4 inhibitors did not cause a 3.2. Novel object recognition task reduction in systolic blood pressure of the 2k-1c hypertensive rats, indicating that PDE-4 inhibitors had no therapeutic action on blood The cognition-enhancing effects of repeated administration of pressure. Clonidine, centrally acting α2 receptor agonist, at the dose PDE-4 inhibitors were shown by its aptitude to delay natural of 25 μg/kg caused statistical significant reductions in SBP in rats on forgetting in rats after a 24 h interval in the NORT. Rats with days 1, 5 and 9 were 149.773.1, 127.2 73.1 and 117.172.4 mm Hg, repeated treatment of PDE-4 inhibitors before acquisition trial (T1) respectively. No significant change in SBP was observed during explored the novel object significantly more than the familiar one these experimental periods in vehicle treated rats. during discrimination trial (T2) respectively, showing an increase 142 S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147

Fig. 3. Effect of PDE4 inhibitors on NORT in hypertensive rats. (A) Discrimination index, and (B) time exploring object the familiar and novel object. Data represents mean7S.E.M. (n¼7–9 per group); *Po0.05, **Po0.01, ***Po0.001 vs. vehicle control; # Po0.05 vs. sham group.

Fig. 4. Effect of PDE4 inhibitors on transfer latency (elevated plus maze) in hypertensive rats. Data represents mean7S.E.M. (n¼6–8 per group); *Po0.05, **Po0.01 vs. vehicle control; and ## Po0.01 vs. sham group. in memory retention. These effects of repeated administration of an improvement in the elevated plus maze learning performance centrally acting anti- hypertensive drug (clonidine) were qualita- (Fig. 4(A) and (B)). tively similar to those obtained with the hypertensive control rats (Fig. 3(A) and (B)). 3.4. Rolipram, roflumilast and roflumilast N-oxide concentrations in rat plasma and brain 3.3. Transfer latency in elevated plus maze The effects of repeated treatment of roflumilast on the exposure Transfer latency of first day (on tenth day of drug treatment) rates of rolipram, roflumilast and roflumilast N-oxide (active meta- reflected the acquisition of learning behavior of animal whereas RL bolite of roflumilast) in plasma and brain samples are represented in of next day reflected improvement of both learning and memory. Fig. 5(A)–(C). Rolipram treated animals showed dose dependent Vehicle and clonidine administered for 10 days orally did not have detectable levels of rolipram in plasma and brain, when the final any significant effect when compared with sham operated group dose was administrated 53 min before sampling. The rolipram on RL of eleventh day in elevated plus maze. However, rolipram concentrations were 87.2778.27, 248.18728.06, 584.55727.27 nM treated intraperitonally at 0.03, 0.1 and 0.3 mg/kg showed remark- in plasma and 148.18720.10, 427.18713.32, 1016 751.57 nM in able reduction (Po0.05 and Po0.01) in RL, indicating significant brain at 0.03, 0.1 and 0.3 mg/kg., i.p respectively. Furthermore, low improvement in learning and memory. In addition, roflumilast at a dose of roflumilast treated animals showed no detectable levels of dose of 0.1, 0.3 and 1 mg/kg., p.o also improved learning and roflumilast in plasma, whereas, detectable concentration of roflumi- memory in RL compared to vehicle treated group, which indicated last N-oxide was found in plasma (14.4871.77 nM). In addition, mid S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147 143

Fig. 5. Plasma and brain concentrations of rolipram, roflumilast and roflumilast N- Oxide. (A) Rolipram, (B) roflumilast and (C) roflumilast N-oxide. Data represents mean7S.E.M. (n¼4 per dose). and high doses of roflumilast showed linear concentrations in plasma (10.4870.89 and14.9472.43 nM) as well as brain (27.9371.88 and 125.96 725.56 nM). The roflumilast N-oxide metabolite concentration was 86.9678.34 and 125.96725.56 nM in plasma and 25.5672.62 and 64.05713.63nM in brain when the final dose was administrated 68 min before sampling, respectively.

3.5. Corticosterone concentration in rat

Fig. 6 shows serum corticosterone concentration in 2k-1c hypertension rats treated with vehicle, clonidine or PDE-4 inhibitors. Clipping significantly increased serum corticosterone levels in 2k-1c hypertension group when compared to sham operated group (3989729 vs 3420716 pg/ml; Po0.001). High doses of rolipram and roflumilast treatment promoted an additional increase in corticosterone levels. One-way ANOVA indicated that there was a significant effect of PDE-4 inhibition on serum concentration of corticosterone with both rolipram and roflumilast showing an increase (Po0.01 and Po0.05). However, clonidine produced no changes in serum corticosterone when compared with vehicle treated group. Fig. 6. Effect of PDE4 inhibitors on corticosterone in hypertensive rats. Data 3.6. Determination of PDE4ABCD mRNA expression represents mean7S.E.M. (n¼5 per group); *Po0.05, **Po0.01 vs. vehicle control; ### Po0.05 vs. sham group. To verify the effects of PDE-4 inhibitors on hypertension- induced changes in PDE-4 subtypes in the hippocampus, we examined mRNA levels in hippocampus tissue when compared to vehicle PDE4A, PDE4B, PDE4C and PDE4D mRNA expression in the hippo- treated group. More specifically, compared to sham operated group, campus using the same rats, decapitated with isoflurane anesthesia 2k-1c induced hypertension group increased PDE4B (Po0.001), and after the behavioral test, by RT-PCR. In the case of PDE4B and D, PDE4D (Po0.01). However, clonidine, centrally acting α2receptor chronic PDE-4 inhibitors treatment dose dependently decreased its agonist, caused no significant suppression of PDE4B and D in 144 S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147

Fig. 7. Effect of PDE4 inhibitors on the expression of PDE4 A, B, C and PDE4D mRNA in hypertensive rat hippocampus tissues. Levels of mRNA were quantified by densitometry and are shown in the bar graph. Data represents mean7S.E.M. (n¼3per group); *Po0.05, **Po0.01, ***Po0.001 vs. vehicle control; ##Po0.05, ###Po0.001 vs. sham group. hypertensive rats. In contrast, PDE4A and C mRNA expression levels Morishita et al., 1991; Derkx and Schalekamp, 1994; Sadjadi were not changed between sham and hypertension as well as et al., 2002; Weissheimer et al., 2012). treatments (Fig. 7(A)–(E)). In the present study, we have demonstrated that 2k-1c induced hypertension produced deficits of learning and memory in novel object recognition task and transfer latency in elevated plus maze compared with sham operated rats. Transfer latency in elevated 4. Discussion plus maze appear to be a spatial memory (Reddy and Kulkarni, 1998) and NORT to be a recognition memory model (Fernandez In the present study we evaluated the possibility of PDE-4 and Garner, 2007). The possible mechanisms by which hyperten- inhibition as a therapeutic agent for cognitive dysfunction by sion could increase risk of cognitive impairment is still elusive. hypertension, we examined the effects of rolipram and roflumilast Perhaps, the possible risk factors causing cognitive impairment on impairment of learning and memory function in the hyperten- could be compromised vasoreactivity and consequent risk of brain sive rats. Compared with sham operated group, systolic blood hypoperfusion, blood–brain barrier dysfunction, amyloid plaques, pressure, right kidney (unclipped), heart weight and the heart increased risk of stroke, occlusion and degeneration of cerebral indices of 2k-1c induced hypertensive rats were found to be capillaries as well as the neuroinflammation (Blossom et al., 2008; significantly increased; with significant elevation of serum corti- Gentile et al., 2009; Peters, 2012; Carnevale et al., 2012). costerone being observed. These results are in agreement with In view of previous data demonstrating that second messengers previous studies which show that serum level of corticosterone such as cAMP play a prominent role in cellular signaling pathways was higher in 2k-1c hypertensive compared with normotensive and neuroplasticity, improve memory performance and based on rats (Zamir et al., 1983), blood pressure, heart weight and right the well established concept that cAMP elevations exert long-term kidney weights were significantly higher in hypertensive group potentiation (LTP) effects in hippocampus, we speculated that compared with sham rats (Hacioglu et al., 2003). The mechanism specific PDE-4 inhibitors may have beneficial effects in HT induced by which chronic hypertension is maintained in 2k-1c induced cognitive impairment by protecting against cerebral vascular dys- RVHT is poorly understood. Possible mechanism could be due to function, cerebral hypoperfusion and neuroinflammation. activation of renin-angiotensin system (RAS) and also an abnormal Here we demonstrate that the effects due to hypertension were activity of the hypothalamic pituitary-adrenal system may play a reversed by repeated treatment with PDE-4 inhibitor- rolipram role in the pathogenesis of 2k-1c model (Zamir et al., 1983; which was confirmed by both memory tests. These results are S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147 145 supported by findings from previous studies, in which rolipram genes in rats: PDE 4A, 4B, 4C and 4D (Houslay and Milligan, 1997), reverses memory impairments in various type of animal models and its subtypes are distributed throughout the brain (Perez- (Imanishi et al., 1997; Zhang and O'Donnell, 2000; Zhang et al., Torres et al., 2000; Bian et al., 2004) which indicate distinct roles 2000; Nagakura et al., 2002; McLean et al., 2009; Li et al., 2011a, of the individual PDE-4 in CNS functions. Previous study reported 2011b; Wang et al., 2012). In addition, a similar response has been that PDE4A is highly expressed in rat hippocampus CA1 sub-region observed in hypertensive rats with roflumilast treatment in which (Zhang et al., 2004). Whereas, PDE4B is the predominant present there was a failure of improvement in the memory functions in the amygdala, hypothalamus, and striatum; it also expressed in induced by hypertension 60 min after oral administration of frontal cortex and olfactory bulb (Zhang et al., 2008) as well as clonidine, which significantly decreased the SBP throughout the hippocampus (Dlaboga et al., 2006). While PDE4C is minimally treatment periods at tested dose. Thus, clonidine showed slight expressed in the CNS (Zhang et al., 2004). In addition, PDE4D is activity on heart weight and heart index, suggesting that it has also highly expressed in hippocampal CA1 region, mediating significant role in blood supply to the brain compared to PDE-4 memory, its major role is cAMP hydrolysis, hippocampal LTP inhibitors. However, more recently, Rutten et al., 2009 demon- (Perez-Torres et al., 2000; Zhang et al., 2002; Rutten et al., strated that intraperitoneal injection of rolipram (0.03 mg/kg) 2008a) and antidepressant activity (Zhang et al., 2002). To date, improved the memory functions of rats without affecting cerebral few studies indicate that PDE4B may be involved in depression blood flow and glucose utilization. To our knowledge, this is the behavior, schizophrenia as well as anxiety (Millar et al., 2005; first report to assess the effects of roflumilast, a selective PDE-4 Zhang et al., 2008). On the finding that pharmacologically induced inhibitor on memory enhancement. Several PDE-4 inhibitors have over expression of PDE4D resulted in memory impairment (Giorgi been identified; unfortunately, development of PDE-4 inhibitors et al., 2004). In addition, mice deficient in PDE4D displayed for therapeutic purposes has been hindered by side effects such as memory enhancement in different type of behavioral tests and emesis (Robichaud et al., 2001, 2002; Rennard et al., 2008; Rabe, these effects were mimicked by repeated administration of roli- 2011). pram in wild-type mice (Burgin et al., 2010; Li et al., 2011a, 2011b). In our present study, we measured the concentrations of Recently, GEBR-7b, a novel PDE4D selective inhibitor, demon- rolipram, roflumilast and roflumilast N-oxide, the active metabo- strated that increased hippocampal cAMP level, improved spatial, lite of roflumilast with similar potency and specificity to the as well as object memory performance in the object recognition parent compound (Bethke et al., 2006) in plasma and brain after tests (Bruno et al., 2011). Here we have demonstrated subtype the NORT experiment. Here we demonstrated the plasma and specific inhibitory properties of rolipram and roflumilast on PDE-4, brain concentrations of rolipram, roflumilast and roflumilast –N- in which PDE4B and PDE4D are significantly inhibited. PDE4A and oxide increased in dose dependent manner. Rolipram was detected PDE4C subtypes are not inhibited by rolipram, roflumilast and in brain as well as plasma at all three doses while the concentra- both are not affected by hypertension as well. Therefore, our tion of roflumilast and its active metabolite levels were not at results provide further evidence that PDE4B and PDE4D plays a detectable in brain at low dose but treatment with low dose of critical role in the mediation of memory processes in brain regions roflumilast resulted in memory enhancement which was observed such as the hippocampus. These results suggest that the anti- from the memory test. This could imply that improvements of amnesic effect of rolipram and roflumilast could be related to cognitive effects of rolipram and roflumilast is probably explained subtypes inhibition as well as elevation of cyclic AMP levels in by differences in potencies (Hatzelmann and Schudt, 2001; hippocampus. Hatzelmann et al., 2010), administration routes, roflumilast is We showed that memory deficits induced by hypertension are rapidly converted by CYP3A4 and CYP1A2 to roflumilast N-oxide likely correlated with increased PDE-4 activity in the hippocam- after oral administration (Bethke et al., 2007), resulting in different pus. These beneficial effects of rolipram and roflumilast appear to degrees of PDE-4 inhibition by these drugs. be directly attributed to its inhibition of PDE-4 in the brain, in It is well established that PDE-4 inhibitors such as denbufylline, particular the hippocampus, leading to subsequent increases in rolipram and BRL 61063 results in an increased plasma corticos- cAMP/PKA/CREB signaling (Impey et al., 1996; Rutten et al., terone from the adrenal glands and also interacts with the 2008b). Further studies are needed to clarify this signaling cascade hypothalamo-pituitary-adrenal (HPA) axis in rat at both the in hypertension associated cognitive impairments. Our findings anterior pituitary and the hypothalamus (Hadley et al., 1996; provide an insight into the involvement of PDE-4 in hypertension Kumari et al., 1997; Kung et al., 2000). In the present study we associated neurochemical and pathological alterations of brain and found that repeated administration of high doses of PDE-4 the therapeutic profile of PDE-4 inhibitors. With respect to inhibitors caused significant increase in serum corticosterone level clonidine treatment, the present study did not allow a definitive in 2k-1c induced hypertensive rats, but had no effect on the conclusion, whether clonidine could resist against cognitive dis- corticosterone at lower doses. Studies on rodents indicate that order associated with HT, because of the limited data as well as stress or corticosterone administration can impair hippocampus contradictory results of clonidine (Jäkälä et al., 1999). specific learning and memory and also block hippocampal long- In conclusion, the rolipram and roflumilast significantly atte- term potentiation (Kim et al., 2001; Alfarez et al., 2002; Woodson nuated memory deficits induced by hypertension. These observa- et al., 2003). In contrast, glucocorticoids have also been associated tions may be attributed to an integrated network of cellular effects with enhanced memory (Sandi et al., 1997; Roozendaal, 2000; of the PDE-4 inhibitors in cognition. The precise role of the PDE-4 Buchanan and Lovallo, 2001). However, in our present study we inhibitors on other cognitive models remains to be explored. observed that elevation of corticosterone due to PDE-4 inhibition failed to affect the behavioral tasks and that rolipram and roflumilast exerts a beneficial effects in HT induced cognitive Acknowledgment impairment model is an important finding. Consistent with these findings, these PDE-4 inhibitors may be therapeutically useful for The authors thank the Drugs and Pharmaceutical Division, the enhancement of cognition associated with hypertension. Department of Science and Technology, Government of India (Ref Depending on the potency, specificity and the chronicity of use, no: VI-D&P/407/2011-12/TDT(G)) for funding the project. Authors PDE-4 inhibitors can be a nootropic. also express their sincere thanks to Dr. S.P. Tyagarajan, Professor of In addition, we investigated the PDE-4 subtypes mRNA expres- Eminence and Dean (Research) and Dr. B.V. David, Test Facility sion in hippocampus tissues. The PDE4 family consists of four Management, Centre for Toxicology and Developmental Research 146 S.G.S.L. Jabaris et al. / European Journal of Pharmacology 746 (2015) 138–147

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