Life Sciences 112 (2014) 90–96

Contents lists available at ScienceDirect

Life Sciences

j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie

Hypotensive and antihypertensive potential of4-[(1-phenyl-1H-pyrazol-4-yl) methyl]1-piperazinecarboxylic acid ethyl ester: A piperazine derivative

James Oluwagbamigbe Fajemiroye a,⁎, Nathalia Oda Amaral a, Elaine Fernanda da Silva a,Pablinny Morreira Galdino a, Thiago Sardinha de Oliveira a, Paulo César Ghedini a, Jordan K. Zjawiony c,Elson Alves Costa a, Gustavo Rodrigues Pedrino a, Ricardo Menegatti b

a Department of Physiological Sciences, Federal University of Goiás, Campus Samambaia, 74001-970 Goiâania, GO, Brazilb Pharmacy Faculty, Federal University of Goiás, Setor Universitário, 74000-000, Goiânia, GO, Brazilc Department of Pharmacognosy and National Center for Natural Product Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University,MS 38677-1848, USA

⁎ Corresponding author. Tel.: +55 62 3521 1491; fax:E-mail address: rm_rj@yahoo.com (J.O. Fajemiroye).

http://dx.doi.org/10.1016/j.lfs.2014.07.0250024-3205/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 22 April 2014Accepted 16 July 2014Available online 26 July 2014

Keywords:LQFM008Spontaneously hypertensive ratNormotensive ratAortic rings

Aims: Clinical complaints on the first-line of cardiovascular medicationsmake continuous search for new drugs anecessity. This study evaluated the cardiovascular effects and mechanism of 4-[(1-phenyl-1H-pyrazol-4-yl)methyl]1-piperazine carboxylic acid ethyl ester (LQFM008).Main methods: Normotensive maleWistar or spontaneously hypertensive rats (anesthetized or conscious) wereused to evaluate the effect of LQFM008 on themean arterial pressure (MAP), heart rate (HR), arterial blood flow(ABF), arterial vascular conductance (AVC), baroreflex effectiveness index (BI), systolic blood pressure (SBP),diastolic blood pressure (DBP) and vascular function.Key findings: In anesthetized normotensive rats, LQFM008 (7.3, 14.3 or 28.6 μmol/kg, iv) reducedMAP (−21.1±2.7;−23.9± 4.7 or−32.4± 8.3mmHg, respectively) and AVC (22%, 32% or 38%) in a dose-dependent manner.

LQFM008 elicited a temporal reduction in the SBP and DBPwithout changes to the BI of conscious normotensiverats. In hypertensive rats, LQFM008 (7.3, 14.3 or 28.6 μmol/kg, iv) reducedMAP (−2.3 ± 2.6;−29.3± 2.7 or −38.4 ± 2.8 mmHg, respectively) and increased HR (1.6 ± 3.7; 15.4 ± 4.9 or 25.5 ± 6.2 bmp, respectively) in adose-dependent manner. A week of oral administration of LQFM008 47.7 μmol/kg elicited a temporal reductionin SBP of hypertensive rats. Pretreatments with atropine, WAY-100635 or L-NAME blocked the effect ofLQFM008. In addition, LQFM008-induced endothelium-dependent vascular relaxationwas inhibited by L-NAME.Significance: Our findings showed hypotensive, antihypertensive and vasorelaxant effects of LQFM008 andsuggest the participation of nitric oxide, 5-HT1A and muscarinic receptors.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Despite major advancements in the development of cardiovasculardrugs (Eric, 2009; Ohlstein, 2010) in recent times, cardiovasculardiseases remain the chief cause of death and disability. Among thesediseases, hypertension has been considered a risk factor for thedevelop-ment of other cardiovascular diseases (Michele et al., 2011; Toney et al.,2010). Both functional and structural modifications of blood vesselshave been associated with hypertension (Intengan and Schiffrin, 2000;Michele et al., 2011). Meanwhile, control of blood pressure is a complexprocess that involves the integration of multiple regulatory systems(Thomas and Steven, 2008; Abrams, 1988; Scher, 1989) such as intrin-sic, reflex, hormonal, renal and vascular regulatory systems.

+55 62 3521 1204.

Given the complexity of hypertension's pathophysiology, differentclasses of drugs are being prescribed for the treatment of hypertensivepatients. Preclinical and clinical studies on antihypertensive drugssuch as prazosin and propranolol have revealed their effectiveness asantianxiety drug (Taylor et al., 2006; Angrini et al., 1998). Since stresscould induce both autonomic and behavioral responses (Ma andMorilak, 2005) to modulate the roles of various limbic structures(Alves et al., 2010; Resstel et al., 2006) that are involved in cardio-vascular regulation, we hypothesized cardiovascular activity for 4-[(1-phenyl-1H-pyrazol-4-yl)methyl]1-piperazine carboxylic acidethyl ester (LQFM008), a newly synthesized piperazine derivativewith promising anxiolytic-like properties (De Brito et al., 2012). The an-tianxiety effect of this compound was associated with the participationof 5-HT1A receptors.

Previous studies have shown that peripheral administration of sero-tonin altered the blood pressure and modulated vagal nerve activity

91J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

(Kalkman et al., 1984). The central administration of this indolamine innormotensive animals induced hypotensive effectwith bradycardia as aresult of 5-HT1A activation (Helke et al., 1993; Miyawaki et al., 2001).The peripheral and central effects of serotonin could be associated tothe wide distribution of serotonin (5-HT) receptors in the organism(Wedzony et al., 2007; Konturek et al., 2011). Despite the fact thatthere are no evidences linking the modulation of adrenergic, muscarin-ic, and nitric oxide (NO) pathways to the pharmacological effects ofLQFM008, several clinical and preclinical reports have demonstratedthe involvement of these pathways in the blood pressure-controllingmechanisms of drugs (Howell and Kovalsky, 1995; Wedzony et al.,2007; Elinor et al., 1983; Hermann et al., 2006).

In the present study, we sought to investigate the hypotensive, anti-hypertensive and vascular function of LQFM008 by using animalmodelsand isolated organs. We also used pharmacological tools like receptorantagonists and enzyme inhibitors in an attempt to unravel the hypo-tensive and vascular mechanisms of LQFM008.

Material and methods

Animals

All protocols described in this study were approved by the EthicsCommittee of the Federal University of Goiás (protocol #172/09) andwere performed in strict adherence to the principle of the Three RsDirective 2010/63/EU. Adult normotensive male Wistar (NMW) orspontaneously hypertensive rats (SHR) (310 ± 40 g, 3 to 4 monthsold) were used in all experiments. Rats with blood pressure above150 mm Hg were considered SHR. The animals were provided by theCentral Animal House of Federal University of Goiás and kept undercontrolled condition (temperature 25 ± 1 °C, light/dark cycle of 12 h),food and water ad libitum.

Drugs and chemicals

LQFM008 with a molecular weight of 314.17 g/mol (Laboratóriode Química FarmacêuticaMedicinal, GO, Brazil), halothane (Cristália,Itapira, SP, Brazil), urethane (Sigma-Aldrich, St. Louis, MO, USA),polysorbate 80 (Tween 80; Synth, Diadema, SP, Brazil), 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; Sigma-Aldrich), N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclohexane-carboxamide (WAY-100635; Sigma-Aldrich), norepinephrine (NOR;União Química, SP, Brazil), acetylcholine chloride (Cristália), NG-nitro-l-arginine methyl ester (L-NAME; Sigma-Aldrich), indometha-cin (Sigma-Aldrich), phenylephrine (PHE; Sigma-Aldrich), and sodiumnitroprusside (SN; Sigma-Aldrich) were used in this study. DMPP wasused as a reference drug in all tests. A mixture of Tween 80 (2%) andsaline was used as the vehicle. A 0.1-mL drug solution or vehicle per0.3 kg body weight (0.3 mL/kg) was administered intravenously(iv) while 0.2 mL per 0.2 kg body weight was administered orally. Forisolated organ assay, LQFM008 was dissolved in alcohol and usedimmediately to prevent oxidation while indomethacin was dissolvedin dimethyl sulfoxide (DMSO). All other drugs were dissolved in 0.9%NaCl. All reagents used were of analytical purity.

Femoral venous and artery catheterizationAccording to the surgical procedure described by Gustavo et al.

(2009), rats were anesthetized with halothane (2–3% halothane in100% O2). The general anesthesia was maintained by intravenousadministration of urethane (1.2 g/kg, Sigma-Aldrich). A catheter wasinserted both into the femoral vein for drug administration and intothe right femoral artery to measure mean arterial pressure (MAP) andheart rate (HR). The catheter was advanced through the back of theneck region to effect its chronic implantation. Body temperature wasmaintained at 37.0 ± 0.5 °C with a thermostatically controlled heatingtable. The trachea was catheterized to reduce airway resistance.

Preparation of aortic ringsThe thoracic aorta of rats was isolated, removed and cleaned of fat

and adherent connective tissues. Removal of the endothelium wasachieved mechanically by gently rubbing a wet cotton swab throughthe luminal surface of ring. The aortas were cut into ring segments of4 mm and mounted on two stainless-steel hooks and suspended in10 mL organ baths filled with modified Krebs–Henseleit solution(pH 7.4; 130 mM NaCl; 14.9 mM NaHCO3; 4.7 mM KCl; 1.18 mMKH2PO4; 1.17 mM MgSO4 · 7H2O; 1.6 mM CaCl2 · 2H2O; 5.5 mM glu-cose). This preparation was maintained with continuous supply of gasmixture (95% O2 and 5% CO2) at 37 °C. Aortic rings were passivelystretched to a resting tension of 1.5 g and allowed to achieve equilibri-um for 1 h, during which the bath fluid was changed every 15 min. Al-terations in tension baseline were recorded by isometric transducersconnected to a data acquisition system (World Precision Instruments,Sarasota, FL, USA). The integrity of the endothelium was examined byadding ACh 10 μM at the plateau of PHE 1 μM induced contraction.The aortic rings were considered endothelium-denuded, if therelaxation response to ACh 10 μM is b10% of the maximal PHE 1 μM-induced contraction while aortic rings were considered endothelium-intact, if the relaxation response to ACh 10 μM is N80% of themaximumPHE-induced contraction.

Recording arterial pressure, heart rate and renal blood flowRats were mounted prone on a stereotaxic apparatus (David Kopf

Instruments, Tujunga, CA, USA) with the incisor bar 11 mm below theinteraural line. The dorsal regions were opened to isolate both aorticand renal artery with a cotton swab. Arterial catheters were connectedto a pressure transducer attached to a bridge amplifier to registerblood pressure. An analog-to-digital converter (PowerLab System,ADInstruments Inc., Colorado Springs, CO, USA) was used for continu-ous recording of pulsatile pressure. Mean arterial pressure (MAP) andheart rate (HR)were determined by the pulsatile signal with Chart soft-ware (version 7.3.1; ADInstruments Inc.). Arterial blood flow (ABF)was measured with a miniature ultrasonic transit-time flow probes(Transonic Systems Inc.) placed around the aorta. Aortic vascularconductance (AVC) was calculated by using the formula ABF/MAP.Values of AVC and ABF were expressed in percentage [(Δ arterialvascular conductance (AVC) × 100 / AVC) and (Δ ABF × 100 / ABF),respectively].

Systolic blood pressure measurement by tail-cuff sphygmomanometer

Rats were acclimatized for approximately 5 to 6 h in the laboratoryat room temperature. Systolic blood pressure (SBP) in conscious NMWand SHR pre-warmed at approximately 37 °C was measured with atail-cuff sphygmomanometer. The apparatus included a restrainer, alatex tube containing a tail-cuff and a dual-channel recorder. A plethys-mographic device was used to detect pulse. This procedure wasrepeated over a period of 15 days during which SBP readings weretaken every 48 h. Readings on the last 7 days were accompanied byoral administration of vehicle (1 mL/kg) or LQFM008 (47.7 μmol/kg).The SBP of each animal per 48 h was a mean of at least three readingsusing the PowerLab System (ADInstruments Inc.).

Effects of LQFM008 on baselines of MAP, HR, SBP and diastolic blood pressure

After oral administration of vehicle (1 mL/kg) or LQFM008(47.7 μmol/kg), NMW rats were subjected to chronic catheterizationas described above to measure changes in the baselines of MAP, HR,SBP and diastolic blood pressure (DBP).

Temporal evaluation of the effects of LQFM008 on MAP and HR

NMW rats were subjected to surgical procedure to implant chroniccatheters as described earlier 24 h prior to oral administration of a single

-10.0-7.5-5.0-2.50.02.55.07.5

10.0Vehicle 1 mL/kg

LQFM008 47.7 µmol/kg

1 3 5 7 9 11 13 15(Days)

* #*

Period of daily oraladministrationof vehicleor LQFM008

Δ SB

P (m

mH

g)

Fig. 1. Time course of systolic blood pressure (SBP) in normotensiveWistar rats. Days 1–7showmeasurements (every 48 h) of SBP baseline valueswhile days 9–15 showSBP valuesfollowing daily oral administration of the vehicle (1 mL/kg) or LQFM008 (47.7 μmol/kg).Data are expressed as mean ± SEM (n = 6–7). *p b 0.05 compared to baseline;#p b 0.05 compared to the vehicle-treated group.

92 J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

dose of LQFM008 (47.7 μmol/kg). Temporal course of alteration in MAPand HR was later investigated.

Evaluation of the effects of LQFM008 on baroreflex sensitivity

Following chronic oral treatment with vehicle (1 mL/kg) orLQFM008 (47.7 μmol/kg), iv infusion of increasing doses of barore-flex activators (PHE 0.30–0.60 μg/kg and SN 3.0–6.0 μg/kg) in NMWrats was performed to test baroreflexive sensitivity. Peak increase anddecrease in MAP and HR were recorded after PHE or SN infusion. Datawere expressed as a function of the baroreflex effectiveness index.

In vivo investigation of the mechanisms of LQFM008 in cardiovascularaction

Femoral venous and artery catheterization were performed prior toiv administration of DMPP (0.2 mg/kg), vehicle (0.3 mL/kg), LQFM008(14.3 μmol/kg), WAY-100635 (0.1 mg/kg), atropine (9.0 mg/kg) andNOR (0.003 mg/kg). L-NAME was infused into the femoral vein witha pump (Insight LTDA, Monte Alto, SP, Brazil) at 3 μg/kg min− 1 for10 min. Drug combinations or pretreatment were used to elucidatepossible cardiovascular mechanisms.

Studies of vascular reactivity and mechanisms of LQFM008

To investigate the vascular effect of LQFM008, steady vessel tensionin endothelium-intact or -denuded aortic rings was induced by PHEprior to the cumulative addition of LQFM008 (10−8, 10−7, 10−6, 10−5

or 10−4 M). Possible mechanisms underlying the vascular function ofLQFM008 were investigated by incubating the aortic rings with indo-methacin (10 μM) or NG-nitro-l-arginine methyl ester (L-NAME)(100 μM) for 30 min. Incubation was followed by PHE (1 μM) inducedaortic ring contraction and the cumulative addition of LQFM008.

Statistical analysisStatistical analyses were performed using GraphPad Prism version

5.0 (GraphPad Software Inc., San Diego, CA, USA). Changes in cardiovas-cular parameters (MAP, HR and SBP) were expressed as the means ofnumber (n) of rats per group ± standard error of mean (SEM). Aorticvascular conductance (AVC) was calculated as the ratio of the ABF byMAP and expressed as a percentage of baseline values (100 [peakincrease − baseline value] / baseline). Unpaired Student's t-tests,ANOVA and appropriate post hoc tests (Dunnett's or Newman–Keuls')were used according to experimental models and data. P values lessthan 0.05 were considered statistically significant (Drummond andTom, 2011a,b).

Results

Effects of IV-infused LQFM008 on MAP, HR, ABF and AVC

The infusion of LQFM008 (7.3, 14.3 or 28.6 μmol/kg) caused a dose-dependent peak decrease in MAP [−21.1 ± 2.7 mm Hg (p b 0.05), −23.9± 4.7mmHg (p b 0.05) or−32.4± 8.3mmHg (p b 0.01), respec-tively; n = 5] without any alteration in HR (p N 0.05, n = 5). The infu-sion of DMPP (0.2 mg/kg) caused a peak increase in MAP (51.3 ±6.3 mm Hg, p b 0.01, n = 5) and a peak decrease in HR (94.02 ±37.7 bpm, p b 0.05, n = 5). LQFM008 elicited a dose-dependentreduction in AVC (22%, 32% or 38%, n = 5) while ABF remainedunchanged (p N 0.05, n = 5).

Effects of LQFM008 on SBP

Fig. 1 shows a temporal decrease in SBP of conscious NMW rats fol-lowingoral treatmentwith either the vehicle or LQFM008 47.7 μmol/kg.The reduction in SBP baseline was recorded on days 13 (p b 0.05) and

15 (p b 0.05). Statistical analysis of SBP values of vehicle-treatedgroup and LQFM008-treated group on day 15 demonstrated significantalteration (p b 0.05).

Effects of LQFM008 on baselines of MAP, HR, DBP and SBP

Chronic oral administration of LQFM008 (47.7 μmol/kg) in con-scious NMW rats, induced slight alteration in the MAP (vehicle-treatedgroup 111.5 ± 2.0 mm Hg versus LQFM008-treated group 104.7 ±1.3 mm Hg, p b 0.05, n = 5). The SBP was reduced by LQFM008treatment (118.2 ± 4.1 mm Hg, p b 0.05, n = 5) as compared to thevehicle-treated group (130.2 ± 2.2 mm Hg) while DBP of the rats wasreduced by LQFM008 (vehicle-treated group 103.7 ± 3.8 mm Hgversus LQFM008-treated group 92.5 ± 3.0 mm Hg, p b 0.05, n = 5).The HR remained unaltered by LQFM008 (LQFM008-treated group377.3 ± 23.6 bpm versus vehicle-treated group 349.6 ± 33.9 bpm,p N 0.05, n = 5).

Temporal effects of LQFM008 on MAP and HR

As shown in Fig. 2A, the acute oral administration of LQFM008(47.7 μmol/kg) in conscious NMW rats reduced MAP baseline(111.8 ± 3.1 mm Hg) from 30 to 150 min as compared to the baseline.Significant reduction in MAP values of the LQFM008-treated groupas compared to vehicle-treated group occurred after 30 min(LQFM008 −11.2 ± 1.0 mm Hg as compared to vehicle −0.3 ±0.8 mm Hg, p b 0.05), 60 min (LQFM008 −11.0 ± 2.0 mm Hg ascompared to vehicle −1.1 ± 1.0 mm Hg, p b 0.05), 90 min(LQFM008 −5.5 ± 0.9 mm Hg as compared to vehicle 2.0 ±0.9 mm Hg, p b 0.05), 120 min (LQFM008 −3.9 ± 1.0 mm Hg ascompared to vehicle 3.6 ± 2.0 mm Hg, p b 0.05) and 150 min(LQFM008 −2.4 ± 1.0 mm Hg as compared to vehicle 2.0 ±1.2 mm Hg, p b 0.05). The baseline HR was increased in the LQFM008-treated group after 60 and 90 min (p b 0.05; Fig. 2B). At 90 min, theLQFM008-treated group (98.0 ± 4.5 bpm) showed significantly in-creased baseline value of HR (327.9 ± 38.0 bpm) as compared to thevehicle-treated group 42.0 ± 12.0 bpm (p b 0.05; Fig. 2B).

Effects of LQFM008 on baroreflexive sensitivity

No significant alteration was detected in the baroreflex effectiveindex of the rats that were treated with LQFM008 (47.7 μmol/kg)prior to intravenous infusion of PHE and SN in conscious NMWrats (BI after PHE infusion: vehicle-treated group −1.9 ± 0.1 versusLQFM008-treated group −2.2 ± 0.2, p N 0.05, n = 5; BI after SNinfusion: vehicle-treated group 1.3 ± 0.3 versus LQFM008-treatedgroup 0.8 ± 0.2, p N 0.05, n = 5).

-15.0-12.5-10.0-7.5-5.0-2.50.02.55.07.5

Vehicle 1 mL/kg

LQFM008 47.7 µmol/kg

1 3 5 7 9 11 13 15

(Days)

Period of daily oraladministration of vehicle or LQFM008#*

Δ SB

P (m

mH

g)

Fig. 3. Time course of systolic blood pressure (SBP) in spontaneously hypertensive rats.Days 1–7 show measurements (every 48 h) of SBP baseline values while days 9–15show SBP values following daily oral administration of vehicle (1 mL/kg) or LQFM008(47.7 μmol/kg). Data are expressed as mean ± SEM (n = 6–7). *p b 0.05 compared tobaseline; #p b p b 0.05 compared to the vehicle-treated group.

DM

PP

Vehi

cle

LQFM

008

Atr

opin

e

WA

Y

NO

R

L N

AM

E

LQ

FM00

8

LQ

FM00

8

08 +

NO

R

LQ

FM00

8

-30 0 30 60 90 120 150-12

-8

-4

0

4

8

12 Vehicle

LQFM008 47.7 µmol/kg

Oral treatment

##

# ##***

**

A

B

Time (min)

-30 0 30 60 90 120 1500

40

80

120 #*

*

Time (min)

Δ M

AP

(mm

Hg)

Δ H

R (m

mH

g)

Fig. 2. Temporal changes (0–150 min) in (A) mean arterial pressure and (B) heart rateafter oral administration of vehicle (1 mL/kg) or LQFM008 (47.7 μmol/kg) at timepoint0 min. Data are expressed as the mean± SEM (n=5). *p b 0.05 compared with baseline;#p b 0.05 compared to the vehicle-treated group.

93J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

Effects of IV-infused LQFM008 on MAP and HR of SHR

SHR showed a basal MAP of 173 ± 5 mm Hg and HR of 422 ±18 bpm. The iv infusion of LQFM008 (7.3, 14.3 or 28.6 μmol/kg) inSHR reduced MAP [−2.3 ± 2.6 mm Hg, −14.3 ± 2.3 mm Hg(p b 0.001, n = 5) or −19.2 ± 3.8 mm Hg (p b 0.001, n = 5),respectively] and increased HR values [1.6 ± 3.7 bmp, 15.4 ± 4.9bmp (p N 0.05, n=5) or 25.5± 6.2 bmp (p N 0.01, n=5), respectively]in a dose-dependent manner.

Atr

opin

e +

WA

Y +

LQFM

0

L N

AM

E +

10

20

30

40

50 ** *

(mm

Hg)

Effects of LQFM008 on SBP of SHR

Oral administration of LQFM008 47.7 μmol/kg elicited a temporalreduction (days 9–15) in the SBP baseline (186 ± 8 mm Hg) of SHR(LQFM008-treated group; day 9, −0.3 ± 4.7; day 11, −2.0 ± 4.5; day13, −9.0 ± 5.5; day 15, −12.1 ± 3.1) compared to the vehicle-treated group (day 9, 0.8 ± 1.7; day 11, 1.7 ± 2.6; day 13, 1.1 ± 2.0;day 15, 0.6 ± 3.7; Fig. 3).

-40

-30

-20

-10

0

** *

#

##

Δ M

AP

Fig. 4. Effects of intravenous treatment of anesthetized normotensive rats with1,1-dimethyl-4-phenylpiperazinium iodide, vehicle (0.3 mL/kg), LQFM008 (14.3 μmol/kg),atropine (0.9 mg/kg), WAY-100635 (0.1 mg/kg) and norepinephrine 0.003 mg/kg onmean arterial pressure (MAP); and pretreatment with atropine (0.9 mg/kg), WAY-100635(0.1 mg/kg) or infusion of L-NAME (3 μg/kg/min for 10 min) on LQFM008 (4.5 mg/kg)induced changes in MAP. The figure also shows the influence of LQFM008 (14.3 μmol/kg)pretreatment on the pressor effect of NOR (0.003 mg/kg). Data are expressed asmean ± SEM (n = 5). *p b 0.05 compared to the vehicle-treated group; #p b 0.05compared to the LQFM008-treated group.

In vivo investigation of mechanism and effects of LQFM008

Fig. 4 shows significant increase in MAP (baseline value of 107.3 ±2.3 mm Hg) following iv infusion of either DMPP (Δ MAP value,41.9 ± 3.3 mm Hg) or NOR (33.0 ± 1.3 mm Hg) in anesthetizedNMW rats, while iv infusion of LQFM008 14.3 μmol/kg reduced thevalue of this parameter (Δ MAP value, −34.4 ± 2.7 mm Hg) as com-pared to the vehicle (Δ MAP value −1.9 ± 4.2 mm Hg). Pretreatmentwith atropine or WAY-100635 reduced MAP value of LQFM008 to −19.7 ± 3.7 mmHg and −18.1 ± 2.8 mmHg, respectively, while the in-fusion of L-NAME completely reversed the effect of LQFM008 on MAP(−2.6 ± 0.6 mm Hg). Treatment of animals with LQFM008 prior toNOR infusion did not affect the pressor effect of noradrenaline (MAP37.9 ± 1.3 mm Hg).

Vasorelaxant effects of LQFM008 and possible mechanisms involved

A representative tracing of the cumulative addition of LQFM008to the bath solution after sustained vascular tension of PHE pre-contracted aortic ring without and with endothelium is shown inFig. 5A and B, respectively. LQFM008 elicited concentration-dependentrelaxation (Fig. 5C) in aortic rings with endothelium [LQFM008 at10−7 M (2.6 ± 0.1%); 10−6 M (17.2 ± 5.6%); 10−5 M (41.3 ± 2.8%);10−4 M (76.0 ± 4.7%)]. Meanwhile, LQFM008 did not elicit any signifi-cant effect (p N 0.05) on the endothelium-denuded rings (Fig. 5C). Re-laxation induced by LQFM008 was not blocked by 30 min incubationof the endothelium-intact aortic rings with indomethacin (Fig. 5D).By contrast, L-NAME incubation significantly inhibited (p b 0.05)LQFM008-induced vasorelaxation at 10−5 M from 39.8 ± 4.3% to1.3 ± 0.5% and 10−4 M from 74.3 ± 6.0% to 24.3 ± 1.4% (Fig. 5D).

A B

C D

Phe (1 µM) 7

LQFM008 (-Log M)

0.3 g

10 min

8 6 5 4Phe (1 µM) 78 6 5 4

LQFM008 (-Log M)

-8 -7 -6 -5 -4

0

20

40

60

80

LQFM008 E+LQFM008 E-Vehicle E-Vehicle E+

*

*

LQFM008, Log [M]

Rel

axat

ion

(% P

HE

10-6

M)

-8 -7 -6 -5 -4

0

20

40

60

80

LQFM008 E++ Indomethacin E++ L-NAME E+

#

#

LQFM008, Log [M]

Rel

axat

ion

(% P

HE

10-6

M)

Fig. 5. Representative cumulative concentration–response tracing of LQFM008 on aortic rings (A) without endothelium (E−) and (B) with endothelium (E+) pre-contracted with phen-ylephrine, 10−6 M; (C) the line diagram shows log of molar concentration versus the response to LQFM008 or vehicle and (D) the effect of indomethacin (10 μM) or L-NAME (100 μM)incubation with endothelium intact aortic rings on LQFM008 induced relaxation. Values are expressed as mean ± SEM. *p b 0.05 compared to the vehicle-treated group; #p b 0.05compared to LQFM008-treated group.

94 J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

Discussion

Synthesis and chemical modification of compounds (Sałaga et al.,2014;White et al., 2014; Polepally et al., 2013) remain correct strategiestowards the development of a novel drugs with great potential for clin-ical application. The synthesis and neuroactivity of LQFM008 have beenextensively discussed in the previous study (De Brito et al., 2012). Inclinical studies, the prevalence of cardiovascular disease has been re-ported to be increased among people with anxiety disorders, thus thispsychiatric disorder is considered a risk factor of coronary heart disease.Overlapping symptoms of anxiety and hypertension could justify theuse of anxiolytic drugs for the treatment of hypertension (Taylor et al.,2006; Angrini et al., 1998). The present study therefore sought toinvestigate the cardiovascular response to LQFM008, an anxiolyticcompound.

The iv administration of LQFM008 in anesthetizedNMWrats eliciteda transient reduction in MAP. Except for the significant reduction inAVC, LQFM008 did not change either HR or ABF values in anesthetizedNMW rats. Temporal changes in SBP were observed in consciousNMW rats by using the tail-cuff method. Reduction in the SBP baselineof the LQFM008-treated group was observed on days 13 and 15, andthe effect of LQFM008 treatment was significant as compared to vehicletreatment on day 15. The process of animal preheating to facilitate vaso-dilation and detection of arterial pulsations (Ruben et al., 1982) couldelevate blood pressure and elicit stress in the tail-cuff method. Inorder to overcome unsuspected thermal stress in this model, consciousNMW rats were subjected to chronic implantation of catheters prior tomeasuring of MAP and HR. The use of animal model with minimumstress to evaluate the cardiovascular effects of an anxiolytic compoundsuch as LQFM008 is necessary since stress-induced changes in hemody-namic parameters may be sensitive to anxiolytic agent. Our findingsdemonstrate temporal reduction in MAP from 30 to 60 min of acute

oral administration of this piperazine derivative. The hypotensive effectdwindled progressively after 60min. Unlike the iv infusion of LQFM008in anesthetized NMW rats, a reduction in MAP was accompanied by asignificant increase in HR (from 60 to 90 min) in this model. As expect-ed, these results indicate that baroreflex is inhibited in anesthetizedrats. Further findings on the cardiovascular activity of LQFM008 suggestthat an overall decrease in MAP could be attributed to a reduction inDBP and SBP. The intensity of the effects of LQFM008 seems to be affect-ed by route of administration, the dose of LQFM008 and the state of theexperimental subject (conscious or anesthetized).

In the present studywe also investigated the antihypertensive effectof LQFM008 in an established model of hypertension by using SHR. Theiv infusion of LQFM008 loweredMAP and increasedHRof SHR in a dose-dependent manner. In the tail-cuff method, chronic oral administrationof LQFM008 elicited temporal reduction in SBP. On day 15, the effect ofLQFM008 was significant as compared to the vehicle-treated group.

The arterial baroreceptor elicits an influence on autonomic control ofcardiovascular function (Ruben et al., 1982; Toney et al., 2010). To in-vestigate any form of alteration in baroreflex sensitivity or the involve-ment of a baroreflex pathway in the cardiovascular effect of LQFM008,conscious NMW rats were subjected to chronic oral administration ofLQFM008 prior to iv infusion of increasing doses of baroreflex activators(PHE and SN). Since our data did not demonstrate any significantalteration in the baroreflex effectiveness index, we assumed that thehypotensive property of LQFM008 did not involve any baroreflexdysfunction or mechanism.

In the previous study, we demonstrated the participation of the5-HT1A receptor in the anxiolytic-like properties of LQFM008. SinceLQFM008 is a psychotropic compound, we hypothesized the involve-ment of the 5-HT1A receptor in its cardiovascular effect and thereforeinvestigated the effect of pretreatment with WAY-100635 (selectiveantagonist of 5-HT1A) on the hypotensive effect of LQFM008. Unlike a

95J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

complete blockade of anxiolytic property of this compoundbyNAN-190(non-selective antagonist of 5-HT1A), the hypotensive effect ofLQFM008 was reduced partially by WAY-100635 (Tancredi et al.,1997; Testa et al., 1999). Though central 5-HT1A receptors have beenimplicated in the hypotensive mechanism of (+) 8-OH-DPAT, DP-5-CT and R 28935, the presence of this receptor in the central and periph-eral nervous systems (Wedzony et al., 2007; Konturek et al., 2011)makes it difficult to conclude that the hypotensive property ofLQFM008 is centrally mediated.

In order to gain insight into the involvement of muscarinic receptorsin the hypotensive activity of LQFM008, NMW rats were pretreatedwith atropine (non-selective muscarinic receptor antagonist) prior toLQFM008 treatment. The effect of this compound was partially blockedatropine. This result suggests that muscarinic receptor is involved in theeffects of LQFM008. Since atropine could block any of the muscarinicM1, M2, or M3 receptor subtypes (Howell and Kovalsky, 1995), ourdata are not sufficient to attribute the cardiovascular property ofLQFM008 to a specific muscarinic receptor. In addition, iv administrationof LQFM008 prior to noradrenaline infusion did not alter the pressoreffect of this catecholamine. Hence, this result suggests that adrenergicreceptors were not involved in the hypotensive effect of LQFM008.

Studies have shown the contribution of NO to the hypotensive effectof drugs and in regulating blood pressure. An impaired NO bioactivity isan important component of hypertension (Hermann et al., 2006).Elevation of blood pressure has been reported in the mice with disrup-tion of the gene for endothelial NO synthase (Hermann et al., 2006). Thebeneficial effect of nebivolol (β-blocker) on endothelial function hasbeen attributed to its NO-releasing activity rather than β-blockade(McEniery et al., 2004). In order to evaluate the involvement of NO inthe hypotensive effect of LQFM008, rats were pretreated with continu-ous infusion of L-NAME (inhibitor of nitric oxide synthase—NOS) priorto the administration of LQFM008. Complete blockade of the hypoten-sive effect of LQFM008 by L-NAME pretreatment suggests the involve-ment of NO in the hypotensive property of LQFM008.

The vascular function of LQFM008was investigated by using the iso-lated organ method. In PHE-induced vascular contraction, LQFM008elicited concentration-dependent vasorelaxant. Results also showedthat the vascular effect of LQFM008 was attenuated by mechanical re-moval of endothelium and suggest that the vasorelaxation effect ofLQFM008 depends on the release of endothelium-derived relaxant fac-tors such as NO, PGI2 and/or an endothelium-derived hyperpolarizingfactor (EDHF). However, the incubation of endothelium-intact aorticrings with indomethacin (non-selective cyclooxygenase inhibitor) didnot block the vasodilation elicited by LQFM008, thus the vasorelaxationinduced by LQFM008 is unlikely to be mediated by prostaglandin re-lease. By contrast, the incubation endothelium-intact aortic rings witha NOS inhibitor L-NAME blocked vasorelaxation induced by LQFM008completely. Though LQFM008 could be involved in endothelial produc-tion and/or release NO, the level of ion channels and other signalingproteins' involvement in the vascular response to LQFM008 remainsto be fully investigated.

Although preclinical studies of LQFM008 demonstrated its anxiolyticproperty, present data indicate its therapeutic potential for the treat-ment of cardiovascular disease. Its short-acting effect may be relevantto diagnosing white coat hypertension (Mancia and Zanchetti, 2006),which has been reported in more than 20% of the hypertensive popula-tion (Pickering, 1996). In addition, this piperazine derivative could offera desirable therapeutic effects to the patient with the comorbidity ofanxiety and hypertension. Future researchwill be focused on the toxico-logical assessment of LQFM008 for possible clinical application.

Conclusion

Our findings demonstrated the hypotensive, antihypertensive andvasorelaxant properties of LQFM008. These cardiovascular effects sug-gest the involvement of serotoninergic, cholinergic and NO pathways.

Conflict of interest statement

The authors declare that no conflict of interest concerns the study described in thisarticle.

Acknowledgments

This study was supported by FAPEG, CAPES and CNPq.

References

AbramsWB. Pathophysiology of hypertension in older persons. Am J Med 1988;85:7–12.Alves FH, Crestani CC, Correa FM. The insular cortex modulates cardiovascular responses

to acute restraint stress in rats. Brain Res 2010;1333:57–63.Angrini M, Leslie JC, Shephard RA. Effects of propranolol, buspirone, pCPA, reserpine, and

chlordiazepoxide on open-field behavior. Pharmacol Biochem Behav 1998;59:387–97.

De Brito FA, Martins JLR, Fajemiroye OJ, Galdino PM, De LimaMCT, Menegatti R, et al. Cen-tral pharmacological activity of a new piperazine derivative: 4-(1-phenyl-1H-pyrazol-4-ylmethyl)-piperazine-1-carboxylic acid ethyl ester. Life Sci 2012;90:910–6.

Drummond GB, Tom BDM. How can we tell if frogs jump further? Br J Pharmacol 2011a;164:209–12.

Drummond GB, Tom BDM. Statistics, probability, significance, likelihood: words meanwhat we define them to mean. Br J Pharmacol 2011b;164:1573–6.

Elinor HC, Shlomo A, Eugene AM, Sydney S. Structure–activity requirements for hypoten-sion and α-adrenergic receptor blockade by analogues of atropine. Eur J Pharmacol1983;90:75–83.

Eric JT. Past the wall in cardiovascular R&D. Nat Rev Drug Discov 2009;8:259.Gustavo RP, Luciana RM, Sergio LC. Renal vasodilation induced by hypernatraemia: role of

alpha-adrenoceptors in the median preoptic nucleus. Clin Exp Pharmacol Physiol2009;36:83–9.

Helke CJ, McDonald CH, Phillips ET. Hypotensive effects of 5-HT1A receptor activation:ventral medullary sites and mechanisms of action in the rat. J Auton Nerv Syst1993;42:177–88.

Hermann M, Flammer A, Lüscher TF. Nitric oxide in hypertension. J Clin Hypertens 2006;8:17–29.

Howell RE, Kovalsky MP. Hypotensive effect of an M2-selective muscarinic antagonist inanaesthetized guinea-pigs. J Auton Pharmacol 1995;15:19–26.

Intengan HD, Schiffrin EL. Structure and mechanical properties of resistance arteries inhypertension: role of adhesion molecules and extracellular matrix determinants.Hypertension 2000;36:312–8.

Kalkman HO, Engel G, Hoyer D. Three distinct subtypes of serotonergic receptors mediatethe triphasic blood pressure response to serotonin in rats. J Hypertens 1984;2:143–5.

Konturek PC, Brzozowski T, Konturek SJ. Gut clock: implication of circadian rhythms inthe gastrointestinal tract. J Physiol Pharmacol 2011;62:139–50.

Ma S, Morilak DA. Norepinephrine release in medial amygdala facilitates activation of thehypothalamic–pituitary–adrenal axis in response to acute immobilisation stress. JNeuroendocrinol 2005;17:22–8.

Mancia G, Zanchetti A. White-coat hypertension: misnomers, misconceptions andmisunderstandings. What should we do next? J Hypertens 2006;14:1049–52.

McEniery CM, Schmitt M, Qasem A, Webb DJ, Avolio AP, Wilkinson IB, et al. Nebivolol in-creases arterial distensibility in vivo. Hypertension 2004;44:305–10.

Michele MC, Jose ET, Raquel FG. Matrix metalloproteinases: targets for doxycycline toprevent the vascular alterations of hypertension. Pharmacol Res 2011;64:567–72.

Miyawaki T, Goodchild AK, Pilowsky PM. Rostral ventral medulla 5-HT1A receptorsselectively inhibit the somatosympathetic reflex. Am J Physiol Regul Integr CompPhysiol 2001;280:1261–8.

Ohlstein EH. The grand challenges in cardiovascular drug discovery and development.Front Pharmacol 2010;1:125.

Pickering TG. White coat hypertension. Curr Opin Nephrol Hypertens 1996;5:192–8.Polepally PR, White K, Vardy E, Roth BL, Ferreira D, Zjawiony JK. Kappa-opioid receptor-

selective dicarboxylic ester-derived salvinorin A ligands. Bioorg Med Chem Lett2013;23:2860–2.

Resstel LB, Joca SR, Guimaraes FG, Correa FM. Involvement of medial prefrontal cortexneurons in behavioral and cardiovascular responses to contextual fear conditioning.Neuroscience 2006;143:377–85.

Ruben D, Bunag MA, Jason B. Tail-cuff blood pressure measurement without externalpreheating in awake rats. Hypertension 1982;4:898–903.

Sałaga M, Polepally PR, Sobczak M, Grzywacz D, Kamysz W, Sibaev A, et al. Novel orallyavailable Salvinorin A analog PR-38 inhibits gastrointestinal motility and reducesabdominal pain in mouse models mimicking irritable bowel syndromes. J PharmacolExp Ther 2014;350:69–78.

Scher A. Textbook of physiology, circulation, respiration, body fluids, metabolism, andendocrinology. Philadelphia, PA: W.B. Saunders Co.; 1989. p. 972–90.

Tancredi A, Conte C, Chaput C, Spedding M, Millan MJ. Inhibition of the constitutiveactivity of human 5-HT1A receptors by the inverse agonist, spiperone but not theneutral antagonist, WAY 100,635. Br J Pharmacol 1997;120:737–9.

Taylor FB, Lowe K, Thompson C, Mc-Fall MM, Peskind ER, Kanter ER, et al. Daytimeprazosin reduces psychological distress to trauma specific cues in civilian traumaposttraumatic stress disorder. Biol Psychiatry 2006;59:577–81.

Testa R, Guarneri L, Poggesi E, Angelico P, Velasco C, Ibba M, et al. Effect of several5-hydroxytryptamine(1A) receptor ligands on the micturition reflex in rats:comparison with WAY 100635. J Pharmacol Exp Ther 1999;290:1258–69.

96 J.O. Fajemiroye et al. / Life Sciences 112 (2014) 90–96

Thomas MC, Steven DC. Kidney in hypertension—guyton redux. Hypertension 2008;51:811–6.

Toney GM, Pedrino GR, Fink GD, Osborn JW. Does enhanced respiratory–sympatheticcoupling contribute to peripheral neural mechanisms of AngII-salt hypertension?Exp Physiol 2010;95:587–94.

Wedzony K, Chocyk A, Kolasiewicz W, Mackowiak M. Glutamatergic neurons of ratmedial prefrontal cortex innervating the ventral tegmental area are positive forserotonin 5-HT1A receptor protein. J Physiol Pharmacol 2007;58:611–24.

White KL, Scopton AP, Rives M, Bikbulatov RV, Polepally PR, Brown PJ, et al. Identification ofnovel functionally selective k-opioid receptor scaffolds. Mol Pharmacol 2014;85:83–90.