The Newly Synthesized Pyrazole Derivative 5-(1-(3 Fluorophenyl)-1H-Pyrazol-4-yl)-2H-Tetrazole Reduces Blood Pressure of Spontaneously Hypertensive Rats via NO/cGMO Pathway.

The search for new antihypertensive drugs has grown in recent years because of high rate of morbidity among hypertensive patients and several side effects that are associated with the first-line medications. The current study sought to investigate the antihypertensive effect of a newly synthesized pyrazole derivative known as 5-(1-(3 fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM-21). Spontaneously hypertensive rats (SHR) were used to evaluate the effect of LQFM-21 on mean arterial pressure (MAP), heart rate (HR), renal vascular conductance (RVC), arterial vascular conductance (AVC), baroreflex sensitivity (BRS) index, and vascular reactivity. Acute intravenous (iv) administration of LQFM-21 (0.05, 0.1, 0.2, and 0.4 mg kg-1) reduced MAP and HR, and increased RVC and AVC. Chronic oral administration of LQFM-21 (15 mg kg-1) for 15 days reduced MAP without altering BRS. The blockade of muscarinic receptors and nitric oxide synthase by intravenous infusion of atropine and L-NAME, respectively, attenuated cardiovascular effects of LQFM-21. In addition, ex vivo experiments showed that LQFM-21 induced an endothelium-dependent relaxation in isolated aortic rings from SHR. This effect was blocked by guanylyl cyclase inhibitor (ODQ) and L-NAME. These findings suggest the involvement of muscarinic receptor and NO/cGMP pathway in the antihypertensive and vasodilator effects of LQFM-21.


INTRODUCTION
Blood pressure control is a complex process that involves variety of organ systems, such as cardiovascular system, central nervous system, adrenal glands, and kidneys (Coffman and Crowley, 2008). Several classes of drugs (beta and alpha blockers, angiotensin converting enzyme inhibitors, calcium channel blockers, diuretics, etc.) are currently being used for the treatment of hypertensive patients in order to reduce cases of morbidity and mortality that are associated with cardiovascular diseases (Law et al., 2009). These classes of drugs are often used in combination for several reasons. However, the cases of side effects that are associated with some of these antihypertensive drugs have led to their use limitations for some patients. Hence, there are needs for the development of new drugs with desirable pharmacological and therapeutic profiles to control arterial hypertension (Manso et al., 2015).
Previous studies have shown that the new LQFM-21 compound has anti-inflammatory, analgesic, and antinociceptive properties (Florentino et al., 2015(Florentino et al., , 2016(Florentino et al., , 2017de Moura et al., 2017). Although this prototype elicited vasorelaxant activity in the isolated arteries (Ramos Martins et al., 2013), the effect of this compound on cardiovascular parameters such as arterial blood pressure, heart rate (HR), and renal and aortic blood flow remains unknown. So, we hypothesized a possible antihypertensive effect of LQFM-21. Therefore, the present study sought to evaluate the antihypertensive and vasodilatory effects of LQFM-21 in spontaneously hypertensive rats (SHR).

Animals
All experiments were conducted on adult male SHRs or Wistar normotensive rats (NTRs) (280-350 g). Rats were obtained from the central animal house of the Federal University of Goiás. Experimental procedures were designed in strict adherence to the National Health Institute Guidelines for Care and Use of Laboratory Animals and approved by the Ethics Committee on the Use of Animals of Federal University of Goiás (protocol number 034/12).

Measurement of Systolic Blood Pressure With Tail-Cuff Sphygmomanometer
Spontaneously hypertensive rat(s) and NTR(s) were acclimatized for about 5-6 h in the laboratory at room temperature. Systolic blood pressure (SBP) and HR were measured with a tail-cuff sphygmomanometer with mercury (Harvard Apparatus, MA, United States) in conscious rats pre-warmed at approximately 37 • C. This is a non-invasive and indirect method of measuring SBP. The apparatus set up include a restrainer, a tail cuff containing latex tube, and a dual channel recorder. It is pertinent to acknowledge the inherent limitations such as stress that is associated with the use of tail-cuff sphygmomanometer to measure the pressure of conscious SHR and NTR(s).

Baroreflex Sensitivity Test
The BRS was evaluated through intravenous infusion of phenylephrine (3, 6, and 12 mg kg −1 ; Sigma-Aldrich) and sodium nitroprusside (16, 32, and 64 mg kg −1 , Sigma-Aldrich) in unanesthetized SHR(s) and NTR(s). BSI index was later calculated as a ratio of changes in HR and MAP.

Surgical Procedures
Spontaneously hypertensive rat(s) and NTR(s) were anesthetized with halothane (2-3% in O2; Tanohalo; Cristália, Brazil). The right femoral vein and artery were catheterized for drug administration and blood pressure recording, respectively. After catheterization, anesthesia was maintained by intravenous administration of urethane (1.2 g kg −1 b.wt., i.v.; Sigma-Aldrich, St. Louis, MO, United States). The trachea was cannulated to facilitate breathing. Subsequently, rats were placed on stereotactic apparatus and retroperitoneal incisions were performed. Miniature ultrasonic transit time flow probes (Transonic Systems Inc., Ithaca, NY, United States) were placed around the left renal artery and abdominal aorta to record renal blood flow (RBF) and aortic blood flow (ABF), respectively. Body temperature was maintained at 37.0 ± 0.5 • C on a thermostatically controlled bench. Following stabilization of cardiovascular parameters, LQFM-21 (0.05; 0.1; 0.2; 0.4 mg kg −1 ) or vehicle (10% DMSO and 2% Tween in 0.9% NaCl) were randomly administered in SHR(s) and NTR(s).

Recording of Arterial Blood Pressure, Renal and Aortic Blood Flow
The pulsatile arterial pressure (PAP) was continuously recorded through the arterial cannula that was connected to a pressure transducer (MLT0380, ADInstruments, Bella Vista, NSW, Australia) with an amplifier (Bridge Amp, ML221, ADInstruments, Bella Vista, NSW, Australia). The miniatures probes were placed around left renal artery and thoracic aorta and connected to T206 flowmeter (Transonic Systems, Inc., Ithaca, NY, United States) to record the RBF and ABF, respectively. Data were digitized at a frequency of 2000 samples per second using an analogue to digital converter (PowerLab 4/25, ML845, ADInstruments, Bella Vista, NSW, Australia).

Effect of L-Name (Nitric Oxide Synthase Inhibitor) and Atropine (Muscarinic Receptors Antagonist) Pretreatment
Spontaneously hypertensive rat(s) group (n = 8) were pretreated intravenously with L-Name (0.3 mg kg −1 , N ω -nitro-L-arginine methyl ester hydrochloride, Sigma-Aldrich, nitric oxide synthase inhibitor) prior to the measurement of cardiovascular parameters. In a separate experiment, SHR(s) group (n = 8) were pretreated intravenously with atropine (0.9 mg kg −1 , muscarinic receptors antagonist) prior to the measurement of cardiovascular parameters.

Data Analysis
Mean arterial pressure (MAP) was calculated with weighted average from PAP signal (2/3 diastolic pressure + 1/3 systolic pressure) using the software PowerLab 4/25 (ML845, ADInstruments, Bella Vista, NSW, Australia). HR was calculated as instantaneous frequency from the PAP signal (PowerLab 4/25, ML845, ADInstruments, Bella Vista, NSW, Australia). Changes in RBF and ABF were calculated as the percentage relative ratio to baseline (% RBF and % ABF). The renal vascular conductance (RVC) and aortic vascular conductance (AVC) were obtained by the ratio of RBF MAP −1 and ABF MAP −1 , respectively. The variations in RVC and AVC were expressed as percentage of change from baseline value (% RVC and % AVC). Statistical analysis was performed using GraphPad Prism version 6.01 (GraphPad Software Inc., San Diego, CA, United States). Data were analyzed by two-way analysis of variance followed by Newman-Keuls post-test. The differences in baseline and BSI between groups were analyzed using unpaired Student's t-test. A value of p < 0.05 was considered significant.

DISCUSSION
The limitations of current antihypertensive therapies have stimulated research and development of new classes of drugs. Some of these new drugs could facilitate better control of blood pressure, high tolerability, and effective prevention of cardiovascular diseases (Ménard, 1993;Tamargo et al., 2015). Synthesis and modification of compounds remain a feasible alternative strategy for new drug development strategies (Wróblewska et al., 2013). Previous data on pyrazole derivatives on voltage-dependent calcium channels (Winters et al., 2014) and vascular function (Carrión et al., 2008;Ramos Martins et al., 2013) suggested potential hemodynamic actions of these class of compounds. However, no study has been conducted in an experimental model of essential hypertension before showed an antihypertensive property of these compounds. So, the present study evaluated the hypotensive property of LQFM-21 and To address these aims, the SHR model of hypertension, a gold standard for experimentally studying essential hypertension, was used to investigate the antihypertensive effect of LQFM-21. The spontaneous rise in the blood pressure of SHR shares common characteristics to human hypertension (Head, 1989;Tsuda and Masuyama, 1991). Hypertension in SHR has been attributed to an increase in sympathetic adrenergic activity (Head, 1989;Behuliak et al., 2011). Arribas et al. (1994) demonstrated that β-adrenoceptor-mediated relaxation of the aortic rings in SHR pre-treated with norepinephrine was less than that of NTRs. This difference was associated with impaired endothelial function.
In the present study, intravenous administration of LQFM-21 caused a decrease in MAP, and an increase in vascular conductance in hypertensive and normotensive animals. In addition, chronic oral treatment this compound decreased SBP significantly as compared to vehicles treated groups. Moreover, in SHR models, the blockade of cardiovascular effects of this compound produced by L-NAME and atropine suggest the involvement of nitric oxide synthase and muscarinic receptors in its antihypertensive mechanisms. Furthermore, ACh/NO/cGMP pathway could be proposed as the underlining mechanism for the hypotensive and antihypertensive effects elicited by LQFM-21. This is in agreement with França-Silva et al. (2012b) where authors demonstrated that the NDBP, a new organic nitrate, also reduces blood pressure via activation of Ach receptors in both the heart and the vessels.
Atropine is a non-selective antagonist of muscarinic receptors with wide applications particularly as a research tool. In the present study, atropine at the dose of 0.15 mg/kg attenuated the effect of LQFM-21 and thereby suggest the involvement of muscarinic receptors. Subtypes of muscarinic receptors may be linked to excitatory G proteins (M1, M3, and M5), as well as to   inhibitory G proteins (M2 and M4). Parasympathetic stimulation and circulating substances often activate muscarinic receptors to elicit reduction of cardiac frequency, force of cardiac contraction, and relaxation of peripheral blood vessels (Ventura et al., 2010). The reduction in these parameters have been associated with the mechanism of antihypertensive drugs. Both cardiac and vascular functions of drugs are important to antihypertensive effects (van Zwieten and Doods, 1995).
The circulating substances and M3 receptors (with greater expression in blood vessels) interaction could activate Gq which in turn induces the activation of phospholipase C. These events promote the hydrolysis of membrane phosphoinositides to diacylglycerol and inositol triphosphate (Sandiford and Slepak, 2009) to induce vasodilation. On the other hand, the activation of inhibitory G protein-bound muscarinic receptor such as M2 (M2 receptor with higher expression in the heart) (Caulfield, 1993) inactivate adenyl cyclase and reduce intracellular levels of cyclic AMP. In addition, studies showed an action of M2 receptors with the activation of endothelium nitric oxide synthase (eNOS) in cardiomyocytes of several mammalian species (Feron et al., 1999;Rocha et al., 2015). Altogether, the signaling pathways involving muscarinic receptors remain important pharmacological targets in the treatment of hypertension.
Hence, our findings, which implicate the involvement of nitric oxide synthase and muscarinic receptors in MAP reduction and vasodilatation effect of LQFM-21, provide potential therapeutic windows for the treatment of some hypertensive patients as a monotherapy or in combination with other classes of antihypertensive drugs. This could be of clinical interest in terms of the dose requirement and faster therapeutic effect with little or no side effects. Rapid therapeutic effects of LQFM-21 could greatly improve patient adherence to prescription.
In addition to the mechanisms involved in the vascular function, some antihypertensive drugs could interfere with normal baroreceptors function (Toney et al., 2010) to regulate blood pressure. The reflex response induced by baroreceptor modulates sympathetic activity to the heart, blood vessels, and adrenal medulla. Stimulation of baroreceptors often results in a decrease in sympathetic tone to heart and blood vessels (Cravo et al., 2006). In the present study, the oral chronic administration of LQFM-21 did not alter baroreceptor reflex sensitivity in SHR. This result rule out the involvement of baroreflex pathways.
Moreover, LQFM-21 induced vascular relaxation in isolated aortic rings of SHR. These data suggest a direct effect of this compound on blood vessel. Of note, the vasorelaxation induced by LQFM-21 was blocked by nitric oxide synthase and guanylate cyclase inhibitors. This result is consistent with current in vivo blockade of cardiovascular effect by L-NAME. Hence, the vascular effect of this compound could be attributed to NO/cGMP mechanism. Other studies seeking for new compounds have also documented that the NO/cGMP pathway is involved in the hypotensive effect of those compounds (França-Silva et al., 2012a;Dantas et al., 2014;Mendes-Júnior et al., 2015).
Taken together, our results present the first in vivo and ex vivo antihypertensive and vasorelaxation effects of LQFM-21. These findings suggest the involvement of nitric oxide and muscarinic receptors in the antihypertensive effects of this compound. However, further studies are required to confirm its therapeutic efficacy and safety profile of LQFM-21.