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. 2019 Sep 4;2(5):353–360. doi: 10.1021/acsptsci.9b00039

Cardiometabolic and Inflammatory Benefits of Sympathetic Down-Regulation with Zamicastat in Aged Spontaneously Hypertensive Rats

Bruno Igreja †, Nuno Pires †, Ana Loureiro †, Lyndon Wright †, Patrício Soares-da-Silva †,‡,§,*
PMCID: PMC7089015  PMID: 32259069

Abstract

graphic file with name pt9b00039_0005.jpg

The hyperactivity of the sympathetic nervous system (SNS) plays a major role in the development and progression of several cardiovascular diseases. One strategy to mitigate the SNS overdrive is by restricting the biosynthesis of norepinephrine via the inhibition of dopamine β-hydroxylase (DBH). Zamicastat is a new DBH inhibitor that decreases norepinephrine and increases dopamine levels in peripherally sympathetic-innervated tissues. The cardiometabolic and inflammatory effects of sympathetic down-regulation were evaluated in 50 week old male spontaneously hypertensive rats (SHRs) receiving zamicastat (30 mg/kg/day) for 9 weeks. After 8 weeks of treatment, the blood pressure (BP) and heart rate (HR) were assessed by tail cuff plethysmography. At the end of the study, 24 h urine, plasma, heart, and kidney were collected for biochemical and morphometric analyses. Zamicastat-induced sympathetic down-regulation decreased the high BP in SHRs, with no observed effect on HR. The heart-to-body weight ratio was lower in SHRs treated with zamicastat, whereas the body weight and kidney-to-body weight ratio were similar between both SHR cohorts. Zamicastat-treated SHRs showed reduced 24 h urine output, but the urinary amount of protein excreted and creatinine clearance rate remained unchanged. Zamicastat treatment significantly decreased plasma triglycerides, free fatty acids, and aspartate aminotransferase levels. Aged SHRs showed higher plasma levels of inflammatory markers as compared with age-matched normotensive Wistar-Kyoto rats. The inflammatory benefits attained with DBH inhibition were expressed by a decrease in CRP, MCP-1, IL-5, IL-17α, GRO/KC, MIP-1α, and RANTES plasma levels as compared with untreated SHRs. In conclusion, DBH inhibition decreased norepinephrine levels, reduced end-organ damage, and improved cardiometabolic and inflammatory biomarkers in aged male SHRs.

Keywords: sympathetic nervous system, dopamine β-hydroxylase, norepinephrine, cardiovascular, hypertension

Cardiovascular diseases (CVDs) are one of the uppermost causes of death in the world, representing ∼31% of all deaths.1 This is in line with the most recent data from the American Heart Association, where ∼1 out of 3 deaths are attributable to CVD.2 The prevalence of CVDs may be reduced by changing behavioral risk factors, like tobacco use, physical inactivity, unhealthy diet, alcohol abuse, and stressful lifestyle, and by treating other risk factors such as hypertension, diabetes, and hyperlipidemia. Although there are several options to treat hypertension, ∼50% of patients remain with high blood pressure (BP).3 Thus there is still an unmet medical need in the treatment of CVDs, namely, hypertension and its comorbidities.

There are several clinical conditions, like essential hypertension, congestive heart failure, obesity, metabolic syndrome, and chronic renal failure, among others, that present as a pathophysiological hallmark an overactivity of the sympathetic nervous system (SNS),4−9 which involves increased spillover of norepinephrine (NE) in specific organs, such as the heart, kidney, and skeletal muscle vasculature.

The modulation of the SNS is a putative approach for the treatment of certain CVDs, such as hypertension and heart failure, where a reduction in the sympathetic tone could be beneficial. The SNS modulates BP by changing the cardiac output, peripheral vascular resistance, and renal function. One strategy to mitigate sympathetic nerve function is decreasing the biosynthesis of NE by inhibiting the enzyme dopamine β-hydroxylase (DBH; EC 1.14.17.1) that catalyzes the conversion of dopamine (DA) into NE in sympathetic nerves.10 This approach also increases DA levels,11,12 which improves the renal function by causing renal vasodilatation and inducing diuresis and natriuresis.13−15 Several DBH inhibitors have been thus far reported,16−18 but none achieved marketing approval because they were found to have low potency, poor selectivity for DBH, or significant adverse effects.19,20

Zamicastat (development code: BIA 5-1058) is a new-generation DBH inhibitor in development by BIAL-Portela & Ca, S.A. as a new putative drug therapy for CVDs that decreases NE levels in sympathetically innervated tissues, and it is currently in Phase-I clinical trials.21 It was previously shown that zamicastat is a reversible DBH inhibitor22 that dose-dependently reduces high BP in spontaneously hypertensive rats (SHRs).23

There are several clinical conditions that arise that are associated with hypertension and that also present, as a pathophysiological hallmark, an overactivity of the SNS.4−9 During the development of hypertension and associated end-organ damage, there is an increased proinflammatory response. For this reason, a wide range of variables associated with inflammation, cardiometabolism, and tissue remodeling were evaluated in aged (i.e., 50 weeks old) SHRs. The aim of the present study was to evaluate the effect of prolonged zamicastat-induced DBH inhibition beyond a BP decrease. For that purpose, we used male aged SHRs, a well-defined preclinical model for human essential hypertension and heart failure24 that has been widely used in the study and development of novel antihypertensive drugs,25−30 and age-matched Wistar Kyoto rats (WKYs) as normotensive counterparts.

Materials and Methods

Animal and Treatment Groups

Fifty week old male SHRs and WKYs (Charles River Laboratories International, Kingston, NY) were individually housed in macrolon cages (Tecniplast, Varese, Italy) with free access to rodent diet (2014 Teklad Global Rodent Diets, Envigo, Barcelona, Spain) and tap water under controlled environmental conditions in a colony room (12 h light/dark cycle, room temperature 22 ± 2 °C, and relative humidity 50 ± 20%). All animal procedures conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, the Portuguese law on animal welfare (Decreto-Lei 113/2013 and 1/2019), and the Guide for the Care and Use of Laboratory Animals (8th ed., 2011). Zamicastat was synthesized in the Laboratory of Chemistry of BIAL-Portela & Ca, S.A. All other chemicals and materials were from Merck (Darmstadt, Germany), unless otherwise noted.

At the start of the experimental protocol, SHRs were randomly assigned to rodent meal (powdered) diet (n = 6) or meal diet supplemented with 30 mg/kg body weight zamicastat (n = 6) group for 9 weeks. The daily dose of zamicastat was chosen based on the pharmacodynamic effect of the single-dose administration of zamicastat23 and on previous work from our laboratory.31 The zamicastat dose was calculated based on individual body weights recorded once a week and on food consumption evaluated three times per week. The age-matched WKY counterparts (n = 6) received rodent meal diet and served as normotensive controls. A second cohort, with five animals per experimental group, was used to evaluate the effect on NE and DA levels in the heart left ventricle and the brain frontal cortex after zamicastat administration under the same dose regimen.

BP and HR Measurements

Animals were handled and placed for 5 min in the restrainer for a period of 5 days prior to the start of noninvasive tail cuff plethysmography BP and HR measurements to minimize distress. After 8 weeks of treatment, BP and HR measurements were made with a Pressure Scanner LE 5700/4 and a Pressure Computer LE 5700 (LETICA Scientific Instruments, Barcelona, Spain); animals were kept warm, and the tail was rinsed with 70% (v/v) ethanol to improve sensitivity. All experiments were carried out during daylight hours, alternating one animal from each group.

Renal Function

After cardiovascular measurements, animals were placed in individual metabolic cages (Tecniplast) on the day before the 24 h urine collection for analysis. Creatinine, urea, and glucose were determined using enzymatic colorimetric tests (HUMAN Diagnostics, Wiesbaden, Germany; refs 10051, 10505, and 10260, respectively). The level of proteins in urine was determined by a photometric assay (Bio-Rad, Hercules, CA). All analyses were run in duplicate according to the manufacturer’s instructions.

Remodeling Evaluation

One week later, at the end of the study, pentobarbital sodium at 60 mg/kg body weight/2 mL was given intraperitoneally. The heart (ventricles + septum) and left kidney were collected, blotted dry, and weighed. The heart- and kidney-to-body weight ratios were calculated and used as an index of cardiac and renal hypertrophy, respectively.

Cardiometabolic and Inflammatory Markers

Blood samples were withdrawn from each animal at the end of study for the quantification of plasma C-reactive protein (CRP, CRP ELISA kit (ERCRP), ThermoFisher Scientific, Waltham, MA), monocyte chemoattractant protein 1 (MCP-1, rat MCP-1 ELISA kit (KRC1012), Invitrogen, Carlsbad, CA), insulin (kit 80-INSRT-E01, Alpco, Salem, NH), and leptin (Mouse/Rat Leptin Quantikine ELISA Kit MOB00, R&D Systems, Minneapolis, MN) by EIA/ELISA kits. Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and glucose were determined using enzymatic colorimetric tests (HUMAN Diagnostics, refs 12012, 12011, and 10260, respectively). A panel of 23 cytokines was evaluated using a Pro Rat Cytokine 23-plex assay from Bio-Rad (ref 171-K1001M). All analyses were run in duplicate according to the manufacturer’s instructions.

Lipids

The lipid profile was evaluated in serum or plasma at the end of the study. Levels of serum triglycerides and total cholesterol were evaluated using enzymatic colorimetric tests (Human Diagnostics, refs 10725 and 10028, respectively). Serum free fatty acids (FFAs; ref 11383175001, Roche Diagnostics, Mannheim, Germany) and plasma keton bodies (KBs, β-HBA; Instruchemie, AB Delfzijl, The Netherlands, ref 2940) were evaluated using enzymatic colorimetric assays. All analyses were run in duplicate according to the manufacturer’s instructions.

Exposure Evaluation

Zamicastat and its metabolites BIA 5-453 and BIA 5-961 were quantified in plasma, and 24 h urine collected at the end of the study. All compounds were quantified by high-pressure liquid chromatography coupled to a tandem mass spectrometer, as previously described.31

Catecholamine Evaluation

A second cohort of animals, under the same dose regimen, was used for the tissue catecholamine evaluation. The heart left ventricle and brain frontal cortex were collected and placed overnight in perchloric acid 0.2 M. The quantification of NE and DA was performed by high-pressure liquid chromatography coupled to electrochemical detection, as previously described.31

Statistical Analysis

Data are reported as the mean ± SEM. Data analyses were performed using Prism 6 (GraphPad Software, San Diego, CA). All data were compared using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test. P values <0.05 were considered statistically significant.

Results

Pharmacokinetic and Pharmacodynamic Evaluation

Zamicastat was supplemented ad libitum in meal food. The average daily dose attained for the entire experimental period was 30.1 ± 1.5 mg/kg body weight/day. The levels of zamicastat and its metabolites, BIA 5-453 and BIA 5-961, were quantified in plasma and urine. As depicted in Figure 1, the major compound found in plasma was zamicastat (157.8 ± 19.1 ng/mL), whereas in urine the most abundant compounds were BIA 5-453 and BIA 5-961 (887.6 ± 179.8 and 871.3 ± 149.1 μg/24 h urine, respectively).

Figure 1.

Figure 1

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Levels of zamicastat and its metabolites BIA 5-453 and BIA 5-961 in (A) plasma and (B) urine at the end of the study. Data are shown as the mean ± SEM (n = 6 per group).

The effect of zamicastat on catecholamine levels was evaluated in the heart left ventricle and the brain frontal cortex. As shown in Figure 2, zamicastat treatment significantly reduced NE and increased DA levels in the heart but not in the frontal cortex.

Figure 2.

Figure 2

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Effect of zamicastat (Zami) on norepinephrine (NE) and dopamine (DA) levels in the (A,C) heart left ventricle and the (B,D) brain frontal cortex. Data are shown as the mean ± SEM (n = 4 to 5 per group). Comparisons against the vehicle-SHR (SHR_Veh) group were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test. P values <0.05 (*) were considered statistically significant.

Cardiovascular Evaluation

Control SHRs showed an increased systolic and diastolic BP as compared with the age-matched WKY group (Figure 3). Eight weeks after the beginning of the experimental protocol, SHRs treated with zamicastat showed a lower systolic (183 ± 8 vs 224 ± 5 mmHg, p = 0.0001) and diastolic (99 ± 11 vs 132 ± 3 mmHg, p < 0.0035) BP as compared with the control SHR group, whereas the HRs were similar (388 ± 7 vs 400 ± 5 beats/min, p = 0.3171) between groups (Figure 3).

Figure 3.

Figure 3

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Effect of zamicastat (Zami) on the (A) systolic and (B) diastolic blood pressure and on the (C) heart rate of SHRs after 8 weeks of treatment. Data are shown as the mean ± SEM (n = 6 per group). Comparisons against the vehicle-SHR (SHR_Veh) group were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test. P values <0.05 (*) were considered statistically significant.

At the end of the study, the heart-to-body weight ratio was assessed in all animals (Figure 4A). Control SHRs had a significant cardiac hypertrophy, defined as a higher heart-to-body weight ratio, as compared with the WKY group (3.66 ± 0.08 vs 2.88 ± 0.05 mg/g body weight, respectively, P < 0.0001). Interestingly, SHRs receiving zamicastat showed a significantly lower heart-to-body weight ratio as compared with the control SHR group (3.45 ± 0.04 vs 3.66 ± 0.08 mg/g body weight, respectively, P = 0.0219). On the contrary, the kidney-to-body weight ratio was significantly higher in both SHR groups as compared with the WKYs, and there were no differences between both SHR groups (Figure 4B).

Figure 4.

Figure 4

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(A) Cardiac and (B) renal hypertrophy observed at the end of the study. Data are shown as the mean ± SEM (n = 6 per group). Comparisons against the vehicle-SHR (SHR_Veh) group were performed using one-way analysis of variance (ANOVA), followed by the Fisher’s least significant difference (LSD) test. P values <0.05 (*) were considered statistically significant.

Renal Function

As compared with control SHRs (13.5 ± 1.6 mL/24 h), 8 weeks of zamicastat dosing significantly reduced the 24 h urine excretion to levels similar to the WKY group (8.9 ± 1.2 vs 8.8 ± 1.2 mL/24 h, respectively). Nevertheless, the creatinine clearance rate was identical in all groups, as shown in Table 1. Both SHR groups showed proteinuria compared with the WKY group (9.1 ± 1.1 mg/24 h), and the difference between treated and nontreated SHR groups did not attain statistical significance (39.3 ± 4.8 vs 45.6 ± 8.2 mg/24 h, respectively). Likewise, both groups of SHRs showed a higher urinary excretion of urea and glucose when compared with the WKY group, with no significant difference between treated and nontreated SHRs, as depicted in Table 1.

Table 1. Renal Function at the End of the Studya.

  vehicle-SHR zamicastat-SHR WKY
urinary output (mL/24 h) 13.5 ± 1.6b 8.9 ± 1.2c 8.8 ± 1.2c
creatinine clearance (mL/min) 1.70 ± 0.13 1.63 ± 0.15 1.44 ± 0.09
urinary protein (mg/24 h) 45.6 ± 8.2b 39.3 ± 4.8b 9.1 ± 1.1c
urinary glucose (mg/24 h) 7.1 ± 0.6b 6.1 ± 0.6b 3.9 ± 0.8c
urinary urea (mg/24 h) 505 ± 21b 572 ± 28b 363 ± 23c
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a

Data are shown as the mean ± SEM (n = 6 per group).

b

P values <0.05 versus WKY.

c

P values <0.05 versus vehicle-SHR. Comparisons between all groups were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test.

Cardiometabolic Biomarkers

As depicted in Table 2, both SHR groups showed higher plasma triglycerides, blood urea nitrogen (BUN), and leptin levels at the end of the study as compared with the WKY group. Nevertheless, SHRs treated with zamicastat presented significantly lower plasma triglyceride, FFA, and ASAT levels as compared with the nontreated SHR group. On the contrary, both SHR groups showed lower plasma total cholesterol and KB levels as compared with the WKY group.

Table 2. Cardiometabolic Biomarkers at the End of the Studya.

  vehicle-SHR zamicastat-SHR WKY
ALAT (U/L) 60.3 ± 6.0 55.7 ± 3.9 47.1 ± 10.4
ASAT (U/L) 91.7 ± 8.0 67.3 ± 4.0b 76.8 ± 7.2
cholesterol (mmol/L) 2.63 ± 0.08c 2.79 ± 0.07c 3.59 ± 0.23b
triglycerides (mmol/L) 0.94 ± 0.05c 0.65 ± 0.02b,c 0.48 ± 0.05b
free fatty acids (mmol/L) 0.23 ± 0.03 0.10 ± 0.01b,c 0.18 ± 0.02
ketone bodies (mmol/L) 0.33 ± 0.04c 0.29 ± 0.03c 0.67 ± 0.15b
BUN (mg/dL) 15.9 ± 0.8c 15.2 ± 0.3c 12.6 ± 0.9b
glucose (mmol/L) 10.0 ± 0.3c 11.5 ± 0.3 12.4 ± 1.2b
insulin (ng/mL) 10.0 ± 2.8 12.1 ± 1.7 6.2 ± 0.9
leptin (ng/mL) 7.57 ± 1.01c 6.51 ± 0.78c 4.01 ± 0.38b
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a

ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; BUN, blood urea nitrogen. Data are shown as the mean ± SEM (n = 6 per group).

b

P values <0.05 versus vehicle-SHR.

c

P values <0.05 versus WKY. Comparisons between all groups were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test.

Inflammatory Profile

In Table 3 are shown the plasma CRP and MCP-1 levels and a selected panel of 23 cytokines and chemokines assayed at the end of the study. The nontreated SHR group revealed higher plasma CRP (447 ± 7 vs 287 ± 22 μg/mL, P < 0.0001), MCP-1 (90.3 ± 10.5 vs 57.5 ± 2.1 pg/mL, P = 0.0035), GRO/KC (294 ± 15 vs 259 ± 6 pg/mL, P = 0.0316), MIP-1α (705 ± 12 vs 653 ± 8 pg/mL, P = 0.0096), and RANTES levels (2409 ± 139 vs 1540 ± 91 pg/mL, P < 0.0001) when compared with the age-matched WKY group. More importantly, the zamicastat-treated SHR group showed a significant reduction in the plasma levels of seven proinflammatory markers: CRP (401 ± 7 vs 447 ± 7 μg/mL, P = 0.0494), MCP-1 (66.8 ± 3.3 vs 90.3 ± 10.5 pg/mL, P = 0.0310), IL-5 (1028 ± 13 vs 1075 ± 9 pg/mL, P = 0.0040), IL-17α (348 ± 7 vs 370 ± 5 pg/mL, P = 0.0119), GRO/KC (294 ± 15 vs 236 ± 6 pg/mL, P = 0.0011), MIP-1α (633 ± 16 vs 705 ± 12 pg/mL, P = 0.0009), and RANTES (2054 ± 105 vs 2409 ± 139 pg/mL, P = 0.0426) as compared with the untreated SHR group (Table 3).

Table 3. Inflammatory Biomarkers at the End of the Studya.

  vehicle-SHR zamicastat-SHR WKY
CRP 447 ± 7b 401 ± 7b,c 287 ± 22c
MCP-1 90.3 ± 10.5b 66.8 ± 3.3c 57.5 ± 2.1c
IL-1α 913 ± 36 839 ± 21 860 ± 17
IL-1β 1027 ± 42 1075 ± 44 1114 ± 46
IL-2 3292 ± 104 3300 ± 26 3375 ± 49
IL-4 605 ± 26 560 ± 10 583 ± 12
IL-5 1075 ± 9 1028 ± 13c 1057 ± 4
IL-6 5328 ± 290 5089 ± 118 4988 ± 110
IL-7 1007 ± 39 950 ± 50 969 ± 18
IL-10 6923 ± 229 6554 ± 190 6983 ± 94
IL-12p70 378 ± 19 359 ± 8 380 ± 3
IL-13 418 ± 11 412 ± 14 438 ± 11
IL-17α 370 ± 5 348 ± 7b,c 367 ± 3
IL-18 2603 ± 69 2644 ± 66 2606 ± 75
EPO 2445 ± 70 2278 ± 73 2330 ± 87
G-CSF 185 ± 13 186 ± 5 182 ± 7
GM-CSF 520 ± 20 475 ± 15 502 ± 7
GRO/KC 294 ± 15b 236 ± 6c 259 ± 6c
INFγ 754 ± 39 721 ± 11 708 ± 23
M-CSF 387 ± 10 354 ± 23b 428 ± 11
MIP-1α 705 ± 12b 633 ± 16c 653 ± 8c
MIP-3α 212 ± 7 198 ± 6 208 ± 2
RANTES 2409 ± 139b 2054 ± 105b,c 1540 ± 91c
TNFα 1766 ± 82 1830 ± 41 1797 ± 68
VEGF 249 ± 9 261 ± 9 269 ± 8
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a

Data are shown as the mean ± SEM (n = 6 per group). All analytes are expressed in pg/mL, except for CRP, which is expressed in μg/mL.

b

P values <0.05 versus WKY.

c

P values <0.05 versus vehicle-SHR. Comparisons between all groups were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test.

Discussion

Aged SHRs show cardiac and renal characteristics, such as reduced coronary flow reserve, increased coronary vascular resistance, impaired cardiac function, proteinuria, glomerular hypertension, and ischemia, as observed in patients with essential hypertension.30 Our results confirm and extend the knowledge that aged hypertensive SHRs present some comorbidities associated with hypertension and heart failure, like cardiac and renal hypertrophy, proteinuria, and elevated plasma levels of inflammatory markers, as opposed to age-matched normotensive WKYs. Despite the reduced number of animals used in each arm of the study, it was possible to clearly see the differences between SHRs and WKYs, as well as the effect of zamicastat. Taken together, one may regard the aged SHRs as a valid model to assess the possible cardiometabolic and inflammatory benefits of sympathetic down-regulation with a DBH inhibitor.

The SNS plays an important role in the development and maintenance of hypertension4,5,32 and congestive heart failure6,9 and is also associated with increased mortality.7,8,33 Nevertheless, there is a discrepancy between the need to target the SNS and treatments for this purpose because α- and β-blockers are not considered as the first-line therapy in essential hypertension.34−36 These facts offer an opportunity for new approaches to modulate the SNS, like DBH inhibition, renal denervation (RDN), and baroreflex activation.

Zamicastat is a reversible DBH inhibitor22 that decreases NE levels in peripheral sympathetically innervated tissues.37 The inhibition of DBH in peripheral organs, such as adrenal glands, the heart, the kidney, and the vasculature, lowers the sympathetic tone by reducing the synthesis of NE and subsequently decreasing the NE available for release in the sympathetic nerves. A reduction in the NE release decreases the stimulation of both α- and β-adrenoceptors, which results in a BP decrease.38,39 In the present study, zamicastat was given daily mixed in the food, and it was possible to confirm the DBH inhibition by a reduction of NE in the heart, which resulted in a decrease in BP. Our laboratory has shown in the past that chronic DBH inhibition results in a sustained decrease in BP that can be reversed by the withdrawal of the DBH inhibitor,39 unlike RDN, whose effect cannot be intentionally reversed. Another possible advantage of the down-regulation of the SNS by the inhibition of DBH is to allow (up or down) titration and dose adjustment, unlike RDN, which causes a gradual sympathetic slowdown instead of acute inhibition, like β-blockers, thus decreasing the hemodynamic negative impact.40

In humans, DBH deficiency results in the inability to synthesize NE and adrenaline in both the central nervous system and the peripheral autonomic neurons, which leads to orthostatic hypotension.41,42 The present study shows that chronic treatment with zamicastat (30 mg/kg/day) does not deplete NE stores but reduces NE levels in the periphery by ∼30%, which leads to a reduction (∼20%) in high BP, albeit the BP is still higher than the observed BP in normotensive WKYs. These findings may explain the reduced risk of orthostatic hypotension observed in the zamicastat Phase-I clinical trial.21

In the present study, we showed that aged SHRs have renal damage and that 8 consecutive weeks of zamicastat treatment (30 mg/kg/day) did not improve renal function. Besides the reduction in the 24 h urine excretion, which may be accounted for by lower glomerular pressure, zamicastat was not able to reduce proteinuria, urea, or glucose levels in urine. This lack of renal effect by zamicastat may be partially justified due to the fact that SHRs do not respond to renal DA,13−15 although it presents an increased renal availability of DA.43−45 Thus the cardiovascular beneficial effects of zamicastat are likely due to a reduction in the synthesis of NE and consequently the lower stimulation of α- and β-adrenergic receptors.

The autonomic nervous system plays an important role in the complex regulation of energy metabolism. The balance between α- and β-adrenergic receptor activity in the liver, skeletal muscle, and adipose tissue is important for the regulation of glucose and lipid metabolism. When comparing body weight and insulin levels between SHRs and WKYs, we observed no differences between these two groups, and no effect of zamicastat on SHRs was observed. Regarding these two parameters, the literature presents conflicting results46,47 that can be related to the animal’s age or the animal’s supplier. In terms of lipid metabolism, serum triglycerides and total cholesterol levels are augmented by β-blockers, whereas α-blockers decrease serum triglycerides and total cholesterol levels.48−50 Because α- and β-blockers have opposite effects on serum triglycerides and total cholesterol levels, it is worthwhile to underscore the effect resulting from decreases in NE availability. The lower levels of NE available for α- and β-adrenergic receptors, resulting from DBH inhibition with zamicastat, did not change plasma cholesterol levels but induced a reduction in plasma triglyceride, FFA, and ASAT levels, which may contribute to improve the cardiovascular function together with the high BP reduction.

The SNS contributes to the regulation of the immune system, namely, through the release of NE from sympathetic nerve endings to α- and β-adrenergic receptors expressed on immune cells.51−55 The amount of NE released depends on the level of sympathetic nerve activity and on the number of cytokines and neuropeptides released by the immune cells. There is an increased proinflammatory response during hypertension development and associated end-organ damage.51−55 In the present study, we observed that aged SHRs have elevated plasma levels of the proinflammatory cytokines/chemokines CRP, MCP-1, GRO/KC, MIP-1α, and RANTES as compared with age-matched normotensive WKYs. On the contrary, a reduction in sympathetic tone, attained by zamicastat treatment in aged SHRs, resulted in a reduction of seven proinflammatory cytokines/chemokines plasma levels (CRP, MCP-1, IL-5, IL-17α, GRO/KC, MIP-1α, and RANTES) as compared with nontreated SHRs. There is increasing evidence that the immune system contributes to hypertension and end-organ damage, leading to heart failure, renal failure, and stroke.51−55 Thus one may hypothesize that instead of being a consequence of lower BP, the observed improved (pro)inflammatory profile may additionally result from the down-regulation of the SNS and contribute to the reduction in the high BP and to the diminished cardiac hypertrophy. These results support the use of peripheral DBH inhibitors as a promising approach for the treatment of cardiovascular disorders, where a reduction in NE levels may be of therapeutic benefit.

Author Contributions

B.I., N.P, L.W., and P.S.S. designed, performed, or provided input for the studies. A.L. performed the pharmacokinetic evaluation. B.I. and N.P wrote the manuscript. All the authors edited and reviewed the manuscript. B.I., N.P, A.L., L.W., and P.S.S. are or were employees of BIAL - Portela & Ca, S.A. (the sponsor of the study) at the time of the study.

BIAL - Portela & Ca, S.A. was the sponsor of the study.

The authors declare no competing financial interest.

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