Burnett, John C.

Significance The particulate guanylyl cyclase A receptor (GC-A), via its endogenous cardiac-derived hormones atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), plays a pivotal role in maintaining intravascular volume, arterial pressure, cardiovascular structure, function, and, more recently, metabolic homeostasis. As such, therapies to potentiate GC-A signaling via peptide augmentation have yielded promising results in clinical trials. A major breakthrough in GC-A therapeutics would be a small molecule that sensitizes the GC-A receptor to endogenous ANP or BNP, which to date does not exist. Here, we report the development of an orally bioavailable, small molecule positive allosteric modulator of GC-A that exhibits therapeutic activity in human hypertension and heart failure ex vivo.

M-atrial natriuretic peptide (MANP) is a novel ANP (atrial natriuretic peptide) analog engineered to be an innovative particulate GC-A (guanylyl cyclase A) receptor activator. The rationale for its design was to develop a best-in-class GC-A activator with enhanced cGMP activating, natriuretic, aldosterone-suppressing, and blood pressure–lowering actions, compared with endogenous ANP, for the treatment of hypertension. Here, we report the first-in-human study on the safety, tolerability, neurohumoral, renal, and blood pressure–lowering properties of MANP in hypertension subjects. This was an open-label sequential single ascending dose design in which all subjects stopped all antihypertensive agents for 14 days before receiving a single subcutaneous injection of MANP. MANP was safe, well tolerated, activated cGMP, induced natriuresis, reduced aldosterone, and decreased blood pressure at or below the maximal tolerated dose. Thus, MANP has a favorable safety profile and produced expected pharmacological effects in human hypertension. Our results support further investigations of MANP as a potential future blood pressure–lowering, natriuretic and aldosterone-suppressing drug for hypertension especially resistant hypertension.

Aldosterone is a critical driver for cardiovascular disease (CVD). We recently discovered that MANP, a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP. MANP is currently entering clinical trials for hypertension and thus understanding its aldosterone suppressing mechanism is important. The mechanism of aldosterone inhibition by natriuretic peptides (NPs) remains to be clearly defined. Conflicting results were reported on the roles of particulate guanylyl cyclase A receptor (pGC-A) and NP clearance receptor (NPRC) in aldosterone inhibition. Furthermore, the functions of protein kinase G (PKG) and phosphodiesterases (PDE) on aldosterone regulation are not clear. Herein, we investigated the molecular mechanism of aldosterone regulation in the human adrenocortical cell line H295R and in mice. We firstly showed that pGC-A mediates aldosterone inhibition. In contrast, with NPRC agonist and antagonist, we showed that NPRC did not inhibit aldosterone. Next, we confirmed that MANP inhibits aldosterone via PDE2, not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer (FRET) experiments. Specifically, MANP suppressed ANGII mediated activation of aldosterone (fold change) MANP+ANGII 3.2±0.1* vs. ANGII 3.8±0.1 (*p<0.05) with IBMX, a PDEs inhibitor and the PDE2 antagonist Bay 60-7550 reversed MANP-mediated aldosterone suppression (IBMX+MANP+ANGII 3.9±0.2 and Bay+MANP+ANGII 4.1±0.1). With PKG agonists and inhibitors, aldosterone levels were not changed. In PDE2 activity FRET studies, aldosterone control was 3.7±0.4 and with MANP 0.9±0.2* supporting PDE2 activation by MANP. Further, the inhibitory effect of PDE2 is mediated by a reduction of intracellular Ca2+ concentration (~22%). We then showed that MANP directly reduced aldosterone synthase CYP11B2 expression in vitro. Lastly, in PDE2 knockout mice (embryonic lethal), embryonic adrenal CYP11B2 expression is markedly increased (wild type: 1±0.2, KO: 2.8±0.5*). Our findings innovatively elucidate the pGC-A/cGMP/PDE2 pathway in aldosterone inhibition by MANP in vitro and in vivo. Additionally, our data also support the development of MANP as a novel ANP analog drug for CVD.

Abstract Mechanism-based therapy centred on the molecular understanding of disease-causing pathways in a given patient is still the exception rather than the rule in medicine, even in cardiology. However, recent successful drug developments centred around the second messenger cyclic guanosine-3′-5′-monophosphate (cGMP), which is regulating a number of cardiovascular disease modulating pathways, are about to provide novel targets for such a personalized cardiovascular therapy. Whether cGMP breakdown is inhibited or cGMP synthesis is stimulated via guanylyl cyclases or their upstream regulators in different cardiovascular disease phenotypes, the outcomes seem to be so far uniformly protective. Thus, a network of cGMP-modulating drugs has evolved that act in a mechanism-based, possibly causal manner in a number of cardiac conditions. What remains a challenge is the detection of cGMPopathy endotypes amongst cardiovascular disease phenotypes. Here, we review the growing clinical relevance of cGMP and provide a glimpse into the future on how drugs interfering with this pathway may change how we treat and diagnose cardiovascular diseases altogether.

Introduction: Blood pressure (BP) control remains low despite combination therapy in resistant hypertension (RH). Thus, there is an unmet need for novel strategies targeting RH. Recently we have reported that M-ANP, a novel Mayo engineered ANP-based peptide, possesses greater and more sustained biological actions compared to ANP and is more resistant to neprilysin degradation. The current study was designed to investigate for the first time the efficacy of a single daily subcutaneous (SQ) injection of M-ANP using a chronic canine model of hypertension induced by angiotensin II (Ang-II). Hypothesis: We hypothesize that a daily SQ M-ANP injection would have potent BP lowering and renal enhancing properties in our chronic hypertensive canine model. Methods: We induced hypertension in canines (n=5) by continuous Ang II (80 ng/kg/min) osmotic mini-pump administration for 24 days. Continuous 24 hour mean BP (MBP) was measured by telemetry and a 24 hour urine collection was used to assess urinary volume, sodium (Na) and cGMP, the second messenger for the ANP system. M-ANP was administered SQ for 5 days (10μg/kg/day). Data are mean±SE. ΨP<0.05 Ang II Day 16 vs. baseline; *P<0.05 Ang II Day 16 vs. M-ANP (Table 1). Results: Chronic Ang II administration resulted in model of severe hypertension, with an increase in MBP from 112±2 to 157±2 mmHg (day 16). As seen in Table 1, M-ANP significantly reduced MBP, significantly increased plasma cGMP as well as urinary cGMP excretion, urinary volume rate (UV) and urinary Na excretion (UNa), without significant changes in heart rate (HR). Furthermore, there was a trend for plasma ANP to increase and plasma aldosterone to decrease with SQ M-ANP injection. Conclusions: This study demonstrates for the first time that chronic SQ administration of M-ANP reduces BP, activates plasma cGMP, and increases UV and UNa, without significant changes in HR in an experimental model of chronic hypertension.

Background: Preclinical diastolic dysfunction (PDD) is defined as subjects with normal systolic function, moderate or severe diastolic dysfunction determined by Doppler criteria but with no symptoms of heart failure (HF). We have previously demonstrated that an impaired cardiorenal endocrine response to stress by acute volume loading (AVL) exists in PDD and that is corrected by acute administration of sq B-type natriuretic peptide (BNP). Type V phosphodiesterase (PDEV) metabolizes cGMP, second messenger of BNP. The objective of the current study is to determine if the combination of PDEV inhibitor (PDEVI) and BNP will result in a greater enhancement of the cardiorenal response to AVL as compared to PDEVI alone in PDD. Methods: Randomized double-blind single placebo-controlled cross-over study in 20 PSD subjects. We determined cardiorenal effects of PDEVI with Tadalafil (T) 5 mg alone and in combination with acute sq BNP (Nesiritide 10 μg/Kg), in response to AVL (normal saline 0.25 ml/kg/min for 1 hour). Each subject had 2 study visits, separated by at least 1 week. At each visit, echocardiogram and renal clearance studies with iothalmate and PAH to measure GFR and renal plasma flow (RPF) were done before and after AVL. Each patient was randomized to receive T at both visits and either placebo or sq-BNP one visit and crossover to the other on the next visit. Results: T+BNP resulted in smaller LA volume index (-4±10 vs 3±7 ml/m 2 , p< 0.05) and lower right ventricular systolic pressure (-4±3 vs 2±6 mmHg, p< 0.05) in response to AVL as compared to T alone. Moreover, T+ BNP resulted in a significant reduction of plasma ANP and LV end-systolic volume in response to AVL while T alone did not. However, the combination to T+BNP did not enhance the renal response to AVL as compared to T alone. Both the change in urine flow (1.7±5 vs 2.4±5 ml/min, p> 0.05) and sodium excretion (129±200 vs 124±270 mEq/min, p>0.05) in response to AVL were similar between T+BNP and T alone. Conclusion: In PDD, the combination of PDEVI and BNP reduced LV filling pressure in response to AVL as compared to PDEVI alone. However, it did not enhance the renal response to AVL. This suggests a differential cardiac versus renal response to the maximization of cGMP system with PDEVI and BNP.

Introduction: Phenome-wide association studies (PheWAS) using electronic medical record (EMR)-linked biobanks have been used not only to identify and replicate known associations of genetic variants with disease phenotypes but have also resulted in the discovery of potentially novel genotype-phenotype relationships. The natriuretic peptide (NP) system plays an important role in a broad range of disease processes including cardiovascular and inflammatory diseases. We hypothesized that performing a PheWAS using previously known functional genetic variants of the NP system may result in novel disease associations that could provide mechanistic insights in an unbiased manner. Methods: We scanned for associations between 9 single-nucleotide polymorphisms (SNPs) in the NP system and 27 EMR-derived chronic disease phenotypes in 3,025 individuals participating in a case-control study of peripheral arterial disease. The EMR phenotypes were identified using two or more ICD-9-CM diagnosis codes based on the AHRQ Clinical Classifications Software (CCS). The relationship of SNPs and phenotypes were modeled using logistic regression adjusting for gender. Results: We identified rs5065, a SNP located in the stop codon of exon 3 of the NPPA gene, to be the strongest associated SNP with rheumatoid arthritis (RA) (OR=0.78, p=0.0008, q-value=0.11). The SNP leads to the extension of atrial natriuretic peptide (ANP) by 2 additional arginines at the C terminus. Cardiovascular disease is known to be the leading cause of death in patients with RA and ANP plays an important immunomodulatory role by inhibiting inducible nitric oxide synthase, reducing TNF-α production and attenuating prostaglandin E2 production in macrophages. Circulating NPs have been used to screen for occult cardiac disease and are associated with mortality in RA. This study demonstrates for the first time the importance of the relationship between genetic variation in the NP system and RA. Conclusions: PheWAS was successfully used as a tool to identify a novel association of functional genetic variation in the NPPA gene with RA. The observation is hypothesis generating and further replication studies are required to determine the role of rs5065 in cardiovascular outcomes of RA.

Introduction: Human and experimental studies have suggested that a decline in renal function after myocardial infarction (MI), possibly due to the activation of adverse molecular pathways, contributes to heart failure (HF) progression and poor outcomes. The apoptosis and the transforming growth factor-beta (TGF-beta) pathways have been implicated in post-MI injury, however it is unclear if these 2 key pathways are activated post-MI in the kidney and may contribute to renal fibrosis and dysfunction. Hypothesis: We hypothesized that MI activates widespread renal apoptotic and TGF-beta related genes and is associated with renal fibrosis and dysfunction. Methods: Wistar rats were randomized into two groups: Sham-operated (S; n=10) and MI (produced by left coronary artery ligation; n=10). Cardiorenal structure and function were assessed at 3 weeks and included mean arterial pressure (MAP), EF, GFR, renal blood flow (RBF), sodium (Na) excretion and renal fibrosis by picrosirius red staining. Microarray analysis (Affymetrix GeneChip Rat Genome) was performed on kidney cortex (KC) and medulla (KM) and focused on apoptotic and TGF-beta related genes. Data presented as mean±SE,*p<0.05. Results: Experimental MI was characterized by a significant decrease in EF (S: 79±1, MI: 49±4 %*) and LV hypertrophy (S: 2.06±0.03, MI: 2.57±0.07 mg/g*), while MAP did not differ between groups. GFR and Na excretion were similar between the groups, however there was a significant decline in post-MI RBF (S: 14±1, MI: 8±1 ml/min*). Notably, after MI the KC (S: 1.3±0.3, MI: 3.4±0.8 %*) and KM (S: 1.0±0.3, MI: 11±2.8 %*) exhibited an increase in fibrosis. Microarray analysis revealed that 138 apoptotic and 15 TGF-beta pathway genes in the KC and 122 apoptotic and 10 TGF-beta pathway genes in the KM were significantly altered (1.5 fold, p<0.05) in the MI group. Conclusions: These findings demonstrate that MI results in adverse renal fibrotic remodeling together with extensive modulation of apoptotic and TGF-beta pathways of which may contribute to post-MI renal dysfunction. This study suggests that therapies should target detrimental molecular pathways in the kidney as well the heart to prevent the progression of post-MI cardiorenal failure.

Introduction: Fibrosis is a hallmark of cardiorenal diseases, including heart failure, for which there are few effective therapies. C-type natriuretic peptide (CNP) is a cardiorenal-derived peptide whose mature active form, CNP-22, possesses potent anti-fibrotic actions through the activation of guanylyl cyclase receptor B (GC-B) and its second messenger, cGMP. However, CNP-22’s therapeutic potential is limited due to its short half-life. We recently identified a 53 amino acid (AA) peptide, CNP-53, whose structure consists of CNP-22 with an additional 31 AA on the N-terminus. Herein, we investigated the cGMP generating actions of CNP-53 (compared to CNP-22) in vivo and in vitro, with the goal of advancing a potential anti-fibrotic strategy for cardiorenal disease. Hypotheses: We hypothesized that CNP-53 would elevate circulating CNP levels as well as have greater cGMP activating actions compared to CNP-22 in vivo. We also hypothesized that CNP-53 would stimulate GC-B specific cGMP production in vitro. Methods: In vivo, two groups of anesthetized normal rats (n=8) received a 75-minute intravenous infusion of an equimolar dose of CNP-53 or CNP-22. Mean arterial pressure (MAP), plasma CNP and plasma and urinary cGMP were assessed. In vitro, HEK 293 cells over-expressing GC-A and GC-B and human fibroblasts (hFs), where GC-B is abundant, were stimulated with CNP-53 or CNP-22 for 10 minutes at a dose of (10-8M). Data are mean ± SE, *p<0.05. Results: In vivo, plasma CNP (CNP-53: 311±66, CNP-22: 153±16 pg/ml*) and cGMP (CNP-53: 21±2, CNP-22: 11±1 pmol/ml*) and urinary cGMP excretion (CNP-53: 75±4, CNP-22: 38±1 pmol/min*) were greater with CNP-53 infusion than CNP-22. MAP was similar between the 2 groups (CNP-53: 85±2, CNP-22: 88±4 mmHg). In vitro, both CNP-53 and CNP-22 activated cGMP in GC-B HEK cells and in hFs, with no activation of cGMP in GC-A HEK cells. Conclusions: These findings demonstrate that CNP-53 is superior to CNP-22 in activating cGMP in vivo, without inducing hypotension. Additionally, CNP-53 is a GC-B specific cGMP activating peptide that potently increases cGMP in hFs. Thus, CNP-53 may represent a novel first in class GC-B agonist with therapeutic potential targeting fibrosis and preventing the progression of cardiorenal disease.