Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

[Atrial natriuretic hormones and metabolic syndrome: recent advances]. / Hormones natriurétiques et syndrome métabolique : mise au point.

Natriuretic peptides are a group of hormones including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C type (CNP), urodilatin and guanilyn. ANP (half-life: 2-4 min), is secreted by the atrium, BNP (half-life: 20 min) by the ventricle, CNP by the vascular endothelium, urodilatin by the kidney and guanylin by the intestine. These natriuretic peptides prevent water and salt retention through renal action, vasodilatation and hormonal inhibition of aldosterone, vasopressin and cortisol. These peptides also have a recently demonstrated metabolic effect through an increase of lipolysis, thermogenesis, beta cell proliferation and muscular sensitivity to insulin. Blood levels of these natriuretic peptides depend on "active NPR-A receptors/clearance NPR-C receptors", the last ones being abundant on adipocytes. Therefore, natriuretic peptides act as adipose tissue regulator and constitute a link between blood pressure and metabolic syndrome. They are used as markers and treatment of cardiac failure. Other applications are on going. BNP and NT-proBNP (inactive portion de la pro-hormone) are used as markers of cardiac failure since they have a longer half-life than ANP. BNP decrease is quicker and more important than that one of NT-ProBNP in case of improvement of cardiac failure. Chronic renal insufficiency and beta-blockers increase BNP levels. BNP measurement is useless under treatment with neprilysine inhibitors such as sacubitril, one of the neutral endopeptidases involved in catabolism of natriuretic peptides. The association sacubitril/valsartan is a new treatment of chronic cardiac failure, acting through the decrease of ANP catabolism.

Design and evaluation of novel natriuretic peptide derivatives with improved pharmacokinetic and pharmacodynamic properties.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor B (NPR-B), are potent positive regulators of endochondral bone growth, making the CNP pathway one of the most promising therapeutic targets for the treatment of growth failure. However, the administration of exogenous CNP is not fully effective, due to its rapid clearance in vivo. Modification of CNP to potentially druggable derivatives may result in increased resistance to proteolytic degradation, longer plasma half-life (T1/2), and better distribution to target tissues. In the present study, we designed and evaluated CNP/ghrelin chimeric peptides as novel CNP derivatives. We have previously reported that the ghrelin C-terminus increases peptide metabolic stability. Therefore, we combined the 17-membered, internal disulfide ring portion of CNP with the C-terminal portion of ghrelin. The resultant peptide displayed improved biokinetics compared to CNP, with increased metabolic stability and longer plasma T1/2. Repeated subcutaneous administration of the chimeric peptide to mice resulted in a significant acceleration in longitudinal growth, whereas CNP(1-22) did not. These results suggest that the ghrelin C-terminus improves the stability of CNP, and the chimeric peptide may be useful as a novel therapeutic agent for growth failure and short stature.

Cenderitide: structural requirements for the creation of a novel dual particulate guanylyl cyclase receptor agonist with renal-enhancing in vivo and ex vivo actions.

AIMS: Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. METHODS AND RESULTS: In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 µEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. CONCLUSION: The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.

Genomic epidemiology of blood pressure salt sensitivity.

High blood pressure (BP) is a complex trait determined by genetic and environmental factors, as well as their interactions. Over the past few decades, there has been substantial progress elucidating the genetic determinants underlying BP response to sodium intake, or BP salt sensitivity. Research of monogenic BP disorders has highlighted the importance of renal salt handling in BP regulation, implicating genes and biological pathways subsequently identified in candidate gene studies of salt sensitivity. Despite these advancements, certain candidate gene findings await replication evidence, and some biological pathways warrant further investigation. Furthermore, results from genome-wide association studies (GWASs) and sequencing work have yet to be reported. GWAS will be valuable for uncovering novel mechanisms underlying salt sensitivity, whereas future sequencing efforts promise the discovery of functional variants related to this complex trait. Delineating the genetic architecture of salt sensitivity will be critical to understanding how genes and dietary sodium interact to influence BP.

B-type natriuretic peptide as a marker of heart failure: new insights from biochemistry and clinical implications.

The mature, biologically active 32-amino acid long B-type natriuretic peptide (BNP(1-32)), is cleaved by corin from the BNP prohormone. Recent data demonstrated that BNP(1-32) might be an ideal substrate for the endogenous aminopeptidase, dipeptidyl-peptidase (DPP) IV. DPP IV removes the two amino-terminal amino acids (Ser and Pro) from BNP(1-32) to produce BNP(3-32), which has been detected in plasma of patients with heart failure. In a canine model, intravenous BNP(3-32) infusion resulted in less natriuresis, diuresis and vasodilation compared to intravenous infusion of BNP(1-32). The clinical relevance of these observations may be important for patients with high plasma BNP concentrations, which can be measured by commercially available immunoassays. Further studies are needed to explore whether DPP IV inhibitors increase the bioavailability of BNP(1-32), delay the progression of heart failure and increase the efficacy of exogenously administered BNP(1-32) in decompensated heart failure.

[Influence of new neurohypophyseal hormone analogs on sodium and water reabsorption in rat kidney].

New analogs of some neurohypophyseal hormones (oxypressin, hydrin, glumitocin, vasotocin) have been synthesized. Experiments with injection of these peptides to rats showed that substitution of C-terminal glycinamide on beta-ethanolamine (glycinol) or ethylamine in 1-deamino-arginine vasotocin resulted in loss of natriuretic but not antidiuretic activity. Analogs of oxypressin and hydrin exhibited neither natriuretic activity nor ability to affect water reabsorption. Glumitocin analog induced renal sodium ion excretion and did not influence potassium ion excretion.

Oral brain natriuretic peptide: a novel strategy for chronic protein therapy for cardiovascular disease.

In 1956, secretory granules were detected via electron microscopy in the mammalian atria by Kisch. This remarkable discovery signaled the beginning of a new field of research that decades later has lead to the concept of the heart as an endocrine organ and the establishment of the natriuretic peptide (NP) system. In 1981, deBold and colleagues identified from the atrial myocardium the first member of the NP family, atrial NP. Thereafter, new members of this growing family of cardiac hormones were identified and investigated. The successful story of B-type or brain NP (BNP), from its discovery to its use in the diagnosis and prognosis of cardiovascular diseases and later as a tool in the treatment of acute congestive heart failure, have since taken place. However, the use of peptides as chronic therapies has been limited by enzymatic degradation. Chronic administration of BNP, particularly in disease states like hypertension and early heart failure, could be effective as an antihypertensive therapy and in delaying progression of cardiac disease. To date, the use of BNP is limited to patients with acute decompensated heart failure, but new strategies are under investigation to extend the use of chronic BNP in less severe stages of cardiovascular diseases. Innovative technologies have been recently developed that allow protection of proteins from enzymatic degradation, making feasible oral administration of small proteins such as BNP. This review will focus on the potential role of BNP as a new chronic therapeutic strategy in the treatment of cardiovascular diseases and will summarize our recent report of the development and in vivo evaluation of orally active human BNP.

Amphiphilic conjugates of human brain natriuretic peptide designed for oral delivery: in vitro activity screening.

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patient-friendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.

Atrial cardiomyocyte-specific expression of Cre recombinase driven by an Nppa gene fragment.

To study the development of the atria, we produced a transgenic mouse line that expresses Cre under the regulatory control of a 7 kbp fragment of the Natriuretic peptide precursor type A gene (Nppa), from -3 kbp to +4 kbp relative to the transcription start site. Crossing this line with the R26R and Z/EG reporter lines revealed recombinase activity specifically in the cardiomyocytes of the atria and to a lesser extent the inflow tract from E10.5 onwards. At E14.5 recombination in the atria is almost complete. No recombination was observed outside the heart. These mice provide a tool to study gene function in the atria.

Stretch-modulation of second messengers: effects on cardiomyocyte ion transport.

In cardiomyocytes, mechanical stress induces a variety of hypertrophic responses including an increase in protein synthesis and a reprogramming of gene expression. Recently, the calcium signaling has been reported to play an important role in the development of cardiac hypertrophy. In this article, we report on the role of the calcium signaling in stretch-induced gene expression in cardiomyocytes. Stretching of cultured cardiomyocytes up-regulates the expression of brain natriuretic peptide (BNP). Intracellular calcium-elevating agents such as the calcium ionophore A23187, the calcium channel agonist BayK8644 and the sarcoplasmic reticulum calcium-ATPase inhibitor thapsigargin up-regulate BNP gene expression. Conversely, stretch-induced BNP gene expression is suppressed by EGTA, stretch-activated ion channel inhibitors, voltage-dependent calcium channel antagonists, and long-time exposure to thapsigargin. Furthermore, stretch increases the activity of calcium-dependent effectors such as calcineurin and calmodulin-dependent kinase II, and inhibitors of calcineurin and calmodulin-dependent kinase II significantly attenuated stretch-induced hypertrophy and BNP expression. These results suggest that calcineurin and calmodulin-dependent kinase II are activated by calcium influx and subsequent calcium-induced calcium release, and play an important role in stretch-induced gene expression during the development of cardiac hypertrophy.

Does the natriuretic peptide system exist throughout the animal and plant kingdom?

Natriuretic peptides (NPs) and their receptors have been identified in vertebrate species ranging from elasmobranchs to mammals. Atrial, brain and ventricular NP (ANP, BNP and VNP) are endocrine hormones secreted from the heart, while C-type NP (CNP) is principally a paracrine factor in the brain and periphery. In elasmobranchs, only CNP is present in the heart and brain and it functions as a circulating hormone as well as a paracrine factor. Four types of NP receptors are cloned in vertebrates. NPR-A and NPR-B are guanylyl cyclase-coupled receptors, whereas NPR-C and NPR-D have only a short cytoplasmic domain. NPs are hormones important for volume regulation in mammals, while they act more specifically for Na(+) regulation in fishes. The presence of NP and its receptor has also been suggested in the most primitive vertebrate group, cyclostomes, and its molecular identification is in progress. The presence of ANP or its mRNA has been reported in the hearts and ganglia of various invertebrate species such as mollusks and arthropods using either antisera raised against mammalian ANP or rat ANP cDNA as probes. Immunoreactive ANP has also been detected in the unicellular Paramecium and in various species of plants including Metasequoia. Furthermore, the N-terminal prosegments of ANP, whose sequences are scarcely conserved even in vertebrates, have also been detected by the radioimmunoassay for human ANP prosegments in all invertebrate and plant species examined including Paramecium. Although these data are highly attractive, the current evidence is too circumstantial to be convincing that the immunoreactivity truly originates from ANP and its prosegments in such diverse organisms. The caution that has to be exercised in identification of vertebrate hormones from phylogenetically distant organisms is discussed.

The structure of the digitalislike and natriuretic factors identified as macrocyclic derivatives of the inorganic carbon suboxide.

The Natriuretic and Endogenous DigitalisLike Factors (EDLFs) are disclosed to be cyclomeric and macroring closed derivatives of the inorganic carbon suboxide. The macrocyclic cyclohexamer with six carbon suboxide units has a molar mass of 408.2 Da, as previously been found for the EDLF of animal origin. The anhydrous cyclohexameric factor is lipophilic but is transformed into more hydrophilic derivatives by the stepwise addition of water. Based on the present findings, it appears that EDLFs exist in solution as an equilibrium mixture of lipophilic and hydrophilic forms and not as a single chemical substance. This structural assumption better accounts for the earlier observed highly anomalous properties of EDLFs. The simultaneously found higher molar mass (4,100 and 4,900 Da) macrocyclic carbon suboxide derivatives are tentatively identified as the Natriuretic factors.

[Study and treatment of heart failure require new approaches. Natriuretic peptides can be safe and simple diagnostic and outcome measures]. / Utredning och behandling av hjärtsvikt söker nya vägar. Natriuretiska peptider kan bli säkert och enkelt mått i diagnostik och behandling.

Analysis of plasma natriuretic peptides and related propeptide fragments may be a cost-effective aid to diagnostic evaluation and treatment follow-up in cases of heart failure. In diagnostic potential such variables may constitute first-line measures of high negative predictive value, allowing further examination, e.g. by echocardiography, in cases where values are above the respective cut-off levels. However, in many cases evaluation of published reports is rendered difficult by their omission of information on such pre-analytical variables as blood sampling and storage, and drug therapy. Moreover, different analytical methods may yield widely divergent results. Thus, before such assays are introduced in general use, their long-term validity needs to be ensured, for instance by consistency in calibration, and measurements need to be made in representative series of unselected patients for the determination of appropriate cut-off levels.

Stomatal guard cell responses to kinetin and natriuretic peptides are cGMP-dependent.

Immunological evidence suggest that plants contain natriuretic peptides (NPs) and furthermore (3-[I]iodotyrosol) rat atrial NP (rANP) binds specifically to plant membranes. rANP and immunoaffinity-purified plant NP analogues also promote concentration-dependent stomatal opening. Here we report that kinetin, a synthetic cytokinin, and rANP induce stomatal opening in Tradescantia albiflora and that the effect of rANP is critically dependent on the secondary structure of the peptide hormone. The native circular molecule is active, whereas the linearized molecule shows no biological activity. Furthermore, kinetin- and rANP-induced stomatal opening is reversibly inhibited by two inhibitors of guanylate cyclase, LY 83583 and methylene blue. Stomatal opening is also induced in a concentration-dependent manner by the cell-permeant cyclic guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, and this effect is prevented by the stomatal closure promoting plant hormone abscisic acid (ABA). We conclude that in guard cells kinetin and rANP pathways operate via guanylate cyclase upregulation, and we propose that ABA-induced closure is not cGMP-dependent.

Localization by photoaffinity labeling of natriuretic peptide receptor-A binding domain.

A portion of the ligand binding domain for atrial natriuretic peptide (ANP) was identified as an affinity cross-linked proteolytic fragment of bovine adrenal natriuretic peptide receptor type-A (NPR-A). Affinity purified NPR-A was UV-cross-linked to the amino terminus of 125I-[Tyr2] rat ANP-(2-27). A chymotryptic fragment of the affinity labeled NPR-A was isolated by chromatography and electrophoresis. This fragment yielded a major microsequence corresponding to a region from Met173 to Phe188 of the receptor extracellular domain and containing one N-glycosylation site at Asn180. Bovine NPR-A receptor was then cross-linked to the carboxy terminus of the highly efficient photoaffinity derivative 125I-[Tyr18,Bpa27] rat ANP(1-27). Proteolysis of the affinity labeled NPR-A with cyanogen bromide and trypsin produced radiolabeled and glycosylated fragments of size 15 and 9 kDa, respectively, which contained the epitope Ile181-Phe188 (CS328) and which were detectable by immunoprecipitation with a monospecific polyclonal antibody against CS328. Proteolysis with cyanogen bromide followed by Glu-C produced a shorter photolabeled 6 kDa fragment which was not immunoprecipitable by anti-CS328 antibody and which was not glycosylated. The results lead to the identification of the short segment Asp191-Arg198 as the site of covalent binding of [Tyr18,Bpa27] rat ANP(1-27). This hydrophilic region is adjacent to the epitope Ile181-Phe188 and to the glycosylation site Asn180. It displays the species variability and the high surface probability expected for a portion of the binding domain of NPR-A in contact with ANP.

Natriuretic hormones.

Natriuretic peptides act as endocrine and paracrine hormones to regulate extracellular fluid volume and blood pressure at all levels of the circulation. Atrial and brain natriuretic peptides, circulating hormones secreted in response to increased stretch within the cardiac atrium and ventricle, respectively, induce comparable natriuresis, vasodepression, and inhibition of aldosterone via the guanylate-cyclase receptor, NPR-A. C-type natriuretic peptide acts via a different guanylate-cyclase receptor, NPR-B, to affect vascular cell growth and remodeling. Possible complex interactions among all three natriuretic peptides are reviewed. Although the importance of natriuretic peptides is still being assessed, data from animal studies strongly support an important role fore these hormones in cardiovascular homeostasis.

Endogenous natriuretic factors 3: isolation and characterization of human natriuretic factors LLU-alpha, LLU-beta 1, and LLU-gamma.

A low molecular weight endogenous substance believed to be responsible for extracellular fluid homeostasis in mammals has been sought for many years. Our goal is to isolate and structurally characterize this putative "natriuretic hormone." We have developed an assay using the conscious rat to measure prolonged natriuresis (Benaksas et al (1993) Life Sciences, 52, 1045-1054), the activity originally described for this putative substance. Using this assay we have identified a number of natriuretic compounds isolated from human uremic urine. The collected urine is processed by ultrafiltration (< or = 3 kDa), gel filtration chromatography (G-25) and extraction with isopropanol and diethyl ether. The organic soluble material is then subjected to sequential high-performance liquid chromatography. We report here the initial characterization of two pure isolates (LLU-alpha and LLU-gamma) obtained by this method, and the structural elucidation of a third pure compound, LLU-beta 1, a natriuretic and previously unreported metabolite of the drug diltiazem.

An intestinal natriuretic factor.

In 1975, reports were published that suggested that the gastrointestinal tract can "taste" the intake of sodium and in some unknown way influence the kidneys to increase sodium excretion. To test whether the intestine contained a natriuretic factor, intestinal tissue from cats was homogenized and fractionated by ultrafiltration to a molecular range of approximately 500-10,000 Da and separated by gel chromatography (Sephadex G25). The fractions were pooled into four large fractions that were assayed for "natriuretic" activity on anesthetized rats. The fraction containing the material with an apparent molecular mass of 500-1,000 Da augmented renal excretion of sodium and water, whereas the other pooled fractions did not exhibit any consistent natriuretic effect. The "natriuretic" fractions from gel filtration were further purified by ion exchange chromatography using a cation exchanger. The natriuretic activity was eluted from the ion exchange chromatography column at a NaCl concentration of 250 mM. Preliminary experiments on Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) suggest that the intestinal influence on renal sodium excretion is more pronounced in SHR than in WKY rats.