Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified Atrial Natriuretic Peptide Derivatives Synthesized Using Chemo-enzymatic Synthesis Approaches.

Atrial natriuretic peptide (ANP) exerts beneficial pharmacological effects in the treatment of various cardiovascular disorders, such as acute congestive heart failure (ADHF). However, the clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemo-enzymatic glyco-engineering using EndoS D233Q/Q303L. The most potent derivative SG-ANP-Fc conjugate extended the half-life to 14.9 d and the duration of blood pressure lowering effect to over 28 d. This new biologic modality provides an opportunity to develop outpatient therapy after ADHF.

Vasonatrin peptide: a unique synthetic natriuretic and vasorelaxing peptide.

This study reports the cardiovascular and renal actions of a novel and newly synthesized 27-amino acid peptide termed vasonatrin peptide (VNP). VNP is a chimera of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). This synthetic peptide possesses the 22-amino acid structure of CNP, which is a cardiovascular selective peptide of endothelial origin and is structurally related to ANP. VNP also possesses the five-amino acid COOH terminus of ANP. The current study demonstrates both in vitro and in vivo that VNP possesses the venodilating actions of CNP, the natriuretic actions of ANP, and unique arterial vasodilating actions not associated with either ANP or CNP.

Effects of intravenously administered beta-human atrial natriuretic polypeptide in humans.

beta-Human atrial natriuretic polypeptide (beta-hANP) is an antiparallel dimer of alpha-human ANP (alpha-hANP) that was isolated from human atria. Using synthetic beta-hANP and a radioimmunoassay for alpha-hANP that also detects beta-hANP, we have previously demonstrated that beta-hANP is converted into alpha-hANP in human plasma in vitro. In the present study, we compared the effects of intravenous administration of beta-hANP (100 micrograms) to five normal human volunteers with those of an equimolar administration of alpha-hANP (50 micrograms) to the same subjects, and we also investigated the possible mechanisms of actions of beta-hANP. Although the administration of alpha-hANP caused a significant decrease in blood pressure with a reactional increase of heart rate, beta-hANP elicited minimal change of blood pressure. In contrast, beta-hANP exerted more potent and longer lasting diuretic and natriuretic activities than did alpha-hANP. Net changes in urine volume and sodium excretion induced by beta-hANP (579 +/- 65 ml, 56.0 +/- 9.9 mEq) were significantly greater than those elicited by alpha-hANP (396 +/- 50 ml, 34.7 +/- 4.9 mEq; p less than 0.05, respectively). The administration of beta-hANP revealed a longer retention of the ANP-like immunoreactivity level in plasma, compared with that of alpha-hANP. High performance gel permeation chromatography coupled with the radioimmunoassay revealed that beta-hANP (Mr = 6000) was also converted into alpha-hANP (Mr = 3000) in human plasma in vivo. The demonstrated conversion of beta-hANP into alpha-hANP could be relevant to the observed effects of beta-hANP in humans.

Synthesis and biological properties of two dimeric forms of human alpha-atrial natriuretic peptide.

Two dimeric forms of human alpha-atrial natriuretic peptide (alpha-ANP) were synthesized by solution methods and compared with monomeric alpha-ANP in terms of some biological and immunochemical properties. The parallel form (beta'-ANP) and the antiparallel form (beta-ANP) were equipotent in smooth muscle relaxant activity in isolated rat aorta and their ED50 values were estimated to be 1.7 X 10(-8) M and 1.6 X 10(-8) M, respectively. Diuretic and natriuretic responses induced by beta-ANP and beta'-ANP anesthetized rats were equally less potent but exhibited a significantly longer duration than those induced by alpha-ANP. beta-ANP and beta'-ANP possessed immunoreactivities of 60-100% and 50-90% (alpha-ANP, 100% on a molar basis), respectively, with three different antisera raised against alpha-ANP-related peptides.

Design and synthesis of metabolically stable atrial natriuretic factor analogs. Amino- and carboxy-terminal stabilization.

Two analogs of rat atrial natriuretic factor, rANF7-28-NH2 and [Mpr7,Ala20,D-Arg27]rANF7-27-NH2, were prepared by the solid-phase method. These peptides had 2-fold and 7-fold less affinity, respectively, than rANF1-28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5-fold less potent than rANF1-28 in relaxing serotonin-contracted rabbit aortic rings. rANF7-28-NH2 was rapidly degraded by rat kidney homogenates but [Mpr7,Ala20,D-Arg27]rANF7-27-NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.

Synthesis and biological activity of atrial natriuretic factor analogues: effect of modifications to the disulfide bridge.

A series of atrial natriuretic factor (ANF) analogues with modifications to the disulfide bridge and lacking the exocyclic N-terminal sequence was synthesized. The native cystine residue was substituted by isofunctional deamino carba, beta,beta-dimethyl carba and dehydro dicarba spanners that bridge residues 106 and 120. The compounds were prepared by segment condensation coupling using the base-labile (9-fluorenylmethyl)carboxyl protecting group. Biological evaluation revealed that the exocyclic N-terminal segment of ANF is not necessary for expression of high biological activity. The compounds retained high affinity for ANF receptors in bovine adrenal zona glomerulosa cells and were found to be potent antihypertensive and diuretic agents, indicating that the native disulfide bridge can be mimicked by isosteric spanning residues. It was noted that the reported analogues, unlike the endogenous hormone, show marked reduced inhibitory activity on PGE1-stimulated aldosterone secretion from adrenal zona glomerulosa cells. This lack of inhibition may be a contributing element to the low saluresis in spite of the high level of diuresis observed with some analogues.

Identification of structural requirements for analogues of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes.

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.

Conformationally restricted analogues of atriopeptin(103-125)amide.

Conformationally restricted analogues of atriopeptin(103-125)amide were prepared by synthesizing novel bicyclic peptides in which a second disulfide bridge linking residues 108 and 117 was introduced. These syntheses were shown to proceed with no significant scrambling of the disulfide bonds and demonstrated that structurally defined bicyclic analogues of atrial peptides could be easily prepared. The conformationally restrained analogues described here were found to be biologically active with potencies (EC50s) ranging from 0.05 to 3 microM. In addition, these bicyclic peptides (and many of the monocyclic precursors) were found to bind selectively to a class of specific tissue binding sites that have not been shown to be associated with any known second messenger system (NVR binding sites). Since affinity for the receptor class linked to vasorelaxation was negatively affected by the conformational restrictions described here, binding of atrial peptides to this class of receptors appears to have more specific conformational requirements than does binding to the NVR sites.

Small atrial natriuretic peptide analogues: design, synthesis, and structural requirements for guanylate cyclase activation.

Structure/activity studies on atrial natriuretic peptide ANP (1-28) have highlighted three portions of the native molecule as necessary for its biological responses. We have linked these three regions and excised the remaining segments to produce a family of small analogues (less than half the size of the parent) which demonstrate the full range of ANP's actions. Importantly, these compounds act at both major types of ANP receptor. Two critical modifications lead to more potent analogues; both involve expanding the cyclic portion of the molecule. Further optimization of one of these modified structures leads to A68828, a full ANP agonist which shows promise as a preventative agent against acute renal failure.

Restriction of a peptide turn conformation and conformational analysis of guanidino group using arginine-proline fused amino acids: application to mini atrial natriuretic peptide on binding to the receptor.

Compounds (2S,4S)- and (2S,4R)-4-(2'-guanidinoethyl)proline have been synthesized as a conformationally restricted arginine. Their backbones fit the i + 1 position in a turn, and the side chains are restricted compared to that of arginine. These analogues were incorporated into mini atrial natriuretic polypeptide, which has an important turnlike conformation at Gly(6)-Arg(7)()-Met(8)-Asp(9). Structural analysis revealed that the size of the conformational space of Arg(7) on binding to the receptor was approximately one-third of the entire conformational space.

Chemical synthesis and structure-activity relations for ANF analogues.

Chemical synthesis of a new peptide hormone allows absolute confirmation of the peptide's structure, allows physiologic and pharmacologic evaluation of the hormone's properties in vivo and in vitro, and permits development of a radioimmunoassay for the hormone. The study of the effects of structure modification on the bioactivity of atrial natriuretic factor (ANF) is at the earliest stages of defining minimum molecular size, aspects of the bioactive conformation, and the contribution of each side chain to receptor binding. A particular problem in the evaluation of structure-function studies with ANF is the diversity of bioassays used by various laboratories.

Rapid isolation and chemical synthesis of two porcine cardiodilatins, the cardiac hormone precursor and related fragment.

To evaluate the chemically synthesized materials, two cardiodilatins, CDD-126 and CDD-88/CDD(39-126), a precursor of atrial hormone and a related fragment, were isolated from porcine atria by immunoaffinity chromatography or alginic acid adsorption followed by an ion exchange high performance liquid chromatography. The chemical synthesis was carried out using an automated peptide synthesizer. After cleavage and refolding, the crude CDDs were directly characterized by the novel method of primary structure determination using electroblotting and microsequencing. The purified synthetic CDDs were identical with the natural ones in physicochemical and immunochemical properties, as well as their biological actions both in vivo and in vitro.

Development of more potent atrial natriuretic factor (ANF) analogs.

Four modified atrial natriuretic factor (ANF) analogs were designed and synthesized by the solid-phase method. Using human ANF-(99-126) as the reference compound the receptor binding affinity and biological activity of these analogs were examined by radioreceptor assay and in vivo experiments. PLO68, a 21 amino peptide with structural modifications des-Ser103,104-[Mpr105,D-Ala107,114]APII-amide exhibited 2.5 and 2.2 fold more activity than human ANF-(99-126) in lowering blood pressure and causing natriuresis in urethane anesthetized rats. Receptor binding assays using rat lung membranes showed that PLO68 had a Kd of 200 +/- 12 pM compared to a Kd of 620 +/- 12 pM for human ANF-(99-126). Similar chemical modifications, except for substitution of glycine and alanine at the positions 115 and 118, and 120 by D-alanine resulted in three analogs PLO63 and PLO64, PLO67 respectively. PLO63, PLO64 and PLO69 had similar affinities and in vivo potency as human ANF-(99-126). These data suggest that the structural modifications made in PLO68 can cause an increase in the receptor binding ability and an enhancement of biological activities.

Receptor binding affinity and thermolysin degradation of truncated and retro-inverso-isomeric ANF analogs.

The peptides H-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-NH2 (rANF8-15-NH2), Ac-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-NH2 (Ac-rANF8-15-NH2), and their corresponding retro-inverso-isomeric peptides H-D-Ile-D-Arg-D-Asp-D-Ile-D-Arg-Gly-Gly-D-Phe-NH2 (D-rANF15-8-NH2), Ac-D-Ile-D-Arg-D-Asp-D-Ile-D-Arg-Gly-Gly-D-Phe-NH2 (Ac-D-rANF15-8-NH2), were evaluated for their ability to compete for the binding of 125I-rANF5-28 to cultured spontaneously hypertensive rat (SHR) aortic smooth muscle cell membranes. Their stability toward hydrolysis by the neutral endopeptidase thermolysin was also studied. The octapeptides rANF8-15-NH2 and Ac-rANF8-15-NH2 bound with IC50's of 367 pM and 1900 pM, respectively, but were rapidly hydrolyzed by thermolysin. Retro-inverso-isomers were prepared to provide molecules with an improved enzymatic stability. The retro-inverso-isomers were completely stable to thermolysin but were virtually inactive in the binding assay (IC50 greater than 1 microM).

Relative natriuretic activities of synthetic atriopeptins I, II and III.

Recent evidence indicates that mammalian atria contain a series of peptides which possess potent natriuretic activity. Using a sensitive and reproducible bioassay developed by this laboratory, the natriuretic and diuretic activities of three peptides, atriopeptins I, II, and III (21, 23 and 24 amino acids, respectively) were compared. Bioassays were conducted in pentobarbital-anesthetized male Sprague-Dawley rats weighing 300-350 grams. At doses ranging from 0.33 to 3.0 micrograms, no significant differences in natriuretic or diuretic activities were observed between the three peptides. The time courses of the natriuretic and diuretic responses to these peptides were also identical. The finding that atriopeptin I (21 amino acids) possesses natriuretic activity equal to that of atriopeptins II and III suggests that the C-terminus residues of atriopeptin III (Phe-Arg-Tyr) are not necessary for full expression of natriuretic activity. However, since several previous reports have indicated that atriopeptin I is considerably less potent as a natriuretic than we report here, perhaps a cautionary note should be sounded concerning the conditions required to produce or to retain full biologic activities of the synthetic atriopeptins.

Complexes of Cu(II) with Asn-Ser-Phe-Arg-Tyr-NH2; an example of metal ion-promoted conformational organization which results in exceptionally high complex stability.

The pentapeptide fragment of ANF, Asn-Ser-Phe-Arg-Tyr-NH2, coordinates to Cu(II) using the same four nitrogen donor centers as simple pentapeptides such as pentaalanine yet the complexes are of much higher stability as a result of a highly organized side-chain structure which is present in the complex but absent from the free ligand.

Structure-function interrelation and clinical effect of atrial natriuretic peptide (ANP).

Atriopeptin III (AP III) and its six analogues were synthesized by solid phase method and their diuretic and hypotensive activities were determined. Among these analogues, analogue [D-Ala-5, D-Arg-23] AP III was nearly 10 times as potent as AP III in diuretic activity while its hypotensive activity increased only 50% of that of AP III. Analogue des [Ser-15Gly-16Leu-17Gly-18Asn-20Ser-21] AP III was 15% as potent as AP III in diuretic activity, but it still maintained about 60% of the hypotensive activity of AP III. Meanwhile, we tried analogue [D-Ala-5, D-Agr-23] AP III for the treatment of hypertensive syndrome in pregnancy and obtained some good results.

[Atrial natriuretic peptides. II. Synthesis of N-terminal fragments]. / Atrial'nye natriiureticheskie peptidy. II. Sintez N-kontsevykh fragmentov.

N-terminal fragments of atrial natriuretic peptides have been synthetized by classical methods of peptide chemistry in solution and characterized by various physicochemical methods. The choice of the scheme and methods of synthesis is discussed.

[Atrial natriuretic peptides. I. Synthesis of C-terminal fragments]. / Atrial'nye natriiureticheskie peptidy. I. Sintez C-kontsevykh fragmentov.

C-terminal fragments of atrial natriuretic peptides have been synthesized by classical methods of peptide chemistry in solution and characterized by various physico-chemical methods. The choice of the scheme and methods of synthesis is discussed.

[Atrial natriuretic peptides. IV. Synthesis and study of shortened analogs]. / Atrial'nye natriiureticheskie peptidy. IV. Sintez i izuchenie ukorochennykh analogov.

New analogues of atrial peptides of rat were synthesized by classical methods of peptide chemistry in solution. They contain a D-amino acid residue in the C-terminal part and a residue of mercaptopropionic acid in the N-terminal part of the molecule. Biological activity of the new analogues was studied.