LHRH antagonists.

After almost two decades, the research on LHRH antagonists has produced a number of decapeptides that are currently in clinical studies. The structures of these antagonists, unlike the agonists, differ substantially from that of LHRH. Five of the ten amino acids are unnatural and of D configuration. The structural combination of a hydrophobic N-terminus (residues 1, 2, and 3) and a basic/hydrophilic C-terminus (residues 6 and 8) was thought to be responsible for some HR reactions encountered with the second generation of LHRH antagonists. This side effect was greatly reduced by substituting the appropriate combination of amino acids at positions 5, 6, and 8. The next hurdle in the drug development of LHRH antagonists was solubility and aggregation. In the case of A-75998, water solubility was improved by 12- to 25-fold via substitution of NMeTyr at position 5. However, based on DLS analysis, the aqueous solutions still contained some large aggregates that were not visible to the naked eye. This formation of aggregates was eliminated on formulating A-75998 in Encapsin. In men, a single s.c. dose of 2 mg of A-75998 suppressed T to the castrate levels for over 30 hr. Other LHRH antagonists including ganirelix and cetrorelix are also in phase I/II clinical studies. Clinical studies with cetrorelix in prostate cancer; in vitro fertilization, and benign prostate hypotrophy have been reported.

In vitro and in vivo activities of reduced-size antagonists of luteinizing hormone-releasing hormone.

A novel series of octapeptide LHRH antagonists was designed on the basis of the structure of the (2-9) fragment of a LHRH agonist. By adopting a systematic SAR study, we were able to improve first the in vitro activity and then the in vivo LH suppression, raising them up to the range of the decapeptide antagonists NalGlu (51) and A-75998 (50), resulting in A-76154 (49). The octapeptide antagonist A-76154 is the most potent reduced-size LHRH antagonist reported. It suppresses LH in the castrated rat by over 80% for a period of 4 h following sc bolus administration of 30 micrograms/kg.

Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists.

Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.

Stabilization of the N-terminal residues of luteinizing hormone-releasing hormone agonists and the effect on pharmacokinetics.

To stabilize leuprolide (1) against chymotrypsin and intestinal degradation several agonists of LHRH (2-12), modified at position 1, 2, or 3 and/or containing N-alpha-methyl at positions 1, 2, or 4, were synthesized by SPPS. These agonists were tested in vitro for (a) rat pituitary LHRH receptor binding, (b) LH release from rat pituitary cells, (c) stability against chymotrypsin, and (d) stability against rat intestinal degradation. The clearances of the compounds in the rat were determined using a RIA. Complete stabilization against chymotrypsin (t1/2) and lumenal degradation (T1/2) was achieved with substitution of NMe-Ser4 in leuprolide; however, with an increase in clearance. Substitution with 1-Nal3 increased both t1/2 and T1/2, while substitution with NAc-Sar1 increased only T1/2. [NAcSar1,NMeSer4,D-Trp6,Pro9NHEt]LHRH (12), the doubly stabilized analogue, was tested in the rat by both iv and id administrations, and its bioavailabilities were measured. No significant improvement in id absorption over leuprolide was observed.

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.

Active reduced-size hexapeptide analogues of luteinizing hormone-releasing hormone.

A series of reduced-size hexapeptide analogues of LH-RH were synthesized that contain the residues corresponding to amino acid positions 4-9 and are linked to various carboxylic acids in place of residue 3. These compounds were tested in vitro in the rat pituitary receptor binding and LH release assays. A wide range of binding affinities was obtained up to and exceeding that of LH-RH. Both agonists and antagonists were obtained. From the SAR studies, it appears that a very precise size, length, and shape of the substituent at position 3 is required to achieve agonist activity, whereas the structural requirements for antagonist activity appear to be much less stringent. Depending on the nature of the substituent at positions 6 and 4, the biological response switches from antagonist to agonist or vice versa. The results suggest that conformational changes at position 6 or 4 feed back to the substituent at position 3, which induces the change from agonist to antagonist. The most potent compounds in the series were tested in vivo and found to be active.

Novel adrenergic compounds. I. Receptor interactions of ABBOTT-54741 [(5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthtyl)imidazoline], an alpha-adrenergic agonist.

ABBOTT-54741 was identified as a full alpha-adrenergic agonist; its interaction with the alpha-adrenergic receptor was compared to that of norepinephrine. ABBOTT-54741 lacks affinity for beta-adrenergic receptors. In radioligand binding studies, the affinity of ABBOTT-54741 for alpha 1-adrenoceptors (as measured against 3H-prazosin binding) was KI = 401 nM, and that for norepinephrine was 388 nM. The affinity of ABBOTT-54741 for alpha 2-adrenoceptors (as measured against 3H-rauwolscine binding) was greater than that of norepinephrine (KIA = 7 nM; KI NE = 37 nM). In vitro, ABBOTT-54741 exhibits high potency in vascular preparations (ED50NE/ED50A in rabbit aorta = 12.9; in phenoxybenzamine-treated dog saphenous vein = 188.5). In rabbit pulmonary artery, it shows greater potency for the presynaptic than postsynaptic receptors, corroborating the observations of selectivity obtained in binding studies. The observations in vivo reflect that in isolated tissues. In different species (dog, rat) and via different routes of administration (i.v., p.o., i.c.v., and nasal), ABBOTT-54741 exhibits cardiovascular effects reflecting the stimulation of both alpha 1- and alpha 2-adrenoceptors consistently with much greater potency than norepinephrine or any other alpha agonist known to the authors.

Biologically active atrial natriuretic peptides selectively activate Na/K/Cl cotransport in vascular smooth muscle cells.

Atrial natriuretic peptides (ANPs) cause vasorelaxation, natriuresis and diuresis. Although the precise mechanism of action for these biological activities is not known, it has been established that ANPs can bind to specific membrane receptors and can cause an increase in intracellular cyclic GMP (cGMP) levels. In previously published studies we have probed the mechanism of action of ANP and have shown that one consequence of ANP receptor-mediated increases in cGMP in vascular smooth muscle cells (VSMC) is stimulation of Na/K/Cl cotransport. Although others have suggested that ANPs may affect Na/H exchange and/or Na/K adenosine triphosphatase (ATPase) activity in various cells and tissues, the effect of ANPs on these other Na transport systems in VSMC is not known. Furthermore, the biological relevance of ANP-stimulation of Na/K/Cl cotransport in VSMC has not been established. The goal of the present study was to investigate whether ANPs selectively stimulate Na/K/Cl cotransport in VSMC and to determine whether effects on cotransport parallel biological activity. We tested the effect of six ANPs on Na/K/Cl cotransport, and of one ANP on Na/H exchange and on Na/K ATPase activity. It was found that ANPs stimulated Na/K/Cl cotransport but had no effect on Na/H exchange or on Na/K ATPase activity in VSMC. Biological activity of the ANPs was assayed by measuring the potencies for producing vasorelaxation of aortic rings and for stimulating an increase in intracellular cGMP in VSMC. The rank orders observed for the two biological activities agreed with the rank order for stimulation of Na/K/Cl cotransport.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of atrial natriuretic factor on Na+-K+-Cl- cotransport of vascular smooth muscle cells.

We previously demonstrated that vascular smooth muscle cells possess a prominent Na+-K+-Cl- cotransport system that can be markedly stimulated by elevations in levels of intracellular cyclic guanosine 3',5'-monophosphate (cGMP). Since others have shown that atrial natriuretic factor (ANF) can bind to specific membrane receptors and can enhance cGMP levels in vascular smooth muscle cells, we asked whether ANF could also stimulate Na+-K+-Cl- cotransport in vascular smooth muscle cells. It was discovered that rat atriopeptin III stimulated Na+-K+-Cl- cotransport of vascular smooth muscle cells in a concentration-dependent manner. In contrast, rat atriopeptin III had no effect on two other sodium transport systems known to be present in vascular smooth muscle cells (i.e., Na+-H+ exchange and Na+-K+-adenosine triphosphatase (ATPase). These studies indicated that ANF selectively stimulates Na+-K+-Cl- cotransport of vascular smooth muscle cells. We then asked whether ANF-stimulated Na+-K+-Cl- cotransport was dependent upon the ability of ANF to enhance intracellular cGMP levels. When rat atriopeptin III-stimulated increases in cGMP were inhibited with the quinolinedione LY 83583, rat atriopeptin III could no longer stimulate Na+-K+-Cl- cotransport of vascular smooth muscle cells. Thus it appeared that the effects of ANF were dependent upon the ability of ANF to elevate intracellular cGMP levels. Finally, we asked whether ANF effects on Na+-K+-Cl- cotransport were related to the biological activity of ANF.(ABSTRACT TRUNCATED AT 250 WORDS)

Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes.

ANF analog potencies in stimulating smooth muscle cell cGMP were compared with the ability to relax histamine-constricted rabbit aorta in vitro. ANF[1-28], [5-28], [5-27] and Lys-11[5-28] elevated cGMP and were potent vasorelaxants. ANF[7-23] and Lys-11[7-23] were potent cGMP stimulators but 1000-fold weaker relaxants. Tyr-8[5-27] did not stimulate cGMP synthesis or antagonize the response of the other peptides, yet was a potent vasorelaxant. Crosslinking with 125I-ANF identified bands at 150 and 65 KD by SDS-PAGE. ANF[1-28], Lys-11[7-23] and Tyr-8[5-27] blocked crosslinking at low concentration despite disparate activities. These data support the existence of ANF receptor subtypes and suggest that cGMP elevation alone is not sufficient to promote atrial peptide-induced vasorelaxation.

Similarity of central and peripheral alpha-1 adrenoceptors in rat and rabbit.

In order to examine species and tissue differences in alpha 1 adrenoceptors, binding experiments were performed using 3H-prazosin and membrane homogenates of central nervous and peripheral tissues of rabbit (cortex and spleen), and rat (cortex, spleen, and liver). Saturation studies indicated one binding site for 3H-prazosin, with apparent log molar dissociation constants (pKD) ranging from 9.43 to 10.20. The rank orders of affinities of three competing antagonists (prazosin much greater than idazoxan greater than rauwolscine) and five agonists (cirazoline greater than clonidine approximately equal to (-)-norepinephrine greater than (-)-phenylephrine greater than (+)-norepinephrine) were typical of alpha 1 receptors in all tissues. There were small but significant differences in the mean affinities of rauwolscine, idazoxan and cirazoline among the five tissues. No significant differences in pseudo-Hill coefficients were observed among tissues, although agonist binding curves were shallow (.7 to .85) and prazosin competition curves were significantly steeper (greater than .85). Guanine nucleotide did not affect the position or slope of the (-)-norepinephrine competition profile in rat cortex. These results demonstrate a qualitative similarity among central and peripheral alpha 1 receptors of the rat and rabbit, with small differences observed between central and peripheral sites in both species.

Adrenergic and dopaminergic properties of dopamine sulfoconjugates.

UNLABELLED: The 3- and 4-sulfate esters of dopamine (DA-3-SO4 and DA-4-SO4, respectively), the two main metabolites of DA, were evaluated for potential intrinsic or indirect catecholaminergic activities. Both compounds lacked any appreciable affinities for alpha 1, alpha 2, beta 1, beta 2 and DA-2 receptors. In the superfused [3H]norepinephrine-preloaded dog saphenous vein, both dopamine sulfates were devoid of any intrinsic inhibitory activity such as observed with dopamine pre- and postsynaptically. In addition, they did not displace the labeled vesicular neurotransmitter as did dopamine. In anesthetized dogs the two compounds failed to stimulate either the dopaminergic receptors (DA-1) of mesenteric vascular beds or the adrenergic receptors mediating the vasodilatory and pressor responses to dopamine. CONCLUSION: In our experimental conditions DA-3-SO4 and DA-4-SO4, the products of dopamine sulfoconjugation, lacked any demonstrable intrinsic affinity and/or efficacy on the dopaminergic and adrenergic receptors directly or indirectly through their metabolic transformation or through displacement of endogenous neurotransmitter.

Conformationally defined adrenergic agents. 1. Design and synthesis of novel alpha 2 selective adrenergic agents: electrostatic repulsion based conformational prototypes.

A previous report of the adrenergic selectivity of 2- and 6-fluoronorepinephrine prompted us to formulate a hypothesis that accounted for this selectivity on the basis of a conformational preference induced by electrostatic repulsion between the aromatic fluorine atom and the side-chain hydroxyl group. A series of nitrogen-substituted catechol (aminomethyl)benzocyclobutenes, indanes, tetralins, and benzocycloheptenes were prepared, and when their radioligand binding affinities were determined, it was found that the overall pattern of binding affinity results supported the electrostatic repulsion hypothesis. The radioligand binding assay also revealed several highly alpha 2 selective adrenergic agents among these compounds, with the binding selectivity maximizing for compounds having nitrogen substituted with a group no larger than methyl and having a five-membered carbocyclic ring (i.e., 16, 17, and 19).

Contribution of beta-adrenergic receptor mediated renin release to the maintenance of blood pressure during nitroprusside infusion in conscious rats.

The extent to which beta-adrenergic receptor mediated renin release contributes to the maintenance of blood pressure during hypotension induced by sodium nitroprusside (SNP, 40 micrograms/kg/min for 30 min) was assessed in conscious Wistar rats fitted with chronic aortic and vena caval catheters. Propranolol, (1.5 mg/kg, i.v.), reduced the basal level of plasma renin activity (PRA) in control rats from 4.4 +/- 0.4 to 2.4 +/- 0.4 ng angiotensin I/ml/min (p less than .05) and decreased PRA obtained during SNP infusion from 35.3 +/- 6.6 to 16.7 +/- 2.2 ng angiotensin I/ml/min (p less than .01). Despite the reduced PRA, the hypotensive response to SNP was not enhanced after propranolol. Treatment of these animals with captopril in order to block the actions of the remaining non-beta-receptor released renin, resulted in augmentation of the SNP hypotension. Captopril also potentiated SNP hypotension in rats that had not received propranolol. The addition of propranolol to the captopril treated rats produced no further change in SNP hypotension. The results of this study indicate that beta-receptor-mediated and beta-receptor-independent mechanisms contribute to the renin released during SNP hypotension. However, if the beta-receptor-mediated component alone is blocked, the remaining renin secretion is adequate to maintain blood pressure during SNP infusion.