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More than 40 % of the world's population is at risk of being infected with malaria. Most malaria cases occur in the countries of sub-Saharan Africa, Central and South America, and Asia. Resistance to standard therapy, including artemisinin combinations, is increasing. There is an urgent need for novel antimalarials with new mechanisms of action. In a phenotypic screen, we identified a series of phenylalanine-based compounds that exhibit antimalarial activity via a new and yet unknown mechanism of action. Our optimization efforts culminated in the selection of ACT-451840 [(S,E)-N-(4-(4-acetylpiperazin-1-yl)benzyl)-3-(4-(tert-butyl)phenyl)-N-(1-(4-(4-cyanobenzyl)piperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)acrylamide] for clinical development. Herein we describe our optimization efforts from the screening hit to the potential drug candidate with respect to antiparasitic activity, drug metabolism and pharmacokinetics (DMPK) properties, and in vivo pharmacological efficacy.

The renin-angiotensin system is a well-known regulator of blood pressure and plays an important role in the pathogenesis of cardiovascular disease and renal damage. Genetic factors, including single nucleotide polymorphisms and sex, are increasingly recognized as potential risk factors for the development of cardiovascular disease. Double transgenic rats (dTGRs), harboring human renin and angiotensinogen genes, were used in this study to investigate potential sex differences influencing renal function and renal gene expression. dTGR males and females had comparable increases in blood pressure, whereas body weight, albuminuria/proteinuria, and urine flow rate were higher in males. At 8 weeks of age, renal plasma flow and glomerular filtration rate were proportionally lower in males, and renal vascular resistance tended to be higher. Males developed more severe tubulointerstitial and vascular lesions. By the end of week 8, 40%of the males but none of the females had died. Genome expression studies were performed with RNA from kidneys of 7-week–old male and female dTGRs and control rats to further investigate the sex-related differences on a molecular level. Forty-five genes showed sex-dependent expression patterns in dTGRs that were significantly different compared to controls. Cathepsin L, one of the genes differentially expressed between the sexes, was also shown to be strongly associated with the degree of renal injury. In dTGRs, urinary cathepsin L at week 7 was higher in males (nanograms per 24 hours: male, 512±163; female, 132±70). These results reveal a potential new biomarker for the personalized diagnosis and management of chronic kidney disease.

The optimization of the 4-position of recently described new 3,4-disubstituted piperidine-based renin inhibitors is reported herein. The synthesis and characterization of compounds leading to the discovery of 11 (ACT-178882, MK-1597), a renin inhibitor with a suitable profile for development is described.

The discovery of a new series of piperidine-based renin inhibitors is described herein. SAR optimization upon the P3 renin sub-pocket is described, leading to the discovery of 9 and 41, two bioavailable renin inhibitors orally active at low doses in a transgenic rat model of hypertension.

Background Artemisinin resistance observed in Southeast Asia threatens the continued use of artemisinin-based combination therapy in endemic countries. Additionally, the diversity of chemical mode of action in the global portfolio of marketed antimalarials is extremely limited. Addressing the urgent need for the development of new antimalarials, a chemical class of potent antimalarial compounds with a novel mode of action was recently identified. Herein, the preclinical characterization of one of these compounds, ACT-451840, conducted in partnership with academic and industrial groups is presented. Method and Findings The properties of ACT-451840 are described, including its spectrum of activities against multiple life cycle stages of the human malaria parasite Plasmodium falciparum (asexual and sexual) and Plasmodium vivax (asexual) as well as oral in vivo efficacies in two murine malaria models that permit infection with the human and the rodent parasites P. falciparum and Plasmodium berghei, respectively. In vitro, ACT-451840 showed a 50% inhibition concentration of 0.4 nM (standard deviation [SD]: ± 0.0 nM) against the drug-sensitive P. falciparum NF54 strain. The 90% effective doses in the in vivo efficacy models were 3.7 mg/kg against P. falciparum (95% confidence interval: 3.3–4.9 mg/kg) and 13 mg/kg against P. berghei (95% confidence interval: 11–16 mg/kg). ACT-451840 potently prevented male gamete formation from the gametocyte stage with a 50% inhibition concentration of 5.89 nM (SD: ± 1.80 nM) and dose-dependently blocked oocyst development in the mosquito with a 50% inhibitory concentration of 30 nM (range: 23–39). The compound’s preclinical safety profile is presented and is in line with the published results of the first-in-man study in healthy male participants, in whom ACT-451840 was well tolerated. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was applied using efficacy in the murine models (defined either as antimalarial activity or as survival) in relation to area under the concentration versus time curve (AUC), maximum observed plasma concentration (Cmax), and time above a threshold concentration. The determination of the dose–efficacy relationship of ACT-451840 under curative conditions in rodent malaria models allowed prediction of the human efficacious exposure. Conclusion The dual activity of ACT-451840 against asexual and sexual stages of P. falciparum and the activity on P. vivax have the potential to meet the specific profile of a target compound that could replace the fast-acting artemisinin component and harbor additional gametocytocidal activity and, thereby, transmission-blocking properties. The fast parasite reduction ratio (PRR) and gametocytocidal effect of ACT-451840 were recently also confirmed in a clinical proof-of-concept (POC) study., Sergio Wittlin and colleagues report in vivo and in vitro modelling of antimalarial activity and dose-efficacy of a new drug candidate., Author Summary Why Was This Study Done? The limited diversity of chemical mode of action in the global portfolio of marketed antimalarials along with recently observed artemisinin resistance that threatens the current first-line treatment highlights the urgent need for development of new antimalarials. In accordance with target product profiles defined by the Medicines for Malaria Venture ([MMV]; www.mmv.org), a new model of not-for-profit public–private partnership providing guidance to the development of new drugs, ACT-451840, a new chemical class of potent compounds with a novel mode of action, was selected for early development. This manuscript integrates a number of studies performed to characterize preclinically ACT-451840 and illustrates the new antimalarial drug development paradigm. What Did the Researchers Do and Find? This study used in vitro models to investigate compound activity on sexual and asexual blood stages and a mouse model to study the human parasite P. falciparum in vivo. While being fully active against artemisinin-resistant strains, ACT-451840 shares many of the favorable properties of artemisinins like its fast onset of action, activity against all asexual blood stage forms, and a PRR of >4 log per parasite cycle. Pharmacodynamic properties of ACT-451840 were interpreted in two mouse models of malaria with respect to the pharmacokinetic properties, with the objective to establish the portable PK/PD parameters to estimate efficacious exposure. What Do These Findings Mean? Modeling the dose–efficacy relationship of ACT-451840 supported prediction of the human efficacious exposure and therefore laid the groundwork for the new clinical development phases. Confirmed in an experimental human malaria infection model, the properties of ACT-451840, including its fast action observed in in vitro and in vivo models and its transmission-blocking activity, suggest this compound may be able to replace the artemisinin component in artemisinin-based combination therapy.

Urotensin-II (U-II) is a cyclic peptide now described as the most potent vasoconstrictor known. U-II binds to a specific G protein-coupled receptor, formerly the orphan receptor GPR14, now renamed urotensin receptor (UT receptor), and present in mammalian species. Palosuran (ACT-058362; 1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt) is a new potent and specific antagonist of the human UT receptor. ACT-058362 antagonizes the specific binding of (125)I-labeled U-II on natural and recombinant cells carrying the human UT receptor with a high affinity in the low nanomolar range and a competitive mode of antagonism, revealed only with prolonged incubation times. ACT-058362 also inhibits U-II-induced calcium mobilization and mitogen-activated protein kinase phosphorylation. The binding inhibitory potency of ACT-058362 is more than 100-fold less on the rat than on the human UT receptor, which is reflected in a pD'(2) value of 5.2 for inhibiting contraction of isolated rat aortic rings induced by U-II. In functional assays of short incubation times, ACT-058362 behaves as an apparent noncompetitive inhibitor. In vivo, intravenous ACT-058362 prevents the no-reflow phenomenon, which follows renal artery clamping in rats, without decreasing blood pressure and prevents the subsequent development of acute renal failure and the histological consequences of ischemia. In conclusion, the in vivo efficacy of the specific UT receptor antagonist ACT-058362 reveals a role of endogenous U-II in renal ischemia. As a selective renal vasodilator, ACT-058362 may be effective in other renal diseases.

Renin is produced by the kidney and secreted into the systemic circulation. However, its biochemical and physiological role of regulating renal blood flow with changing renal perfusion pressure (RPP) is not fully understood. In this study, the function of the intrarenal renin for production of angiotensin (Ang) I and maintenance of vascular tone was evaluated in dogs under normal conditions and when the kidney was perfused at low RPP. The dog left kidney was perfused first at normal (100 mm Hg) and then at low (30 mm Hg) RPP in the presence or absence of the renin inhibitor ciprokiren (3 mg/kg, i.v.). Both hemodynamic and biochemical parameters were measured. Lowering RPP markedly reduced left renal blood flow and elevated left renal vascular resistance. These effects were prevented by ciprokiren, which blocked the intrarenal production of Ang I. Lowering RPP increased the renal venous/ arterial ratio from 1.4+/-0.1 to 3.6+/-0.3 for plasma renin activity and from 2.4+/-0.2 to 9.8+/-1.1 for Ang I, but did not change the venous/arterial ratio for Ang II. The net renal venous conversion rate of Ang I to Ang II decreased from 0.22 to 0.09 after RPP was lowered, whereas the conversion rate in arterial blood was 1.35 and did not decrease significantly. Our results demonstrated the importance of intrarenal renin-angiotensin system for Ang I production and for the maintenance of the vascular tone, especially at low RPP. Our study also shows the limited capacity for Ang I conversion in the renal vasculature in vivo.

The identification, synthesis and activity of a novel class of piperidine renin inhibitors is presented. The most active compounds show activities in the picomolar range and are among the most potent renin inhibitors ever identified.

Blockade of the renin-angiotensin system by inhibition of angiotensin-converting enzyme (ACE) is beneficial for the treatment of hypertension and congestive heart failure. However, it is unclear how complete the blockade by ACE inhibitors is and if there is continuing angiotensin II (Ang II) formation during chronic treatment with ACE inhibitors. Indeed chymase, a serine protease, which is able to form angiotensin II from angiotensin I (Ang I) and cannot be blocked by ACE inhibitors, has been shown to be present in human heart. The goal of the present study was to evaluate the extent of renin-angiotensin system blockade and the Ang II–forming pathways in cardiac tissue of patients chronically treated with ACE inhibitors or in patients without ACE inhibition therapy. Our studies indicate an incomplete ACE inhibition in human heart tissue after chronic ACE inhibitor therapy. Moreover, ACE contributes only a small portion to the total Ang I conversion, as shown in biochemical studies in ventricular and coronary homogenates or functionally as Ang I contractions in isolated rings of coronary arteries. A serine protease was responsible for the majority of Ang II production in both the membrane preparation and Ang I–induced contractions of isolated coronary arteries. In humans, the serine protease pathway is likely to play an important role in cardiac Ang II formation. Thus, drugs such as renin inhibitors and Ang II receptor blockers might be able to induce a more complete blockade of the renin-angiotensin system, providing a more efficacious therapy.

Human renin has been expressed in Sf9 and CHO cells using two different gene constructs. The first construct contained a foreign signal peptide fused directly to the sequence encoding mature renin, whereas the second construct harbors the sequence for preprorenin. Prorenin was produced in significantly higher amounts than the mature enzyme expressed without its propeptide in both expression systems. Both directly expressed mature renin and proteolytically derived active renin have been purified and cocrystallized with the renin inhibitor Ro 42-5892. The 3D structure has been solved for both versions and demonstrates identity despite different glycosylation and different N termini.

1. An hplc method with fluorescence derivatization was developed for the quantification of remikiren in plasma (limit of quantification 2 ng/ml). This was used to determine the pharmacokinetics in various species of primate, in which the compound is a potent inhibitor of renin, as well as in the rat and dog in which it is less active. 2. After intravenous administration the mean residence time wasor = 1.5 h in all species, and the plasma clearance approached the corresponding hepatic blood flows. 3. Studies in the bile-duct cannulated rat and dog demonstrated that the high clearance was due to a combination of rapid metabolism, plus biliary and renal excretion of intact drug. 4. Consistent with the high hepatic clearance, oral bioavailability was low (or = 6%) in each species. However, all of the species tested absorbed a small proportion of an oral dose extremely rapidly, to give peak concentrations generally within 5 min of administration. 5. 'Simultaneous' collection of blood samples from the hepatic portal vein and aorta of rat confirmed that shortly after oral dosing the intact drug did cross the liver; however, the later collections contained predominantly more polar metabolites. 6. The rapid absorption of intact remikiren is consistent with the transient blockade of plasma renin activity, previously observed in primates after oral administration. However, the high clearance appears inconsistent with the subsequent prolonged decrease in blood pressure, suggesting that the latter effect is mediated through a 'tissue' compartment.

ACE inhibition has been shown to prevent neointima formation after vascular injury. However, it is not known if this effect is due to a specific inhibition of the renin angiotensin system or to another mechanism such as the accumulation of bradykinin. In order to answer this question we compared the effects of maximal effective doses of cilazapril, an angiotensin converting enzyme (ACE) inhibitor, and ciprokiren, a new renin inhibitor, in guinea pigs. Vascular injury was induced by endothelial denudation of the right carotid artery of guinea pigs treated either by saline (control group), cilazapril (30 mg/kg/day) or ciprokiren (24 mg/kg/day). Twelve days after the ballooning, the guinea pigs were sacrificed, the carotid arteries were perfused fixed and neointima formation was evaluated by quantitative morphometry. Both, ciprokiren and cilazapril prevented neointima formation to the same extent (inhibition by 42 and 49%, respectively, p These results suggest that, in guinea pigs, renin inhibition prevents neointima formation to a similar extent as ACE inhibition. Therefore, ACE inhibitors seem to act in this model by inhibiting the renin angiotensin system and not by other effects such as accumulation of bradykinin.

BACKGROUND Angiotensin converting enzyme (ACE) inhibition has been shown to prevent neointima formation after vascular injury in rats. However, clinical results evaluating restenosis after angioplasty have been negative. The goal of the present study was to evaluate the influence of the renin-angiotensin system (RAS) status on the effect of ACE inhibition on neointima formation. METHODS AND RESULTS Arterial injury was produced by ballooning the left carotid artery of rats, and neointima formation was evaluated by morphometry 2 weeks after ballooning. The effects of cilazapril were assessed in four experimental groups: normotensive rats, spontaneously hypertensive rats, hypertensive rats with a renal artery stenosis induced by clipping (two-kidney, one-clip rats), and hypertensive rats with uninephrectomy, high salt intake, and administration of deoxycorticosterone (DOCA). In parallel groups of rats, measurement of plasma renin activity was made in order to characterize (at least at the plasma level) the status of the RAS. As expected, renal artery stenosis markedly increased plasma renin activity, and DOCA decreased it to undetectable levels. Cilazapril had a marked preventive effect on neointima formation in normotensive rats, spontaneously hypertensive rats, and two-kidney, one-clip rats but was ineffective in DOCA rats. CONCLUSIONS We conclude that the status of the RAS has a major influence on the effect of cilazapril on neointima formation after vascular injury.

The purpose of the present study was to evaluate whether in addition to its myocardial effects, chronic beta-adrenergic stimulation with isoproterenol could modify the coronary vasculature. For this purpose, rats were treated for 2 weeks with isoproterenol, and the hearts were isolated for determination of the minimal coronary vascular resistance under maximal vasodilation and then perfused-fixed for morphometry of the coronary arteries. In addition, because isoproterenol stimulates the renin-angiotensin system (RAS), the effects of angiotensin-converting enzyme (ACE) inhibition on the coronary vasculature were assessed. Isoproterenol increased heart weight by 65% and minimal coronary resistance by 55%. This effect was associated with medial thickening in intermediate (32%) and large coronary arterioles (26%). Cilazapril, a long-acting ACE inhibitor, completely prevented the medial thickening induced by isoproterenol and normalized the minimal coronary resistance. We conclude that impairment of the rat coronary vasculature induced by chronic beta-adrenergic stimulation might be in part mediated by activation of the RAS.

The goal of the present study was to compare the hemodynamic and biochemical effects of the renin inhibitor Ro 42-5892, the angiotensin converting enzyme inhibitor cilazapril, and the angiotensin II receptor blocker EXP132, the aldehyde derivative of DuP 753. The three drugs were evaluated in guinea pigs, previously treated with furosemide, using their maximal effective doses. Cilazapril decreased arterial blood pressure more than Ro 42-5892 and EXP132. In contrast, Ro 42-5892 and EXP132 had similar effects. The larger decrease of arterial pressure induced by cilazapril was not due to a larger decrease of angiotensin II in plasma and was not influenced by cyclooxygenase inhibition with indomethacin or by bradykinin antagonism with Hoe 140. After binephrectomy, most of the blood pressure-lowering effect of Ro 42-5892 disappeared. In contrast, cilazapril was still markedly effective, pointing to extrarenal effects. We conclude that in furosemide-treated guinea pigs, as opposed to previously published animal models, the decrease of arterial pressure induced by angiotensin converting enzyme inhibitors may be partly due to extrarenal effects not related to the renin-angiotensin system.

Starting from known piperidine renin inhibitors, a new series of 3,9-diazabicyclo[3.3.1]nonene derivatives was rationally designed and prepared. Optimization of the positions 3, 6, and 7 of the diazabicyclonene template led to potent renin inhibitors. The substituents attached at the positions 6 and 7 were essential for the binding affinity of these compounds for renin. The introduction of a substituent attached at the position 3 did not modify the binding affinity but allowed the modulation of the ADME properties. Our efforts led to the discovery of compound (+)-26g that inhibits renin with an IC(50) of 0.20 nM in buffer and 19 nM in plasma. The pharmacokinetics properties of this and other similar compounds are discussed. Compound (+)-26g is well absorbed in rats and efficacious at 10 mg/kg in vivo.

The goal of the present study was to characterize the new renin inhibitor Ro 42-5892 in vitro and in vivo. In vitro, Ro 42-5892 inhibited purified human renin and human plasma renin specifically with an IC50 of 0.7 nM and 0.8 nM, respectively. In vivo, Ro 42-5892 reduced mean arterial blood pressure in sodium-depleted marmosets and squirrel monkeys with as low a dose as 0.1 mg/kg orally. Higher doses reduced pressure by 30-35 mm Hg in both species. The duration of blood pressure decrease with 3 mg/kg orally was more than 24 hours. Maximal changes of plasma renin activity, immunoreactive angiotensin I, and immunoreactive angiotensin II were observed at 15 minutes. Renin was reduced by 74 +/- 31%, angiotensin I by 85 +/- 14%, angiotensin II by 89 +/- 17%, and immunoreactive active renin was increased by 70 +/- 39%. However, unlike pressure, these maximal effects were only transient with complete recovery of renin at 60 minutes under still reduced levels of angiotensin I (61 +/- 24%) and angiotensin II (71 +/- 38%) and increased concentrations of active renin (86 +/- 30%). The blood pressure lowering was due to specific renin inhibition as exemplified by the influence of the kidney, sodium status, species, or stereoselectivity. Moreover, the reduction of arterial blood pressure was similar to the action of the angiotensin converting enzyme inhibitor cilazapril and was not associated with reflex tachycardia in contrast to the pure vasodilator minoxidil. We conclude that Ro 42-5892 is a potent orally active renin inhibitor acting mainly by inhibition of renin in an extraplasmatic compartment.

Anantin, a peptide binding to the receptor of the atrial natriuretic factor (ANF) was isolated from a strain of Streptomyces coerulescens. The molecule consists of 17 natural L-amino acids which form a peptidic ring system. It has a MW of 1,871.0. The chemical composition is C90H111N21O24. The compound was found to bind competitively to ANF-receptors from bovine adrenal cortex (Kd = 0.61 microM). Furthermore, it dose-dependently inhibited the ANF-induced intracellular cyclic guanosine monophosphate accumulation in bovine aorta smooth muscle cells. At the same concentration no agonistic effects were detectable in these cells. Thus, anantin is considered to be the first microbially produced antagonist of the cardiac hormone, ANF.

Chronic hypoxia is known to be associated with a thickening of the media of pulmonary arteries. The goal of the present study was to assess if cilazapril, a novel long-acting angiotensin converting enzyme (ACE) inhibitor, could prevent this thickening. For this purpose, three groups of rats were studied. One group was kept in normal room air. Two other groups were exposed to chronic hypoxia (inspired fraction of oxygen equal to 8% during 4 weeks). One group of hypoxic rats was treated with placebo and the other group received cilazapril (as food admixture of approximately 3 mg/kg/day). After 4 weeks, rats were anesthetized and pulmonary artery pressure and hematocrit measured. Then, the lungs were perfused and fixed and morphometry of the pulmonary arteries was performed. Hypoxia induced an increase in pulmonary artery pressure and hematocrit associated with a dramatic increase in the thickness of the media of the pulmonary arteries. Cilazapril completely prevented the thickening of the media of the pulmonary arteries but did not significantly decrease the pulmonary artery pressure or right ventricular weight.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.

The mutant mouse strain Mpv17−/−, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17−/ − mice might be hypertensive. Indeed, our study revealed that Mpv17−/ − mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concommittantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17−/− mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

The poor interspecies conservation of the renin-angiotensin system prevents the use of nonprimate in vivo models to test renin inhibitors. Thus the small New-World monkey marmoset is used in many instances as a model. However, large differences between the potencies of renin inhibitors as measured in human and marmoset plasma were observed. To understand this phenomenon, we cloned marmoset renin and angiotensinogen. They were highly homologous to their human counterparts, except for a six-residue deletion in the marmoset renin propeptide. Human and marmoset recombinant renins were found in vitro to display comparable activities, suggesting that the observed differences in plasma apparent affinity of inhibitors could be due to different plasma protein binding of the inhibitors.

Piperidine renin inhibitors with heterocyclic core modifications or hydrophilic attachments show improved physical properties (lower lipophilicity, improved solubility). Tetrahydroquinoline derivative rac-30 with a molecular weight of 517 and a log D(pH 7.4) of 1.9 displays potent and long lasting blood pressure lowering effects after oral administration to sodium depleted conscious marmosets.

Background The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. Results High-throughput screening of the Roche compound library identified a simple 3,4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. Conclusions The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

1. Following intravenous administration of 14C-remikiren to the male rat, 78% of the administered radioactivity was recovered in faeces, indicating high biliary elimination. Of the 25 +/- 0.1% of the dose recovered in urine, the majority (16.5% of dose) was intact drug. 2. After oral administration to the male rat the urinary recovery was markedly reduced (8.5 +/- 2.0% of dose), and virtually all of the material was excreted as an inactive hydrolysis product. Intact drug was non-detectable, suggesting extensive first-pass metabolism. 3. Perfusion of isolated rat liver confirmed high biliary elimination, coupled with extensive metabolism. Although intact remikiren was the major component in bile (20% of the 'dose'), the majority of the radioactivity was recovered as a series of mono- and di-hydroxylated metabolites. 4. When screened against human renin, only one of the metabolites in bile and urine (mono-hydroxylated in the t-butyl side chain, and synthesized as Ro 44-0444) showed comparable activity to remikiren. The remaining ten metabolites tested were at least one order of magnitude less active than the parent drug. 5. In comparative in vitro studies Ro 44-0444 was formed by rat, but not human or cynomolgus monkey, liver microsomes. The primate microsomes also produced more of the remaining mono- and di-hydroxy products, suggesting that metabolites make little contribution to the oral activity of remikiren which is observed in these species in vivo.

Abstract Transgenic rats, which express the human angiotensinogen gene, provide a unique model for studying local vascular effects of human renin. We examined the cleavage of human angiotensinogen to angiotensin I (Ang I) by human renin and its inhibition by a human renin inhibitor in an isolated perfused hindlimb preparation from such rats. Perfusion resulted in the sustained release of human angiotensinogen, which decreased from 19.4 to 11.8 pmol/mL over 45 minutes. Active human renin at doses of 3, 10, and 30 ng/mL perfusate for 15 minutes increased Ang I release from undetectable levels (mean±SEM) to 31.9±3.3, 147.1±26.2, and 206.4±17.1 fmol/mL, respectively, by 9 minutes. In separate experiments aimed at the quantification of renin-induced vasoconstriction, captopril decreased the perfusion pressure and lowered Ang II concentrations to nondetectable levels, whereas Ang I values increased sharply. When renin (30 ng/mL) was infused for 15 minutes, renin values in the perfusate decreased to barely detectable levels within minutes after termination of the infusion. However, Ang I values remained high for at least 30 minutes thereafter. The addition of a human renin inhibitor during renin infusion caused Ang I values to promptly decrease within minutes to undetectable levels. Hindlimbs from nontransgenic control rats released no detectable amounts of Ang I, with or without human renin. Finally, by in situ hybridization we documented the presence of human angiotensinogen message in the vessels of the hindlimb. We conclude that renin acts on angiotensinogen at a site in the vascular wall. The cleavage depends on renin and not on other lysosomal proteases. Transgenic rats are a novel model that may be used to test the functional importance of the local human renin-angiotensin system in experimental animals.

The goal of the present study was to compare the effects of three potent reference renin inhibitors (remikiren, CGP 38560A, and enalkiren) in sodium-depleted normotensive squirrel monkeys. In these monkeys, arterial pressure was measured in the conscious state with a telemetry system. Oral and intravenous maximal effective doses of the three renin inhibitors were compared in parallel groups of monkeys. In additional experiments, remikiren was given on top of either CGP 38560A or enalkiren in the same animals. Finally, the three drugs were compared with the angiotensin converting enzyme inhibitor cilazapril. The effects of the three drugs on the plasma components of the renin-angiotensin system (plasma renin activity, immunoreactive renin, and immunoreactive angiotensin II concentrations) were also measured. Our results show that remikiren was as effective as cilazapril and markedly more effective than CGP 38560A or enalkiren in reducing arterial pressure in our monkey model. Interestingly, these differences in arterial pressure could not be explained by differences of in vitro potency or different biochemical changes of the plasma components of the renin-angiotensin system, because the inhibitors all reduced immunoreactive angiotensin II to similarly low levels. One possible explanation is that, in our model, remikiren in contrast to CGP 38560A and enalkiren is able to inhibit renin in a functionally important extraplasmatic compartment.

OBJECTIVE To compare the acute hypotensive effects of three different methods of inhibiting the renin-angiotensin system in a primate model of cyclosporin-induced hypertension. DESIGN The effects of maximally effective doses of an angiotensin I converting enzyme inhibitor, antihuman renin antibodies or a renin inhibitor (Ro 42-5892) on arterial pressure were evaluated in cyclosporin-treated monkeys. METHODS Squirrel monkeys were made hypertensive by a 4-week treatment with oral cyclosporin (30 mg/kg) and were equipped with a telemetry system in order to measure arterial blood pressure in the conscious state. RESULTS Each inhibitor induced a complete suppression of plasma angiotensin II 1 h after administration. The renin antibodies did not decrease blood pressure. Cilazapril decreased blood pressure and Ro 42-5892 was more effective than cilazapril. Moreover, when the renin inhibitor was administered after injection of renin antibodies (rabbit antiserum, 0.4 ml intravenously) or after cilazapril (10 mg/kg orally), it induced a supplementary fall in blood pressure. CONCLUSIONS These data demonstrate that in this experimental model of hypertension the three different methods of maximal inhibition of the renin-angiotensin system do not lead acutely to the same blood pressure decrease. The results also suggest that renin inhibition might be more effective than angiotensin converting enzyme inhibition in certain forms of hypertension.

OBJECTIVE: The goal of the present study was to show that, in contrast to an angiotensin converting enzyme (ACE) inhibitor, Ro 42-5892, a new renin inhibitor, can block the renin-angiotensin system without potentiating skin reactions induced by bradykinin. DESIGN: Potentiation of skin reaction to i.d. injections of bradykinin and histamine was evaluated in guinea pigs in the presence and absence of the drug (placebo, Ro 42-5892 or cilazapril). The elimination rate of radioactive bradykinin in blood was measured in other groups of guinea pigs treated with the same drugs. Maximal effective doses of each drug were used. METHODS: Measurements of erythema area induced by bradykinin and histamine injection were performed using a digital planimeter. Radioactive bradykinin was measured in blood by high-performance liquid chromatography and followed over 40 min. RESULTS: The ACE inhibitor cilazapril increased the area of erythema induced by bradykinin but not that induced by histamine. In contrast, Ro 42-5892 did not potentiate the effect of bradykinin. In addition, cilazapril did not change the elimination rate of i.v. radioactive bradykinin in blood. CONCLUSION: These results suggest that potentiation of bradykinin-induced skin reaction by cilazapril is due to a tissular (and not systemic) inhibition of ACE and does not occur with Ro 42-5892. Thus, side effects such as rash, angioneurotic edema or cough, which have been attributed to bradykinin accumulation by ACE inhibitors, may not occur with the use of specific renin inhibitors such as Ro 42-5892.

The full primary structure of the very potent opioid peptide dynorphin, from porcine pituitary, has been determined. It is (H)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln(OH). The synthetic peptide with this sequence behaves identically to natural dynorphin in a number of ways, and it has the same potency in the guinea pig ileum myenteric plexus--longitudinal muscle bioassay. The potency is accounted for by the first 13 residues.

A highly hydrophobic analogue of angiotensin II AT, [Sar 1 , (2′,3′,4′,5′,6′-Br5)Phe 8 ]AT exhibited strong and persistent specific antagonism against AT, both in vitro and in vivo . This peptide exhibited 32% of the binding affinity of [Sar 1 ]AT towards membranes of bovine adrenal cortex, it was a specific AT antagonist of irreversible character on smooth muscle assays, and it also suppressed for over 120 min at 7.10 −8 M/kg the blood pressure response towards AT in the rat blood pressure assay. This compound harbours therefore the potential of a new class of AT-specific antihypotensive drugs.

To establish a model of hypertension in marmosets, increasing doses of the immune-modulator cyclosporin (from 3 to 30 mg/kg/day) were given orally to marmosets for 31 days. Cyclosporin consistently induced hypertension with doses greater than or equal to 10 mg/kg/day. Time course and level of hypertension reached were dose-dependent. Hypertension was not associated with an increase of plasma renin activity (PRA) or plasma creatinine concentration and was sensitive to angiotensin-converting enzyme (ACE) inhibition by cilazapril (10 mg/kg p.o.). After cyclosporin treatment was stopped, hypertension regressed. We conclude that hypertension can consistently be induced in marmosets by chronic treatment with cyclosporin.

Cilazapril is a new angiotensin-converting-enzyme inhibitor. In conscious renal-hypertensive dogs, cilazapril (2 X 10 mg/kg/day p.o.) caused a long-lasting (greater than 24 h) decrease in systolic arterial blood pressure, the magnitude of which was potentiated by pretreatment with furosemide. A maximal fall in systolic blood pressure of 39 +/- 6 mm Hg (from 145 +/- 5 to 106 +/- 7 mm Hg) was recorded. The antihypertensive effect did not decline with repeated administration and was accompanied by only a slight increase in heart rate. Cilazapril also reduced systolic blood pressure in furosemide-pretreated normotensive dogs. Hemodynamic studies in anesthetized dogs revealed that cilazapril (0.03-1 mg/kg i.v.) caused a fall in mean arterial and left ventricular systolic pressures. At the highest dose of 1 mg/kg i.v., the blood-pressure-lowering effect (-27%) was due to a decrease in total peripheral resistance (-12%) and cardiac output (-16%). Intravenous administration of cilazapril to anesthetized dogs resulted in a rise in plasma renin activity and a significant fall in plasma angiotensin II levels. In conscious normotensive dogs, cilazapril (0.3-10 mg/kg p.o.) exerted diuretic and saluretic effects, which were accompanied by a significant increase in renal plasma flow (46%), but only a slight rise in the glomerular filtration rate. These results characterize cilazapril as an effective and long-lasting antihypertensive drug, with diuretic activity and, possibly, preload- as well as afterload-reducing properties.

In order to test the effects of the replacement of phenylalanine by carboranylalanine (Car) in biological ligand-acceptor interactions, Z · Ala-Ala-Car · OH (1) and Ac · Car · OEt (2) were synthesized and their reactions with chymotrypsin studied. The two compounds proved to be good inhibitors with K(i) values of 3 · 10−4M(1) and 8.6 · 104M(2); the K(i) of Z · Ala-Ala-Phe · OH (1a) is 1 · 10−3M. The inhibition constants were determined by a new photolytic technique, inhibition of photoaffinity labelling by Z · Ala-Ala-Phe(pN3) · OH. Ac · Car · OEt is not hydrolysed by chymotrypsin. The findings indicate that the carboranyl group can interact with the ‘phenyl recognition site’ of the enzyme to produce the binding that is characteristic of aromatic amino acid residues. However, some kind of distortion in the region of the ‘mechanistic site’ must be postulated in order to account for the failure of hydrolysis. Some possible effects of the replacement of aromatic amino acids by Car in peptide hormones on hormone-receptor interactions are discussed.

Chronic daily administration of cilazapril (1 X 10 mg/kg/day p.o., from age 4 to 14 weeks) to young spontaneously hypertensive rats (SHR) prevented the development of hypertension. The antihypertensive effect of a single dose of cilazapril persisted greater than 24 h. Discontinuation of long-term treatment resulted in an increase of systolic arterial blood pressure (SAP) to control hypertensive levels within 4 days. Following 10 weeks of drug administration, comparative hemodynamic studies were carried out on age-matched (14 weeks) control SHR and cilazapril-treated SHR. Cilazapril-treated SHR had a significantly lower mean arterial blood pressure (MAP) and total peripheral vascular resistance than did control SHR. The antihypertensive effect of cilazapril was not associated with changes in heart rate (HR). The myocardial performance parameters, cardiac output, and stroke volume, were similar in treated and control SHR, suggesting that the antihypertensive effect of cilazapril following chronic administration to SHR is mainly due to a reduction in peripheral vascular resistance. Vasopressor responses to angiotensin I were significantly lower in cilazapril-treated SHR than in control SHR. By contrast, pressor responses to angiotensin II and a high dose of norepinephrine (1.0 microgram/kg i.v.) were significantly enhanced. Isoproterenol elicited a fall in blood pressure in both groups, the extent of which was dependent upon the magnitude of basal blood pressure levels. Chronic cilazapril treatment resulted in a reduction of heart weight, suggesting that the drug may prevent development of cardiac hypertrophy in SHR. Kidney and adrenal weights were unaffected by the chronic treatment. Specific renin activities (SRA) in tissues of SHR were increased by factors of 20 (plasma) or 2 (kidney and adrenal) following cilazapril administration.(ABSTRACT TRUNCATED AT 250 WORDS)