Orally active aminopeptidase A inhibitors reduce blood pressure: A new strategy for treating of hypertension
Laurence Bodineau1,2,3, Alain Frugière1,2,3, Yannick Marc1,2,3, Nicolas Inguimbert4,5, Céline Fassot1,2,3, Fabrice Balavoine6, Bernard Roques4,5 and Catherine Llorens-Cortes1,2,3.
1 Inserm, U 691, Paris, F-75231 France; 2 Collège de France, Paris, F-75231 France; 3 Université Pierre et Marie Curie-Paris VI, Paris, F-75231 France;4 Inserm, U640, Paris, F-75000 France; 5 Université Paris V, Paris, F-75000 France, 6 Quantum Genomics, Massy, F-91300, France.
Orally active APA inhibitors and hypertension
Word count of manuscript, including title page, abstract, references, legends, tables, and figures: 6556
Word count of abstract: 250
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Corresponding author:
Catherine LLORENS-CORTES, PhD
Inserm U 691
Collège de France
11, place Marcelin Berthelot
75231 PARIS CEDEX 05, FRANCE
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Tel: 33 1 44 27 16 63
Fax: 33 1 44 27 16 67
Materials and Methods
Drugs
2-L-glutamyl-β-naphthylamide (GluβNA), bestatin and all other chemicals were obtained from Bachem, Weil am Rhein, Germany and Sigma Aldrich, Saint Quentin Fallavier, France. The APA inhibitors, RB150 and EC33, were synthesized by the team of B.P. Roques (Institut National de la Santé et de la Recherche Médicale, Unité 640) as previously described.1,2. RB150 was dissolved in saline and the pH adjusted to 7.4 with 0.1 N NaOH for in vivo administration. RB150 or EC33 were dissolved in 50 mmol/L Tris-HCl (pH 7.4) supplemented with 100 eq DTT per eq of inhibitor for the in vitro measurement of APA activity.
Animals
Experiments were performed on male normotensive Wistar Kyoto (WKY), sham and hypertensive DOCA-salt rats weighing about 350 g (Charles River Laboratories, L’Arbresle, France). Sham rats corresponded to unilaterally nephrectomized WKY rats. Hypertensive DOCA-salt rats corresponded to unilaterally nephrectomized WKY rats in which a DOCA pellet had been implanted subcutaneously (50 mg; Innovative Research of America, Florida, USA). After surgery, DOCA-salt rats received a standard rat chow diet and tap water supplemented with 0.9% NaCl and 0.2% KCl. Hypertension was detected three weeks after surgery. All animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
APA activity measurement
APA activity was determined using GluβNA as a synthetic substrate, in initial velocity conditions, as previously described.2,3 The inhibitory potency of RB150, was first evaluated in parallel with that of EC33, against recombinant mouse APA.4 Purified recombinant APA was incubated for 30 minutes at 37°C in the presence of 200 µmol/L GluβNA, with or without various concentrations of RB150 or EC33 (1 nmol/L to 3×103 nmol/L) prepared in 50 mmol/L Tris-HCl buffer (pH 7.4) supplemented with 100 eq DTT per eq of inhibitor, in a total volume of 100 µL of 50 mmol/L Tris-HCl buffer (pH 7.4) supplemented with 4 mmol/L CaCl2 (n = 15). The metabolic product, 2-naphthylamine (βNA) was detected by a colorimetric method, as previously described.2
For ex vivo APA enzymatic activity measurements, 12 hours before administration of the drug, rats were deprived of food. They were then treated orally by gavage with saline or RB150 at several doses (7.5-50.0 mg/kg) in a volume 300 µL. Animals were decapitated 3.5 or 24 hours after po administration and the brains were immediately collected and one half of the brain was homogenized by sonication in 10 volumes of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4). APA activity was measured on brain homogenates.3,5 It was measured with the same assay described at the top of the paragraph. However, as brain homogenate contains many other aminopeptidases able to cleave the substrate, GluβNA, we added bestatin, an inhibitor of non specific aminopeptidases, at a concentration of 1 μmol/L, to prevent degradation of the substrate by aminopeptidases such as B, N, W and cytosolic leucine aminopeptidases.6,7 At this concentration, bestatin does not inhibit APA activity.3 Furthermore, assays were performed in presence or absence of of EC33 (5 µmol/L), for the specific estimation of APA activity. Briefly, aliquots of brain homogenate (16 µL) were incubated for 30 minutes at 37°C with 200 µmol/L GluβNA, 4 mmol/L CaCl2 and 1 µmol/L bestatin inhibitor, with or without EC33 (5 µmol/L) in a total volume of 100 µL of 50 mmol/L Tris-HCl buffer (pH 7.4).8 The reaction was stopped by adding 10 μL of 3 N HCl. Samples were centrifuged 15,000 x g for 10 minutes. Aliquots of supernatant fractions were dispensed into a 96-well microplate, and the metabolic product determined, as previously described.3
Activities are expressed in nmol of βNA/mg protein/h. Protein concentrations were determined as described by Lowry et al.,9 using bovine serum albumin as the standard.
In vitro pharmacological profile
Binding affinities and inhibition activities of RB150 and EC33 for receptors and against enzymes involved in blood pressure control were assessed in various in vitro assays performed at Cerep (Celle l’Evescault, France). For receptor binding assays on AT1(h), AT2(h), endothelin ETA(h) and ETB(h), results were expressed as a percent inhibition of control specific binding (100-((measured specific binding/control specific binding) x 100)) obtained in the presence of RB150 (1µM) and EC33 (10 µM). Specific ligand binding was defined as the difference between the total binding and the nonspecific binding determined in the presence of an excess of unlabelled ligand. Labeled ligand and nonspecific ligand respectively used for binding assays were [125I][Sar1, Ile8]-ATII (0.05 nM) and angiotensin II (10 µM) for AT1(h), [125I]CGP 42221A (0.05 nM) and angiotensin II (1 µM) for AT2(h), and [125I]endothelin-1 (0.03 nM) and endothelin-1 (0.1 µM) for ETA(h) and ETB(h). For enzyme assays on ACE(h), ACE2(h), ECE-1(h) and NEP(h), results were expressed as a percent inhibition of control specific activity (100-((measured specific activity/control specific activity) x 100)) obtained in the presence of RB150 (1 µM) and EC33 (10 µM). Substrate respectively used for enzyme assays were MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-(DNP)-OH (10 µM) for ACE(h), MCA-Tyr-Val-Ala-Asp-Pro-Ala-Lys-(DNP)-OH (10 µM) for ACE2(h), ECE-1 fluorescent substrate (15 µM) for ECE-1(h) and DAGNPG (50 µM) for NEP(h).
Surgical methods and BP recording
BP was recorded in conscious, unrestrained rats. For this purpose, at least two days before the experiment, animals were anesthetized with pentobarbital sodium (60 mg/kg i.p.; Sanofi-Aventis, France) and femoral arterial catheters were implanted surgically, as previously described.10 The catheters were tunneled subcutaneously, to exit from the neck. Animals were treated with penicillin (100,000 units, i.p.), and their temperature was maintained at 37°C throughout their recovery from anesthesia and surgery. We checked that the observed effect of RB150 was central, rather than peripheral. We assessed the capacity of the selective active APA inhibitor, EC33, which does not cross the blood-brain barrier, to decrease BP in hypertensive DOCA-salt rats. For this purpose, rats were catheterized in the femoral vein for the iv injection of EC33.
As previously described,5,11 BP was recorded continuously. One hour after the start of BP recording, saline or RB150 (300 µL) and EC33 (300 µL) were administered orally by gavage and iv, respectively). RB150 (0.1-30.0 mg/kg po), EC33 (15 mg/kg iv) or saline (po), were administered in conscious unrestrained rats.
After po acute treatment with RB150 or saline, BP was monitored during 7 hours on the first day of experiment. For DOCA-salt rats given salin or RB150 (15 mg/kg), an other recording of one hour was performed at 24 and 48 hours post-administration. For DOCA-salt rats treated iv with EC33 (15 mg/kg), BP was continuously recorded for five hours after the injection. Mean BP (MBP) and heart rate (HR) were calculated with the MacLab system (Phymep, Paris, France), consisting of a MacLab hardware unit and CHART software, running on a Macintosh computer. HR was triggered by the BP signal.
Urine and electrolyte output and fluid consumption measurements
Normotensive WKY, and hypertensive DOCA-salt rats were individually housed in metabolic cages (Tecniplast®, Limonest, France), with a constant temperature (23±2°C) and photoperiod (12 hours light, 12 hours dark), with drinking fluid and food freely available. The rats were allowed to acclimatize to the metabolic cages over a period of three days. Fluid consumption and the urinary excretion of water and electrolytes were then measured for each rat after saline (300 µL on the fourth day at 7:00 pm) or RB150 (15 mg/kg; 300 µL on the fifth day at 7:00 pm) administration po. Urinary Na+ and K+ concentrations were determined using a flame photometer (model 243, Instrumentation Laboratory, France) on a non diluted 20 µL sample.
Arginine-vasopressin RIA
Plasma AVP concentrations were estimated from 0.2 ml of plasma using a specific RIA kit (Peninsula Laboratories) using (3-[125I]iodotyrosyl-2)vasopressin- [Arg8], 2000 Ci/mmol) as a tracer and a polyclonal antiserum specific for vasopressin-[Arg8] with no cross reactivity with oxytocin. AVP was extracted from the plasma with C18 Sep-Pak cartridges and measured in duplicate according to the protocol of the kit. The limit of detection of the AVP was 0.2 pg per tube.
Data analysis and statistics
Data are presented as means ± SEM. Comparisons between groups of APA activities, baseline MBP and HR (values corresponding to data obtained at least one hour before po administration), MBP and HR after po administration of RB150or EC33, plasma AVP concentration, drinking fluid, urine flow rate and urinary electrolytes were made with paired or unpaired Student’s t test or one-way analysis of variance (ANOVA) for repeated measures and ANOVA followed by Fisher’s post hoc least squares differences (PLSD) correction for multiple comparisons. Differences were considered significant if P<0.05.
References
1 Fournie-Zaluski MC, Coric P, Turcaud S, Lucas E, Noble F, Maldonado R, Roques BP. "Mixed inhibitor-prodrug" as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes. J Med Chem. 1992;35:2473-2481.
2 Chauvel EN, Coric P, Llorens-Cortes C, Wilk S, Roques BP, Fournie-Zaluski MC. Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors. J Med Chem. 1994;37:1339-1346.
3 Zini S, Masdehors P, Lenkei Z, Fournie-Zaluski MC, Roques BP, Corvol P, Llorens-Cortes C. Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats. Neuroscience. 1997;78:1187-1193.
4 Iturrioz X, Vazeux G, Celerier J, Corvol P, Llorens-Cortes C. Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A. Biochemistry. 2000;39:3061-3068.
5 Fournie-Zaluski MC, Fassot C, Valentin B, Djordjijevic D, Reaux-Le GA, Corvol P, Roques BP, Llorens-Cortes C. Brain renin-angiotensin system blockade by systemically active aminopeptidase A inhibitors: a potential treatment of salt-dependent hypertension. Proc Natl Acad Sci U S A. 2004;101:7775-7780.
6 Schalk C, d'Orchymont H, Jauch MF, Tarnus C. 3-Amino-2-tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-M (EC 3.4.11.2). Arch Biochem Biophys. 1994;311:42-46.
7 Tieku S, Hooper NM. Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme. Biochem Pharmacol. 1992;44:1725-1730.
8 Zini S, Fournie-Zaluski MC, Chauvel E, Roques BP, Corvol P, Llorens-Cortes C. Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci U S A. 1996;93:11968-11973.
9 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
10 Gaudet E, Blanc J, Elghozi JL. Role of angiotensin II and catecholamines in blood pressure variability responses to stress in SHR. Am J Physiol. 1996;270:R1265-R1272.
11 Reaux A, Fournie-Zaluski MC, David C, Zini S, Roques BP, Corvol P, Llorens-Cortes C. Aminopeptidase A inhibitors as potential central antihypertensive agents. Proc Natl Acad Sci U S A. 1999;96:13415-13420.