Research Article Correction of endothelial dysfunction by arginase type II inhibitors and tadalafil in rats Ivan C. Koklin*, Mikhail V. Pokrovskiy, Lyudmila М. Danilenko, Konstantin S. Trunov, Anton P. Danilenko, Vladislav O. Soldatov, Natalia N. Bystrova, Natalia N. Bunatyan ABSTRACT Background: Endothelial dysfunction (ED) is one of the most cardiovascular disease pathogenesis leaders. This fact discloses a large number of potential molecular targets for novel pharmacological drugs. Among these targets, enzymes arginase type II and phosphodiesterase type 5 can be distinguished. Materials and Methods: The study included 120 male Wistar rats weighting 250–300 g, which were randomly divided into 12 equal groups. The screening was carried out among compounds with laboratory codes C239–0844, L207–0208, L207–0210, L207–0322, L207–0404, L207–0525, and L327–0346. N-nitro- L-arginine methyl ether (L-NAME)-induced ED was modeled by daily intraperitoneal injection of L-NAME at a dose of 25 mg/kg to male rats for 7 days. The degree of coefficient of ED (CED) was determined on the 8th day from the beginning of the experiment. In parallel, a test was performed for adrenoreactivity and myocardial reserve exhaustion, as well as an assessment of the values of biochemical markers (total nitric oxide [NO] and endothelial NO synthase [eNOS] expression). Results and Discussion: Compounds L207–0525 and L327–0346 at a dose of 1 mg/kg showed the highest activity and were showing the values of CED 1.5 ± 0.3 and 1.9 ± 0.4 c.u., respectively, whereas in the control, it was 5.4 ± 0.4 c.u. At the same time, studied compounds showed cardioprotective effect, what expressed in adrenoreactivity decrease and myocardial reserve exhaustion, as well as a lowering of NO metabolites (NOx) values and expression of eNOS. The combined use of tadalafil and arginase II inhibitors increased the endothelioprotective effect but did not enhance the cardioprotective effect. Conclusion: The study proved the possibility of arginase inhibitors using to prevent the ED development. Among arginase II inhibitors, the most promising compounds are L207–0525 and L327–0346. KEY WORDS: Arginase II inhibitors, Endothelial dysfunction, N-nitro-L-arginine methyl ether, Tadalafil BACKGROUND They catalyze the same biochemical reactions but differ in sites of tissue expression. There are many A large number of accumulated studies demonstrate studies confirming the relationship of arginase the relationship between endothelial dysfunction increased activity with the development of ED in [1] (ED) and cardiovascular diseases. A number of various vascular and heart diseases. The decrease studies have convincingly shown that an increase in the arginase activity leads to gain of nitric oxide in arginase activity leads to the development of (NO) production and due to this positive effect on [2] ED in experimental models of hypertension, the vascular function. The necessity to use arginase [3] [4] [5] atherosclerosis, diabetes, and aging. It was inhibitors to increase NO synthesis and prevent the established that arginase is an enzyme of the urea development of ED becomes obvious.[6,7] cycle, which is highly active, thousands of times higher than that of endothelial NO synthase (eNOS). Arginase inhibitors can be selective and nonselective. In the human organism, arginase is presented in L-norvaline is the most studied of the non-selective the two isoforms: Arginase I is the hepatic form arginase inhibitors.[8,9] However, its effectiveness and arginase II is the extrahepatic form, most often does not fully meet the requirements of clinical localized in the kidneys, prostate, and small intestine. practice. In this regard, it seems appropriate to search for compounds with high cardio- and endothelio- Access this article online protective activity among various pharmacological groups of promising endothelioprotectors[10-17] Website: jprsolutions.info ISSN: 0975-7619 including selective arginase II inhibitors.[18-20] Department of Pharmacology and Clinical Pharmacology, Medical Institute, Belgorod State University, 85, Pobedy St., Belgorod 308015, Russia *Corresponding author: Ivan C. Koklin, Department of Pharmacology and Clinical Pharmacology, Medical Institute, Belgorod State National Research University, 85, Pobedy St., Belgorod 308015, Russia. E-mail: [email protected] Received on: 13-07-2019; Revised on: 15-08-2019; Accepted on: 19-09-2019 Drug Invention Today | Vol 12 • Issue 11 • 2019 2731 Ivan C. Koklin, et al. MATERIALS AND METHODS to its molecular weight, as compared with the tap proteins. The film was dried in air, the strips were Animals scanned, and the AUC was calculated using the total The study included 120 adult male Wistar rats laboratory program. weighting 250–300 g. The animals were kept in a standard conventional vivarium in individually The level of NO metabolites (the total concentration ventilated cages (Tecniplast, USA). Twelve hours of nitrates and nitrites, NOx) was determined by a before the experiment, animals were deprived of access colorimetric method for the development of color to feed and randomly divided into 12 equal groups in in the diazotization reaction of sulfanilamide nitrite, accordance with the type of pharmacotherapy. which is a part of the Griess reagent. To construct a calibration curve, a 1 M solution of NaNO2 in water Procedures was used, which was stored at −20°C; before use, it N-nitro-L-arginine methyl ether (L-NAME)-induced was diluted 1000 times and a series of dilutions were NO deficiency was simulated by injecting L-NAME prepared to construct a curve. daily for 7 days once a day, intraperitoneally, at a Statistical Assay dose of 25 mg/kg. Then, the animals were taken in an experiment for the quantification of the coefficient The data were checked for normal distribution. The of ED (CED), as described previously.[21] In brief, type of distribution was determined by the criterion of the common carotid artery was catheterized and Shapiro–Wilk test. In the case of a normal distribution, connected to a pressure transducer (Biopac Systems, the mean value (M) and standard error of the mean USA). Then, a solution of acetylcholine at a dose of (m) were calculated. In cases of non-parametric 40 µg/kg (endothelium-dependent vasodilation) and a distribution, the median (Me) and quartile range were solution of sodium nitroprusside at a dose of 30 µg/kg calculated. Intergroup differences were analyzed (endothelium-dependent vasodilatation) were injected by Dunnett’s test or Mann–Whitney U-test with into the femoral vein. The area under curve (AUC) Bonferroni correction test, depending on the type of of the pressure drop in response to nitroprusside distribution. Statistical analysis was performed using divided by the AUC of pressure drop in response to Statistica 10.0 software. acetylcholine was taken as CED. RESULTS AND DISCUSSION Testing the myocardium function due to the evaluation Arginase II inhibitors moderately reduced blood of adrenoreactivity and myocardial reserve was carried pressure (BP) and CED. Compounds L207–0525 out as described before.[22] and L327–0346 showed the highest activity in a Western Blot and Biochemical Assay dose of 1 mg/kg, where the values of CED were 1.5 ± 0.3 c.u. and 1.9 ± 0.4 c.u., against 5.4 ± 0.4 c.u. The level of eNOS expression was determined in in control group. The combined use of tadalafil a cell lysate, as previously described.[23] After the (0.1 mg/kg) and compounds L207–0525 and L327– incubation, cell was washed 3 times with 5 mM 0346 has a protective effect, preventing a CED phosphate buffer. The cells from one well were increase and lowering BP [Table 1]. collected in 100 μl of lysis buffer, centrifuged for 10 min at 1000 g, and subjected to polyacrylamide At the same time, the compounds attenuated L-NAME- gel electrophoresis. Each plate was applied a mixture induced heart injury, lowering the adrenoreactivity, of pre-stained protein markers with a molecular increasing the myocardial reserve as well as the values weight range (Mm) 7000–200,000 Yes (Bio-Rad of the NOx and eNOS expression [Tables 2 and 3]. Kaleidoscope Prestained Standards, USA). At the end of electrophoresis, the proteins were transferred It is known that pharmacological inhibition of arginase from the gel to a nitrocellulose membrane – Western activity can potentially have a beneficial effect in a blot (60 V, 1 h). After washing the membrane 3 times number of pathological cardiovascular diseases.[24] The with 5 mM phosphate buffer pH 7.4, containing therapeutic effect of arginase inhibition was investigated 150 mM NaCl and 0.05% Tween-200, it was treated in many experimental models of cardiovascular overnight with a solution of polyclonal rabbit diseases with positive results. There are clinical antibodies against human eNOS (BD Transduction studies where arginase inhibitors were studied through Laboratories, USA) diluted (ratio=1:500). Then, cutaneous microdialysis in patients with ischemic heart the membrane was washed and incubated for an disease and type 2 diabetes mellitus[25] and heart failure hour with diluted at a ratio of 1:300 secondary and hypertension.[26] These observations suggest that antibodies conjugated with horseradish peroxidase arginase activity gain is important in the development (goat anti-rabbit IgG antibodies, Sigma, USA). The of human cardiovascular diseases. An important strip corresponding to eNOS was detected according limitation of currently available arginase inhibitors is 2732 Drug Invention Today | Vol 12 •