European Journal of Pharmacology, 156 (1988) 95-103 95 Elsevier EJP 50498 Cardiovascular effects of dl-nebivolol and its enantiomers - a comparison with those of atenolol A. Van de Water *, R. Xhonneux, R.S. Reneman 1 and P.A.J. Janssen Cardiovascular Department, Janssen Research Foundation, Beerse, Belgium and ! Department of Physiology, University of Limbur~ Maastricht, The Netherlands Received 2 May 1988, revised MS received 11 July 1988, accepted 2 August 1988 In the present study, we investigated the cardiovascular effects of dl-nebivolol, a newly synthetized, chemically novel, flFadrenoceptor antagonist and its enantiomers, d-nebivolol (SRRR) and 1-nebivolol (RSSS), in closed-chest anesthetized dogs, using atenolol as a reference substance. Results from preliminary studies in vitro indicate that d-nebivolol is the fll-adrenoceptor antagonist and that 1-nebivolol is practically devoid of fl-adrenoceptor-blocking properties. Unlike atenolol, dl-nebivolol does not depress left ventricular function and slightly, but significantly, reduces peripheral vascular resistance over the dose range from 0.0025 to 0.04 mg. kg-1 i.v. These observations are likely to be clinically relevant because one daily oral dose of 5 mg dl-nebivolol effectively lowers arterial blood pressure in patients with hypertension. The favorable hemodynamic profile of dl-nebivolol can be ascribed to the l-enantiomer because the cardiovascular effects of this enantiomer are similar to those of the racemate. The cardiovascular profile of the d-enantiomer is similar to that of atenolol, albeit that its depressant effect on left ventricular function occurs at higher doses. flx-Adrenoceptor antagonists (selective); dl-Nebivolol; Atenolol; Left ventricular function; Closed-chest dog; (Enantiomers, Hemodynamics) 1. Introduction dl-Nebivolol is a chemically novel, potent and selective fll-adrenoceptor antagonist. (Van de Water et al., 1988). It is a racemic mixture of two enantiomers: d-nebivolol (SRRR) and 1-nebivolol (RSSS) (fig. 1), dl-Nebivolol acutely lowers blood pressure in hypertensive humans and in sponta- neously hypertensive rats. The compound de- creases peripheral vascular resistance but does not depress, or even enhance, left ventricular function. Its selectivity in vivo is comparable to that of atenolol (Van de Water et al., 1988). It was the aim of the present study to further investigate the hemodynamic profile of dl-nebivo- 1ol. Since the results of preliminary studies in vitro indicate that d-nebivolol is the fll-adrenoceptor antagonist and that l-nebivolol has practically no fl-adrenoceptor-blocking properties, the cardio- vascular effects of both enantiomers were also investigated. Atenolol was used as a reference compound, because of its comparable selectivity in vivo. The study was performed on closed-chest anesthetized dogs. 2. Materials and methods * To whom all correspondence should be addressed: Cardio- vascular Department, Life Science Building, Janssen Re- search Foundation, B-2340 Beerse, Belgium. 2.1. Experimental set-up and data aquisition The experiments were performed on 28 mongrel dogs of either sex and varying age, ranging in 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-1
`IPR2016-00379
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`96 F o. o.f. yF F ~ 0 .,~-CH-CH2-NH- CH~-CH .'~'-, 0 RSSS ~F oQo o F N E B I VOLOt. Fig. 1. Chemical structure and perspective drawing of dl-nebivolol. SRRR: d-enantiomer; RSSS: 1-enantiomer. body weight from 18 to 34 kg (median 27 kg). The dogs were divided into four groups. The animals received dl-nebivolol in one group (n = 7), d- nebivolol (n = 7) and 1-nebivolol (n = 7) in the second and third group, respectively, and atenolol in the last group (n = 7). The animals were i.v. anesthetized with a mixture of scopolamine (0.015 mg- kg -]) and lofentanil (0.05 mg. kg -1) and in- tubated with a cuffed endotracheal tube. Intermit- tent positive pressure ventilation with a mixture of pressurized air and oxygen (60/40) was carried out with a volume-controlled ventilator (Siemens Elema). In the control period the CO 2 concentra- tion in the expired air (ET CO2), as determined with a capnograph (Gould Godart), was kept at 5% by adjustment of the respiratory volume (re- spiratory rate = 20 breaths, rain- ]). A continuous i.v. infusion of etomidate (0.5 mg. kg -1 • h -1) was started immediately after induction. Body temper- ature was monitored with a thermistor positioned in the pulmonary artery and kept at 37-38°C. Heparin (1 000 IU • kg- 1 i.v.) was administered to prevent the blood from clotting. The electrocardiogram (ECG) was derived from limb leads (standard lead 2). Left ventricular (LVP) and ascending aortic blood pressure (AoP) were measured by retrograde catheterization via the femoral arteries with high fidelity catheter-tip mi- cromanometers (PPG Biomedical Systems). Zero- line calibration was achieved by simultaneously recording the pressure signal through the lumen of the catheter with an external pressure transducer (Gould P23ID) positioned at the mid-chest level and equating both zero lines on the physiological recorder. A Swan-Ganz balloon guided thermistor catheter was placed via a femoral vein in the pulmonary artery to measure pulmonary artery blood pressure (PAP) with an external pressure transducer (Gould P23ID) positioned at the mid- chest level. The cardiac output (CO) was mea- sured using the ECG (Snoeckx et al., 1976) and the respiration triggered thermodilution technique (Janssen Scientific Instruments). The other femoral vein was cannulated for injection of saline (at room temperature) into the right atrium and for injection of the test compounds. Peak ascending aortic blood flow velocity was measured through the right carotid artery with an electromagnetic catheter-tip probe connected to a square wave electromagnetic flow meter (Janssen Scientific In- struments). To continuously measure cardiac out- put (stroke volume times heart rate), calibration of the electromagnetic velocity catheter was accom- plished by equating the area under the velocity
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-2
`IPR2016-00379
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`97 curve to the average stroke volume, calculated from the heart rate and cardiac output values. These values were assessed with the use of the thermodilution technique at the beginning of, and at various time intervals during, each experiment. All catheters were placed in position under fluoro- scopic control. Continuous registration of the ana- log signals was performed on a 8-channel ink-jet recorder (Mingograph 800). The blood pressure, thermistor, velocity and ECG signals were, via transducer amplifiers, fed into a digital minicomputer (PDP 11/23), con- nected to a dual diskette drive (RX02) and a display console (VT105). The following variables were calculated on-line and printed on a matrix printer (Facit 4512B), usually at 1 min intervals: heart rate (HR), the duration of the PQ, QRS and QT intervals, the QTc interval (QT interval cor- rected for heart rate with the Bazett formula, 1920), the R-wave amplitude, systolic (AoPs) and diastolic (AoPd) aortic blood pressure, systolic (PAPs) and diastolic (PAPd) pulmonary artery blood pressure, left ventricular end-diastolic pres- sure (LVEDP), the maximum positive rate of change of isovolumic LVP (LV dp/dtmax), this maximum first derivative divided by the actually developed pressure in the left ventricle (LV dp/dt max/Pd), maximal aortic blood flow velocity (AOVm~ ,) and acceleration (Ao dv/dt max), cardiac output (CO), stroke volume (SV), and pressure rate product (PRP). The systemic vascular (SVR) and pulmonary vascular resistance (PVR) were calculated according to standard formulas. All values were simultaneously transmitted to a dis- kette and were plotted after the experiments on a digital x-y plotter (HP 7475A). 2.2. Protocol dl-Nebivolol was dissolved in 10% hydroxy- propyl-fl-cyclodextrine ether in a concentration of 2.5 mg-m1-1 (pH 6.1). Its enantiomers were dis- solved in the same solvent acidified with tartaric acid in a concentration of 1 mg. m1-1 (pH 2.5). Atenolol was dissolved in distilled water (con- centration: 2.5 mg-ml-1; pH 8.1). The com- pounds were injected i.v. after a recorded control period of 20 rain in cumulative doses of 0.0025, 0.01, 0.04, 0.16 and 0.63 mg.kg -~ at 30 rain intervals. 2.3. Statistical analysis The changes in the values of the various varia- bles 1, 2, 5, 10, 20 and 30 min after administration of the various doses of the compounds (these values were compared to the control values, i.e. the median value of the values obtained just prior to, and 5 and 10 min before the first injection of the compound), were evaluated for statistical sig- nificance by applying the Wilcoxon m.p.s.r, test. Differences between control values and the results obtained with atenolol, dl-nebivolol and its enan- tiomers at various time intervals were evaluated for statistical significance by using Mann-Whit- ney's U-test. Two-tailed probabilities less than or equal to 0.05 were considered to be significant. 3. Results 3.1. Changes induced by dl-nebivolol Cumulative i.v. administration of dl-nebivolol did not induce overt changes in the ECG. In the lower to median dose range (0.0025-0.04 mg. kg- 1 ) the compound did not affect the pressure needed for the volume controlled respirator to ventilate the dog at a constant minute volume. The SVR decreased significantly and the CO and SV signifi- cantly increased following i.v. injection of 0.0025 and 0.01 mg-kg -1 dl-nebivolol. The AoPd de- creased slightly, but significantly, starting after injection of 0.0025 mg. kg -1 i.v., while the LV dp/dt m~x/Pd increased slightly, but significantly, after administration of this dose. The PAPd, PVR and LVEDP increased significantly, starting after injection of 0.01 mg-kg -1 i.v. A significant de- crease was noted in the HR after administration of 0.04 and 0.16 mg- kg -1 i.v. The LV dP/dtm~ x and PRP also decreased significantly following administration of 0.04 mg. kg -~ i.v., as did the LV dP/dtma~/Pd, AOVmax, Ao dv/dtma ~ and CO after 0.16 mg-kg -1 i.v., and the SV after the injection of 0.63 mg. kg-1 i.v. The SVR increased significantly after administration of 0.16 and 0.63
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-3
`IPR2016-00379
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`98 TABLE 1 The median values and 95% confidence limits dl-nebivolol (N). of the hemodynamic variables before and after various doses of atenolol (A) and Variable Drug Control Dose (mg. kg- 1) 0.0025 0.01 0.04 0,16 0.63 Heart rate N 75 72 71 65 a 62 a 68 (beats- rain- 1) (53-94) (51-93) (54-91) (45-88) (40-87) (41-96) A 70 64 ~ 61 ~ 60 56 55 (55-110) (54-89) (50-96) (44-104) (41-115) (40-118) AoP syst. N 16.9 16.8 16.6 16.3 16.3 16.4 (kPa) (16.3-19.9) (16.3-19.6) (16.1-19.2) (15.2-18.7) (14.5-17.5) (13.9-17.7) A 16.1 15.7 a,b 15.4 ~ 15.1 a 14,7 a 14.1 ~ (15,9-20,4) (14.8-20.4) (14.4-20.2) (13.1-20.1) (13.2-19.0) (12.1-19.0) AoP diast. N 12.5 12.0 ~ 11.8 a 11.7 a 12.0 12.0 ~ (kPa) (11.6-14,3) (11.6-14.4) (9.9-14.3) (10.1-14.1) (10.1-13.7) (9.9-13.7) A 10.8 10.4 a 10.3 a 10.0 a 9.7 a 9.3 a (8.8-14.5) (8.3-14.5) (8.4-13.2) (8.1-13.3) (7.2-13.3) (6.0-13.0) Cardiac output N 3.5 3.8 a 3.7 a 3.4 3.0 ~ 2.3 a (1- min- 1) (2.0-6.9) (2.0-7.6) (2.1-6.8) (1.9-5.8) (1.5-5.2) (1.2-3,8) A 4.1 3.7 a,b 3.5 a,b 3.1 ~,b 2.5 a,b 2.6 a (2.4-5.4) (2.3-5.0) (2.1-4.8) (1.8-3.5) (1.3-3.2) (1.4-3.2) Stroke volume N 48 55 a 53 a 54 45 36 a (cm 3- beat- a) (38-75) (39-85) (39-77) (42-72) (32-56) (31-42) A 55 51 b 50 b 44 a,b 41 ~,b 39 a (36-86) (31-77) (29-91) (25-80) (17-69) (20-63) SVR N 3.57 3.34 ~ 3.24 a 3.85 4.24 ~ 5.29 ~ (kaa. l - ~. rain) (2.32-6.63) (2.09-6.80) (2.35-6.39) (2.75-5.47) (2.93-9.65) (4.04-11.29) A 3.05 3.28 b 3.74 a,b 4.56 ~,b 5.44 ~,b 5.73 ~ (2.37-5.05) (2.33-5.60) (3.25-6.31) (3.16-7.31) (2.84-8.67) (2.45-7.80) LV dp/dt max N 301 297 292 261 a 209 ~ 190 ~ (kPa. s- ~ ) (237-431) (240-501) (250-470) (202-336) (169-260) (139-209) A 322 290 a,b 234 a,b 192 a,b 166 a 159 a (302-445) (286-414) (197-340) (166-305) (135-300) (116-245) LV dp/dt max/Pd N 34 36 ~ 35 33 26 ~ 23 ~ (s- ~) (29-48) (29-50) (32-51) (28-49) (22-41) (21-31) A 44 41 a,b 38 a,b 34 a 32 a 31 a (36-53) (33-52) (28-52) (28-47) (26-40) (26-36) LVEDP N 0,65 0.78 0.80 ~ 0.96 ~ 0.92 a 1.08 ~ (kPa) (0.29-0.87) (0,07-0.99) (0.36-1.15) (0.35-1.89) (0.51-2.12) (0,71-1.36) A 1.05 1.29 1.48 ~ 1.40 1.40 ~ 1.70 a (0.16-1.36) (0.40-1.45) (0.85-1.91) (0.88-2.01) (0.83-2.05) (1.03-2.13) AOVma x N 51 52 53 49 45 a 42 a (cm-s- 1 ) (41-67) (48-69) (46-68) (33-76) (30-64) (25-26) A 56 53 a,b 50 a,b 47 a,b 44 a 43 ~ (41-69) (37-85) (35-84) (30-73) (24-62) (22-57) AO dv/dt m,~ N 1335 1408 1485 1385 1085 ~ 860 ~ (cm-s -2) (1130-1907) (1264-2326) (1153-2529) (872-2608) (685-1997) (553-1563) A 1695 1603 ~,b 1 455 ~,b 1215 ~,b 945 ~,b 844 ~ (1 413-2 515) (1 344-1 743) (1 128-1 800) (1 022-2 033) (898-1 383) (561-1 320)
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-4
`IPR2016-00379
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`TABLE 1 (continued) 99 Variable Drug Control Dose (mg. kg - 1 ) 0.0025 0.01 0.04 0.16 0.63 PRP N 1333 1253 1213 1160 a 1080 ~ 1120 a (1053-1787) (867-1800) (907-1693) (747-1640) (680-1520) (693-1573) A 1347 1227 ~ 1160 ~ 1134 a 1054 1014 a (920-1680) (760-1547) (787-1413) (707-1693) (640-1547) (587-1533) PAP syst. N 2.5 2.6 2.8 3.3 2.9 2.8 (kPa) (1.9-3.6) (2.4-4.0) (2.5-4.5) (2.1-4.7) (2.1-4.7) (2.5-4.7) A 3.6 3.6 3.7 3.7 3.7 3.7 (2.3-4.4) (2.1-4.0) (2.0-4.0) (2.7-4.0) (2.5-3.7) (2.8-3.9) PAP diast. N 0.7 1.0 1.1 a 1.2 ~ 1.2 a 1.3 a (0.4-1.2) (0.5-1.3) (0.5-1.5) (0.7-1.5) (0.8-2.1) (1.1-1.6) A 0.9 0.9 1.1 ~ 1.2 1.2 1.2 (0.3-1.5) (0.3-1.5) (1.0-1.5) (0.9-1.3) (0.7-1.3) (0.7-1.3) PVR N 0.37 0.41 0.45 a 0.61 ~ 0.69 ~ 0.86 ~ (kPa- 1 - 1. rain) (0.27-0.63) (0.25-0.67) (0.28-0.69) (0.29-0.89) (0.33-1.16) (0.52-1.33) A 0.43 0.47 a.b 0.58 a.b 0.74 ~ 0.76 a 0.79 ~ (0.25-0.83) (0.29-0.93) (0.37-0.99) (0.53-1.04) (0.64-1.44) (0.67-1.16) a Significantly different from the control value; b significant difference between both groups. mg. kg -1 i.v. No significant changes were ob- served in the AoPs and PAPs. The median values and 95% confidence limits of the haemodynamic variables before and after administration of vari- ous doses of dl-nebivolol are presented in table 1. 3.2. Changes induced by atenolol The pressure needed for the volume-controlled respirator to ventilate the lungs of the animals at a constant minute volume did not change after ad- ministration of various doses of atenolol. A sig- nificant decrease was observed in the AoPs, AoPd, LV dp/dtm~x, LV dp/dtmax/Pd , AOVmax, Ao dv/dtm~ x, CO, after all doses. The SV decreased significantly only at the higher doses (0.04 through 0.63 mg- kg-1 i.v.) and the HR only decreased in the lower dose range (0.0025-0.04 mg. kg -1 i.v.). The PRP decreased significantly after most of the doses injected. A significant increase was observed in the LVEDP, starting after i.v. injection of 0.01 mg-kg-~ atenolol. The SVR and PVR increased significantly after injection of 0.01 and 0.0025 mg-kg -1 i.v., respectively. The ECG showed a significant increase in the duration of the PQ interval, starting after 0.01 mg-kg -1 i.v., and of the QT interval following administration of the lowest dose. However, the QT interval did not show significant changes when corrected for the heart rate. The other measured or calculated varia- bles did not show consistent, significant changes. The median values and 95% confidence limits of the variables before and after administration of doses of atenolol are presented in table 1. 3.3. Changes induced by d-nebivolol (SRRR) The time intervals of the ECG did not change significantly after administration of this enanti- omer, except for a slight, but significant decrease, in the duration of the QTc interval following administration of 0.63 mg. kg -1 d-nebivolol. A significant decrease was observed in the LV dp/dtmax/Pd and Ao dv/dt .... starting after i.v. injection of 0.01 mg-kg -1 and in the AoPs, AOVmax, SV and CO, starting after i.v. administra- tion 0.16 mg. kg -1 of the compound, A significant increase was observed in the PAPd, PVR and SVR after i.v. injection of 0.01 and 0.04 mg.kg -1, respectively. These changes were also observed after i.v. injection of the remaining doses. The HR increased slightly, but significantly, after i.v. ad-
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-5
`IPR2016-00379
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`lOO 120 o) lOO 80 c 60 ,~ 40 .t:: o 20 >, (.) 3 "5 .rE O 3 120 .e * '? Hi 2 c * o l.li . 4O 2 d 14o- ~ 12o. -~ 100 9 80" _= 60- 40" 0 20- 8 0 e 200 '~ 150 ~>, 100 - 8, .5 50- 6 8 &i o A • 0025 .01 .04 .16 .63 mg.kg ' i.v. ministration of 0.63 mg. kg -1, while the other measured or calculated variables did not show consistent, significant changes. The changes in the most relevant variables are shown in fig. 2. 3.4. Changes induced by l-nebivolol (RSSS) The time intervals of the ECG did not change after administration of the various doses of this enantiomer. A significant decrease was observed in the SVR after i.v. injection of 0.0025 and 0.01 mg-kg -1, in the AoPd and PRP following 0.01, 0.04 and 0.16 mg. kg -1, and in the AoPs and LV dP/dtma x after 0.63 mg-kg -1 1-nebivolol. A sig- nificant increase was seen in the LVEDP, LV dP/dtmaJPd , Ao dv/dt .... SV and CO after injection of the lowest dose and was also seen at doses of 0.01 or 0.04 mg-kg -1. The AOVm~ x was significantly increased after i.v. injection of 0.01- 0.16 mg-kg -1 1-nebivolol and the PAPs was in- creased by 1-nebivolol over the dose range 0.01-0.63 mg. kg -1. The PAPd and PVR increased signifi- cantly after injection of the higher doses of 1- nebivolol. The HR did not show consistent, sig- nificant changes over the dose range tested. The changes in the most relevant variables are shown in fig. 2. 3.5. Comparison of the effects induced by atenolol, dl-nebivolol and its enantiomers The changes in the AoPd, LVEDP, HR and PRP induced by atenolol and dl-nebivolol were Fig. 2. Percentage changes in the heart rate (HR), the maxi- mum positive derivate of isovolumic left ventricular pressure divided by the actually developed pressure in the left ventricle (dP/dt/Pd), cardiac output (CO), stroke volume (SV) and systemic vascular resistance (SVR) after administration of vari- ous doses of dl-nebivolol (n), its d- (SRRR) ([]) and 1- (RSSS) ([]) enantiomers and atenolol (m). (*) Significantly different from its own control value. (o) Significantly different from the effect induced by dl-nebivolol. ( + ) Significantly different from the effect induced by the l-enantiomer. (zx) Significantly differ- ent from the effect induced by dl-nebivolol. (O) Significantly different from the effect induced by atenolol.
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-6
`IPR2016-00379
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`not significantly different from each other. On the contrary, the changes in the ECG and the con- tractility indices (SV, CO, SVR, AoPs and PVR) induced by atenolol were significantly different from those induced by dl-nebivolol at various dose levels (table 1). The differences between the effects of both compounds on the HR, CO, SV, SVR and LV dp/dtm~x/Pd are shown in fig. 2. The d-nebivolol-induced changes in the LVEDP, LV dP/dt m~x, LV dP/dtm~x/Pd, Aovma x, Ao dv/dt max, SV, CO, SVR and HR were signifi- cantly different from those induced by l-nebivolol at various dose levels. Some of these differences are shown in fig. 2. No consistent, significant differences between the effects of either enanti- omer on the AOPs, PAPs, PAPd, TPR and PRP were detected. The changes induced by 1-nebivolol were barely different from those induced by dl-nebivolol. Only the effect on most of the variables related to left ventricular function was different. The LV dp/dtma~, LV dp/dtm,JPd and Aovma x de- creased slightly, but significantly, after adminis- tration of 0.04 and 0.16 mg. kg- 1 i.v. dl-nebivolol, but did not change following injection of the same doses of 1-nebivolol. A similar difference was ob- served in the effect of these compounds on the SV following administration of 0.16 and 0.63 mg- kg-1 i.v. (fig. 2). There was little difference between the effects of d-nebivolol and atenolol and, except for the effect on the LVEDP, it was only significant after injection of the lowest doses. Following adminis- tration of 0.0025 mg. kg -1 i.v., d-nebivolol did not induce significant changes in the AOPs, AoPd, LV dp/dtma ~, LV dP/dtm,~/Pd, Aovmax, Ao dv/dt max, HR, CO and PRP, but atenolol induced a decrease in these variables. A decrease in the AoPs, AoPd, HR and PRP was also observed after i.v. injection of 0.01 mg. kg -1 atenolol, but not after administration of d-nebivolol. The LVEDP increased after administration of atenolol (0.01, 0.16 and 0.63 mg.kg -~ i.v.) but not after the same doses of d-nebivolol. The differences be- tween the effects of atenolol and d-nebivolol on some variables are shown in fig. 2. 101 4. Discussion dl-Nebivolol is a recently synthetized, chem- ically novel fll-adrenoceptor antagonist with a selectivity in vivo comparable to that of atenolol (Van de Water et al., 1988). In preliminary studies dl-nebivolol was found to accutely lower blood pressure in hypertensive patients and rats without having a negative influence on left ventricular function. There are indications that the blood pressure-lowering effect of the compound might be explained by a decrease in the peripheral vascu- lar resistance. The findings in the present study show that the cardiovascular effects of dl-nebivolol are different from those of atenolol, despite their comparable selectivity in vivo (Van de Water et al., 1988). While atenolol negatively influences the variables related to left ventricular performance, starting at the lowest dose injected (0.0025 mg-kg -1 i.v.), dl-nebivolol does not affect these variables, except for the two highest doses administered (0.16 and 0.63 mg. kg-1 i.v.), dl-Nebivolol also has negative inotropic properties at these doses. These dif- ferences between their effects on left ventricular function were observed in the absence of signifi- cant differences between their effects on the heart rate and left ventricular end-diastolic pressure. dl-Nebivolol caused a slight decrease in the sys- temic vascular resistance and a small increase in the cardiac output after the two lowest doses injected, while atenolol reduced the cardiac output at all doses and increased the systemic vascular resistance at doses greater than 0.01 mg. kg-1 i.v. These effects of dl-nebivolol could be compatible with an intrinsic sympathomimetic activity of fl- adrenoceptor antagonists (Man in 't Veld and Schalekamp, 1983). However, recent studies in our laboratories have shown that dl-nebivolol is de- void of such activity. It does not exert a stimulat- ing effect on the heart rate and left ventricular function in reserpinized dogs or on the heart rate of reserpinized spontaneously hypertensive rats and on isolated atria of reserpinized Wistar rats. Besides, dl-nebivolol does not induce relaxation of isolated coronary arteries and saphenous veins at
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-7
`IPR2016-00379
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`102 concentrations that exert fl-adrenoceptor blockade (Janssens et al., submitted). The absence of an increase in the systemic vascular resistance, when a fl-adrenoceptor antagonist does not depress left ventricular function, supports the idea that the increase in the systemic vascular resistance ob- served after the acute administration of other fl- adrenoceptor antagonists, is secondary to their depressant effect on left ventricular performance (Man in 't Veld, 1987). Both compounds increase pulmonary vascular resistance, an effect which is significantly more pronounced after administra- tion of atenolol, at least at the two lowest doses. The cardiovascular profile of d-nebivolol is sim- ilar to that of atenolol, albeit that the depressant effect on left ventricular function is observed at higher doses of d-nebivolol. On the contrary, the negative chronotropic effect of atenolol was more pronounced at the lower doses than that of d- nebivolol. The cardiovascular effects of 1-nebivolol were rather similar to those of the racemate, only the depressant effect on left ventricular function was slightly more pronounced with dl-nebivolol than with its 1-enantiomer. These findings indicate that the beneficial cardiovascular profile of dl- nebivolol over the lower dose range tested can be explained by the mode of action of the 1-enanti- omer and that the d-enantiomer induces cardio- vascular effects consistent with those observed after i.v. administration of fll-adrenoceptor antagonists without intrinsic sympathomimetic ac- tivity (Man in 't Veld and Schalekamp, 1983). The mode of action of l-nebivolol is not completely understood at present. Preliminary studies in our laboratory suggest that the fl-adrenoceptor-block- ing properties of this enantiomer are only one percent of those of the d-enantiomer. It is interest- ing to note that, unlike other fl-adrenoceptor antagonists, d-nebivolol has the fl-adrenoceptor- blocking properties. The increase in the left ventricular end-diastolic pressure observed after i.v. injection of dl-nebivo- lol and l-nebivolol, over the lower to median dose range, cannot be explained by a depressant effect on left ventricular function because this increase was not associated with a decrease in the variables related to left ventricular performance. It is not very likely that this increase in left ventricular end-diastolic pressure is caused by an enhanced venous return, because stimulation of fl-receptors in vivo induces venoconstriction (Eckstein and Hamilton, 1959). The increase in left ventricular end-diastolic pressure probably results from the decrease in heart rate. Whether 1-nebivolol induces an increase in venous return is unknown. The increase of left ventricular end-diastolic pressure after injection of atenolol can most likely be explained by the decrease in heart rate together with the depressant effect on left ventricular func- tion. The differences between the effects of atenolol on the one hand, and dl-nebivolol and its enanti- omers on the other, cannot be explained by the different solvents used because previous studies in our laboratories have shown that hydroxy-propyl- fl-cyclodextrine ether is devoid of cardiovascular effects in the amounts used in the present study. The beneficial cardiovascular effects of dl- nebivolol at the lower to median doses (0.0025-0.04 mg. kg -1 i.v.) are likely to be chnically relevant, because one daily oral dose of 5 mg dl-nebivolol effectively lowers arterial blood pressure in hyper- tensive patients (Van de Water et al., 1988). In conclusion, unlike other fll-adrenoceptor antagonists, dl-nebivolol does not depress left ventricular function an slightly, but significantly, lowers peripheral vascular resistance over the dose range in which it effectively lowers arterial blood pressure in hypertensive patients. These cardio- vascular effects can be ascribed to the 1-enanti- omer of nebivolol, a substance practically devoid of fl-adrenoceptor-blocking properties. Acknowledgements The authors are indebted to Jos Heemskerk, Karin Van Brussel and Lucienne De Boer for their help in preparing the manuscript and to Bob Joossen and Henri Vermeer for making the illustrations. References Bazett, H.C., 1920, An analysis of the time-relations of electro- cardiograms, Heart J. 7, 353. Eckstein, J.W. and W. Hamilton, 1958, Effects of isoproterenol
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-8
`IPR2016-00379
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`on peripheral venous tone and transmural right atrial pres- sure in man, J. Clin. Invest. 38, 342. Man in 't Veld, A.J., 1987, Effect of beta blockers on vascular resistance in systemic hypertension, Am. J. Cardiol. 38, 21F. Man in 't Veld, A.J. and A.D.H. Schalekamp, 1983, How fl-blockers lower blood pressure: divulgence of the non-es- sentiality of cardiodepression and renin suppression by partial agonism and the key role of vascular resistance changes, Vasc. Med. 1, 195. 103 Snoeckx, L.H.E.H., J.L. Verheyen, A. Van de Water, P. Lewi and R.S. Reneman, 1976, On-line computation of cardiac output with the thermodilution method using a digital minicomputer, Cardiovasc. Res. 10, 556. Van de Water, A., W. Janssens, J. Van Nueten, R. Xhormeux, J. De Cree, H. Verhagen, R.S. Reneman and P.A.J. Jans- sen, 1988, The pharmacological and hemodynamic profile of dl-nebivolol, a chemically novel, potent and selective fll-adrenoceptor antagonist, J. Cardiovasc. Pharmacol. 11, 552.
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1031-9
`IPR2016-00379