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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1043-1
`IPR2016-00379
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`262 ~-~ ].,L.~.fl--CH-CH-NH-CH-CH-L^ ~k. /J F v ~J * x_ 2 2-X- -X-'U- v ~0 RSSS ~~ :~OF o o o~ ~ I~ F NEBIVOLOL Fig. 1. Chemical structure and three-dimensional drawing of nebivolol. SRRR: d-enantiomer; RSSS: l-enantiomer. that the blood pressure lowering effect of d,1- nebivolol was significantly more pronounced than that of the d-enantiomer, which alone has fl- adrenoceptor blocking properties. This observa- tion indicates that 1-nebivolol potentiates the anti- hypertensive effect of d-nebivolol. The aim of the present study was to investigate the effect of 1-nebivolol on the antihypertensive action of d-nebivolol. 2. Materials and methods The study was performed with adult (6 months old) SHR. The animals were anesthetized with ether and a femoral artery was dissected and can- nulated with a polyethylene catheter connected to a strain gauge blood pressure transducer. When the animals were fully awake, they were restrained in Bollman cages (Bollman, 1948), and lidocaine (20%) was administered to the wound around the femoral canula. Systolic and diastolic arterial blood pressure and heart rate were continuously recorded. An observation period of at least 30 min preceded the intraperitoneal administration of the compounds. The enantiomers of nebivolol were dissolved in 20% polypropylene glycol at a con- centration of 1 mg- ml-1. In a first series of experiments, 1- or d-nebivolol was injected in doses of 0.63, 1.25, 2.5 and 5 mg.kg -1 i.p. (n = 18 per dose). A group of 24 SHR, received placebo, served as control. In another series of experiments, seven groups of SHR (n = 12 per group) were given d-nebivolol, 1.25 mg-kg -1 i.p., alone, or combined with the following doses of 1-nebivolol: 0.16, 0.31, 1.25, 2.5 and 5 mg- kg-1. In these experiments the changes recorded after d-nebivolol alone were taken as controls. The effect of the compounds or placebo on systolic and diastolic blood pressure, and on heart rate was assessed by averaging the changes in these variables over a period of 120 min. The statistical significance of the different effects com- pared to controls was assessed with the Mann- Whitney U-test. Two-tailed probabilities ~< 0.05 were considered to be significant. 3. Results In control experiments, the systolic and di- astolic blood pressure as well as the heart rate slightly but significantly increased during the 120- min observation period (fig. 2; see also control values fig. 3A, B). These increases were probably
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1043-2
`IPR2016-00379
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`300 - 600 250 :I 200- E o 150- . . . ~==~ 100- 50 0 5e 500 "400 '.£ E • 300 -2oo 0 - 100 placebo 0 (~ 5'0 100 150 Time (rnin.) Fig. 2. Systolic (SBP) and diastolic blood pressure (DBP), and heart rate (HR) in awake spontaneously hypertensive rats in Bollman cages before and after administration of placebo. * Significantly different from the control situation (P ~< 0.05; Student's t-test). caused by stress due to confinement of the animals in the Bollman cages. Over the dose range 0.63 to 5 mg.kg -1, 1- nebivolol did not significantly decrease the di- astolic blood pressure. At 2.5 and 5 mg. kg -~ 1-nebivolol slightly but significantly decreased the systolic blood pressure, whereas the heart rate was significantly reduced after 5 mg. kg -1 (fig. 3A). Over the dose range 0.63 to 5 mg.kg -1, d- nebivolol significantly decreased the systolic blood pressure and heart rate; the diastolic blood pres- sure was significantly reduced by doses of 1.25 mg. kg-a and higher (fig. 3B). When d-nebivolol, 1.25 rag. kg -1, was com- bined with 1-nebivolol (0.16 to 5 mg. kg-1), the decrease in the systolic blood pressure was signifi- cantly more pronounced than after the adminis- tration of d-nebivolol alone (fig. 3C). The decrease in the diastolic blood pressure was also signifi- cantly more pronounced when d-nebivolol was combined with 1-nebivolol in doses from 0.32 to 5 mg- kg-~ (fig 3C). The reduction in the heart rate was also more pronounced, but only when 1.25 mg- kg-a d-nebivolol was combined with 2.5 or 5 rag-kg -1 1-nebivolol, the two highest doses in- jected (fig. 3C). After the administration of 1.25 rag. kg -1 d-nebivolol and 2.5 or 5 mg-kg -1 1- nebivolol, the systolic and diastolic blood pressure decreased further, but these changes were not sig- 263 nificantly different from those observed when the d- and 1-enantiomer were given at doses of 1.25 mg-kg-1 each. The heart rate also showed a fur- ther reduction, starting at these doses, although the decrease after 1.25 mg. kg-1 d-nebivolol and 2.5 mg. kg -1 1-nebivolol did not reach signifi- cance (P = 0.08). 4. Discussion The findings in the present study indicate that 1-nebivolol potentiates the antihypertensive effect of d-nebivolol. Doses of 1-nebivolol which do not affect blood pressure when given alone potenti- ated the decrease in systolic and diastolic blood pressure induced by 1.25 rag-kg -~ d-nebivolol, when both drugs were administered simulta- neously. This potentiating effect started at doses of 0.16 and 0.31 mg. kg -1 for systolic and di- astolic pressure, respectively. At 1.25 mg-kg -~, d-nevibolol significantly reduced the heart rate, an effect which was not potentiated by 1-nebivolol in doses up to 1.25 mg. kg -~. Although the blood pressure tended to decrease further after the ad- ministration of 2.5 or 5 mg. kg -1 l-nebivolol com- bined with 1.25 mg- kg-1 d-nebivolol, this is prob- ably not beneficial because at these doses the 1-enantiomer potentiates the bradycardia caused by the d-enantiomer. The latter observation indi- cates that, at these doses of the 1-enantiomer, the combination of d- and l-nebivolol exerts more pronounced fll-adrenoceptor blocking properties. This can be considered to be disadvantageous because d,l-nebivolol has been shown to effec- tively decrease arterial blood pressure before there is a pronounced decrease in heart rate, indicating that the blood pressure lowering effect of this compound is, at least partly, dissociated from its /3~-adrenoceptor blocking properties. This may ex- plain the beneficial pharmacological profile of d,l-nebivolol (Van de Water et al., 1988a). There- fore, based on our findings with spontaneously hypertensive rats, the blood pressure lowering dose of 1.25 mg-kg -~ d-nebivolol combined with 1.25 mg-kg -1 1-nebivolol is recommended so as to limit the side-effects otherwise seen with fll-adren- oceptor blocking agents.
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1043-3
`IPR2016-00379
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`264 lO A 5 0 -5 10 10 B 5 0- -5 -10 -15 • -20 • -25 -30 -35 % changes control (n =24) I- Nebivolol d - Nebivolol z, 5 C o -5 -lO -15 -20 -25 [] -30. [] [] -35 d - Neblvolol -I- ~ 0 1.25 mg/kg i.p. (n =18) SBP DBP HR 0.63 1.25 (n =18) (n =18) d - Nebivolol + 2.5 5 (n =18) (n =18) - Nebivolol mg/kg i.p. 1 . 0.16 0.31 0.63 1.25 2.5 5 mg/kg i,p. (n =12) (n=12) (n=12) (n=12) (n=12) (n =12) I - Nebivolol Fig. 3. (A, B) Median'percentage changes and 95% confidence limits in systolic (SBP) and diastolic blood pressure (DBP), and heart rate (HR) in spontaneously hypertensive rats, following the i.p. administration of placebo (control) and after the intraperitoneal administration of various doses of 1-nebivolol (A) or d-nebivolol (B). * Significantly different from the control rats (P ~< 0.05). (C) Median percentage changes and 95% confidence limits in systolic (SBP) and diastolic blood pressure (DBP), and heart rate (HR) in spontaneously hypertensive rats after the intraperitoneal injection of d-nebivolol alone (0) or combined with various doses of 1-nebivolol. * Significantly different from the effect induced by d-nebivolol alone (P ~< 0.05).
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1043-4
`IPR2016-00379
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`It is interesting that doses of l-nebivolol which alone did not lower arterial blood pressure poten- tiated the antihypertensive effect of d-nebivolol. The mechanism involved is unknown. It is un- likely that metabolites of l-nebivolol are responsi- ble for the potentiating effect of this enantiomer, because its metabolites are inactive as far as their fl-adrenoceptor blocking properties are concerned (Heykants, personal communication). An interac- tion between the metabolism of the two enanti- omers can probably also be excluded, because 1-nebivolol does not influence the metabolism of d-nebivolol and vice versa (Heykants, personal communication). It remains to be seen whether presynaptic fl-blockade plays a role in this interac- tion because Heimberger and colleagues (1989) recently showed that, although differences in potency have to be appreciated, the relative potencies of d,l-nebivolol and its enantiomers were the same on presynaptic and postsynaptic recep- tors. At therapeutic doses the 1-enantiomer is practi- cally devoid of fl-adrenoceptor blocking properties (Van de Water et al., 1988a), but in the lower dose range (0.0025 to 0.16 rag-kg -1) it improves left ventricular function and reduces systemic vascular resistance (0.0025 to 0.04 mg. kg -1) in dogs (Van de Water et al., 1988b). The doses required to lower arterial pressure are higher in rats than in dogs. The data obtained for the lower doses in dogs are likely to be clinically relevant because one daily oral dose of 5 mg d,l-nebivolol has been 265 shown to lower arterial blood pressure in patients with hypertension. Acknowledgements The authors are indebted to Lambert Leijssen for making the illustrations, to Rita De Reese and Leo Van Dael for their technical assistance and to Karin Van Brussel and Jos Heems- kerk for their help in preparing the manuscript. References Bollman, J.L., 1948, Cage which limits the activity of rats, J. Lab. Clin. Med. 33, 1348. Heimberger, M., M.J. Montero, V. Fougeres, F. Beslot, M. Davy, M. Midol-monnet and Y. Cohen, 1989, Presynaptic fl-adrenoceptors in rat atria: evidence for the presence of stereoselective fll-adrenoceptors, Br. J. Pharmacol. 98, 211. Janssens, W.J., A. Van de Water, R. Xhonneux, R.S. Reneman, J.M. Van Nueten and P.A.J. Janssen, 1989, Nebivolol is devoid of intrinsic sympathomimetic activity, European J. Pharmacol. 159, 89. Rahn, K.H., 1983, Haemodynamic effects of the optical iso- mers of beta-receptor blocking agents, European Heart J. 4 (Suppl. D), 27. Van de Water, A., W. Janssens, J. Van Nueten, R. Xhonneux, J. De Cree, H. Verhaegen, R.S. Reneman and P.A.J. Jans- sen, 1988a, Pharmacological and hemodynamic profile of nebivolol, a chemical novel, potent and selective fll-adren- ergic antagonist, J. Cardiovasc. Pharmacol. 11, 552. Van de Water, A., R. Xhonneux, R.S. Reneman and P.A.J. Janssen, 1988b, Cardiovascular effects of d,l-nebivolol and its enantiomers - a comparison with those of atenolol, European J. Pharmacol. 156, 95.
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1043-5
`IPR2016-00379