Nitric oxide mediated venodilator effects of nebivolol

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1 Br J lin Pharma 1994; 38: Nitri oxide mediated venodilator effets of nebivolol A. J. BOWMAN, C. P. L.-H. CHEN' & G. A. FORD Departments of Mediine and Pharmaologial Sienes, The University, Newastle upon Tyne NE2 4HH and 'Janssen Researh Foundation, Grove, Oxon OX12 ODQ 1 Nebivolol, a seletive f31-adrenoeptor antagonist with antihypertensive effets, has haemodynami effets suggestive of a diret vasodilator ation. 2 The dorsal hand vein tehnique was used to determine whether nebivolol has venodilator ation in vivo in man. 3 Nebivolol and atenolol were infused into the phenylephrine preonstrited superfiial hand veins of 11 healthy male volunteers. In separate studies L-NMMA (.1 gg min-) was pre- and o-infused with nebivolol to determine whether nitri oxide (NO) mediated mehanisms were present. Further studies with prostaglandin F2a (PGF2a) preonstrition were performed to exlude an at-adrenergi antagonisti effet of nebivolol. Effets of L-NMMA infusion on nitroglyerin venodilation were also determined. 4 Nebivolol produed a dose dependent venodilation, (72 ± 18% maximum), whereas atenolol produed no signifiant venodilation. At doses of nebivolol produing plasma onentrations omparable with plasma levels ahieved after standard oral dosing (1' mol min-') small (14 ± 6% and 23 ± 8%) but signifiant (P <.5) venodilation was observed. 5 The venodilator response to nebivolol was signifiantly redued by infusion of L-NMMA (maximum dilation 18% vs 72%, P <.1). Venodilator responses to nitroglyerin were unaffeted by L-NMMA infusion. A venodilator effet to nebivolol was also seen following preonstrition with PgF2, (4 ± 2% maximum). 6 Nebivolol has nitri oxide mediated, venodilator effets in man. Keywords nebivolol,-adrenoeptor bloker veins endothelium nitri oxide Introdution Nebivolol, a raemi drug, is a seletive,i-adrenoeptor antagonist, of whih the (+)-isomer is one hundred times more potent a PI-adrenoeptor antagonist than the (-)-isomer [1]. Nebivolol maintains ardia output by inreasing stroke volume, whilst dereasing systemi vasular resistane in human volunteers and in animal studies [1]. This effet on peripheral vasular resistane does not appear to be due to effets on ax-adrenoeptors, potassium or alium hannel blokade, peripheral sympatheti nervous system ativity or by diret ation on endothelial ells at low onentrations [2, 3]. Some work suggests that the vasodilatory effets are mediated through nitri oxide. Nebivolol indues an endothelial dependent relaxation of anine oronary arteries and potentiates ADP indued endothelial dependent relaxation via endothelial dependent relaxation fator (EDRF), now known to be nitri oxide [5]. The aims of the present study were to determine whether nebivolol has venodilator properties in man and whether suh effet is mediated through nitri oxide. Methods Subjets Eleven healthy male volunteers, mean age 3 years (range years) were studied. All had a normal history, physial examination, full blood ount, urea Correspondene: Dr G. A. Ford, Wolfson Unit of Clinial Pharmaology, The University, Newastle upon Tyne NE2 4HH 199

2 2 A. J. Bowman, C. P. L.-H. Chen & G. A. Ford and eletrolytes, liver funtion tests, random blood gluose, urinalysis and resting ECG. Mean height was 18 m (range m) and mean weight 82 kg (range kg). Mean blood pressure was 123/78 (range /64-9) mm Hg. Subjets refrained from affeine for 24 h prior to eah study. Written informed onsent was obtained from all subjets and studies were approved by the Newastle Joint Ethis Committee. Dorsal hand vein tehnique The dorsal hand vein tehnique as modified by Aellig [6] and previously desribed in detail was used [7]. Studies were performed in a temperature ontrolled (23 ± 2 C) laboratory. Subjets lay semi-supine with the study arm resting at a 3 slope to ensure venous drainage. A 23 gauge butterfly needle was inserted into a suitable vein, on the dorsum of the hand and a ontinuous infusion of.9% saline at.3 ml min-1 ommened. A linear variable differential transduer (LVDT, Shaevitz Engineering) was then plaed over the study vein 1 mm downstream from the needle tip. The position of the ore, linearly related to voltage output was reorded on a strip hart reorder. Readings of the position of the ore were made before and during inflation of a sphygmomanometer uff on the same arm to 4 mm Hg to produe venous filling. The baseline venodilation during saline infusion with the uff inflated was defined as 1% relaxation, the reording with the uff not inflated was defined as 1% onstrition. Venodilator responses to nebivolol, atenolol or nitroglyerin following preonstrition with phenylephrine or PGF2a* was defined as a perentage of the baseline obtained during initial saline infusion by the formula [(V-X)/ (X-Y)] x 1 where V is vein diameter with vasodilator, X is baseline (saline) vein diameter and Y is onstrited (phenylephrine pre vasodilator) vein diameter. Blood pressure and heart rate were monitored at 1 min intervals on the opposite arm. All drug solutions were infused at.3 ml min-m. Studies Studies were separated by not less than 1 week. Nebivolol effets on phenylephrine preonstrited hand veins The ation of nebivolol on preonstrited hand veins was determined in eight subjets. Saline was infused for 3 min followed by inremental doses of phenylephrine (range ng min-) until the vein was onstrited to 7-8% of baseline. The diluent arrier solution and then nebivolol (MW 442) at inreasing doses (1-1, ng min-1, 2 x 1-12 to 2 x 1-8 mol min-') were infused onomitantly with the phenylephrine onstritor dose. All responses were reorded after at least 7 min infusion time. Dilator responses to nebivolol were observed to have plateaued at 7 min as responses reorded at 1 min in initial studies were not signifiantly different. L-NMMA studies The effet of L-NMMA on the vasodilator effet of nebivolol was determined in seven of eight subjets studied in the first phenylephrine studies. One subjet who had no venodilator response to nebivolol was exluded. Saline was infused for 3 min followed by phenylephrine until the vein was onstrited to 7-8% of baseline. L-NMMA was then infused onomitantly at a dose of.1,ug min-' for 3 min and then during inreasing nebivolol doses (2 x 1-12 to 2 x 1-8 mol min-'). Control studies of L-NMMA effets in nitroglyerin venodilation were performed in four subjets. Following phenylephrine preonstrition nitroglyerin was infused at doses of 1-1 pg min-'. L-NMMA was then infused for 3 min, during whih time baseline preonstrition returned and responses to nitroglyerin were again determined. Atenolol effets on phenylephrine preonstrited hand veins A 3 min saline infusion was followed by inremental doses of phenylephrine until the study vein was onstrited to 7-8% of baseline. Inreasing doses of atenolol (MW 266) were infused (.5-5, ng min-', 2 x 1-12 to 2 x 1-8 mol min-1) onomitantly with the phenylephrine onstritor dose. The response to eah atenolol dose was reorded after 7 min infusion. Lower dose and duration of effet of nebivolol To assess the response of human hand veins to lower doses of nebivolol and to haraterise the duration of venodilator effets, further studies were performed in nine subjets. Following 3 min infusion of saline, a phenylephrine preonstrition dose was established, as desribed above. Nebivolol was then infused inrementally (2 x 1-13 to 2 x 1-9 mol min-1) with the onomitant infusion of the phenylephrine preonstritor dose. When a maximum dilation at 2 x 1-9 mol min-1 was reahed, the nebivolol infusion was stopped and the preonstrition dose of phenylephrine was infused for a further 6 min. Hand vein dilation was measured every 1 min. Nebivolol effet on PGF2a preonstrited hand veins To exlude ox-adrenergi blokade as the ause for the nebivolol indued venodilation of phenylephrine onstrited veins, the effets of nebivolol in PGF2a onstrited veins were determined, in nine subjets. Following 3 min infusion of saline, a preonstrition dose of PGF2, that produed 7-8% onstrition of baseline distension was established and nebivolol was then infused onomitantly (2 x 1-12 to 2 x 1-8 mol min- 1) Statistial analysis Statistial analysis was performed using Student's t-test for paired data, P values <.5 were on-

3 NO mediated venodilator effets of nebivolol 21 sidered signifiant. Dose-response urves to nebivolol and atenolol were analysed by repeated measures ANOVA. Results Ation of nebivolol on phenylephrine preonstrited hand veins The mean preonstrition dose of phenylephrine infused was 62 ± 598 ng min-' (mean ± s.d., range ng min-). The degree of preonstrition was 79 ± 14% expressed relative to the baseline distension during saline infusion. The diluent did not signifiantly affet the phenylephrine preonstrition (mean hange 2 ± 7%, P =.44). A dose-dependent venodilator response to nebivolol was seen in seven of eight subjets (Figure 1). Maximum venodilation was 72 ± 18%. In three subjets, a biphasi response was seen with the maximum venodilator response ourring at doses lower than the highest infused dose of nebivolol used. L-NMMA effets Nebivolol indued venodilation was signifiantly redued following L-NMMA infusion, at all doses of nebivolol infused, with a maximum dilation of 18 ± 19% (Figure 1). Mean phenylephrine preonstrition dose was 893 ± 62 (range ng min-) whih was not signifiantly different from the initial nebivolol study (P =.19). The degree of preonstrition was 79 ± 5%. L-NMMA infused alone in phenylephrine onstrited hand veins did not affet the degree of preonstrition (72 ± 19 vs 74 ± 15%, P =.77). Nitroglyerin indued venodilation was unaltered by L-NMMA; mean venous dilation before/ following L-NMMA in four subjets (1 pg min-' 17 vs 2%, 1 pg min-' 49 vs 42, 1 pg min-' 61 vs 71%). Atenolol ation on phenylephrine preonstrited hand veins Mean phenylephrine preonstrition dose was 361 ± 238 (range 19-6) ng min-'. The degree of preonstrition was 85 ± 6% whih was not signifiantly different from the degree of preonstrition obtained in studies with nebivolol (P =.19). There was no signifiant venodilatation at any dose of atenolol (Figure 2). Lower dose and duration of nebivolol ation Signifiant venodilation was observed at all doses of nebivolol (Figure 3a). Small but signifiant venodilation ourred (mean ± s.d. dilation ompared with baseline; 14 ± 6% at 2 x 1-13, P <.5; 23 ± 8% at 2 x 1-13 mol min-', P <.5) at infusions equivalent to the plasma onentrations ahieved with standard oral dosing for hypertension and angina (.1 to 1 ng ml-') assuming blood flow of 1 ml min-' through a single hand vein. There was no signifiant differene between the mean dilation (51 ± 17%) at nebivolol _ o 4 -o io Nebivolol dose (mol min-) 1-7 Figure 1 Venodilator effets of nebivolol in phenylephrine onstrited hand veins of healthy volunteers without (A, n = 8) and during L-NMMA infusion (O1, n = 7). Data are mean ± s.e. mean. lo Dose (mol min-') 1-7 Figure 2 Venodilator effets of nebivolol (A) and atenolol (Ol) in phenylephrine onstrited hand veins. Data are mean ± s.e. mean (n = 9).

4 22 A. J. Bowman, C. P. L.-H. Chen & G. A. Ford dose 2 x 1-9 mol min-1 obtained in the low dose nebivolol studies (Figure 1) and the mean dilation of (33% ± 1%) seen at nebivolol dose 2.3 x 1-9 mol min-1 obtained in the nebivolol studies onduted with L-NMMA (P =.39) (Figure 3a). Persistene of m._ 4 I. a 1 r 8 F- 6 F- 4 F- signifiant venodilator effet was seen beyond 6 min, however, there was a notieable derease in the degree of dilation in most individuals after 3 min (Figure 3b). The degree of preonstrition was 84 ± 6% with a phenylephrine preonstrition dose of 341 ± 122 (range ) ng min-'. There was no signifiant differene in either the dose of phenylephrine (P =.45) or the degree of preonstrition (P =.24) ompared with the previous nebivolol studies. Nebivolol effet on PGF2a onstrited hand veins Mean PGF2a onstrition dose was 96 ± 4 (range ) ng min-'. The degree of preonstrition was 74 ± 11%. This was not signifiantly different from the mean preonstrition of the first study where phenylephrine was the onstritor (P =.74). A statistially signifiant nebivolol indued venodilation was seen at all doses of nebivolol infused (Figure 4). There was no signifiant differene in the degree of nebivolol indued venodilation with either phenylephrine or PGF2a as the preonstritor (P =.74). A biphasi response was observed in some subjets as seen in the phenylephrine studies. 2 F 1-13 b 1 r 8 F lo-11 1o-1 i-9 Nebivolol dose (mol min-1) Disussion These results onfirm that nebivolol has a venodilator 18 ation in human hand veins and that this effet is not shared by atenolol. The inhibitory effet of L-NMMA indiates that venodilation due to nebivolol is primarily mediated through nitri oxide. This nitri oxide mediated, endothelial dependent venodilator effet desribed has not previously been desribed in human 1r._i Cu o 1-6 F C :6 ol 8 F / 2 [ Time (min) Figure 3 a) Venodilator effets of nebivolol at lower dose infusions on phenylephrine onstrited hand veins and b) Duration of nebivolol venodilator effet during 6 min following disontinuation of 2 x 1-9 mol min-' nebivolol infusion. Data are mean ± s.e. mean (n = 9). I u- Nev-o lo1 ol-9 1o-8 Nebivolol dose (mol min-') Figure 4 Venodilator effet of nebivolol with PGF2a as venoonstritor. Data are mean ± s.e. mean (n = 8). 1-7

5 NO mediated venodilator effets of nebivolol 23 vasulature with other j-adrenoeptor bloking drugs. The observations that atenolol has no signifiant venodilator effet in human hand veins, indiate that nitri oxide mediated venodilation is not a lass effet of all P-adrenoeptor antagonists and may be unique to nebivolol. Studies of other vasodilatory P- adrenoeptor antagonists using the hand vein model and L-NMMA would be of interest. L-NMMA infusion did not modify the venoonstritor ation of phenylephrine, an observation in keeping with previous observations that L-NMMA does not alter vasoonstritor effet of noradrenaline [8]. This and the lak of effet of L-NMMA on venodilator responses to nitroglyerin exludes a non-speifi venoonstritor effet of L-NMMA and suggests that nebivolol is ating through endogenous nitri oxide. The venodilator responses with PGF2a as the venoonstritor exlude an t-adrenoeptor antagonist ation of nebivolol as the ause of the venodilation observed in the phenylephrine studies. This is in keeping with previous work whih indiates that nebivolol has no effets at the a-adrenoeptor [2, 3]. Some animal work has found that,b-adrenoeptor mediated vasular relaxation is mediated via nitri oxide release from the endothelium, although this has not been a onsistent finding and appears to be speifi to ertain speies and vasular beds [9-11]. Nebivolol has a relatively weak affinity for P2-ompared with PI-adrenoeptors, and one possible explanation for our findings is that nebivolol is ating as a partial agonist at vasular P2-adrenoeptors whih mediate venodilation via nitri oxide. Raemi nebivolol laks intrinsi sympathomimeti ativity in isolated right atria of reserpinised rats and in reserpinised anaesthetised dogs, suggesting that suh an ation is unlikely [2]. Also evidene that f-adrenoeptor mediated vasodilatation in human hand veins is mediated via nitri oxide is laking. Alternatively nebivolol may be ating through a non,b-adrenoeptor mediated mehanism. It would be important to investigate the mehanism underlying this effet as it ould lead to the development of seletive modulators of nitri oxide synthase. Further studies examining the vasodilatory effets of individual nebivolol isomers and the effet of oinfusion of propranolol on nebivolol indued venodilation would be of interest. In three of seven subjets a biphasi venodilator of nebivolol on phenyleprhine onstrited veins was reorded. This may be due to tahyphylaxis but is perhaps more likely to be a non-speifi venoonstritor effet of the higher doses infused. Non-speifi venodilator effets to the preservative sodium metabisulphite have been previously desribed [7]. The results of the study using nebivolol at lower doses show that venodilation ours at infusions equivalent to the plasma onentrations ahieved with standard oral dosing for hypertension and angina (.1 to 1 ng ml-') suggesting that a vasodilatory effet through nitri oxide may aount for the haemodynami profile of nebivolol when administered in these doses. The persistene of venodilator ation was seen to our beyond 6 min in some individuals, however, there was a marked derease in venodilation in most subjets after 3 min. The nature of this prolonged effet is unlear however, it is possibly a result of persistent tissue binding of nebivolol but may represent a sustained effet on vasular nitri oxide synthase. Other,3-adrenoeptor antagonists have vasodilator effets, although the modes of ation of most of these are not thought to be endothelial dependent, and those that have been shown to at on the endothelium have not been shown to at via nitri oxide. Janzewski et al. [12] suggest that arteolol failitates the abluminal release of nitri oxide aused by a2-adrenergi ativation and auses the intraluminal release of vasodilator prostaglandin in animal in vitro studies but studies on human vasulature have not been performed. Vasodilation by the,-adrenoeptor blokers, suh as labetalol, timolol and propranolol, have been shown to be mediated via their weak ation on a- adrenoeptors [13]. Nipradilol is another non-seletive,-adrenoeptor bloker with vasodilator ativities whih are thought to be due to its weak a-adrenoeptor bloking ations [14]. However, it may also have a nitroglyerin like ation, as it ontains nitroxy residues in its moleule [15]. A reent study showed that in rat arterioles and venules it appeared to have a dilator effet when applied topially, whih did not our with propranolol, atenolol and labetalol, The study did not however, use endothelial denuded tissue or nitri oxide synthase inhibitors and hene an endothelial dependent venodilation has not been established [16]. Carvedilol has been shown to have vasodilator ativity mediated primarily via al-adrenergi blokade [17]. However, a more reent study [18] suggests that an additional, as yet undefined, mehanism ould be involved.,b-adrenoeptor bloking drugs suh as xamoterol, have in the past been used in the treatment of ongestive heart failure. Most of these drugs have a signifiant negative inotropi effet whih severely restrits their use. Reently there has been renewed interest in this form of therapy [19]. The nitri oxide mediated venodilation indued by nebivolol, may derease preload and therefore be of benefit in the treatment of heart failure, partiularly, when it is this ation that is thought to be responsible for the maintenane of ardia output found with nebivolol. Further linial trials investigating the haemodynami effets on nebivolol in animal and linial studies would larify this. Endothelial dysfuntion is well desribed in the peripheral vasulature of patients with hypertension and in the oronary arteries of patients with angina [2-23]. This may be a pathophysiologial fator in oronary and erebral vessel thrombosis. Further evidene is emerging that interferene with the L- arginine/nitri oxide synthesis pathway is important in the aetiology of hypertension and that orretion of nitri oxide levels, either diretly or by addition of L-arginine, results in lowering of the blood pressure [24-27]. Enhaning vasular endothelial response in these situations ould result in nebivolol having greater linial benefits in terms of stroke and myoardial infartion than other P-adrenoeptor blokers. We thank Janssen Researh Foundation for providing the nebivolol used in these studies.

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