Journal of Human Hypertension (2000) 14, Suppl 1, S91–S95  2000 Macmillan Publishers Ltd All rights reserved 0950-9240/00 $15.00 www.nature.com/jhh Effect of lacidipine and GITS on platelet function in patients with essential hypertension

MC Armas-Padilla1, MJ Armas-Herna´ndez1, R Herna´ndez-Herna´ndez1, M Velasco2, B Pacheco1, AR Carvajal1 and A Castillo-Moreno1 1Clinical Pharmacology Unit, School of Medicine, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela; 2Clinical Pharmacology Unit, Vargas Medical School, Central University of Venezuela, Caracas, Venezuela

With the aim of evaluating the effects on blood pressure, Both drugs reduced systolic and diastolic blood press- platelet function and insulin sensitivity of the dihydro- ure at the same level, however there were observable piridines lacidipine and nifedipine GITS, a parallel dou- differences in the rate of reduction. The nifedipine GITS ble-blind study was carried out in a group of 20 patients reduced supine systolic blood pressure by 25 mm Hg in with mild to moderate essential hypertension. They the first week, while the lacidipine did so by 11 mm Hg. received a placebo for 4 weeks; then were divided at At the end of the study period nifedipine reduced supine random into two groups of 10 patients each. Nifedipine systolic blood pressure by 28 mm Hg and lacidipine by GITS, 30 mg and lacidipine, 4 mg, were given during 16 20 mm Hg. Heart rate was increased slightly but signifi- weeks of active treatment. Blood pressure and heart cantly in the nifedipine GITS group only in the standing rate were measured at the clinic in supine, sitting and position. Both drugs reduced platelet aggregation ex -standing positions, 24 ؎ 1 h after the last dose. After the vivo only marginally but they modified the malondial placebo and active phases were carried out, a platelet dehyde production, indicating an action on the arachi- aggregation test was performed to determine platelet donic acid metabolic pathway. Journal of Human Hyper- malondialdehyde production and a tolerance to 100 g of tension (2000) 14, Suppl 1, S91–S95. glucose by measuring glucaemia and plasma insulin.

Keywords: lacidipine; nifedipine GITS; malondialdehyde; insulin sensitivity

Introduction ipine GITS allows the drug to be released through the intestinal tract over a long enough period to Calcium antagonists reduce arterial blood pressure maintain stable plasma levels for 24 h with a single by relaxing the smooth arteriole muscle and dimin- daily dose.11 1 ishing peripheral vascular resistance. Platelets play an important role in the The pharmacodynamic and pharmacokinetic atherosclerotic process in the hypertensive patient, characteristics of different calcium antagonists vary not only in initial vessel damage but also in the pro- 2,3 considerably. According to the agent, the effect cess of acute lumen obstruction.12,13 Eicosanoids may last from a short to a very long time period, produced by platelets are important to regulate their which has important clinical implications in the activity.12 Either tromboxano A-2 or malondial- protection of the hypertensive patient throughout 4,5 dehyde production can be determined to estimate the 24 h period. and prostaglandin metabolism at Lacidipine, a dihydropyridine derived calcium the platelet level.14 Antihypertensive treatment antagonist, has relative prolonged action: the admin- should ideally not only modify blood pressure to istration of 4 mg once a day reduces blood pressure 6,7 control level, but also contribute to modify other over 24 h with one, daily dosage. In comparison risk factors. with short-acting dihydropyridine drugs which The aim of the present study was to evaluate the resulted in a fluctuation in blood pressure and some effects on blood pressure, platelet aggregation and side effects associated with rapid vasodilatation and insulin and glucose levels by lacidipine and nifedip- 3,8,9 sympathetic activation. ine GITS given once daily, in a parallel, double- Regular nifedipine requires three or four doses per blind, randomised study on patients with mild to day to have a sustained effect over 24 h. The osmotic moderate hypertension. slow release formulation (GITS) has been developed to allow administration in one dose per day.10 Nifed- Patients and methods

Correspondence: Dr Marı´a Cristina Armas Padilla, Unidad de Ter- Twenty patients with mild to moderate hyperten- apia Me´dica, Calle 55 entre Ave. PL Torres Y Carrera 21, Edificio sion, who also fulfilled pre-established selection cri- El Bosque, Apto. A01, Barquisimeto, Venezuela teria, were the subjects of this study. The patients Effect of lacidipine and nifedipine GITS on platelet function MC Armas-Padilla et al S92 average age was 50 ± 2.3 years. The patient’s charac- Measurement of platelet malondialdehyde pro- teristics can be seen in Table 1. duction: Malondialdehyde was measured by the All patients initially received placebo tablets in a thiobarbituric acid methods,15,16 using N-ethylmalei- simple blind test for 4 weeks. Then patients were mide (Sigma Chemical Co) as activator, at the end allocated at random either in lacidipine or nifedip- of placebo and active therapy. Venous blood was ine GITS groups; 10 patients to, each group. Patients collected from the antecubital vein and mixed with received two tablets daily (one placebo and one 3.8% sodium citrate solution. The platelet-rich active tablet), taken between 7 and 8 am over the 16- plasma and platelet-poor plasma were obtained as week treatment. described previously. The platelet-rich plasma Patients were evaluated every 2 weeks at the adjusted at 300 000 platelet/␮L, was divided into 2- hypertension clinic. Systolic and diastolic blood ml aliquots and centrifuged at 250 g for 18 min to pressure were measured using phase I and V Korot- obtain platelet buttons. The supernatant was dis- koff sounds in supine, seated and standing pos- carded. itions. Heart rate was taken in the same positions. Either 2 ml of N-ethylmaleimide (NEM) in 1 mM All measurements were carried out 24 ± 1 h from the or 2 ml of isotonic saline were added, then incu- last dose of the tablets. Side effects were recorded. bated at 37°C for 60 min in a water bath. Malondial- Compliance of treatment was assessed by counting dehyde was determined using 2 ml of a mixer of tablets, every visit. thiobarbituric acid (0.53% in perchloric acid and 2.3% in PCA 7%, ratio 2:1). The solution was heated in a boiling water bath Platelet function evaluation for 15 min, cooled off to room temperature and cen- Platelet aggregation: At the end of the placebo and trifuged. The optical density was read at 534 nM in active phases a 20-ml antecubital blood a Spectronic 20D (Milton Roy Company, USA) sample was taken and mixed with a solution of against a blank. sodium citrate at 3.8% in a ratio of 9:1. The blood To calculate the malondialdehyde concentration, was centrifuged to obtain platelet-rich and platelet- the molar coefficient of extinction was 1.47 × 105. poor plasma. The concentration of platelet-rich The ratio MDA-basal (isotonic saline)/MDA-acti- plasma was adjusted in every case to 300 000 vated (NEM) was calculated as described by Makris platelets/␮L using the platelet-poor plasma. Platelet et al.16 aggregation test was carried out with a Biodata PAP- 4 Aggregometer (Biodata Corporation, Hatboro, PA, USA) using the following proaggregation agents: Statistical analysis (1) Adenosine Diphosphate (ADP) (Sigma Chemical Co, St Louis, MO, USA) in concentrations of 2.0; The data were grouped and the mean and standard 5.0 and 10 ␮M. error calculated. Statistical significance was (2) Adrenaline (Sigma Chemical Co) in concen- obtained using the Student’s t-test (P Ͻ 0.05, two- trations of 2.0, 5.0 and 10.0 ␮M. tailed) for paired and unpaired data: as well as for (3) Collagen (Horm Chemie, West Germany) in con- the analysis of variance (ANOVA). centrations of 0.5, 1.0 and 2.0 ␮g/ml. Platelet-rich plasma, adjusted to 300 000 platelets/␮L was placed in the test tube with a mag- Results netic agitator at a velocity of 1100 rotations per minute. The paper was allowed to run for a minute Blood pressures and heart rate to check spontaneous aggregation after which the pro-aggregate agent was added and the test was In the lacidipine group supine blood pressures allowed to run for 5 min. At the end of this period (systolic/diastolic) during the placebo were 168/103 the percentage aggregation produced was measured. mm Hg; after weeks 1 and 16 of active treatment The test was repeated twice and the values averaged blood pressure was reduced to 157/94 (P Ͻ 0.02) in each case. and 148/91 (P Ͻ 0.001), respectively. Statistical comparison between weeks 1 and 16 in active treat- Table 1 Patients characteristics ment was significant (Table 2). In the nifedipine GITS group the results for the Age Sex Hypertension Basal blood supine position were 165/105 mm Hg in the placebo (years) (male/ evolution pressure phase, 140/90 mm Hg (P Ͻ 0.001) in the first week female) time (years) (SBP/DBP) (mm Hg) and 137/88 mm Hg after 16 weeks of treatment (P Ͻ 0.001). Statistical comparison between week 1 Lacidipine Group and week 16 showed equivalent results Mean 52.36 7/3 8.3 168/103 In the supine position, the heart rate in the lacidi- Standard Error 3.29 – 1.4 4.5/1.10 pine group varied from 74 bpm to 82 bpm and to 75 bpm in the placebo phase between the first week Nifedipine OROS Ͼ Group and week 16 of active treatment (P 0.05). In the Mean 47.1 5/5 8.41 165/105 nifedipine GITS group, heart rate changed from Standard Error 1.78 – 1.5 4.64/1.32 73 bpm in the placebo phase to 84 and 77 bpm in the first and 16th weeks of active medication (P Ͼ 0.05).

Journal of Human Hypertension Effect of lacidipine and nifedipine GITS on platelet function MC Armas-Padilla et al S93 Table 2 Lacidipine and nifedipine GITS on systolic and diastolic Table 3 Effect of nifedipine GITS and lacidipine on platelet blood pressure and heart rate during placebo and active phases aggregation induced by adenosine diphosphate (ADP), adrenaline during placebo, 1 and 16 weeks after active treatment (ADR) and collagen (COL) at different concentrations

Placebo Week 1 Week 16 ADP 5. ADP ADR ADR COL COL 0(␮M) 10.0 5.0 10.0 0.5 1.0 ␮ ␮ ␮ ␮ ␮ Lacidipine Group (n = 10) ( M) ( M) ( M) ( M) ( M) Supine SBP ± s.d. 168 ± 4.5 157 ± 5.7* 148 ± 5.88**† Lacidipine Group (n = 10) DBP ± s.d. 103 ± 1.1 94 ± 2.8* 91 ± 3.22**† Placebo HR ± s.d. 74 ± 3.7 82 ± 5.7 75 ± 3.86 Mean (%) 72 89.2 79.8 82.2 87.4 93.2 Standard 7.2 1.9 6.3 5.7 2.8 2.9 Sitting Error SBP ± s.d. 161 ± 4.3 148 ± 4.7 143 ± 5.06**† DBP ± s.d. 103 ± 1.1 95 ± 2.44* 89 ± 2.59**† Active HR ± s.d. 76 ± 3.6 86 ± 5.3 79 ± 3.58 Mean (%) 79.5 86.2 68.9 68.9* 88.4 90.4 Standard 6.2 3.8 10.04 9.6 3.8 3.9 Standing Error SBP ± s.d. 163 ± 4.0 147 ± 5.66* 140 ± 4.68**† DBP ± s.d. 104 ± 1.2 95 ± 2.9* 90 ± 3.01*† Nifedipine Osmotic Group (n = 10) HR ± s.d. 80 ± 3.9 89 ± 5.1 83 ± 3.70 Placebo Mean (%) 84.8 90.8 82.8 86.1 88.6 89.3 Nifedipine GITS Group (n = 10) Standard 4.5 4.06 6.0 5.7 4.5 4.6 Supine Error SBP ± s.d. 165 ± 4.64 140 ± 4.84** 137 ± 3.95** Active DBP ± s.d. 105 ± 1.32 90 ± 3.87** 88 ± 3.77** Mean (%) 79.6 87.9 77.2 78.4* 78.9 86.4 HR ± s.d. 73 ± 3.00 84 ± 2.75 77 ± 3.44 Standard 8.0 4.2 8.1 8.2 8.8 4.4 Error Sitting SBP ± s.d. 160 ± 4.76 136 ± 3.82** 131 ± 4.05** DBP ± s.d. 103 ± 0.93 92 ± 3.50** 87 ± 3.34** *Paired Student’s t-test: P Ͻ 0.05. HR ± s.d. 74 ± 2.43 81 ± 2.96 81 ± 2.62

Standing SBP ± s.d. 161 ± 5.03 134 ± 3.56** 131 ± 5.02** Table 4 Basal and activated malondialdehyde production and DBP ± s.d. 105 ± 1.03 93 ± 3.91** 89 ± 4.59** the ratio basal: activated in the lacidipine and nifedipine GITS HR ± s.d. 77 ± 2.86 88 ± 3.42* 88 ± 3.29* groups in patients with hypertension

*P Ͻ 0.02; **P Ͻ 0.001 Student’s t-test for paired data vs placebo MDA Basal MDA Ratio ␮ 9 in each group. †P Ͻ 0.05 ANOVA comparisons between week 1 g/10 Activated basal:activated ␮ 9 and week 16. platelets g/10 platelets

Lacidipine Group (n = 10) Platelet aggregation Placebo Mean 2.35 4.56 0.50 Platelet aggregation test during the study can be seen Standard Error 0.28 0.35 0.038 in Table 3. Platelet aggregation induced by adenos- ␮ ␮ Active ine diphosphate (ADP) (10 M), adrenaline (10 M), Mean 1.37 3.71 0.38 and collagen (1.0 ␮g/ml) at the end of the placebo Standard Error 0.08 0.288 0.03 phase were 89.2%, 82.2% and 93.2%, respectively; P* value 0.01 0.10 (NS) 0.01 this changed to 86.2% (P Ͼ 0.05, NS), 68.9% % Change in 41.8 18.65 23.35 (P Ͻ 0.05), and 90.4% (P Ͼ 0.05, NS) respectively at relationship to placebo the end of active treatment in the lacidipine group Nifedipine GITS Group (n = 10) of patients. Placebo In the nifedipine GITS group of patients, platelet Mean 1.95 4.31 0.45 aggregation changed from 90.8%, 86.1% and 89.3% Standard Error 0.14 0.22 0.03 to 87.9% (P Ͼ 0.05, NS), 78.4% (P Ͻ 0.05) and Active 86.4% (P Ͼ 0.05, NS) for the three inducers, at the Mean 1.34 3.61 0.378 Standard Error 0.14 0.28 0.04 same concentrations respectively. P* value 0.0003 0.12 (NS) 0.07 (NS) % Change in 31.4 16.19 16.78 relationship to placebo Platelet malondialdehyde The results of basal and activated malondialdehyde *P value based on Student’s t-test for paired data (two columns) and the ratio MDA basal:activated can be seen in in relation to placebo for each group. Table 4, in the lacidipine group basal malondial- dehyde was reduced in 41.8% (P Ͻ 0.01); MDA acti- vated in 18.65% (NS) and the ratio basal:activated Both drugs were well tolerated, in the lacidipine in 23.35% (P Ͻ 0.01). group some patients complained of headache and In the nifedipine GITS group of patients, basal one of dizziness; on nifedipine GITS two patients malondialdehyde production was reduced in 31.4% complained of headache and one patient of mild (P Ͻ 0.001), activated MDA in 16.19% (NS), and the ankle oedema. Side effects were mild and tolerated ratio basal:activated in 16.78% (P = 0.07, NS). by patients; no one dropped out from the study.

Journal of Human Hypertension Effect of lacidipine and nifedipine GITS on platelet function MC Armas-Padilla et al S94 Discussion centrations but not with ADP or collagen. Calcium antagonists are known to share the common pro- From the results obtained, both calcium antagonist perty of inhibiting the entrance of calcium into the drugs studied, nifedipine GITS and lacidipine cell,21 and in higher concentrations may interfere administered in a single daily dose of 30 mg and with storage and release of calcium intracellularly. 4 mg respectively, significantly reduced systolic and In 1981 Ideka22 demonstrated that diastolic blood pressure in patients with mild to reduced platelet aggregation, especially at low con- moderate hypertension. However, the speed of centrations of ADP and adrenaline when patients reduction on blood pressure was different with each took 240 mg/daily of the drug. Dihydropyridine drug. Nifedipine GITS reduced blood pressure to derivatives have also been shown to have an effect control levels after the first week of treatment; at the on platelet aggregation, especially in in vitro tests, supine position the blood pressure at the end of the that is, when concentrations of the agent are added first week had decreased to 25/15 mm Hg in relation to the test tube before the inductors agent. Thus, to placebo values, statistically equivalent to a nifedipine23 and nitrendipine24 reduce the platelet reduction of 28/17 mm Hg obtained in week 16 aggregation in vitro at higher concentrations than (ANOVA test, P Ͼ 0.05, NS). However, lacidipine those used in antihypertensive treatment. Other reduced blood pressure at the end of the first week dihydropyridine derivatives, such as , to 11/9 mm Hg in relation to placebo values reduce platelet aggregation during antihypertensive (P Ͻ 0.025). At the end of week 16 the decrease therapy, as reported by Hernandez-Hernandez et al25 reached 20/12 mm Hg (P Ͻ 0.001). Thus a compari- when in a group of 12 patients with essential hyper- son of the results of the values of the different weeks tension a 20–45% reduction in platelet aggregation of active medication according to ANOVA showed was obtained using the same inducers tested in the them to be statistically significant (P Ͻ 0.05), a present study. slower and more progressive reduction of blood Activation of platelets can be evaluated by the pressure being observable with lacidipine in com- measurement of platelet malondialdehyde pro- parison to nifedipine GITS. duction, in basal conditions or after the stimulation A rapid reduction in blood pressure by vasodilat- of arachidonic acid metabolic pathway. Both drugs, ation induces sympathetic reflex discharge in the lacidipine and nifedipine GITS, induced a reduction hypertensive patients, with release of catecholam- in the production of both basal and activated malon- ines and increasing heart rate by stimulation of beta- dialdehyde; as well as in the ratio MDA basal:activ- 1 adrenoceptors. ated. Lacidipine reduced these values by 41.8%, When regular nifedipine release is administered 16.65% and 23.35% respectively. Meanwhile nifedi- orally, a fast reduction in blood pressure is found in pine GITS did so by 31.4%, 16.19% and 16.78% 20 min and maximum is reached in 1 h. But the respectively. There was no statistically significant effect does not last more than 8 h,17 at the same time difference between the groups, either for placebo a significant increase in heart rate, indicating an values or at the end of active medication. This immediate sympathetic discharge as a response to would indicate that both drugs have a similar effect the decrease in peripheral vascular resistance. on platelet malondialdehyde production, indicating Leenen,18 comparing the effect of nifedipine GITS to these agents tend to normalise the balance of eicos- amlodipine, found that nifedipine GITS slightly but anoids, especially in the production of thromboxane significantly increases levels of adrenaline, norad- A2 which has been reported to be high in patients renaline and renin serum, indicating a neuro- with arterial hypertension.26 Other dihydropyridine humoral response even in long term treatment with calcium antagonists, such as amlodipine, reduce this drug prepared for gradual long-term release, platelet aggregation and malondialdehyde pro- while amlodipine did not produce this effect. Lacid- duction, in contrast to enalapril.27 ipine, on the other hand, has been reported not to modify catecholamine levels, renin serum levels or aldosterone.18,19 References In the present study lacidipine increased heart rate in the standing position by 9 bpm (P Ͼ 0.05, NS) 1 Lehmann H, Hochreun H, Witt E, Mies H. Hemodyn- amic effects of calcium antagonists. Review. in the first week of treatment and by 3 bpm on aver- Hypertension 1983; 5: 1166–1173. age by the end of week 16. This was not statistically 2 Reid JL, Meredith PA, Donnely R, Elliott HT. Pharma- significant in either case. The nifedipine GITS cokinetics of calcium antagonist. J Cardiovasc Pharma- increased heart rate by 11 bpm both in the first and col 1988; 12 (Suppl 7): S22–S26. 16th week of active treatment (P Ͻ 0.02); indicating 3 Van Zwieten PA. The pharmacological properties of a slight sympathetic activation, as reported by Leen- lipophilic calcium antagonists. Blood Press Suppl en.18 1998; 2: 5–9. 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