A Comparison of Isradipine and Atenolol on Ex Vivo Platelet Function in Hypertensive Subjects

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ORIGINAL ARTICLE Pro-haemorrhagic effects of calcium antagonists: a comparison of isradipine and atenolol on ex vivo platelet function in hypertensive subjects

A Smith1, J McPherson2, M Taylor1, A Mason2, S Carney2 and A Gillies2 Disciplines of 1Clinical Pharmacology and 2Medicine, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, NSW, Australia

It has been suggested that long term treatment with cal- neither drug produced a detectable effect on ex vivo cium antagonist drugs might inhibit platelet function platelet aggregation, platelet retention, or thromboxane and lead to an anti-atheromatous effect. However recent generation with adrenaline, collagen, adenosine- data have also suggested that such an effect might di-phosphate, or platelet activating factor. However a increase mortality due to an increased incidence of gas- decrease in plasma ␤-thromboglobulin levels was trointestinal bleeding. observed which reached statistical significance (P Ͻ We identified 43 subjects from general practice with 0.05) after 12 weeks treatment in the isradipine but not uncomplicated mild to moderate hypertension to com- the atenolol group. A 39% reduction with isradipine pare the effects of the calcium antagonist isradipine compared with 34% following atenolol treatment. Euglo- with that of the ␤-blocker atenolol on platelet function, bulin clot lysis time was not altered by either drug. plasma ␤-thromboglobulin levels, fibrinolysis, and Serum cholesterol concentrations were also unaltered serum lipids in a randomised double-blind parallel by drug treatment. group study. After careful evaluation to exclude con- Therapeutic doses of the calcium antagonist isradipine comitant aspirin use, only 24 subjects were eligible to may produce a minor indirect effect on platelet function enter the study. While isradipine and atenolol produced after several weeks of treatment. However, this is of doubt- comparable and clinically significant falls in blood ful clinical importance and may simply reflect an effect of .pressure (167 ؎ 2 /102 ؎ 1 to 153 ؎ 3/91 ؎ 2 mm Hg, and lowered blood pressure on platelet function ,(to 156 ؎ 4/91 ؎ 2 mm Hg, respectively 1 ؎ 2/101 ؎ 165

Keywords: calcium channel blocking drugs; ␤-blocking drugs; platelet function

Introduction lation is sparse.5 Although calcium antagonists have been demonstrated to inhibit platelet aggregation in While calcium antagonists are frequently used in the vitro,6–8 the doses required have been very high and treatment of hypertension and coronary heart dis- would be difficult to achieve in the clinical situ- ease, several recent studies have questioned their ation. Results of in vivo studies have been difficult long term safety. Apart from a meta-analysis and a to interpret because of unsatisfactory experimental case control study which suggested a greater mor- controls.5,9 tality rate with calcium antagonists1,2 and evidence Our study was designed to examine the compara- of a possible greater risk of cancer,3 analysis of the tive effects of the dihydropyridine isradipine and same database of elderly patients has suggested an the ␤-blocker atenolol on ex vivo platelet function increased risk of gastrointestinal haemorrhage for hypertensive patients on calcium antagonists when in hypertensive patients whose blood pressure (BP) compared to ␤-blockers.4 The potential for calcium had been reduced by either drug as monotherapy. antagonists, particularly dihydropyridines, to The design was a randomised, parallel group, dou- inhibit platelet function and promote vasodilation ble-blind comparison over 12 weeks of active treat- were the suggested mechanisms. However, evidence ment with a preliminary 4-week placebo ‘run-in’ ␤ that calcium channel blockers in general, and dihyd- and 4-week placebo ‘washout’ period. The -blocker ropyridines in particular, can significantly inhibit atenolol was used since it does not appear to alter platelet function in vivo in the hypertensive popu- platelet function. Moreover, recent studies on haemorrhagic, vascular and cancer mortality rates relied on comparisons between calcium antagonists and Correspondence: SL Carney, Discipline of Medicine, Locked Mail ␤-blockers. Bag No 1, Hunter Region Mail Centre, NSW 2300, Australia Received 18 November 1996; revised 28 February 1997; accepted 28 February 1997 Pro-haemorrhagic effects of Ca antagonists A Smith et al 784 Patients and methods NSW, Australia); and platelet activating factor (PAF, 0.19 mM) from Boehringer Mannheim (Mannheim, Patients aged between 40 and 60 years with mild to Germany). The velocity slopes of primary and sec- moderate hypertension (diastolic BP 95–109 ondary aggregation (mm/min) were measured; for mm Hg) were recruited from general practice. collagen the lag phase (seconds) was also measured. Patients were excluded if they were taking any Thromboxane B2 (TXB2) was measured by radioim- medications known to influence lipids, platelet munossay. TXB standard and TXB antiserum were у 2 2 function, or BP; had DBPs 110 mm Hg and/or sys- obtained from Sigma Chemical Co and tritiated tolic pressures у180 mm Hg at any phase of the TXB2 from New England Nuclear (Du Pont NEN, study; had a history of vascular disease (angina, Boston, MA, USA). TXB2 generation was measured myocardial infarction, transient ischaemic attack, in response to adrenaline (5.5 mM), ADP (1.25 ␮M), stroke, claudication or any current disturbance in collagen (0.5 ␮g/mL) and PAF (0.19 mM). Aggre- cardiac rhythm); had a history of diabetes mellitus, gation was allowed to proceed for 6 min after which asthma or history of drug or alcohol abuse. Female an equal volume of 70% ice cold ethanol was added patients in the reproductive age group were also to the cuvette. The sample was immediately mixed excluded. A research nurse visited patients ident- and transferred to polypropylene tubes on ice until ified by participating doctors who confirmed their centrifugation at 2000 g for 30 min at 4°C. The suitability for inclusion and obtained informed con- supernatants were stored at −80°C until assay. The sent. The patient then entered a 4-week run-in pla- interassay variability was 12%; range of assay 350– cebo period. At randomisation, fasting venous blood 560 pg/mL; results expressed as mg/108 platelets. was collected for platelet function studies, plasma Plasma ␤-thromboglobulin assay was performed ␤ -thromboglobulin and euglobulin lysis time, lipids, using the Amersham kit (Amersham, UK) and blood electrolytes, renal, and hepatic function. Supine (10 samples were collected into the tubes provided. Pla- min) and erect (5 min) BP was measured prior to telet retention was measured using heparinised the blood test with a mercury sphygmomanometer. whole blood collected in 10 mL syringes without Patients were then allocated to either isradipine or disturbance; retention was measured progressively atenolol. Starting doses were 1.25 mg BD isradipine in the first 5 mL emerging from a glass bead col- or 50 mg atenolol (plus one placebo tablet to main- umn.10 Haemoglobin, total and differential white tain the blind), and all patients agreed to take their cell count, platelet count and red cell indices were medications between 06.00 and 08.00 hours. measured on a Coulter S Plus Jr; blood films were Patients were reviewed at 2 and then 4 weeks. If the reviewed if indicated. Biochemical analyses were Ͼ DBP was 95 mm Hg, the drug dose was doubled. performed on a Technicon SMAC-2 (Bayer Diagnos- At weeks 4 and 12, all blood studies were repeated. tics, Australia). Serum total cholesterol, high density Following completion of 12 weeks of active treat- lipoprotein and triglycerides were performed using ment, all patients entered a final 4-week placebo a Roche Cobas-Bio (Roche Diagnostics, Basel, (washout) phase and the blood tests were repeated. Switzerland). All blood samples were collected in the laboratory at approximately the same time (am). This study was approved by the Hunter Area Research Ethics Com- mittee. Results Of 43 patients identified, 24 patients (15 male, 9 Laboratory methods female) were eligible for study entry. There were no significant differences in BP, age, weight or height All blood samples were collected from an antecubi- between the two groups at entry (Table 1). Two tal vein without stasis through a 19 gauge needle. patients failed to complete the 12-week active treat- Blood for platelet aggregation, platelet retention, ment period; one withdrew from the isradipine thromboxane generation, and euglobulin lysis time, group at week 3 because of headaches and lethargy was collected into tubes containing 0.1 volume of and one withdrew from the atenolol group at week 3.13% tri-sodium citrate dihydrate. Platelet-rich 8 due to lethargy and impotence. All other patients plasma (PRP) and platelet-poor plasma (PPP) were ° completed the study without significant adverse prepared by centrifugation at 22 C at 120 g for 10 events. min and 3000 g for 20 min, respectively. The PRP was divided into 4 –5 mL aliquots and stored under ° 5% CO2 in oxygen in sealed tubes at 22 C until tested. Platelet aggregation was performed at 37°C BP responses using a dual channel Chronolog aggregometer (Chronolog Corporation, Havertown, PA, USA) and Three patients achieved satisfactory BP control with was completed within 4 h of blood collection. PRP 2.5 mg of isradipine daily while eight required 5 mg was not corrected to a standard platelet count. Agon- daily. Five atenolol patients achieved control with ists tested were: adenosine-5′-diphosphate (ADP, 50 mg while six required 100 mg daily. BP falls with final concentration 1.25 and 2.5 ␮M) from Sigma drug treatment were similar in each treatment group Chemical Co (St Louis, MO, USA); collagen reagent (supine and erect) and returned towards pretreat- Horm (0.5 and 1.0 ␮g/mL) from Paton Scientific ment levels during the washout period. The pulse (Victor Harbour, South Australia); adrenalin (5.5 rate was significantly reduced with atenolol (Table and 110 ␮M) from Parke Davis Co (Carringbah, 1). Pro-haemorrhagic effects of Ca antagonists A Smith et al 785 Table 1 Effect of isradipine and atenolol on standing BP (mean ± s.e.m.)

End placebo Active treatment Placebo washout

4 wks 12 wks

Isradipine: Age 53 ± 3 years; weight 87 ± 5 kg; height 170 ± 3cm Systolic mm Hg 167 ± 2 153 ± 3* 153 ± 3* 160 ± 2 Diastolic mm Hg 102 ± 192±2* 91 ± 2* 101 ± 2 Pulse bpm 85 ± 487±282±382±2

Atenolol: Age 56 ± 4 years; weight 85 ± 5 kg; height 171 ± 3cm Systolic mm Hg 165 ± 2 153 ± 4* 156 ± 4* 167 ± 4 Diastolic mm Hg 101 ± 186±2* 91 ± 2* 100 ± 2 Pulse bpm 78 ± 471±5* 70 ± 3* 83 ± 3

*Signiﬁcantly different from control (and placebo) P Ͻ 0.05 (paired t-test).

Platelet function There were no significant differences during the study period or between the two treatment groups in the slopes of primary aggregation with ADP or adrenaline or the slopes of aggregation with PAF or collagen. The lag time for collagen-induced aggregation was similar in both treatment groups and did not alter over the treatment period. Similarly, no significant differences in agonist-induced thromboxane generation were observed over the study period or between treatment groups. Platelet retention did not alter significantly in either group throughout the study. Platelet numbers were also similar in both groups and did not change over the study period.

Plasma ␤-thromboglobulin (Table 2, Figure 1) In the isradipine patients, mean pre-randomised levels were almost identical to those measured at the end of washout. Values measured at 4 and 12 weeks of active treatment were lower and this difference was statistically signiﬁcant at 12 weeks. A similar but nonsigniﬁcant trend was observed in the atenolol group where, although baseline levels were somewhat higher (although still within the reference interval), they also tended to fall over the active treatment period. The mean reduction was 35% compared to a 39% reduction during isradipine treatment.

Euglobulin clot lysis time Results were consistently within the reference interval, apart from those patients who admitted to Figure 1 Effect of isradipine and atenolol on plasma ␤-thromboglobulin. P = placeo, R = treatment time (weeks).

Table 2 Effect of isradipine and atenolol on plasma ␤-thrombo- globin (ng/ml) strenuous exercise prior to blood collection. There was no difference between the two treatment groups End Active treatment Placebo or over the study period. placebo washout 4 wks 12 wks Other laboratory data Isradipine 69 ± 16 59 ± 14 42 ± 8* 65 ± 15 Atenolol 95 ± 22 81 ± 14 62 ± 864±15 No significant changes were observed in the standard haematological or biochemical parameters *Significantly different from control P Ͻ 0.05. Laboratory refer- including total and high density lipoprotein (HDL) ence interval 82 ± 24 ng/ml. cholesterol. The total to HDL cholesterol ratio was Pro-haemorrhagic effects of Ca antagonists A Smith et al 786 4.5 and 4.4 before and after isradipine treatment seen in patients treated with atenolol, although the respectively and 5.3 and 5.9 with atenolol. A small levels did not return to baseline after the 4-week nonsignificant increase in triglycerides was noted washout period. As other laboratories have also with atenolol. observed a reduction in baseline ␤-thromboglobulin levels in hypertensive patients receiving isradip- 5,14–16 16 Discussion ine, as well as in patients receiving atenolol, this could be interpreted as representing a reduction The aim of the study was to achieve an acceptable in the level of in vivo platelet activation. However, and similar control of BP in both the isradipine and in a study where plasma ␤-thromboglombulin was atenolol groups, so that any differences found measured as a marker of in vivo platelet activation,18 between the groups in platelet function, fibrinolytic untreated hypertensive patients had significantly activity or lipid profile could not be attributed to higher levels than normotensive controls. Angioten- differences in the level of BP control which was ach- sion-converting enzyme (ACE) inhibitors appeared ieved. The strict entry criteria excluded a number of to be more effective than calcium channel blockers hypertensive patients who would have been in reducing plasma levels in this study. It is there- included in a simple comparative study of efficacy. fore likely that the ␤-thromboglobulin changes noted Although patients were rigorously selected, and in our study and others reflect the fall in BP rather clearly instructed to avoid aspirin, platelet function than a direct drug effect. That these effects of isradi- tests had to be repeated on 10 occasions (11% of pine, other calcium channel blockers, and other visits) because patients had inadvertently taken antihypertensive drugs are related to BP reduction aspirin containing compounds, usually as over-the- is supported by evidence that hypertension and counter medications in which the presence of hypercholesterolaemia increase the sensitivity of aspirin was not clearly shown. platelets to activating agents.19 Also, platelet aggre- In the two groups of patients matched for initial gation during treatment with diverse calcium chan- BP elevation, BP was reduced to a similar and satis- nel blockers (nisoldipine, nifedipine, diltiazem, and factory extent in both groups. A mean dose of 4.3 verapamil) was related to their BP lowering ability mg isradipine in divided doses proved to have an and platelet function was normalised, not equivalent effect to a mean dose of 81.8 mg/day depressed.20 atenolol and there were no serious adverse effects. In vivo studies have evaluated the effects of other No significant haematological or biochemical abnor- calcium channel blockers on platelet function. How- mality was noted during the course of the study and ever, again, the changes reported are inconsistent plasma lipid parameters were not significantly alt- and trial designs often suboptimal. A comparison of ered in either group. nifedipine with cilazapril21 and propranolol and Blood was collected and platelet studies perfor- verapamil 22 failed to demonstrate any effect on plat- med under strictly controlled conditions and there elet function or blood rheology although both pro- was no evidence of a significant drug effect on aggre- pranolol and verapamil reduced plasma ␤-throm- gation or thromboxane generation with a range of boglobulin levels. However the same laboratory agonists. Previous reports of a platelet effect of israd- found that felodipine treatment for 12 weeks ipine were based on in vitro addition of relatively reduced ADP-induced platelet aggregation.23 In con- high drug concentrations to the PRP,6,7,11 rather than trast Wilson et al24 could not demonstrate any effect ex vivo studies of drug treated patients. Ding et al9 of nifedipine or felodipine on platelet responsive- reported inhibition of adrenaline-induced aggre- ness to collagen or ADP, despite a fall in platelet gation after 12 weeks of isradipine therapy (2.5–5 basal cytoplasmic calcium. Zucker et al25 have mg daily) but did not include a control group. Fet- reported that treatment with the non-dihydropyrid- kovska et al12,13 reported that isradipine reduces pla- ine diltiazem for 1 week reduced platelet aggre- telet aggregation induced by serotonin in combi- gation and nucleotide release induced by sub-thres- nation with low density lipoprotein (LDL), and hold concentrations of paired agonists (arachidonate while they used a 4-week run-in placebo period, the and adrenaline) in normal volunteers. However the study group was not controlled. Three other studies in vitro ability of ditiazem to potentiate the inhibi- that reported a reduction in ADP or serotonin- tory effect of aspirin on platelet aggregation8 with induced platelet aggregation5,14,15 also did not this drug could not be demonstrated in vivo. Amlo- include a control group, nor was there evidence that dipine and quinalapril did not appear to alter plate- concomitant and unintentional aspirin use was avo- let function.26 ided. Gleerup et al16 found that both isradipine and Dihydopyridines bind to the ␣1-subunit of the L- atenolol decreased platelet activity following ther- type voltage-activated calcium channel and in this apy. Fitscha et al5 also reported a reduction in serum way exert their influence on calcium-mediated

TXB2 levels, however, they did not examine agonist- events such as smooth muscle contraction and car- induced thromboxane generation. Interestingly, diac excitation. Recently, putative dihydropyridine- Klauser et al17 in a small, blinded, cross-over con- sensitive channels were described in platelets27 giv- trolled study, reported that isradipine had no effect ing a credible target for platelet interaction with cal- on collagen or ADP-induced platelet aggregation. cium channel blocking drugs. Many actions and While changes in baseline plasma ␤-thromboglobu- interactions of platelets are calcium-dependant. lin, a marker of platelet ␣-granule release, appeared This is not to say that they are calcium-channel- consistent and drug-related with isradipine in our dependant but rather that they depend on local con- study, a lesser fall over the study period was also centrations of calcium either around or within the Pro-haemorrhagic effects of Ca antagonists A Smith et al 787 platelet. Binding of primary agonists, thromboxane 2 Psaty BM et al. The risk of myocardial infarction asso- generation and the release reaction are all to some ciated with antihypertensive drug therapies. JAMA extent calcium-dependant and, therefore, putative 1995; 274: 620–625. processes where manipulation of the calcium 3 Pahor M et al. Do calcium channel blockers increase environment might produce changes in platelet the risk of cancer? Am J Hypertens 1996; 9: 695–699. function. While in vitro studies have suggested that 4 Pahor M et al. Risk of gastrointestinal haemorrhage with calcium antagonists in hypertensive persons over platelet aggregation may be reduced by calcium 67 years old. Lancet 1996; 347: 1061–1065. channel blocking drugs, the concentrations of drug 5 Fitscha P et al. Effects of isradipine on platelet func- used have often been greatly in excess of those achi- tion in hypertension at rest and during exercise. Am J evable in plasma in normal clinical practice and in Hypertens 1991; 4 (Suppl): 1785–1805. vitro studies may be a poor guide to the clinical 6 Addonizio VP et al. Effects of verapamil and diltiazem effects of these drugs. on human platelet function. Am J Physiol 1986; 250: The euglobulin clot lysis time is a global indicator H366–371. of the level of baseline fibrinolytic activity and as 7 Anfossi G et al. Calcium-channel blocking agents vera- such is of relevance to a study of patients at risk of pamil and diltiazem are inhibitors of vasopressin- arterial disease and of gastrointestinal bleeding. Our induced human platelet activation. Clin Exp Pharma- results remained within the reference interval with col Physiol 1991; 18: 767–773. placebo and both forms of drug therapy, suggesting 8 Altman R, Scazziota A, Dujovne C. Diltiazem that these agents do not have a significant effect on potentiates the inhibitory effect of aspirin on platelet aggregation. Clin Pharmacol Therapeut 1988; 44: basal fibrinolytic activity. 320–325. In conclusion, our results provide little support 9 Ding Y-A et al. Effect of isradipine on platelet aggre- for the proposition that platelet function will be sig- gation and oxygen free radicals in hypertension. J Hyp- nificantly altered in vivo when isradipine is used as ertens 1991; 9: S370–371. an antihypertensive agent. Our patients achieved an 10 McPherson J, Zucher MB. Platelet retention in glass adequate fall in BP, and therefore experienced bead columns: adhesion to glass and subsequent plate- plasma concentrations of isradipine which are likely let-platelet interactions. Blood 1976; 47: 55–67. to occur in normal clinical practice. While the sig- 11 Sinzinger H, Weiss K, Fitscha P. Isradipine is a potent nificant decrease in plasma ␤-thromboglobulin calcium antagonist on platelet function and the prosta- appeared to relate particularly to the phase of active glandin system. Thromb Haemost 1990; 62: 551. treatment with isradipine, it probably reflects BP 12 Fetkovska N et al. Serotonin and platelet activation reduction rather than a direct drug effect on platelet during treatment with isradipine. J Cardiovasc Pharm- function. Particularly since the decrease with ateno- acol 1991; 18: S31–33. 13 Fetkovska N et al. Platelet activating effect of low-den- lol treatment was 34% compared with a 39% fall sity lipoprotein and its reversal by isradipine. Am J following 12 weeks of isradipine. Other in vivo stud- Hypertens 1991; 4: 175S–177S. ies using isradipine and other calcium channel 14 Tison P et al. Effects of dihydropyridines and their blockers have produced contradictory results and combination with aspirin on blood pressure and cir- provide little evidence that these drugs significantly cadian platelet activity in patients with essential alter platelet function. However aspirin anti-platelet hypertension. Am J Hypertens 1994; 7: 46S–49S. therapy is consistently associated with improved 15 Sinzinger H et al. Isradipine improves platelet func- cardiovascular morbidity and mortality in large tion in hypertensives. Eur J Clin Pharmacol 1992; 42: studies, and even in low concentrations produces 43–46. major changes in platelet function. From our data 16 Gleerup G, Mehlsen J, Winther K. Does calcium chan- it would not be expected that isradipine (or other nel blockade and beta-adrenergic blockade affect plate- calcium antagonists) in therapeutic doses could pro- let function and fibrinolysis to a varying degree? J Car- diovasc-Pharmacol 1995; 25: 87–89. duce in vivo effects of a magnitude even approach- 17 Klauser R et al. Platelet aggregation and metabolic con- ing those of low dose aspirin. It is therefore surpris- trol are not affected by calcium antagonist treatment ing that calcium antagonists were more likely to be in type II diabetes mellitus. J Cardiovasc Pharmacol associated with gastrointestinal bleeding than 1990; 15: S93–96. aspirin in the recently published non-randomised, 18 Islim IF, Beevers DG, Bareford D. The effect of antihy- retrospective, observational analysis.4 However their pertensive drugs on in vivo platelet activity in essen- vasodilator effect or some other currently unrecog- tial hypertension. J Hypertens 1992; 10: 379–383. nised effect may account for these unexpected find- 19 Mazeavd MM et al. Hypercholesterolaemia modulates ings. the effects of nitrendipine on blood pressure and platelet function in essential hypertension. J Cardiovasc Pharmacol 1991; 18 (Suppl 10): S32–35. Acknowledgement 20 Belousov Y, Kushnaryov V. The effects of nisoldipine, diltiazem, nifedipine, and verapamil on haemody- This study was carried out with the collaboration namic parameters and red blood cells – platelet inter- of Drs Dennis Harvey and Ian Ratcliffe, Newcastle actions in patients with arterial hypertension. J Car- General Practitioners. diovasc Pharmacol 1991; 18 (Suppl 9): S32–35. 21 Ding YA, Law HW, Chou TL. Comparison of cilazapril References and nifedipine retard on ambulatory blood pressure, metabolic, rheological and platelet function in hyper- 1 Furberg CD, Psaty BM and Meyer JV. Nifedipine: dose tensive patients. J Hum Hypertens 1994; 8: 137–143. related increase in mortality in patients with coronary 22 Ding YA, Chou TC, Lin KC. Effects of long acting pro- heart disease. Circulation 1995; 92: 1326–1331. pranolol and verapamil on blood pressure, platelet Pro-haemorrhagic effects of Ca antagonists A Smith et al 788 function metabolic and rheological properties in aspirin on platelet aggregation and ATP release hypertension. J Hum Hypertens 1994; 8: 273–278. induced by paired agonists. Thromb Haemost 1993; 23 Chou TZ, Lee KW, Ding YA. Effect of felodipine-ER 70: 332–335. on blood pressure, platelet function, and rheological 26 Ding YA, Chang SM, Chou TC. Comparison of amlodi- properties in hypertension. Can J Cardiol 1993; 9: pine and quinalapril on ambulatory blood pressure 423–427. and platelet function in hypertension. J Hum Hypert- 24 Wilson J et al. Treatment of hypertension induces a ens 1995; 9: 637–641. fall in platelet cytoplasmic calcium concentration 27 Pales J et al. Effects of dihydropyridines and inorganic without influencing platelet aggregation. Thromb calcium blockers on aggregation and on intracellular Haemost 1992; 68: 683–686. free calcium in platelets. Biochem Biophys Acta 1991; 25 Zucker ML et al. Effect of diltiazem and low dose 1064: 169–174.