Differential lipogenic effects of cilostazol and pentoxifylline in patients with intermittent claudication: potential role for interleukin-6

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1 Atherosclerosis 158 (2001) Differential lipogenic effects of cilostazol and pentoxifylline in patients with intermittent claudication: potential role for interleukin-6 Tsung-Ming Lee a, Sheng-Fang Su b, Juey-Jen Hwang a, Chuen-Den Tseng a, Ming-Fong Chen a, Yuan-Teh Lee a, Shoei-Shen Wang c, * a Department of Internal Medicine, Cardiology Section, National Taiwan Uni ersity Hospital, 7, Chung-Shan S. RD., Taipei, 10002, Taiwan b Department of Clinical Pharmacy, College of Medicine, National Cheng Kung Uni ersity, Tainan, Taiwan c Department of Surgery, Cardiology Section, National Taiwan Uni ersity Hospital, Taipei, Taiwan Received 15 September 2000; received in revised form 7 December 2000; accepted 24 January 2001 Abstract Cilostazol, a novel oral phosphodiesterase inhibitor, has shown consistent improvement in exercise tolerance in patients with intermittent claudication (IC). In addition to this effect, cilostazol has previously been shown to have beneficial effects on the dyslipidemia, i.e., combination of high triglycerides with low high-density-lipoprotein cholesterol (HDL-C) levels. Interluekin-6 (IL-6) suppresses the activity of lipoprotein lipase, which modulates the metabolism of triglycerides and HDL-C. To determine whether a reduction of IL-6 contributes to the improvement of lipid profiles, we prospectively investigated the effect of cilostazol (n=16, 100 mg, twice daily) on the changes of lipid profiles and on the association with the changes of IL-6 compared with those of pentoxifylline (n=16, 400 mg, bid) in patients with IC. After eight weeks of administration of cilostazol to patients with IC, walking distances were increased, associated with a 29% decrease in plasma triglycerides and a 13% increase in HDL-C. No significant changes of lipid profiles in the pentoxifylline and placebo groups were observed although a similar improvement in walking distances was achieved in the pentoxifylline group. IL-6 levels were significantly reduced in patients receiving cilostazol as compared with those receiving placebo or pentoxifylline. The cilostazol-induced changes in the IL-6 were positively related to those of triglycerides in the cilostazol group (r=0.63, P 0.05) and negatively related to those of HDL-C (r= 0.55, P 0.05). These findings suggest that in addition to consistent improvement of exercise tolerance, cilostazol may improve lipid profiles by reducing IL-6 release. However, pentoxifylline did not affect lipid profiles although a similar improvement of maximal walking distance (MWD) was achieved Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cilostazol; Interleukin-6; Intermittent claudication; Lipid; Pentoxifylline; Peripheral vascular disease; Phosphodiesterase inhibitor 1. Introduction The prevalence of peripheral arterial occlusive disease (PAOD) of the lower extremities increases with age. Hemodynamically significant PAOD manifests as intermittent claudication (IC) in 1% 7% of men years old [1]. Numerous drugs have been studied for the treatment of IC, including prostaglandins, vasodilating There are neither financial nor other relations that could lead to a conflict of interest. * Corresponding author. Tel.: /5074; fax: address: sswang@ha.mc.ntu.edu.tw (S.-S. Wang). agents, anticoagulants, and antiplatelets [2]. Cilostazol is a novel phosphodiesterase (PDE) inhibitor that has been used for treatment of PAOD. In addition to its antiplatelet and vasodilator properties, cilostazol may also favorably modulate plasma lipoproteins by increasing HDL cholesterol and decreasing triglycerides [3]. Patients with IC are at a high risk of disability from atherosclerotic vascular diseases. Therefore, patients may benefit from lipoprotein modification to alter the clinical course of concomitant atherosclerotic vascular disease. However, the exact mechanisms involved in the ability of cilostazol to improve lipid profiles remain unknown /01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S (01)

2 472 T.-M. Lee et al. / Atherosclerosis 158 (2001) The IL-6 gene was overexpressed in hypoperfused skeletal muscle of patients with PAOD [4]. IL-6 is considered to be one of the proinflammatory cytokines [5], produced by various cell types including macrophages [6], lymphocytes [7], and endothelial cells [8]. IL-6 inhibits lipoprotein lipase and stimulates hepatic trigylceride secretion [9]. IL-6 gene polymorphism has been hypothesized to be responsible for the lipid abnormalities [10]. Epidemiological studies have shown that higher IL-6 levels were associated with raised plasma triglyceride concentrations and low HDLcholesterol [11]. However, the role of IL-6 in the lipoprotein is not understood in patients treated with cilostazol. Cilostazol and pentoxifylline are the only two drugs approved by the US Food and Drug Administration for pharmacological treatment of IC. Thus, we tested whether the improvement of lipid profiles after cilostazol is mediated by reduced levels of IL-6 release compared with that with pentoxifylline. 2. Patients and methods 2.1. Patient selection Eligible patients were more than 40 years of age and had had IC with no symptomatic changes in the previous 3 months. The presence of PAOD was defined by a Doppler-measured ankle-brachial index (ABI) 0.9. Eligible patients had baseline maximal walking distances (MWD) between 30 and 200 m. Patients with Burger s disease, category II and III chronic lower extremity ischemia [12], or arterial surgery/angioplasty or sympathectomy within the previous 3 months were excluded. Patients were instructed to avoid lipid-lowering drugs, aspirin-containing products, non-steroid anti-inflammatory agents, anticoagulant, and ticlopidine. The patients were not given specific risk factor modification instructions such as diet control, exercise, or smoking cessation. All medications were kept constant throughout the study Study design The study was designed to be a prospective and randomized trial conducted in a single medical center. After a 2-week placebo run-in phase, eligible patients were randomly assigned to receive cilostazol (n=16, 100 mg, bid), pentoxifylline (n=16, 400 mg, bid), or placebo (n=16) for 8 weeks in a double-blind manner. The dose of pentoxifylline was chosen from prior studies which have shown that pentoxifylline was effective in improving IC at a dose of 800 mg/day [13]. The patient received a randomized code number, according to which the sponsor supplied the study drug. Special drug packaging was used to maintain blindness of the treatment code. A sealed envelope, with information on the treatment allocated, was kept in the clinical file of each patient. In an emergency, the seal could be broken and the treatment code disclosed, at the discretion of the case physician. For comparison, ten normal control subjects were selected and matched according to age, sex, height, and body weight. All subjects were free of chest pain, had no history of IC, and had normal rest blood pressure, and results of cardiac physical examination and a resting electrocardiogram were normal. The study was conducted in accordance with good clinical practices and local regulations. The protocol was approved by an ethical committee, and patients were required to sign an informed consent form before undergoing screening procedures Study procedures Clinical examination, vital signs, and blood tests were recorded at D0 and at the end of the study. Treadmill exercise tests were performed at the two screening visits, and at the end of the study. Treadmill exercise tests were performed at 3.2 km/h with a 12.5% gradient by the same operator and at the same time of the day for a given patient throughout the trial. The distance walked when the patient could not proceed was defined as MWD. Randomization and initiation of study drug treatment were allowed only if treadmill testing at the two screening tests demonstrated a variance of 20% in MWD. The change in MWD was the main outcome Lipid measurements Blood samples were taken after an 8-h overnight fasting at the start of the experiment and after 8-week treatment. Plasma concentrations of total, high-density lipoprotein (HDL) cholesterol and triglycerides were measured by enzymatic methods as previously described [14,15]. Low-density lipoprotein (LDL) cholesterol was calculated by the Friedewald equation [16] Determination of plasma IL-6 le els To avoid diurnal variation in IL-6, venous blood was taken from all subjects in the morning after an overnight fast. The heparinized plasma was stored at 70 C until assayed. Plasma IL-6 levels in each sample were measured in duplicate by a human IL-6 ELISA high sensitivity kit (QuantiGlo human IL-6, R&D System, Minneapolis, Minnesota, USA) according to the manufacture s instruction. The interassay variability was 5%. The detection limit was 0.2 pg/ml. IL-6 ELISA gave a linear response in the range pg/ml (r=0.998).

3 T.-M. Lee et al. / Atherosclerosis 158 (2001) Statistics The analysis was performed on the set of patients who completed the trial and had a valid treadmill test and blood tests at baseline and at the end of the study. All statistical analyses were performed by using SAS (SAS Institute, version 6.12; Cary, N.C.). The continuous variables are expressed as mean SD. Because many variables were not normally distributed, nonparameteric statistical tests were used throughout. The differences of continuous parameters among the three groups were compared by the Kruskal Wallis test. For the categorical parameters, the differences were compared by the chi-square test and Fisher exact test if case number 5. Linear regression models were used to compute the association between the changes of lipid profiles and IL-6 in the cilostazol or pentoxifylline groups. A P value 0.05 was considered statistically significant. 3. Results Cilostazol was very well tolerated by all patients and none had any significant subjective side effects. Table 1 shows the clinical and biochemical characteristics of the three groups studied. There were no differences in baseline characteristics among these three groups. Cilostazol- or pentoxifylline-treated patients walked significantly farther distances than did placebo-treated patients (Table 2). The mean change from baseline after 8 weeks was 33, 32, and 5 m for the patients who received cilostazol, pentoxifylline, or placebo, respectively. The levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides at baseline and after 8 weeks are listed in Table 2. Over the 8 weeks of treatment, total triglycerides remained stable in the placebo group and in the pentoxifylline group, whereas it decreased by 29% in the cilostazol group, a significant difference (P=0.04). HDL-C levels were significantly increased from 42 8 to47 10 mg/dl (P=0.004) in response to the administration of cilostazol. No significant changes in the serum levels of total cholesterol and LDL cholesterol were observed in the three groups. IL-6 levels were significantly increased in patients with IC ( pg/ml) compared with normal controls ( pg/ml, P ). IL-6 levels were significantly reduced in patients receiving cilostazol as compared with those receiving placebo or pentoxifylline (Table 2). The cilostazol-induced changes in the IL-6 were positively related to those of triglycerides (r=0.63, P 0.05, Fig. 1) and negatively related to those of HDL-C (r= 0.55, P 0.05, Fig. 1). Patients treated with either pentoxifylline or placebo showed no significant changes of IL Discussion In the present trial, 8 weeks of treatment with cilostazol in patients with IC led to increased walking distances, associated with a 29% plasma triglyceride decrease and a 13% increase in HDL-C. The effects of cilostazol on reducing IL-6 production have not been documented previously. Serum IL-6 levels correlated positively with fasting triglycerides and negatively with Table 1 Demographic data of patients a Parameter Cilostazol (n=16) Pentoxifylline (n=16) Placebo (n=16) Age (year) Sex (male/female) 13/3 13/3 14/2 Weight (kg) Duration of IC (years) Hypertension (%) 14 (88) 11 (69) 11 (69) Diabetes mellitus (%) 9 (56) 9 (56) 9 (56) Smokers (%) Never 4 (25) 4 (25) 4 (25) Previous 3 (19) 2 (13) 4 (25) Current 9 (56) 10 (63) 8 (50) Antihypertensive agents ACE b inhibitor, n (%) 7 (44) 8 (50) 6 (38) AT 1c receptor blocker, n (%) 5 (31) 7 (44) 9 (56) Calcium blocker, n (%) 11 (69) 12 (75) 10 (63) Diuretics, n (%) 7 (44) 8 (50) 7 (44) Vasodilators, n (%) 8 (50) 9 (56) 9 (56) a Data is presented as mean SD or number (%). b ACE: angiotensin-converting enzyme. c AT 1 : angiotensin II.

4 474 T.-M. Lee et al. / Atherosclerosis 158 (2001) Table 2 Effects of different treatments on exercise capacity, IL-6, and lipid profiles a Cilostazol (n=16) Pentoxifylline (n=16) Placebo (n=16) Parameters Baseline 8 Week Baseline 8 Week Baseline 8 Week MWD (m) * * ** IL-6 (pg/ml) * ** ** TC b (mg/dl) HDL-C (mg/dl) * LDL-C (mg/dl) TG c (mg/dl) * a Data presented as mean SD. b TC, total cholesterol. c TG, total triglycerides. * P 0.05 compared with respective baseline. ** P 0.05 compared with 8 week from the cilostazol group. HDL-cholesterol. These results indicate that cilostazol modulates the lipid profiles through an IL-6-dependent mechanism and circulating IL-6 changes may be more than just markers of atherosclerosis, but actual participants in the modulation of lipid profiles. In contrast, although the similar degree of MWD improvement in the pentoxifylline group was noted, no favorable lipid modulation was associated. The mechanisms responsible for reduction of IL-6 protein expression in patients treated with cilostazol remain unknown. IL-6 is produced by a great variety of normal cell types. Analysis of IL-6 promoters revealed the existence of several potential binding sites for transcription factors camp response element-binding protein, activator protein-1, nuclear factor for IL-6, and nuclear factor-kappab [17]. Consistent with the presence of these elements, IL-6 expression is regulated by camp. Cilostazol is a PDE III inhibitor which upregulates intracellular camp. Zitnik et al. [18] have demonstrated that increased intracellular camp inhibits IL-1-induced IL-6 production in human lung fibroblasts. In contrast, Zhang et al. [19] have shown that an increase in intracellular camp triggered the synthesis of IL-6 in human fibroblasts. The discrepancy could be explained by the fact that in in vivo systems and in different cell lines, different mediators could be involved which are responsible for the upregulatory effect of IL-6 release. It has been suggested that pentoxifylline may modulate IL-6 production in whole blood at serum concentrations of 10 3 M [20]. However, serum levels of pentoxifylline including active metabolites do not exceed 10 5 Mto10 4 M in a clinical dose [21]. Thus, the beneficial effects of pentoxifylline on IL-6 are difficult to keep effective with ordinary therapeutic doses. Besides, we cannot exclude the possibility that other actions of cilostazol may be involved via non-il-6-dependent mechanisms such as inhibition of lipoprotein lipase activity [22], that may also modulate lipid profile. Lipoprotein lipase plays an important role in lipid profiles by hydrolyzing circulating triglycerides to fatty acids. Previous studies have shown that IL-6 reduced heparin-releasable lipoprotein lipase activity in adipocytes in a dose-dependent manner [9]. Thus, it is possible that the effects of IL-6 on lipid profiles observed in this study might be mediated by the decreased reduction of lipoprotein lipase. Whether alone or in conjunction with lipoprotein lipase, the data clearly suggest a role for IL-6 in the improvement of lipid profiles in patients treated with cilostazol. Patients with IC had significantly higher circulating IL-6 levels both at baseline and after 8 weeks treatment of cilostazol compared with those in the normal control group. This finding is in contrast with that of Fiotti et al. [23], who have demonstrated similar IL-6 levels in patients with PAOD and normal controls. The discrepancy could be, at least in part, due to the use of non-steroidal anti-inflammatory drugs (aspirin) in patients with PAOD in the latter study. The use of aspirin is a possible confounder, because the non-steroidal anti-inflammatory drug inhibits IL-6 synthesis by preventing degradation of IkappaB-alpha [24] Clinical implications The cilostazol-induced decrease in IL-6 in patients with IC is an important finding. Inflammatory cells during pathogenesis of atherosclerosis release a wide range of cytokines, including IL-1 and TNF-. However, the role of IL-6 in the process is primary. Evidence comes from gene knockout animal models, where only IL-6 knockout animals show an impaired acute phase response, while IL-1 and TNF- knockout animals did not show impairment [25,26]. IL-6 might play a crucial role in the development of vascular atheroscle-

5 T.-M. Lee et al. / Atherosclerosis 158 (2001) Fig. 1. Correlation of the changes between interleukin (IL)-6 and total cholesterol (A), high-density lipoprotein (HDL) cholesterol (B), low-density lipoprotein (LDL) cholesterol (C), and total triglycerides (D) in the cilostazol-treated patients presenting with intermittent claudication. rosis through a number of different mechanisms (metabolic, endothelial, and anticoagulant). Increased levels of IL-6 indicate not only current disease but also the development of adverse events in the future. Higher circulating levels of IL-6 have been associated with an increased risk of future myocardial infarction [27] and mortality [28] in apparently healthy men Study limitation The main limitation of this study is the small number of patients studied, so caution is recommended until other studies confirm the results. Secondly, some drugs have influences on transcriptional factors, which will affect IL-6 expression. For example, calcium channel blockers upregulate the IL-6 gene in human vascular muscle cells [29]. However, it seems unlikely that the effect of calcium channel blockers on IL-6 will produce different results because medications were kept constant throughout the study. Finally, in this study the diagnosis was based on clinical history and ABI 0.9. Peripheral angiography was not routinely performed for ethical reasons. When compared with angiographic studies of the lower extremities, an ABI of 0.9 has been demonstrated to have a sensitivity and specificity of 96% for occlusive lesions [30]. In conclusion, this novel PDE inhibitor, cilostazol, with favorable modulation of lipid profiles represents a promising adjuvant in the management of the overall burden of PAOD, of which atherosclerotic cardiovascular disease is the major component. Cilostazol modulates the lipid profiles through an IL-6-dependent mechanism. In contrast, although patients administered with pentoxifylline had a similar improvement in walking distances, pentoxifylline did not improve the lipid profiles. Larger prospective studies are needed to determine whether the decrease in cilostazol-induced IL-6 can be used as a predictive and prognostic marker in patients with PAOD. References [1] Fowkes FGR. Epidemiology of atherosclerotic disease in the lower limbs. Eur J Vasc Surg 1988;2: [2] Radaack K, Wyderski RJ. Conservative management of intermittent claudication. Ann Intern Med 1990;113: [3] Elam M.B., Heckman J., Crouse J.R., Hunningghake D.B., Herd J.A., Davidson M., Gordon I.L., Bortey E.B., Forbes W.P., for the Cilostazol Lipid Investigators Study Group. Effect of the novel antiplatelet agent cilostazol on plasma lipoproteins in patients with intermittent claudication. Arterioscler Throm Vasc Biol 1998;18:

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