- Email: [email protected]
A randomized, double-blind, placebo-controlled study of the efficacy and tolerability of policosanol in adolescents with type II hypercholesterolemia
~l
| I~l t{ , l ' l I ',i,
Ri 'sl .',~{
VOLUME 63, NO. 4, APRIL 2002
A Randomized, Double-Blind, Placebo-Controlled Study of the Efficacy and Tolerability of Policosanol in Adolescents with Type II Hypercholesterolemia Gladys Casta~o, MD, PhD, 1 Rosa Mils, PhD, 2 Lilia Fernfindez, PhD, 2 Jos~ IIInait, MD, PhD, 2 Eric Hernfindez, MD, 1 Julio C. Fernfindez, 2 Rafael Gfimez, 2 Carlos Guti6rrez, MD, PhD, 2 and Estrella Alvarez, PhD 2
I Medical Surgical Research Center, and 2Center for Natural Products, National Center for Scientific Research, Havana City, Cuba
ABSTRACT
Background: Atherosclerosis begins in childhood and is influenced by risk factors for coronary heart disease (CHD), of which hypercholesterolemia is crucial. The rationale for treating hypercholesterolemia in childhood is to limit atherosclerosis development, for which adherence to a cholesterol-lowering diet is the first-choice therapy. Nevertheless, pharmacological intervention with bile acid-binding resins may be prescribed for patients older than 10 years, mainly those with family history of CHD, multiple risk factors, and/or severe hypercholesterolemia. Resins are effective and tolerable in this population, but their clinical use has been limited because of poor compliance due to unpalatability; other effective cholesterol-lowering drugs have not been recommended in this population because of the potential impact of drug-related adverse effects such as increases in transaminases, myopathies, and gastrointestinal disturbances. Thus, the need for safer, easy-to-take, and effective cholesterollowering agents for this population continues. Policosanol is a mixture of higher primary aliphatic alcohols purified from sugar cane wax with cholesterollowering effects proven in patients with type II hypercholesterolemia and dyslipidemia due to type 2 diabetes mellitus. Policosanol shows good safety and tolerability profiles, with no evidence of drug-related adverse events (AEs) to date. This background supports the idea that policosanol could be a good candidate for treating hypercholesterolemia in children and adolescents, but it requires clinical demonstration. Objective: This 12-week study was undertaken to investigate the cholesterollowering effects and tolerability of policosanol in hypercholesterolemic patients aged 11 to 19 years. Accepted for publication February 12, 2002. Printed in the USA. Reproduction in w h o l e or part is n o t p e r m i t t e d .
286
0011-393X/02/$19.00
G. Casta~o et aL
Methods: In this randomized, double-blind, placebo-controlled study, after 4 weeks of dietary stabilization, adolescents with type II hypercholesterolemia were randomly assigned (1:1 ratio) to receive placebo or policosanol 5-mg tablets once daily for 12 weeks. Physical examinations were performed, and lipid profiles and blood samples were obtained at baseline and after 6 and 12 weeks of therapy. The treatment was considered effective if mean reductions of low-density lipoprotein cholesterol (LDL-C) were >15%. In addition, the percentages of patients reaching final values of LDL-C <3.4 mmol/L and optimal values of <2.8 mmol/L were also evaluated. The doses were doubled if LDL-C values were ->3.4 mmol/L after 6 weeks of therapy. The incidence of AEs and compliance with study medications were also evaluated after 6 and 12 weeks of treatment. Results: Fifty-five patients were enrolled in the study (28 policosanol, 27 placebo). Twenty-three patients (17 placebo, 6 policosanol) required dose titration at 6 weeks. After 12 weeks of therapy, policosanol significantly decreased LDL-C with respect to baseline and placebo (both P < 0.001), showing a mean reduction of 32.6%. Total cholesterol (TC) and TC/high-density lipoprotein cholesterol (I-IDL-C) and LDL-C/HDL-C ratios were reduced by 21.9%, 27.8%, and 37.2%, respectively, in the policosanol group (P < 0.001, compared with baseline and placebo). HDL-C rose 10.1% (P < 0.001), compared with baseline and placebo. Triglycerides were unaffected by policosanol. LDL-C, TC, and both atherogenic ratios were reduced significantly in the policosanol group (P < 0.001), and significant increases in HDL-C values were observed at the 6-week interim checkup (P < 0.001 vs baseline, P < 0.01 vs placebo). Twenty-five (89.3%) of 28 patients in the policosanol group showed LDL-C reductions >15% compared with 2 (7.4%) of 27 patients in the placebo group (P < 0.001). In addition, 26 (92.8%) of 28 policosanol patients reached LDL-C values <3.4 mmol/L compared with 4 (14.8%) of 27 patients in the placebo group (P < 0.001). Likewise, the response rate for achievement of optimal values (LDL-C <2.8 mmol/L) was also larger in the policosanol group (20/28; 71.4%) than in the placebo group (0/27; 0.0%) (P < 0.001). Policosanol was well tolerated, with no drug-related effects found on physical examination. Blood biochemistry determinations revealed significantly lower alanine aminotransferase levels in the policosanol group after 6 weeks of therapy compared with placebo (P < 0.05), as well as significant reductions in aspartate aminotransferase levels at 6 weeks (P < 0.01) and 12 weeks (P < 0.05) compared with baseline. No patients withdrew from the study, and only 3 patients (2 placebo, 1 policosanol) experienced mild AEs during the study; the placebo patients reported abdominal pain and constipation (1 each), and the policosanol patient reported polyphagia. Conclusions: Policosanol 5 mg/d appears to be well tolerated and effective as short-term treatment of hypercholesterolemia in adolescents. Keg words: policosanol, cholesterol-lowering drugs, lipid-lowering therapy, hypercholesterolemia, adolescents. (Curr Ther Res Clin Exp. 2002;63:286-
3O3) 287
CURRENT THERAPEUTIC RESEARCH®
INTRODUCTION Coronary heart disease (CHD) is the leading cause of morbidity and mortality in middle-aged and elderly people.1 Expert guidelines have been established for the prevention of CHD,2-5 which has been recognized as a disease of multiple causes, arising from the concomitant effects of several coronary risk factors. 2-7 The direct relationship between CHD and elevated serum levels of lowdensity lipoprotein cholesterol (LDL-C) and total cholesterol (TC) has been demonstrated. ~-1° In addition, landmark clinical studies have demonstrated, beyond any doubt, the benefits of lowering LDL-C levels in both primary and secondary prevention of CHD.11-15 Studies of the early stages of atherosclerosis in children and young adults have shown the occurrence of arterial deposits of fats and early plaques at young ages. The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study 16 was undertaken to assess whether atherosclerosis begins in childhood and how recognized atherosclerotic risk factors influence this process. The results support the occurrence of early atherosclerotic lesions; the extent of fatty and fibrous arterial lesions increased with age, and the major risk factor associated with the arterial lesions was the postmortem concentration of LDL-C.16 Of the 3 principal coronary risk factorsmhypercholesterolemia, hypertension, and smoking7--hypercholesterolemia has been documented most extensively. Previous studies revealed the occurrence of lipid-profile abnormalities in children. 17-19 Hypercholesterolemia in childhood and adolescence may increase the risk of CHD later in life. 17A9 Because atherosclerosis is a continuous process that starts in childhood, guidelines for screening and treating young hypercholesterolemic people aged 2 to 19 years have been developed. 2° Indeed, the best primary prevention must start in childhood and adolescence; screening recommendations must address young patients with parents at high atherosclerotic risk. Early guidelines were criticized by pediatricians who believed that the putative risks and benefits of a long-term, low-fat diet had not been properly evaluated; as a result, these guidelines recommend more modest or gradual restriction of total dietary fat in pediatric patients. 21 The major concern has been whether a low-fat, low-cholesterol diet will support the special needs of children and adolescents with respect to growth, energy, and development. Nevertheless, recent studies demonstrate that normal growth was seen in children regardless of whether or not they received a low- or normal-fat diet. 22'23 F u r t h e r e v i d e n c e s u p p o r t s t h a t an American Heart Association Step I cholesterol-lowering diet can be safely recommended for patients aged 2 to 19 years. 24,25 Pharmacotherapy has been considered in pediatric patients ---10 years of age when LDL-C levels remain high despite lifestyle intervention, mainly when family history of CHD and/or multiple risk factors are present. To date, the bile-acid
288
G. Casta~o et al.
sequestrant resins are the only approved drugs for pharmacologic intervention of hypercholesterolemia in children and adolescents, with a US National Cholesterol Education Program recommended daily dose of 16 g.26 Taking into account the lack of a linear dose response, lower doses of colestipol (10 g/d) and cholestyramine (8 g/d) have been used to treat hypercholesterolemia in childhood and adolescence. The most serious limitation for the clinical use of resins in any age group is low compliance because of unpalatability. In addition, resins must be avoided in children and adolescents with elevated triglyceride levels because these drugs tend to increase triglycerides in these cases, 27 as occurs normally when administered to patients with combined dyslipidemia or dyslipidemia associated with type 2 diabetes. 28 Nicotinic acid has been used in a few young adults, but its administration requires careful medical follow-up.26 Combined therapy with nicotinic acid and resins may be indicated in cases of severe and refractory hypercholesterolemia, especially when other atherosclerotic risk factors are present. 26 Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) are the most effective of the cholesterol-lowering drugs that have been used extensively in the past decade28; some data suggest that they can improve lipid profiles in children and adolescents. 26 The statins can be useful in patients who have failed to respond to resins and/or nicotinic acid, especially when evidence of high atherosclerotic risk is present, but the use of statins to treat hypercholesterolemia in children and adolescents has not yet been approved. Several drug interactions 29 and statin-related adverse events (AEs), such as myopathy and elevations of serum levels of liver transaminases and creatine kinase, 3° are potentially dangerous for children and adolescents. Taking this background into account, the search for effective, easy-to-take, and safer cholesterollowering agents to manage hypercholesterolemia in children and adolescents is supported. Policosanol is a mixture of higher aliphatic alcohols isolated and purified from sugar cane (Saccharum officinarum L) wax 31 with cholesterol-lowering effects proven in patients with type II hypercholesterolemia32--47 and in patients with dyslipidemia due to type 2 diabetes mellitus. 48-5° Policosanol reduces cholesterol levels by inhibiting cholesterol biosynthesis at a step between acetate consumption and mevalonate production, although the inhibition of HMGCoA reductase as a possible mechanism of such an effect has been disproved. 51-54 Policosanol also increases LDL receptor-dependent processing in fibroblast cultures 51 as well as the clearance of ~2Sl-labeled LDL-C administered to rabbits with hypercholesterolemia induced by a cholesterol-free, casein-rich diet. 53 On the other hand, extensive experimental toxicologic evaluation of policosanol, including long-term and carcinogenicity studies, have not demonstrated any drug-related toxicity,s5-6~ Short- and long-term clinical studies have shown that policosanol is well tolerated, and no drug-related AEs have been demonstrated. 32-~°
289
CURRENT THERAPEUTIC RESEARCH®
Thus, studies conducted in populations more susceptible to drug-related AEs due to their impaired metabolism and/or frequent use of concomitant medications, such as patients at high coronary risk, 37-4° patients with hepatic disturbances, 41 and elderly people, 4247 have also failed to demonstrate any policosanol-related AEs. In addition, long-term (4- to 5-year) pharmacologic postmarketing surveillance studies, including more than 30,000 policosanoltreated patients, 62'63 have corroborated that the potential risk of policosanol treatment is negligible. Taking this background into account, policosanol could be a good candidate for treating hypercholesterolemia in children and adolescents. Thus, the objective of the present study was to investigate the cholesterol-lowering effects and tolerability of policosanol 5 to 10 mg administered once daily for 12 weeks to hypercholesterolemic patients aged 11 to 19 years. PATIENTS AND METHODS This study was conducted at the Medical Surgical Research Center (Havana City, Cuba), and the protocol was approved by the Ethics Committee of this institution and by the State Center for Quality Drug Control. Patients 11 to 19 years of age were enrolled in the study after providing informed personal assent and familial written consent (visit 1). A complete clinical history, physical examination, and sampling for lipid-profile determinations were performed at this time. Patients with LDL-C levels ->3.4 mmol/L were encouraged to follow a healthy lifestyle, including adherence to a standard cholesterol-lowering diet, for 4 weeks. Mter this period, a serum lipid profile was again determined and safety laboratory tests were conducted. At this time, the following additional laboratory variables were measured: glucose, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Compliance with cholesterol-lowering diet was assessed by interviews with patients and their families about follow-up on the recommended weekly menu options. Control of body weight was an additional, indirect way to corroborate diet compliance. Patients were included in the study if they had LDL-C levels ->3.4 mmol/L. Triglyceride levels had to be <4.52 mmol/L so that the Friedewald equation 64 could be used to calculate LDL-C levels. Patients with active renal or hepatic disease, thyroid dysfunction, neoplastic disease, severe hypertension (diastolic pressure ->120 mm Hg), uncontrolled diabetes (fasting glucose >7 mmol/L), serum creatinine >140 pmol/L, or transaminases ->2 times the upper-normal limit were excluded. In addition, patients who had had any serious AEs within 6 months of study enrollment, young women who were or intended to become pregnant, and those who were breastfeeding were excluded. Eligible patients were randomly assigned, under double-blind conditions, to receive policosanol 5 mg or placebo tablets (visit 2). Policosanol and placebo tablets identical in appearance were prepared; the active ingredient was re-
290
G. Casta~o et aL
placed by similar quantities of lactose in the tablets, and the rest of the formulation was left unchanged. Packages were properly identified with code numbers by a member of the quality assurance unit, independent from all study investigators. Individual envelopes with codes corresponding to each patient were placed under controlled access in the quality assurance unit in case of any severe adverse effect that justified the opening of the envelope to ensure proper treatment. Study medications were randomized by a fixed method using an allocation ratio of 1:1. At this visit, patients underwent a complete physical examination, received coded packages containing policosanol tablets or identical placebo, and were instructed to take 1 tablet once daily with the evening meal for the next 6 weeks and to maintain the same dietary conditions. After 6 weeks of therapy, laboratory tests were repeated and patients underwent an interim checkup (visit 3), wherein physical examination, inquiries about AEs, and assessment of therapy compliance were performed. At this visit, patients received tablets for the remaining 6 weeks. Study protocol established that doses would be doubled in cases with LDL-C values ->3.4 mmol/L at the interim checkup; those patients were instructed to take 2 tablets once a day at the same time each day. Final evaluation was performed after 12 weeks of therapy, when the physical examination, laboratory tests, and assessments were repeated. Physical examinations, laboratory tests, and assessments of AEs and compliance with study medications were repeated after 12 weeks of therapy (visit 4). Drug compliance was assessed by tablet counts and interviews with patients and their families. All blood samples were drawn between 8 and 8:30 AMafter an evening fast of 12 hours, and aliquots were obtained for laboratory determinations. TC and triglycerides were determined by enzymatic methods using reagent kits (Roche Diagnostics GmbH, Mannheim, Germany). Use of lipid-lowering drugs was not allowed from enrollment until study completion. High-density lipoprotein-cholesterol (HDL-C) levels were determined according to the cholesterol content present in the supernatant obtained after beta-lipoprotein precipitation. 65 LDL-C values were calculated using the Friedewald equation. 64 Glucose, creatinine, AST, and ALT measurements were made following enzymatic methods using reagent kits. Laboratory tests were performed using the Hitachi 712 autoanalyzer (Tokyo, Japan) at the laboratory of the Medical Surgical Research Center (Havana City, Cuba). A systematic quality control was performed throughout the study. Precision was assessed according to repeatability (r, within-day variations) and reproducibility (R; between-day variations); accuracy was evaluated against standard references for each parameter. The coefficients of variation (95% CIs) were TC (r = 2.4; R = 2.7), triglycerides (r = 3.9; R = 4.0), and HDL-C (r = 2.9; R = 3.4). The differences from the standard reference were <4% for TC and <5% for triglycerides. The assay bias of these parameters was constant throughout the study.
291
CURRENT THERAPEUTICRESEARCH®
The primary efficacy end point was the change in LDL-C values relative to baseline. The treatment was considered effective if mean LDL-C levels were reduced by >15% compared with baseline at study completion. 66 The frequency of patients who reached percent decreases of LDL-C >15%, final LDL-C values <3.4 retool/L, and optimal values <2.8 mmol/L were also analyzed. 6 Secondary efficacy variables included changes in TC, HDL-C, triglycerides, and LDL-C/HDL-C and TC/HDL-C ratios. Data from physical examinations, laboratory tests, and patient interviews were included in the analysis of drug safety and tolerability. Serious AEs were defined as fatal or disabling events or those that led to or prolonged hospitalization; moderate AEs were those requiring specific treatment and discOntinuation of therapy according to the medical criteria. AEs that did not require discontinuation of treatment were classified as mild.
Statistical Analysis Study protocol required that all data be analyzed according to the intent-totreat approach, so that available data from patients who withdrew from the study must be included in all analyses. Within-group comparisons of continuous variables were performed using the Wilcoxon test for paired samples; between-group comparisons were done using the Mann-Whitney Utest. A Bonferroni adjustment for repeated comparisons in a single test was applied. 67 Categorical variables were compared using the Fisher exact test. All tests were 2 tailed. An alpha level of 0.05 was assumed for statistical significance. Statistical analyses were performed using Statistica (Stat) for Windows, version 4.2 (StatSoft, Inc, Tulsa, Okla).
RESULTS Ninety patients were recruited. After the 4-week diet-only period, 55 of the 90 patients (20 girls and 35 boys) were enrolled, all of whom completed the study. The remaining 35 patients were not included because of LDL-C levels <3.4 mmol/L at the end of the baseline diet-only period. Table I summarizes the baseline characteristics of the study population, showing that both groups were statistically similar at randomization. Although study protocol established that study patients could be 11 to 19 years of age, most of the enrolled subjects were between 14 and 19 years, a characteristic well matched in both groups. A high percentage of the enrolled patients (85.4%) had parents with atherosclerotic risk factors, such as dyslipidemia, hypertension, and diabetes mellitus, including 43.6% who had suffered premature myocardial infarction (before age 50), unstable angina, or stroke. Consumption of concomitant medications was also similar in both groups, with antiasthmatic drugs being consumed most often; overall, however, consumption of drugs other than the study medications was low.
292
G. Casta~o et al.
Table I. Baseline characteristics of study patients.* Characteristic Age, y, mean ___ SD Patients aged 11-13 years, no. (%) Patients older than 13 years, no. (%) Sex, no. (females/males) Body mass index, kg/m 2, mean __+ SD Subtype of type II hypercholesterolemia, no. (%) Primary (lla) Combined (lib) Personal history, no. (%) Hypertension HDL-C <0.9 mmol/L Family history, no. (%) Coronary heart disease (CHD) Stroke Total (CHD + stroke) Dyslipidemia Arterial hypertension Diabetes mellitus Parents showing 1 RF Parents showing ---2 RFs Parents showing RF Concomitant medications (CMs), no. (%) Antiasthmatics Diuretics ACE inhibitors Anxiolytics Antihistaminics Patients consuming CMs
Policosanol (n = 28)
Placebo (n = 27)
Total (N = 55)
18 + 2 1 (3.6) 27 (96.4) 10/18 23.40 + 2.35
17 + 2 1 (3.7) 26 (96.3) 10/17 23.77 + 2.60
18 + 2 2 (3.6) 53 (96.4) 20/35 23.58 + 2.46
28 (100.0) 0 (0.0)
26 (96.3) 1 (3.7)
54 (98.2) 1 (1.8)
1 (3.6) 5 (1 7.9)
0 (0.0) 5 (18.5)
1 (1.8) 10 (18.2)
11 (39.3) 1 (3.6) 12 (42.9) 13 (46.4) 12 (42.9) 11 (39.3) 7 (25.0) 1 7 (60.7) 24 (85.7)
11 (40.7) 1 (3.7) 12 (44.4) 11 (40.7) 15 (55.6) 12 (44.4) 7 (25.9) 16 (59.3) 23 (85.2)
4 (14.3) 1 (3.6) 1 (3.6) 1 (3.6) 0 (0.0) 7 (25.0)
4 (14.8) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.7) 5 (18.5)
22 2 24 24 27 23 14 33 47
(40.0) (3.6) (43.6) (43.6) (49.1) (41.8) (25.4) (60.0) (85.4)
8 (14.5) 1 (1.8) 1 (1.8) 1 (1.8) 1 (1.8) 12 (21.8)
RF = risk factors; ACE = angiotensin-converting enzyme. *All comparisons were not significant.
Efficacy Table II summarizes treatment effects on lipid profiles. Both groups showed similar values of all lipid-profile variables at randomization. After 12 weeks of therapy, policosanol significantly decreased LDL-C, the primary efficacy variable, with respect to baseline and placebo, showing a mean reduction of 32.6% (P < 0.001); TC and TC/HDL-C and LDL-C/HDL-C ratios were reduced by 21.9%, 27.8%, and 37.2%, respectively (all P < 0.001, compared with baseline and placebo). HDL-C was increased by 10.1% (P < 0.001 with respect to baseline and placebo). Triglycerides did not change significantly with respect to baseline or placebo.
293
CURRENT THERAPEUTIC RESEARCH®
Table II. Effects of policosanol on lipid profile (mean ± SD) of adolescents with type II hypercholesterolemia. Treatment LDL-C, mmol/L Policosanol Placebo TC, mmol/L Policosanol Placebo HDL-C, mmol/L Policosanol Placebo Triglycerides, mmol/L Policosanol Placebo LDL-C/HDL-C Ratio Policosanol Placebo TC/HDL-C Ratio Policosanol Placebo
Baseline
Week 6
Week 12
% Change
3.85 + 0.22 3.89 ___ 0.26
2.83 ± 0.55 *t~ 3.50 ± 0.46"
2.59 +_ 0.55 *§ 3.82 + 0.44
-32.6 § -1.7
5.30 ___0.32 5.27 ± 0.33
4.35 ___ 0.59 *§ 4.95 ± 0.49"
4.13 + 0.54 *§ 5.18 ___0.48
-21.9 § -1.7
1.04 ± 0.17 0.98 _ 0.11
1.13 ± 0.15 *~ 1.02 ___0.20
1.14 + 0.25 *§ 0.94 ± 0.10
10.1 § -3.1
1.10 ± 0.32 1.10 ± 0.50
1.06 ± 0.31 1.16 ± 0.55
1.06 ± 0.32 1.14 ___0.45
-0.3 6.5
3.89 ___0.68 4.03 ___ 0.53
2.57 ± 0.70 *§ 3.58 ± 0.94 il
2.39 ___0.75 *§ 4.12 ± 0.72
-37.2 § 3.4
5.20 ± 0.75 5.46 ___ 0.66
3.92 ± 0.76 *§ 5.03 ± 1.14 mR
3.75 _+ 0.82 *§ 5.58 _+ 0.81
-27.8 § 3.0
*P < 0.001, compared with baseline 0Nilcoxon test for paired samples). tp < 0.05, compared with baseline (Wilcoxon test for paired samples). ~P < 0.05, compared with placebo (Mann-Whitney U test). §P < 0.001, compared with placebo (Mann-Whitney U test). lip < 0.01, compared with baseline (Wilcoxon test for paired samples). ~P < 0.01, compared with placebo (Mann-Whitney U test).
Significant decreases in LDL-C, TC, and both atherogenic ratios (P < 0.001) and significant increases in HDL-C levels (P < 0.001) from baseline were observed in the policosanol group at the 6-week checkup. Twenty-three patients (17 placebo, 6 policosanol) required dose adjustments after 6 weeks of therapy, so that 6 (21.4%) of 28 policosanol-treated patients received 10 mg/d during the second step of active treatment. At study completion, the placebo group did not demonstrate any significant change in lipid profile variables compared with baseline, but significant decreases in LDL-C, TC, and LDL-C/HDL-C and TC/HDL-C ratios were observed after 6 weeks of therapy (all P < 0.01). Nevertheless, because the extent and significance of the reductions reached with policosanol were greater than those reached with placebo, and between-group comparisons at this time revealed significant differences from the policosanol group, we believe that a drug effect on these parameters was evident at 6 weeks. Efficacy assessment, according to the percentage of responders (Table liD, showed that 25 (89.3%) of 28 policosanol patients showed LDL-C reductions >15% compared with 2 (7.4%) of 27 placebo patients (P < 0.001). In addition, at
294
G. Casta~o et aL
study completion, 26 (92.8%) of 28 policosanol patients reached LDL-C values <3.4 mmol/L compared with 4 (14.8%) of 27 patients receiving placebo, the difference in responders in both groups being statistically significant (P < 0.001). Likewise, the percentage of responders who achieved optimal values (LDL-C <2.8 mmol/L) was also larger in the policosanol group (20/28; 71.4%) than in the placebo group (0/27; 0%; P < 0.001).
Tolerability Regarding the effects of policosanol on physical examination and blood biochemistry safety indicators, a mild but significant decrease in AST was detected in the policosanol group in comparisons with baseline (P = 0.003 at 6 weeks, and P < NS at 12 weeks), whereas the interim value of ALT was significantly lower in the policosanol group (P = 0.04 vs placebo) than in the placebo group. No other significant differences were detected with respect to any variable, and all individual values remained within normal limits (Table IV). No patients withdrew from the study, and only 3 patients (2 placebo, 1 policosanol) experienced mild AEs during the study; the placebo patients reported abdominal pain and constipation (1 each), and the policosanol patient reported polyphagia.
Compliance Compliance, as assessed by tablet count and patient interviews, was >95% in both groups, with 54 of the 55 patients consuming all doses of treatment. DISCUSSION
Pharmacological recommendations for the treatment of hypercholesterolemia in patients aged 10 to 19 years have been limited to ionic exchange resins; these agents, even though effective and safe, present problems in terms of long-term compliance with treatment because of unpalatability and some gastrointestinal drug-related adverse effects, such as constipation. Thus, the evaluation of new effective, easy-to-take, tolerable agents is needed for the treatment of hyper-
Table III. Number (%) of responses to active treatment, according to LDL-C reduction compared with baseline and to LDL-C goals.
Patients reaching compared with Patients reaching Patients reaching
LDL-C reductions >15%, baseline LDL-C values <3.4 mmol/L LDL-C values <2.8 mmol/L
Policosanol (n -- 28)
Placebo (n -- 27)
25 (89.3)* 26 (92.8)* 20 (71.4)*
2 (7.4) 4 (14.8) 0 (0.0)
*P < 0.001, compared with placebo (Fisher exact test).
295
CURRENTTHERAPEUTICRESEARCH®
Table IV. Effects of policosanol on safety indicators (mean +_ SD) of adolescents with type II
hypercholesterolemia.
Physical examination Body weight, kg Policosanol Placebo Pulse rate, beats/min Policosanol Placebo SBP, mm Hg Policosanol Placebo DBP, mm Hg Policosanol Placebo Blood biochemistry ALT, U/L Policosanol Placebo AST, U/L Policosanol Placebo Glucose, mmol/L Policosanol Placebo Creatinine, IJmol/L Policosanol Placebo
Baseline
Week 6
Week 12
64.29 _ 9.02 65.25 ___ 12.28
64.21 + 9.05 65.81 + 12.12
63.93 + 8.84 65.19 ___ 12.19
69.64 _ 7.19 70.41 ___ 8.98
69.61 ___ 5.63 72.26 ___6.62
70.54 + 4.74 72.22 ___ 7.15
111.79 ___ 10.20 110.37 _+ 10.91
114.64 _+ 11.38 113.52 ___9.18
112.50 + 9.28 112.59 ___ 7.64
70.00 ___ 6.67 71.11 ___ 7.51
72.57 ___ 6.68 73.41 +_ 5.10
71.43 ___ 6.51 72.78 _ 5.25
18.57 _ 8.22 19.70 ± 9.35
16.00 _+ 5.57* 22.96 + 9.29
16.43 + 6.57 20.70 + 9.70
29.43 _ 11.27 31.48 + 11.04
24.15 +_ 10.46 t 29.67 ± 10.16
26.18 ___ 10.52 ~ 28.33 ± 10.52
4.10 ± 0.40 3.98 _+ 0.41
4.11 ± 0.63 3.97 _+ 0.46
4.20 + 0.51 4.16 + 0.47
78.68 ___ 12.15 79.22 ___ 12.52
81.86 ___ 14.34 81.67 _ 12.33
80.18 _+ 11.60 80.78 + 12.65
SBP = systolic blood pressure; DBP = diastolic blood pressure; ALT = alanine aminotransferase; AST = aspartate aminotransferase. *P < 0.05, compared with placebo (Mann-Whitney U test). tp < 0.01, compared with baseline (Wilcoxon test for paired samples). ~P < 0.05, compared with baseline (Wilcoxon test for paired samples).
cholesterolemia in adolescent patients in whom the condition is not sufficiently controlled by diet alone. The present results represent the first published data of the effects of policosanol on hypercholesterolemic adolescents, and support the idea that policosanol administered at 5 mg/d for 12 weeks effectively lowers LDL-C and TC in these patients. As stated, the age range for inclusion was wide (11 to 19 years), but most of the enrolled patients were aged 14 to 19 years. This suggests that older adolescents and parents may have been motivated to participate in this study,
296
G. Casta~o et aL
which is a logical reaction for the first study of this drug in children and adolescents. The risk factors of study patients were mainly based on family history corroborated by family members; LDL-C, TC, and HDL-C levels were determined by laboratory analysis. Only 1 patient had a personal history of hypertension, but did not have other nonlipid risk factors. This characteristic was not predefined; however, it attenuates the possible influence of other risk factors on the present results. The mean LDL-C values of study patients at baseline actually reflect a high level of this lipoprotein, which has been considered the main target for hypercholesterolemia treatment in children and adolescents, according to recent guidelines. 6 The choice of once-daily administration in the evening was based on the fact that policosanol lowers cholesterol levels by inhibiting cholesterol biosynthesis and increasing receptor-dependent LDL catabolism. 5~-53 It might be expected that the best time for a once-a-day regimen is when cholesterol biosynthesis is inherently increased, which occurs at night in humans. 68 The study design was placebo-controlled because, to date, only resins have been approved as pharmacotherapy for such cases. Thus, a placebo group represented no additional risk for study patients and served as a parallel control to detect any systematic factor that could affect the results and to properly evaluate the drug-related effects. Although policosanol is thought to be well tolerated, we selected a dose of 5 mg/d because children and adolescents are still developing organisms and have relatively immature detoxifying mechanisms that may be affected by any drug exposure. This dose is also effective for reducing LDL-C levels. Thus, exposing them to the minimal effective dose is a logical, conservative approach for the first study in such populations; the increase to 10 mg/d was only indicated for cases with LDL-C ->3.4 mmol/L at the interim checkup. The reductions in LDL-C, the primary efficacy variable in this study, show that policosanol was effective in this population. The efficacy profile observed is consistent, but is only moderately enhanced when compared with that obtained with the same dosage in previous short-term studies of policosanol administered at the same doses to middle-aged and elderly people. 31 The treatment response was also evaluated according to the percentage of patients who reached efficacy goals, using LDL-C percent reductions of >15%, with respect to baseline, and final values of LDL-C <3.4 mmol/L and <2.8 mmol/ L.6 The results suggest that the percentage of patients reaching their LDL-C goals with policosanol is comparable to that reported for the first-line cholesterol-lowering drugs used to treat hypercholesterolemia in adults, including middle-aged and elderly people. 66'69 The effects observed on other lipid-profile variables, such as HDL-C and triglycerides, are also consistent with previous data on the use of policosanol in other population subsets. The beneficial effect of treatment on lipid-profile variables was observed from the interim checkup, suggesting that the cholesterol-lowering effects of
297
CURRENT THERAPEUTIC RESEARCH®
policosanol 5 mg/d can occur after 6 weeks of therapy. Nevertheless, the responses of LDL-C and TC, but not of HDL-C, suggest that this effect was partially masked by a small downward trend in the placebo group, indicating that a dietary effect may have still been present at the interim checkup. As determined by interviews with patients and their families, dietary compliance was high, with 85% of all patients declaring complete adherence to the recommended diet. Changes in body weight among study patients were consistent with such claims. However, a slight, although not statistically significant, increase in triglycerides was noted in the placebo group, which could indicate that adherence to the recommended diet was not as high as indicated by interviews and body-weight monitoring. Triglyceride levels did not change significantly in the policosanol group compared with baseline or placebo. This finding is consistent with the proposed mechanism of action of policosanol. Thus, policosanol induces a moderate inhibition of cholesterol biosynthesis, not through direct competitive inhibition of enzyme activity, but rather by increasing LDL receptor--dependent processing, thereby increasing LDL catabolism and then lowering LDL-C and TC levels. Policosanol was well tolerated by study patients. No drug-related effects on clinical or blood biochemistry parameters were observed, and no patients withdrew from the study. The significant decrease in AST values agrees with results obtained in some previous clinical studies performed in other population subsets. 37'41'47 The reason why policosanol tends to reduce transaminase levels must be further investigated, because this result has been observed in different clinical studies. 37'41'47 Nevertheless, the policosanol values were similar to the placebo values, and the individual values remained within normal limits. The efficacy and tolerability results obtained in this study are promising enough to justify further studies in this population subset. Investigations are needed to determine the effects of longer treatment periods and some pleiotropic properties of policosanol, such as its capacity to prevent LDL from undergoing lipid peroxidation in this group. 7°'71 Thus, because of the increased susceptibility of LDL to oxidation in children of high-risk families, 72 the role of consumption of a compound not only ensuring beneficial changes on lipid profiles, but also preventing LDL oxidation, could play an important role in preventing the development of CHD during childhood.
CONCLUSIONS
The results of this study show that policosanol 5 mg/d is effective in lowering LDL-C, TC, and both atherogenic ratios in adolescents with type II hypercholesterolemia, also increasing HDL-C levels in these cases. Because most of the study patients were >13 years of age, the demonstration of the benefits of policosanol are restricted to this age group. In addition, policosanol was well tolerated in this study. These findings suggest that policosanol could be a suitable addition to healthy lifestyle measures in the management of hyper-
298
G. Casta~o et al.
c h o l e s t e r o l e m i a in p a t i e n t s a g e d 13 to 19 years, t h u s c o n t r i b u t i n g to c o n t r o l of s u c h risk f a c t o r s in t h e e a r l y s t a g e s of life. B e c a u s e this is t h e first s t u d y of p o l i c o s a n o l in this p o p u l a t i o n a n d t h e s a m p l e size is relatively limited, f u r t h e r s t u d i e s are n e e d e d to d e t e r m i n e t h e risk-to-benefit ratio of p o l i c o s a n o l in t h e t r e a t m e n t of h y p e r c h o l e s t e r o l e m i a in this p o p u l a t i o n .
REFERENCES 1. Epstein FH. Cardiovascular disease epidemiology. A journey from the past into the future. Circulation. 1996;93:1755-1764. 2. Pyorala K, De Backer G, Graham I, et al. Prevention of coronary heart disease in clinical practice: Recommendations of the Task Force of the European Society of Cardiology, European Atherosclerosis Society and European Society of Hypertension. Eur Heart J. 1994;15:1300-1331. 3. Prevention of coronary heart disease in clinical practice: Recommendations of the Second Joint Task Force of European and other Societies on coronary prevention. Eur Heart J. 1998;19:1434-1503. 4. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (adult treatment panel IIl). JAMA. 2001;285:2486-2497. 5. Wood D. European and American recommendations for coronary heart disease prevention. Eur Heart J. 1998;19(Suppl A):A12-A19. 6. Gotto A, Assmann G, Carmena R, et al. Lipid Handbook for Clinical Practice. Blood Lipids and Coronary Heart Disease. 2nd ed. New York, NY: International Lipid Information Bureau; 2000:106-156. 7. Anderson KM, Wilson PW, Odell PM, Kannel WB. An updated coronary risk profile: A statement for health professionals. Circulation. 1991;83:356-362. 8. The Lipid Research Clinics Coronary Primary Prevention Trial results I. Reduction in incidence of coronary heart disease. JAMA. 1984;251:351-364. 9. The Lipid Research Clinics Coronary Primary Prevention Trial results II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA. 1984;251:365-374. 10. Frick MH, Elo O, Haapa K, et al. Helsinki Heart Study: Primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987;317:12371245. 11. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383-1389. 12. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Meal. 1996;335:1001-1009. 13. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med. 1998;339:1349-1357. 14. Shepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301-1307. 15. Weis S, Clearfield M, Downs JR, Gotto A. The Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS): Primary Prevention of Acute Major Coro-
299
CURRENT THERAPEUTICRESEARCH®
nary Events in Women and Men with Average Cholesterol. In: Grundy SM, ed. Cholesterol-Lowering Therapy." Evaluation of Clinical Trial Evidence. New York, NY: Marcel Dekker lnc; 2000:151-172. 16. Relationship of atherosclerosis in young men to serum lipoprotein cholesterol concentrations and smoking: A preliminary report from the Pathobiological Determinants of Atherosclerosis in Youth 0°DAY) Research Group. JAMA. 1990;264:30183024. 17. Lee J, Lauer RM, Clarke WR. Lipoproteins in the progeny of young men with coronary artery disease: Children with increased risl¢ Pediatrics. 1986;78:330-337. 18. Lauer RM, Lee J, Clarke WR. Factors affecting the relationship between childhood and adult cholesterol levels: The Muscatine Study. Pediatrics. 1988;82:309-318. 19. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Parental history of cardiovascular disease as an indication for screening for lipoprotein abnormalities in children. J Pediatr. 1989;115:186-194. 20. American Academy of Pediatrics. National Cholesterol Education program: Report of the expert panel on blood cholesterol levels in children and adolescents. Pediatrics. 1992;89:525-584. 21. Kleinman RN, Finberg LF, Klish WJ, Lauer RM. Dietary guidelines for children: U.S. recommendations. J Nutr. 1996;126(4 Suppl):1028S-1030S. 22. Efficacy and safety of lowering dietary intake of fat and cholesterol in children with elevated low-density lipoprotein cholesterol: The Dietary Intervention Study in Children (DISC). JAMA. 1995;273:1429-1435. 23. Lapinleimu H, Viikari J, Jokinen E, et al. Prospective randomised trial in 1062 infants of diet low in saturated fat and cholesterol. Lancet. 1995;345:471--476. 24. Thompson GR. Lipid lowering in the young. Heart. 1996;76:1-2. 25. Fisher EA, Van Horn L, McGill HC Jr. Nutrition and children: A statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation. 1997;95:2332-2333. 26. Tonstad S. A rational approach to treating hypercholesterolaemia in children: Weighing the risks and benefits. Drug Saf. 1997;16:330-341. 27. Liacouras CA, Coates PM, Gallagher PR, Cortner JA. Use of cholestyramine in the treatment of children with familial combined hyperlipidemia. JPediatr. 1993;122:477482. 28. Farmer JA, Gotto AM. Choosing the right lipid-regulating agent: A guide to selection. Drugs. 1996;52:649-661. 29. Bottorff M. "Fire and forget?" Pharmacological considerations in coronary care. Atherosclerosis. 1999;147(Suppl 1):$23--$30. 30. Farmer JA, Torre-Amione G. Comparative tolerability of the HMG-CoA reductase inhibitors. Drug Saf. 2000;23:197-213. 31. M~s R. Policosanol. Drugs Future. 2000;25:569-586. 32. Pons P, Rodrfguez M, Robaina C, et al. Effects of successive dose increases of policosanol on the lipid profile of patients with type II hypercholesterolaemia and tolerability to treatment. Int J Clin Pharmacol Res. 1994;14:27-33. 33. Aneiros E, Mils R, Calderon B, et al. Effect of policosanol in lowering cholesterol levels in patients with type lI hypercholesterolemia. Curr Ther Res Clin Exp. 1995;56: 176-182.
300
G. Casta~o et al.
34. Casta~o G, M~s R, Nodarse M, et ai. One-year study of the efficacy and safety of policosanol (5 mg twice daily) in the treatment of type II hypercholesterolemia. Curr Ther Res Clin Exp. 1995;56:296-304. 35. Canetti M, Moreira M, M~s R, et al. A two-year study on the efficacy and tolerability of policosanol in patients with type II hyperlipoproteinaemia. Int J Clin Pharmacol Res. 1995;15:159-165. 36. Canetti MM, Moreira M, M~s R, et al. Effects of policosanol on primary hypercholesterolemia: A 3-year open-extension follow-up. Curr TherRes Clin Exp. 1997;58:868875. 37. M~s R, Castafio G, llinait J, et al. Effects of policosanol in patients with type I1 hypercholesterolemia and additional coronary risk factors. Clin Pharmacol Ther. 1999;65:439--447. 38. Castafio G, Mils R, Fernandez JC, et al. A long-term, open-label study of the efficacy and tolerability of policosanol in patients with high global coronary risk. Curr Ther Res Clin Exp. 1999;60:379-391. 39. Castafio G, M~s R, Fernandez JC, et al. Efficacy and tolerability of policosanol compared with lovastatin in patients with type II hypercholesterolemia and concomitant risk factors. Curr Ther Res Clin Exp. 2000;61:137-147. 40. Castafio G, M~s R, Fernandez L, et al. Effect of policosanol on postmenopausal women with type II hypercholesterolemia. Gynecol Endocrinol. 2000;14:187-195. 41. Zardoya R, Tula L, Castafio G, et al. Effects of policosanol on hypercholesterolemic patients with abnormal serum biochemical indicators of hepatic function. Curr Ther Res Clin Exp. 1996;57:568--577. 42. Castafio G, Canetti M, Moreira M, et al. Efficacy and tolerability of policosanol in elderly patients with type II hypercholesterolemia: A 12-month study. Curr Ther Res Clin Exp. 1995;56:819--828. 43. Ortensi G, Gladstein J, Valli H, Tesone PA. A comparative study of policosanol versus simvastatin in elderly patients with hypercholesterolemia. Curr Ther Res Clin Exp. 1997;58:390-401. 44. Castafio G, M~s R, Arruzazabala ML, et al. Effects of policosanol and pravastatin on lipid profile, platelet aggregation and endothelemia in older hypercholesterolemic patients, lnt J Clin Pharmacol Res. 1999;19:105-116. 45. Castafio G, Mils R, Fernandez JC, et al. Effect of policosanol in older patients with type II hypercholesterolemia and high coronary risk. J Gerontol A Biol Sci Med Sci. 2001 ;56:819-828. 46. Fern,~ndez JC, Mils R, Castaho G, et al. Comparison of the efficacy, safety and tolerability of policosanol versus fluvastatin in elderly hypercholesterolaemic women. Clin Drug Invest. 2001;21:103-113. 47. Mils R, Castafio G, Fernfindez L, et al. Effects of policosanol on lipid profile and cardiac events in older hypercholesterolemic patients with coronary disease. Clin Drug Invest. 2001 ;21:485-497. 48. Torres O, Agramonte AJ, Ilinait J, et al. Treatment of hypercholesterolemia in NIDDM with policosanol. Diabetes Care. 1995;18:393-397. 49. Crespo N, Alvarez R, M~s R, et al. Effect of policosanol on patients with non-insulindependent diabetes mellitus and hypercholesterolemia: A pilot study. Curr Ther Res Clin Exp. 1997;58:44-51. 50. Crespo N, Illnait J, M~s R, et al. Comparative study of the efficacy and tolerability of policosanol and lovastatin in patients with hypercholesterolemia and noninsulin dependent diabetes mellitus. Int J Clin Pharrnacol Res. 1999;19:117-127.
301
CURRENT THERAPEUTIC RESEARCH®
51. Men~ndez R, Fernandez Sl, Del Rio A, et al. Policosanol inhibits cholesterol biosynthesis and enhances low density lipoprotein processing in cultured human fibroblasts. Biol Res. 1994;27:199--203. 52. Men~ndez R, Amor AM, Gonz~lez RM, et al. Effect of policosanol on the hepatic cholesterol biosynthesis of normocholesterolemic rats. Biol Res. 1996;29:253-257. 53. Men~ndez R, Arruzazabala L, M~s R, et al. Cholesterol-lowering effect of policosanol on rabbits with hypercholesterolaemia induced by a wheat starch-casein diet. Br J Nutr. 1997;77:923-932. 54. Men~ndez R, Amor A, Rodeiro I, et al. Policosanol modulates HMG-CoA reductase activity in cultured fibroblasts. Arch Meal Res. 2001;32:8-12. 55. Alemfin CL, Mils R, Hernfindez C, et al. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol Lett. 1994;70:77-87. 56. Mesa AR, M~s R, Noa M, et al. Toxicity of policosanol in beagle dogs: One-year study. Toxicol Lett. 1994;73:81-90. 57. Rodrfguez MD, Garcia H. Teratogenic and reproductive studies of policosanol in the rat and rabbit. Teratog Carcinog Mutag. 1994;14:107-113. 58. Alemfin CL, Mils R, Noa M, et al. Carcinogenicity of policosanol in Sprague Dawley rats: A 24-month study. Teratog Carcinog Mutag. 1994;14:239-249. 59. Alem~n C, Puig MN, Elias EC, et al. Carcinogenicity of policosanol in mice: An 18month study. Food Chem Toxicol. 1995;33:573-578. 60. Rodriguez MD, S~nchez M, Garcia H. Multigeneration reproduction study of policosanol in rats. Toxicol Lett. 1997;90:97-106. 61. G~mez R, Alem~n CL, Mas R, et al. A 6-month study on the toxicity of high doses of policosanol orally administered to Sprague-Dawley rats. JMed Food. 2001;4:57-66. 62. M~s R, Rivas P, Izquierdo JE, et al. Pharmacoepidemiologic study of policosanol. Curr Ther Res Clin Exp. 1999;60:458-467. 63. Fernandez L, M~s R, Illnait J, Fern6ndez JC. Policosanol: Results of a postmarketing surveillance study of 27,879 patients. Curt Ther Res Clin Exp. 1998;59:717-722. 64. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502. 65. Seigler L, Wu WT. Separation of serum high-density iipoprotein for cholesterol determination: Ultracentrifugation vs precipitation with sodium phosphotungstate and magnesium chloride. Clin Chem. 1981;27:838-841. 66. Schectman G, Hiatt J. Drug therapy for hypercholesterolemia in patients with cardiovascular disease: Factors limiting achievement of lipid goals. Am JMed. 1996;100: 197-204. 67. O'Brien PC, Shampo MA. Statistical considerations for performing multiple tests in a single experiment. 5. Comparing two therapies with respect to several endpoints. Mayo Clin Proc. 1988;63:1140-1143. 68. Parker TS, McNamara DJ, Brown C, et al. Mevalonic acid in human plasma: Relationship of concentration and circadian rhythm to cholesterol synthesis rates in man. Proc Natl Acad Sci U S A. 1982;79:3037-3041. 69. Jones P, Kafonek S, Laurora l, Hunninghake D. Comparative dose efficacy study of atorvastatin versus simvastatin, pravastatin, lovastatin, and fluvastatin in patients with hypercholesterolemia (the CURVES study). Am J Cardiol. 1998;81:582-587.
302
G. Casta~o et aL
70. Men~ndez R, M~s R, Amor AM, et al. Effects of policosanol treatment on the susceptibility of low density lipoprotein (LDL) isolated from healthy volunteers to oxidative modification in vitro. Br J Clin PharmacoL 2000;50:255-262. 71. Men~ndez R, Mas R, Amor AM, et al. Effects of policosanol on the susceptibility of low-density lipoprotein isolated from hypercholesterolemic patients at high coronary risk to in vitro copper-mediated lipid peroxidation: A randomized, double-blind pilot study. Curr Ther Res Clin Exp. 2000;61:609-620. 72. Kelishadi R, Asgary S, Naderi G, et al. Evaluation of LDL oxide metabolites in children of high risk families. Jpn J Cardiovasc Dis Prev. 2001;36(Suppl):69.
Address correspondence to: R o s a Mils, PhD C e n t e r for Natural P r o d u c t s National C e n t e r for Scientific R e s e a r c h PO Box 6880 Playa, H a v a n a City Cuba E-mail: [email protected]
303
| I~l t{ , l ' l I ',i,
Ri 'sl .',~{
VOLUME 63, NO. 4, APRIL 2002
A Randomized, Double-Blind, Placebo-Controlled Study of the Efficacy and Tolerability of Policosanol in Adolescents with Type II Hypercholesterolemia Gladys Casta~o, MD, PhD, 1 Rosa Mils, PhD, 2 Lilia Fernfindez, PhD, 2 Jos~ IIInait, MD, PhD, 2 Eric Hernfindez, MD, 1 Julio C. Fernfindez, 2 Rafael Gfimez, 2 Carlos Guti6rrez, MD, PhD, 2 and Estrella Alvarez, PhD 2
I Medical Surgical Research Center, and 2Center for Natural Products, National Center for Scientific Research, Havana City, Cuba
ABSTRACT
Background: Atherosclerosis begins in childhood and is influenced by risk factors for coronary heart disease (CHD), of which hypercholesterolemia is crucial. The rationale for treating hypercholesterolemia in childhood is to limit atherosclerosis development, for which adherence to a cholesterol-lowering diet is the first-choice therapy. Nevertheless, pharmacological intervention with bile acid-binding resins may be prescribed for patients older than 10 years, mainly those with family history of CHD, multiple risk factors, and/or severe hypercholesterolemia. Resins are effective and tolerable in this population, but their clinical use has been limited because of poor compliance due to unpalatability; other effective cholesterol-lowering drugs have not been recommended in this population because of the potential impact of drug-related adverse effects such as increases in transaminases, myopathies, and gastrointestinal disturbances. Thus, the need for safer, easy-to-take, and effective cholesterollowering agents for this population continues. Policosanol is a mixture of higher primary aliphatic alcohols purified from sugar cane wax with cholesterollowering effects proven in patients with type II hypercholesterolemia and dyslipidemia due to type 2 diabetes mellitus. Policosanol shows good safety and tolerability profiles, with no evidence of drug-related adverse events (AEs) to date. This background supports the idea that policosanol could be a good candidate for treating hypercholesterolemia in children and adolescents, but it requires clinical demonstration. Objective: This 12-week study was undertaken to investigate the cholesterollowering effects and tolerability of policosanol in hypercholesterolemic patients aged 11 to 19 years. Accepted for publication February 12, 2002. Printed in the USA. Reproduction in w h o l e or part is n o t p e r m i t t e d .
286
0011-393X/02/$19.00
G. Casta~o et aL
Methods: In this randomized, double-blind, placebo-controlled study, after 4 weeks of dietary stabilization, adolescents with type II hypercholesterolemia were randomly assigned (1:1 ratio) to receive placebo or policosanol 5-mg tablets once daily for 12 weeks. Physical examinations were performed, and lipid profiles and blood samples were obtained at baseline and after 6 and 12 weeks of therapy. The treatment was considered effective if mean reductions of low-density lipoprotein cholesterol (LDL-C) were >15%. In addition, the percentages of patients reaching final values of LDL-C <3.4 mmol/L and optimal values of <2.8 mmol/L were also evaluated. The doses were doubled if LDL-C values were ->3.4 mmol/L after 6 weeks of therapy. The incidence of AEs and compliance with study medications were also evaluated after 6 and 12 weeks of treatment. Results: Fifty-five patients were enrolled in the study (28 policosanol, 27 placebo). Twenty-three patients (17 placebo, 6 policosanol) required dose titration at 6 weeks. After 12 weeks of therapy, policosanol significantly decreased LDL-C with respect to baseline and placebo (both P < 0.001), showing a mean reduction of 32.6%. Total cholesterol (TC) and TC/high-density lipoprotein cholesterol (I-IDL-C) and LDL-C/HDL-C ratios were reduced by 21.9%, 27.8%, and 37.2%, respectively, in the policosanol group (P < 0.001, compared with baseline and placebo). HDL-C rose 10.1% (P < 0.001), compared with baseline and placebo. Triglycerides were unaffected by policosanol. LDL-C, TC, and both atherogenic ratios were reduced significantly in the policosanol group (P < 0.001), and significant increases in HDL-C values were observed at the 6-week interim checkup (P < 0.001 vs baseline, P < 0.01 vs placebo). Twenty-five (89.3%) of 28 patients in the policosanol group showed LDL-C reductions >15% compared with 2 (7.4%) of 27 patients in the placebo group (P < 0.001). In addition, 26 (92.8%) of 28 policosanol patients reached LDL-C values <3.4 mmol/L compared with 4 (14.8%) of 27 patients in the placebo group (P < 0.001). Likewise, the response rate for achievement of optimal values (LDL-C <2.8 mmol/L) was also larger in the policosanol group (20/28; 71.4%) than in the placebo group (0/27; 0.0%) (P < 0.001). Policosanol was well tolerated, with no drug-related effects found on physical examination. Blood biochemistry determinations revealed significantly lower alanine aminotransferase levels in the policosanol group after 6 weeks of therapy compared with placebo (P < 0.05), as well as significant reductions in aspartate aminotransferase levels at 6 weeks (P < 0.01) and 12 weeks (P < 0.05) compared with baseline. No patients withdrew from the study, and only 3 patients (2 placebo, 1 policosanol) experienced mild AEs during the study; the placebo patients reported abdominal pain and constipation (1 each), and the policosanol patient reported polyphagia. Conclusions: Policosanol 5 mg/d appears to be well tolerated and effective as short-term treatment of hypercholesterolemia in adolescents. Keg words: policosanol, cholesterol-lowering drugs, lipid-lowering therapy, hypercholesterolemia, adolescents. (Curr Ther Res Clin Exp. 2002;63:286-
3O3) 287
CURRENT THERAPEUTIC RESEARCH®
INTRODUCTION Coronary heart disease (CHD) is the leading cause of morbidity and mortality in middle-aged and elderly people.1 Expert guidelines have been established for the prevention of CHD,2-5 which has been recognized as a disease of multiple causes, arising from the concomitant effects of several coronary risk factors. 2-7 The direct relationship between CHD and elevated serum levels of lowdensity lipoprotein cholesterol (LDL-C) and total cholesterol (TC) has been demonstrated. ~-1° In addition, landmark clinical studies have demonstrated, beyond any doubt, the benefits of lowering LDL-C levels in both primary and secondary prevention of CHD.11-15 Studies of the early stages of atherosclerosis in children and young adults have shown the occurrence of arterial deposits of fats and early plaques at young ages. The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study 16 was undertaken to assess whether atherosclerosis begins in childhood and how recognized atherosclerotic risk factors influence this process. The results support the occurrence of early atherosclerotic lesions; the extent of fatty and fibrous arterial lesions increased with age, and the major risk factor associated with the arterial lesions was the postmortem concentration of LDL-C.16 Of the 3 principal coronary risk factorsmhypercholesterolemia, hypertension, and smoking7--hypercholesterolemia has been documented most extensively. Previous studies revealed the occurrence of lipid-profile abnormalities in children. 17-19 Hypercholesterolemia in childhood and adolescence may increase the risk of CHD later in life. 17A9 Because atherosclerosis is a continuous process that starts in childhood, guidelines for screening and treating young hypercholesterolemic people aged 2 to 19 years have been developed. 2° Indeed, the best primary prevention must start in childhood and adolescence; screening recommendations must address young patients with parents at high atherosclerotic risk. Early guidelines were criticized by pediatricians who believed that the putative risks and benefits of a long-term, low-fat diet had not been properly evaluated; as a result, these guidelines recommend more modest or gradual restriction of total dietary fat in pediatric patients. 21 The major concern has been whether a low-fat, low-cholesterol diet will support the special needs of children and adolescents with respect to growth, energy, and development. Nevertheless, recent studies demonstrate that normal growth was seen in children regardless of whether or not they received a low- or normal-fat diet. 22'23 F u r t h e r e v i d e n c e s u p p o r t s t h a t an American Heart Association Step I cholesterol-lowering diet can be safely recommended for patients aged 2 to 19 years. 24,25 Pharmacotherapy has been considered in pediatric patients ---10 years of age when LDL-C levels remain high despite lifestyle intervention, mainly when family history of CHD and/or multiple risk factors are present. To date, the bile-acid
288
G. Casta~o et al.
sequestrant resins are the only approved drugs for pharmacologic intervention of hypercholesterolemia in children and adolescents, with a US National Cholesterol Education Program recommended daily dose of 16 g.26 Taking into account the lack of a linear dose response, lower doses of colestipol (10 g/d) and cholestyramine (8 g/d) have been used to treat hypercholesterolemia in childhood and adolescence. The most serious limitation for the clinical use of resins in any age group is low compliance because of unpalatability. In addition, resins must be avoided in children and adolescents with elevated triglyceride levels because these drugs tend to increase triglycerides in these cases, 27 as occurs normally when administered to patients with combined dyslipidemia or dyslipidemia associated with type 2 diabetes. 28 Nicotinic acid has been used in a few young adults, but its administration requires careful medical follow-up.26 Combined therapy with nicotinic acid and resins may be indicated in cases of severe and refractory hypercholesterolemia, especially when other atherosclerotic risk factors are present. 26 Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) are the most effective of the cholesterol-lowering drugs that have been used extensively in the past decade28; some data suggest that they can improve lipid profiles in children and adolescents. 26 The statins can be useful in patients who have failed to respond to resins and/or nicotinic acid, especially when evidence of high atherosclerotic risk is present, but the use of statins to treat hypercholesterolemia in children and adolescents has not yet been approved. Several drug interactions 29 and statin-related adverse events (AEs), such as myopathy and elevations of serum levels of liver transaminases and creatine kinase, 3° are potentially dangerous for children and adolescents. Taking this background into account, the search for effective, easy-to-take, and safer cholesterollowering agents to manage hypercholesterolemia in children and adolescents is supported. Policosanol is a mixture of higher aliphatic alcohols isolated and purified from sugar cane (Saccharum officinarum L) wax 31 with cholesterol-lowering effects proven in patients with type II hypercholesterolemia32--47 and in patients with dyslipidemia due to type 2 diabetes mellitus. 48-5° Policosanol reduces cholesterol levels by inhibiting cholesterol biosynthesis at a step between acetate consumption and mevalonate production, although the inhibition of HMGCoA reductase as a possible mechanism of such an effect has been disproved. 51-54 Policosanol also increases LDL receptor-dependent processing in fibroblast cultures 51 as well as the clearance of ~2Sl-labeled LDL-C administered to rabbits with hypercholesterolemia induced by a cholesterol-free, casein-rich diet. 53 On the other hand, extensive experimental toxicologic evaluation of policosanol, including long-term and carcinogenicity studies, have not demonstrated any drug-related toxicity,s5-6~ Short- and long-term clinical studies have shown that policosanol is well tolerated, and no drug-related AEs have been demonstrated. 32-~°
289
CURRENT THERAPEUTIC RESEARCH®
Thus, studies conducted in populations more susceptible to drug-related AEs due to their impaired metabolism and/or frequent use of concomitant medications, such as patients at high coronary risk, 37-4° patients with hepatic disturbances, 41 and elderly people, 4247 have also failed to demonstrate any policosanol-related AEs. In addition, long-term (4- to 5-year) pharmacologic postmarketing surveillance studies, including more than 30,000 policosanoltreated patients, 62'63 have corroborated that the potential risk of policosanol treatment is negligible. Taking this background into account, policosanol could be a good candidate for treating hypercholesterolemia in children and adolescents. Thus, the objective of the present study was to investigate the cholesterol-lowering effects and tolerability of policosanol 5 to 10 mg administered once daily for 12 weeks to hypercholesterolemic patients aged 11 to 19 years. PATIENTS AND METHODS This study was conducted at the Medical Surgical Research Center (Havana City, Cuba), and the protocol was approved by the Ethics Committee of this institution and by the State Center for Quality Drug Control. Patients 11 to 19 years of age were enrolled in the study after providing informed personal assent and familial written consent (visit 1). A complete clinical history, physical examination, and sampling for lipid-profile determinations were performed at this time. Patients with LDL-C levels ->3.4 mmol/L were encouraged to follow a healthy lifestyle, including adherence to a standard cholesterol-lowering diet, for 4 weeks. Mter this period, a serum lipid profile was again determined and safety laboratory tests were conducted. At this time, the following additional laboratory variables were measured: glucose, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Compliance with cholesterol-lowering diet was assessed by interviews with patients and their families about follow-up on the recommended weekly menu options. Control of body weight was an additional, indirect way to corroborate diet compliance. Patients were included in the study if they had LDL-C levels ->3.4 mmol/L. Triglyceride levels had to be <4.52 mmol/L so that the Friedewald equation 64 could be used to calculate LDL-C levels. Patients with active renal or hepatic disease, thyroid dysfunction, neoplastic disease, severe hypertension (diastolic pressure ->120 mm Hg), uncontrolled diabetes (fasting glucose >7 mmol/L), serum creatinine >140 pmol/L, or transaminases ->2 times the upper-normal limit were excluded. In addition, patients who had had any serious AEs within 6 months of study enrollment, young women who were or intended to become pregnant, and those who were breastfeeding were excluded. Eligible patients were randomly assigned, under double-blind conditions, to receive policosanol 5 mg or placebo tablets (visit 2). Policosanol and placebo tablets identical in appearance were prepared; the active ingredient was re-
290
G. Casta~o et aL
placed by similar quantities of lactose in the tablets, and the rest of the formulation was left unchanged. Packages were properly identified with code numbers by a member of the quality assurance unit, independent from all study investigators. Individual envelopes with codes corresponding to each patient were placed under controlled access in the quality assurance unit in case of any severe adverse effect that justified the opening of the envelope to ensure proper treatment. Study medications were randomized by a fixed method using an allocation ratio of 1:1. At this visit, patients underwent a complete physical examination, received coded packages containing policosanol tablets or identical placebo, and were instructed to take 1 tablet once daily with the evening meal for the next 6 weeks and to maintain the same dietary conditions. After 6 weeks of therapy, laboratory tests were repeated and patients underwent an interim checkup (visit 3), wherein physical examination, inquiries about AEs, and assessment of therapy compliance were performed. At this visit, patients received tablets for the remaining 6 weeks. Study protocol established that doses would be doubled in cases with LDL-C values ->3.4 mmol/L at the interim checkup; those patients were instructed to take 2 tablets once a day at the same time each day. Final evaluation was performed after 12 weeks of therapy, when the physical examination, laboratory tests, and assessments were repeated. Physical examinations, laboratory tests, and assessments of AEs and compliance with study medications were repeated after 12 weeks of therapy (visit 4). Drug compliance was assessed by tablet counts and interviews with patients and their families. All blood samples were drawn between 8 and 8:30 AMafter an evening fast of 12 hours, and aliquots were obtained for laboratory determinations. TC and triglycerides were determined by enzymatic methods using reagent kits (Roche Diagnostics GmbH, Mannheim, Germany). Use of lipid-lowering drugs was not allowed from enrollment until study completion. High-density lipoprotein-cholesterol (HDL-C) levels were determined according to the cholesterol content present in the supernatant obtained after beta-lipoprotein precipitation. 65 LDL-C values were calculated using the Friedewald equation. 64 Glucose, creatinine, AST, and ALT measurements were made following enzymatic methods using reagent kits. Laboratory tests were performed using the Hitachi 712 autoanalyzer (Tokyo, Japan) at the laboratory of the Medical Surgical Research Center (Havana City, Cuba). A systematic quality control was performed throughout the study. Precision was assessed according to repeatability (r, within-day variations) and reproducibility (R; between-day variations); accuracy was evaluated against standard references for each parameter. The coefficients of variation (95% CIs) were TC (r = 2.4; R = 2.7), triglycerides (r = 3.9; R = 4.0), and HDL-C (r = 2.9; R = 3.4). The differences from the standard reference were <4% for TC and <5% for triglycerides. The assay bias of these parameters was constant throughout the study.
291
CURRENT THERAPEUTICRESEARCH®
The primary efficacy end point was the change in LDL-C values relative to baseline. The treatment was considered effective if mean LDL-C levels were reduced by >15% compared with baseline at study completion. 66 The frequency of patients who reached percent decreases of LDL-C >15%, final LDL-C values <3.4 retool/L, and optimal values <2.8 mmol/L were also analyzed. 6 Secondary efficacy variables included changes in TC, HDL-C, triglycerides, and LDL-C/HDL-C and TC/HDL-C ratios. Data from physical examinations, laboratory tests, and patient interviews were included in the analysis of drug safety and tolerability. Serious AEs were defined as fatal or disabling events or those that led to or prolonged hospitalization; moderate AEs were those requiring specific treatment and discOntinuation of therapy according to the medical criteria. AEs that did not require discontinuation of treatment were classified as mild.
Statistical Analysis Study protocol required that all data be analyzed according to the intent-totreat approach, so that available data from patients who withdrew from the study must be included in all analyses. Within-group comparisons of continuous variables were performed using the Wilcoxon test for paired samples; between-group comparisons were done using the Mann-Whitney Utest. A Bonferroni adjustment for repeated comparisons in a single test was applied. 67 Categorical variables were compared using the Fisher exact test. All tests were 2 tailed. An alpha level of 0.05 was assumed for statistical significance. Statistical analyses were performed using Statistica (Stat) for Windows, version 4.2 (StatSoft, Inc, Tulsa, Okla).
RESULTS Ninety patients were recruited. After the 4-week diet-only period, 55 of the 90 patients (20 girls and 35 boys) were enrolled, all of whom completed the study. The remaining 35 patients were not included because of LDL-C levels <3.4 mmol/L at the end of the baseline diet-only period. Table I summarizes the baseline characteristics of the study population, showing that both groups were statistically similar at randomization. Although study protocol established that study patients could be 11 to 19 years of age, most of the enrolled subjects were between 14 and 19 years, a characteristic well matched in both groups. A high percentage of the enrolled patients (85.4%) had parents with atherosclerotic risk factors, such as dyslipidemia, hypertension, and diabetes mellitus, including 43.6% who had suffered premature myocardial infarction (before age 50), unstable angina, or stroke. Consumption of concomitant medications was also similar in both groups, with antiasthmatic drugs being consumed most often; overall, however, consumption of drugs other than the study medications was low.
292
G. Casta~o et al.
Table I. Baseline characteristics of study patients.* Characteristic Age, y, mean ___ SD Patients aged 11-13 years, no. (%) Patients older than 13 years, no. (%) Sex, no. (females/males) Body mass index, kg/m 2, mean __+ SD Subtype of type II hypercholesterolemia, no. (%) Primary (lla) Combined (lib) Personal history, no. (%) Hypertension HDL-C <0.9 mmol/L Family history, no. (%) Coronary heart disease (CHD) Stroke Total (CHD + stroke) Dyslipidemia Arterial hypertension Diabetes mellitus Parents showing 1 RF Parents showing ---2 RFs Parents showing RF Concomitant medications (CMs), no. (%) Antiasthmatics Diuretics ACE inhibitors Anxiolytics Antihistaminics Patients consuming CMs
Policosanol (n = 28)
Placebo (n = 27)
Total (N = 55)
18 + 2 1 (3.6) 27 (96.4) 10/18 23.40 + 2.35
17 + 2 1 (3.7) 26 (96.3) 10/17 23.77 + 2.60
18 + 2 2 (3.6) 53 (96.4) 20/35 23.58 + 2.46
28 (100.0) 0 (0.0)
26 (96.3) 1 (3.7)
54 (98.2) 1 (1.8)
1 (3.6) 5 (1 7.9)
0 (0.0) 5 (18.5)
1 (1.8) 10 (18.2)
11 (39.3) 1 (3.6) 12 (42.9) 13 (46.4) 12 (42.9) 11 (39.3) 7 (25.0) 1 7 (60.7) 24 (85.7)
11 (40.7) 1 (3.7) 12 (44.4) 11 (40.7) 15 (55.6) 12 (44.4) 7 (25.9) 16 (59.3) 23 (85.2)
4 (14.3) 1 (3.6) 1 (3.6) 1 (3.6) 0 (0.0) 7 (25.0)
4 (14.8) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.7) 5 (18.5)
22 2 24 24 27 23 14 33 47
(40.0) (3.6) (43.6) (43.6) (49.1) (41.8) (25.4) (60.0) (85.4)
8 (14.5) 1 (1.8) 1 (1.8) 1 (1.8) 1 (1.8) 12 (21.8)
RF = risk factors; ACE = angiotensin-converting enzyme. *All comparisons were not significant.
Efficacy Table II summarizes treatment effects on lipid profiles. Both groups showed similar values of all lipid-profile variables at randomization. After 12 weeks of therapy, policosanol significantly decreased LDL-C, the primary efficacy variable, with respect to baseline and placebo, showing a mean reduction of 32.6% (P < 0.001); TC and TC/HDL-C and LDL-C/HDL-C ratios were reduced by 21.9%, 27.8%, and 37.2%, respectively (all P < 0.001, compared with baseline and placebo). HDL-C was increased by 10.1% (P < 0.001 with respect to baseline and placebo). Triglycerides did not change significantly with respect to baseline or placebo.
293
CURRENT THERAPEUTIC RESEARCH®
Table II. Effects of policosanol on lipid profile (mean ± SD) of adolescents with type II hypercholesterolemia. Treatment LDL-C, mmol/L Policosanol Placebo TC, mmol/L Policosanol Placebo HDL-C, mmol/L Policosanol Placebo Triglycerides, mmol/L Policosanol Placebo LDL-C/HDL-C Ratio Policosanol Placebo TC/HDL-C Ratio Policosanol Placebo
Baseline
Week 6
Week 12
% Change
3.85 + 0.22 3.89 ___ 0.26
2.83 ± 0.55 *t~ 3.50 ± 0.46"
2.59 +_ 0.55 *§ 3.82 + 0.44
-32.6 § -1.7
5.30 ___0.32 5.27 ± 0.33
4.35 ___ 0.59 *§ 4.95 ± 0.49"
4.13 + 0.54 *§ 5.18 ___0.48
-21.9 § -1.7
1.04 ± 0.17 0.98 _ 0.11
1.13 ± 0.15 *~ 1.02 ___0.20
1.14 + 0.25 *§ 0.94 ± 0.10
10.1 § -3.1
1.10 ± 0.32 1.10 ± 0.50
1.06 ± 0.31 1.16 ± 0.55
1.06 ± 0.32 1.14 ___0.45
-0.3 6.5
3.89 ___0.68 4.03 ___ 0.53
2.57 ± 0.70 *§ 3.58 ± 0.94 il
2.39 ___0.75 *§ 4.12 ± 0.72
-37.2 § 3.4
5.20 ± 0.75 5.46 ___ 0.66
3.92 ± 0.76 *§ 5.03 ± 1.14 mR
3.75 _+ 0.82 *§ 5.58 _+ 0.81
-27.8 § 3.0
*P < 0.001, compared with baseline 0Nilcoxon test for paired samples). tp < 0.05, compared with baseline (Wilcoxon test for paired samples). ~P < 0.05, compared with placebo (Mann-Whitney U test). §P < 0.001, compared with placebo (Mann-Whitney U test). lip < 0.01, compared with baseline (Wilcoxon test for paired samples). ~P < 0.01, compared with placebo (Mann-Whitney U test).
Significant decreases in LDL-C, TC, and both atherogenic ratios (P < 0.001) and significant increases in HDL-C levels (P < 0.001) from baseline were observed in the policosanol group at the 6-week checkup. Twenty-three patients (17 placebo, 6 policosanol) required dose adjustments after 6 weeks of therapy, so that 6 (21.4%) of 28 policosanol-treated patients received 10 mg/d during the second step of active treatment. At study completion, the placebo group did not demonstrate any significant change in lipid profile variables compared with baseline, but significant decreases in LDL-C, TC, and LDL-C/HDL-C and TC/HDL-C ratios were observed after 6 weeks of therapy (all P < 0.01). Nevertheless, because the extent and significance of the reductions reached with policosanol were greater than those reached with placebo, and between-group comparisons at this time revealed significant differences from the policosanol group, we believe that a drug effect on these parameters was evident at 6 weeks. Efficacy assessment, according to the percentage of responders (Table liD, showed that 25 (89.3%) of 28 policosanol patients showed LDL-C reductions >15% compared with 2 (7.4%) of 27 placebo patients (P < 0.001). In addition, at
294
G. Casta~o et aL
study completion, 26 (92.8%) of 28 policosanol patients reached LDL-C values <3.4 mmol/L compared with 4 (14.8%) of 27 patients receiving placebo, the difference in responders in both groups being statistically significant (P < 0.001). Likewise, the percentage of responders who achieved optimal values (LDL-C <2.8 mmol/L) was also larger in the policosanol group (20/28; 71.4%) than in the placebo group (0/27; 0%; P < 0.001).
Tolerability Regarding the effects of policosanol on physical examination and blood biochemistry safety indicators, a mild but significant decrease in AST was detected in the policosanol group in comparisons with baseline (P = 0.003 at 6 weeks, and P < NS at 12 weeks), whereas the interim value of ALT was significantly lower in the policosanol group (P = 0.04 vs placebo) than in the placebo group. No other significant differences were detected with respect to any variable, and all individual values remained within normal limits (Table IV). No patients withdrew from the study, and only 3 patients (2 placebo, 1 policosanol) experienced mild AEs during the study; the placebo patients reported abdominal pain and constipation (1 each), and the policosanol patient reported polyphagia.
Compliance Compliance, as assessed by tablet count and patient interviews, was >95% in both groups, with 54 of the 55 patients consuming all doses of treatment. DISCUSSION
Pharmacological recommendations for the treatment of hypercholesterolemia in patients aged 10 to 19 years have been limited to ionic exchange resins; these agents, even though effective and safe, present problems in terms of long-term compliance with treatment because of unpalatability and some gastrointestinal drug-related adverse effects, such as constipation. Thus, the evaluation of new effective, easy-to-take, tolerable agents is needed for the treatment of hyper-
Table III. Number (%) of responses to active treatment, according to LDL-C reduction compared with baseline and to LDL-C goals.
Patients reaching compared with Patients reaching Patients reaching
LDL-C reductions >15%, baseline LDL-C values <3.4 mmol/L LDL-C values <2.8 mmol/L
Policosanol (n -- 28)
Placebo (n -- 27)
25 (89.3)* 26 (92.8)* 20 (71.4)*
2 (7.4) 4 (14.8) 0 (0.0)
*P < 0.001, compared with placebo (Fisher exact test).
295
CURRENTTHERAPEUTICRESEARCH®
Table IV. Effects of policosanol on safety indicators (mean +_ SD) of adolescents with type II
hypercholesterolemia.
Physical examination Body weight, kg Policosanol Placebo Pulse rate, beats/min Policosanol Placebo SBP, mm Hg Policosanol Placebo DBP, mm Hg Policosanol Placebo Blood biochemistry ALT, U/L Policosanol Placebo AST, U/L Policosanol Placebo Glucose, mmol/L Policosanol Placebo Creatinine, IJmol/L Policosanol Placebo
Baseline
Week 6
Week 12
64.29 _ 9.02 65.25 ___ 12.28
64.21 + 9.05 65.81 + 12.12
63.93 + 8.84 65.19 ___ 12.19
69.64 _ 7.19 70.41 ___ 8.98
69.61 ___ 5.63 72.26 ___6.62
70.54 + 4.74 72.22 ___ 7.15
111.79 ___ 10.20 110.37 _+ 10.91
114.64 _+ 11.38 113.52 ___9.18
112.50 + 9.28 112.59 ___ 7.64
70.00 ___ 6.67 71.11 ___ 7.51
72.57 ___ 6.68 73.41 +_ 5.10
71.43 ___ 6.51 72.78 _ 5.25
18.57 _ 8.22 19.70 ± 9.35
16.00 _+ 5.57* 22.96 + 9.29
16.43 + 6.57 20.70 + 9.70
29.43 _ 11.27 31.48 + 11.04
24.15 +_ 10.46 t 29.67 ± 10.16
26.18 ___ 10.52 ~ 28.33 ± 10.52
4.10 ± 0.40 3.98 _+ 0.41
4.11 ± 0.63 3.97 _+ 0.46
4.20 + 0.51 4.16 + 0.47
78.68 ___ 12.15 79.22 ___ 12.52
81.86 ___ 14.34 81.67 _ 12.33
80.18 _+ 11.60 80.78 + 12.65
SBP = systolic blood pressure; DBP = diastolic blood pressure; ALT = alanine aminotransferase; AST = aspartate aminotransferase. *P < 0.05, compared with placebo (Mann-Whitney U test). tp < 0.01, compared with baseline (Wilcoxon test for paired samples). ~P < 0.05, compared with baseline (Wilcoxon test for paired samples).
cholesterolemia in adolescent patients in whom the condition is not sufficiently controlled by diet alone. The present results represent the first published data of the effects of policosanol on hypercholesterolemic adolescents, and support the idea that policosanol administered at 5 mg/d for 12 weeks effectively lowers LDL-C and TC in these patients. As stated, the age range for inclusion was wide (11 to 19 years), but most of the enrolled patients were aged 14 to 19 years. This suggests that older adolescents and parents may have been motivated to participate in this study,
296
G. Casta~o et aL
which is a logical reaction for the first study of this drug in children and adolescents. The risk factors of study patients were mainly based on family history corroborated by family members; LDL-C, TC, and HDL-C levels were determined by laboratory analysis. Only 1 patient had a personal history of hypertension, but did not have other nonlipid risk factors. This characteristic was not predefined; however, it attenuates the possible influence of other risk factors on the present results. The mean LDL-C values of study patients at baseline actually reflect a high level of this lipoprotein, which has been considered the main target for hypercholesterolemia treatment in children and adolescents, according to recent guidelines. 6 The choice of once-daily administration in the evening was based on the fact that policosanol lowers cholesterol levels by inhibiting cholesterol biosynthesis and increasing receptor-dependent LDL catabolism. 5~-53 It might be expected that the best time for a once-a-day regimen is when cholesterol biosynthesis is inherently increased, which occurs at night in humans. 68 The study design was placebo-controlled because, to date, only resins have been approved as pharmacotherapy for such cases. Thus, a placebo group represented no additional risk for study patients and served as a parallel control to detect any systematic factor that could affect the results and to properly evaluate the drug-related effects. Although policosanol is thought to be well tolerated, we selected a dose of 5 mg/d because children and adolescents are still developing organisms and have relatively immature detoxifying mechanisms that may be affected by any drug exposure. This dose is also effective for reducing LDL-C levels. Thus, exposing them to the minimal effective dose is a logical, conservative approach for the first study in such populations; the increase to 10 mg/d was only indicated for cases with LDL-C ->3.4 mmol/L at the interim checkup. The reductions in LDL-C, the primary efficacy variable in this study, show that policosanol was effective in this population. The efficacy profile observed is consistent, but is only moderately enhanced when compared with that obtained with the same dosage in previous short-term studies of policosanol administered at the same doses to middle-aged and elderly people. 31 The treatment response was also evaluated according to the percentage of patients who reached efficacy goals, using LDL-C percent reductions of >15%, with respect to baseline, and final values of LDL-C <3.4 mmol/L and <2.8 mmol/ L.6 The results suggest that the percentage of patients reaching their LDL-C goals with policosanol is comparable to that reported for the first-line cholesterol-lowering drugs used to treat hypercholesterolemia in adults, including middle-aged and elderly people. 66'69 The effects observed on other lipid-profile variables, such as HDL-C and triglycerides, are also consistent with previous data on the use of policosanol in other population subsets. The beneficial effect of treatment on lipid-profile variables was observed from the interim checkup, suggesting that the cholesterol-lowering effects of
297
CURRENT THERAPEUTIC RESEARCH®
policosanol 5 mg/d can occur after 6 weeks of therapy. Nevertheless, the responses of LDL-C and TC, but not of HDL-C, suggest that this effect was partially masked by a small downward trend in the placebo group, indicating that a dietary effect may have still been present at the interim checkup. As determined by interviews with patients and their families, dietary compliance was high, with 85% of all patients declaring complete adherence to the recommended diet. Changes in body weight among study patients were consistent with such claims. However, a slight, although not statistically significant, increase in triglycerides was noted in the placebo group, which could indicate that adherence to the recommended diet was not as high as indicated by interviews and body-weight monitoring. Triglyceride levels did not change significantly in the policosanol group compared with baseline or placebo. This finding is consistent with the proposed mechanism of action of policosanol. Thus, policosanol induces a moderate inhibition of cholesterol biosynthesis, not through direct competitive inhibition of enzyme activity, but rather by increasing LDL receptor--dependent processing, thereby increasing LDL catabolism and then lowering LDL-C and TC levels. Policosanol was well tolerated by study patients. No drug-related effects on clinical or blood biochemistry parameters were observed, and no patients withdrew from the study. The significant decrease in AST values agrees with results obtained in some previous clinical studies performed in other population subsets. 37'41'47 The reason why policosanol tends to reduce transaminase levels must be further investigated, because this result has been observed in different clinical studies. 37'41'47 Nevertheless, the policosanol values were similar to the placebo values, and the individual values remained within normal limits. The efficacy and tolerability results obtained in this study are promising enough to justify further studies in this population subset. Investigations are needed to determine the effects of longer treatment periods and some pleiotropic properties of policosanol, such as its capacity to prevent LDL from undergoing lipid peroxidation in this group. 7°'71 Thus, because of the increased susceptibility of LDL to oxidation in children of high-risk families, 72 the role of consumption of a compound not only ensuring beneficial changes on lipid profiles, but also preventing LDL oxidation, could play an important role in preventing the development of CHD during childhood.
CONCLUSIONS
The results of this study show that policosanol 5 mg/d is effective in lowering LDL-C, TC, and both atherogenic ratios in adolescents with type II hypercholesterolemia, also increasing HDL-C levels in these cases. Because most of the study patients were >13 years of age, the demonstration of the benefits of policosanol are restricted to this age group. In addition, policosanol was well tolerated in this study. These findings suggest that policosanol could be a suitable addition to healthy lifestyle measures in the management of hyper-
298
G. Casta~o et al.
c h o l e s t e r o l e m i a in p a t i e n t s a g e d 13 to 19 years, t h u s c o n t r i b u t i n g to c o n t r o l of s u c h risk f a c t o r s in t h e e a r l y s t a g e s of life. B e c a u s e this is t h e first s t u d y of p o l i c o s a n o l in this p o p u l a t i o n a n d t h e s a m p l e size is relatively limited, f u r t h e r s t u d i e s are n e e d e d to d e t e r m i n e t h e risk-to-benefit ratio of p o l i c o s a n o l in t h e t r e a t m e n t of h y p e r c h o l e s t e r o l e m i a in this p o p u l a t i o n .
REFERENCES 1. Epstein FH. Cardiovascular disease epidemiology. A journey from the past into the future. Circulation. 1996;93:1755-1764. 2. Pyorala K, De Backer G, Graham I, et al. Prevention of coronary heart disease in clinical practice: Recommendations of the Task Force of the European Society of Cardiology, European Atherosclerosis Society and European Society of Hypertension. Eur Heart J. 1994;15:1300-1331. 3. Prevention of coronary heart disease in clinical practice: Recommendations of the Second Joint Task Force of European and other Societies on coronary prevention. Eur Heart J. 1998;19:1434-1503. 4. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (adult treatment panel IIl). JAMA. 2001;285:2486-2497. 5. Wood D. European and American recommendations for coronary heart disease prevention. Eur Heart J. 1998;19(Suppl A):A12-A19. 6. Gotto A, Assmann G, Carmena R, et al. Lipid Handbook for Clinical Practice. Blood Lipids and Coronary Heart Disease. 2nd ed. New York, NY: International Lipid Information Bureau; 2000:106-156. 7. Anderson KM, Wilson PW, Odell PM, Kannel WB. An updated coronary risk profile: A statement for health professionals. Circulation. 1991;83:356-362. 8. The Lipid Research Clinics Coronary Primary Prevention Trial results I. Reduction in incidence of coronary heart disease. JAMA. 1984;251:351-364. 9. The Lipid Research Clinics Coronary Primary Prevention Trial results II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA. 1984;251:365-374. 10. Frick MH, Elo O, Haapa K, et al. Helsinki Heart Study: Primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987;317:12371245. 11. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383-1389. 12. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Meal. 1996;335:1001-1009. 13. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med. 1998;339:1349-1357. 14. Shepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301-1307. 15. Weis S, Clearfield M, Downs JR, Gotto A. The Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS): Primary Prevention of Acute Major Coro-
299
CURRENT THERAPEUTICRESEARCH®
nary Events in Women and Men with Average Cholesterol. In: Grundy SM, ed. Cholesterol-Lowering Therapy." Evaluation of Clinical Trial Evidence. New York, NY: Marcel Dekker lnc; 2000:151-172. 16. Relationship of atherosclerosis in young men to serum lipoprotein cholesterol concentrations and smoking: A preliminary report from the Pathobiological Determinants of Atherosclerosis in Youth 0°DAY) Research Group. JAMA. 1990;264:30183024. 17. Lee J, Lauer RM, Clarke WR. Lipoproteins in the progeny of young men with coronary artery disease: Children with increased risl¢ Pediatrics. 1986;78:330-337. 18. Lauer RM, Lee J, Clarke WR. Factors affecting the relationship between childhood and adult cholesterol levels: The Muscatine Study. Pediatrics. 1988;82:309-318. 19. Dennison BA, Kikuchi DA, Srinivasan SR, et al. Parental history of cardiovascular disease as an indication for screening for lipoprotein abnormalities in children. J Pediatr. 1989;115:186-194. 20. American Academy of Pediatrics. National Cholesterol Education program: Report of the expert panel on blood cholesterol levels in children and adolescents. Pediatrics. 1992;89:525-584. 21. Kleinman RN, Finberg LF, Klish WJ, Lauer RM. Dietary guidelines for children: U.S. recommendations. J Nutr. 1996;126(4 Suppl):1028S-1030S. 22. Efficacy and safety of lowering dietary intake of fat and cholesterol in children with elevated low-density lipoprotein cholesterol: The Dietary Intervention Study in Children (DISC). JAMA. 1995;273:1429-1435. 23. Lapinleimu H, Viikari J, Jokinen E, et al. Prospective randomised trial in 1062 infants of diet low in saturated fat and cholesterol. Lancet. 1995;345:471--476. 24. Thompson GR. Lipid lowering in the young. Heart. 1996;76:1-2. 25. Fisher EA, Van Horn L, McGill HC Jr. Nutrition and children: A statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation. 1997;95:2332-2333. 26. Tonstad S. A rational approach to treating hypercholesterolaemia in children: Weighing the risks and benefits. Drug Saf. 1997;16:330-341. 27. Liacouras CA, Coates PM, Gallagher PR, Cortner JA. Use of cholestyramine in the treatment of children with familial combined hyperlipidemia. JPediatr. 1993;122:477482. 28. Farmer JA, Gotto AM. Choosing the right lipid-regulating agent: A guide to selection. Drugs. 1996;52:649-661. 29. Bottorff M. "Fire and forget?" Pharmacological considerations in coronary care. Atherosclerosis. 1999;147(Suppl 1):$23--$30. 30. Farmer JA, Torre-Amione G. Comparative tolerability of the HMG-CoA reductase inhibitors. Drug Saf. 2000;23:197-213. 31. M~s R. Policosanol. Drugs Future. 2000;25:569-586. 32. Pons P, Rodrfguez M, Robaina C, et al. Effects of successive dose increases of policosanol on the lipid profile of patients with type II hypercholesterolaemia and tolerability to treatment. Int J Clin Pharmacol Res. 1994;14:27-33. 33. Aneiros E, Mils R, Calderon B, et al. Effect of policosanol in lowering cholesterol levels in patients with type lI hypercholesterolemia. Curr Ther Res Clin Exp. 1995;56: 176-182.
300
G. Casta~o et al.
34. Casta~o G, M~s R, Nodarse M, et ai. One-year study of the efficacy and safety of policosanol (5 mg twice daily) in the treatment of type II hypercholesterolemia. Curr Ther Res Clin Exp. 1995;56:296-304. 35. Canetti M, Moreira M, M~s R, et al. A two-year study on the efficacy and tolerability of policosanol in patients with type II hyperlipoproteinaemia. Int J Clin Pharmacol Res. 1995;15:159-165. 36. Canetti MM, Moreira M, M~s R, et al. Effects of policosanol on primary hypercholesterolemia: A 3-year open-extension follow-up. Curr TherRes Clin Exp. 1997;58:868875. 37. M~s R, Castafio G, llinait J, et al. Effects of policosanol in patients with type I1 hypercholesterolemia and additional coronary risk factors. Clin Pharmacol Ther. 1999;65:439--447. 38. Castafio G, Mils R, Fernandez JC, et al. A long-term, open-label study of the efficacy and tolerability of policosanol in patients with high global coronary risk. Curr Ther Res Clin Exp. 1999;60:379-391. 39. Castafio G, M~s R, Fernandez JC, et al. Efficacy and tolerability of policosanol compared with lovastatin in patients with type II hypercholesterolemia and concomitant risk factors. Curr Ther Res Clin Exp. 2000;61:137-147. 40. Castafio G, M~s R, Fernandez L, et al. Effect of policosanol on postmenopausal women with type II hypercholesterolemia. Gynecol Endocrinol. 2000;14:187-195. 41. Zardoya R, Tula L, Castafio G, et al. Effects of policosanol on hypercholesterolemic patients with abnormal serum biochemical indicators of hepatic function. Curr Ther Res Clin Exp. 1996;57:568--577. 42. Castafio G, Canetti M, Moreira M, et al. Efficacy and tolerability of policosanol in elderly patients with type II hypercholesterolemia: A 12-month study. Curr Ther Res Clin Exp. 1995;56:819--828. 43. Ortensi G, Gladstein J, Valli H, Tesone PA. A comparative study of policosanol versus simvastatin in elderly patients with hypercholesterolemia. Curr Ther Res Clin Exp. 1997;58:390-401. 44. Castafio G, M~s R, Arruzazabala ML, et al. Effects of policosanol and pravastatin on lipid profile, platelet aggregation and endothelemia in older hypercholesterolemic patients, lnt J Clin Pharmacol Res. 1999;19:105-116. 45. Castafio G, Mils R, Fernandez JC, et al. Effect of policosanol in older patients with type II hypercholesterolemia and high coronary risk. J Gerontol A Biol Sci Med Sci. 2001 ;56:819-828. 46. Fern,~ndez JC, Mils R, Castaho G, et al. Comparison of the efficacy, safety and tolerability of policosanol versus fluvastatin in elderly hypercholesterolaemic women. Clin Drug Invest. 2001;21:103-113. 47. Mils R, Castafio G, Fernfindez L, et al. Effects of policosanol on lipid profile and cardiac events in older hypercholesterolemic patients with coronary disease. Clin Drug Invest. 2001 ;21:485-497. 48. Torres O, Agramonte AJ, Ilinait J, et al. Treatment of hypercholesterolemia in NIDDM with policosanol. Diabetes Care. 1995;18:393-397. 49. Crespo N, Alvarez R, M~s R, et al. Effect of policosanol on patients with non-insulindependent diabetes mellitus and hypercholesterolemia: A pilot study. Curr Ther Res Clin Exp. 1997;58:44-51. 50. Crespo N, Illnait J, M~s R, et al. Comparative study of the efficacy and tolerability of policosanol and lovastatin in patients with hypercholesterolemia and noninsulin dependent diabetes mellitus. Int J Clin Pharrnacol Res. 1999;19:117-127.
301
CURRENT THERAPEUTIC RESEARCH®
51. Men~ndez R, Fernandez Sl, Del Rio A, et al. Policosanol inhibits cholesterol biosynthesis and enhances low density lipoprotein processing in cultured human fibroblasts. Biol Res. 1994;27:199--203. 52. Men~ndez R, Amor AM, Gonz~lez RM, et al. Effect of policosanol on the hepatic cholesterol biosynthesis of normocholesterolemic rats. Biol Res. 1996;29:253-257. 53. Men~ndez R, Arruzazabala L, M~s R, et al. Cholesterol-lowering effect of policosanol on rabbits with hypercholesterolaemia induced by a wheat starch-casein diet. Br J Nutr. 1997;77:923-932. 54. Men~ndez R, Amor A, Rodeiro I, et al. Policosanol modulates HMG-CoA reductase activity in cultured fibroblasts. Arch Meal Res. 2001;32:8-12. 55. Alemfin CL, Mils R, Hernfindez C, et al. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol Lett. 1994;70:77-87. 56. Mesa AR, M~s R, Noa M, et al. Toxicity of policosanol in beagle dogs: One-year study. Toxicol Lett. 1994;73:81-90. 57. Rodrfguez MD, Garcia H. Teratogenic and reproductive studies of policosanol in the rat and rabbit. Teratog Carcinog Mutag. 1994;14:107-113. 58. Alemfin CL, Mils R, Noa M, et al. Carcinogenicity of policosanol in Sprague Dawley rats: A 24-month study. Teratog Carcinog Mutag. 1994;14:239-249. 59. Alem~n C, Puig MN, Elias EC, et al. Carcinogenicity of policosanol in mice: An 18month study. Food Chem Toxicol. 1995;33:573-578. 60. Rodriguez MD, S~nchez M, Garcia H. Multigeneration reproduction study of policosanol in rats. Toxicol Lett. 1997;90:97-106. 61. G~mez R, Alem~n CL, Mas R, et al. A 6-month study on the toxicity of high doses of policosanol orally administered to Sprague-Dawley rats. JMed Food. 2001;4:57-66. 62. M~s R, Rivas P, Izquierdo JE, et al. Pharmacoepidemiologic study of policosanol. Curr Ther Res Clin Exp. 1999;60:458-467. 63. Fernandez L, M~s R, Illnait J, Fern6ndez JC. Policosanol: Results of a postmarketing surveillance study of 27,879 patients. Curt Ther Res Clin Exp. 1998;59:717-722. 64. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502. 65. Seigler L, Wu WT. Separation of serum high-density iipoprotein for cholesterol determination: Ultracentrifugation vs precipitation with sodium phosphotungstate and magnesium chloride. Clin Chem. 1981;27:838-841. 66. Schectman G, Hiatt J. Drug therapy for hypercholesterolemia in patients with cardiovascular disease: Factors limiting achievement of lipid goals. Am JMed. 1996;100: 197-204. 67. O'Brien PC, Shampo MA. Statistical considerations for performing multiple tests in a single experiment. 5. Comparing two therapies with respect to several endpoints. Mayo Clin Proc. 1988;63:1140-1143. 68. Parker TS, McNamara DJ, Brown C, et al. Mevalonic acid in human plasma: Relationship of concentration and circadian rhythm to cholesterol synthesis rates in man. Proc Natl Acad Sci U S A. 1982;79:3037-3041. 69. Jones P, Kafonek S, Laurora l, Hunninghake D. Comparative dose efficacy study of atorvastatin versus simvastatin, pravastatin, lovastatin, and fluvastatin in patients with hypercholesterolemia (the CURVES study). Am J Cardiol. 1998;81:582-587.
302
G. Casta~o et aL
70. Men~ndez R, M~s R, Amor AM, et al. Effects of policosanol treatment on the susceptibility of low density lipoprotein (LDL) isolated from healthy volunteers to oxidative modification in vitro. Br J Clin PharmacoL 2000;50:255-262. 71. Men~ndez R, Mas R, Amor AM, et al. Effects of policosanol on the susceptibility of low-density lipoprotein isolated from hypercholesterolemic patients at high coronary risk to in vitro copper-mediated lipid peroxidation: A randomized, double-blind pilot study. Curr Ther Res Clin Exp. 2000;61:609-620. 72. Kelishadi R, Asgary S, Naderi G, et al. Evaluation of LDL oxide metabolites in children of high risk families. Jpn J Cardiovasc Dis Prev. 2001;36(Suppl):69.
Address correspondence to: R o s a Mils, PhD C e n t e r for Natural P r o d u c t s National C e n t e r for Scientific R e s e a r c h PO Box 6880 Playa, H a v a n a City Cuba E-mail: [email protected]
303