Effectiveness and Safety of Low-Dose

Pravastatin and Squalene, Alone and in

Combination, in Elderly Patients
with Hypercholesterolemia
Paul Chan, MD, PhD, Brian Tomlinson, MD, Ching-Bing Lee, MD, and Ying-Shiung Lee

A double-blind, placebo-controlled study was conducted to compare the efficacy and

safety of low-dose (10 mg) pravastatin and squalene (860 mg), either alone or in combina-
tion therapy, with placebo in the treatment of elderly patients with hypercholesterolemia.
Ambulatory elderly patients (N = 102) were assigned in randomized fashion to receive
active treatment or placebo for 20 weeks after a single-blind placebo lead-in period of 8
weeks. Total cholesterol and triglyceride levels in plasma were at least 250 mgjdL and
less than 300 mgjdL, respectively. Concentrations of lipids and lipoproteins were mea-
sured, and clinical laboratory tests included liver function and creatine kinase determi-
nations. Pravastatin 10 mg daily was more effective than squalene in reducing total cho-
lesterol, low-density lipoprotein (LDL) cholesterol, and trig1ycerides and in increasing lev-
els of high-density lipoprotein (HDL) cholesterol. Combination therapy signijicant1y
reduced total cholesterol and LDL cholesterol and increased HDL cholesterol to a greater
extent than either drug alone. Adverse events and clinical laboratory abnormalities were
generally mild and transient in all groups, and all but two patients jinished the study. The
incidence of side effects was low; myopathy did not occur. Coadministration of pravas-
tatin and squalene combined the specijic effects of the two drugs on lipoprotein concen-
trations. This combination may be useful and more cost-effective in elderly patients with
hypercholesterolemia, who might have a higher incidence of side effects when using
larger doses of pravastatin alone.

he role of elevated total cholesterol, and in par-
T ticular low-density lipoprotein (LDL) cholesterol,
in plasma as important factors contributing to the de-
velopment of atherosclerosis is well established.'
Clinical trials have shown that decreasing choles-
terol concentrations in plasma can contribute to pri-
mary and secondary prevention of coronary events
From the Clinical Pharmacology and Therapeutics Unit, Department of
Cardiology, Taipei Municipal Chung-Hsiao Hospital and the Institute of
Clinical Medicine of National Yang-Ming University, Taipei, Taiwan (Dr.
Chan), the Department of Medicine, Chang-Gung Medical Centre at
Linkou, Taipei, Taiwan (Drs. Lee and Lee); and the Department of Clin-
ical Pharmacology, Chinese University of Hong Kong (Dr. Tomlinson).
Submitted for publication October 10, 1995; accepted in revised form
February 20, 1996. Address for reprints: Paul Chan, Department of
Cardiology, Taipei MuniciPal Chung-Hsiao Hospital, No. 87, Tung Teh
Road, Nan Kang, Taipei, Taiwan 115.
and mortality.v" Although earlier studies have
shown a dose-dependent reduction in LDL choles-
terol with doses of pravastatin up to 40 mg daily,4.5 a
recent study by Celis et al" showed-that pravastatin
10 mg daily was effective in reducing LDLcholesterol
and that increasing the dosage above 10 mg daily did
not result in any further significant reduction of LDL
cholesterol levels. Considering the cost and risk-
benefit ratio of increasing the dosage of pravastatin
in elderly patients, who may experience a higher in-
cidence of side effects from drug therapy, combina-
tion therapy with another drug with mild or negligi-
ble adverse effects may be a better alternative.
Miettinen" has demonstrated that a diet containing
squalene-rich olive oil could effectively reduce se-
rum cholesterol levels, although it markedly in-
creased levels of squalene and other cholesterol pre-
cursors. Squalene, a 30-carbon chain hydrocarbon
with 6 double bonds, is structurally similar to beta-

422 • J CllnPh.rmacoI1996;36:422-427
 PRAVASTATIN AND SQUALENE IN HYPERCHOLESTEROLEMIA

TABLE I
Demolraphy and Baseline Characteristics of the Patients Whose Efficacy Data Were Analyzed
Pravastalln Squalene Combination Placebo Over.1I
(n = 25) (n = 26) (n = 26) (n= 25) (n = 102)

Sex (M/F) 11/14 10/15 11/15 12/13 44/58

Age (Yrs)
mean±SD 74.3 ± 5.7 73.6 ± 5.6 72.6± 6.2 73.8 ± 5.0 73.6± 5.6
range 65-85 65-82 65-89 67-86 65-88
BMI (kgfm 2 ) 24.4 ± 2.5 23.9±2.0 24.2±0.9 23.8±2.6 24.1 ± 2.2
Heart rate (beats/min) 65± 14 67± 13 68± 15 66± 12 67± 13
SittingSBP/DBP (mmHg) 136/82 127/84 131/83 133/85 133/82
Lipidor lipoprotein (mgJdL)
TC 284 ± 14 279 ± 17 279 ± 17 283 ± 20 281 ± 17
LDL-C 207 ± 20 205±20 202 ± 22 208± 24 205 ± 21
HDL-C 46.4 ± 8.1 45.7 ±7.9 46.5±8.4 45.6±8.8 46.1 ± 8.3
TG 135"±46 141 ±40 153 ±42 144 ± 56 148±43
Unlessindicated, valuesare presented as the mean ± standard deviation. BMI, body massindex (weight/heigh~); SBP,
systolic blood pressure; DBP, diastolic blood pressure; Te, total cholesterol; LDL-C, low-density lipoprotein cholesterol;
HDL-C,high-density lipoprotein cholesterol; TG,triglyceride.

carotene and is produced by metabolism of mevalo-
nate in the cholesterol synthesis pathway. It may
have an inhibitory effect on the source of the en-
zymes in this pathway.
This study was undertaken to evaluate whether
the combination of 10 mg pravastatin and 860 mg
squalene daily might have additive properties to
effectively and safely reduce cholesterol levels in el-
derly patients.
PATIENTS AND METHODS
Study Design and Patient Characteristics
The study protocol conformed to the ethical guide-
lines of the 1989 Declaration of Helsinki and was ap-
proved by the investigational review board of Taipei
Chung-Hsiao Hospital and Chang-Gung Hospital. All
patients gave written informed consent for participa-
tion.
The study group included 102 elderly (>65 years
of age) patients with primary hypercholesterolemia
(total cholesterol >250 mgjdL, triglycerides <300
mgjdL) but who were otherwise apparently healthy.
These patients were enrolled after a 3-month period
of dietary intervention with the American Heart As-
sociation (AHA) Step I Diet. t Patients with homozy-
gous familial hypercholesterolemia or type I, ill, IV,
or V hyperlipoproteinemia were excluded, as were
patients with significant cardiovascular, renal, gas-
trointestinal, hepatic, or metabolic diseases, such as
diabetes mellitus, malignancies, or those expected to
to have a life span limited to less than 3 years.
This study was a double-blind, randomized, pla-
cebo-controlled trial conducted at Chung-Hsiao Hos-
pital and Chang-Gung Hospital, Taipei, Taiwan. Any
previous treatment with lipid-lowering drugs was
discontinued for 2 months before randomization.
Placebo tablets of pravastatin and placebo squalene
capsules were subsequently administered for a min-
imum of 4 weeks along with dietary treatment. All
patients then began taking one of the following with
their evening meal for a 20-week period: 1) 10 mg
pravastatin; 2) 2 capsules (430 mg each) of squalene
(Goldian Co., Singapore); 3) a combination of pravas-
tatin and squalene; or 4) matching placebo. Plasma
samples obtained after fasting were analyzed for total
cholesterol, high-density lipoprotein (HDL) choles-
terol, and triglycerides. Levels of LDL cholesterol
were calculated using the Friedewald equation."
Efficacy, Safety Measurement, and Compliance
Total cholesterol, HDL cholesterol, and triglyceride
levels were determined at baseline after the 4-week
lead-in placebo treatment and again after 4, 8, 12,
and 20 weeks of the active treatment (LDL choles-
terollevels were determined by calculation). During
each visit, blood also was obtained for full laboratory
testing, including liver function and creatine kinase
tests. During each visit the patients were encouraged
to report any side effects and were given a complete

CARDIOVASCULAR 423
 CHAN ET AL

TABLE II
Lipid Concentrations at Baseline and after 20 Weeks Treatment, and Percentale Chanle
Treatment Group
Pravastatln Squalene Combination Placebo
(n = 25) (n = 26) (n = 26) (n = 25)

TC(mgfdL)
baseline 284± 14 279 ± 17 279± 17 283 ± 20
week 20 217 ± 15 231 ± 13 177± 9 274± 13
percentage change -23.5*tt§ -17.3*tt~ -36.6*t§~ -3.0
LDL-C (mgfdL)
baseline 207 ± 20 205 ± 20 202 ± 23 208±22
week 20 148 ± 16 159 ± 16 108 ± 11 200± 13
percentage change -28.5*tt§ -22.2*tt~ -46.5*t§~ -3.1
TG(mgfdL)
baseline 135.0±46 142±40 153 ±42 144±46
week 20 123.0± 28 134.6 ± 24 132 ± 22 139± 27
percentage change -9.1* -5.3* -10.9*t -0.2
HDL-C(mgfdL)
baseline 46.4 ± 8.1 45.7 ± 7.9 46.4±8.4 45.6±8.8
week 20 49.5 ± 7.3 46.3 ± 7.5 SO.S ± 7.2 4S.2 ± 8.2
percentage change 6.S*t I.S 8.S*t -O.S
Percentage change given as geometric means; concentrations are presented as the mean ± standard deviation. TC,
total cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein choles-
terol.
• P < 0.05 versus baseline.
t P < 0.05 versus placebo.
* P < 0.05 versus combination.
§ P < 0.05 versus squalene.
, P < 0.05 versus pravastatin.

physical examination, including chest roentgeno- RESULTS

gram and 12-lead electrocardiogram. Urine and he-
matology analyses were performed at the same in-
tervals during the study. Adherence to the pre-
scribed drugs was assessed by counting of tablets.
Statistics
All P values and significance levels were generated
by two-sided comparisons. Between-group compari-
sons of demographic and baseline lipid variables
were performed by means of analysis of variance
(ANQVA) for continuous variables such as age and
chi-square tests for discrete variables such as gender.
Analysis of covariance (ANCQVA) was used to assess
percentage change from baseline after log transfor-
mation. Percentage change from baseline is reported
as the geometric mean adjusted for the effects of the
model. Clinical laboratory data were analyzed by
means of ANCOVA after log transformation. Sub-
stantiallaboratory abnormalities and adverse events
were compared using chi-square tests.
The 102 patients (44 men, 58 women) were divided
into the following four treatment groups: pravastatin
(n = 25), squalene (n = 26), combination (n = 26), or
placebo (n = 25). No significant differences were
found between treatment groups in any of the base-
line demographic data (Table I). The mean age of all
the patients in the 4 groups was 73.6 years. Two pa-
tients (one taking placebo and one taking pravas-
tatin) discontinued treatment prematurely because
of musculoskeletal pain and moderate elevation of
creatinine kinase levels (less than three times the up-
per limit of normal).
Baseline lipoprotein and lipid levels did not differ
significantly between groups (Table I). After treat-
ment with pravastatin 10 mg daily for 20 weeks, total
cholesterol was reduced by 23.5% (P <0.05), LDL
cholesterol by 28.5%, and triglycerides by 9.1%; HDL
cholesterol levels were increased by 6.5% (P <0.05;
Table II). After treatment with squalene 860 mg
daily, total cholesterol was decreased by 17.3%, LDL
cholesterol by 22.2°k, and triglycerides by 5.30/0; HDL

424 • J Clln PharmacoI1996;36:422-427

 PHAVASTA TIN AND SQUALENE IN HYPERCHOLESTEROLEMIA

TABLE III
Most Common Adverse Events (by No. of Patients)
Tre.tment Group
Pr.v.lt8tln Squalene Com&ln.tlon PIKebo
AdverseEvents (n = 25) (n = 26) (n = 26) (n = 25)

Musculoskeletal pain 2 (1*) o 1 2 (1*)

Abdominal pain 1 o 1 1
Diarrhea 1 1 1 1
Gastritis o o o o
Dizziness 1 o 1 1
Epigastric pain o o o 1
Dyspepsia/heartburn 1 o 1 1
Fatigue 1 o 1 1
Headache 1 o 1 1
Malaise 1 o 1 1
Markedcreatine kinase abnormalitiest o o o o
• Premature withdrawal.
t Marked abnormality = >3 X pretreatment values on one or more occasions.

cholesterol only tended to increase by 1.8%. After
treatment with both pravastatin and squalene, total
cholesterol was decreased by 36.6 % and LDLcholes-
terol by 46.5%, a greater effect than with either drug
alone, and triglycerides were decreased by 10.9%.
Levels of HDL cholesterol increased 8.8% with com-
bination treatment, but this increase was not signifi-
cantly greater than the increases seen with either
drug alone. No significant changes in lipid profile
were noted in the group taking placebo except for a
small reduction in LDLcholesterol levels.
Low-dose (10 mg) pravastatin and 860 mg squalene
were well tolerated, both alone and in combination,
and almost all of the patients completed the study.
Although side effects were recorded from each treat-
ment group, the effects were minor and infrequent,
and no different from those that occurred with pla-
cebo. Premature withdrawal from the study was nec-
essary for one patient taking pravastatin and one tak-
ing placebo who experienced musculoskeletal pain
and slight elevation of creatine kinase levels «3
times higher than baseline levels).
DISCUSSION
These data show that low-dose (10 mg) pravastatin
was well tolerated by the elderly patients with hy-
percholesterolemia in this study, and that efficacy
also was satisfactory. In a previous study of the 3-hy-
droxyl-3-methyl-glutaryl coenzyme A (HMG-CoA)
reductase inhibitor lovastatin in the treatment ofhy-
percholesterolemia in younger patients (mean age
58.4 years), increasing the dosage from 20 mg to 80
mg daily resulted in only a minor increase in side
effects." Special attention is required with many
medications when prescribed to elderly patients be-
cause of age-related changes in pharmacokinetics
and pharmacodynamics," and the risk of untoward
effects in these patients can be lessened by reducing
the dose, sometimes without much impact on thera-
peutic effect.
A dose-dependent reduction in total cholesterol
(up to 26%) and LDL cholesterol (up to 34%) levels
has been demonstrated with doses of 5 to 40 mg pra-
vastatin daily in short-term, dose-response studies.v"
However, a recent study found that this effect did not
increase further when the dose was increased be-
yond 10 mg daily in patients with hypercholesterol-
emia taking antihypertensive treatment." Similarly,
our previous experience in a similar group of elderly
Chinese patients showed 10 mg pravastatin to be
effective and well tolerated." Moreover, the West of
Scotland Coronary Prevention Study" showed a
long-term reduction in total cholesterol of 20% and
in LDL cholesterol of 26% with' pravastatin 40 mg
daily, a decrease that was less than that observed in
the present study with 10 mg daily. Elderly patients
appear to respond similarly to younger patients to
doses of 20 or 40 mg daily of pravastatin, as described
in pooled data from multicenter short-term studies."
There was no increase in side effects in older subjects
or with higher doses in this report, but increases in
liver transaminases during pravastatin treatment
have been found to be dose-dependent."

CARDIOVASCULAR 425
 CHAN ET AL
Low-dose, combination cholesterol-lowering drug
therapy has been advocated to reduce the risk of the
toxic effects of higher doses, and the use of a low dose
of statin alone with another less-expensive agent
would increase the cost-effectiveness of therapy."
The addition of a different agent to a low dose of
statin may have a greater lipid-lowering effect than
increasing the dose of statin. The overall reductions
in cholesterol or LDL cholesterol levels with combi-
nation treatment found in this study are similar to or
greater than those found in studies combining pra-
vastatin with cholestyramine, nicotinic acid, or gem-
fibrozil, and the incidence of side effects was much
higher with these other agents.":":" The combina-
tion of a statin with fish oil may have additional ben-
efits without major adverse effects, but is unlikely to
reduce LDL cholesterol levels to the extent seen with
our combination of pravastatin with squalene."
The dosage of squalene chosen was the standard
recommended by the manufacturer, and this partic-
ular combination of doses may not be the ideal for
maximum cost-effectiveness; further dose-ranging
studies are needed to examine this in more detail. It
is likely that the interaction between these two
agents is pharmacodynamic, although we have not
studied whether pharmacokinetic interactions oc-
cur.
People living in Mediterranean countries have a
low incidence of coronary heart disease (CHD), and
this phenomenon has been linked with the high in-
take of olive oil in this region." In addition to its
monosaturated fatty acids, olive oil also contains a
substantial amount of squalene; dietary intake of
squalene can be as high as 200 mg daily." Squalene
is a 30-hydrocarbon nonsteroid intermediate of cho-
lesterol synthesis preceding the formation of the ste-
roid nucleus, and when taken orally may be con-
verted to cholesterol. 21 Although a substantial
amount of dietary squalene is absorbed and con-
verted to cholesterol in humans, this squalene-in-
duced increase in synthesis is not associated with
consistent increases in serum cholesterol levels."
Interestingly, dietary squalene can actually reduce
serum levels of total cholesterol and LDL cholesterol,
and increase levels ofHDL cholesterol." The mecha-
nism by which dietary squalene can reduce serum
cholesterol levels may be due to the down-regulation
of HMG-CoA reductase activity by enhanced squa-
lene-derived synthesis of cholesterol, depending on
the amount and absorption' of dietary squalene."
This phenomenon may contribute to the low inci-
dence of CHD in Mediterranean populations.
This study indicates that a combination of 860 mg
squalene and low-dose (10 mg) pravastatin is a safe
and effective method of treating elderly patients with
426 • J Clln Ph.rmacoI1996;36:422-427
hypercholesterolemia. To our knowledge, this is the
first clinical report to demonstrate the benefits of this
combination.
REFERENCES
1. Cholesterol Adult Treatment Panel: Report of the National
Cholesterol Education Program expert panel on detection, evalu-
ation, and treatment of high blood cholesterol in adults. Arch In-
tern Med 1988;148:36-69.
2. Frick MH, Elo 0, Haapa K, Heinonen OP, Helo P, Huttunen JK,
et al, for the Helsinki Heart Study: Primary prevention trial with
gemfibrozil in middle-aged men with dyslipidemia: safety of treat-
ment, changes in risk factors, and incidence of coronary heart dis-
ease. N Engl JMed 1987;317:1237-1245.
3. Pederson TR: Randomised trial of cholesterol lowering in 4444
patients with coronary heart disease: the Scandinavian Simvas-
tatin Survival Study (4S). Lancet 1994;344:1383-1389.
4. Hunninghake DB, Knopp RH, Schonfeld G, Goldberg AC,
Brown WV, Schaefer EJ,et al: Efficacy and safety ofpravastatin in
patients with primary hypercholesterolemia. I. A dose-response
study. Atherosclerosis 1990;85:81-89.
5. Jones PH, Farmer JA, Cressman MD, McKenny JM, Wright fT,
Proctor JD, et al: Once-daily pravastatin in patients with primary
hypercholesterolemia: a dose-response study. Clin Cardiol1991;
14:146-151.
6. Celis H, Lijnen P, Fagard R, Fagard R, Staessen J,Thijs L,Amery
A: Efficacy and safety of pravastatin in hypertensive hypercholes-
terolemic patients on antihypertensive therapy. J Hum Hypertens
1994;8:525-530.
7. Miettinen TA: Dietary squalene related to cholesterol and lipo-
protein metabolism. In, Fidge NH, Nestel PJ (eds.): Atherosclerosis
VII: Proceedings of the 7th International Atherosclerosis Sympo-
sium, Melbourne, Australia, 1985. Amsterdam: Elsevier, 1986;
671-674.
8. Fredrickson OS:Estimation of the concentration of low-density
lipoprotein cholesterol in plasma, without use of preparative ul-
tracentrifuge. Clin Chern 1972;18:499-502.
9. Bradford RH, Downton M, Chremos AN, Langendorfer A, Stin-
nett S, et al: Efficacy and tolerability of lovastatin in 3390 women
with moderate hypercholesterolemia. Ann Intern Med 1993;118:
850-855.
10. Greenblatt OJ,Sellers EM, Shader RI: Drug disposition in old
age. N Engl J Med 1982;306:1081-1088.
11. Chan P, Lee CB, Ko JT, Lin TZ, WH Pan, Lee YS: The
effectiveness and safety of low dose pravastatin in elderly hyper-
cholesterolemic subjects on antihypertensive therapy. Am J Hy-
pertens 1995;8:1099-1104.
12. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, Macfar-
lane PW, et al: Prevention of coronary heart disease with pravas-
tatin in men with hypercholesterolemia. N Engl J Med 1995;333:
1301-1307.
13. Mellies MJ, DeVault AR, Kassler-Taub K, McGovern ME, Pan
HY: Pravastatin experience in elderly and non-elderly patients.
Atherosclerosis 1993;101:97-110.
14. Newman TJ, Kassler-Taub KB, Gelarden RT, Korzin EG, De-
Vault AR, McGovern ME, Pan HY: Safety of pravastatin in long-
term clinical trials conducted in the United States. J Drug Dev
1990;3(suppI1):274-281.
15. Denke MA, Grundy SM:Efficacyof low-dose, cholesterol-low-
ering drug therapy in men with moderate hypercholesterolemia.
Arch Intern Med 1995;155:393-399.
 PRAVASTATIN AND SQUALENE IN HYPERCHOLESTEROLEMIA
18. Davignon J, Roederer G, Montigny M, Hayden MR, Tan M-H,
Connelly PW, et al: Comparative efficacy and safety of pravastatin,
nicotinic acid and the two combined in patients with hypercho-
lesterolemia. Am JCardioI1994; 73:339-345.
17. Wiklund 0, Angelin B, Bergman M, Berglund L, Bonders G,
Carlsson A, et al: Pravastatin and gemfibrozil alone and in combi-
nation for the treatment of hypercholesterolemia. Am J Med
1993;94:13-20.
18. Sanset PM, Lund H, Norseth J, Abidgaard U, Ose L:Treatment
with hydroxylmethylglutaryl-coenzyme A reductase inhibitors in
hypercholesterolemia induces changes in the components of the
extrinsic coagulation system. Arterioscler Thromb 1991;11:138-
145.
CARDIOVASCULAR
19. Keys A, Menotti A, Karvonen J, Aravanis C, Blackburn H, Buz-
ina R, et al: The diet and 15-year death rate in the seven countries
study. Am JEpidemio11986;124:903-915.
20. Liu GCK,Ahrens EH Jr, Schreibman PH, Crouse JR: Measure-
ment of squalene in human tissues and plasma: validation and ap-
plication. J Lipid Res 1976;17:38-45.
21. Gylling H, Miettinen TA: Postabsorptive metabolism of di-
etary squalene. Atherosclerosis 1994;106:169-178.
22. Stranberg TE, Tilvis RS, Miettinen TA: Metabolic variables of
cholesterol during squalene feeding in humans: comparison with
cholestyramine treatment. J Lipid Res 1990;31:1637-1643.
23. Miettinen TA, Vanhanen H:Serum concentration and metab-
olism of cholesterol during rapeseed oil and squalene feeding. Am
J Clin Nutr 1994;59:356-363.
427