The Hypolipidemic
Effects of Lovastatin and Clofihrate Alone and in Combination in Patients With Type III Hyperlipoproteinemia D. Roger lllingworth and Jean P. O’Malley
The hypolipidemic effects of lovastatin and clofibrate have been evaluated in 12 patients with type Ill hyperlipoproteinemia. In these patients plasma concentrations of total cholesterol decreased from 500 ? 56 mg/dL (mean + SEMI at baseline to 276 + 23 mg/dL on lovastatin (20 mg twice daily), and were 299 + 15 mg/dL during treatment with clofibrate (1 g twice daily). Nine patients were treated sequentially with lovastatin at doses of 20 and 40 mg twice daily and clofibrate; in these patients total plasma cholesterol concentrations decreased from 549 * 67 mg/dL at baseline to 291 _t 24 mg/dL on lovastatin (20 mg twice daily), 247 k 20 mg/dL (40 mg twice daily) and were 297 + 16 mg/dL on monotherapy with clofibrate. Concentrations of very-low-density lipoprotein (VLDL) cholesterol were similar on clofibrate and the higher dose of lovastatin, whereas concantrations of low-density lipoprotein (LDL) cholesterol were significantly lower on lovastatin. In six patients who remained hyperlipidemic on monotherapy with either drug, combination drug therapy with lovastatin (20 mg twice daily) plus clofibrete reduced plasma concentrations of total cholesterol from 635 f 79 mg/dL to 205 * 11 mg/dL. No patients were discontinued from single or combined drug therapy and no significant biochemical abnormalities were observed. The results of this study demonstrate the potential usefulness of lovastatin in the therapy of type Ill hyperlipoproteinemia and indicate that, in selected patients who remain hypercholesterolemic on monotherapy with either clofibrate or lovastatin, combination drug therapy with both of these drugs is effective in further reducing plasma concentrations of total, VLDL, and LDL cholesterol. Although none of the six patients treated with lovastatin plus clofibrate developed myopathy, we would urge caution in the use of this combination in view of the known increased risk of myopathy associated with the use of lovastatin plus gemfibrozil. 0 1990 by W.B. Saunders Company.
YPE III hyperlipoproteinemia (dysbetalipoproteinemia) is an uncommon disorder of lipid metabolism characterized by the accumulation of very-low-density lipoprotein (VLDL) remnants in plasma.‘*’ Patients with type III hyperlipoproteinemia are at substantially increased risk for the premature development of coronary and peripheral vascular disease and frequently develop palmar and tuberous xanthomas.’ The latter regress in response to effective hypolipidemic therapy. The pathophysiologic mechanisms responsible for the development of type III hyperlipoproteinemia have been elucidated and the majority of patients with this disorder are homozygous for apoprotein E2, which has a diminished affinity for binding to low-density lipoprotein ([LDL] and presumably apoprotein E) receptors.’ Although homozygosity for apoprotein E2 is present in 1% of the population,3 development of type III hyperlipoproteinemia requires the concurrent presence of an apoprotein E variant with a reduced receptor binding affinity together with an increase in the hepatic production of VLDL. Increases in the hepatic production of VLDL can be due to secondary factors such as obesity or type II diabetes or may be due to the concurrent presence of a separate monogenic lipid disorder such as familial combined hyperlipidemia. Treatment of patients with type III hyperlipoproteinemia who remain hyperlipidemic following the correction of secondary factors and maximal dietary therapy invariably involves the use of lipid-lowering drugs. Previous studies have indicated that fibrate drugs (clofibrate, gemfibrozil, or fenofibrate) are effective in the treatment of patients with type III hyperlipoproteinemia,4’6 as is nicotinic acid.’ Two recent report&g also have indicated that lovastatin, a specific inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase, is effective in the therapy of patients with type III hyperlipoproteinemia. In the present report, we have evaluated the comparative efficacy of lovastatin and clofi-
T
Metabolism, Vol39, No 4 (April), 1990: pp 403-409
brate in the treatment of 12 patients with well-characterized type III hyperlipoproteinemia and, in six patients who remained hyperlipidemic on monotherapy with either drug, have assessed the potential efficacy of combined drug therapy with lovastatin plus clofibrate. Our results indicate that both drugs are effective in the treatment of patients with type III hyperlipoproteinemia, but in those patients who remain hyperlipidemic on single-drug therapy with either agent, combination drug therapy with lovastatin and clofibrate produces an additive lipid-lowering effect. MATERIALS AND METHODS
Subjects
The efficacyof lovastatin and clofibrate was evaluated in 12 adult patients with well-characterized type III hyperlipoproteinemia (Table 1). The ratio VLDL cholesterol divided by total plasma triglycerides exceeded 0.3 (mean * SEM, 0.41 * 0.03) in all patients. Ten of the 12 patients were homozygous for apoprotein E2, whereas one patient each had the apoprotein E3, E2 and apoprotein E4, E2 phenotypes (Table 1). All patients were habituated to a low fat, low cholesterol diet containing less than 30% of calories in total fat and less than 300 mg/d of cholesterol; dietary compliance was stable throughout the period of $udy as assessed by diet questionnaires. All patients had normal tests of thyroid, renal, and hepatic function; three patients had stable diabetes (one on insulin [patient no. 41, the
From the Division of Endocrinology, Metabolism, and Clinical Nutrition, Department of Medicine, The Oregon Health Sciences University, Portland, OR. Supported in part by National Institutes of Health Research Grants No. HL28399 and HL37940 and by the General Clinical Research Center’s Program (RR334). Address reprint requests to D. Roger Illingworth. MD, PhD, Department of Medicine, L465, The Oregon Health Sciences University, Portland, OR 97201. Q1990 by W.B. Saunders Company. 00260495/90/3904-OOl2$3.00/0
403
ILLINGWORTH AND O’MALLEY
404
Table 1. Characteristics
of the Study Subjects
Plasma Cholesterol Patient
No.
Age/Sex
(yr)
Weight
(kg)
1
62/M
79
2
43/F
55.3
Percent
IBW
115
91
Total
VLDL
(mg/dL)
TG
LDL
HDL
lmg/dLl
Ratio VLDL Cholesterol:TG
Apoprotein Phenotype
E CAD/ PVD
700
434
115
26
1,395
0.311
EZ/EZ
-I+
612
342
192
37
786
0.435
EZ/EZ
-I-
3
61/F
77.1
127
520
394
83
31
1,028
0.383
EZ/EZ
+I-
4
64/F
55.8
100
360
164
172
31
288
0.569
EZ/EZ
iI+
5
49/M
83.0
119
385
235
117
31
745
0.315
EZ/EZ
+I-
6
46/M
85.61
127
364
134
212
37
416
0.322
E3/E2
+/-
7
65/F
78.2
135
406
261
88
48
710
0.367
EZ/EZ
+I-
8
44/M
85.3
120
446
248
145
53
510
0.486
EZ/EZ
-I+
9
41/M
98.4
130
572
444
93
31
810
0.548
EZ/EZ
-i-
10
52/F
81.6
137
1,015
752
152
46
1,880
0.400
EZ/EZ
-/-
276
143
31
11
42/M
109.1
144
478
12
36/M
119.7
151
318
Mean r SEM 50.4
+ 2.9
84.0
k 5.4
124.8
f 5.0
108 515 * 56 316 L 51
196 30 142.3 t 13 36 * 2.5
676 320 797 r 133
0.408
EZ/EZ
-/-
0.338 0.41 + .03
E4/E2
-i-
Abbreviations: IBW, ideal body weight; TG, triglycerides; CAD, coronary artery disease; PVD, peripheral vascular disease.
other two diet controlled [patients no. 3 and 51). Four patients were taking drugs known to effect lipid metabolism (estrogens [patients no. 2 and 71, &blockers [patients no. 6 and 71). but these remained unchanged throughout the period of study. Informed consent was obtained from all patients and the protocols were approved by the Human Research Committee of The Oregon Health Sciences University and by the US Food and Drug Administration. All patients were seen as outpatients in the Clinical Research Center or Lipid Disorders Clinic at The Oregon Health Sciences University.
Study Protocol Prior to entry into the study, all patients underwent a complete physical examination and an eye examination, and blood samples were obtained to exclude secondary causes of hyperlipidemia. Lipid-lowering drugs were withdrawn for a period of 1 month before entry into the baseline period of the study. During the baseline period, which lasted 1 month, two blood samples were obtained for lipid and lipoprotein studies; patients were then begun on therapy with lovastatin at a dose of 20 mg twice daily. Patients were seen at monthly intervals for a period of 3 months. At each visit, brief physical examinations were conducted, patient compliance to medications assessed by verbal questions, and blood samples obtained for lipid and lipoprotein studies and to assess clinical chemistry parameters and hematologic studies. In nine of the 12 patients, the dose of lovastatin was increased to 40 mg twice daily, whereas in the other three patients treatment with lovastatin was discontinued for 1 month and the patients then begun on clofibrate at a dose of 1 g twice daily. The nine patients who remained on monotherapy with lovastatin at a dose of 40 mg twice daily were seen at monthly intervals for a period of 3 months, at which time they were evaluated as on the lower dose of lovastatin. Lovastatin therapy in these nine patients was then discontinued for a period of 1 month, after which treatment with clofibrate was initiated at a dose of 1 g twice daily. Due to the fact that several patients who participated in this trial lived a considerable distance from Portland, OR, blood samples were not routinely obtained during the washout period and patients were seen again after 1 month on single-drug therapy with clofibrate. However, based on previous studies with lovastatin,‘a,” the period off therapy with this drug should have been sufficient to ensure a return to baseline, whereas the time period on clofibrate was sufficient to enable a new steady-state to be obtained. Six of the 12 patients showed persistent hypercholesterolemia (>220 mg/dL) on monotherapy with lovastatin at doses of 20 or 40 mg twice daily on clofibrate and all six of these patients were subsequently treated with combined drug therapy using lovastatin (20 mg twice daily) and clofibrate.
Family studies were compatible with a diagnosis of familial combined hyperlipidemia in three of the six patients in whom sufficient family information could be obtained to further characterize the genotypic reasons responsible for their severe hyperlipidemia (patients no. 1,2, and 10 in Table 1). The hypolipidemic effects of combined drug therapy with lovastatin plus clofibrate were assessed over a 3-month period in which patients were seen at monthly intervals and blood tests obtained as previously described. Patients were advised to take lovastatin and clofibrate with breakfast and with their evening meal and, in patients treated with both drugs in combination therapy, they were advised to take the drugs at the same time. Patients were seen in the morning after having fasted for at least 12 hours, and had not taken their medications prior to being seen.
Laboratory Methods Blood samples for lipid and lipoprotein analysis were obtained in tubes containing 1 mg/dL of EDTA anticoagulant and plasma was separated at 4“C in a refrigerated centrifuge. Total concentrations of plasma cholesterol and triglyceride were determined with an autoanalyzer,’ and lipoproteins were separated by a combination of ultracentrifugation and heparin manganese precipitation of plasma.” In all cases, VLDL was initially removed by ultracentrifugation of plasma at density 1.006 g/mL and the concentration of cholesterol and triglycerides in the lipoprotein measured directly. High-density lipoprotein (HDL) concentrations were then measured in the supernatant following precipitation of apoprotein B-containing lipoproteins with heparin manganese and the concentration of LDL cholesterol was calculated.” Lipid determinations were standardized with samples of known composition obtained from the Center for Disease Control, Atlanta, GA. Biochemical assessments included total bilirubin, alkaline phosphatase, aspartate amino transferase, creatinine, total serum protein, uric acid, serum glucose, and electrolytes. In addition, a complete blood cell count was performed periodically. Apoprotein E phenotypes were determined using a modification of the slab gel isoelectric focusing of method of Assmann et al” on delipidated VLDL. After isolation from plasma, VLDL samples were delipidated with 1:l chloroform:methanol and the protein pellets washed with an additional aliquot of these solvents. The pellets were dried under nitrogen and solubilized for 1 hour at 37’C in 200 rL of fresh sample buffer (20% wt/vol sucrose, 1% wt/vol decyl sodium sulfate, 2% vol/vol Serva pH4-7 ampholines (Serva Fine Chemicals, Westbury, NY), 2% vol/vol fl-mercaptoethanol). Samples were subjected to isoelectric focusing with pH4:7 ampho-
LOVASTATIN
IN TYPE III HYPERLIPOPROTEINEMIA
405
lines in a Hoefer flat-bed electrophoresis chamber (Hoefer Scientific Institute, San Francisco, CA). The gels were run overnight at 250 V and stained for 4 hours in 0.05 wt/vol Crocein Scarlet, 0.04% wt/vol Coomassie brilliant blue, 16 mmol/L cupric sulfate in 10% acetic acid, 27% isopropanol.
Table 2. The Influence of Lovastatin and Clofibrate on Plasma Lipids and Lipoproteins in 12 Patients With Type 81Hyperlipoproteinemia Plasma Cholesterol (mg/dL) Total
Statistical Analysis
Baseline (A)
Statistical analysis was performed by one-way ANOVA with repeated measures followed by the Neuman Keuls post hoc testI
Lovastatin (B)
concentrations of cholesterol, triglyceride, and VLDL decreased rapidly when patients were started on drug therapy with lovastatin, and stable values were reached after 1 month on single-drug therapy. The stability of cholesterol and triglyceride concentrations during three successive monthly visits on lovastatin at doses of 20 and 40 mg twice daily, three visits at monthly intervals during therapy with clofibrate, and three visits on combination drug therapy with lovastatin and clofibrate are illustrated in Fig 1. ANOVA with repeated measures of the data from these six patients shows no significant differences between the concentrations of cholesterol and triglycerides during three consecutive visits on the same treatment regimen. This analysis provides strong evidence that the decreases that occur in response to changes in drug dosage or with combination drug therapy represent the effects of this therapeutic change rather than a carry-over effect due to progressive further decreases in lipid and lipoprotein concentrations during prolonged drug therapy on a given treatment regimen. The comparative efficacy of lovastatin (20 mg twice daily) and clofibrate (1 g twice daily) on plasma concentrations of lipids and lipoproteins in 12 patients with type III hyperlipoproteinemia is shown in Table 2. For each patient, the baseline data represent the results from blood samples obtained on two outpatient visits on diet only, whereas the values for each patient on lovastatin and clofibrate are from three outpatient visits on each drug, respectively. Significant Plasma
Lovastatin 40 mg BID
20 mg BID
HDL
TG lme/dL)
139 + 13 35 t 2 788 + 136
(20 mg 278
r 23
142 + 83
94 zt 6
44 + 3 454
+ 83
Clofibrate (Cl
RESULTS
Base In?
LDL
500 t 56 301 f 48
BID)
A
VLDL
Clofibrate
(1 g BID)
299 + 15
116 + 16 138 f
10 46 + 3 378 + 54
P value AvB
Effects of Lovastatin and Clofihrate Alone and in Combination in Patients With Type III Hyperlipoproteinemia D. Roger lllingworth and Jean P. O’Malley
The hypolipidemic effects of lovastatin and clofibrate have been evaluated in 12 patients with type Ill hyperlipoproteinemia. In these patients plasma concentrations of total cholesterol decreased from 500 ? 56 mg/dL (mean + SEMI at baseline to 276 + 23 mg/dL on lovastatin (20 mg twice daily), and were 299 + 15 mg/dL during treatment with clofibrate (1 g twice daily). Nine patients were treated sequentially with lovastatin at doses of 20 and 40 mg twice daily and clofibrate; in these patients total plasma cholesterol concentrations decreased from 549 * 67 mg/dL at baseline to 291 _t 24 mg/dL on lovastatin (20 mg twice daily), 247 k 20 mg/dL (40 mg twice daily) and were 297 + 16 mg/dL on monotherapy with clofibrate. Concentrations of very-low-density lipoprotein (VLDL) cholesterol were similar on clofibrate and the higher dose of lovastatin, whereas concantrations of low-density lipoprotein (LDL) cholesterol were significantly lower on lovastatin. In six patients who remained hyperlipidemic on monotherapy with either drug, combination drug therapy with lovastatin (20 mg twice daily) plus clofibrete reduced plasma concentrations of total cholesterol from 635 f 79 mg/dL to 205 * 11 mg/dL. No patients were discontinued from single or combined drug therapy and no significant biochemical abnormalities were observed. The results of this study demonstrate the potential usefulness of lovastatin in the therapy of type Ill hyperlipoproteinemia and indicate that, in selected patients who remain hypercholesterolemic on monotherapy with either clofibrate or lovastatin, combination drug therapy with both of these drugs is effective in further reducing plasma concentrations of total, VLDL, and LDL cholesterol. Although none of the six patients treated with lovastatin plus clofibrate developed myopathy, we would urge caution in the use of this combination in view of the known increased risk of myopathy associated with the use of lovastatin plus gemfibrozil. 0 1990 by W.B. Saunders Company.
YPE III hyperlipoproteinemia (dysbetalipoproteinemia) is an uncommon disorder of lipid metabolism characterized by the accumulation of very-low-density lipoprotein (VLDL) remnants in plasma.‘*’ Patients with type III hyperlipoproteinemia are at substantially increased risk for the premature development of coronary and peripheral vascular disease and frequently develop palmar and tuberous xanthomas.’ The latter regress in response to effective hypolipidemic therapy. The pathophysiologic mechanisms responsible for the development of type III hyperlipoproteinemia have been elucidated and the majority of patients with this disorder are homozygous for apoprotein E2, which has a diminished affinity for binding to low-density lipoprotein ([LDL] and presumably apoprotein E) receptors.’ Although homozygosity for apoprotein E2 is present in 1% of the population,3 development of type III hyperlipoproteinemia requires the concurrent presence of an apoprotein E variant with a reduced receptor binding affinity together with an increase in the hepatic production of VLDL. Increases in the hepatic production of VLDL can be due to secondary factors such as obesity or type II diabetes or may be due to the concurrent presence of a separate monogenic lipid disorder such as familial combined hyperlipidemia. Treatment of patients with type III hyperlipoproteinemia who remain hyperlipidemic following the correction of secondary factors and maximal dietary therapy invariably involves the use of lipid-lowering drugs. Previous studies have indicated that fibrate drugs (clofibrate, gemfibrozil, or fenofibrate) are effective in the treatment of patients with type III hyperlipoproteinemia,4’6 as is nicotinic acid.’ Two recent report&g also have indicated that lovastatin, a specific inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase, is effective in the therapy of patients with type III hyperlipoproteinemia. In the present report, we have evaluated the comparative efficacy of lovastatin and clofi-
T
Metabolism, Vol39, No 4 (April), 1990: pp 403-409
brate in the treatment of 12 patients with well-characterized type III hyperlipoproteinemia and, in six patients who remained hyperlipidemic on monotherapy with either drug, have assessed the potential efficacy of combined drug therapy with lovastatin plus clofibrate. Our results indicate that both drugs are effective in the treatment of patients with type III hyperlipoproteinemia, but in those patients who remain hyperlipidemic on single-drug therapy with either agent, combination drug therapy with lovastatin and clofibrate produces an additive lipid-lowering effect. MATERIALS AND METHODS
Subjects
The efficacyof lovastatin and clofibrate was evaluated in 12 adult patients with well-characterized type III hyperlipoproteinemia (Table 1). The ratio VLDL cholesterol divided by total plasma triglycerides exceeded 0.3 (mean * SEM, 0.41 * 0.03) in all patients. Ten of the 12 patients were homozygous for apoprotein E2, whereas one patient each had the apoprotein E3, E2 and apoprotein E4, E2 phenotypes (Table 1). All patients were habituated to a low fat, low cholesterol diet containing less than 30% of calories in total fat and less than 300 mg/d of cholesterol; dietary compliance was stable throughout the period of $udy as assessed by diet questionnaires. All patients had normal tests of thyroid, renal, and hepatic function; three patients had stable diabetes (one on insulin [patient no. 41, the
From the Division of Endocrinology, Metabolism, and Clinical Nutrition, Department of Medicine, The Oregon Health Sciences University, Portland, OR. Supported in part by National Institutes of Health Research Grants No. HL28399 and HL37940 and by the General Clinical Research Center’s Program (RR334). Address reprint requests to D. Roger Illingworth. MD, PhD, Department of Medicine, L465, The Oregon Health Sciences University, Portland, OR 97201. Q1990 by W.B. Saunders Company. 00260495/90/3904-OOl2$3.00/0
403
ILLINGWORTH AND O’MALLEY
404
Table 1. Characteristics
of the Study Subjects
Plasma Cholesterol Patient
No.
Age/Sex
(yr)
Weight
(kg)
1
62/M
79
2
43/F
55.3
Percent
IBW
115
91
Total
VLDL
(mg/dL)
TG
LDL
HDL
lmg/dLl
Ratio VLDL Cholesterol:TG
Apoprotein Phenotype
E CAD/ PVD
700
434
115
26
1,395
0.311
EZ/EZ
-I+
612
342
192
37
786
0.435
EZ/EZ
-I-
3
61/F
77.1
127
520
394
83
31
1,028
0.383
EZ/EZ
+I-
4
64/F
55.8
100
360
164
172
31
288
0.569
EZ/EZ
iI+
5
49/M
83.0
119
385
235
117
31
745
0.315
EZ/EZ
+I-
6
46/M
85.61
127
364
134
212
37
416
0.322
E3/E2
+/-
7
65/F
78.2
135
406
261
88
48
710
0.367
EZ/EZ
+I-
8
44/M
85.3
120
446
248
145
53
510
0.486
EZ/EZ
-I+
9
41/M
98.4
130
572
444
93
31
810
0.548
EZ/EZ
-i-
10
52/F
81.6
137
1,015
752
152
46
1,880
0.400
EZ/EZ
-/-
276
143
31
11
42/M
109.1
144
478
12
36/M
119.7
151
318
Mean r SEM 50.4
+ 2.9
84.0
k 5.4
124.8
f 5.0
108 515 * 56 316 L 51
196 30 142.3 t 13 36 * 2.5
676 320 797 r 133
0.408
EZ/EZ
-/-
0.338 0.41 + .03
E4/E2
-i-
Abbreviations: IBW, ideal body weight; TG, triglycerides; CAD, coronary artery disease; PVD, peripheral vascular disease.
other two diet controlled [patients no. 3 and 51). Four patients were taking drugs known to effect lipid metabolism (estrogens [patients no. 2 and 71, &blockers [patients no. 6 and 71). but these remained unchanged throughout the period of study. Informed consent was obtained from all patients and the protocols were approved by the Human Research Committee of The Oregon Health Sciences University and by the US Food and Drug Administration. All patients were seen as outpatients in the Clinical Research Center or Lipid Disorders Clinic at The Oregon Health Sciences University.
Study Protocol Prior to entry into the study, all patients underwent a complete physical examination and an eye examination, and blood samples were obtained to exclude secondary causes of hyperlipidemia. Lipid-lowering drugs were withdrawn for a period of 1 month before entry into the baseline period of the study. During the baseline period, which lasted 1 month, two blood samples were obtained for lipid and lipoprotein studies; patients were then begun on therapy with lovastatin at a dose of 20 mg twice daily. Patients were seen at monthly intervals for a period of 3 months. At each visit, brief physical examinations were conducted, patient compliance to medications assessed by verbal questions, and blood samples obtained for lipid and lipoprotein studies and to assess clinical chemistry parameters and hematologic studies. In nine of the 12 patients, the dose of lovastatin was increased to 40 mg twice daily, whereas in the other three patients treatment with lovastatin was discontinued for 1 month and the patients then begun on clofibrate at a dose of 1 g twice daily. The nine patients who remained on monotherapy with lovastatin at a dose of 40 mg twice daily were seen at monthly intervals for a period of 3 months, at which time they were evaluated as on the lower dose of lovastatin. Lovastatin therapy in these nine patients was then discontinued for a period of 1 month, after which treatment with clofibrate was initiated at a dose of 1 g twice daily. Due to the fact that several patients who participated in this trial lived a considerable distance from Portland, OR, blood samples were not routinely obtained during the washout period and patients were seen again after 1 month on single-drug therapy with clofibrate. However, based on previous studies with lovastatin,‘a,” the period off therapy with this drug should have been sufficient to ensure a return to baseline, whereas the time period on clofibrate was sufficient to enable a new steady-state to be obtained. Six of the 12 patients showed persistent hypercholesterolemia (>220 mg/dL) on monotherapy with lovastatin at doses of 20 or 40 mg twice daily on clofibrate and all six of these patients were subsequently treated with combined drug therapy using lovastatin (20 mg twice daily) and clofibrate.
Family studies were compatible with a diagnosis of familial combined hyperlipidemia in three of the six patients in whom sufficient family information could be obtained to further characterize the genotypic reasons responsible for their severe hyperlipidemia (patients no. 1,2, and 10 in Table 1). The hypolipidemic effects of combined drug therapy with lovastatin plus clofibrate were assessed over a 3-month period in which patients were seen at monthly intervals and blood tests obtained as previously described. Patients were advised to take lovastatin and clofibrate with breakfast and with their evening meal and, in patients treated with both drugs in combination therapy, they were advised to take the drugs at the same time. Patients were seen in the morning after having fasted for at least 12 hours, and had not taken their medications prior to being seen.
Laboratory Methods Blood samples for lipid and lipoprotein analysis were obtained in tubes containing 1 mg/dL of EDTA anticoagulant and plasma was separated at 4“C in a refrigerated centrifuge. Total concentrations of plasma cholesterol and triglyceride were determined with an autoanalyzer,’ and lipoproteins were separated by a combination of ultracentrifugation and heparin manganese precipitation of plasma.” In all cases, VLDL was initially removed by ultracentrifugation of plasma at density 1.006 g/mL and the concentration of cholesterol and triglycerides in the lipoprotein measured directly. High-density lipoprotein (HDL) concentrations were then measured in the supernatant following precipitation of apoprotein B-containing lipoproteins with heparin manganese and the concentration of LDL cholesterol was calculated.” Lipid determinations were standardized with samples of known composition obtained from the Center for Disease Control, Atlanta, GA. Biochemical assessments included total bilirubin, alkaline phosphatase, aspartate amino transferase, creatinine, total serum protein, uric acid, serum glucose, and electrolytes. In addition, a complete blood cell count was performed periodically. Apoprotein E phenotypes were determined using a modification of the slab gel isoelectric focusing of method of Assmann et al” on delipidated VLDL. After isolation from plasma, VLDL samples were delipidated with 1:l chloroform:methanol and the protein pellets washed with an additional aliquot of these solvents. The pellets were dried under nitrogen and solubilized for 1 hour at 37’C in 200 rL of fresh sample buffer (20% wt/vol sucrose, 1% wt/vol decyl sodium sulfate, 2% vol/vol Serva pH4-7 ampholines (Serva Fine Chemicals, Westbury, NY), 2% vol/vol fl-mercaptoethanol). Samples were subjected to isoelectric focusing with pH4:7 ampho-
LOVASTATIN
IN TYPE III HYPERLIPOPROTEINEMIA
405
lines in a Hoefer flat-bed electrophoresis chamber (Hoefer Scientific Institute, San Francisco, CA). The gels were run overnight at 250 V and stained for 4 hours in 0.05 wt/vol Crocein Scarlet, 0.04% wt/vol Coomassie brilliant blue, 16 mmol/L cupric sulfate in 10% acetic acid, 27% isopropanol.
Table 2. The Influence of Lovastatin and Clofibrate on Plasma Lipids and Lipoproteins in 12 Patients With Type 81Hyperlipoproteinemia Plasma Cholesterol (mg/dL) Total
Statistical Analysis
Baseline (A)
Statistical analysis was performed by one-way ANOVA with repeated measures followed by the Neuman Keuls post hoc testI
Lovastatin (B)
concentrations of cholesterol, triglyceride, and VLDL decreased rapidly when patients were started on drug therapy with lovastatin, and stable values were reached after 1 month on single-drug therapy. The stability of cholesterol and triglyceride concentrations during three successive monthly visits on lovastatin at doses of 20 and 40 mg twice daily, three visits at monthly intervals during therapy with clofibrate, and three visits on combination drug therapy with lovastatin and clofibrate are illustrated in Fig 1. ANOVA with repeated measures of the data from these six patients shows no significant differences between the concentrations of cholesterol and triglycerides during three consecutive visits on the same treatment regimen. This analysis provides strong evidence that the decreases that occur in response to changes in drug dosage or with combination drug therapy represent the effects of this therapeutic change rather than a carry-over effect due to progressive further decreases in lipid and lipoprotein concentrations during prolonged drug therapy on a given treatment regimen. The comparative efficacy of lovastatin (20 mg twice daily) and clofibrate (1 g twice daily) on plasma concentrations of lipids and lipoproteins in 12 patients with type III hyperlipoproteinemia is shown in Table 2. For each patient, the baseline data represent the results from blood samples obtained on two outpatient visits on diet only, whereas the values for each patient on lovastatin and clofibrate are from three outpatient visits on each drug, respectively. Significant Plasma
Lovastatin 40 mg BID
20 mg BID
HDL
TG lme/dL)
139 + 13 35 t 2 788 + 136
(20 mg 278
r 23
142 + 83
94 zt 6
44 + 3 454
+ 83
Clofibrate (Cl
RESULTS
Base In?
LDL
500 t 56 301 f 48
BID)
A
VLDL
Clofibrate
(1 g BID)
299 + 15
116 + 16 138 f
10 46 + 3 378 + 54
P value AvB
