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Inhibitor of proliferation of arterial smooth-muscle cells by fluvastatin
THE LANCET
Inhibitor of proliferation of arterial smoothmuscle cells by fluvastatin SIR—Smooth-muscle-cell (SMC) proliferation is a feature of atherogenesis and restenosis after angioplasty.1 Mevalonate and other intermediates of cholesterol biosynthesis (isoprenoids) are essential for cell growth,2 hence drugs affecting this metabolic pathway, such as 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), may reduce SMC proliferation. The ability of fluvastatin to interfere with SMC proliferation in vitro3 at therapeutic concentrations (0·1–1 µmol/L)4 may thus have potential clinical significance. The more hydrophilic pravastatin, with a lipid-lowering effect similar to fluvastatin but without in vitro effect on SMC proliferation,3 provides a suitable control for this non-lipid related effect of fluvastatin. These observations and the similar pharmacokinetics of fluvastatin and pravastatin prompted investigation of the pharmacological activity of sera from patients treated with either statin on the proliferation of cultured human SMC, and their effects on cholesterol biosynthesis. 15 patients of both sexes, age 41–66 yr, with a diagnosis of type IIa hypercholesterolaemia (8–39 [SD 1·5] and 6·23 [1·5] mmol/L for total and LDL cholesterol) were randomly allocated to receive either 40 mg fluvastatin or 40 mg pravastatin once a day for 6 days. None had a history of cardiovascular disease. Blood samples were collected before, 0·5, 1, 2·5, and 6 h after the last dose. The effect of wholeblood sera from statin-treated patients on SMC proliferation was evaluated by human SMC growth assay.3 Treatment with fluvastatin or pravastatin resulted in similar effects on plasma lipids and lipoprotein concentrations (total cholesterol –16% and –11%, LDLcholesterol –20% and –19%, HDL cholesterol –2% and –10%, triglycerides –15% and +19%, for fluvastatin and pravastatin, respectively). The addition of 15% whole-blood sera from patients treated with fluvastatin to the culture medium caused an inhibition of cholesterol synthesis (figure), that mirrored the pharmacokinetic profile of fluvastatin, as previously determined (Tmax between 0·5 and 1 h with a t β of approximately 1·5 h).4 The strongest inhibition (43% decrease; p<0·01) was detected with sera collected 1 h after the dose. When SMC proliferation was investigated, a significant inhibition of cell growth (28% A
B
% of control
100
100
80
80 *
60 Proliferation Synthesis
40 0 1 0·5
2·5
60
*
40 60 1 0·5 Time (h)
2·5
6
Figure: Effect of sera from pravastatin (A) and fluvastatin (B) treated people on cholesterol synthesis and proliferation of human arterial myocytes Target cells (human femoral artery myocytes) were incubated for 72 h in a medium containing 15% whole blood sera from statin-treated patients. Abscissa is time of sera collection after the last dose. 14C-acetate incorporation was used to assay cholesterol synthesis, and the cell number provided an index of the effect on cell proliferation. Means and SEMs of 15 ex periments, each run in triplicate, with different sera. The mean value of control (100%) for cell number, after subtracting t=0, was 786 (47)⫻103 cells/plate and for cholesterol synthesis was 14·3 (1·7 pmol/mg) cell protein per h. *Duncan’s test (p<0·01) 1 h vs 6 h; 6 h vs 0·5, 1, 2·5 h.
1584
A Corsini, F Pazzucconi, P Pfister, R Paoletti, *C R Sirtori *Institute of Pharmacological Sciences, University of Milan; Institute of Pharmacological Sciences and Center Enrica Grossi, Paoletti, University of Milan, 20133 Milan, Italy; and Sandoz Pharma Ltd, Basel, Switzerland
1
2 3
4
5
Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol 1990; 15: 1667–87. Maltese WA. Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J 1990; 4: 3319–28. Corsini A, Raiteri M, Soma MR, Bernini F, Fumagalli R, Paoletti R. Pathogenesis of atherosclerosis and the role of 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors. Am J Cardiol 1995; 76: 21A-28A. Tse FLS, Jaffe JM, Troendle A. Pharmacokinetics of fluvastatin after single and multiple doses of normal volunteers. J Clin Pharmacol 1992; 32: 630–38. Herd JA, West MS, Ballantyne C, Farmer J, Gotto AM Jr. Baseline characteristics of subjects in the Lipoprotein and Coronary Atherosclerosis Study (LCAS) with fluvastatin. Am J Cardiol 1994; 73: 42D–49D.
120
% of control
120
decrease; p<0·01) was detected with sera obtained 6 h after the last dose, when fluvastatin concentrations are below the detection limit (figure).4 This effect appears, therefore, not to be directly related to the presence of the drug but rather the end result of an inhibition of the mevalonate-derived isoprenoids, such as farnesol or geranylgeraniol. The latter compounds, when added to rapidly growing cells, prevent the inhibitory effect of statins.3 The inhibition of HMG-CoA reductase and isoprenoids biosynthesis by fluvastatin may thus prevent isoprenylation and membrane attachment of key G proteins involved in the signal transduction of mitogenic stimuli.2 No effect was observed on either SMC proliferation or cholesterol biosynthesis with sera from patients treated with pravastatin. These present findings are the first demonstration in man of the antiproliferative activity of an HMG-CoA reductase inhibitor. These findings may provide a basis for the potential effect of fluvastatin in the ongoing clinical trials (Lipoprotein and Coronary Atherosclerosis Study [LCAS]) investigating the progression and/or regression of atherosclerotic lesions,5 and (fluvastatin angioplasty restenosis [FLARE]) on the prevention of coronary restenosis after angioplasty. In addition, the presented model may provide a simple and useful test for evaluating the potential of different drugs in reducing cell proliferation in man.
3-adrenoceptor gene variant in obesity and insulin resistance SIR—Mauriège and Bouchard (Sept 14, p 698)1 state that the Trp64Arg variant in the 3-adrenoceptor receptor is of doubtful importance for obesity and insulin resistance. On the contrary, we believe that this variant is a modest but important contributor to these polygenic disorders. Several susceptibility genes, each with modest effect—in addition to physiological provocations (ie, caloric excess and sedentary lifestyle) and time—are likely to be required for expression of the obesity and non-insulin dependent diabetes mellitus (NIDDM) phenotypes. The Trp64Arg 3adrenoceptor variant is probably one of these susceptibility genes. First, association studies in several cohorts (including Pima Indians, French, Finns, Mexican Americans, Danes, Australians, and Japanese) link the Trp64Arg 3-adrenoceptor variant to features of the insulin-resistance syndrome (central obesity, hyperinsulinaemia with insulin resistance, and hypertension), and an earlier onset of NIDDM. In these studies (for review, see ref 2) the effect of
Vol 348 • December 7, 1996
Inhibitor of proliferation of arterial smoothmuscle cells by fluvastatin SIR—Smooth-muscle-cell (SMC) proliferation is a feature of atherogenesis and restenosis after angioplasty.1 Mevalonate and other intermediates of cholesterol biosynthesis (isoprenoids) are essential for cell growth,2 hence drugs affecting this metabolic pathway, such as 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), may reduce SMC proliferation. The ability of fluvastatin to interfere with SMC proliferation in vitro3 at therapeutic concentrations (0·1–1 µmol/L)4 may thus have potential clinical significance. The more hydrophilic pravastatin, with a lipid-lowering effect similar to fluvastatin but without in vitro effect on SMC proliferation,3 provides a suitable control for this non-lipid related effect of fluvastatin. These observations and the similar pharmacokinetics of fluvastatin and pravastatin prompted investigation of the pharmacological activity of sera from patients treated with either statin on the proliferation of cultured human SMC, and their effects on cholesterol biosynthesis. 15 patients of both sexes, age 41–66 yr, with a diagnosis of type IIa hypercholesterolaemia (8–39 [SD 1·5] and 6·23 [1·5] mmol/L for total and LDL cholesterol) were randomly allocated to receive either 40 mg fluvastatin or 40 mg pravastatin once a day for 6 days. None had a history of cardiovascular disease. Blood samples were collected before, 0·5, 1, 2·5, and 6 h after the last dose. The effect of wholeblood sera from statin-treated patients on SMC proliferation was evaluated by human SMC growth assay.3 Treatment with fluvastatin or pravastatin resulted in similar effects on plasma lipids and lipoprotein concentrations (total cholesterol –16% and –11%, LDLcholesterol –20% and –19%, HDL cholesterol –2% and –10%, triglycerides –15% and +19%, for fluvastatin and pravastatin, respectively). The addition of 15% whole-blood sera from patients treated with fluvastatin to the culture medium caused an inhibition of cholesterol synthesis (figure), that mirrored the pharmacokinetic profile of fluvastatin, as previously determined (Tmax between 0·5 and 1 h with a t β of approximately 1·5 h).4 The strongest inhibition (43% decrease; p<0·01) was detected with sera collected 1 h after the dose. When SMC proliferation was investigated, a significant inhibition of cell growth (28% A
B
% of control
100
100
80
80 *
60 Proliferation Synthesis
40 0 1 0·5
2·5
60
*
40 60 1 0·5 Time (h)
2·5
6
Figure: Effect of sera from pravastatin (A) and fluvastatin (B) treated people on cholesterol synthesis and proliferation of human arterial myocytes Target cells (human femoral artery myocytes) were incubated for 72 h in a medium containing 15% whole blood sera from statin-treated patients. Abscissa is time of sera collection after the last dose. 14C-acetate incorporation was used to assay cholesterol synthesis, and the cell number provided an index of the effect on cell proliferation. Means and SEMs of 15 ex periments, each run in triplicate, with different sera. The mean value of control (100%) for cell number, after subtracting t=0, was 786 (47)⫻103 cells/plate and for cholesterol synthesis was 14·3 (1·7 pmol/mg) cell protein per h. *Duncan’s test (p<0·01) 1 h vs 6 h; 6 h vs 0·5, 1, 2·5 h.
1584
A Corsini, F Pazzucconi, P Pfister, R Paoletti, *C R Sirtori *Institute of Pharmacological Sciences, University of Milan; Institute of Pharmacological Sciences and Center Enrica Grossi, Paoletti, University of Milan, 20133 Milan, Italy; and Sandoz Pharma Ltd, Basel, Switzerland
1
2 3
4
5
Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol 1990; 15: 1667–87. Maltese WA. Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J 1990; 4: 3319–28. Corsini A, Raiteri M, Soma MR, Bernini F, Fumagalli R, Paoletti R. Pathogenesis of atherosclerosis and the role of 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors. Am J Cardiol 1995; 76: 21A-28A. Tse FLS, Jaffe JM, Troendle A. Pharmacokinetics of fluvastatin after single and multiple doses of normal volunteers. J Clin Pharmacol 1992; 32: 630–38. Herd JA, West MS, Ballantyne C, Farmer J, Gotto AM Jr. Baseline characteristics of subjects in the Lipoprotein and Coronary Atherosclerosis Study (LCAS) with fluvastatin. Am J Cardiol 1994; 73: 42D–49D.
120
% of control
120
decrease; p<0·01) was detected with sera obtained 6 h after the last dose, when fluvastatin concentrations are below the detection limit (figure).4 This effect appears, therefore, not to be directly related to the presence of the drug but rather the end result of an inhibition of the mevalonate-derived isoprenoids, such as farnesol or geranylgeraniol. The latter compounds, when added to rapidly growing cells, prevent the inhibitory effect of statins.3 The inhibition of HMG-CoA reductase and isoprenoids biosynthesis by fluvastatin may thus prevent isoprenylation and membrane attachment of key G proteins involved in the signal transduction of mitogenic stimuli.2 No effect was observed on either SMC proliferation or cholesterol biosynthesis with sera from patients treated with pravastatin. These present findings are the first demonstration in man of the antiproliferative activity of an HMG-CoA reductase inhibitor. These findings may provide a basis for the potential effect of fluvastatin in the ongoing clinical trials (Lipoprotein and Coronary Atherosclerosis Study [LCAS]) investigating the progression and/or regression of atherosclerotic lesions,5 and (fluvastatin angioplasty restenosis [FLARE]) on the prevention of coronary restenosis after angioplasty. In addition, the presented model may provide a simple and useful test for evaluating the potential of different drugs in reducing cell proliferation in man.
3-adrenoceptor gene variant in obesity and insulin resistance SIR—Mauriège and Bouchard (Sept 14, p 698)1 state that the Trp64Arg variant in the 3-adrenoceptor receptor is of doubtful importance for obesity and insulin resistance. On the contrary, we believe that this variant is a modest but important contributor to these polygenic disorders. Several susceptibility genes, each with modest effect—in addition to physiological provocations (ie, caloric excess and sedentary lifestyle) and time—are likely to be required for expression of the obesity and non-insulin dependent diabetes mellitus (NIDDM) phenotypes. The Trp64Arg 3adrenoceptor variant is probably one of these susceptibility genes. First, association studies in several cohorts (including Pima Indians, French, Finns, Mexican Americans, Danes, Australians, and Japanese) link the Trp64Arg 3-adrenoceptor variant to features of the insulin-resistance syndrome (central obesity, hyperinsulinaemia with insulin resistance, and hypertension), and an earlier onset of NIDDM. In these studies (for review, see ref 2) the effect of
Vol 348 • December 7, 1996