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Selectivity of ZD4522 for inhibition of cholesterol synthesis in hepatic versus non-hepatic cells
Monday June 26, 2000: Poster Abstracts P: W6 New Aspects of Stalin Treatment changes in, and achieved levels of, LDL-C, in the subjects treated with a statin. The methodology used by each study was different making comparisons difficult. Here, the Prospective Pravastatin Pooling project investigators present the results for WOSCOPS, LIPID and CARE using the 4S methodology. Methods: In the pravastatin treated groups, subjects were divided into quintiles of achieved levels of and % changes in LDL-C at 12 months. Future coronary event rates were calculated and compared statistically after adjusting for baseline risk factors and lipids and on-treatment HDL cholesterol and triglycerides. Results and Discussion: There was no evidence of heterogeneity of risk associated with variations in % change in LDL cholesterol. In CARE achieved LDL-C was not associated with risk of a coronary event. In LIPID and WOSCOPS, where baseline LDL-C levels were higher, there was a positive association, primarily due to higher risk in subjects in the quintile with highest on-treatment LDL-C (> 126 mg/dl in LIPID and >164 mg/dl in WOSCOPS).
I MoP27:W6 1 Prospective pravastatin pooling project: Relative risk reduction by baseline HDL and TG concentrations E Sacks I , A. Tonkin2, J. Shepherd3 , E. Braunwald 1, S. Cobbe3 , M. Hawkins4, A. Kcech5, C. Packard 3, J. Simes~, R. Byington6, C. Furberg6. For the Pravastatin Pooling Project Investigators;/Harvard Medical Sch Boston, MA; 2Nat'l Heart Foundation, Melbourne; 3 Univ Glasgow, UK 4Univ Texas, Houston, TX; 5Univ Sydney, Australia; 6Wake Forest Univ, Winston-Salem, NC, USA; Objective: To study the relationship between baseline plasma HDL-C and triglyceride (TG) levels, recurrent coronary events, and the efficacy of pravaslatin 40 mg in reducing coronary events in 3 major studies, WOSCOPS, CARE, and LIPID. Methods: Plasma HDL and TG were divided into prespecified categories, and into quinfiles. Results: Pravastatin produced a uniform, significant relative risk reduction for the primary end points (EP), CAD death and nonfatal MI and the secondary EP, CAD death, nonfatal MI, and CABG/PTCA. in prespecified categories of baseline HDL (<1 mmoUl, >1 retool/l), and of TG (<1.5 1.5-2.5, >2.5 retool/l). Baseline HDL and TG had a similar relationship to coronary events in patients treated with pravastatin or placebo, and there was no evidence of modification of event reduction. CARE and LIPID
.
Q
3L34
I
.19-414
region which affects a transcription factor binding site. Simvastatin treatment was associated with a significant increase in serum PON activity (292.2 (30.0) v 267.6 (35.9) U/nil; p < 0.01). Conclusion: Simvastatin increased plasma PON activity through transcriptional mechanisms. The study demonstrates a novel anti-atherogenic mechanism of simvastatin linked to a beneficial influence on serum levels of an anti-oxidant enzyme.
I MoP29:W6 ] Selectivity of ZD4522 for inhibition of cholesterol synthesis in hepatic versus non-hepatic cells L. Buckett, P. Ballard, R. David,son, C. Dunkley, L. Martin, J, Stafford, E McTaggart. AstraZeneca, Alderley Park, Cheshire, UK Objectives: To measure the potency and selectivity of ZD4522 (rosuvastatin) - a new HMG-CoA reductase inhibitor - as an inhibitor of cholesterol synthesis in hepatic and non-bepatic cells in comparison with 5 other statins. Methods: Primary rat hepatocytes, a rat fihroblast cell line (NRK-49F) and human umbilical vein endothelial cells (HUVECs) were used. The statins, at a range of concentrations, were pre-incnhated with the cells in serum-free medium for 30 rain before addition of 14C-acetate for 3 hr and measurement of the incorporation of 14C into cholesterol. ICso values were calculated from the dose-response curves. Results: In primary rat bepatocytes, ZD4522 was the most potent inhibitor of cholesterol synthesis. This result was confirmed using shorter incubation times to minimise any effect of differential metabolism of the compounds. Both ZD4522 and pravastatin were selective for hepatocytes but the others were relatively non-selective between the cell types. The ICs0 of ZD4522 in hepatocytes was unaffected by the presence of lipoprotein-deficient serum. 1C5o (nM) Hepatocytes ZD4522 Atorvastatin Cerivastatin Fluvastatin Pravastatin Simvastatin
0.30 0.82 2.5 4.8 5.0 5.2
NRK-49F
HUVECs
310 340 1.2 3.4 14,0 x 103 7.9
41 5.5 3.1 0.56 1.9 × 103 1.0
Conclusions: ZD4522 is a very potent inhibitor of cholesterol synthesis in hepatocytes and is highly selective for liver cells, consistent with its relatively hydrophilic properties.
MoP30:W6 J Increased levels
of oxidized LDL in obese patients are normalised by treatment with atorvastatin
~
I,=tt.m=tke.~ t
41
~44
:l 1ill
B~Jdine HDL..Cbelester~I~dntle "CAD dnth, m m ~ m d
•
I...,rmlo,=,lu,.,.O.U
. I f.- i l l . I I I .. I I I . I I ~ I 1 7 ~IIT MI,
8hmflmeT~lJ3~ur'JdcsQuh~Je CABG,PTCA
Conclusions: Pravastatin effectively reduces coronary events across a wide range of baseline HDL and TG concentrations.
I MoP28:W6 J Simvastatin increases plasma levels of the anti-oxidant enzyme paraoxonase by PONI gene activation Leviev, R. James. Clinical Diabetes Unit, University Hospital, Geneva, Switzerland Objective: To investigate the effects of simvastatin on biosynthesis and plasma levels of the antioxidative enzyme paraoxonase (PON). Method I: The PON1 gene promoter region was cloned and reporter gene constructs containing promoter fragments of different lengths were transfected into HepG2 cells. The effects of simvastatin on transcriptional activities were analysed. Method II: Plasma levels of PON were measured in patients (n = 25) before and after treatment (mean = 6.3 weeks) with simvastatin. Results: Simvastatin increased the transcriptional activity of the PON1 gene promoter 2.5 times. Co-incubation with mevalonate prevented stafin-mediated increases in transcriptional activity, indicating that the statin effect was mediatexi by changes in cell cholesterol. Using different promoter constructs, the stalin-sensitive element was located within a 200 bp DNA fragment adjacent to the coding region of the gene. The simvastatin effect was impaired by a common C to T polymorphism (T present in 51.9% of population) in this
Ann Mertens I , Erik Muls 2, Greet Vansant2, Dtsir6 Collen 1, Paul Holvoet1. t Centerfor Molec. and Vascul. Biol.; 2Dept. Endocrinol; Univ. of Leuven, Belgium Objective: Obesity is a major risk factor for cardiovascular disease (CVD). The incidence of obesity is gradually increasing. Previously, the association between CVD and the oxidation of LDL has been demonstrated. Therefore, the association between obesity and circulating oxidized LDL has been studied. Methods and Results: In a blinded cohort study 198 patients (73 male/125 female) without clinical evidence of CVD were included. Mean body mass index (BMI) was 28 4- 9 kg/m 2. Plasma levels of oxidized LDL were 0.74 40.48 mg/dl in patients with BMI < 25 kg/m 2 (n = 90), 1.02 4- 0.87 mg/dl in patients with BM125-29.9 kg/m 2 (n = 50) and 1.46 4- 0.72 mg/dl (p < 0.001) in patients with BMI > 30 kg/m2 (n = 58). Levels of total cholesterol were 157 4- 35 mg/dl, 172 4- 33 mg/dl (p < 0.05) and 174 4- 32 mg/dl (p < 0.01), respectively. Levels of HDL cholesterol were 52 q- 14 mg/dl, 49 4- 13 mg/dl and 43 4- 12 mg/dl (p < 0.001), respectively. Levels of triglycerides were 93 4- 32 mg/dl, 116 4- 85 mg/dl and 140 4- 69 mg/dl (p < 0.001), respectively. In a multivariate stepwise regression model BMI (F = 62; p < 0.001), LDL cholesterol (F = 12; p = 0.001) and male gender (F = 6.9; p = 0.009) predicted independently levels of circulating oxidized LDL. Subsequently, in a double-banded int~-vention study, 40 consecutive female patients with BMI > 30 kg/m2 and with a similar lipid profile as above were randomized either to placebo or to atorvastatin (20 mg/dl) during 1 month after a 1 month placebo run-in period. In the placebo-treated group, lipid values and levels of oxidized LDL did not change. In the atorvastatin-treated group, total cholesterol decreased to 133 4- 27 mg/dl (p < 0.01), LDL cholesterol to 66 + 27 mg/dl (p < 0.001) and oxidized LDL to 0.81 5= 0.38 mg/dl (p < 0.001). Body weight, triglyceride and HDL cholesterol levels did not change.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
I MoP27:W6 1 Prospective pravastatin pooling project: Relative risk reduction by baseline HDL and TG concentrations E Sacks I , A. Tonkin2, J. Shepherd3 , E. Braunwald 1, S. Cobbe3 , M. Hawkins4, A. Kcech5, C. Packard 3, J. Simes~, R. Byington6, C. Furberg6. For the Pravastatin Pooling Project Investigators;/Harvard Medical Sch Boston, MA; 2Nat'l Heart Foundation, Melbourne; 3 Univ Glasgow, UK 4Univ Texas, Houston, TX; 5Univ Sydney, Australia; 6Wake Forest Univ, Winston-Salem, NC, USA; Objective: To study the relationship between baseline plasma HDL-C and triglyceride (TG) levels, recurrent coronary events, and the efficacy of pravaslatin 40 mg in reducing coronary events in 3 major studies, WOSCOPS, CARE, and LIPID. Methods: Plasma HDL and TG were divided into prespecified categories, and into quinfiles. Results: Pravastatin produced a uniform, significant relative risk reduction for the primary end points (EP), CAD death and nonfatal MI and the secondary EP, CAD death, nonfatal MI, and CABG/PTCA. in prespecified categories of baseline HDL (<1 mmoUl, >1 retool/l), and of TG (<1.5 1.5-2.5, >2.5 retool/l). Baseline HDL and TG had a similar relationship to coronary events in patients treated with pravastatin or placebo, and there was no evidence of modification of event reduction. CARE and LIPID
.
Q
3L34
I
.19-414
region which affects a transcription factor binding site. Simvastatin treatment was associated with a significant increase in serum PON activity (292.2 (30.0) v 267.6 (35.9) U/nil; p < 0.01). Conclusion: Simvastatin increased plasma PON activity through transcriptional mechanisms. The study demonstrates a novel anti-atherogenic mechanism of simvastatin linked to a beneficial influence on serum levels of an anti-oxidant enzyme.
I MoP29:W6 ] Selectivity of ZD4522 for inhibition of cholesterol synthesis in hepatic versus non-hepatic cells L. Buckett, P. Ballard, R. David,son, C. Dunkley, L. Martin, J, Stafford, E McTaggart. AstraZeneca, Alderley Park, Cheshire, UK Objectives: To measure the potency and selectivity of ZD4522 (rosuvastatin) - a new HMG-CoA reductase inhibitor - as an inhibitor of cholesterol synthesis in hepatic and non-bepatic cells in comparison with 5 other statins. Methods: Primary rat hepatocytes, a rat fihroblast cell line (NRK-49F) and human umbilical vein endothelial cells (HUVECs) were used. The statins, at a range of concentrations, were pre-incnhated with the cells in serum-free medium for 30 rain before addition of 14C-acetate for 3 hr and measurement of the incorporation of 14C into cholesterol. ICso values were calculated from the dose-response curves. Results: In primary rat bepatocytes, ZD4522 was the most potent inhibitor of cholesterol synthesis. This result was confirmed using shorter incubation times to minimise any effect of differential metabolism of the compounds. Both ZD4522 and pravastatin were selective for hepatocytes but the others were relatively non-selective between the cell types. The ICs0 of ZD4522 in hepatocytes was unaffected by the presence of lipoprotein-deficient serum. 1C5o (nM) Hepatocytes ZD4522 Atorvastatin Cerivastatin Fluvastatin Pravastatin Simvastatin
0.30 0.82 2.5 4.8 5.0 5.2
NRK-49F
HUVECs
310 340 1.2 3.4 14,0 x 103 7.9
41 5.5 3.1 0.56 1.9 × 103 1.0
Conclusions: ZD4522 is a very potent inhibitor of cholesterol synthesis in hepatocytes and is highly selective for liver cells, consistent with its relatively hydrophilic properties.
MoP30:W6 J Increased levels
of oxidized LDL in obese patients are normalised by treatment with atorvastatin
~
I,=tt.m=tke.~ t
41
~44
:l 1ill
B~Jdine HDL..Cbelester~I~dntle "CAD dnth, m m ~ m d
•
I...,rmlo,=,lu,.,.O.U
. I f.- i l l . I I I .. I I I . I I ~ I 1 7 ~IIT MI,
8hmflmeT~lJ3~ur'JdcsQuh~Je CABG,PTCA
Conclusions: Pravastatin effectively reduces coronary events across a wide range of baseline HDL and TG concentrations.
I MoP28:W6 J Simvastatin increases plasma levels of the anti-oxidant enzyme paraoxonase by PONI gene activation Leviev, R. James. Clinical Diabetes Unit, University Hospital, Geneva, Switzerland Objective: To investigate the effects of simvastatin on biosynthesis and plasma levels of the antioxidative enzyme paraoxonase (PON). Method I: The PON1 gene promoter region was cloned and reporter gene constructs containing promoter fragments of different lengths were transfected into HepG2 cells. The effects of simvastatin on transcriptional activities were analysed. Method II: Plasma levels of PON were measured in patients (n = 25) before and after treatment (mean = 6.3 weeks) with simvastatin. Results: Simvastatin increased the transcriptional activity of the PON1 gene promoter 2.5 times. Co-incubation with mevalonate prevented stafin-mediated increases in transcriptional activity, indicating that the statin effect was mediatexi by changes in cell cholesterol. Using different promoter constructs, the stalin-sensitive element was located within a 200 bp DNA fragment adjacent to the coding region of the gene. The simvastatin effect was impaired by a common C to T polymorphism (T present in 51.9% of population) in this
Ann Mertens I , Erik Muls 2, Greet Vansant2, Dtsir6 Collen 1, Paul Holvoet1. t Centerfor Molec. and Vascul. Biol.; 2Dept. Endocrinol; Univ. of Leuven, Belgium Objective: Obesity is a major risk factor for cardiovascular disease (CVD). The incidence of obesity is gradually increasing. Previously, the association between CVD and the oxidation of LDL has been demonstrated. Therefore, the association between obesity and circulating oxidized LDL has been studied. Methods and Results: In a blinded cohort study 198 patients (73 male/125 female) without clinical evidence of CVD were included. Mean body mass index (BMI) was 28 4- 9 kg/m 2. Plasma levels of oxidized LDL were 0.74 40.48 mg/dl in patients with BMI < 25 kg/m 2 (n = 90), 1.02 4- 0.87 mg/dl in patients with BM125-29.9 kg/m 2 (n = 50) and 1.46 4- 0.72 mg/dl (p < 0.001) in patients with BMI > 30 kg/m2 (n = 58). Levels of total cholesterol were 157 4- 35 mg/dl, 172 4- 33 mg/dl (p < 0.05) and 174 4- 32 mg/dl (p < 0.01), respectively. Levels of HDL cholesterol were 52 q- 14 mg/dl, 49 4- 13 mg/dl and 43 4- 12 mg/dl (p < 0.001), respectively. Levels of triglycerides were 93 4- 32 mg/dl, 116 4- 85 mg/dl and 140 4- 69 mg/dl (p < 0.001), respectively. In a multivariate stepwise regression model BMI (F = 62; p < 0.001), LDL cholesterol (F = 12; p = 0.001) and male gender (F = 6.9; p = 0.009) predicted independently levels of circulating oxidized LDL. Subsequently, in a double-banded int~-vention study, 40 consecutive female patients with BMI > 30 kg/m2 and with a similar lipid profile as above were randomized either to placebo or to atorvastatin (20 mg/dl) during 1 month after a 1 month placebo run-in period. In the placebo-treated group, lipid values and levels of oxidized LDL did not change. In the atorvastatin-treated group, total cholesterol decreased to 133 4- 27 mg/dl (p < 0.01), LDL cholesterol to 66 + 27 mg/dl (p < 0.001) and oxidized LDL to 0.81 5= 0.38 mg/dl (p < 0.001). Body weight, triglyceride and HDL cholesterol levels did not change.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000