Abstract
The cholesterol‐lowering drug simvastatin (SIMV, Zocor®) reduced heart attacks by 42% in patients who had high cholesterol levels and suffered from heart disease. Upon oral administration, SIMV is quickly hydrolyzed to its β‐hydroxyacid and other acid metabolites, which are potent inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. A Tecan‐based enzyme inhibition assay has been developed to improve the existing Zymark‐based assay for the determination of both active and total concentrations of HMG‐CoA reductase inhibitors in human plasma. A Tecan Genesis 200 robotic workstation equipped with eight probes and customized hardware was utilized to achieve higher sample throughput and improve assay reproducibility and mechanical stability. The developed enzyme inhibition assay was validated over two concentration ranges of 0.4–20 ng equivalent/mL, and 2–50 ng equivalent/mL. Intra‐ and interday precision data (coefficient of variation (CV)) for both concentration ranges were less than 9%, with an accuracy of 93–107%. The interday precision for the determination of quality control (QC) samples was less than 2% and 8%, respectively. The respective interday QC accuracy values were 93–103% and 97–104%. Good linearity across the two concentration ranges was observed, with acceptable reproducibility. This improved enzyme inhibition assay has been utilized to analyze human plasma samples from several clinical studies. J. Clin. Lab. Anal. 16:209–215, 2002. © 2002 Wiley‐Liss, Inc.
Keywords: Robotic Inhibition Assay, HMG‐CoA reductase inhibitors, cholesterol‐lowering drug
REFERENCES
- 1. Alberts A. Discovery, biochemistry and biology of lovastatin. Am J Cardiol 1988;62:10J. [DOI] [PubMed] [Google Scholar]
- 2. The Scandinavian Simvastatin Survival Study Group . Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease. Lancet 1994;344:1382–1389. [PubMed] [Google Scholar]
- 3. Kjekshus J, Pedersen TR. Reducing the risk of coronary events: evidence from the Scandinavian simvastatin survival study (4S). Am J Cardiol 1995;76:64C–68C. [DOI] [PubMed] [Google Scholar]
- 4. Pedersen TR, Berg K, Cook TJ. Safety and tolerability of cholesterol lowering with simvastatin during 5 years in the Scandinavian simvastatin survival study. Arch Int Med 1996;156:2085–2092. [PubMed] [Google Scholar]
- 5. Pedersen TR, Tobert JA. Benefits and risks of HMG‐CoA reductase inhibitors in the prevention of coronary heart disease. Drug Safety 1996;14:11–24. [DOI] [PubMed] [Google Scholar]
- 6. Pyorala K, Pedersen TR, Kjekshus J. Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease. Diabetic Care 1997;20:614–620. [DOI] [PubMed] [Google Scholar]
- 7. Heart Protection Study Collaborative Group . MRC/BHF heart protection study of cholestrol‐lowering drug therapy and of antioxidant vitamin supplementation in patients at increased risk of coronary heart disease: design, study population and early experience. Eur Heart J 1999;20:705–741. [Google Scholar]
- 8. Vickers S, Duncan CA, Chen I‐Wu, Rosegary A, Duggan DE. Metabolic disposition studies on simvastatin, a cholesterol‐lowering prodrug. Drug Metab Dispos 1990;18:138–145. [PubMed] [Google Scholar]
- 9. Prueksaritanont T, Gorham LM, Ma B, Liu L, Yu X, Zhao J. In vitro metabolism of simvastatin in humans: identification of metabolizing enzymes and effect of drug on hepatic P450s. Drug Metab Dispos 1997;25:1191–1199. [PubMed] [Google Scholar]
- 10. Schwartz MS, Maglietto BK, Stubbs RJ. An automated assay for plasma HMG‐CoA reductase inhibitors. Joint Regional Meeting of the AAPS, Atlantic City, 1988. p31.
- 11. Schwartz MS, Stubbs RJ, Thornton TJ. Comparison of the active HMG‐CoA reductase inhibitors in human and dog plasma following oral doses of lovastatin and simvastatin. AAPS Third Annual Meeting and Exposition, Orlando, 1988. P1494.