title: Development of activity assays for high-volume evaluation of human immunodeficiency virus (HIV) protease inhibitors in rat serum: results with ditekiren.
abstract: We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.
authors: Drug Delivery Systems Research, Upjohn Co., Kalamazoo, Michigan 49001.
authors: K F
authors: KF
authors: Wilkinson
authors: B D
authors: BD
authors: Rush
authors: S K
authors: SK
authors: Sharma
authors: D B
authors: DB
authors: Evans
authors: M J
authors: MJ
authors: Ruwart
authors: J M
authors: JM
authors: Friis
authors: M J
authors: MJ
authors: Bohanon
authors: P K
authors: PK
authors: Tomich
mesh_terms: D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D017320:HIV Protease Inhibitors / Q000097:blood*; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012083:Renin / Q000097:blood; D000092502:Renin Inhibitors
chemical_list: D000803:Angiotensin I; D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D012083:Renin; D000092502:Renin Inhibitors; C050898:ditekiren