Human physiologically based pharmacokinetic model for ACE inhibitors: ramipril and ramiprilat

BACKGROUND: The angiotensin-converting enzyme (ACE) inhibitors have complicated and poorly characterized pharmacokinetics. There are two binding sites per ACE (high affinity "C", lower affinity "N") that have sub-nanomolar affinities and dissociation rates of hours. Most inhibitors are given orally in a prodrug form that is systemically converted to the active form. This paper describes the first human physiologically based pharmacokinetic (PBPK) model of this drug class. METHODS: The model was applied to the experimental data of van Griensven et. al for the pharmacokinetics of ramiprilat and its prodrug ramipril. It describes the time course of the inhibition of the N and C ACE sites in plasma and the different tissues. The model includes: 1) two independent ACE binding sites; 2) non-equilibrium time dependent binding; 3) liver and kidney ramipril intracellular uptake, conversion to ramiprilat and extrusion from the cell; 4) intestinal ramipril absorption. The experimental in vitro ramiprilat/ACE binding kinetics at 4°C and 300 mM NaCl were assumed for most of the PBPK calculations. The model was incorporated into the freely distributed PBPK program PKQuest. RESULTS: The PBPK model provides an accurate description of the individual variation of the plasma ramipril and ramiprilat and the ramiprilat renal clearance following IV ramiprilat and IV and oral ramipril. Summary of model features: Less than 2% of total body ACE is in plasma; 35% of the oral dose is absorbed; 75% of the ramipril metabolism is hepatic and 25% of this is converted to systemic ramiprilat; 100% of renal ramipril metabolism is converted to systemic ramiprilat. The inhibition was long lasting, with 80% of the C site and 33% of the N site inhibited 24 hours following a 2.5 mg oral ramipril dose. The plasma ACE inhibition determined by the standard assay is significantly less than the true in vivo inhibition because of assay dilution. CONCLUSION: If the in vitro plasma binding kinetics of the ACE inhibitor for the two binding sites are known, a unique PBPK model description of the Griensven et. al. experimental data can be obtained

A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer ( FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. the FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK( Dnp) P-OH for N-domain, Abz-LFK( Dnp)-OH for C-domain and Abz-FRK(Dnp) P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.Univ Cape Town, Inst Infect Dis & Mol Med, Div Med Biochem, ZA-7925 Cape Town, South AfricaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilWeb of Scienc