Solid-phase microextraction for assessment of plasma protein binding, a complement to rapid equilibrium dialysis

Aim: Determination of plasma protein binding ( PPB) is considered vital for better understanding of pharmacokinetic and pharmacodynamic activities of drugs due to the role of free concentration in pharmacological response. Methodology & results: Solid-phase microextraction (SPME) was investigated for measurement of PPB from biological matrices and compared with a gold standard approach (rapid equilibrium dialysis [RED]). Discussion & conclusion: SPME-derived values of PPB correlated well with literature values, and those determined by RED. Respectively, average protein binding across three concentrations by RED and SPME was 33.1 and 31.7% for metoprolol, 89.0 and 86.6% for propranolol and 99.2 and 99.0% for diclofenac. This study generates some evidence for SPME as an alternative platform for the determination of PPB.

A Steady-State Algebraic Model for the Time Course of Covalent Enzyme Inhibition

This report describes an algebraic equation for the time course of irreversible enzyme inhibition following a two-step mechanism. In the first step, the enzyme and the inhibitor associate reversibly to form a non-covalent complex. In the second step, the noncovalent complex is irreversibly converted to the final covalent conjugate. Importantly, the algebraic derivation was performed under the<i> steady-state approximation</i>. Under the previously invoked <i>rapid-equilibrium approximation</i> [Kitz & Wilson (1962) <i>J. Biol. Chem.</i> <b>237</b>, 3245] it is by definition assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. In contrast, the steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional “kobs” method for evaluating the potency of covalent inhibitors in drug discovery is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, even for inhibitors that have very high binding affinity in the reversible noncovalent step.

A Steady-State Algebraic Model for the Time Course of Covalent Enzyme Inhibition

This report describes an algebraic equation for the time course of irreversible enzyme inhibition following a two-step mechanism. In the first step, the enzyme and the inhibitor associate reversibly to form a non-covalent complex. In the second step, the noncovalent complex is irreversibly converted to the final covalent conjugate. Importantly, the algebraic derivation was performed under the<i> steady-state approximation</i>. Under the previously invoked <i>rapid-equilibrium approximation</i> [Kitz & Wilson (1962) <i>J. Biol. Chem.</i> <b>237</b>, 3245] it is by definition assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. In contrast, the steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional “kobs” method for evaluating the potency of covalent inhibitors in drug discovery is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, even for inhibitors that have very high binding affinity in the reversible noncovalent step.

A Steady-State Algebraic Model for the Time Course of Covalent Enzyme Inhibition

This report describes an algebraic equation for the time course of irreversible enzyme inhibition following a two-step mechanism. In the first step, the enzyme and the inhibitor associate reversibly to form a non-covalent complex. In the second step, the noncovalent complex is irreversibly converted to the final covalent conjugate. Importantly, the algebraic derivation was performed under the<i> steady-state approximation</i>. Under the previously invoked <i>rapid-equilibrium approximation</i> [Kitz & Wilson (1962) <i>J. Biol. Chem.</i> <b>237</b>, 3245] it is by definition assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. In contrast, the steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional “kobs” method for evaluating the potency of covalent inhibitors in drug discovery is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, even for inhibitors that have very high binding affinity in the reversible noncovalent step.

A steady-state algebraic model for the time course of covalent enzyme inhibition

This report describes a double-exponential algebraic equation for the time course of irreversible enzyme inhibition following the two-step mechanism E + I ⇌ E·I → EI, under the steady-state approximation. Under the previously invoked rapid-equilibrium approximation [Kitz & Wilson (1962) J. Biol. Chem. 237, 3245] it was assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. The steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional “kobs” method is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, E + I → EI, even for inhibitors that have very high binding affinity in the reversible noncovalent step.

A Steady-State Algebraic Model for the Time Course of Covalent Enzyme Inhibition

This report describes an algebraic equation for the time course of irreversible enzyme inhibition following a two-step mechanism. In the first step, the enzyme and the inhibitor associate reversibly to form a non-covalent complex. In the second step, the noncovalent complex is irreversibly converted to the final covalent conjugate. Importantly, the algebraic derivation was performed under the<i> steady-state approximation</i>. Under the previously invoked <i>rapid-equilibrium approximation</i> [Kitz & Wilson (1962) <i>J. Biol. Chem.</i> <b>237</b>, 3245] it is by definition assumed that the rate constant for the reversible dissociation of the initial noncovalent complex is very much faster than the rate constant for the irreversible inactivation step. In contrast, the steady-state algebraic equation reported here removes any restrictions on the relative magnitude of microscopic rate constants. The resulting formula was used in heuristic simulations designed to test the performance of the standard rapid-equilibrium kinetic model. The results show that if the inactivation rate constant is significantly higher than the dissociation rate constant, the conventional “kobs” method for evaluating the potency of covalent inhibitors in drug discovery is incapable of correctly distinguishing between the two-step inhibition mechanism and a simpler one-step variant, even for inhibitors that have very high binding affinity in the reversible noncovalent step.