Studies of imipramine binding to human serum albumin by high-performance affinity chromatography

Abstract

Binding by the drug imipramine to the protein human serum albumin (HSA) was studied by using high-performance affinity chromatography. The association equilibrium constants and number of binding sites for imipramine with HSA were first estimated by utilizing frontal analysis. Imipramine was found to have one major binding site on HSA with an association equilibrium constant of 1.6 × 10 5 M −1 at pH 7.4 and 37°C, as well as a second group of weaker and non-specific binding regions (8-9 mol/mol HSA) with an average association equilibrium constant of 1.5 × 10 3 M −1 . Competition studies based on zonal elution were performed to identify the location of the major binding site for imipramine on HSA. Imipramine was found to have direct competition with L-tryptophan, which indicated that imipramine was interacting with Sudlow site II, or the indole-benzodiazepine site of HSA. No competition or allosteric effects were noted between imipramine and warfarin, a probe for Sudlow site I or the warfarin-azapropazone site of HSA. The association equilibrium constant found for imipramine at its site of competition with L-tryptophan also agreed with the value that was obtained for the major binding site of imipramine in the frontal analysis studies. These results confirmed that Sudlow site II was the location of the major binding site for imipramine on HSA. These results gave good agreement with previous observations made in the literature and should provide a more detailed description of how imipramine is transported in blood and of how it may interact with other drugs in the body.

Importance of Relating Efficacy Measures to Unbound Drug Concentrations for Anti-Infective Agents

“…For example, high-performance affinity chromatography (HPAC), a technique coupling high-performance liquid chromatography (HPLC) with affinity columns containing proteins of interest, has been applied to measure the extent of protein binding in blood and characterize the binding process through estimation of equilibrium constants (53)(54)(55)(56)(57). Moreover, immunoaffinity chromatography, a chromatographic method that uses drug binding antibodies, has been applied to measure the free drug fraction of warfarin in a sample containing human serum albumin (58).…”

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

“…Bulky heterocyclic molecules (e.g., warfarin) bind preferentially to Sudlow's site I, whereas Sudlow's site II is preferred by aromatic carboxylates with an extended conformation (e.g., ibuprofen) [1][2][3][5][6][7][8][9][10][11][12][13][14]26,[31][32][33][34][35][36].…”

Affinity Chromatography as a Tool for Quantification of Interactions Between Drug Molecules and Their Protein Targets

“…Also number of injections at different flow rates should be done to confirm that the processes of association/dissociation are fast enough (compared to how much time analyte spends in the column) to create inside the column local state of a binding equilibrium (Loun & Hage, 1995). Considerations on the factors that need to be pointed out using the zonal QAC are described in publications by prof. D. S. Hage (2002 & Hage, 1995;Yoo et al, 2009, Mallik et al, 2008.…”

“…Such solution has been used in the study of binding of both; hormones (Loun & Hage, 1995) and drugs Yoo et al, 2009;Mallik et al, 2008) to different places in the HSA molecule. Simultaneous injection of imipramine with L-tryptophan on the column with immobilized HSA, showed a competitive interaction between those two drugs, confirming that imipramine specifically connects to the indole-benzodiazepine binding site on the molecule of albumin (Sudlow site II).…”