Search

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 micrograms mL-1. It took approximately 8 h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the 'buffer') using a Spectra/Por 2 membrane (molecular weight cut-off, 12000-14000) in a water bath shaker kept at 37 degrees C and a rate of 50 oscillations min-1. Azosemide was fairly stable both in 4% HSA and in the 'buffer' for up to 24 h. The binding of azosemide to 4% HSA was constant (95.5 +/- 0.142%) at azosemide concentrations ranging from 5 to 100 micrograms mL-1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6%, respectively. The binding was also dependent on incubation temperature: the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37 degrees C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9 and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 U mL-1), AAG (up to 0.16%), sodium azide (NaN3, up to 5%), its metabolite, M1 (up to 10 micrograms mL-1), and anticoagulants (EDTA and citrate).

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000 g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (i.v.) administration of azosemide, 20 mg kg-1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 micrograms mL-1; more than 93.4% of azosemide was recovered, and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH1 solution: 75.8% of azosemide was recovered and 2.16 micrograms mL-1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 micrograms mL-1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 microgram mL-1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 micrograms mL-1. However, all rat tissues studied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic activity: approximately 22% of azosemide disappeared and 9.32 micrograms of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 micrograms of azosemide in 9000 g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after i.v. administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78-4.25 at azosemide blood concentrations of 1, 10, and 20 micrograms mL-1 in three rabbits. There was negligible 'blood storage effect' of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 micrograms mL-1.