A quantitative circular dichroic investigation of the binding of the enantiomers of ibuprofen and naproxen to human serum albumin
Introduction
Albumin drug interactions have been investigated by circular dichroism for over 25 years. If a chromophore in the drug molecule becomes optically active when it is perturbed by an asymmetric centre in the human serum albumin (HSA) molecule, as the perturbation is from a second molecule then the resultant Cotton effect is termed extrinsic. Early studies of this phenomenon were used in a qualitative manner to support quantitative data from dialysis and ultrafiltration studies 1, 2, 3. Since the work of Rosen [4]many studies have estimated binding constants for the formation of drug HSA complexes using the proportionality between the intensity of the induced Cotton effect and the concentration of complex assuming a single binding phenomenon is involved 5, 6, 7, 8, 9. Not all drug molecules with suitable chromophores give measurable Cotton effects following interaction with albumin. In 1973, Perrin and Nelson estimated binding constants of drug HSA complexes by using a probe giving a large extrinsic Cotton effect on binding to albumin and then measuring the amounts displaced by drugs competing for the same binding site but which produced no measurable circular dichroic signals following interaction with albumin [10]. Ibuprofen and naproxen have been reported to bind to the same binding site on HSA as diazepam 11, 12. Diazepam gives a quantifiable Cotton effect following binding to HSA with a maximum near 260 nm. On the other hand ibuprofen and naproxen display no measurable extrinsic Cotton effects under the experimental conditions found to give suitable ellipticities for measurement of the diazepam–HSA interaction. It was therefore decided to determine binding constants for the interaction between the enantiomers of ibuprofen and naproxen with HSA by measuring their ability to displace diazepam from HSA using the circular dichroic technique.
Section snippets
Materials
Essentially fatty acid free HSA (lot no. 42H9313 prepared from Fraction V albumin) was obtained from Sigma (St. Louis, MO), and used without further treatment. S(+) and R(−) naproxen were gifts from Syntex Research (Palo Alto, CA). S(+) ibuprofen was a gift from Sepracor (Marlborough, MA) and R(−) ibuprofen was obtained as a gift from Research Biochemicals (Natick, MA). Diazepam was kindly supplied by Dr Richard E. Tessel, Dept. of Pharmacology and Toxicology, University of Kansas. The drugs
Results and discussion
The enantiomers of ibuprofen and naproxen displayed no intrinsic ellipticity in the wavelength range 250–360 nm under the current experimental conditions, and so no corrections to the spectra were necessary. Both molecules have absorption maxima in the low UV range, where measurements in the presence of HSA are of little interest because of the relatively large ellipticity of the HSA molecule. Similarly at drug to albumin ratios of up to 10, no extrinsic Cotton effects following interaction of
Acknowledgements
This work is taken from a thesis accepted by the University of Kansas as partial fulfillment of the requirements for a Ph.D. degree (VKC 1994).
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