A method for estimation of plasma protein binding using diffusion ordered NMR spectroscopy (DOSY)

Abstract

The plasma protein binding (PPB) of a drug plays a key role in both its pharmacokinetic and pharmacodynamic properties. During lead optimisation, medium and high throughput methods for the early determination of PPB can provide important information about potential PKPD profile within a chemotype or between different chemotype series. Diffusion ordered spectroscopy (DOSY) is an NMR spectroscopic technique that measures the diffusion of a molecule through the magnetic field gradient, according to its molecular size/weight. Here, we describe the use of DOSY for a rapid and straightforward method to evaluate the PPB of drug molecules, using their binding to bovine serum albumin (BSA) as a model.

Fig. 1. (A) Changes in diffusion coefficient, D, of caffeine over a concentration range in the absence of BSA (blue crosses) and in the presence of BSA (black crosses). (B) Comparison between the diffusion coefficients of caffein/BSA mixtures in the presence or absence of 3% v/v DMSO (C) the maximum value for diffusion coefficient, D_max, of a mixture of caffeine and BSA steadily rises towards the value for caffeine alone as the ratio of caffeine : BSA increases.

Fig. 2. (A) Changes in diffusion coefficient, D, of propranolol (blue) and diclofenac (green) over a concentration range in the presence of BSA (blue crosses). Control values for absence of BSA are also shown. (B) The maximum value for diffusion coefficient, D_max, of a mixture of caffeine and BSA steadily rises towards the value for diclofenac alone as the ratio of diclofenac : BSA increases.

Fig. 3. (A) Similarity in changes in diffusion coefficient, D, of theophylline (blue) and caffeine (black) over a concentration range in the presence of BSA. (B) Changes in diffusion coefficient, D, of theophylline (blue), acetylsalicylic acid (black) and glucosamine over a concentration range in the presence of BSA. Control values for absence of BSA are not shown for clarity.

Fig. 4. Differences in changes to the averaged diffusion coefficient of different molecules in the presence of BSA: (A) diffusion coefficient, D, of ribose over a concentration range in the presence of BSA. (B) Diffusion coefficient, D, of cimetidine over a concentration range in the presence of BSA. (C) Diffusion coefficient, D, of esomeprazole over a concentration range in the presence of BSA. (D) Changes in diffusion coefficient, D, of ribose, cimetidine and esomeprazole over a concentration range in the presence of BSA. Control values for absence of BSA are not shown for clarity.

Fig. 5. Estimation of the relative values of f_b at a given drug : BSA ratio.