Short communication

On the interaction of diazepam with human, rat and mouse plasma proteins and erythrocytes

Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO₂ condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

Allometrically scaled data sets (138 compounds) used for predicting human clearance were obtained from the literature. Our analyses of these data have led to four observations. (1) The current data do not provide strong evidence that systemic clearance (CL_s; n = 102) is more predictable than apparent oral clearance (CL_po; n = 24), but caution needs to be applied because of potential CL_po prediction error caused by differences in bioavailability across species. (2) CL_s of proteins (n = 10) can be more accurately predicted than that of non-protein chemicals (n = 102). (3) CL_s is more predictable for compounds eliminated by renal or biliary excretion (n = 33) than by metabolism (n = 57). (4) CL_s predictability for hepatically eliminated compounds followed the order: high CL (n = 11) > intermediate CL (n = 17) > low CL (n = 29). All examples of large vertical allometry (% error of prediction greater than 1000%) occurred only when predicting human CL_s of drugs having very low CL_s. A qualitative analysis revealed the application of two potential rules for predicting the occurrence of large vertical allometry: (1) ratio of unbound fraction of drug in plasma (f_u) between rats and humans greater than 5; (2) C logP greater than 2. Metabolic elimination could also serve as an additional indicator for expecting large vertical allometry.

There is evidence that the apparent association constant (K) and/or the number of binding sites (n) are inversely dependent upon protein concentration for a number and variety of ligands with no obvious structure-activity relationships. A model recently shown to explain this effect with an inorganic ligand has now been applied to 2-(4'-hydroxybenzeneazo) benzoic acid (HABA) and also explains the inverse protein dependence of the binding of this compound to human albumin. The model explains this inverse dependence on the basis of a highly bound contaminant which competes with the ligand for the high affinity site. HABA was found to interact with human albumin at three or more sites, the high affinity site which was about 95% contaminated had an association constant of 2 × 10⁵/M, an order of magnitude higher than that found previously when the effect of a contaminant was not considered. The association constant of the competitive contaminant was estimated to be about 5 × 10⁶/M. Since the model accounts for the phenomenon in terms of a property of the protein, rather than of the ligand, it could provide a general explanation for this effect with other ligand-acceptor combinations including a wide variety of drug and hormone receptor preparations.

Valproate-Diazepam association is not unusual in the treatment of epilepsies. The present paper investigates in an experimental study the effect of sodium valproate (VPA) on the regional cerebral level of diazepam (DZP), and the relationship with plasma or erythrocytes amounts after subchronic administration. The VPA addition increases the DZP levels in peripheral and central compartments. The results shows a linear correlation between the drug concentration in all areas studied and the plasmatic or erythrocytic amounts during the CNS-impregnation phase. The VPA influence is greater in the CNS where the DZP impregnation is selectively increased, specially in cortex and cerebellum.

Drug–serum protein binding was evaluated in genetically obese Zucker rats, their lean littermates, and lean Sprague–Dawley rats. The free fraction (f_p) of phenytoin was significantly higher in the obese rat (f_p = 0.177) compared to its lean littermate (f_p = 0.136), apparently due to displacement by free fatty acids. Conversely, diazepam and propranolol f_p values were decreased in the obese Zucker rat (f_p = 0.107 and f_p = 0.122, respectively) compared to the lean Zucker rat (f_p = 0.140 and f_p = 0.174, respectively). Evidence strongly suggests that the increased binding of propranolol was not due to elevations in the serum concentrations of α₁-acid glycoprotein (as is the case in the human obese population). Rather, the decreased f_p for both diazepam and propranolol was a result of increased lipoprotein partitioning. Strain differences between the lean Zucker rat and lean Sprague–Dawley rat were also evident, with the serum binding of the Sprague–Dawley rat more closely resembling the obese Zucker rat.