It is a commonly accepted hypothesis that central nervous system (CNS) activity is determined by the unbound brain drug concentration. However, limited experimental data are available in the literature to support this hypothesis. The objective of this study was to test this hypothesis by examining the relationship between in vitro binding affinity (K_I) and in vivo activity quantified as the drug concentration occupying 50% of the transporters (OC₅₀) for 18 serotonin (SERT) and dopamine transporter (DAT) inhibitors. In vivo rat OC₅₀ was determined by autoradiography using [³H]N,N-dimethyl-2,2-amino-4-cyanophenylthiobenzylamine and [³H](–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) as the ligands to assess SERT and DAT occupancy, respectively. The unbound brain concentrations were calculated from total brain concentrations and the unbound brain fraction, which was determined by the brain homogenate method. The in vivo total brain SERT and DAT OC₅₀ values (mean ± S.D.) were 408 ± 368- and 410 ± 395-fold greater than the K_I values, respectively. In contrast, the in vivo unbound brain SERT and DAT OC₅₀ values were only 3.3 ± 2.1- and 4.1 ± 4.0-fold different from the K_I values. Therefore, prediction of the biophase drug concentration by using the unbound brain concentration rather than the total brain concentration results in an approximately 100-fold improvement for the accuracy. In the present study, a 10-fold improvement was also observed by using the unbound plasma concentration rather than the total plasma concentration to predict the biophase concentration in the brain. This study supports the hypothesis that CNS activity is more accurately determined by the unbound brain drug concentration and not by the total brain drug concentration.

The objective of the present study was to examine the cassette dosing method in determination of brain-to-plasma concentration ratio (area under the concentration-time profiles for plasma/area under the concentration-time profiles for brain, K_p). Eleven model compounds, amprenavir, citalopram, digoxin, elacridar, imatinib, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), loperamide, prazosin, quinidine, sulfasalazine, and verapamil, were selected to compare their K_p determined from discrete dosing in wild-type mice and their K_p from cassette dosing in wild-type, Mdr1a/1b(−/−), Bcrp1(−/−), and Mdr1a/1b(−/−)/Bcrp1(−/−) mice at 1 to 3 mg/kg. The mice brain and plasma were collected at 0.25, 1, and 3 h and were analyzed using high-performance liquid chromatography-tandem mass spectrometry methods. The K_p determined from discrete dosing versus cassette dosing in the wild-type mice were within 2-fold for all the compounds except sulfasalazine and Ko143. The brain concentrations of sulfasalazine and Ko143 and the plasma concentrations of Ko143 were below the lower limit of quantitation. In addition, the K_p values estimated by mass spectrometry responses, namely the ratio of compound peak area to internal standard peak area, were within 2-fold of the K_p observed from the actual concentrations. Furthermore, the ratios of K_p in Mdr1a/1b(−/−), Bcrp1(−/−), and Mdr1a/1b(−/−)/Bcrp1(−/−) mice versus the K_p in the wild-type mice from cassette dosing were consistent with the ones reported in the literature where the compounds were dosed discretely. These results demonstrate that drug-drug interactions at the blood-brain barrier are unlikely at a subcutaneous dose of 1 to 3 mg/kg and support the use of the cassette dosing approach to assess brain penetration in drug discovery.

It is a commonly accepted assumption that only unbound drug molecules are available to interact with their targets. In order to achieve high unbound plasma drug concentration, it seems obvious to design compounds with low plasma protein binding. Similarly to achieve high unbound tissue concentration, we apparently need compounds with low tissue binding. Our theoretical analysis and experimental data demonstrate that unbound plasma concentration is not determined by plasma protein binding but by hepatic intrinsic clearance after oral dose, and unbound tissue concentration is not determined by tissue binding but determined by unbound plasma concentration and transport properties at the blood-tissue barrier. Reduction of plasma and tissue protein binding for a compound will increase the unbound concentration in vitro but may not increase its unbound plasma or tissue concentration in vivo after oral administration. We conclude that plasma protein and tissue binding are essential parameters to understand pharmacokinetics and pharmacodynamics but they should not be optimized independently in drug discovery. Instead we should focus on reducing clearance and efflux at the blood-tissue barrier to increase in vivo plasma and tissue unbound concentration.

Industrialization in the northwest provinces of the People’s Republic of China is accelerating rapid increases in early life environmental exposures, yet no publications have assessed health care provider capacity to manage common hazards.