EFFECT OF PLASMA-PROTEIN BINDING ON IN-VIVO ACTIVITY AND BRAIN PENETRATION OF GLYCINE NMDA RECEPTOR ANTAGONISTS/

A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in t he DBA/2 mouse anticonvulsant assay, but improvements in in vitro affi nity were not mirrored by corresponding increases in anticonvulsant ac tivity. Here we show that binding of the compounds to plasma protein l imits their brain penetration. Relative binding to the major plasma pr otein, albumin, was measured in two different ways: by a radioligand b inding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensive ly to quantify plasma protein binding. For the 4-hydroxyquinolone seri es, binding to plasma protein correlates (r = 0.92) with log P (octano l/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticon vulsant activity increases with in vitro affinity, but the slope of a plot of pED(50) versus pIC(50) is low (0.40); taking plasma protein bi nding into account in this plot increases the slope to 0.60. This show s that binding to albumin in plasma reduces the amount of compound fre e to diffuse across the blood-brain barrier. Further evidence comes fr om three other experiments: (a) Direct measurements of brain/blood rat ios for three compounds (2, 16, 26) show the ratio decreases with incr easing log P. (b) Warfarin, which competes for albumin binding sites d ose-dependently, decreased the ED50 of 26 for protection against seizu res induced by NMDLA. (c) Direct measurement of brain penetration usin g an in situ brain perfusion model in rat to measure the amount of dru g crossing the blood/brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this i s greatly reduced when the drug is delivered in plasma. In the 4-hydro xyquinolones glycine site binding affinity increases with lipophilicit y of the 3-substituent up to a maximum at a log P around 3, then does not improve further. When combined with increasing protein binding, th is gives a parabolic relationship between predicted in vivo activity a nd log P, with a maximum log P value of 2.39. Finally, the plasma prot ein binding studies have been extended to other series of glycine site antagonists, and it is shown that for a. given log P these have simil ar protein binding to the 4-hydroxyquinolones, except for compounds th at are not acidic. The results have implications for the design of nov el glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pK(a) high to obtain good central nervous system activity.