Effect of Plasma Protein Binding on in Vivo Activity and Brain Penetration of Glycine/NMDA Receptor Antagonists

Abstract

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 the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding 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 extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity increases with lipophilicity 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, this gives a parabolic relationship between predicted in vivo activity and log P, with a maximum log P value of 2.39. Finally, the plasma protein binding studies have been extended to other series of glycine site antagonists, and its is shown that for a given log P these have similar protein binding to the 4-hydroxyquinolones, except for compounds that are not acidic. The results have implications for the design of novel glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pKa high to obtain good central nervous system activity.

A Prodrug of NMDA/Glycine Site Antagonist, L-703,717, with Improved BBB Permeability. 4-Acetoxy Derivative and Its Positron-Emitter Labeled Analog.

“…5) Rowley and co-workers have shown that the binding of L-701,324 to albumin can be effectively inhibited by pre-treatment with warfarin, a drug that resembles L-701,324 in structure and probably competes with L-701,324 for albumin binding. 5) We have recently shown that simultaneous injection of warfarin dose-dependently increases the brain uptake of a positron-emitter labeled L-703,717 ([ 11 C]L-703,717) in mice.…”

“…5) Rowley and co-workers have shown that the binding of L-701,324 to albumin can be effectively inhibited by pre-treatment with warfarin, a drug that resembles L-701,324 in structure and probably competes with L-701,324 for albumin binding. 5) We have recently shown that simultaneous injection of warfarin dose-dependently increases the brain uptake of a positron-emitter labeled L-703,717 ([ 11 C]L-703,717) in mice. 6) It was further found that, after the increase in brain uptake by warfarin, the glycine site antagonist was more highly localized in the rodent cerebellum than in regions with high-density NMDA glycine receptors.…”

“…The ionization of a drug in blood usually reduces its ability to penetrate the BBB, and the acidic compounds are more amenable to bind with albumin than non-acidic compounds. 5) Therefore protection of the ionizable hydroxyl groups of the 4-hydroxyquinolones by esters might prevent their ionization in vivo and eventually alter their binding characteristic to serum albumin, leading to the enhancement of brain penetration by the 4-hydroxyquinolones. The esters taken up by the brain can be expected to be hydrolyzed to L-703,717 by ubiquitous esterase in the brain.…”

Thermal reaction of 3aH,5H-thiazolo[5,4-c]quinoline-2,4-diones – an easy pathway to 4-amino-1H-quinolin-2-ones and novel 6H-thiazolo[3,4-c]quinazoline-3,5-diones

“…Only a small number of 4-amino-3-alkyl/aryl-1H-quinolin-2-ones 5 have been described so far. They were prepared starting from 4-hydroxy-2-quinolones, 10-12 4-halogeno-2-quinolones, 11, 13-14 2-aminobenzenecarbonitriles, [15][16][17][18][19] and 2-bromobenzonitrile. 20 Compounds 5 have been employed in the synthesis of indolo[3,2-c]quinolin-6-ones and dibenzonaphtyridine-6,11-diones.…”

“…21 To date, few 4-aminoquinolin-2-ones have been tested for biological activity. 7-Chloro-3-phenylsubstituted compounds 5 and their N-alkyl and N-acyl derivatives exhibited in vivo anticonvulsant activity in the DBA/2 strain of mouse 18 and in vitro antagonist activity at the glycine site of the N-methyl-D-aspartate receptor. 17 We believe that the most significant contribution of the present work is the synthesis of novel thiazolo [3,4- …”