Characterization of the transport, metabolism, and pharmacokinetics of the dopamine D3 receptor-selective fluorenyl- and 2-pyridylphenyl amides developed for treatment of psychostimulant abuse

Abstract

The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse. Additional screening with a panel of human and rat Supersomes was performed for NGB 2904 and PG 01037. Drug-stimulated ATPase activation assays and bidirectional transport and efflux assays were used to test for substrate specificity of NGB 2904 and PG 01037 for human and rat efflux transporters. All compounds exhibited moderate elimination half-lives, ranging from 1.49 to 3.27 h, and large volumes of distribution (5.95-14.19 l/kg). The brain-to-plasma ratios ranged from 2.93 to 11.81 and were higher than those previously reported for cocaine. Brain exposure levels of NGB 2904 and PG 01037 were significantly reduced after intraperitoneal administration compared with intravenous administration. The metabolism of these compounds was mediated primarily by CYP3A subfamilies. PG 01037 was a P-glycoprotein-transported substrate. Higher doses of these compounds are often required for in vivo action, suggesting decreased bioavailability via extravascular administration that may be attributed to high drug efflux and hepatic metabolism. These studies provide important preclinical information for optimization of next-generation D3R selective agents for the treatment of drug addiction.

Fig. 1.

Structures of the fluorenyl amides NGB 2904 (A) and JJC 4-077 (B) and the 2-pyridylphenyl amides CJB 090 (C), PG 01037 (D), and PG 01030 (E).

Fig. 2.

Plasma and brain pharmacokinetic profiles of D3R analogs in male Sprague-Dawley rats (n = 3/time point). A, observed (mean ± S.D.) and predicted plasma concentration versus time profiles of the fluorenyl amides based on the naive averaging analysis conducted with WinNonlin. B, observed brain concentrations (mean ± S.D.) versus time profiles of the fluorenyl amides. C, observed (mean ± S.D.) and predicted plasma concentration versus time profiles of the 2-pyridylphenyl amides. D, observed brain concentrations (mean ± S.D.) versus time profiles of the 2-pyridylphenyl amides.

Fig. 3.

B/P ratio versus time profiles of the fluorenyl amides NGB 2904 and JJC 4-077 (n = 3) (A) and 2-pyridylphenyl amides CJB 090 and PG 01037 (n = 3) (B).

Fig. 4.

Metabolism of NGB 2904 (A and B) and PG 01037 (C and D) by the human recombinant P450s (A and C, respectively) and the rat recombinant P450s (B and D, respectively). Compounds (10 μM) were incubated with Supersomes (50 pmol/ml) and cofactors for 60 min followed by termination of reactions and determination of unchanged substrate. Data are presented as mean percentage of the average control ± S.D. from triplicate reactions. *, p < 0.05 based on Dunnett's test.

Fig. 5.

Stimulation of human P-gp (A), rat P-gp (B), human BCRP (C), rat BCRP (D), human MRP2 (E), and rat MRP2 (F) ATPase activity by increasing concentrations of NGB 2904 and PG 01037. Stimulation of ATPase activity was estimated by measuring inorganic phosphate released from ATP in the presence or absence of their respective controls or D3R analogs (NGB 2904 and PG 01037; 5–100 μM). The drug-stimulated ATPase activity was reported as fold-stimulation relative to the basal ATPase activity in the absence of drug (DMSO control). Data are represented as mean fold stimulation of the average control from triplicate reactions mean ± S.D. *, p < 0.05.

Fig. 6.

Effect of the competitive inhibitor verapamil (200 μM) on the permeability and P-gp-mediated efflux of PG 01037 across MDCK-MDR1 monolayers. A, P-gp expression levels over the period in which transport studies were conducted (passages 18–24). B, efflux ratios (mean ± S.D., n = 3) across MDCK-MDR1 monolayers in the absence (−) and presence (+) of verapamil. C, apical-to-basolateral permeability in the absence (−) and presence (+) of verapamil across MDCK-MDR1 monolayers (data represented as mean ± S.D., n = 3). *, p < 0.05.