Design and profiling of GS-9148, a novel nucleotide analog active against nucleoside-resistant variants of human immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate prodrug, GS-9131

Abstract

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (K(i) = 0.8 muM) and exhibits low inhibitory potency against host polymerases including DNA polymerase gamma. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 microM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

FIG. 1.

Structures of GS-9148 and GS-9131.

FIG. 2.

Structures of the nucleotide analogs tested. Compounds: 1, nucleoside phosphonate analog d4AP; 2, l-d4AP; 3, Cd4AP; 4, Fd4AP; 5, Fd4GP; 6, Fd4IP; and 7, Fd4DAPP.

FIG. 3.

Structures of GS-9148 amidate prodrugs. Compounds: 8, mono(ethyl-alanine) amidate; 9, diastereomer A (designated GS-9131); 10, diastereomer B; 11, mono(isopropyl-alanine) prodrug; 12, mono(isobutyl-phenylalanine) amidate; 13, bis(n-butyl-alanine) amidate.

FIG. 4.

(A) Comparison of the resistance profile of GS-9148 with those of clinically approved NRTIs. The bars represent the change in the susceptibility of each mutant virus relative to that of the wild-type control strain, using a PhenoSense assay (Monogram Biosciences). GS-9148 (green), TFV (yellow), d4T (red), FTC (light blue), ddI (orange), ABC (purple), AZT (dark blue). To improve the resolution of lower resistance, a greater than 30-fold change is not depicted. The exact values of each virus’ resistance to the NRTIs tested are provided in the supplemental material. (B) List of HIV-1 strains used for the resistance analysis, with primary resistance mutations present in RT.

FIG. 5.

Plasma and PBMC pharmacokinetic profiles for GS-9131 and its metabolites following the oral administration of 3 mg/kg to male beagle dogs. GS-9131 is rapidly absorbed and has a short t_1/2 in plasma (solid circles). Following the disappearance of the prodrug, a concomitant increase in plasma levels of GS-9148 is observed (open circles). Analysis of intracellular PBMC drug levels indicates that GS-9148 reaches a maximal concentration at the first measurement following a slow disappearance (open triangles). GS-9148-DP (solid triangles) forms over time and reaches a persistent maximal level at between 8 and 24 h postdose. All values represent the mean ± standard deviations of data from eight dogs dosed in two independent studies.