Comparative study of the anti-human cytomegalovirus activities and toxicities of a tetrahydrofuran phosphonate analogue of guanosine and cidofovir

Abstract

Cidofovir is the first nucleoside monophosphate analogue currently being used for the treatment of human cytomegalovirus (HCMV) retinitis in individuals with AIDS. Unfortunately, the period of therapy with the use of this compound may be limited due to the possible emergence of serious irreversible nephrotoxic effects. New drugs with improved toxicity profiles are needed. The goal of this study was to investigate the anticytomegaloviral properties and drug-induced toxicity of a novel phosphonate analogue, namely, (-)-2-(R)-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran (compound 1), in comparison with those of cidofovir. The inhibitory activities of both compounds on HCMV propagation in vitro were similar against the AD 169 and Towne strains, with 50% inhibitory concentrations ranging from 0.02 to 0.17 microgram/ml for cidofovir and < 0.05 to 0.09 microgram/ml for compound 1. A clinical HCMV isolate that was resistant to ganciclovir and that had a known mutation within the UL54 DNA polymerase gene and a cidofovir-resistant laboratory strain derived from strain AD 169 remained sensitive to compound 1, whereas their susceptibilities to ganciclovir and cidofovir were reduced by 33- and 10-fold, respectively. Both compound 1 and cidofovir exhibited equal potencies in an experimentally induced murine cytomegalovirus (MCMV) infection in mice, with a prevention or prolongation of mean day to death at dosages of 1.0, 3.2, and 10.0 mg/kg of body weight/day. In cytotoxicity experiments, compound 1 was found to be generally more toxic than cidofovir in cell lines Hs68, HFF, and 3T3-L1 (which are permissive for HCMV or MCMV replication) but less toxic than cidofovir in MRC-5 cells (which are permissive for HCMV replication). Drug-induced toxic side effects were noticed for both compounds in rats and guinea pigs in a 5-day repeated-dose study. In guinea pigs, a greater weight loss was noticed with cidofovir than with compound 1 at dosages of 3.0 and 10.0 mg/kg/day. An opposite effect was detected in rats, which were treated with the compounds at relatively high dosages (up to 100 mg/kg/day). Compound 1 and cidofovir were nephrotoxic in both rats and guinea pigs, with the epithelium lining the proximal convoluted tubules in the renal cortex being the primary target site. The incidence and the severity of the lesions were found to be dose dependent. The lesions observed were characterized by cytoplasm degeneration and nuclear modifications such as karyomegaly, the presence of pseudoinclusions, apoptosis, and degenerative changes. In the guinea pig model, a greater incidence and severity of lesions were observed for cidofovir than for compound 1 (P < 0.001) with a drug regimen of 10 mg/kg/day.

FIG. 1

Chemical structure of cidofovir (A) and chemical synthesis of (−)-2-(R)-dihydroxyphosphinoyl-5-(S)-(guanin-9′-yl-methyl) tetrahydrofuran (compound 1) (B). a, CBr₄, PPh₃, MeCN, 84%; b, diisobutylaluminum hydride, toluene, −78°C, 77%; c, Ac₂O, pyr, 4-(dimethylamino)pyridine, CH₂Cl₂, 86%; d, TiCl₄, P(OEt)₃, CH₂Cl₂, 86%; e, Cs₂CO₃, 2-amino-6-chloropurine, dimethylformamide 95°C, 42%; f, bromotrimethylsilane, CH₂Cl₂ and then water, 100°C, 60%.

FIG. 2

Anti-HCMV activities of compound 1, cidofovir, GCV, and PFA in the virus yield reduction assay. HFF cells were infected at an MOI of 0.5 and were then treated with compound 1 (▴), cidofovir (▵), GCV (○), and PFA (●) at concentrations ranging from 0.01 to 100 μg/ml for a period of 7 days at 37°C. Thereafter, infected-treated cells were subjected to one freeze-thaw cycle and then the virus titer in the cell lysates was determined as described in Materials and Methods.

FIG. 3

(A) Compound 1-related weight changes in rats. Rats were injected i.p. once a day for 5 days with a dose of 0 (■), 25 (□), 50 (▴), 75 (▵), or 100 (●) mg of compound 1 per kg. The averages of quadriplicates are presented. The standard deviation was less than 10% for each point. (B) Cidofovir-related weight changes in rats. Rats were injected i.p. once a day for 5 days with a dose of 0 (■), 25 (□), 50 (▴), or 100 (●) mg of cidofovir per kg. The averages of quadriplicates are presented. The standard deviation was less than 10% for each point.

FIG. 4

(A) Compound 1-related weight changes in guinea pigs. Guinea pigs were injected subcutaneously once a day for 5 days with a dose of 0 (■), 0.3 (□), 1.0 (▴), 3.0 (▵), or 10 (●) mg of compound 1 per kg. The averages of quadriplicates are presented. The standard deviation was less than 10% for each point. (B) Cidofovir-related weight changes in guinea pigs. Guinea pigs were injected subcutaneously once a day for 5 days with a dose of 0 (■), 0.3 (□), 1.0 (▴), 3.0 (▵), or 10 (●) mg of cidofovir per kg. The averages of quadriplicates are presented. The standard deviation was less than 10% for each point.

FIG. 5

Histopathology analysis of kidneys from guinea pigs treated with cidofovir and compound 1. The animals were administered a solution of saline (A), a dose of 10 mg of cidofovir per kg (B), or a dose of 10 mg of compound 1 per kg (C) i.p. once daily for 5 consecutive days. (B) The severe tubular nephropathy was characterized by widespread necrosis and sloughing of the lining epithelium (➧) and denudation of the basal lamina (✻). (C) The tubular nephropathy was characterized by cytoplasmic eosinophilic droplets (➱) and nuclear changes consisting of karyomegaly (➞), eosinophilic inclusions (✻), and apoptosis (✻✻).

FIG. 6

Histopathology analysis of kidneys from rats treated with cidofovir and compound 1. The animals were administered a solution of saline (A), a dose of 100 mg of cidofovir per kg (B), or a dose of 100 mg of compound 1 per kg (C) subcutaneously once daily for 5 consecutive days. (B) Cytoplasmic changes included effacing of the fine structural details (➧) and shedding into the lumen (✻). Nuclear changes consisted of karyomegaly and eosinophilic pseudoinclusions (➱) and apoptosis (➞). (C) Cytoplasmic changes included mild vacuolation and perinuclear hydropic degeneration (✻). Nuclear changes consisted of karyomegaly (➞) and apoptosis (➧).