In vitro pharmacodynamic parameters of sordarin derivatives in comparison with those of marketed compounds against Pneumocystis carinii isolated from rats

Abstract

Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E(max)), the drug concentrations to reach 50% of E(max) (EC(50)), and the slope of the dose-response curve were then estimated by the Hill equation (E(max) sigmoid model). Sordarin derivatives were the most potent agents against P. carinii, with EC(50)s of 0.00025, 0.0007, 0.0043, and 0. 025 microg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC(50)s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 microg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo-GM 191519, GM 237354, and GM 219771-showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED(50)], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED(50)s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.

FIG. 1

Typical growth curve of rat-derived P. carinii cultivated in DMEM supplemented with 10% FCS.

FIG. 2

Influence of incubation time on the in vitro activities of GM 237354 and pentamidine for 5 consecutive days of culture. Both compounds were tested at three different concentrations. □, 10 μg/ml; ▨, 1 μg/ml; ▧, 0.01 μg/ml. The effect of each compound concentration on P. carinii was expressed as percent inhibition versus the drug-free control.

FIG. 3

In vitro activity of GM 237354 against P. carinii. (A) Culture medium with 0.5 μg of GM 237354 per ml. A dramatic reduction in the number of microorganisms was observed. (B) Culture medium with 5 × 10⁻⁴ μg of GM 237354 per ml. A marked decrease in the number of microorganisms was observed. (C) Drug-free control culture. Trophozoites (open arrowhead) and trophozoite clusters (solid arrowhead) were observed. RAL-555 staining was used. Magnification, ×1,100.

FIG. 4

Concentration-in vitro activity relationships of the three commercial compounds against P. carinii. ●, pentamidine; ▴, atovaquone; ⧫, TMP-SMX. Results were calculated after 4 days of incubation.

FIG. 5

Concentration-in vitro activity relationships of the four sordarin derivatives against P. carinii. ●, GM 191519; ■, GM 237354; ▴, GM 193663; ▾, GM 219771. Results were calculated after 4 days of incubation.

FIG. 6

In vivo efficacies of GM 191519 (●) and GM 219771 (▾) on the P. carinii burden in lung. The data are means with standard deviations for six animals. The dose-effect relationships were calculated according a simple E_max model.