A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii agents

Abstract

A series of over 60 agents representing several different classes of compounds were evaluated for their effects on the ATP pools of Pneumocystis carinii populations derived from immunosuppressed rats. A cytotoxicity assay based on an ATP-driven bioluminescent reaction was used to determine the concentration of agent which decreased the P. carinii ATP pools by 50% versus untreated controls (IC50). A ranking system based on the IC50 value was devised for comparison of relative responses among the compounds evaluated in the cytotoxic assay and for comparison to in vivo efficacy. With few exceptions, there was a strong correlation between results from the ATP assay and the performance of the compound in vivo. Antibiotics, with the exception of trimethoprim-sulfamethoxazole (TMP-SMX), were ineffective at reducing the ATP pools and were not active clinically or in the rat model of P. carinii pneumonia. Likewise, other agents not expected to be effective, e.g., antiviral compounds, did not show activity. Standard anti-P. carinii compounds, e.g., TMP-SMX, pentamidine, and dapsone, dramatically reduced ATP levels. Analogs of the quinone and topoisomerase inhibitor groups were shown to reduce ATP concentrations and hold promise for further in vivo investigation. The cytotoxicity assay provides a rapid assessment of response, does not rely on replicating organisms, and should be useful for assessment of structure-function relationships.

Anti–Human Immunodeficiency Virus Drugs Are Ineffective againstPneumocystis cariniiIn Vitro and In Vivo

“…In vitro studies. The techniques used here have been reported elsewhere [5,6]. In brief, P. carinii organisms were purified from infected rats, characterized genetically by contour-clamped homogenous electric field analysis (the predominant form was P. carinii f sp carinii form 1), cryopreserved and stored in liquid nitrogen, and tested for contaminants by culture before use.…”

“…We have a long-term program to develop new anti-P. carinii drugs. Compounds are first studied in an ATP cytotoxicity assay and then in immunosuppressed rats or mice with pneumocystosis [5][6][7][8][9]. A high degree of correlation has been found among these systems; drugs that exhibit anti-P. carinii activity in vitro and in vivo are likely to be active against the organism in humans.…”

Functional Characterization of Pneumocystis carinii Inositol Transporter 1

“…We evaluated the effects of these drugs on the uptake of radiolabeled myo- inositol (Fig. 6) and on the viability of cultured P. carinii using an ATP-driven assay (Table 1) (27). As shown by the results in Fig.…”

Comparison of in vivo and in vitro inactivation of endospores of Rhinosporidium seeberi following dapsone treatment

“…Even after the endospores are liberated from the sporangium, they will still be within the down-growths of hyperplastic epithelium or entrapped in the mucoid matrix in the stroma of the rhinosporidial tissue Such interference with the access of drugs in vivo might account for the great difference between the rapidity of the in vitro inactivation and the response of the pathogen in diseased tissues in vivo. Rex et al (1993) and Cushion et al (1997) referred to correlations between the results from in vitro assays and in vivo responses in the testing of antimicrobial drug activity in terms of the effective concentrations of the drugs in vitro and in vivo. The phenomenon of differences in time courses demonstrated for in vitro activity compared with in vivo responses discussed in this paper refer to an additional dimension of this correlation: the pharmacodynamics of drug action in vitro and pharmacokinetics in vivo, rather than to drug concentrations.…”