Jul 16, 1990 - In Vitro Activities of Acridone Alkaloids against Pneumocystis carinii. SHERRY F. QUEENER,1* HISASHI FUJIOKA,2 YUKIHIRO NISHIYAMA,3 ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1991, 0066-4804/91/020377-03$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 35, No. 2
In Vitro Activities of Acridone Alkaloids against Pneumocystis carinii SHERRY F. QUEENER,1* HISASHI FUJIOKA,2 YUKIHIRO NISHIYAMA,3 HIROSHI FURUKAWA,4 MARILYN S. BARTLETT,' AND JAMES W. SMITH5 Department of Pharmacology & Toxicology1 and Department of Pathology,5 Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202-5120, and Department of Medical Zoology2 and Research Institute for Disease Mechanism and Control,3 Nagoya University School of Medicine, Showa-ku, Nagoya 466, and Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468,4 Japan Received 16 July 1990/Accepted 20 November 1990
Acridone alkaloids isolated from plants of the family Rutaceae have antiplasmodial activity in rodent models of malaria. Because a variety of antimalarial agents have also been shown to have activity against Pneumocystis carinii, we tested six of these alkaloids in an established culture model for P. carinii. Atalaphillinine and glycobismine A inhibited growth of cultured P. carinii at concentrations of 2.7 and 1.7 ,uM, respectively. This potency of effect is similar to that of chloroquine (3 ,uM) but somewhat less than that of primaquine (0.4 ,uM), which was previously evaluated in the same system.
cm2 of a slide, fixed with 100% methanol, and stained with Giemsa stain. These slides were examined at x 1,000 magnification for quantification of organisms. The data are reported as the number of trophozoites per field; multiplication
Acridone alkaloids have been isolated from plants of the family Rutaceae, including Citrus, Glycosmis, and Severinia species (5). These compounds, as well as their analogs and derivatives, have been tested for their antimicrobial activities and effects on mammalian cells (4). Acronycine has been tested for antitumor properties (9, 10), and acronycine analogs were reported to be effective against Trichomonas vaginalis (8). A series of 30 acridone alkaloids was evaluated for their antiplasmodial activities in a rodent model (4). Seven of the alkaloids in the series had activities equivalent to that of chloroquine against Plasmodium yoelii. Atalaphillinine was effective as a prophylactic agent against Plasmodium berghei and Plasmodium vinckie infections in mice. In our effort to expand the range of potentially useful drugs against Pneumocystis carinii, we sought to test a variety of compounds. In particular, we and others have noted that antimalarial drugs often also have activity against P. carinii (1, 3, 6, 7). Therefore, on the basis of the activities cited above, six acridone alkaloids with antimalarial activities were selected for testing in an established in vitro model for P. carinii. Test procedure for Pneumocystis cultures. Cultures of P. carinii were prepared and evaluated as described previously (7). Briefly, human embryonic lung fibroblastic cells (WI-38) were cultured in 24-well tissue culture plates with minimum essential medium containing 10% fetal bovine serum. Confluent monolayers were inoculated with homogenates of rat lungs infected with P. carinii. The infected rat lungs were obtained from rats immunosuppressed with cortisone acetate by using previously published procedures (2). Lungs from these animals were ground in minimum essential medium, and the inoculum was adjusted to give a final concentration in culture of 3 x 105 to 7 x 105 trophozoites per ml. Drugs were then diluted in culture medium and added to the cultures. The drug solution added was 10 ,ul or less per ml of culture medium; the inoculum was 0.1 ml/ml of culture medium. Plates were incubated at 35°C in 5% oxygen and 5 to 10% carbon dioxide, with the balance being nitrogen. Separate plates were harvested for analysis at 1, 3, 5, and 7 days after inoculation. At these times, 10 ,ul of the culture supernatant was removed from each well, air dried onto 1
H0 IO 0
FIG. 1. Structures of the acridone alkaloids used in this study. 377
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Activities of acridone alkaloids against P. carinii in vitro Culture condition (concn
Ratio of counts
(day 7/day 1)
% of control at: Da1 Dy7
A 10 0 -
0 0 0
Atalaphillinine 1 10
Atalaphillidine 1 10
Des-N-methylnoracronycine 1 10
Glycocitrine I 1 10
0 0 N
0 0 L.
2Atalaphillinine 10 gmI
Glycobismine A 1 10
5-OH-N-Methylseverifoline 1 10
Glycocitrine 1 pg/mI
of these counts by 105 (calculated from the magnification factor and size of the field) yielded the number of organisms per milliliter of culture supernatant. The test compounds were added in dimethyl sulfoxide to give a final concentration of 1 or 10 ,ug/ml. The final concentration of dimethyl sulfoxide (1 RI/ml) was shown in control experiments to have no significant effect on growth. Each assay was performed in quadruplicate, and results were plotted as means. Sources of acridone alkaloids. The compounds used in this study (Fig. 1) were either synthesized or isolated from the root barks of Citrus, Glycosmis, or Severinia plants. Cytotoidcity tests. The lymphocytic mouse leukemia L1210 cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. Diluted drugs were dispensed into plastic petri dishes (diameter, 35 mm) to make appropriate concentrations. Two milliliters of a suspension of L1210 cells (1 x 105 to 2 x iOs cells per ml) was added to each dish and cultured for 42 h at 37°C in a 5% CO2 humidified incubator. After incubation, the number of cells per milliliter was determined with a hemacytometer, and the percentage of growth inhibition with respect to that by nontreated controls was calculated. The 50% effective concentration (ED50) for each drug was determined by probit a factor of 4 x
analysis. The six acridone alkaloids that showed the most activity against plasmodia in vitro were selected for testing against P. carinii by an in vitro culture method (Table 1). Growth curves of cultures treated with one of the most active and one of the least active compounds are shown (Fig. 2) relative to those of untreated cultures (negative control)
0 0 c
0 0 N 0
4. Glycocitrne 10 pg/ml
Days FIG. 2. Growth curves for P. carinii in culture. Control curves show growth in the absence of any additives. The dilution control is growth with only 0.1 ml of drug diluent added to the culture. Trimethoprim-sulfamethoxazole was present at 50 and 250 >ig/ml, respectively; this curve serves as a positive control. (A) Atalaphillinine at 1 and 10 ,ug/ml in the final medium was achieved by adding 0.1 ml of the appropriate dilution of stock. (B) Glycocitrine at 1 and 10 ,ug/ml in the final medium was achieved by adding 0.1 ml of the appropriate dilution of stock.
VOL. 35, 1991
and cultures treated with trimethoprim-sulfamethoxazole. The host WI-38 cells remained normal in appearance during these experiments and did not detach from the surface of the culture dish. The cytotoxicities of five of these agents were evaluated against another cell line, L1210. The order from most to least cytotoxic, based on ED50s, was 5-OH-N-methylseverifoline (ED50, 1 ,ug/ml) > atalaphillinine (ED50, 1.25 ,ug/ml) > des-N-methylnoracronycine (ED50, 2 jig/ml) > atalaphillidine (ED50, 3.1 ,ug/ml) > glycocitrine I (ED50, 4.75 ,ug/ml). All six acridone alkaloids tested were active against P. carinii in culture at a concentration of 10 ,ug/ml, which corresponded to 17 to 34 ,M for the various compounds (Table 1). Only two of the compounds were active at 1 ,ug/ml; these were atalaphillinine (2.7 ,uM) and glycobismine A (1.7 ,uM). By comparison, from previous studies with this model system, we have reported minimal effective concentrations of 0.4 ,uM for primaquine and 3 ,uM for chloroquine (1, 7). Of the two most active compounds, one (atalaphillinine) was also one of the most cytotoxic compounds tested. However, another highly cytotoxic agent (5-OH-N-methylseverifoline) was not active against P. carinii at 1 ,ug/ml, suggesting that cytotoxicity alone may not predict anti-P. carinii activity. Glycobismine A was not tested in the system against L1210 cells. In summary, atalaphillinine and glycobismine A showed significant activities (2.7 and 1.7 ,uM, respectively) against P. carinii in a culture model. The potencies of these compounds in the culture model are similar to that of chloroquine, but slightly less than that of primaquine, which was evaluated previously in the same system (7). The excellent laboratory skills of Margaret Shaw and Michelle Durkin are acknowledged. This work was supported in part by Public Health Service
contract AI-72647 from the Division of AIDS, National Institutes of Health. REFERENCES 1. Bartlett, M. S., R. Eichholtz, and J. W. Smith. 1985. Antimicrobial susceptibility of Pneumocystis carinii in culture. Diagn. Microbiol. Infect. Dis. 3:381-387. 2. Bartlett, M. S., J. A. Fishman, M. M. Durkin, S. F. Queener, and J. W. Smith. 1990. Pneumocystis carinii: improved models to study efficacy of drugs for treatment or prophylaxis of Pneumocystis pneumonia in the rat (Rattus spp.). Exp. Parasitol. 70:100-106. 3. Frenkel, J. K., J. T. Good, and J. A. Shultz. 1966. Latent Pneumocystis infection of rats, relapse and chemotherapy. Lab. Invest. 15:1559-1577. 4. Fujioka, H., Y. Nishiyama, H. Furukawa, and N. Kumada. 1989. In vitro and in vivo activities of atalaphillinine and related acridone alkaloids against rodent malaria. Antimicrob. Agents Chemother. 33:6-9. 5. Grundon, M. F. 1988. Quinoline, quinazoline, and acridone alkaloids. 3. Acridone alkaloids. Nat. Prod. Rep. 5:302-307. 6. Pesanti, E. 1980. In vitro effects of antiprotozoan drugs and immune serum on Pneumocystis carinii. J. Infect. Dis. 141:775780. 7. Queener, S. F., M. S. Bartlett, J. D. Richardson, M. M. Durkin, M. A. Jay, and J. W. Smith. 1988. Activity of clindamycin with primaquine against Pneumocystis carinii in vitro and in vivo. Antimicrob. Agents Chemother. 32:807-813. 8. Schneider, J., E. L. Evans, E. Grunberg, and R. I. Fryer. 1972. Synthesis and biological activity of acronycine analogs. J. Med. Chem. 15:266-270. 9. Svoboda, G. H., G. A. Poore, P. J. Simpson, and G. B. Boder. 1966. Alkaloids of Acronychia baueri Schott. I. Isolation of the alkaloids and a study of the antitumor and other biological properties of acronycine. J. Pharm. Sci. 55:758-768. 10. Tan, P., and N. Auersperg. 1973. Effects of the antineoplastic alkaloid acronycine on the ultrastructure and growth patterns of cultured cells. Cancer Res. 33:2320-2329.