Abstract
Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 μg/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 μg/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.
Dihydrofolate reductase (DHFR) is an essential enzyme involved in folate metabolism for both mammalian and microbial cells. DHFR inhibitors, such as pyrimethamine (PYR) and trimethoprim, have been shown to have useful antimicrobial activity. PYR with sulfadiazine is used most frequently to treat and prevent relapses in central nervous system toxoplasmosis (5). Combinations of trimethoprim-dapsone and trimethoprim-sulfamethoxazole have been used for the treatment of Pneumocystis carinii pneumonia (2–4). Folate antagonists have also been shown to have in vitro activity against mycobacteria (7–9).
In an attempt to identify therapeutically useful agents with increased potency and greater selectivity towards Toxoplasma gondii and P. carinii, several analogs were synthesized (6). Ten analogs were screened for their in vitro activities against three isolates of Mycobacterium avium complex (MAC). The three most active compounds, 6-[[(3,5-dimethoxyphenyl)amino] methyl]-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (Chemical Abstracts [C.A.]registration no. 163629-16-1), 6-[[(2,5-dimethoxyphenyl)amino]-methyl]2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (C.A. registration no. 159210-76-1), and 6-[[[(2-methoxy-5-tri-fluoromethyl)phenyl]amino]methyl]-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (C.A. registration no. 174654-98-9), were chosen for more extensive in vitro evaluation against MAC isolates. The three candidates were converted to water-soluble hydrochloride derivatives designated SoRI 8890, 8895, and 8897 (respective to the order listed above for the free-base forms).
Stock solutions of SoRI 8890, SoRI 8895, and SoRI 8897 were prepared by dissolving them in double-distilled water. Dapsone (provided by Jacobus Pharmaceutical Co., Princeton, N.J.) was dissolved in 50% methanol. Solutions were sterilized by passage through a 0.22-μm-pore-size membrane filter and were diluted 20-fold with modified 7H10 broth (7H10 agar formulation with agar and malachite green omitted), pH 6.6, with 10% OADC (oleic acid, albumin, dextrose, and catalase) enrichment and 0.05% Tween 80. Serial twofold dilutions were prepared in modified 7H10 broth, with concentrations of drug ranging from 16 to 0.06 μg/ml. In the combination drug assay, the concentration of dapsone was held constant at 0.5 μg/ml, while the concentration of the DHFR inhibitor ranged from 8 to 0.008 μg/ml.
Twenty-five clinical MAC isolates were used in the study. Isolates were kindly provided by Leonid B. Heifets (Mycobacteriology Reference Laboratory, National Jewish Center for Immunology and Respiratory Diseases, Denver, Colo.) and Lowell S. Young (Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, Calif.). The remaining isolates were obtained from patients with AIDS at the State University of New York Health Science Center at Syracuse. Isolates were grown at 37°C in modified 7H10 broth for 3 to 5 days on a rotary shaker and were then diluted in modified 7H10 broth to yield a final concentration of approximately 2.5 × 104 CFU/ml (range, 1.5 × 104 to 9 × 104 CFU/ml). The final volume in each tube was 2 ml. The inoculum size was measured by titration and counting from duplicate 7H10 agar plates supplemented with 5% OADC. The isolates maintained a predominant smooth-transparent phenotype (>90%) on the titer plates. For each isolate a tube without drug was included as a positive control. Tubes were incubated in ambient air on a rotary shaker (150 rpm) at 37°C for 5 to 7 days, until good growth was present in the control tubes. The MIC was defined as the lowest drug concentration that yielded no visible turbidity.
The isolates were studied in groups of five to eight organisms, with ATCC 49601 as an internal control. The MICs of SoRI 8890, 8895, and 8897 against this isolate were 2, 8, and 4 μg/ml, respectively.
The MICs of SoRI 8890, 8895, and 8897 against all 25 isolates ranged from 0.125 to 4, 0.5 to 8, and ≤0.06 to 16 μg/ml, respectively (Table 1). The MICs at which 50 and 90% of isolates were inhibited (MIC50 and MIC90) were, respectively, 1 and 2 μg/ml for SoRI 8890, 4 and 8 μg/ml for SoRI 8895, and 4 and 8 μg/ml for SoRI 8897 (Table 1).
TABLE 1.
MICs of SoRI 8890, 8895, and 8897 against 25 MAC isolates
| MAC isolate | MIC (μg/ml) of:
|
||
|---|---|---|---|
| SoRI 8890 | SoRI 8895 | SoRI 8897 | |
| DW4 | 2 | 4 | 0.5 |
| DW5 | 2 | 2 | 8 |
| DW6 | 1 | 8 | 8 |
| DW7 | 0.5 | 4 | 4 |
| DW9 | 2 | 8 | 8 |
| DW10 | 1 | 2 | 1 |
| DW11 | 0.5 | 4 | 2 |
| DW12 | 1 | 4 | 2 |
| DW14 | 1 | 8 | 4 |
| DW15 | 2 | 8 | 2 |
| DW16 | 1 | 8 | 8 |
| DW17 | 2 | 4 | 1 |
| DW18 | 2 | 2 | 8 |
| DW20 | 0.5 | 4 | 2 |
| DW23 | 0.5 | 2 | 4 |
| DW25 | 2 | 8 | 8 |
| DW26 | 2 | 8 | 4 |
| DW27 | 4 | 8 | 16 |
| DW29 | 1 | 4 | 2 |
| DW30 | 1 | 4 | 2 |
| DW31 | 2 | 4 | 8 |
| DW32 | 1 | 2 | 0.5 |
| 101 | 0.25 | 1 | 0.125 |
| 3404.4 | 0.125 | 0.5 | ≤0.06 |
| ATCC 49601 | 2 | 8 | 4 |
| MIC50 | 1 | 4 | 4 |
| MIC90 | 2 | 8 | 8 |
Several of these isolates had been tested previously for their in vitro susceptibilities to two other DHFR inhibitors, PYR and WR99210 (8, 9). The MIC50 and MIC90 of PYR were each 32 μg/ml, and those of WR99210 were 2 and 8 μg/ml, respectively. When the dihydropteroate synthase inhibitor dapsone (0.5 μg/ml) was combined with PYR or WR99210, the MIC50 and MIC90 decreased to 4 and 8 μg/ml for PYR and to 0.25 and 4 μg/ml for WR99210. The addition of dapsone at a fixed concentration of 0.5 μg/ml to SoRI 8890, 8895, or 8897 yielded no significant enhancement of in vitro activity (data not shown).
These in vitro results suggest that SoRI 8890, 8895, or 8897 may have useful activity for the treatment of MAC infections. Although the addition of dapsone provided no enhancement of in vitro activity, the MICs of these drugs against MAC were lower than those of other DHFR inhibitors such as PYR or WR99210. Evaluation of the in vivo activities of these compounds in the beige mouse model of disseminated MAC infection would be of interest (1).
Acknowledgments
We acknowledge the technical and editorial assistance of Michelle DeStefano.
This study was supported by the NCDDG-OI program and by cooperative agreement U19-AI40972 with NIAID, and the compounds tested were synthesized with support from PHS grant UOI-AI30279 from NIAID, NIH.
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