Abstract
BAL0030543, BAL0030544, and BAL0030545 are dihydrophthalazine inhibitors with in vitro potency against gram-positive pathogens. The MIC50s for methicillin (meticillin)-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, hetero-vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant Staphylococcus aureus (VISA) range from 0.015 to 0.25 μg/ml (MIC90s ≤ 0.5 μg/ml). MIC50s for beta-hemolytic streptococci range from 0.03 to 0.06 μg/ml, MIC50s for Streptococcus pneumoniae range from 0.06 to 0.12 μg/ml, MIC50s for Listeria monocytogenes range from 0.015 to 0.06 μg/ml, and MIC50s for Streptococcus mitis are ≤0.015 μg/ml. These three dihydrophthalazine antifolates have improved potency compared to that of trimethoprim and activity against gram-positive pathogens resistant to other drug classes.
(This work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008 [1].)
Dihydrofolate reductase (DHFR) is an enzyme with a pivotal role in the synthesis of intracellular tetrahydrofolic acid, which is essential in the synthesis of purines, some amino acids, and thymidine (6). DHFR is the sole source of tetrahydrofolic acid, and its inhibitors are employed in anti-infective and antitumor chemotherapy, most notably trimethoprim and methotrexate. Differences between mammalian and bacterial DHFR can be exploited in terms of the affinity of antibacterials for DHFR; for example, trimethoprim binds 5 log10 more tightly to bacterial DHFR than to vertebrate DHFR (7). Trimethoprim is the most widely used antimicrobial DHFR inhibitor in clinical practice and is employed as monotherapy to treat urinary tract infections, as therapy to treat tissue-based bacterial infections in the skin and chest in combination with sulfamethoxazole as co-trimoxazole, and as therapy to treat Pneumocystis jirovecii pneumonia.
In the last decade, new DHFR inhibitors have been developed and progressed to phase II and III clinical trials; most recently, iclaprim has completed phase III clinical studies in complicated skin and skin structure infections (9). A second class of DHFR inhibitors, the dihydrophthalazine antifolates, is currently in preclinical development. Three compounds, BAL0030543, BAL0030544, and BAL0030545, have demonstrated in vitro activity against multiresistant staphylococci and Streptococcus pneumoniae (2). The dihydrophthalazine substituent confers potent inhibitory activity against the more prevalent staphylococcal DHFR variants responsible for resistance to trimethoprim. Similar to other DHFR inhibitors, they can be administered both intravenously and orally (10).
In this study, we assessed the in vitro potency of three dihydrophthalazine antifolates, BAL0030543, BAL0030544, and BAL0030545, and a range of comparator agents against clinical isolates of gram-positive pathogens.
The antibacterial agents used in the study were obtained from the following sources: BAL0030543, BAL0030544, and BAL0030545, Basilea Pharmaceutica AG, Switzerland; daptomycin (lot no. 095703A), Cubist Pharmaceuticals, Inc., Lexington, MA; linezolid (lot no. 05003), Pfizer Ltd., Surrey, United Kingdom; moxifloxacin (lot no. BX01US1), Bayer plc, Berkshire, United Kingdom; vancomycin (lot no. 1671543B), Alpha Pharma, Devon, United Kingdom; minocycline (lot no. 014K1207) and trimethoprim (lot no. 68H140), Sigma Ltd., Dorset, United Kingdom. A total of 225 clinically significant gram-positive isolates from the collection held in the Department of Medical Microbiology, Southmead Hospital, Bristol, United Kingdom (1994 to 2008) were used (Table 1). Hetero-vancomycin-resistant Staphylococcus aureus (hVISA) and vancomycin-resistant Staphylococcus aureus (VISA) strains were identified by population analysis profiling (12). MICs were determined using the Clinical and Laboratory Standards Institute agar dilution method for staphylococcal and listerial strains (3), using Mueller-Hinton broth supplemented with 5% lysed horse blood for S. pneumoniae, Streptococcus mitis, and beta-hemolytic streptococci, and using Corynebacteria sp. Mueller-Hinton broth supplemented with 50 mg/liter calcium for daptomycin. S. aureus ATCC 29213 and S. pneumoniae ATCC 49619 were used as control strains. The percentage of susceptible strains using Clinical and Laboratory Standards Institute breakpoints was calculated (4).
TABLE 1.
Antibacterial activities of BAL0030543, BAL0030544, and BAL0030545
| Organism (no. of strains) | Compound | MIC (μg/ml) |
% Susceptiblea | ||
|---|---|---|---|---|---|
| Range | 50% | 90% | |||
| MSSA (25) | BAL0030543 | 0.015-0.06 | 0.03 | 0.03 | 100 |
| BAL0030544 | 0.015-0.25 | 0.03 | 0.03 | 100 | |
| BAL0030545 | 0.015-0.25 | 0.12 | 0.25 | 100 | |
| Trimethoprim | 0.25-32 | 1 | 2 | 92 | |
| Daptomycin | 0.12-0.25 | 0.25 | 0.25 | 100 | |
| Linezolid | 2-2 | 2 | 2 | 100 | |
| Minocycline | 0.25-0.25 | 0.25 | 0.25 | 100 | |
| Moxifloxacin | 0.12-4 | 0.12 | 0.25 | 92 | |
| Vancomycin | 0.5-1 | 1 | 1 | 100 | |
| MRSA (25) | BAL0030543 | 0.015-0.06 | 0.015 | 0.06 | 100 |
| BAL0030544 | 0.03-0.25 | 0.06 | 0.25 | 100 | |
| BAL0030545 | 0.06-0.06 | 0.06 | 0.06 | 100 | |
| Trimethoprim | 0.12-0.25 | 0.25 | 32 | 80 | |
| Daptomycin | 0.12-0.25 | 0.12 | 0.25 | 100 | |
| Linezolid | 2-2 | 2 | 2 | 100 | |
| Minocycline | 0.25-0.25 | 0.25 | 0.25 | 100 | |
| Moxifloxacin | 0.12-4 | 4 | 8 | 4 | |
| Vancomycin | 0.5-1 | 1 | 1 | 100 | |
| hVISA (25) | BAL0030543 | 0.015-0.06 | 0.03 | 0.06 | 100 |
| BAL0030544 | 0.03-0.25 | 0.25 | 0.25 | 100 | |
| BAL0030545 | 0.03-0.06 | 0.06 | 0.06 | 100 | |
| Trimethoprim | 0.06-32 | 1 | 16 | 80 | |
| Daptomycin | 0.25-2 | 0.5 | 0.5 | 96 | |
| Linezolid | 1-1 | 1 | 2 | 100 | |
| Minocycline | 0.12-8 | 0.25 | 0.5 | 92 | |
| Moxifloxacin | 0.12-4 | 4 | 4 | 8 | |
| Vancomycin | 1-4 | 1 | 2 | 96 | |
| VISA (17) | BAL0030543 | 0.015-0.5 | 0.003 | 0.25 | 100 |
| BAL0030544 | 0.03-0.5 | 0.25 | 0.5 | 100 | |
| BAL0030545 | 0.06-8 | 0.06 | 0.5 | 100 | |
| Trimethoprim | 0.12-32 | 1 | 32 | 71 | |
| Daptomycin | 0.25-2 | 1 | 2 | 71 | |
| Linezolid | 0.5-2 | 1 | 2 | 100 | |
| Minocycline | 0.12-8 | 1 | 4 | 96 | |
| Moxifloxacin | 8-8 | 8 | 8 | 0 | |
| Vancomycin | 1-4 | 2 | 4 | 52 | |
| Coagulase-negative | BAL0030543 | 0.015-16 | 0.06 | 8 | 86 |
| staphylococci (29) | BAL0030544 | 0.03-32 | 0.06 | 8 | 86 |
| BAL0030545 | 0.03-16 | 0.06 | 4 | 86 | |
| Trimethoprim | 0.12-32 | 16 | 32 | 48 | |
| Daptomycin | 0.06-0.5 | 0.25 | 0.5 | 100 | |
| Linezolid | 0.25-2 | 1 | 2 | 100 | |
| Minocycline | 0.25-8 | 0.5 | 2 | 96 | |
| Moxifloxacin | 0.12-8 | 0.25 | 8 | 55 | |
| Vancomycin | 0.5-4 | 1 | 2 | 100 | |
| Group A streptococci (17) | BAL0030543 | 0.015-0.06 | 0.06 | 0.06 | 100 |
| BAL0030544 | 0.06-0.06 | 0.06 | 0.06 | 100 | |
| BAL0030545 | 0.03-0.12 | 0.06 | 0.12 | 100 | |
| Trimethoprim | 0.12-0.5 | 0.5 | 1 | N/A | |
| Daptomycin | 0.015-0.03 | 0.015 | 0.03 | 100 | |
| Linezolid | 0.5-1 | 1 | 1 | 100 | |
| Minocycline | 0.12-0.25 | 0.12 | 0.25 | 100 | |
| Moxifloxacin | 0.25-0.5 | 0.25 | 0.5 | N/A | |
| Vancomycin | 0.12-0.25 | 0.25 | 0.25 | 100 | |
| Group B streptococci (17) | BAL0030543 | 0.015-0.06 | 0.03 | 0.06 | 100 |
| BAL0030544 | 0.06-0.12 | 0.06 | 0.12 | 100 | |
| BAL0030545 | 0.06-0.12 | 0.06 | 0.12 | 100 | |
| Trimethoprim | 0.25-2 | 1 | 1 | N/A | |
| Daptomycin | 0.015-0.06 | 0.015 | 0.06 | 100 | |
| Linezolid | 1-2 | 1 | 2 | 100 | |
| Minocycline | 0.12-32 | 16 | 32 | 29 | |
| Moxifloxacin | 0.25-0.5 | 0.25 | 0.5 | N/A | |
| Vancomycin | 0.12-0.25 | 0.25 | 0.25 | 100 | |
| Group C streptococci (13) | BAL0030543 | <0.008-0.12 | 0.015 | 0.06 | 100 |
| BAL0030544 | <0.008-0.25 | 0.06 | 0.06 | 100 | |
| BAL0030545 | 0.03-0.25 | 0.06 | 0.12 | 100 | |
| Trimethoprim | <0.008-8 | 0.5 | 1 | N/A | |
| Daptomycin | 0.015-0.12 | 0.03 | 0.12 | 100 | |
| Linezolid | 1-1 | 1 | 2 | 100 | |
| Minocycline | 0.12-16 | 0.25 | 4 | 84 | |
| Moxifloxacin | 0.25-0.5 | 0.25 | 0.5 | N/A | |
| Vancomycin | 0.12-0.5 | 0.12 | 0.5 | 100 | |
| Group G streptococci (12) | BAL0030543 | 0.015-0.06 | 0.03 | 0.06 | 100 |
| BAL0030544 | 0.06-0.25 | 0.06 | 0.06 | 100 | |
| BAL0030545 | 0.03-0.12 | 0.03 | 0.06 | 100 | |
| Trimethoprim | 0.5-1 | 1 | 1 | N/A | |
| Daptomycin | 0.015-0.03 | 0.015 | 0.03 | 100 | |
| Linezolid | 1-2 | 2 | 2 | 100 | |
| Minocycline | 0.12-16 | 0.25 | 16 | 75 | |
| Moxifloxacin | 0.25-4 | 0.25 | 0.5 | N/A | |
| Vancomycin | 0.12-0.25 | 0.12 | 0.12 | 100 | |
| Streptococcus pneumoniae (15) | BAL0030543 | 0.015-0.12 | 0.06 | 0.12 | 100 |
| BAL0030544 | 0.03-0.25 | 0.12 | 0.25 | 100 | |
| BAL0030545 | 0.03-0.25 | 0.12 | 0.12 | 100 | |
| Trimethoprim | 0.5-8 | 1 | 4 | N/A | |
| Daptomycin | <0.008-0.5 | 0.06 | 0.12 | N/A | |
| Linezolid | 0.03-2 | 1 | 2 | 100 | |
| Minocycline | 0.12-8 | 0.25 | 0.25 | 93 | |
| Moxifloxacin | 0.12-0.5 | 0.25 | 0.25 | 100 | |
| Vancomycin | 0.06-0.5 | 0.12 | 0.25 | 100 | |
| Corynebacterium spp. (12) | BAL0030543 | <0.008-8 | 0.06 | 4 | 83 |
| BAL0030544 | 0.015-16 | 0.12 | 4 | 83 | |
| BAL0030545 | <0.008-6 | 0.12 | 4 | 83 | |
| Trimethoprim | 0.12-16 | 16 | 16 | N/A | |
| Daptomycin | 0.03-0.12 | 0.06 | 0.12 | N/A | |
| Linezolid | 0.12-1 | 0.06 | 0.12 | N/A | |
| Minocycline | 0.06-32 | 2 | 16 | N/A | |
| Moxifloxacin | 0.25-16 | 1 | 8 | N/A | |
| Vancomycin | 0.12-0.5 | 0.25 | 0.5 | N/A | |
| Listeria monocytogenes (10) | BAL30543 | 0.015-0.015 | 0.015 | 0.015 | 100 |
| BAL30544 | 0.03-0.03 | 0.03 | 0.03 | 100 | |
| BAL30545 | 0.06-0.06 | 0.06 | 0.06 | 100 | |
| Trimethoprim | 0.06-0.06 | 0.06 | 0.06 | N/A | |
| Daptomycin | 0.5-2 | 1 | 2 | N/A | |
| Linezolid | 2-2 | 2 | 2 | N/A | |
| Minocycline | 0.25-16 | 0.25 | 16 | N/A | |
| Moxifloxacin | 0.5-1 | 0.5 | 1 | N/A | |
| Vancomycin | 1-1 | 1 | 1 | N/A | |
| Streptococcus mitis (8) | BAL0030543 | <0.008-0.06 | <0.008 | 100 | |
| BAL0030544 | <0.008-0.06 | 0.03 | 100 | ||
| BAL0030545 | <0.008-0.12 | 0.015 | 100 | ||
| Trimethoprim | <0.008-8 | 4 | |||
| Daptomycin | 0.006-0.5 | 0.25 | |||
| Linezolid | <0.008-1 | 1 | |||
| Minocycline | 0.25-8 | 0.25 | |||
| Moxifloxacin | 0.12-1 | 0.25 | |||
| Vancomycin | <0.008-1 | 1 | |||
N/A, breakpoint not available. A breakpoint of 0.5 mg/liter was used for BAL compounds for comparison.
The antibacterial activities of BAL0030543, BAL0030544, BAL0030545, and the comparator drugs are shown in Table 1. BAL0030543 and BAL0030544 had the lowest MIC50s (0.03 μg/ml) of the four DHFR inhibitors tested against methicillin (meticillin)-sensitive S. aureus (MSSA) strains, and the maximum MIC for the BAL compounds was 0.25 μg/ml (BAL0030544 and BAL0030545). As expected, the MSSA strains were susceptible mainly to the comparator agents. BAL0030543 had the lowest MIC50 of the DHFR inhibitors against methicillin-resistant S. aureus (MRSA) strains, that is, 0.015 μg/ml, while BAL0030544 and BAL0030545 were equipotent (MIC50, 0.06 mg/ml). Fluoroquinolone resistance was common among these MRSA isolates (moxifloxacin MIC50, 4 μg/ml). BAL0030543, BAL0030544, and BAL0030545 had MIC50s of 0.03, 0.25, and 0.06 μg/ml, respectively, against the hVISA and VISA strains, values similar to those of the MSSA and MRSA isolates. MIC90s for the BAL compounds were 0.25 to 0.5 μg/ml against the VISA strains but lower against the hVISA strains, being in the range of 0.06 to 0.25 μg/ml. The MIC50 for all three BAL compounds was 0.06 μg/ml against coagulase-negative staphylococci; the MIC90s ranged from 4 to 8 μg/ml. In contrast, the trimethoprim MIC90 was 32 μg/ml.
The BAL0030543, BAL0030544, and BAL0030545 MIC50 values for beta-hemolytic streptococci (Lancefield groups A, B, C, and G) ranged from 0.015 to 0.06 μg/ml, and no isolates had a MIC of >0.25 μg/ml for the BAL compounds. These strains were also susceptible to the comparator agents tested, with the exception of minocycline against group B streptococci. The BAL compounds were markedly more potent against S. pneumoniae strains than trimethoprim. BAL0030543, BAL0030544, and BAL0030545 MIC50s for Corynebacteria spp. were 0.06, 0.12, and 0.12 μg/ml, respectively, with the MIC90 being 4 μg/ml for all three drugs. This is significantly more potent than the trimethoprim MIC50/MIC90 at 16/16 μg/ml. All five DHFR inhibitors had excellent activity against Listeria monocytogenes, with MIC50s of ≤0.06 μg/ml and MIC90s of ≤0.06 μg/ml. BAL0030543, BAL0030544, and BAL0030545 had lower MIC50s of ≤0.03 μg/ml against S. mitis than trimethoprim (MIC50, 4 μg/ml).
Five MRSA and two MSSA strains were resistant to trimethoprim, but none had a MIC of >0.5 mg/liter to the BAL compounds. Six of the VISA strains were trimethoprim resistant, of which five strains had BAL MICs of <0.5 mg/liter. One VISA strain had a raised MIC to the BAL compounds (≥8 mg/liter). Similarly, of the four hVISA strains that were trimethoprim resistant, all had BAL MICs of <0.5 mg/liter. Of the 15 coagulase-negative staphylococcus strains that were trimethoprim resistant, four strains had BAL MICs of >0.5 mg/liter.
The present study confirms the in vitro potency of BAL0030543, BAL0030544, and BAL0030545 against S. aureus isolates. Previously, it has been shown that the MIC50s of all three compounds were 0.03 μg/ml against MSSA strains, MRSA strains, and strains with reduced vancomycin susceptibility (5). Our data indicate that these compounds had MIC50s in the range of 0.015 to 0.25 μg/ml, depending on the compound and the S. aureus resistance phenotype. All the MSSA, MRSA, and hVISA isolates had MICs of ≤0.25 μg/ml. VISA strains had higher MIC90s than other S. aureus stains, which has been described before and is probably related to the higher trimethoprim MICs in this group in general. BAL0030543, BAL0030544, and BAL0030545 are about fourfold less active against trimethoprim-resistant S. aureus than susceptible strains (5). BAL0030543, BAL0030544, and BAL0030545 have been shown in time-kill experiments to produce a 3-log reduction in viable counts of most S. aureus isolates and are more bactericidal against S. aureus than minocycline, linezolid, or clindamycin (8).
Our data extends the data available on the BAL DHFR inhibitors against beta-hemolytic streptococci, indicating that all three compounds are highly active against Lancefield group A, B, C, and G streptococci. The previously reported geometric mean MIC for Streptococcus pyogenes was 0.25 μg/ml, fourfold higher than the MIC50s obtained for our strains (11). Some Corynebacteria sp. isolates had MICs above 1 μg/ml, while all three compounds had MICs of ≤0.25 μg/ml against S. pneumoniae, ≤0.06 μg/ml against L. monocytogenes, and ≤0.12 μg/ml against S. mitis.
In conclusion, the improved in vitro potency of BAL0030543, BAL0030544, and BAL0030545 against gram-positive bacterial organisms compared to that of other DHFR inhibitors and their activity against isolates resistant to other drug classes justify further assessment of their utility in the therapy of gram-positive bacterial infection. The ability of dihydrophthalazine antifolates to be administered orally and parentally is an additional therapeutic benefit.
Acknowledgments
We thank Malcolm Page of Basilea Pharmaceutica for his help in performing the study.
This study was funded by a grant from Basilea Pharmaceutica AG.
Footnotes
Published ahead of print on 8 September 2009.
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