In vitro and Intracellular Activities of Peptide Deformylase. Inhibitor GSK against Legionella pneumophila Isolates

Transcription

1 AAC Accepts, published online ahead of print on 27 October 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 In vitro and Intracellular Activities of Peptide Deformylase Inhibitor GSK against Legionella pneumophila Isolates Jacques Dubois 1, Maïtée Dubois 1, Jean-François Martel 1, Kelly Aubart 2 and Deborah Butler 2# 1 M360, Sherbrooke, Québec, Canada; 2 Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, PA, USA 8 9 Running Title: GSK activity against L. pneumophila # corresponding author, address, Deborah.L.Butler@gsk.com

2 ABSTRACT GSK , a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 L. pneumophila strains, with MICs ranging from 1 to 16 μg/ml, and an MIC 90 of 16 μg/ml, but very potent intracellular activity, with MIECs ranging from 0.12 to 2 μg/ml, and 98% of the strains being inhibited by concentrations 1 μg/ml

3 TEXT The opportunistic Gram-negative bacterium Legionella pneumophila, which can grow free-living or as a facultative intracellular organism in amoebae or human alveolar macrophages, is the major causative agent of Legionnaire s disease, a severe form of pneumonia (1). Although there are at least 15 serogroups identified, L. pneumophila serogroup 1 accounts for over 80% of the cases worldwide (2). Except in the event of a Legionnaire s disease outbreak, L. pneumophila is treated empirically as part of the therapy used for hospitalized pneumonia and, given the severity of the disease, it is important to ensure that the antibacterial agent to be utilized shows good activity against this pathogen. GSK is a novel peptide deformylase (PDF) inhibitor with good safety and pharmacokinetic properties (3-5) currently in Phase II development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. GSK has demonstrated good antibacterial activity against other bacterial pneumonia causative agents, such as Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae (6) and it was, therefore, important to investigate its potency against this atypical pathogen. In order to assess the in vitro activity of GSK , susceptibility studies were performed against 50 L. pneumophila strains: 25 from serogroup 1, and 5 each from serogroups 2, 3, 4, 5 and 6. L. pneumophila isolates were collected from 1992 to present, mostly from human respiratory tract, and grown on buffered charcoal yeast extract (BCYE) agar to produce pure cultures. Minimum inhibitory concentration (MIC) endpoints were determined by broth microdilution according to Clinical and Laboratory 3

4 Standards Institute (CLSI) guidelines (7). MIC plates, containing approximately 5x10 5 colony forming units (CFU)/mL in BYE broth (with Legionella BCYE Growth supplement), were incubated at 35 o C in aerobic conditions for 48 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible growth after the appropriate incubation time. GSK showed variable activity against this panel of 50 L. pneumophila strains, with MICs ranging from 1 to 16 μg/ml, and MIC 50 and MIC 90 values of 8 and 16 μg/ml respectively (Table 1). No significant differences could be observed in the activity of GSK against strains from different serogroups, with 8 μg/ml being the most frequent MIC in all sets (Table 1). Both, azithromycin and levofloxacin showed more potent extracellular activity with MIC 50 /MIC 90 values of 0.06/0.5 μg/ml, and 0.008/0.016 μg/ml, respectively (Table 1). The results of this study indicate that GSK is substantially less active in vitro against L. pneumophila serogroups 1 to 6 than azithromycin or levofloxacin, the drugs normally used for the treatment of Legionellosis. The GSK MIC for S. aureus ATCC29213 tested in Mueller- Hinton broth was identical or within a 2-fold dilution of the MIC obtained using BYE, indicating that there is not drug inactivation by the test medium. As L. pneumophila is a facultative intracellular bacterium that multiplies within phagocytic cells, the ability of GSK to effectively inhibit growth of L. pneumophila in human monocytes was investigated. In these studies, human mononuclear cells (U-937) (8) cultured at a final concentration of approximately 10 4 cell/well in Dulbecco's modified Eagle's medium (DMEM) containing 10% heatinactivated fetal bovine serum, were infected as a suspension with of each of the 4

5 L. pneumophila strains grown to logarithmic phase, by incubating together for 1 h in a shaking incubator. Infected cultures were transferred to flat-bottomed wells and maintained without shaking thereafter at 37 o C in 5% CO 2 and 95% air for 7 days. After 24 h, infected cultures were washed three times with drug-free broth to remove extracellular bacteria, and GSK (1 to 16 μg/ml), azithromycin (0.03 to 0.05 μg/ml), or erythromycin (0.06 to 1 μg/ml), were added to the wells at a concentration equal to the corresponding MIC/strain combination. At Day 3, cultures were washed, split into two groups, and incubated for an additional 4 days with or without antibiotic. CFUs were determined in duplicate using BCYE agar at time 0, and then every 24 h until Day 7. The results demonstrated that MIC concentrations of GSK not only prevented growth of L. pneumophila inside macrophages but also reduced intracellular bacterial counts by an average of 1.21og 10 with respect to the original inocula (Figure 1A). Although some growth could be observed after Day 5, bacterial counts never reached those present in the culture prior to the addition of the antibiotic. Azithromycin also reduced bacterial counts by an average of 1.5log 10 by Day 3, but a significantly less reduction of growth was observed with erythromycin (Figure 1A). Neither of the macrolides could prevent bacterial growth during the last 4 days of exposure, perhaps due to metabolism or changes in uptake, by either the bacterium or the monocytes. None of the three drugs demonstrated a strong post-antibiotic effect, and re-growth could be observed in all cases after the removal of the antibiotic at Day 3 (Figure 1B) although exposure to GSK seemed to delay re-growth slightly more. Given the clear intracellular activity demonstrated by GSK , the minimum extracellular concentration capable of inhibiting intracellular proliferation (MIEC) of 5

6 these 50 L. pneumophila strains was determined in human mononuclear cells (U-937) (8, 9) using the conditions described above except that, in this case, the infected cultures were maintained without shaking for 5 days. After 24 h, extracellular bacteria were removed, and GSK , azithromycin, or levofloxacin, were added at fractions of their MIC to determine the precise MIEC. The MIEC was defined as the lowest extracellular concentration of compound capable of preventing L. pneumophila intracellular proliferation and was calculated at Day 3 and at Day 5 of antibiotic exposure. Against the 50 strains of L. pneumophila with extracellular MICs ranging from 1 to 16 μg/ml, the GSK MIEC at Day 3 ranged from 0.12 to 2 μg/ml, with 98% of the strains being inhibited by concentrations 1 μg/ml (Table 2). MIECs of 2, 1, 0.5, 0.25 and 0.12 μg/ml, with a mean reduction of 73% in bacterial counts, were demonstrated against 1/50 (2%), 29/50 (58%), 9/50 (18%), 10/50 (20%) and 1/50 (2%) of the strains respectively (Table 2). In all cases the compound was at least 4-fold more potent intracellularly than extracellularly. Most of the strains showed GSK MIECs 8-fold (35/50; 70%) or 16-fold (11/50; 22%) below their extracellular MIC, with the remaining 3/50 and 1/50 strains demonstrating a 4-fold and a 32-fold difference between both values, respectively (Table 2). No significant differences were observed between the serogroups tested and in all cases MIECs were maintained at Day 5 of exposure. GSK was clearly much more effective at inhibiting proliferation of L. pneumophila within macrophages than in extracellular conditions. The azithromycin MIEC at Day 3 exposure was identical to its extracellular MIC in 94% of the strains (47/50) and it was 2-fold lower in the other 3 strains (Table 2). The levofloxacin MIEC 6

7 was 2-fold below its extracellular MIC in most of the strains (41/50; 82%), with 1/50 strains showing a MIEC 4-fold lower than the MIC and the other 7/50 (14%) demonstrating identical MIEC and MIC values (Table 2). It is not the first time that the extracellular activity of an antimicrobial agent against L. pneumophila does not correlate with its intracellular potency. β-lactams, for example, are highly active in vitro but do not penetrate the intracellular compartment and are therefore inactive in vivo. On the other hand, tetracyclines have poor in vitro activity against L. pneumophila, as they are inactivated by iron in the BYEα growth media, but are active in animal models of Legionnaires s disease and have been proven an effective treatment in humans (10). However, in the case of PDF inhibitors, the difference in extracellular and intracellular activities could be due to a change in the primary target of the antibacterial agent when the bacterium growths inside macrophages. L. pneumophila has three functional PDF enzymes that show different substrate specificities and catalytic efficiencies (11). Recently, it has been shown that PdfA plays an essential role in the extracellular growth of L. pneumophila and that activity against this enzyme is the main determinant of the compound s in vitro antibacterial activity. On the other hand, PdfC seems to play a critical role in L. pneumophila replication within macrophages and is indispensable for the bacterium to cause a productive infection in a guinea pig model of Legionellosis (Huang et al., submitted for publication). Therefore, the antibacterial activity shown by GSK against intracellular L. pneumophila could be due, at least in part, to its potent inhibition of PdfC (Glen Van Aller, unpublished results), although its accumulation inside macrophages may also contribute to it (12). 7

8 In conclusion, GSK demonstrates good intracellular activity against 50 L. pneumophila strains from serogroups 1 to 6, with an MIEC 90 of 1 μg/ml. In fact, its intracellular activity is similar to that demonstrated against other typical pneumonia causative agents which have GSK MIC 90 s of 2 μg/ml (S. pneumoniae) or 4 μg/ml (S. aureus and H. influenzae) (6). Moreover, plasma and intrapulmonary pharmacokinetic studies with GSK dosed intravenously twice daily for 4 days shows that its time course in epithelial lining fluid (ELF) and alveolar macrophages (AM) mirrors the plasma concentration-time profile. In fact, the AUC0-τ ratios of ELF and AM to total plasma were 1.2 and 2.5, respectively (12). Therefore, GSK should be active against intracellular L. pneumophila at the concentrations necessary to achieve efficacy versus the other bacterial respiratory tract pathogens and could be an effective treatment for lower respiratory tract infections caused by this atypical pathogen

9 ACKNOWLEDGEMENTS Editorial support in the form of development of manuscript first draft, collating author comments, assembling tables and preparing final version for publication was provided by Dr. Magdalena Zalacain at Zala Drug Discovery Consulting and was funded by GSK

10 REFERENCES 1. Edelstein PH, Meyer RD Legionnaires' disease. A review. Chest 85: Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, Summersgill J, File T, Heath CM, Paterson DL, Chereshsky A Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186: Naderer OJ, Dumont E, Zhu J, Kurtinecz M, Jones LS Safety, tolerability and pharmacokinetics of repeat dosing of the antibiotic GSK , a peptide deformylase inhibitor: a randomized placebocontrolled study. J Antimicrob Chemother 68: Naderer OJ, Dumont E, Zhu J, Kurtinecz M, Jones LS Effect of H2 blockade and food on single-dose pharmacokinetics of GSK , a peptide deformylase inhibitor antibacterial. Antimicrob Agents Chemother 57: Naderer OJ, Dumont E, Zhu J, Kurtinecz M, Jones LS Single-dose safety, tolerability, and pharmacokinetics of the antibiotic GSK , a novel peptide deformylase inhibitor. Antimicrob Agents Chemother 57: O'Dwyer K, Hackel M, Hightower S, Hoban D, Bouchillon S, Qin D, Aubart K, Zalacain M, Butler D Comparative analysis of the antibacterial 10

11 activity of a novel peptide deformylase inhibitor, GSK Antimicrob Agents Chemother 57: Clinical and Laboratory Standards Institute Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 8th ed. Approved standard M07-A8. Clinical and Laboratory Standards Institute, Wayne, Pa, USA. 8. Horwitz MA Legionella, vol. Proceedings of the 2nd International Symposium of the American Society for Microbiology. ASM Press, Washington DC. 9. Higa F, Kusano N, Tateyama M, Shinzato T, Arakaki N, Kawakami K, Saito A Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila. J Clin Microbiol 36: Edelstein PH Antimicrobial chemotherapy for legionnaires' disease: a review. Clin Infect Dis 21 Suppl 3:S Huang J, Van Aller GS, Taylor AN, Kerrigan JJ, Liu WS, Trulli JM, Lai Z, Holmes D, Aubart KM, Brown JR, Zalacain M Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases. J Bacteriol 188: Naderer OJ, Rodvold KA, Jones LS, Zhu JZ, Bowen CL, Chen L, Dumont E Penetration of GSK into epithelial lining fluid and alveolar macrophages as determined by bronchoalveolar lavage. Antimicrob Agents Chemother 58:

12 FIGURE LEGENDS Fig. 1. Intracellular activity of GSK , azithromycin and erythromycin against L. pneumophila strains. At Day 1, drug was added to the infected cultures, at the appropriate concentration for each MIC/strain combination (GSK at 1 to 16 μg/ml; azithromycin at 0.03 to 0.05 μg/ml, and erythromycin at 0.06 to 1 μg/ml). At Day 3, cultures were washed, split into two groups and incubated until Day 7 with antibiotic (A) or without antibiotic (B). Each point represents the mean of the results obtained from duplicate or triplicate experiments (and duplicate quantitative plating) with each of the 50 L. pneumophila strains. Black arrows indicate the point at which the drugs were added

13 Table 1. Summary of the extracellular activity of GSK , azithromycin and levofloxacin against 50 L. pneumophila strains from serogroups 1 to 6 Antibiotic L. pneumophila (n) Number of strains inhibited by antibiotic at MIC* (μg/ml) All (50) Serogroup 1 (25) Serogroup 2 (5) 3 2 GSK Serogroup 3 (5) 4 1 Serogroup 4 (5) Serogroup 5 (5) Serogroup 6 (5) All (50) Serogroup 1 (25) Serogroup 2 (5) Azithromycin Serogroup 3 (5) Serogroup 4 (5) Serogroup 5 (5) 4 1 Serogroup 6 (5) 4 1 All (50) Serogroup 1 (25) Serogroup 2 (5) 5 Levofloxacin Serogroup 3 (5) 1 4 Serogroup 4 (5) 4 1 Serogroup 5 (5) 3 2 Serogroup 6 (5) 5 *All experiments were performed in duplicate or triplicate MIC 50s are in italics; MIC 90s are in bold 13

14 Table 2. Summary of the intracellular activity of GSK , azithromycin, and levofloxacin at Day 3 of exposure against 50 L. pneumophila strains with different MICs Antibiotic GSK Azithromycin L. pneumophila (# strains) MIC (μg/ml) Number of strains inhibited by antibiotic at MIEC* (μg/ml) Levofloxacin *All experiments were performed in duplicate or triplicate MIC 50s are in italics; MIC 90s are in bold 14

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