Fluorocycline TP-271 Is Potent against Complicated Community-Acquired Bacterial Pneumonia Pathogens

Rising resistance rates for macrolides, fluoroquinolones, and β-lactams in the most common pathogens associated with community-acquired bacterial pneumonia (CABP) are of concern, especially for cases of moderate to severe infections in vulnerable populations such as the very young and the elderly. New antibiotics that are active against multidrug-resistant Streptococcus pneumoniae and Staphylococcus aureus are needed for use in the empirical treatment of the most severe forms of this disease. TP-271 is a promising new fluorocycline antibiotic demonstrating in vitro potency and nonclinical efficacy by intravenous and oral administration against the major pathogens associated with moderate to severe CABP.

IMPORTANCE Rising resistance rates for macrolides, fluoroquinolones, and ␤-lactams in the most common pathogens associated with community-acquired bacterial pneumonia (CABP) are of concern, especially for cases of moderate to severe infections in vulnerable populations such as the very young and the elderly. New antibiotics that are active against multidrug-resistant Streptococcus pneumoniae and Staphylococcus aureus are needed for use in the empirical treatment of the most severe forms of this disease. TP-271 is a promising new fluorocycline antibiotic demonstrating in vitro potency and nonclinical efficacy by intravenous and oral administration against the major pathogens associated with moderate to severe CABP. KEYWORDS TP-271, community-acquired bacterial pneumonia, fluorocycline C ommunity-acquired bacterial pneumonia (CABP) is a serious condition associated with mortality rates estimated to be as high as 12 to 14% for hospitalized individuals and 25 to 40% for those admitted to intensive care units (1)(2)(3). Lower respiratory tract infections were the second greatest cause of deaths and years of life lost in 2013 as reported by The Global Burden of Disease Study, with the highest incidence occurring in children Ͻ5 years and adults Ͼ65 years (1,4). The Centers for Disease Control estimated that 30% of severe Streptococcus pneumoniae infections are fully resistant to one or more antibiotics; drug-resistant S. pneumoniae infections complicate treatment and cause approximately 7,000 deaths per year (5). Excess medical costs associated with treating drug-resistant S. pneumoniae infections were estimated at approximately $96 million per year.
The bacterial etiology of CABP varies with severity of disease; however, S. pneumoniae is the most frequent cause of CABP across all levels of severity (6). The incidence of infections by Staphylococcus aureus and Legionella spp. increases with more-severe CABP, whereas Haemophilus influenzae, Mycoplasma pneumoniae, and Chlamydia pneumoniae are generally associated with mild to moderate CABP (6,7). Recently, large medical centers in the United States saw a dramatic increase in the incidence of infections caused by S. aureus, including those caused by methicillin-resistant S. aureus (MRSA) strains, once considered rare in CABP (8,9). A survey of 59 United States hospitals, involving 4,543 patients with culture-positive pneumonias between January 2002 and January 2004, identified S. aureus and MRSA as potential pathogens in 25.5% and 8.9% of the cases, respectively (8). Further, in this study, S. aureus was identified by logistic regression analysis as the only pathogen independently associated with mortality. Most of the MRSA strains causing health care-associated pneumonia, hospitalacquired bacterial pneumonia, and ventilator-associated bacterial pneumonia are hospital-acquired MRSA (HA-MRSA) strains containing staphylococcal cassette chromosome mec type I (SCCmec I) to SCCmec III (10). A new variant of MRSA, identified as community-acquired MRSA (CA-MRSA) containing SCCmec IV, has emerged globally as a potent pulmonary pathogen (10). This strain type typically carries a bacteriophage encoding Panton-Valentine leukocidin (PVL), a toxin that destroys polymorphonuclear leukocytes and is associated with tissue necrosis and increased virulence (11,12). The increased incidence of CA-MRSA infections in more-severe cases of CABP limits the empirical use of fluoroquinolones, macrolides, and most currently marketed ␤-lactams due to high resistance rates (13,14).
Although there are numerous therapies for S. pneumoniae infection, including oral drugs in the macrolide, fluoroquinolone, and ␤-lactam classes, resistance to ␤-lactams and macrolides is increasing (6). Resistance to currently approved antibiotics in other respiratory pathogens is also increasing. Global surveillance by the Tigecycline Evaluation and Surveillance Trial (TEST) in 2004 to 2013 found that penicillin-resistant S. pneumoniae (PRSP) comprised 14.8% of the S. pneumoniae isolates and that the percentages were highest in the Middle East (24.7%), Africa (28.1%), and the Asia-Pacific Rim (30.1%) (15). Erythromycin resistance during the same period was 32.7% and was highly correlated with penicillin resistance. TEST also found that the global rate of ␤-lactamase production among H. influenzae isolates collected from 2004 to 2013 was 20.1%. In the AWARE ceftaroline surveillance program, 96.4% of Moraxella catarrhalis isolates collected in the United States from 2008 to 2010 were ␤-lactamase producers (16). The increasing prevalence of antimicrobial resistance in S. pneumoniae, H. influenzae, and M. catarrhalis significantly impacts the utility of currently available antibiotics (17). Macrolide resistance in M. pneumoniae has increased worldwide, with Ͼ90% of clinical isolates from Japan and China and~20% of isolates in some European countries having high-level resistance to azithromycin (18). A surveillance study performed in the United States from 2012 to 2014 reported a macrolide resistance rate of 13.2% (19). Macrolide resistance in M. pneumoniae is clinically significant, often requiring switching to other drug classes such as tetracyclines or fluoroquinolones (18).
Tetracycline antibiotics are well known for their broad spectrum, which includes a wide range of Gram-positive and Gram-negative bacteria, spirochetes, and obligate intracellular bacteria. TP-271, a novel fluorocycline antibiotic of the tetracycline class ( Fig. 1), is a candidate for the treatment of serious infections, including those caused by multidrug-resistant pathogens. On the basis of in vitro and in vivo evaluations, TP-271 shows potential to treat key susceptible and resistant organisms associated with moderate to severe CABP.

RESULTS
TP-271 mechanism of action. The site of action of tetracycline drugs such as TP-271 is the 30S ribosomal subunit; drug binding interferes with access of aminoacyl-tRNA to the A-site on the mRNA-ribosome complex, preventing new amino acid addition and peptide chain growth (20,21). TP-271 showed potent mechanism-based antitranslational activity in an in vitro coupled transcription/translation (TnT) assay in the presence and absence of the tetracycline-specific ribosomal protection protein Tet(M), which confers high-level resistance to tetracycline in bacteria (22). The halfmaximal inhibitory concentration (IC 50 ) determined in the TnT assay for TP-271 was 0.18 Ϯ 0.08 g/ml; in comparison, the IC 50 for tetracycline was 1.1 Ϯ 0.07 g/ml and for the nontetracycline translation inhibitor linezolid the IC 50 was 1.3 Ϯ 0.28 g/ml. In the presence of Tet(M), the IC 50 for TP-271 was unaffected (0.13 Ϯ 0.04 g/ml) whereas the IC 50 for tetracycline increased by~5-fold (5.8 Ϯ 1.1 g/ml), consistent with the tetracycline resistance seen with Tet(M)-expressing organisms. Thus, TP-271 is distinguished by its being a Tet(M)-insensitive novel tetracycline.
Activity of TP-271 against CABP pathogens in vitro. TP-271 was active against key community respiratory Gram-positive pathogens (Table 1), including S. pneumoniae (MIC 90 ϭ 0.03 g/ml), methicillin-sensitive S. aureus (MSSA; MIC 90 ϭ 0.25 g/ml), MRSA (MIC 90 ϭ 0.12 g/ml), and Streptococcus pyogenes (MIC 90 ϭ 0.03 g/ml). TP-271 was active (MIC 90 ϭ 0.12 g/ml) against CA-MRSA expressing PVL. As shown by the MIC 90 data, TP-271 was Ն1,000-fold more potent than tetracycline against S. pneumoniae and S. pyogenes and 128-fold more potent against S. aureus. TP-271 was also potent against  For hospitalized CABP patients on the general wards, the Infectious Diseases Society of America/American Thoracic Society guidelines currently recommend a respiratory fluoroquinolone or the combination of a ␤-lactam and a macrolide (7). For patients with severe CABP requiring intensive care unit admission, the guidelines recommend a ␤-lactam plus either azithromycin or a respiratory fluoroquinolone; if MRSA is a concern, either vancomycin or linezolid should be added (7). TP-271 retained good antibacterial potency against subsets of S. pneumoniae resistant to penicillin (MIC 90 ϭ 0.03 g/ml) and macrolides (MIC 90 ϭ 0.03 g/ml) and against MRSA displaying resistance to fluoroquinolones (MIC 90 ϭ 0.25 g/ml), macrolides (MIC 90 ϭ 0.25 g/ml), and linezolid (MIC range, Յ0.016 to 0.5 g/ml) ( Table 2). TP-271 was also active against MRSA isolates displaying nonsusceptibility to daptomycin (MIC range, Յ0.016 to 0.25 g/ml), a drug used in the treatment of serious Gram-positive infections, excluding pneumonia (Table 2).
As recommended in the August 2016 FDA publication of guidance for microbiological data for systemic antibacterial drug products (23), TP-271 was tested for retention of microbiological activity in the presence of 5% bovine pulmonary surfactant and found to be equally active against S. aureus ATCC 29213 (MIC ϭ 0.063 g/ml in cation-adjusted Mueller-Hinton broth [ca-MHB]; MIC ϭ 0.031 g/ml in ca-MHB ϩ 5% bovine pulmonary surfactant). The MIC of the positive control, daptomycin, was elevated by 512-fold in ca-MHB ϩ 5% bovine pulmonary surfactant, as expected (24).
Activity of TP-271 against atypical respiratory pathogens. TP-271 was tested against atypical pathogens commonly associated with CABP: L. pneumophila, M. pneumoniae, and the obligate intracellular pathogen C. pneumoniae (Table 3). TP-271 showed an MIC 90 of 1 g/ml against a panel comprised of 20 isolates of L. pneumophila serogroup 1 and 10 isolates each of serogroups 2 to 6; TP-271 was found to be similarly potent against all serogroups when assessed individually (25). When tested by agar dilution in buffer yeast extract (BYE), it was noted that the TP-271 and tetracycline MIC values for the quality control strain S. aureus ATCC 29213 were 128-fold and 64-fold higher, respectively, compared to results in cation-adjusted Mueller-Hinton agar after 48 h of incubation, the duration of incubation for L. pneumophila testing (data not shown). This finding suggested that the BYE medium used in the testing of L. pneumophila may have reduced the activity of TP-271 and inflated the MIC values for L. pneumophila.
TP-271 was tested against 10 human isolates of C. pneumoniae in an intracellular infection assay with HEp-2 cells and showed an MIC 90 of 4 g/ml (Table 3). Similar to the interference from media observed in L. pneumophila assays, the MIC values of TP-271 were 4-to 32-fold higher in Eagle's minimum essential medium (EMEM) than in ca-MHB for the quality control organisms S. aureus ATCC 29213, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, and Pseudomonas aeruginosa ATCC 27853 tested by broth microdilution assay (data not shown). These results suggested that the use of EMEM reduced the activity of TP-271 and artificially raised MIC values.
The effect of common tetracycline resistance determinants on in vitro activity of TP-271. Given the widespread dissemination of tetracycline resistance determinants in clinical isolates (26), it is important that a new tetracycline-class antibiotic be active against the major tetracycline resistance mechanisms. TP-271 was tested against a previously described panel of isogenic strains overexpressing major tetracycline resistance genes from an arabinose-inducible promoter on a plasmid in laboratory E. coli strain DH10B (22). The overexpressed genes encoded two major efflux mechanisms found in Gram-negative bacteria, Tet(A) and Tet(B); a major Gram-positive efflux mechanism, Tet(K); a major ribosomal protection mechanism, Tet(M); and Tet(X), a secreted flavin-dependent monooxygenase, originally identified in Bacteroides fragilis, capable of covalently inactivating tetracycline (22,27,28). Compared to the negativecontrol strain expressing lacZ, overexpression of all tet resistance genes had a pronounced effect on tetracycline and minocycline susceptibility, increasing MIC values by Ն32-fold and by 2-fold to Ͼ64-fold, respectively ( Table 4)   the presence of an overexpressed tet gene was 4 g/ml. As expected, the activity of the nontetracycline control, ceftriaxone, was not impacted by tet gene expression. The effect of upregulated chromosomally encoded efflux pumps in S. aureus on TP-271 activity. TP-271 was evaluated against previously described S. aureus clinical isolates with upregulated expression of norA (29,30) and mepA (31,32), genes encoding intrinsic efflux pumps conferring resistance to quinolones (NorA and MepA) and tigecycline (MepA). The MIC value of TP-271 for both norA-overexpressing mutant SA982 and the corresponding SA981 parental strain was 0.06 g/ml; however, an 8-fold increase in the MIC was noted for TP-271 in mepA-overexpressing mutant SA984 (MIC ϭ 0.12 g/ml) versus the corresponding parental SA983 strain (MIC ϭ 0.016 g/ ml) ( Table 5). The MIC value for the tigecycline comparator was 4-fold higher in the norA mutant and 16-fold higher in the mepA mutant than in the corresponding parental strain.
Activity of TP-271 in murine respiratory infection models. TP-271 administered either intravenously (i.v.) or orally (p.o.) reduced the bacterial burden in the lung, versus the control group results at the start of dosing, in murine pneumonia models with MRSA, S. pneumoniae, and H. influenzae.

DISCUSSION
Recent FDA guidelines (23) indicate that new antibacterial drugs for CABP must have nonclinical data showing activity against the most commonly implicated pathogens, i.e., S. pneumoniae, H. influenzae, S. aureus, and M. catarrhalis, and for moderate to serious CABP, coverage of L. pneumophila is critical for empirical use. TP-271 meets these criteria, demonstrating good in vitro and in vivo potency against key susceptible and drug-resistant causative pathogens for this indication. The activity of TP-271 was minimally affected, or unaffected, by tetracycline-specific, fluoroquinolone, or macrolide resistances. MIC 90 values for TP-271 were 0.03 g/ml for all streptococci, the most common CABP pathogens, regardless of resistance phenotype. TP-271 was also active against MSSA and MRSA (MIC 90 ϭ 0.12 to 0.25 g/ml), including community-acquired MRSA expressing PVL toxin. Against H. influenzae and M. catarrhalis¸TP-271 MIC 90 values were 0.12 and Յ0.016 g/ml, respectively. Good activity was also demonstrated against M. pneumoniae, with all MICs being Յ0.008 g/ml, including those against macrolide-resistant organisms. TP-271 was active against C. pneumoniae (MIC 90 ϭ 4 g/ml) and L. pneumophila (MIC 90 ϭ 1 g/ml) despite potential interference from assay conditions. TP-271 was efficacious when administered i.v. and p.o. versus MRSA, S. pneumoniae, and H. influenzae in rodent pneumonia models, demonstrating potential as both an i.v. treatment and an oral treatment for CABP. The spectrum of activity of TP-271, along with the results from the animal infection models of pneumonia, makes it a promising candidate for development as an antibiotic for treatment of moderate to severe CABP.