In vitro Potency of Novel Tetracyclines against Pseudomonas aeruginosa and Other Major Gram-Negative Pathogens

W. O'Brien, C. Fyfe, T. Grossman, C. Chen, R. Clark, Y. Deng, M. He, D. Hunt, C. Sun, X. Xiao, J. Sutcliffe*

Tetraphase Pharmaceuticals, Watertown, US22nd ECCMID31 March – 3 April 2012

London, United Kingdom

P 1448

Contact:Leland Webster

Tetraphase Pharmaceuticalslwebster@tphase.com

Revised Abstract

Introduction

Results

Methods

Conclusions

Objectives: To discover novel tetracyclines with enhanced Pseudomonas aeruginosa activity while maintaining in vitro activity against other important gram-negative pathogens.

Methods: The guidances and breakpoints of the Clinical Laboratory Standards Institute were used to determine the susceptibility of new compounds and comparators in microtiter-based cation-supplemented Mueller Hinton broth or in time-kill assays using 5 milliliter cultures. In the case of tigecycline, FDA breakpoints (if available) were used. In vitro potency against Escherichia coli DH10B strains genetically engineered to express tet(A), tet(B), tet(K), tet(M), tet(X) or blaNDM-1 was assessed. Compounds were also assessed for mechanism of action (MOA) using a coupled transcription/translation assay (TnT) fueled with S30 ribosomal extracts from either E. coli or P. aeruginosa.

Results: Nine novel scaffolds were found that produced compounds with MIC90 values of 8-16 μg/ml against recent P. aeruginosa clinical isolates, including isolates from cystic fibrosis (CF) patients (n=96 total, including 20 CF isolates). In vitro activity against panels of other organisms, including Acinetobacter baumannii and extended-spectrum beta-lactamase producing Klebsiella pneumoniae and E. coli was also retained by many compounds, with MIC90 values of ≤1 μg/ml for several compounds and comparator MIC90 values of ≥32 μg/ml for tetracycline, ceftriaxone, meropenem, levofloxacin, gentamicin, and tobramycin for P. aeruginosa, Enterobacteriaceae and A. baumanii. Two compounds were profiled in 24-hour time-kill studies using 4 isolates of P. aeruginosa and were generally found to be bactericidal at 4-8x the MIC. The new scaffolds retained activity against strains expressing genes encoding tetracycline-specific efflux pumps (Tet(A), Tet(B), Tet(K)), a ribosomal protection mechanism (Tet(M)), and a monooxygenase that inactivates tetracyclines (Tet(X)). The compounds inhibited protein synthesis in both TnT assays, with IC50 values 5-10x lower than conventional tetracyclines (1-2 μM).

Conclusions: This is the first report of novel tetracyclines with improved potency against contemporary P. aeruginosa isolates. These compounds retain activity against other major gram-negative pathogens and merit additional work to advance into development.

MIC assays. Compounds were tested against panels of unbiased sets (except where noted, i.e. “ESβL+”) of recent clinical isolates, including quality control strains according to methods published by Clinical and Laboratory Standards Institute (CLSI) (5). Isolate collections include strains from Eurofins Medinet (Chantilly, VA) and IHMA (Schaumburg, IL). PCR-characterization of extended spectrum β-lactamases was done at IHMA or by standard PCR methodology at Tetraphase Pharmaceuticals using published primers to confirm the presence of ESβL enzymes (6).

Time-kill assays. The minimal inhibitory concentrations (MIC) were determined for antibiotic stocks as per CLSI standardized methodology prior to running time-kill assays. Time-kill assays were performed as described by CLSI guidelines (7), with the following modifications: five milliliter cultures inoculated to a final starting density of ~1 x 105 – 1 x 106 colony forming units (CFU) /ml were shaken vigorously (300 rpm) at 35oC in 50 ml polypropylene conical tubes. Cultures were sampled at various time points, serially diluted in sterile saline, and plated on tryptic soy agar. The lower limit of detection per culture was 100 CFU/ml. Antibacterial activity against E. coli DH10B recombinantly expressing tetracycline-resistance genes. Genes encoding tet(A), tet(B), tet(K), tet(M), tet(X), and E. coli β-galactosidase (lacZ) as a control were amplified by PCR from clinical isolates confirmed by prior sequencing to have these tetracycline-resistant determinants and cloned into an L-arabinose inducible expression system without any affinity tags (pBAD-Myc-His, Invitrogen, Carlsbad, CA). The class B carbapenemase NDM-1 was also cloned into the same inducible expression system. Plasmids were transformed and expressed in E. coli DH10B cells (Invitrogen, Carlsbad, CA). Cloned inserts were sequenced to verify the resistance gene sequence and compared to reported sequences in GenBank (accession numbers; tet(A), AJ419171; tet(B), AP0961; tet(K), AJ888003; tet(M), X90939.1; tet(X), M37699; and NDM-1, HQ162469). Cells were grown in Mueller Hinton Broth containing ampicillin, 50 μg/ml, pre-induced for 30 minutes with 1% arabinose (tet(A), tet(B), tet(M), tet(X), NDM-1) or 0.3% arabinose (tet(K)) at 30C prior to use as inocula in MIC assays containing ampicillin, 50 g/ml. Susceptibility assays were incubated at 35C as per CLSI guidelines.

Transcription-Translation assays. E. coli assays were run using commercially available S30 for circular DNA (Promega Cat # L1130). P. aeruginosa extracts were prepared as described previously (8) and were supplemented with S30 buffer from Promega (Cat # L512A) and plasmid pBESTluc purchased from Promega (Cat. # L492A). Reactions were carried out in a total volume of 20 µl in black-walled 96-well flat-bottom assay plates (Costar Cat. # 3915) for one hour at 37C. Each reaction contained 5 to 5.23 µl of extract, 0.05 to 0.6 µl of plasmid, 8 µl of S30 buffer and 3 µl of compound dilution. Reactions were stopped by placing on ice for 5 minutes followed by the addition of 25 µl of luciferase substrate (Promega Cat. # E1500) per well. Luminescence was read using a LUMIStar Optima (BMG Labtech) with gain at 3600, 0.2 second read time, 0 seconds between wells. Percent luminescence was plotted against inhibitor concentration and the compound concentration producing 50% inhibition (IC50) was determined. Inhibitor IC50 values were calculated as an average of a minimum of three independent IC50 determinations.

Pseudomonas aeruginosa is an old foe, replete with 37 intrinsic multidrug-resistant (MDR) pumps as well as metabolic pathways to digest a variety of different substrates. It remains a key challenge as a nosocomial pathogen, with strains resistant to all classes of antibiotics. Increasing resistance of P. aeruginosa to fluoroquinolones and other antimicrobial agents has greatly impacted management decisions in patients with this infection (1). Oral therapy is no longer a treatment option in many patients. And in others, there may be no safe and active parenterally administered antibiotic available for use. Since it accounts for ~15-20% each of all acute bacterial skin and skin structure infections (ABSSSI; 2) and hospital- and ventilator-associated pneumonia (HAP/VAP; 3), and 8-10% of complicated urinary tract infections (cUTI; 4), a drug effective against P. aeruginosa would be life-saving. The antibacterial activity of classic tetracycline antibiotics against P. aeruginosa is limited by low permeability and intrinsic efflux. Tetraphase’s program is aimed at optimizing tetracyclines for anti-pseudomonal activity. Ideally, compounds would also have intrinsic activity against other key MDR organisms so that the drug could be used as empiric monotherapy to treat infections caused by other difficult-to-treat organisms. In exploration of fully synthetic novel scaffolds with gram-negative activity, a number of distinct classes were found to posses promising anti-pseudomonal activity. This is the first report of their antimicrobial activity against P. aeruginosa and other key MDR pathogens.

References

1.Sader, HS, R. Mallick, A. Kuznik, T.R. Fritsche, and R.N. Jones. 2007. Use of in vitro susceptibility and pathogen prevalence data to model the expected clinical success rates of tigecycline and other commonly used antimicrobials for empirical treatment of complicated skin and skin-structure infections. Int. J. Antimicro.b Agents 30:514-520. Epub 2007 Oct 23.2.Itani, K.M.F., S. Merchant, S.-J. Lin, K. Akhras, J.C. Alandete, and H.T. Hatoum. 2011. Outcomes and management costs in pastients hospitalized for skin and skin-structure infections. Am. J. Infect. Contro.l 39:42-49.3.Jones, R.N. 2010. Microbial etiologies of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia. Clin. Infect. Di.s 51 Suppl 1:S81-87.4.Wagenlehner, F.M.E., W. Weidner, G. Perletti, and K. G. Naber. 2010. Emerging drugs for bacterial urinary tract infedtions. Expert Opin. Emerging Drugs 15:375-397.5.Clinical and Laboratory Standards Institute (CLSI). 2009. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA. 6.Dallenne, C., A. Da Costa, D. Decre, C. Favier, and G. Arlet. 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 65:490-495.7.CLSI. 1999. Methods for determining bactericidal activity of antimicrobial agents; approved guideline. CLSI document M26-A., vol. 19, CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, USA.8.Fyfe, C., T. Farrell, J. Sutcliffe, and T. Grossman. 2011. Abstr. 51st Annual Intersci. Conf. Antimicrob. Agents Chemother,. abstr. F2-158. Development and characterization of a coupled transcription/translation (TNT) assay from P. aeruginosa.

In Vitro Activity against Recombinantly Expressed Resistance Mechanisms in E. coli DH10B

Compound Inhibition of In Vitro Transcription/Translation in E. coli

and P. aeruginosa S30 Extracts

Activity of Compounds against Panels of Recent Gram-negative and Gram-positive Isolates

TP-433 and TP-559 are Bactericidal In Vitro

The Tetraphase synthetic chemistry platform has enabled the synthesis of multiple distinct classes of novel tetracyclines with potent, mechanism-based in vitro activity against P. aeruginosa which translates to in vivo efficacy (see posters P1425 and P1426).

Several of these lead classes show broad-spectrum potency against panels of multidrug-resistant organisms and bactericidal activity against P. aeruginosa.

This is the first demonstration of novel tetracyclines with potential for use against P. aeruginosa and other difficult-to-treat Gram-negative and Gram-positive infections.

Compound

MIC (µg/ml)

E. coli DH10B strain expressing:

lacZ tet(M) tet(K) tet(A) tet(B) tet(X) NDM-1

TP-433 0.0312 0.5 0.0312 4 2 4 ≤0.016

TP-559 0.0625 0.5 0.0312 4 0.125 0.5 0.0625

TP-389 0.25 0.5 0.125 1 0.5 1 0.25

TP-214 0.5 4 0.5 1 1 4 0.25

TP-726 0.25 4 0.125 1 0.5 1 0.25

TP-819 0.125 0.25 0.125 4 0.25 1 0.125

TP-950 0.25 4 0.0625 16 0.5 1 0.25

TP-469 0.25 2 0.125 32 1 2 0.5

TP-512 0.25 0.25 0.25 1 0.25 1 ND

Tetracycline 4 128 128 >128 >128 128 ND

Ceftriaxone 0.125 0.125 0.0625 0.125 0.0625 0.125 >32

Compound

MIC50/MIC90

(range)

Pseudomonas aeruginosa

(n=96)

Pseudomonas aeruginosa

cystic fibrosis

(n=20)

Acinetobacter baumannii

(n=29)

Stenotrophomonas maltophila (n=15)

Burkholderia cenocepacia

(n=10)

Enterobacter cloacae

(n=19)

Proteus mirabilis (n=20)

Escherichia coli

ESBL+

(n=27)

Klebsiella pneumoniae

ESBL+

(n=25)

Staphylococcus aureus (MRSA)

(n=20)

Enterococcus faecalis

(n=21)

Enterococcus faecium

(n=14)

TP-4334/8 4/8 0.25/1 1/2 4/8 0.13/0.5 0.5/0.5 0.25/1 0.063/1 0.031/0.13 0.5/1 0.031/0.5

(0.13-32) (0.13-8) (≤0.016 - 2) (0.25-4) (0.063-16) (≤0.016-2) (0.13-1) (≤0.016-1) (≤0.016-2) (≤0.016-1) (≤0.016-1) (≤0.016-1)

TP-5594/8 4/8 0.25/1 0.5/1 8/32 0.25/1 1/2 0.25/1 0.25/1 0.063/0.25 1/2 0.25/1

(0.25-32) (0.25-8) (≤0.016 - 2) (0.13-1) (0.13-32) (0.063-2) (0.25-2) (≤0.016-1) (0.063-2) (0.063-0.25) (0.031-2) (≤0.016-2)

TP-3898/16 4/8 0.5/1 0.5/2 8/32 0.25/1 0.5/1 0.25/0.5 0.25/0.5 0.063/0.25 0.13/0.13 0.031/0.13

(0.25-32) (0.25-16) (0.031-4) (0.25-2) (0.25->32) (0.13-1) (0.25-1) (0.063-0.5) (0.13-0.5) (0.063-0.25) (0.063-0.13) (≤0.016-0.13)

TP-2148/32 8/16 2/8 4/8 32/>32 1/8 2/4 0.5/1 1/2 0.25/0.5 2/4 0.13/2

(0.5-32) (0.5-32) (0.25-16) (1-8) (2->32) (0.5-8) (1-4) (0.25-2) (0.5-2) (0.13-1) (0.25-4) (0.063-4)

TP-7268/16 8/16 2/4 2/8 8/32 0.5/2 0.5/1 0.5/0.5 0.5/1 0.13/0.25 4/8 2/8

(0.25-32) (0.25-16) (0.031-16) (0.5-8) (0.5->32) (0.25-2) (0.25-1) (0.13-1) (0.25-1) (0.13-1) (0.13-8) (0.031-16)

TP-8198/16 8/16 0.5/1 1/2 8/32 0.25/2 2/4 0.5/1 0.25/0.5 0.13/0.25 0.25/0.25 0.063/0.25

(0.5-32) (0.5-32) (0.031-4) (0.25-4) (0.25->32) (0.13-2) (2-4) (0.13-1) (0.13-1) (0.063-0.25) (0.063-0.25) (≤0.016-0.25)

TP-9508/16a 4/8 1/4 1/4 8/32 0.5/2 0.5/1 2/4 0.5/4 0.13/0.25 4/8 2/8

(0.5->32) (0.5-16) (0.031-8) (0.5-4) (0.5->32) (0.25-2) (0.25-1) (0.13-4) (0.5-8) (0.063-0.5) (0.13-8) (≤0.016-8)

TP-4698/16a 8/16 2/8 4/8 16/>32 1/4 0.5/1 4/8 1/16 0.25/0.5 4/8 0.5/8

(0.25-32) (1-16) (0.063-32) (1-16) (1->32) (0.5-4) (0.25-1) (0.25-16) (0.5->16) (0.13-0.5) (0.25-8) (0.031-8)

TP-5128/16b 8/16 0.5/2 0.5/1 8/32 0.5/1 2/2 0.5/0.5 0.5/1 0.13/0.25 0.25/0.25 0.063/0.25

(1-16) (1-16) (0.016-4) (0.063-2) (0.25->32) (0.25-2) (1-2) (0.063-1) (0.25-2) (0.063-0.25) (0.063-0.25) (0.031-0.5)

Tetracycline>32/>32d

ND16/>32 32/32 >32/>32 8/>32 >32/>32 >32/>32 8/>32 0.5/0.5 >32/>32 32/>32

(>32->32) (0.25->32) (4->32) (16->32) (2->32) (32->32) (2->32) (2->32) (0.25-32) (0.13->32) (0.031->32)

Tigecycline16/32c

ND0.5/4 1/2 8/32 1/4 4/8 0.13/0.5 0.5/1 0.13/0.13 0.063/0.13 ≤0.016/0.063

(1->32) (≤0.016-4) (0.25-4) (0.25->32) (0.25-4) (2-8) (0.063-0.5) (0.25-1) (0.063-0.25) (0.031-0.13) (≤0.016-0.25)

CXA-1012/4a 1/2 32/>32 >32/>32 8/32 8/>32 1/2 >16/>16 >16/>16 >32/>32 >32/>32 >32/>32

(0.25-8) (1-8) (1->32) (4->32) (2->32) (0.5->32) (0.5-4) (0.5->16) (1->16) (>32->32) (>32->32) (>32->32)

Tobramycin1/>32 1/4

ND>32/>32 32/>32

ND ND ND ND ND ND ND(0.13->32) (0.5-16) (2->32) (>32->32)

Gentamicin ND ND16/>32

ND>32/>32 1/>32 2/8 4/>32 16/>32 1/>32 32/>32 >32/>32

(2->32) (>32->32) (0.5->32) (0.5->32) (1->32) (1->32) (0.5->32) (8->32) (32->32)

Meropenem0.5/16 0.5/8 2/>32 >32/>32 8/32 0.063/0.5 0.25/1 0.031/0.13 0.063/32

ND ND ND(0.063->32) (0.63->32) (0.13->32) (8->32) (0.5->32) (0.031-32) (0.063-1) (≤0.016-2) (0.031->32)

Levofloxacin1/32c

ND4/32 2/8 4/8 1/16 0.13/32 16/>32 32/>32 8/>32 2/>32 >32/>32

(0.25->32) (0.063->32) (0.25-8) (0.5->32) (0.031->32) (0.063-32) (0.031->32) (0.031->32) (0.25->32) (1->32) (>32->32)

Ceftazidime4/>32c

ND ND>32/>32 8/16

ND0.13/0.13 32/>32 >32/>32

ND ND ND(1->32) (4->32) (2->32) (0.063-0.25) (0.5->32) (2->32)

Ceftriaxone ND ND ND ND ND32/>32

ND>32/>32 >32/>32 >32/>32

ND>32/>32

(0.13->32) (4->32) (2->32) (>32->32) (>32->32)

Piperacillin/ Tazobactam

16/>128c

ND ND ND16/>128

ND ND>128/>128 16/>32

ND ND ND(0.5->128) (0.5->128) (>128->128) (2/>32)

Colistin1/1 1/2 0.5/1 8/>32 >32/>32 0.25/2 >32/>32 0.25/0.25 0.25/1

ND ND ND(0.13-4) (0.13-4) (0.13-2) (0.13->32) (>32->32) (0.13->32) (>32->32) (0.13-4) (0.13->16)

Vancomycin ND ND ND ND ND ND ND ND ND1/1 1/>32 >32/>32

(0.5-1) (1->32) (32->32)a 55 P. aeruginosa isolates (35 unbiased + 20 cystic fibrosis isolates); b 20 P. aeruginosa isolates (cystic fibrosis isolates); c76 P. aeruginosa isolates (no cystic fibrosis isolated included); d35 isolates (no cystic fibrosis isolates); ND, not done

Compound

P. aeruginosa E. coli

IC50 (µg/ml)

SDIC50 (µg/ml)

SD

TP-433 0.12 0.04 0.25 0.08

TP-559 0.11 0.03 0.24 0.05

TP-389 0.25 0.13 0.26 0.05

TP-214 0.23 0.09 0.37 0.02

TP-726 0.25 0.07 0.36 0.08

TP-819 0.27 0.12 0.37 0.05

TP-950 0.30 0.11 0.65 0.06

TP-469 0.25 0.01 0.60 0.00

TP-512 0.21 0.03 0.33 0.04

Tetracycline 1.40 0.86 1.87 0.21

Linezolid 0.94 0.39 1.53 0.06