Antimicrobial activity of POL7306 tested against clinical isolates of Gram-negative bacteria collected worldwide Free

Abstract

Background

POL7306 belongs to a new class of peptidomimetic outer-membrane-protein-targeting antibiotics with a novel mechanism of action. POL7306 is in development for the treatment of infections caused by antimicrobial-resistant Gram-negative bacteria and has demonstrated low cytotoxicity and nephrotoxicity.

Methods

A total of 891 isolates were collected by the SENTRY Antimicrobial Surveillance Program from 134 medical centres in Europe (n = 424; 41 centres in 18 nations), the USA (n = 411 isolates from 67 centres), the Asia-Pacific region (n = 24; 15 centres in 6 nations) and Latin America (n = 32; 11 centres in 9 nations) and included 558 Enterobacterales, 310 non-fermenters and 23 fastidious organisms. Susceptibility testing was performed using the reference broth microdilution method and the medium was supplemented with 0.002% polysorbate-80 for testing POL7306. Resistant subsets were characterized by WGS.

Results

POL7306 demonstrated potent in vitro activity against Enterobacterales [including carbapenem-resistant (MIC_50/90, 0.06/0.25 mg/L), ESBL-producing (MIC_50/90, 0.06/0.12 mg/L), KPC-producing (MIC_50/90, 0.12/0.25 mg/L), MBL-producing (MIC_50/90, 0.06/0.25 mg/L), colistin-non-susceptible, mcr-negative (MIC_50/90, 0.5/2 mg/L) and mcr-positive (MIC_50/90, 0.12/0.25 mg/L) Enterobacterales], Pseudomonas aeruginosa (MIC_50/90, 0.25/0.25 mg/L), Acinetobacter baumannii (MIC_50/90, 0.06/0.12 mg/L) and Stenotrophomonas maltophilia (MIC_50/90, 0.06/0.25 mg/L).

Conclusions

POL7306 demonstrated potent activity against a large collection of Gram-negative organisms collected worldwide that included colistin-resistant, XDR and ESBL- and carbapenemase-producing isolates for which there are currently limited treatment options.

Introduction

The outer membrane of Gram-negative bacteria contains a variety of β-barrel outer membrane proteins (OMPs), which serve many functions for cell survival and virulence, and includes an asymmetric bilayer with glycerophospholipids in the inner leaflet and LPS in the outer leaflet.¹ Targeting OMPs by macrocyclic peptidomimetic antibiotics is a new strategy to discover and develop urgently needed new antimicrobial agents active against Gram-negative ESKAPE pathogens.² Murepavadin is a Pseudomonas-specific peptidomimetic antibacterial agent targeting the essential β-barrel OMP LptD and is the first OMP-targeting antibiotic to enter late-stage clinical development.^1,3

POL7306 belongs to a new class of chimeric peptidomimetic antibiotics with broad-spectrum Gram-negative activity in which the β-hairpin macrocycle is linked to the peptide macrocycle of polymyxin (Figure 1).⁴ Recent biophysical and genetic studies provide evidence that these new compounds, exemplified here by POL7306, act by a novel mechanism involving binding to LPS and the essential OMP BamA.⁴ BamA is the main component of the β-barrel folding complex (BAM) that promotes folding and insertion of β-barrel proteins into the outer membrane in Gram-negative bacteria.⁴ Structural investigations indicate that POL7306 interacts with the BamA β-barrel domain, changes the conformational ensemble in the β-barrel lateral gate between open and closed states, and locks BamA in its closed state.⁴ POL7306 and related compounds have shown promising activity and tolerability in mouse models of infection such as the peritonitis model and the thigh model.⁴ Regarding safety, POL7306 showed no haemolysis at up to 200 mg/L, very low cytotoxicity against HeLa and HepG2 cells (IC₅₀ 200 and >200 mg/L, respectively) and low nephrotoxicity.⁴ Here we report the in vitro activity of POL7306 against a large well-characterized collection of contemporary Gram-negative bacterial isolates collected from 134 medical centres worldwide.

Materials and methods

The organism collection was comprised of 891 isolates collected by the SENTRY Antimicrobial Surveillance Program.⁵ The isolates were from 134 medical centres in Europe (n = 424; 41 centres in 18 nations), the USA (n = 411 isolates from 67 centres), the Asia-Pacific region (n = 24; 15 centres in 6 nations) and Latin America (n = 32; 11 centres in 9 nations) and included 558 Enterobacterales, 310 non-fermenters and 23 fastidious organisms. Approximately 90% of the collection, including all randomly selected Enterobacterales and all non-fermentative Gram-negative bacilli and fastidious organisms, were collected during 2017–18, whereas some of the molecularly characterized Enterobacterales were collected during 2009–16. The following organism subsets were evaluated:

• Enterobacterales (n = 558)

  • Randomly selected non-intrinsically resistant Enterobacterales (272): included Escherichia coli (50), Klebsiella pneumoniae (50), Klebsiella oxytoca (32), Klebsiella aerogenes (30), Citrobacter freundii species complex (31), Enterobacter cloacae species complex (30), Salmonella spp. (25) and Shigella spp. (24). Species/genera intrinsically resistant to colistin and polymyxin B, such as Morganella morganii, Proteus spp., Providencia spp. and Serratia spp., were included in the ‘intrinsically resistant’ group.

  • Third-generation-cephalosporin-resistant, carbapenem-susceptible Enterobacterales (52): included C. freundii species complex (10), Citrobacter koseri (1), E. cloacae species complex (5), E. coli (16), K. aerogenes (5) and K. pneumoniae (15) isolates resistant to ceftriaxone and ceftazidime, and susceptible to meropenem and imipenem.

  • Carbapenem-resistant Enterobacterales (CRE; 53): isolates displaying imipenem, doripenem and/or meropenem MIC values >2 mg/L. Included E. coli (7), E. cloacae species complex (14), C. freundii species complex (3), K. aerogenes (7), K. oxytoca (2) and K. pneumoniae (20).

  • ESBL-producing Enterobacterales (24): selected based on results from WGS. Included E. cloacae species complex (1), E. coli (15) and K. pneumoniae (8). The β-lactamases encoded by these isolates included CTX-M-1, CTX-M-2-like, CTX-M-3, CTX-M-9, CTX-M-15, CTX-M-14, CTX-M-24, CTX-M-27, CTX-M-55, OXA-1/OXA-30, SHV-5, SHV-7, SHV-12, SHV-27, SHV-30, TEM-19 and TEM-169.

  • KPC-producing Enterobacterales (23): included C. freundii species complex (1), E. cloacae species complex (1), E. coli (2), K. aerogenes (1), K. oxytoca (2) and K. pneumoniae (16). These isolates produced either KPC-2 (14) or KPC-3 (9).

  • MBL-producing Enterobacterales (12): included E. cloacae species complex (3), E. coli (3), K. pneumoniae (5) and Serratia marcescens (1). These isolates produced NDM-1, NDM-5, NDM-7, VIM-1, VIM-4 and/or VIM-12.

  • mcr-positive Enterobacterales (75): included E. coli (71) and K. pneumoniae (4). The following mcr genes were identified in these isolates: mcr-1, mcr-1.5, mcr-2 and mcr-3.1.

  • Colistin-resistant, mcr-negative K. pneumoniae (24). Resistance mechanisms included mgrB disruption/mutations (16), phoPQ mutations (4), prmA domain alteration (1), prmB mutation (1), prmD disruption (1) and crrAB deletion (1).

  • Intrinsically resistant Enterobacterales (23): included M. morganii (5), Proteus mirabilis (4), Proteus vulgaris (1), P. vulgaris group (1), Providencia rettgeri (3), Providencia stuartii (1), Serratia liquefaciens (1), S. marcescens (5), Serratia rubidaea (1) and unspeciated Providencia (1).

• Pseudomonas aeruginosa (154): included 52 randomly selected, non-MDR isolates and 102 XDR isolates.

• Acinetobacter baumannii-calcoaceticus species complex (105): all XDR isolates.

• Stenotrophomonas maltophilia (51).

• Haemophilus influenzae (12).

• Moraxella catarrhalis (11).

Isolates were categorized as MDR or XDR according to criteria published by Magiorakos et al.,⁶ defining MDR as ‘non-susceptible to ≥1 agent in ≥3 antimicrobial classes’ and XDR as ‘non-susceptible to ≥1 agent in all but ≤2 antimicrobial classes’. CLSI interpretive criteria were used to categorize isolates as CRE, MDR or XDR.⁷

Isolates were tested against POL7306 and comparator agents by the reference broth microdilution method⁸ using CAMHB. CLSI and EUCAST interpretive criteria were used to determine susceptibility and resistance for comparator agents.^7,9 MIC panels were manufactured at JMI Laboratories and medium was supplemented with 0.002% polysorbate-80 for POL7306 tests. Quality control was tested daily and inoculum density was monitored by colony counts. The quality control strains were: E. coli ATCC 25922 and NCTC 13846; K. pneumoniae ATCC 700603 and BAA 1705; and P. aeruginosa ATCC 27853.

Results

POL7306 demonstrated potent activity against all resistant subsets evaluated and Enterobacterales organisms that are not intrinsically resistant to colistin and polymyxin B (Tables 1 and 2). POL7306 MIC₅₀ values were 0.06 or 0.12 mg/L and MIC₉₀ values were 0.12 or 0.25 mg/L for all subsets, except the group of colistin-resistant, mcr-negative K. pneumoniae isolates that showed MIC_50/90 values of 0.5/2 mg/L (Table 1). Only four Enterobacterales isolates (excluding the intrinsically resistant Enterobacterales) had POL7306 MIC values >1 mg/L: three colistin-resistant mcr-negative K. pneumoniae isolates with POL7306 MIC values of 2 mg/L (two isolates) and 4 mg/L (one isolate) and an MBL-producing S. marcescens isolate, a species that is intrinsically resistant to POL7306, colistin and polymyxin B, with a POL7306 MIC value of >8 mg/L (Table 1). Among the collection of randomly selected non-intrinsically resistant isolates, POL7306 MIC_50/90 values ranged from 0.06/0.12 mg/L for E. coli, K. oxytoca and Shigella spp. to 0.25/0.25 mg/L for Salmonella spp. (data not shown).

The most active comparator agents tested against the non-intrinsically resistant Enterobacterales group were ceftazidime/avibactam (MIC_50/90, 0.12/0.5 mg/L; 100.0% susceptible per CLSI and EUCAST), meropenem (MIC_50/90, ≤0.12/≤0.12 mg/L; 98.9%/99.3% susceptible per CLSI/EUCAST) and amikacin (MIC_50/90, 2/4 mg/L; 98.9%/98.5% susceptible per CLSI/EUCAST). Colistin (MIC_50/90, 0.12/0.25 mg/L) inhibited 96.0% of isolates using the EUCAST susceptible breakpoint of ≤2 mg/L (Table 2).

CRE isolates were very susceptible to POL7306 (MIC_50/90, 0.06/0.25 mg/L; highest MIC, 0.5 mg/L), but showed low susceptibility to comparator agents, including colistin (MIC_50/90, 0.12/0.25 mg/L; 90.6% susceptible per EUCAST), ceftazidime/avibactam (MIC_50/90, 2/8 mg/L; 90.6% susceptible per CLSI and EUCAST) and amikacin (MIC_50/90, 4/32 mg/L; 81.1%/66.0% susceptible per CLSI/EUCAST; Table 2). POL7306 was also highly active against ESBL-producing (MIC_50/90, 0.06/0.12 mg/L), KPC-producing (MIC_50/90, 0.12/0.25 mg/L) and MBL-producing (MIC_50/90, 0.06/0.25 mg/L) Enterobacterales (Table 1). The only β-lactamase-producing Enterobacterales isolate with an elevated POL7306 MIC result was a VIM-producing S. marcescens.

POL7306 (MIC_50/90, 0.12/0.25 mg/L; highest MIC, 0.25 mg/L) was 32-fold more potent than colistin (MIC_50/90, 4/8 mg/L) when tested against mcr-positive Enterobacterales isolates (Table 2). POL7306 activity against mcr-positive Enterobacterales was similar to activity against the non-intrinsically resistant Enterobacterales group (MIC_50/90, 0.06/0.25 mg/L; highest MIC, 1 mg/L), indicating that the mcr genes do not adversely affect POL7306 activity (Tables 1 and 2). Moreover, POL7306 retained good activity (MIC_50/90, 0.5/2 mg/L) against colistin-resistant (MIC >2 mg/L), mcr-negative K. pneumoniae isolates, a subset that exhibited low susceptibility to many comparator agents, including meropenem (MIC_50/90, 8/>32 mg/L; 33.3%/41.7% susceptible per CLSI/EUCAST), gentamicin (MIC_50/90, 8/>8 mg/L; 45.8% susceptible per CLSI and EUCAST) and amikacin (MIC_50/90, 16/>32 mg/L; 58.3%/45.8% susceptible per CLSI/EUCAST; Table 2).

The P. aeruginosa collection (n = 154) included 102 XDR isolates and POL7306 (MIC_50/90, 0.25/0.25 mg/L; highest MIC, 0.5 mg/L) was 2–4-fold more active than colistin (MIC_50/90, 0.5–1/1 mg/L) against these organisms (Table 2). The most active comparator agents tested against XDR P. aeruginosa were colistin (MIC_50/90, 1/1 mg/L; 98.0% susceptible per CLSI and EUCAST), ceftazidime/avibactam (MIC_50/90, 8/>32 mg/L; 66.7% susceptible per CLSI and EUCAST) and amikacin (MIC_50/90, 16/>32 mg/L; 67.6%/48.0% susceptible per CLSI/EUCAST; Table 2).

POL7306 exhibited potent activity against XDR A. baumannii (n = 105; MIC_50/90, 0.06/0.12 mg/L; highest MIC, 0.25 mg/L) and it was 4- to 16-fold more active than colistin (MIC_50/90, 0.25/2 mg/L; 91.4% susceptible per CLSI and EUCAST) against these organisms (Table 2). All other compounds showed very limited activity against XDR A. baumannii (≤25.7% susceptibility; Table 2).

S. maltophilia (n = 51) was also very susceptible to POL7306, with MIC_50/90 values of 0.06/0.25 mg/L and a highest MIC value of 0.25 mg/L. Among the comparators, only trimethoprim/sulfamethoxazole (MIC_50/90, ≤0.5/≤0.5 mg/L; 96.1%/98.0% susceptible per CLSI/EUCAST) and levofloxacin (MIC_50/90, 1/4 mg/L; 78.4% susceptible per CLSI) exhibited some in vitro activity against S. maltophilia (Tables 1 and 2).

POL7306 exhibited potent in vitro activity against M. catarrhalis, with MIC values of either 0.12 or 0.25 mg/L (MIC_50/90, 0.25/0.25 mg/L; Table 1), but only moderate activity against H. influenzae (MIC_50/90, 2/4 mg/L; Table 1).

Discussion

The results of this study demonstrated the potent in vitro activity and broad spectrum of POL7306 against clinically relevant Gram-negative bacteria. POL7306 activity was not adversely affected by resistance to antimicrobial agents currently used to treat Gram-negative infections and retained activity against isolates resistant to cephalosporins, carbapenems, β-lactamase/inhibitor combinations (including ceftazidime/avibactam), aminoglycosides and/or fluoroquinolones. POL7306 also retained activity against isolates with acquired colistin resistance and was not affected by the presence of mcr genes that confer resistance to polymyxins. POL7306 MIC values were slightly higher (4- to 8-fold) for K. pneumoniae isolates expressing other mechanisms of resistance to polymyxins, such as LPS modification mediated by mgrB mutations,¹⁰ compared with other Enterobacterales subsets, but MIC values were ≤2 mg/L for 95.8% of those isolates. Therefore, POL7306’s spectrum of activity included colistin-resistant, XDR and ESBL- and carbapenemase-producing Enterobacterales isolates for which there are currently limited treatment options. It is important to emphasize, however, that some Enterobacterales species are intrinsically resistant to POL7306, including P. mirabilis, indole-positive Proteeae and Serratia spp.

POL7306 also showed potent activity against difficult-to-treat non-fermentative Gram-negative organisms, including P. aeruginosa, A. baumannii and S. maltophilia with MDR and XDR phenotypes, notably carbapenem-resistant strains. The number of these organisms has increased continuously in some geographical areas and therapeutic options are very limited.^11,12 In summary, the results of this study coupled with results from ongoing in vitro and in vivo studies^13–15 support preclinical development of POL7306 for treating Gram-negative infections.

Funding

This study was supported by Polyphor Ltd (Switzerland) and the Novo REPAIR Impact Fund.

Transparency declarations

JMI Laboratories was contracted to perform services in 2018 for Achaogen, Inc., Albany College of Pharmacy and Health Sciences, Allecra Therapeutics, Allergan, AmpliPhi Biosciences Corp., Amplyx, Antabio, American Proficiency Institute, Arietis Corp., Arixa Pharmaceuticals, Inc., Astellas Pharma, Inc., Athelas, Basilea Pharmaceutica Ltd, Bayer AG, Becton, Dickinson and Company, bioMerieux SA, Boston Pharmaceuticals, Bugworks Research, Inc., CEM-102 Pharmaceuticals, Cepheid, Cidara Therapeutics, Inc., CorMedix, Inc., DePuy Synthes, Destiny Pharma, Discuva Ltd, Dr. Falk Pharma GmbH, Emery Pharma, Entasis Therapeutics, Eurofarma Laboratorios SA, US FDA, Fox Chase Chemical Diversity Center, Inc., Gateway Pharmaceutical LLC, GenePOC, Inc., Geom Therapeutics, Inc., GlaxoSmithKline plc, Harvard University, Helperby, HiMedia Laboratories, F. Hoffmann-La Roche Ltd, ICON plc, Idorsia Pharmaceuticals Ltd, Iterum Therapeutics plc, Laboratory Specialists, Inc., Melinta Therapeutics, Inc., Merck & Co., Inc., Microchem Laboratory, Micromyx, MicuRx Pharmaceuticals, Inc., Mutabilis Co., Nabriva Therapeutics plc, NAEJA-RGM, Novartis AG, Oxoid Ltd, Paratek Pharmaceuticals, Inc., Pfizer, Inc., Pharmaceutical Product Development, LLC, Prokaryotics, Inc., Qpex Biopharma, Inc., Ra Pharmaceuticals, Inc., Roivant Sciences Ltd, Safeguard Biosystems, Scynexis, Inc., SeLux Diagnostics, Inc., Shionogi and Co. Ltd, SinSa Labs, Spero Therapeutics, Summit Pharmaceuticals International Corp., Synlogic, T2 Biosystems, Inc., Taisho Pharmaceutical Co. Ltd, TenNor Therapeutics Ltd, Tetraphase Pharmaceuticals, The Medicines Company, Theravance Biopharma, University of Colorado, University of Southern California-San Diego, University of North Texas Health Science Center, VenatoRx Pharmaceuticals, Inc., Vyome Therapeutics, Inc., Wockhardt, Yukon Pharmaceuticals, Inc., Zai Lab, Zavante Therapeutics, Inc. There are no speakers’ bureaus or stock options to declare. H.H.L. is employed by Polyphor Ltd. G.E.D. was employed by Polyphor Ltd and is currently employed by BioVersys AG.

References

Author notes