DS86760016, a Leucyl-tRNA Synthetase Inhibitor with Activity against Pseudomonas aeruginosa

DS86760016 is a new leucyl-tRNA-synthetase inhibitor at the preclinical development stage. DS86760016 showed potent activity against extended-spectrum multidrug-resistant Pseudomonas aeruginosa isolated from clinical samples and in vitro biofilms.

ABSTRACT

DS86760016 is a new leucyl-tRNA-synthetase inhibitor at the preclinical development stage. DS86760016 showed potent activity against extended-spectrum multidrug-resistant Pseudomonas aeruginosa isolated from clinical samples and in vitro biofilms. In a murine catheter-associated urinary tract infection model, DS86760016 treatment resulted in significant eradication of P. aeruginosa from the kidney, bladder, and catheter without developing drug resistance. Our data suggest that DS86760016 has the potential to act as a new drug for the treatment of Pseudomonas infections.

INTRODUCTION

Pseudomonas aeruginosa is the most common bacterial pathogen that causes biofilm-mediated infections, including catheter-associated urinary tract infection (CAUTI), ventilator-associated pneumonia, mechanical heart valve-related infections, grafts, and contact lens-associated corneal infections (1–4). Within nosocomial infections, the most prevalent are urinary tract infections (UTIs), which account for around 49% of all hospital setting infections and are directly related to the duration of hospitalization (4). The problem is further aggravated in critically ill patients who are subjected to catheterization of the urinary tract, which predisposes them to bacterial infections (5, 6). P. aeruginosa has an innate propensity to attach to the surfaces of catheters to form biofilms, which leads to a higher incidence of urinary tract infections (UTIs) in patients with indwelling bladder catheterization (7, 8).

The antibiotics of choice for UTI treatment are parenteral carbapenems, such as imipenem and meropenem, or colistin and quinolones (9). However, the poor activity of those available therapies on bacterial biofilms and the growing prevalence of multidrug-resistant (MDR) P. aeruginosa (10–13) emphasize the need for new and improved therapies.

Benzoxaboroles are a novel class of antibiotics that exhibit excellent activity against Gram-negative pathogens by inhibiting the editing site of the leucyl-tRNA-synthetase (LeuRS) enzyme (14–17). GSK2251052 was the first benzoxaborole compound with potent activity against drug-resistant Gram-negative organisms (16, 17); however, its development was halted during phase II clinical trials because of the rapid selection of resistant mutants (18). DS86760016 is a new investigational benzoxaborole compound (see Fig. S1 in the supplemental material) which showed a potent activity against Gram-negative pathogens, excellent pharmacokinetics (PK), and a reduced risk of resistance in mouse ascending UTI models compared to GSK2251052 (15). In this study, we evaluated the activity of DS86760016 against MDR P. aeruginosa with the prospect of its potential use in treating Pseudomonas infections.

All experimental animal procedures were performed in accordance with the protocols approved by the institutional animal ethics committee at Daiichi Sankyo India Pharma Pvt. Ltd., Gurugram. DS86760016 was synthesized at Daiichi Sankyo Indian Pharma Pvt. Ltd., Gurugram, India. All antibiotics and other reagents were purchased from Sigma-Aldrich, India. Clinical isolates of P. aeruginosa used in this study were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), International Health Management Associates, Inc., (IHMA, IL, USA), and from our proprietary collection of clinical isolates.

MIC values of DS86760016, ciprofloxacin, meropenem, and tobramycin were determined against 350 clinical isolates of P. aeruginosa using the broth dilution method as described in Clinical and Laboratory Standards Institute (CLSI) guidelines (19). The panel used in this study consisted of P. aeruginosa clinical isolates harboring extended-spectrum beta-lactamases (ESBLs), carbapenemases (KPC), IMP-type carbapenemases (IMP), and Verona integron-encoded metallo-β-lactamase (VIM) and exhibiting fluoroquinolone resistance and aminoglycoside resistance. DS86760016 showed a superior activity with an MIC_50/90 of 1/2 μg/ml compared to the standard antipseudomonas agents, meropenem, ciprofloxacin, and tobramycin (MIC₉₀ > 32 μg/ml) (Table 1). DS86760016 showed a narrow MIC range and >8-fold lower mutant prevention concentrations (MPC) against P. aeruginosa than GSK2251052 (15). Reduced MPC for DS86760016 might be the result of a differential interaction of the compound with the Pseudomonas LeuRS enzyme, as suggested by a difference in mutation sites in GSK2251052- and DS86760016-resistant mutants (15).

TABLE 1.

Antimicrobial activity of DS86760016 and comparator agents tested against clinical isolates of P. aeruginosa

Antimicrobial agent	No. of isolates at MIC (μg/ml):a										MIC50 (μg/ml)
	≤0.06	0.125	0.25	0.5	1	2	4	8	16	≥32	50%	90%
DS86760016	0	0	4	48	219	57	18	4	0	0	1	2
Meropenem	16	15	25	38	35	24	29	48	27	93	4	>32
Ciprofloxacin	38	45	24	20	22	11	42	42	18	88	4	>32
Tobramycin	2	18	33	102	48	19	11	7	9	101	1	>32

^aFrom a total of 350 P. aeruginosa isolates.

The effect of DS86760016 on P. aeruginosa 1594965 (multidrug-resistant clinical isolate) and P. aeruginosa ATCC 700829 (standard strain for biofilm) biofilm formation was assessed as described previously (20). The overnight-grown bacterial cultures were pelleted and resuspended in LB plus 0.2% glucose (10⁷ CFU/ml). Culture suspensions were spiked with different concentrations (64 μg/ml to 0.06 μg/ml) of DS86760016 in 96-well plates and incubated at 28 ± 2°C. The adherent biofilm was stained with 200 μl of 1% crystal violet for 30 min and rinsed 3 times with distilled water. Finally, the adherent biofilm in the wells was dissolved in 30% acetic acid, and the optical density at 600 nm (OD₆₀₀) was measured to quantify the formed biofilms. DS86760016 showed a potent antibiofilm activity with 50% inhibition of in vitro biofilms produced by strains MDR P. aeruginosa 1594965 and P. aeruginosa ATCC 700829 at 4 μg/ml (Fig. 1). The biofilm inhibitory concentration (BIC₅₀) value (4 μg/ml) of DS86760016 was 2-fold higher than the MIC (2 μg/ml). This difference might be due to the bacteriostatic nature of DS86760016. Similar differences in BIC and MIC values were reported for other antipseudomonal antibiotics in independent studies (21, 22).

FIG 1.

Activity of DS86760016 against in vitro biofilm of P. aeruginosa 1594965 (A) and P. aeruginosa ATCC 700829 (B). The adherent biofilm in the presence of DS86760016 or in a control well (drug free) was stained with 1% crystal violet and the OD₆₀₀ was measured after it was dissolved in 30% acetic acid. The inhibition of biofilm in the presence of DS86760016 was calculated by comparing the biofilm formation to that in the control well. Values shown in the figures are averages from two independent experiments.

To evaluate the in vivo efficacy of DS86760016 against biofilm, a murine CAUTI model was established in female Swiss Webster mice (Vivo BioTech, Ltd., Hyderabad, India) as described by Kurosaka et al. (23) with some modifications. Female mice weighing 26 to 32 g received 1.0-cm indwelling catheter pieces in their bladders under anesthetic conditions. To cause the infection, P. aeruginosa 1594965 was grown overnight on Mueller-Hinton agar (MHA; Becton, Dickinson and Company, NJ) plates at 36 ± 1°C. Using a 25-gauge blunt needle, 50 μl of freshly prepared bacterial suspension was injected transurethrally, resulting in ∼10⁷ CFU/animal. The urethral opening was clamped for 4 h to prevent urine flow. Treatment with DS86760016 and meropenem was initiated 4 h postinfection and administered by a subcutaneous (s.c.) route every 6 h (q.i.d.) for 7 days. Mice were euthanized 8 h from the last administration, and CFU counts in the kidneys, bladders, and catheter pieces were determined by plating diluted samples on MHA. The numbers of bacteria resistant to DS86760016 were also determined by plating samples on MHA plates containing a 32-μg/ml concentration of DS86760016.

To determine whether the bacteria within the infected bladders were just located on the catheter surface or started forming the biofilm by the time of initiation of DS86760016 treatment, kidneys, bladders, and associated catheters were removed from the infected mice at 4 h postinfection and assessed for bacterial counts. The catheter pieces were also sectioned and processed for scanning electron microscopy (SEM) as described previously (14). The catheter pieces showed biofilm formation within 4 h of infection (Fig. 2A), with visible bacteria in the form of elongated and septated rods on the surfaces of implanted catheters (Fig. 2B and C). The higher magnification (×2,000) images of catheters revealed that P. aeruginosa 1594965 was embedded in a matrix material and formed solid biofilms on the catheters (Fig. 2C).

FIG 2.

Scanning electron micrographs revealing biofilm formation on catheters isolated from mouse bladders. Catheters were isolated from mice infected with P. aeruginosa 1594965 sacrificed at 4 h postinfection; ×200 (A), ×590 (B), and ×2,000 (C) magnification.

Treatment with DS86760016 showed efficacy in the murine CAUTI model (Fig. 3). DS86760016 at a dose of 220 mg/kg, q.i.d., resulted in 2.4, 2.3, and 1.6 log reductions in bacterial counts in kidneys, bladders, and catheters, respectively. On the other hand, the 30-mg/kg q.i.d. dose showed 2.2 and 1.2 log reductions in kidneys and bladders, respectively, compared to the pre-control. Meropenem was found to be inefficacious in this study, with high bacterial counts in kidneys as well as on catheters, probably because of the high MIC of P. aeruginosa 1594965 to meropenem (MIC, 16 μg/ml). One animal in the meropenem-treated group and two animals in the vehicle-treated group died during the experimental period, while other animals from vehicle-treated group showed anorexia and weight loss, possibly due to higher bacterial loads.

FIG 3.

Efficacy of DS86760016 in mouse catheter-associated UTI model infected via the transurethral route with P. aeruginosa 1594965. Bacterial counts recovered from bladders (A), kidneys (B), and catheters (C) of mice treated with either DS86760016 or meropenem. Each circle represents the number of CFU derived from an individual animal, and horizontal lines represents the means (n = 5). ○, bacterial load from live animal; •, bacterial load from dead animal. The numbers of CFU in each treated group were compared with those in the control group before treatment (pre-control) using a nonparametric Mann-Whitney analysis. Asterisks indicate a significant difference compared with the result for the control group (**, P < 0.001).

To determine the urine excretion of DS86760016, the mice were dosed intravenously with 4 mg/kg of DS86760016. Blood and urine were collected at different time intervals up to 24 h and 72 h postinjection, respectively. Plasma samples were harvested, and PK parameters were calculated as described previously (15). The human intravenous urine profile was simulated according to the allometry of mouse, rat, monkey, and dog PK parameters and human renal and plasma clearance (15). The graphs show that at an intravenous dose of 220 mg/kg/dose q.i.d. in mice or 1,500 mg every 12 h (b.i.d.) in humans, the simulated urine concentrations were higher than the mutant prevention concentration (MPC) level (Fig. 4A and B).

FIG 4.

Simulation studies to evaluate human equivalent exposure of DS86760016 in mice. Simulated human urine concentrations at 1,500 mg/dose, b.i.d. (A), adapted from Purnapatre et al. (15), and simulated mouse urine concentrations at 220 mg/kg/dose, q.i.d. (B).

DS86760016 has shown better metabolic stability, good tolerance in mice (50% lethal dose [LD₅₀] > 1,000 mg/kg), and potent activity in urine, with a 2-fold higher DS86760016 urine excretion in mice and rats, which was significantly higher in monkeys at 80% compared to 8.6% for GSK2251052 (15). Based on our allometric analysis and Dedrick plots, a DS86760016 dose of 220 mg/kg q.i.d. in mice, equivalent to 1,500 mg b.i.d. in humans, was predicted to maintain the urinary concentrations above the MPC₁₀₀ for P. aeruginosa throughout the treatment period, suggesting reduced risk of emergence of drug-resistant mutants in the urinary tract.

To confirm this hypothesis, we used a nonsurgical mouse CAUTI model, which mimics catheter-associated UTI in humans. SEM studies of in vivo catheter biofilms confirmed that P. aeruginosa 1594965 forms biofilms within 4 h postinfection (Fig. 2). Hence, treatment was initiated 4 h postinfection with 220 mg/kg, q.i.d., of DS86760016 for 7 days by the s.c. route. This treatment regimen resulted in significant reduction in the bacterial counts in the kidney, bladder, and catheter compared to the pre-control. In addition, DS86760016-resistant mutants were not detected in any of the treated animals. Although a 30-mg/kg dose of DS86760016 showed static activity on the catheter biofilm, it prevented the migration of P. aeruginosa infection from the bladder to the kidneys. Our data suggest there is reduced risk of resistance development to DS86760016 when an adequate concentration greater than the MPC is maintained in the urinary tract system. An extended spectrum study with 30 clinical isolates of P. aeruginosa confirmed the MPC₁₀₀ of 256 μg/ml (see Table S1). Furthermore, our stimulated studies suggest that a dose of 1,500 mg (intravenous, every 12 h) of DS86760016 in humans would keep drug concentrations greater than the MPC throughout the treatment regimen; hence, it will reduce the risk of developing a DS86760016 resistance mutant (Fig. 4). Additional future studies will be required to access the efficacy of this compound against multistep mutants with an MPC greater than 256 μg/ml.

In conclusion, DS86760016 showed potent antibacterial activity against MDR P. aeruginosa strains, retained activity against P. aeruginosa in vitro biofilms, and was efficacious against P. aeruginosa in the mouse CAUTI model with reduced risk of resistance. Furthermore, improved metabolic properties of DS86760016, including reduced metabolic clearance, higher stability, and higher renal excretion, could allow longer interactions with bacterial pathogens, resulting in better eradications of P. aeruginosa in UTI patients. Our findings suggest that DS86760016 is a promising investigational new drug candidate for the treatment of UTIs caused by MDR P. aeruginosa strains and support its further investigation.