In Vitro and In Vivo Activities of a Bi-Aryl Oxazolidinone, RBx 11760,

against Gram-Positive Bacteria

Tarani Kanta Barman,a,g Manoj Kumar,a Tarun Mathur,a Tridib Chaira,b G. Ramkumar,a Vandana Kalia,a,d Madhvi Rao,a
Manisha Pandya,a Ajay Singh Yadav,c Biswajit Das,c Dilip J. Upadhyay,a Hamidullah,e Rituraj Konwar,e V. Samuel Raj,a,f Harpal Singhg
Department of Microbiology,a Department of Pharmacokinetics & Metabolism,b and Department of Medicinal Chemistry,c Daiichi Sankyo India Pharma Private Limited,
Gurgaon, Haryana, India; Department of Scientific and Industrial Research, Government of India, New Delhi, Indiad ; Central Drug Research Institute (CDRI), Lucknow, Uttar
Pradesh, Indiae ; Centre for Drug Design Discovery and Development (C4D), SRM University Haryana, Sonepat, Haryana, Indiaf; Centre for Biomedical Engineering, Indian
Institute of Technology, New Delhi, Indiag

RBx 11760, a bi-aryl oxazolidinone, was investigated for antibacterial activity against Gram-positive bacteria. The MIC90s of RBx
11760 and linezolid against Staphylococcus aureus were 2 and 4 mg/liter, against Staphylococcus epidermidis were 0.5 and 2 mg/
liter, and against Enterococcus were 1 and 4 mg/liter, respectively. Similarly, against Streptococcus pneumoniae the MIC90s of RBx
11760 and linezolid were 0.5 and 2 mg/liter, respectively. In time-kill studies, RBx 11760, tedizolid, and linezolid exhibited
bacteriostatic effect against all tested strains except S. pneumoniae. RBx 11760 showed 2-log10 kill at 4X MIC while tedizolid and
linezolid showed 2-log10 and 1.4-log10 kill at 16X MIC, respectively, against methicillin-resistant S. aureus (MRSA) H-29.
Against S. pneumoniae 5051, RBx 11760 showed bactericidal activity, with 4.6-log10 kill at 4X MIC compared to 2.42-log 10
and 1.95-log10 kill for tedizolid and linezolid, respectively, at 16X MIC. RBx 11760 showed postantibiotic effects (PAE) at 3
h at 4 mg/liter against MRSA H-29, and linezolid showed the same effect at 16 mg/liter. RBx 11760 inhibited biofilm pro-
duction against methicillin-resistant S. epidermidis (MRSE) ATCC 35984 in a concentration-dependent manner. In a for-
eign-body model, linezolid and rifampin resulted in no advantage over stasis, while the same dose of RBx 11760 demon-
strated a significant killing compared to the initial control against S. aureus (P < 0.05) and MRSE (P < 0.01). The
difference in killing was statistically significant for the lower dose of RBx 11760 (P < 0.05) versus the higher dose of lin-
ezolid (P > 0.05 [not significant]) in a groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis
at 20 mg/kg of body weight, whereas tedizolid showed the same effect at 40 mg/kg. These data support RBx 11760 as a
promising investigational candidate.

B acterial infections due to methicillin-resistant Staphylococcus

aureus (MRSA), methicillin-resistant coagulase-negative Staph-
ylococcus (MRCoNS), and vancomycin-resistant enterococci (VRE)
have emerged as a major public health problem throughout the
world. Over the past few years, there has been a dramatic increase
in the number of cases of MRSA and VRE infections in hospitals
and communities. In 2014, the Agency for Healthcare Research &
Quality reported 23,000 deaths due to MRSA in the United States
alone. The prevalence of Streptococcus pneumoniae strains resis-
tant to -lactam and macrolide antibiotics has increased world-
wide (16). Gram-positive bacterial pathogens, particularly
hospital-acquired MRSA, have become increasingly resistant to
more than one antibiotic, such as vancomycin, trimethoprim-
sulfamethoxazole, -lactams, tetracycline, clindamycin, quino-
lones, and aminoglycosides (7, 8). In addition to drug resistance,
the presence of high-virulence determinants in the pathogens
makes the situation in the hospital and community worse. Ac-
cording to the Centers for Disease Control and Prevention (CDC)
and others, the bacteria causing nosocomial infections are often
associated with biofilms that cause treatment failure and result in
high mortality (9, 10). Although vancomycin is considered the
drug of choice for MRSA treatment, MIC creep of vancomycin
contributed to treatment failure and highlights the need for alter-
ative therapy against MRSA (11, 12). Moreover, coinfection with
VRE and MRSA in hospitalized patients is a concern (13, 14).
Linezolid (Fig. 1A) is the first FDA-approved oxazolidinone
introduced for the treatment of complicated skin and skin struc-
ture infections (cSSSI) due to MRSA and infections associated
with VRE, including bloodstream infection and hospital-acquired
pneumonia. However, linezolid resistance in S. aureus and VRE
has already been observed (15, 16). In addition, linezolid is not
approved for the treatment of patients with catheter site or cath-
eter-related bloodstream infections due to the potential coculture
of Gram-negative infection or infections due to coagulase-nega-
tive Staphylococcus (CoNS) (17). Tedizolid phosphate (Fig. 1B),
an expanded-spectrum oxazolidinone, has 4- to 16-fold more ac-
tivity than linezolid against MRSA and was approved for clinical
use by the FDA in June 2014 (18). However, there were safety and
efficacy concerns for tedizolid in patients with neutropenia (neu-
trophil counts of <1,000 cells/mm3) (18). In an animal model of
infection, the antibacterial activity of tedizolid was reduced in the
Received 29 February 2016 Returned for modification 5 April 2016
Accepted 7 September 2016
Accepted manuscript posted online 19 September 2016
Citation Barman TK, Kumar M, Mathur T, Chaira T, Ramkumar G, Kalia V, Rao M,
Pandya M, Yadav AS, Das B, Upadhyay DJ, Hamidullah, Konwar R, Raj VS, Singh H.
2016. In vitro and in vivo activities of a bi-aryl oxazolidinone, RBx 11760, against
Gram-positive bacteria. Antimicrob Agents Chemother 60:7134 7145.
doi:10.1128/AAC.00453-16.
Address correspondence to V. Samuel Raj, samuelrajv@yahoo.com, or
Harpal Singh, harpal2000@yahoo.com.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.00453-16.
Copyright 2016, American Society for Microbiology. All Rights Reserved.

7134 aac.asm.org Antimicrobial Agents and Chemotherapy December 2016 Volume 60 Number 12
 Barman et al.
FIG 1 Structures of linezolid (A), tedizolid (B), and a novel bi-aryl oxazolidi-
none, RBx 11760 (C).
absence of granulocytes (19). It has been suggested that alternative
therapies should be considered when treating patients with neu-
tropenia and acute bacterial skin and skin structure infections
(ABSSSI) (18, 19).
A series of aryl-oxazolidinone compounds was synthesized and
checked for antibacterial activity by our group (20, 21). RBx 11760
(Fig. 1C) is a bi-aryl oxazolidinone identified in our laboratory.
The objective of the present study was to evaluate RBx 11760 for in
vitro activity against Gram-positive isolates and in vivo efficacy in
a skin and soft-tissue infection model and in foreign-body biofilm
models. The pharmacokinetics (PK) of RBx 11760 was investi-
gated to substantiate the pharmacodynamics (PD) in animal
models. In addition, in vitro transcription/translation assays and
resistance development against linezolid and RBx 11760 by serial
passaging and molecular modeling were performed to confirm the
mechanism of action (MOA).
MATERIALS AND METHODS
Bacterial strains and growth conditions. A total of 378 clinical isolates,
including Staphylococcus aureus (n = 126), Staphylococcus epidermidis
(n = 40), Enterococcus faecalis (n = 63), Enterococcus faecium (n = 55),
and S. pneumoniae (n = 94), were used in this study. These isolates include
American Type Culture Collection (ATCC; Manassas, VA, USA) strains,
and clinical isolates obtained from Indian hospitals were also used in in
vitro screening. For the identification of clinical strains, we subcultured
the strains on growth and selective media. Based on Gram-positive and
-negative identification, we performed biochemical characterization us-
ing the catalase, coagulase test (Gram positive) and IMViC test (Gram
negative) for presumptive identification. Following the biochemical tests,
we confirmed strain identifications using bioMrieuxs API system and
stored them at -80C in 20% glycerol in Trypticase soy broth (TSB)
(Becton, Dickinson and Company, Cockeysville, MD). MRSA Xen-29 was
purchased from Xenogen Corporation, USA (now PerkinElmer). MRSA
Xen-29 showed very good reproducible infection in mouse, so we in-
cluded it in our evaluation system.
Media, reagents, and antimicrobial agents. All media and reagents
were purchased from Becton Dickinson and Company and Sigma-Al-
December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy
drich Co. LLC, USA, respectively, unless otherwise mentioned. Mueller-
Hinton broth (MHB) was used for MIC testing and growing bacteria for
the mouse thigh infection model. TSB was used for biofilm experiments,
and brain heart infusion (BHI) broth was used for growing bacteria for
groin abscess. RBx 11760 and tedizolid were synthesized in-house (18) in
Gurgaon, India. Linezolid was obtained from the National Chemical Lab-
oratory, Pune, India. Vancomycin, rifampin, penicillin, and erythromy-
cin were purchased from commercial sources. For in vitro experiments,
RBx 11760 and tedizolid were prepared in dimethyl sulfoxide (DMSO)
with the final concentration of DMSO being below 1%; rifampin and
erythromycin were prepared in methanol (maximum concentration, 640
11g/ml) and 95% ethanol, respectively. Vancomycin, penicillin, and lin-
ezolid were dissolved in sterile water and then further diluted in medium
to the desired concentrations.
In vitro antimicrobial susceptibility testing. MICs of RBx 11760,
tedizolid, linezolid, and vancomycin were determined in MHB against
378 Gram-positive bacterial isolates using the broth microdilution
method recommended by the Clinical and Laboratory Standards Institute
(CLSI) (22). The MIC50/90 was defined as the MIC for 50% and 90% of the
bacterial isolates. The inoculum and pH effect on the activity of RBx 11760
and standard drugs were also studied using the same broth microdilution
method. In order to determine the effect of plasma proteins on the activity
of RBx 11760, the MIC in the presence of 50% human plasma in MHB was
determined. All experiments were performed in triplicate, and higher
MIC values were considered the final results. The in vitro postantibiotic
effect (PAE) and frequency of resistance were also determined (see the
supplemental material for methods) (22, 23).
Time-kill kinetics. The time-kill kinetics of RBx 11760, tedizolid, and
linezolid against MRSA H-29, MRSE 35984, E. faecalis 427, and S. pneu-
moniae 5051 were determined as described earlier (24). Briefly, 10 ml
cation-adjusted Mueller-Hinton broth (CAMHB) was prewarmed at
37C in reaction tubes, and the drug was added at multiple concentrations
ranging from 1X to 8X MIC. Exponential-phase cultures were diluted to
1 McFarland standard (Densimat; bioMrieux, SA, France) and added to
the reaction tubes to give final inocula of -5 X 106 CFU/ml. For S. pneu-
moniae 5051, tubes containing 10 ml of CAMHB with 5% lysed horse
blood and doubling drug concentrations were inoculated with -5 X
106
CFU/ml, and the tubes were incubated at 35C. The viable cell count was
determined at predefined time points by serial dilution plating onto Tryp-
ticase soy agar (TSA) or blood agar plates for S. pneumoniae 5051. The
time-kill kinetics assays were performed three independent times. Anti-
microbials that reduced the original inoculum by :::3 log10 CFU/ml
(99.9% killing) were considered bactericidal, and they were considered
bacteriostatic if the inoculum was reduced by 0 to <3 log10 CFU/ml. The
reduction of 1 log10 and 2 log10 CFU/ml from original inocula was defined
as 90% and 99% killing.
Effect of RBx 11760 on biofilm-producing and glass-adherent bac-
teria. The effects of RBx 11760, linezolid, and vancomycin on biofilm
production were assessed as described by previously with slight modifica-
tions (25). Briefly, the overnight growth of MRSE ATCC 35984 in TSB was
diluted, adjusted to -106 CFU/ml in TSB supplemented with 2% glucose,
and incubated in polystyrene microtiter flat-bottom 96-well plates. After
4 h of incubation, the bacterial culture was exposed to various concentra-
tions of RBx 11760, linezolid, and vancomycin, ranging from 0.03 to 16
mg/liter. The effects of RBx 11760 and comparators on inhibition of bio-
film formation were assayed after 24 h of exposure at 37C. Wells were
emptied and washed three times with phosphate-buffered saline (PBS)
(pH 7.3). Adherent bacteria were stained with 1% crystal violet for 20 min,
excess stain was rinsed with distilled water, and adherent materials were
solubilized with 200 11l of 0.2 M sodium hydroxide for 1 h at 85C. The
optical density at 544 nm (OD544) was measured. Each experiment was
performed thrice, and the mean percent inhibition and standard devia-
tions were calculated. The relative inhibition of biofilms expressed as
mean percentage was determined by the following formula: percent inhi-
aac.asm.org 7135

bition = 100 - [(OD544 of drug well/OD544 of positive-control well) X
100].
The killing effects of RBx 11760, linezolid, and vancomycin were also
assessed on glass-adherent bacteria as described earlier (21). The adherent
inocula were prepared using sintered glass beads with pore sizes of 60 to
300 11m. The adherent inocula (relative biomass coated on the bead) were
5 X 106 to 5 X 107 CFU/bead. RBx 11760, linezolid, and vancomycin were
prepared from 0.125 to 32 mg/liter in 5 ml TSB in triplicate. Three beads
were tested per concentration. These tested concentrations (0.125 to 32
mg/liter) for RBx 11760, linezolid, and vancomycin were 0.25X to 64X,
0.125X to 32X, and 0.0625X to 16X MIC, respectively, against MRSE
ATCC 35984. Each tube was incubated with one sintered glass bead coated
with adherent bacteria at 37C for 24 h. After incubation, nonadherent
bacteria were washed from the bead and the number of CFU per bead was
determined. The final concentration of each drug required to kill adherent
bacteria was calculated after performing three independent experiments.
The mean log10 killing and standard deviations were calculated for each
concentration.
The mean log10 killing of adherent bacteria was calculated as the geo-
metric mean log10 CFU of untreated beads minus the geometric mean
log10 CFU of drug-treated beads. Three independent experiments were
done to determine the killing concentration of adherent bacteria. The
mean log killing and standard deviations were calculated for each concen-
tration, and the data were presented for 1X to 16X MIC for head-to-head
comparison.
Generation of linezolid-resistant strains. In vitro development of re-
sistance for linezolid was carried out against clinical and ATCC strains of
S. aureus (n = 11) and Enterococcus (n = 22). Staphylococcus and Entero-
coccus strains were cultivated in Mueller-Hinton II broth, containing sub-
MICs and MICs of linezolid on day 1, and were subsequently observed
daily for visual turbidity. Each day, the cultures growing in the highest
drug concentration were repeatedly passaged on the same and next higher
concentration of drug along with a drug-free control for up to 30 passages.
The selected resistant isolates were checked for stability by subculturing
on drug-free medium plates. MICs for linezolid, tedizolid, and RBx 11760
were checked by the broth microdilution method against these resistant
cultures to confirm the development of resistance.
In vitro transcription/translation assay. Cell-free in vitro translation
of the luciferase gene was performed with a bacterial or mammalian tran-
scription/translation system as described earlier (26). The assay was per-
formed in the presence of various concentrations of RBx 11760 and lin-
ezolid using commercially available bacterial (Escherichia coli S30) and a
mammalian (rabbit TNT) kit (Promega, USA) per the manufacturers
protocol. The percent inhibition of luciferase activity by RBx 11760 or
linezolid was used to calculate the 50% inhibitory concentrations (IC50).
Experimental animals. Normal immunocompetent Swiss albino
mice were used for all in vivo efficacy experiments except for the mouse
thigh infection model, wherein neutropenic Swiss albino mice were used.
For pharmacokinetics (PK) studies, normal immunocompetent Swiss al-
bino mice and Wistar rats were utilized. Ethical review of this study was
carried out by the Institutional Animal Ethics Committee (IAEC) of Daii-
chi Sankyo India Pharma Private Limited and Ranbaxy Research Labora-
tories, Gurgaon, India, and the IAEC approved all experimental protocols
for the use of animals. The study was conducted under the strict guidelines
set out by the Committee for the Purpose of Control and Supervision on
Experiments on Animals (CPCSEA), India, for the correct implementa-
tion of animal care and experimentation. Specific-pathogen-free animals
were used for the in vivo studies, and animals were allowed 7 days of
acclimatization before commencing experiments.
Mouse groin abscess model. The in vivo efficacy of RBx 11760 against
MRSA H-29 was determined in the murine groin abscess infection model.
Each experimental group was allocated with 6 animals per group. Animals
were allocated to each group by random selection based on body weight.
The hair was removed from the groin area of the animals using an electric
hair remover (TSE Systems, Germany) a day before infection. During the
7136 aac.asm.org Antimicrobial Agents and Chemotherapy
Activity of RBx 11760 against Gram-Positive Bacteria
development and optimization of mouse groin abscess, we tried inocula
starting from 104, 105, 106, 107, and 108 per mouse. Finally, the optimum
inocula for MRSA H-29 in mouse groin infection was found to be <105 to
107 CFU/mouse. We used 0.6% semisolid nutrient agar, an abscess-pro-
moting agent which helps in development of abscess by activating the host
defense and immune infiltration. The overnight-grown culture of MRSA
H-29 was diluted 1:20 in normal saline and mixed with 0.6% semisolid
nutrient agar at a 1:10 ratio. The bacterial suspension was subcutaneously
injected into the groin area at a volume of 0.5 ml/mouse for a total of
4.5 X 106 CFU per mouse. The formulations of RBx 11760 and linezolid
were prepared in 0.25% (wt/vol) methylcellulose (Sigma-Aldrich Co.
LLC) for in vivo efficacy studies. Treatment with RBx 11760 (10, 20, or 40
mg/kg of body weight) and linezolid (20 or 40 mg/kg) was started 2 h
postinfection and given twice a day orally for 4 consecutive days. Twelve
hours after the last dose, animals were euthanized; the abscess content
along with skin was excised aseptically in 1 ml normal saline and homog-
enized. The resultant homogenate was used for the determination of via-
ble bacterial count.
Foreign-body mouse biofilm infection model. The foreign-body bio-
film infection model in mice was performed as described by others (27,
28). Briefly, overnight-grown culture of S. aureus Xen-29 and MRSE
ATCC 35984 was reinoculated in TSB supplemented with 2%, wt/vol,
glucose (TSBG) and grown for 2 h at 200 rpm at 37C. The turbidity of
cultures was adjusted to an OD600 of 0.25 and further diluted 1:10 in fresh
TSB supplemented with 2% glucose. Fourteen-gauge Teflon intravenous
catheter pieces (Abbocath-T; Vet Supply, Vancouver, Canada) were incu-
bated with culture for 3 h at 37C to form biofilm as described previously
(23). In order to remove the nonadherent bacteria, the catheter pieces
were washed thrice with fresh TSB. The number of bacteria coated per
catheter piece was 1 X 105 to 5 X 105 CFU/catheter. Preanesthetized mice
(0.05-ml mixture of 5 mg/kg xylazine and 100 mg/kg ketamine) were
infected in the flank region (n = 6 mice/group) with two catheters per
animal through a trocar and cannula. The skin opening was closed with
skin adhesive (J & J, USA). Treatment started 3 days postimplant, and
mice were treated with RBx 11760, linezolid, or rifampin at a dose of 40
mg/kg twice a day orally for three consecutive days. At the end of treat-
ment, mice were euthanized and the catheter was surgically removed for
enumeration of bacteria by a conventional viable count method. To re-
move the adherent bacteria, the catheter piece was placed in a tube con-
taining 3 ml PBS, placed in an ultrasonic bath, and sonicated for 5 min,
followed by vortexing for 1 min (27).
Murine pyelonephritis model. In vivo efficacy of RBx 11760 against E.
faecalis 427 was evaluated in the murine pyelonephritis model as de-
scribed earlier (29). Overnight-grown culture of E. faecalis 427 on a TSA
plate was suspended in PBS and adjusted to 0.5 McFarland. This inoculum
was further diluted to get 2 X 106 to 5 X 106 CFU per 0.2 ml. Seven days
prior to infection, mice were given a single 200-11l intravenous dose of 2%
carrageenan (Sigma, USA). Mice (n = 6/group) were infected intrave-
nously with 2 X 106 to 5 X 106 CFU/mouse (0.2 ml/mouse) of E. faecalis
427, and oral treatment was started 2 h postinfection either twice or four
times a day for 2 consecutive days with RBx 11760 (20 mg/kg) and lin-
ezolid (20 mg/kg). Approximately 12 h after the last dose, mice were
sacrificed and their kidneys were excised and homogenized in 1 ml PBS.
The homogenate was diluted and plated on bile esculin agar (BEA; Difco,
BD) to determine the bacterial load. Data from two independent experi-
ments were pooled and analyzed.
Efficacy of RBx 11760, tedizolid, and linezolid in neutropenic mouse
thigh infection. The method described earlier for mouse thigh infection
was followed for this model (30). Mice were rendered neutropenic (neu-
trophil count of <100 cells/mm3) with two intraperitoneal (i.p.) injec-
tions of cyclophosphamide 4 days and 1 day prior to infection at dose rates
of 150 mg and 100 mg per kg. Overnight-grown culture of MRSA 562 on
MHB was adjusted to 0.5 McFarland. The suspension was then diluted
1:100 in fresh MHB, and 100 11l (5 X 105 CFU/thigh) was injected intra-
muscularly in the thigh muscles of the mice (n = 5/group). The bacterial
December 2016 Volume 60 Number 12
 Barman et al.


TABLE 1 Mutation rate of Gram-positive pathogens to RBx 11760, tedizolid, linezolid, and vancomycin
MIC (mg/liter) of g :

RBx 11760 Tedizolid Linezolid Vancomycin

Passage 50% 90% Range 50% 90% Range 50% 90% P val. 50% 90% Range
0 1 2 0.254 0.5 0.5 0.251 2 4 6 1 2 0.54
5 0.25 0.5 0.250.5 0.25 0.5 0.251 2 32 0.2 1 2 0.54
10 1 1 0.51 0.5 0.5 0.251 1 32 0.54 4 8 48
15 2 8 28 2 8 28 16 32 864 ND ND ND
20 0.5 1 0.252 0.5 1 0.252 2 60 18 2 4 14
25 0.5 1 0.252 0.5 1 0.252 2 90 14 32 32 1664
30 4 8 28 ND ND ND 16 128 864 ND ND ND
a
MSSA (n = 54) and MRSA (n = 46).
b
Methicillin-sensitive Staphylococcus epidermidis (MSSE) (n = 11) and MRSE (n = 29).
inocula were confirmed by quantitative culture analyses. Treatment ribosome. MGLTools 1.5.4 was used to prepare the receptor and ligands
started 2 h postinfection, the pretreatment control group was immediately (http://mgltools.scripps.edu). Prior to the docking, three-dimensional
sacrificed, and tissue homogenates of thigh muscles were quantitatively (3D) structures of linezolid and RBx 11760 were generated with the help
cultured to determine the bacterial density in the thigh muscle. RBx 11760 of Marvin Sketch and were subjected to optimization for their geometry
and tedizolid were administered at 10, 20, and 40 mg/kg, and linezolid was using the energy minimization in UCSF Chimera software. All ligands
administered at 80 mg/kg twice a day. RBx 11760 was administered by the were treated as being flexible. Gasteiger Huckel charges were assigned to
oral route, and tedizolid was administered by the i.p. route. RBx 11760 ligands as well as rRNA. The Autogrid 4.2 program of the Autodock suite
and linezolid were suspended in 0.25% methyl cellulose, and tedizolid was (http://autodock.scripps.edu/) was used to generate the grid maps. Grid
prepared in phosphate-buffered saline. dimensions of 90 by 82 by 86, enclosing the reported important residues
PK study. Male Swiss albino mice and Wistar rats were fasted over- involved in inhibitor binding, were constructed in x, y, and z directions,
night and then administered a single dose of RBx 11760 at 10 mg/kg by respectively. The Lamarkian genetic algorithm (LGA) was used to gener-
oral (per os [p.o.] and intravenous (i.v.) routes in mouse and at 5 mg/kg ate 50 conformations of each ligand with the help of Autodock 4.2. Dock-
i.v. and 25 mg/kg p.o. in Wistar rat. Noninfected animals were used in PK ing results were analyzed using MGLTools and UCSF Chimera 1.6.
studies. Sparse blood sampling in mouse (use of multiple animals to gen- Statistical analysis. All data were analyzed using GraphPad Prism
erate a complete PK) and serial blood sampling in rat were carried out at (version 5.03; GraphPad Software, San Diego, CA). The statistical signif-
different time points up to 24 h postdose. Plasma was harvested by cen- icance of treated (RBx 11760 and comparator antimicrobial agents) and
trifugation of blood and kept at -80C until analysis. RBx 11760 concen- untreated control (preinfection control or postinfection, untreated con-
trations in plasma samples were determined using liquid chromatogra- trol) were calculated by nonparametric Mann-Whitney analysis. The sta-
phy-tandem mass spectrometry (LC-MS/MS). RBx 11760 was extracted tistical significance of killing of adherent bacteria and percent inhibition
from plasma samples by acetonitrile precipitation. Briefly, 50 11l Milli- were determined and data were compared using one-way analysis of vari-
Q-grade water was added to 20 11l of plasma samples. To this, 50 11l ance (ANOVA) with Dunnetts multiple-comparison posttest. A P value
acetonitrile and 350 11l internal standard solution (1 11M niflumic acid in of <0.05 was considered statistically significant. The bar chart and scatter
acetonitrile) were added. The contents were mixed with 96-well shakers line graph (for kill kinetics) data, presented as means SD (standard
and filtered through a Captiva 96-well polypropylene filter plate. The deviations), were calculated using Microsoft Office Excel. The pharmaco-
supernatant (8 11l) was injected into LC-MS/MS (AB Sciex API 4000 with kinetics data were analyzed using the NCA module of WinNonlin profes-
electrospray ionization-MS probe kept at 450C) coupled with an Acquity sional software (version 4.1). The pharmacokinetic parameters, i.e., max-
ultraperformance LC (UPLC; Waters) system in gradient mobile flow. A imum concentration of drug in serum (Cmax), time to maximum
Sunniest HT C18, 50- by 2.1-mm, 2-11m column was used at a flow rate of concentration of drug in serum (Tmax), plasma clearance (CLp), volume
0.8 ml/min. The mobile phase consisted of (i) 100 mM ammonium ace- of distribution at steady state (Vss), half-life (t1/2), and area under the
tate-acetonitrile-formic acid (50:950:2) and (ii) 100 mM ammonium ac- concentration-time curve (AUC), were determined. Percent bioavail-
etate-Milli-Q-grade water-acetonitrile-formic acid (50:900:50:2). The se- ability (F) was calculated as [(AUCp.o. X dosei.v.) X 100]/[(AUCi.v. X
lected precursor and product ion for RBx 11760 were m/z 398.2 and 268. dosep.o.)].
Analyst software, version 1.4, was used to process the sample in quantita-
tion mode using a (1/x) weighted linear regression analysis. RESULTS
Structural modeling studies for interaction with ribosome. The In vitro antimicrobial susceptibility testing. RBx 11760 showed
crystal structure of the E. coli 50S ribosome (PDB entry 2AW4) was used
in vitro antibacterial activity against S. aureus, S. epidermidis, E.
for molecular docking of linezolid and RBx 11760 (31). The proposed
structural effects of the ribosomal mutations in this study were inferred by faecalis, E. faecium, and S. pneumoniae. The MIC50s, MIC90s, and
using the coordinates of the Deinococcus radiodurans and Haloarcula MIC ranges against these pathogens are presented in Table 1.
marismortui linezolid-bound 50S ribosomal subunit crystal structures Linezolid-resistant (LZDr) mutants of S. aureus and enterococci
(PDB entries 3DLL and 3CPW) (26). We first validated the docking sim- were generated from wild-type strains after 30 passages in lin-
ulation by reproducing the reported binding mode of linezolid in the ezolid-containing growth medium. MIC50s and MIC90s of RBx

December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy aac.asm.org 7137

FIG 2 Time-kill kinetics study of RBx 11760, tedizolid, and linezolid against MRSA H-29 (A), MRSE ATCC 35984 (B), E. faecalis 427 (C), and S. pneumoniae
5051 (D). Graphs represent means from three independent experiments standard deviations (SD).
11760 were 4- to 8-fold lower than those of linezolid against LZD r
strains of S. aureus and enterococci. The MIC90s of RBx 11760 and
linezolid against laboratory-generated LZDr S. aureus (n = 11) as
well as Enterococcus (n = 22) strains were 8 and 32 mg/liter, re-
spectively. RBx 11760 also showed in vitro activity against VISA
strains. MIC ranges of RBx 11760 and vancomycin against VISA
(n = 15) strains were 0.5 to 1 and 4 to 8 mg/liter, respectively. RBx
11760 did not show inoculum effects at 105 (low) and 107 (high)
inocula; however, linezolid and vancomycin showed moderate in-
creases in MIC values at high inocula of 107 CFU/ml (see Table S1
in the supplemental material). The pH effect study of RBx 11760
and linezolid showed minimal variation in MIC at an inoculum of
105 CFU/ml across a medium pH range of 5.2 to 8.7 against S.
aureus or Enterococcus strains. However, vancomycin showed 2-
to 4-fold variation in MIC under the same experimental condi-
tions, and erythromycin showed high MIC at low pH even at a low
inoculum of 105 CFU/ml (see Table S2 in the supplemental mate-
rial). RBx 11760 and linezolid displayed equipotent in vitro activ-
ity in the presence of 50% human plasma (see Table S3). RBx
11760 showed 3-h PAE at 4X MIC (4 mg/liter), while linezolid
showed the same effect at 8X MIC (16 mg/liter) against MRSA
H-29 (see the supplemental material for methods). RBx 11760 (4
mg/liter) exhibited PAE at 3.2 h against MRSE, whereas linezolid
(8 mg/liter) showed PAE at 2.5 h at 8X MIC (see Table S4).
RBx 11760, tedizolid, and linezolid showed very low frequen-
cies of resistance, i.e., <3 X 10-10 against MRSA 562 at 4X
MIC, and the positive control, rifampin, showed high
frequencies of resistance of 1.1 X 10-7 and 2.5 X 10-7 at
4X and 8X MIC, respectively (see Table S5 and methods in the
supplemental ma- terial).
7138 aac.asm.org Antimicrobial Agents and Chemotherapy
Activity of RBx 11760 against Gram-Positive Bacteria
Time-kill kinetics. The time-kill kinetics studies of RBx 11760,
tedizolid, and linezolid were performed against MRSA H-29,
MRSE ATCC 35984, E. faecalis 427, and S. pneumoniae 5051. The
results from three independent experiments are summarized in
Fig. 2A to D. All three oxazolidinones exhibited bacteriostatic ef-
fect against MRSA, MRSE, and E. faecalis but not S. pneumoniae.
RBx 11760 showed 2-log10 kill at 4X MIC, while tedizolid and
linezolid showed 2-log10 and 1.4-log10 kills, respectively, at 16X
MIC (P < 0.05) against MRSA H-29. Against S. pneumoniae 5051,
RBx 11760 showed cidal potential with 4.6-log10 kill at 4X MIC,
compared to a 2.42-log10 and 1.95-log10 reduction for tedizolid
and linezolid at 16X MIC as early as 6 h (P < 0.05). Similarly, RBx
11760, linezolid, and tedizolid showed 2.4-log10, 2.1-log10, and
2.0-log10 kills at 16X MIC against MRSE ATCC 35984. However,
they did not show significant bacterial killing beginning at 0 h at
any concentration against E. faecalis 427 (P > 0.05), indicating
stasis.
Effect of RBx 11760 on biofilm-producing and glass-adher-
ent bacteria. The effect of RBx 11760 on the biofilms producing
MRSE ATCC 35984 is presented in Fig. 3A. RBx 11760 inhibited
biofilm production in a concentration-dependent manner and
exhibited a better effect than linezolid and vancomycin at 4X (P <
0.01) and 8X MIC (P < 0.001). RBx 11760 displayed activity
similar to that of vancomycin and linezolid at 1X MIC and 2X
MIC against glass-adherent MRSE ATCC 35984; however, at 4X,
8X, and 16X MIC, it showed significantly better inhibition of
glass-adherent bacteria than linezolid and vancomycin (Fig. 3B).
In vitro transcription/translation assay. The sub-MIC level of
RBx 11760 resulted in selective inhibition of protein synthesis (see
Fig. S1A and methods in the supplemental material). RBx 11760
December 2016 Volume 60 Number 12
 Barman et al.
FIG 3 Percent inhibitory effect of RBx 11760, linezolid, and vancomycin on biofilm production (A) and killing effect of RBx 11760, linezolid, and vancomycin
on glass-adherent bacteria (mean log10 kill of glass-adherent bacteria SD) (B) when drug exposure was initiated at 4 h postinoculation of MRSE ATCC 35984.
The statistical significance of the indicated P values was determined as <0.05 (*), <0.01 (**), <0.001 (***), and >0.05 (not significant [ns]).
and linezolid specifically inhibited bacterial protein synthesis, and
we determined their IC50s against bacterial (E. coli S30) and mam-
malian (rabbit TNT) protein synthesis. The IC50s of RBx 11760
and linezolid against bacterial and mammalian protein synthesis
and the safety window are presented in Table 2.
Mouse groin abscess model. In the mouse groin abscess
model, the mean (SD) log10 bacterial count of the untreated
control (96 h) mice was 7.60 0.37, which was an increase from
the value for the untreated control group infected for 2 h (termed
the 2-h control), 6.39 0.39 (Fig. 4A). The treatment with lin-
ezolid at 20 and 40 mg/kg achieved a static effect, while RBx 11760
treatment showed 1-log10 killing for the 2-h control even at 10
mg/kg. Moreover, treatment with RBx 11760 at 20 and 40 mg/kg
resulted in significant decreases in bacterial loads of 1.36 and 1.76
log10 CFU/groin abscess tissue, respectively (Fig. 4A). The simu-
lated concentration-time profile of RBx 11760 at 10 mg/kg, twice
a day by oral dose for 4 days, yielded an AUC 0 24 of 66.9 11g h/ml
and an AUC/MIC ratio (MIC of MRSA H-29, 1 mg/liter) of 66.9
(see Fig. S2 in the supplemental material).
We have also investigated the efficacy of RBx 11760 against a
Panton valentine leukocidin (PVL)-positive community-ac-
quired MRSA (CA-MRSA) strain. The bacterial load recovered at
the beginning of treatment at 2 h after infection was 6.22 log10,
which increased to 8.28 log10. RBx 11760 showed significant re-
duction of 1.63 log10 compared to the 2-h control (P < 0.01), and
linezolid showed a reduction of 0.503 compared to the 2-h control
(P > 0.05). However, compared to the 96-h late control, RBx
TABLE 2 IC50s of RBx 11760 and linezolid against bacterial and
mammalian ribosomes in a cell-free in vitro transcription/translation
system
a Safety window
IC 50 (11g/ml)
(mammalian IC50/
Compound Bacterial Mammalian bacterial IC50)
RBx 11760 7.94 3.97 6,476.96 2,483.5 815.7
Linezolid 47.68 10.33 5,125.94 437.09 107.5
a
Values are means SD from three independent experiments.
December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy
11760 and linezolid showed reductions of 3.67 log10 and 2.56
log10, respectively. RBx 11760 also showed less abscess formation
even at half the dose of linezolid (see Fig. S3A to D in the supple-
mental material).
Foreign-body mouse biofilm infection model. The mean bac-
terial log10 count of S. aureus Xen-29 and MRSE ATCC 35984 on
each implanted catheter was 5.57 0.38 and 6.61 0.20 when the
treatment was started on day 3. After 6 days postinfection, average
log10 CFU counts of 7.48 0.37 and 9.08 0.46 were recovered
from S. aureus Xen-29- and MRSE ATCC 35984-coated catheters,
respectively, from untreated control animals. Treatment with lin-
ezolid and rifampin at 40 mg/kg, twice a day for 3 days, resulted in
no advantage over stasis, while the same dose of RBx 11760 dem-
onstrated a significant killing (*, P < 0.05) of approximately
1-log10 CFU/catheter from the initial bacterial load (precontrol
day 3 group) against both organisms (Fig. 4B and C).
Murine pyelonephritis model. In the pyelonephritis model,
RBx 11760 and linezolid showed comparable efficacy (Fig. 4D). At
20 mg/kg, twice-daily treatment with both RBx 11760 and lin-
ezolid did not show any significant reduction in bacterial count
compared to the initial 2-h control group. In contrast, treatment
with RBx 11760 at 20 mg/kg four times a day yielded a 0.5-log10
reduction from value for the initial control while linezolid resulted
in stasis (Fig. 4D). Compared to 48-h postcontrol RBx 11760 at 20
mg/kg four times a day, a 3.82-log10 reduction (P < 0.05) was seen
compared to a 3.27-log10 reduction for linezolid (P < 0.05). When
results were statistically compared using Mann-Whitney U test,
the groups did not differ significantly (P > 0.05), indicating com-
parable efficacy.
Efficacy of RBx 11760, tedizolid, and linezolid in neutropenic
mouse thigh infection. In a neutropenic mouse thigh infection
model, the mean (SD) log10 bacterial count for mice was 6.03
0.26, which increased to 8.98 0.38 at 26 h postinfection. RBx 11760
showed static effect at half the dose, i.e., 20 mg/kg, compared to tedi-
zolid, which showed stasis at 40 mg/kg in this experiment. On the
contrary, linezolid displayed a 0.53-log10 increase in bacterial count
compared to the 2-h controls even at 80 mg/kg (Fig. 5).
aac.asm.org 7139
 Activity of RBx 11760 against Gram-Positive Bacteria

FIG 4 (A) In vivo efficacy of RBx 11760 and standard drugs against MRSA H-29 in murine groin abscess model. Treatment was started 2 h postinfection and
administered twice a day (BID) for 4 consecutive days. All treated groups were compared to the 2-h control. (B and C) In vivo efficacy of RBx 11760 and standard
drugs against MRSA Xen-29 (B) and MRSE ATCC 35984 (C) in a murine foreign-body biofilm infection model. Treatment was started 3 days postinfection and
administered at 40 mg/kg twice a day for 3 consecutive days. All treated groups were compared with precontrol (3 days) and postcontrol (6 days) values. (D) In
vivo efficacy of RBx 11760 and standard drugs against E. faecalis 427 in a murine pyelonephritis model. Treatment was started 2 h postinfection and administered
at 20 mg/kg BID or four times a day (QID) for 2 consecutive days. Each bar represents the means standard deviations for 6 mice. For statistical analysis, all
treated groups were compared to the untreated controls infected for 2 h or 48 h. The statistical significance of the indicated P values was determined as <0.05 (*),
<0.01 (**), <0.001 (***), and >0.05 (ns). BID, twice daily; QID, four times a day.


Pharmacokinetic studies. RBx 11760 showed low plasma G2505 (Fig. 6B); however, a few other conformations (with
clearance of 11.5 and 3.6 ml/min/kg with Vss of 0.74 and 0.4 li- slightly less binding energy) from the same cluster showed H
ters/kg in mouse and rat, respectively (Table 3). Long terminal bonds with 5= orientation oxygen of U2504 (Fig. 6D). The binding
half-lives of 9.3 h and 3.2 h in mouse and rat, respectively, suggest energy of RBx 11760 (-8.80 kcal/mol) was found to be more than
long duration of action. RBx 11760 showed 60% and 72% oral that of linezolid (-6.62 kcal/mol). The preferred and top-ranked
bioavailability in mouse and rat, respectively, following adminis- conformation indicates the H-bonding interaction with ribose
tration of suspension formulation. sugar moiety U2584.
Structural modeling studies for interaction with ribosome.
Initial docking of linezolid produced conformation similar to that DISCUSSION
of the previously reported binding mode (25). As reported for the The emergence of multidrug resistance in Gram-positive bacteria
crystal structure, nucleotide residues G2061, A2451, A2503, has dramatically limited the therapeutic options for hospital- as
U2504, G2505, and U2506 are important residues which are in- well as community-acquired infections. Despite extensive antimi-
volved in interaction with linezolid (Fig. 6A). Further, docking of crobial research in the last 50 years, the drugs used for hospital-
RBx 11760 in the active site of the 50S ribosomal subunit revealed acquired infections are still limited (32, 33). In the present study,
a binding conformation similar to that of linezolid (Fig. 6C and 7). antibacterial activity of RBx 11760, a bi-aryl oxazolidinone, was
We found that a few selected top conformations of RBx 11760 evaluated against a panel of Gram-positive bacteria. The MIC 50s
make an H bond with ribose moiety of U2584 and OP2 oxygen of and MIC90s of RBx 11760 were significantly lower than those of

7140 aac.asm.org Antimicrobial Agents and Chemotherapy December 2016 Volume 60 Number 12
 Barman et al.
FIG 5 Comparative efficacy of RBx 11760, tedizolid, and linezolid against
MRSA-562 in neutropenic mouse thigh infection model. Treatment was
started 2 h postinfection and administered twice a day for 1 day. Each bar
represents the means standard deviations for 6 mice. For statistical analysis,
all treated groups were compared to the untreated control group that was
infected for 2 h. The statistical significance of the indicated P values was deter-
mined as <0.05 (*) and >0.05 (ns).
linezolid against S. aureus (2-fold), S. epidermidis (4- to 8-fold),
Enterococcus (4-fold), S. pneumoniae (4- to 8-fold), and LZD r
strains of S. aureus and Enterococcus (4- to 8-fold), indicating im-
proved activity of RBx 11760 against methicillin-susceptible S.
aureus (MSSA), MRSA, MRSE, Enterococcus, S. pneumoniae, VISA,
and VRE. RBx 11760 displayed comparatively potent activity
against MSSA, MRSA, E. faecalis, and E. faecium even at higher
inoculum levels. When checked at different pH levels, RBx 11760
and linezolid did not show pH effect on the activity; however,
vancomycin and erythromycin did show pH effect on activity.
RBx 11760 showed more potent activity against S. pneumoniae
than that of tedizolid and linezolid. RBx 11760 and linezolid dis-
played no shift in MIC in the presence of human plasma against
staphylococci. RBx 11760 maintained a balanced and potent an-
tibacterial profile and demonstrated significant advantages over
linezolid against a variety of antimicrobial-resistant strains.
In order to assess the killing potential of RBx 11760, the time-
kill kinetics studies were performed against MRSA, MRSE, E.
faecalis, and S. pneumoniae, owing to their importance in hospital-
acquired infections. RBx 11760, tedizolid, and linezolid exhibited
bacteriostatic activity, except that 4X MIC of RBx 11760 and 16X
MIC of tedizolid and linezolid against MRSA H-29 at 8 h exhibited
an -2-log10 kill. RBx 11760 showed better killing potential at 4X
under in vivo conditions; therefore, it is presumed that the results
obtained are clinically relevant. However, in vivo PAE, which is
longer than in vitro PAE, needs to be generated in animal models
before making a conclusive determination of its effect on dosing
regimens (34, 35).
RBx 11760, tedizolid, and linezolid displayed a very low fre-
quency of resistance against MRSA 562, while the positive control,
rifampin, showed a high frequency of resistance at 4X and 8X
MIC. These data suggest that there is little chance for spontaneous
resistance development to the oxazolidinone class of compounds,
including RBx 11760 (36). However, in our serial passage study for
the potential for resistance development, linezolid in an S. aureus
mutant generated MICs of 16 to 32 mg/liter, whereas the same
analyses performed with RBx 11760 yielded mutants with MICs of
2 to 8 mg/liter. This indicated that the lower cross-resistance po-
tentials of RB11760 than those of linezolid biofilm-mediated ad-
herence and bacterial biofilms are critical factors in the pathogen-
esis of S. epidermidis infections of medical devices (37, 38). RBx
11760 showed significant reduction in biofilm production by
MRSE ATCC 35984 compared to linezolid and vancomycin (P <
0.05). RBx 11760 not only displayed potent biofilm inhibitory
activity but also exhibited superior activity against glass-adherent
bacteria (P < 0.01). This is one of the unique characteristics of
RBx 11760 compared to linezolid.
The effect of RBx 11760 on macromolecular synthesis inhibi-
tion of MRSA H-29 exhibited that the sub-MIC level of RBx 11760
resulted in selective inhibition of protein synthesis, while all other
macromolecular syntheses, such as DNA, RNA, cell wall, and lipid
syntheses, were almost unaffected (see Fig. S1 and methods in the
supplemental material). Therefore, the inhibitory target of RBx
11760 is bacterial protein biosynthesis. In addition, RBx 11760
and linezolid act specifically on bacterial protein synthesis and
RBx 11760 showed significantly better IC50s than linezolid against
bacterial protein synthesis, while RBx 11760 displayed very poor
activity against mammalian protein synthesis. Therefore, RBx 11760
may show comparatively better safety profiles against mammalian
protein synthesis.
Three types of resistance mechanisms, including mutations in
the domain V region of the 23S rRNA gene, acquisition of the
ribosomal methyltransferase gene cfr, and mutations in the genes
encoding 50S ribosomal proteins, have been reported against oxa-
zolidinones (39). In this study, we have shown through serial pas-
TABLE 3 Pharmacokinetic study of RBx 11760 in Swiss albino mouse
and Wistar rat by oral and intravenous routes
Value fora :

MIC than tedizolid and linezolid at the same concentration Mouse Rat
against S. pneumoniae 5051. PAE is an important pharmacody-
namic parameter that can influence the dosing regimens of an 10 mg/kg 10 mg/kg 5 mg/kg 25 mg/kg
antibiotic (20). In PAE experiments, RBx 11760 showed pro-
Parameter i.v. p.o. i.v. p.o.

longed PAE against MRSA and MRSE compared to linezolid at the Cmax (11g/ml) 22.4 4.50 15.5 2.8 6.6 1.3
same concentration. However, RBx 11760 and linezolid showed Tmax (h)
0.5
2.7 1.2
comparable PAE against E. faecalis. The long PAE of aminoglyco-
AUC024 (11g h/ml) 14.4 8.6
23.8 4.8 86.1 34.7

sides are thought to contribute to clinical efficacy with once-daily

t1/2 (h) 9.3
3.2 1.1
dosing (34). Linezolid exhibits moderate concentration-depen-
CLP (ml/min/kg) 11.5
3.6 0.8
dent in vitro PAE against Gram-positive pathogens, supporting its
Vss (liter/kg) 0.74 0.4 0.1
MRTb (h) 1.1 2.0 0.1
twice-daily dosing in humans. The long PAE of RBx 11760 could F (%) 60 72
be a valuable parameter in human clinical settings. The concen- a
Values are presented either as means or means SD for n = 3 animals.
trations selected for in vitro PAE determination are achievable b
MRT, mean residence time.

December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy aac.asm.org 7141
 Activity of RBx 11760 against Gram-Positive Bacteria

FIG 6 Interaction of linezolid and RBx 11760 with active-site residues of 50S ribosomes of E. coli. Overlay of docked conformation (yellow) and cocrystal
conformation (magenta) of linezolid (A) and RBx 11760 (green) (B) into active sites of 50S ribosomes of E. coli. Merged conformation of linezolid with RBx 11760
(C) and other low-scoring conformations of RBx 11760 in different orientations (D) within the active site of 50S ribosomes of E. coli. H bonds are shown by dotted
green lines.


sage experiments of linezolid selection that the MIC of linezolid
increased 32-fold (2 to 64 mg/liter) compared to 4-fold (1 to 4
mg/liter) for RBx 11760 (see Table S6 and methods in the supple-
mental material). The concentration of 4 mg/liter is not very high,
and this concentration of RBx 11760 can easily be maintained in
human plasma to reach clinical efficacy against linezolid-resistant
strains by optimization of pharmacodynamic parameters. How-
ever, in RBx 11760 selection, the MIC of both RBx 11760 and
linezolid increased to 64 mg/liter. This indicated that the fre-
quency of a linezolid selection by a mutant was greater than that of
RBx 11760 selection, which is in agreement with another report
(40). In mutant sequence analysis (see Table S6), domain V 23S
rRNA gene mutations revealed a novel mutant, T2374C, in rrn 6
in addition to the already reported mutations. The mutation
G2576T in linezolid selection mutants observed in this study was

FIG 7 Surface image of docked conformation of linezolid and RBx 11760 in also reported to be the common mechanism for linezolid resis-
the same active site of 50S ribosomes of E. coli. tance through 23S rRNA, with 63.5% frequency (15). This mutant

7142 aac.asm.org Antimicrobial Agents and Chemotherapy December 2016 Volume 60 Number 12
 Barman et al.
study is a preliminary one, and detailed studies in the future will
help to understand the fitness of RBx 11760 resistance reported in
this study.
We evaluated the in vivo efficacy of RBx 11760 in a reproduc-
ible murine groin abscess model caused by MRSA, which resem-
bles human localized skin and soft-tissue infections. The infection
was maintained throughout the 4 days of the experiment in un-
treated control groups and also showed groin abscess formation
similar to clinical abscess formation. In the groin abscess model,
the bacterial count of the untreated control increased from that of
precontrol animals and was maintained until day 4. This increase
of bacterial load was also substantiated by the gross pathology of
the abscess. RBx 11760 showed killing potential at half the dose of
linezolid. This indicated that RBx 11760 was more efficacious than
linezolid against skin and soft-tissue infection, and it showed ex-
cellent activity in a dose range study. The in vivo efficacy of RBx
11760 was AUC 0 24 driven in the mouse model (data not shown).
Linezolid has also been evaluated in patients who have bacteremia,
complicated skin and skin structure infections, and pneumonia
that is associated with MRSA or VRE. The time the concentration
of an antibiotic remains above the MIC (t>MIC) and AUC 0 24/
MIC ratios are reported to be highly correlated with cure. As re-
ported previously, the static effect of linezolid against S. aureus
was achieved at a mean 24-h AUC/MIC ratio of 83, which was
almost equivalent to the finding of human clinical trials of a mean
24-h AUC/MIC ratio of 110 (30, 4143). For the recently FDA-
approved tedizolid phosphate, it was reported that the pharmaco-
dynamic index most closely linked with efficacy was AUC over 24
h divided by the MIC in the mouse thigh infection model of MRSA
(40). PVL is a cytotoxin produced by some strains of Staphylococ-
cus which is associated with severe virulence and necrosis of skin
and soft tissues and necrotic pneumonia. They are commonly
associated with community-acquired MRSA (CA-MRSA). In
order to determine the spectrum of in vivo efficacy of RBx
11760, we investigated the efficacy against an PVL-positive
MRSA strain which causes severe necrosis. We observed that
even at half the dose, treatment with RBx 11760 showed signif-
icant bacterial count reduction from the initial 2-h control
(P < 0.001) compared to linezolid treatment. In addition, no
abscess formation was observed in RBx 11760-treated animals,
whereas significant abscess formation and gross tissue damage
were observed in the untreated control mice followed by mild
abscess formation in the linezolid-treated group (see Fig. S3 in the
supplemental material).
In the foreign-body mouse biofilm infection model, the level
of bacteria recovered from the implant catheters of postcontrol
animals after 6 days was significantly higher than that recov-
ered from the catheters of precontrol animals (3 days postin-
fection at the time of first dose). In the implanted catheter
experiments, we observed that there was a significant advan-
tage for RBx 11760. The treatment of linezolid and rifampin at
40 mg/kg twice a day for 3 days resulted in no improvement
over stasis, while the same dose of RBx 11760 demonstrated a
significant killing of approximately 1 log10 CFU/catheter from
the initial bacterial load against both organisms. Moreover, the
recovered bacteria from treated animals were also checked for
drug susceptibility. There was no change in MIC in the case of
RBx 11760. In contrast, MRSE 35984, recovered from the lin-
ezolid- and rifampin-treated group, showed 2- and 8-fold
higher MICs, respectively. These results demonstrated that
December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy
while only two organisms were studied and more studies are
needed, the relative in vitro performance of RBx 11760 was
recapitulated in vivo. Linezolid resistance was reported in
Gram-positive organisms mainly due to biofilm formation in
blood and catheters (44). However, it will be interesting to
observe the antibiofilm activities of RBx 11760 in the clinic.
The increasing incidence of complicated urinary tract infec-
tions (cUTIs) in hospitals due to drug resistance of Staphylococcus
and Enterococcus at present is a matter of concern. Oxazolidi-
nones active against Gram-positive bacteria may be considered
for use in the treatment of cUTIs because of the presence of
Gram-positive uropathogens (45, 46). In a mouse pyelonephri-
tis infection model, RBx 11760 and linezolid showed compara-
ble efficacy, resulting in stasis when administered at 20 mg/kg
four times a day.
The low plasma clearance suggests good plasma stability of
RBx 11760. Low to moderate volumes of distribution suggest
that RBx 11760 is less distributed in organs and therefore is less
likely to cause organ toxicity. Long terminal half-lives in mouse
and rat, respectively, suggests long duration of action. Both
long PAE and long terminal half-lives of RBx 11760 contrib-
uted to the prolonged activity of the drug. The long half-lives
delayed the initiation of the PAE and prolonged the activity of
the drug because it was cleared more slowly. High oral bioavail-
ability of 60% and 72% was observed in mouse and rat, respec-
tively, following administration of suspension formulation,
supporting further development of RBx 11760. Pharmacoki-
netics in higher species would help to understand the molecule
better.
In a molecular modeling study, RBx 11760 and linezolid occu-
pied almost identical orientations in the active site of the 50S ri-
bosome of E. coli. However, the formation of additional van der
Waals interactions and H bonding by RBx 11760 contributed to
better stability and strong interactions in the active site compared
to those of linezolid, which may provide an explanation for its
better antibacterial activity than that of linezolid.
In conclusion, RBx 11760 demonstrated better in vitro activity
against the clinically relevant bacterial isolates by exhibiting 2- to
4-fold lower MICs than linezolid and MICs comparable to those
of tedizolid. Pharmacokinetics of RBx 11760 exhibited good oral
bioavailability with low plasma clearance and low to moderate
volumes of distribution in mouse and rat. RBx 11760 showed
superior activity against biofilm-producing bacteria in vitro
and also translated the activity in a foreign-body-associated
mouse biofilm model. This is one of the unique characteristics
of RBx 11760 compared to linezolid (44). RBx 11760 showed a
1-log10 kill from initial control at a 4-fold lower dose than that
of linezolid in a mouse groin abscess model. In neutropenic
mouse thigh infection, RBx 11760 showed static effect at half
the dose of tedizolid. These data indicate that RBx 11760 can be
explored as a new therapeutic agent against Gram-positive bac-
terial infections.
ACKNOWLEDGMENTS
We thank Kenji Namba and Nobuhisa Masuda (Daiichi Sankyo India
Pharma Private Limited, Gurgaon, India) for helpful discussion, support,
and suggestions during the preparation of the manuscript.
We have no conflicts of interest to declare.
aac.asm.org 7143

FUNDING INFORMATION
This work, including the efforts of Tarani Kanta Barman, was funded by
Daiichi Sankyo India Pharma Private Limited.
This study was supported by Daiichi Sankyo India Pharma Private Lim-
ited (DSIN), Gurgaon, India. The funder had no role in study design, data
collection and interpretation, or the decision to submit the work for pub-
lication.
REFERENCES
1. Kavanagh KT, Calderon LE, Saman DM, Abusalem SK. 2014. The use of
surveillance and preventative measures for methicillin-resistant Staphylo-
coccus aureus infections in surgical patients. Antimicrob Resist Infect Con-
trol 3:18. http://dx.doi.org/10.1186/2047-2994-3-18.
2. Van Bambeke F, Reinert RR, Appelbaum PC, Tulkens PM, Peetermans
WE. 2007. Multidrug-resistant Streptococcus pneumoniae infections: cur-
rent and future therapeutic options. Drugs 67:23552382. http://dx.doi
.org/10.2165/00003495-200767160-00005.
3. MacKenzie FM, Bruce J, Struelens MJ, Goossens H, Mollison J, Gould
IM, ARPAC Steering Group. 2007. Antimicrobial drug use and infection
control practices associated with the prevalence of methicillin-resistant
Staphylococcus aureus in European hospitals. Clin Microbiol Infect 13:
269 276. http://dx.doi.org/10.1111/j.1469-0691.2006.01592.x.
4. Chua K, Laurent F, Coombs G, Grayson ML, Howden BP. 2011.
Antimicrobial resistance: not community-associated methicillin-resistant
Staphylococcus aureus (CA-MRSA)! A clinicians guide to community
MRSAits evolving antimicrobial resistance and implications for therapy.
Clin Infect Dis 52:99 114. http://dx.doi.org/10.1093/cid/ciq067.
5. Widerstrom M, Wistrom J, Sjostedt A, Monsen T. 2012. Coagulase-
negative staphylococci: update on the molecular epidemiology and clinical
presentation, with a focus on Staphylococcus epidermidis and Staphylococ-
cus saprophyticus. Eur J Clin Microbiol Infect Dis 31:720. http://dx.doi
.org/10.1007/s10096-011-1270-6.
6. Leavis HL, Willems RJ, Top J, Spalburg E, Mascini EM, Fluit AC,
Hoepelman A, de Neeling AJ, Bonten MJ. 2003. Epidemic and nonepi-
demic multidrug-resistant Enterococcus faecium. Emerg Infect Dis
9:1108 1115. http://dx.doi.org/10.3201/eid0909.020383.
7. Bozdogan B, Esel D, Whitener C, Browne FA, Appelbaum PC. 2003.
Antibacterial susceptibility of a vancomycin-resistant Staphylococcus au-
reus strain isolated at the Hershey Medical Center. J Antimicrob Che-
mother 52:864 868. http://dx.doi.org/10.1093/jac/dkg457.
8. Rice LB. 2006. Antimicrobial resistance in Gram positive bacteria. Am J
Med 119:S11S19. http://dx.doi.org/10.1016/j.amjmed.2006.03.012.
9. Donlan RM. 2001. Biofilms and device-associated infections. Emerg In-
fect Dis 7:277281.
10. Loffler CA, MacDougall C. 2007. Update on prevalence and treatment of
methicillin-resistant Staphylococcus aureus infections. Exp Rev Anti Infect
Ther 5:961981. http://dx.doi.org/10.1586/14787210.5.6.961.
11. Steinkraus G, White R, Friedrich L. 2007. Vancomycin MIC creep in
non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomy-
cin susceptible clinical methicillin-resistant S. aureus (MRSA) blood iso-
lates from 2001 05. J Antimicrob Chemother 60:788 794. http://dx.doi
.org/10.1093/jac/dkm258.
12. Sader HS, Fey PD, Fish DN, Limaye AP, Pankey G, Rahal J, Rybak MJ,
Snydman DR, Steed LL, Waites K, Jones RN. 2009. Evaluation of
vancomycin and daptomycin potency trends (MIC creep) against methi-
cillin-resistant Staphylococcus aureus isolates collected in nine U.S. medi-
cal centers from 2002 to 2006. Antimicrob Agents Chemother 53:4127
4132. http://dx.doi.org/10.1128/AAC.00616-09.
13. Warren DK, Nitin A, Hill C, Fraser VJ, Kollef MH. 2004. Occurrence of
co-colonization or co-infection with vancomycin-resistant enterococci
and methicillin-resistant Staphylococcus aureus in a medical intensive care
unit. Infect Control Hosp Epidemiol 25:99 104. http://dx.doi.org/10
.1086/502357.
14. Zhu W, Murray PR, Huskins WC, Jernigan JA, McDonald LC, Clark
NC, Anderson KF, McDougal LK, Hageman JC, Olsen-Rasmussen M,
Frace M, Alangaden GJ, Chenoweth C, Zervos MJ, Robinson-Dunn B,
Schreckenberger PC, Reller LB, Rudrik JT, Patel JB. 2010. Dissemina-
tion of an Enterococcus Inc18-like vanA plasmid, associated with vanco-
mycin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 54:
4314 4320. http://dx.doi.org/10.1128/AAC.00185-10.
15. Wilson P, Andrews JA, Charlesworth R, Walesby R, Singer M, Farrell
7144 aac.asm.org Antimicrobial Agents and Chemotherapy
Activity of RBx 11760 against Gram-Positive Bacteria
DJ, Robbins M. 2003. Linezolid resistance in clinical isolates of Staphylo-
coccus aureus. J Antimicrob Chemother 51:186 188. http://dx.doi.org/10
.1093/jac/dkg104.
16. Gu B, Kelesidis T, Tsiodras S, Hindler J, Humphries RM. 2013. The
emerging problem of linezolid-resistant Staphylococcus. J Antimicrob
Chemother 68:4 11. http://dx.doi.org/10.1093/jac/dks354.
17. Pharmacia & Upjohn Co. 2015. Zyvox (linezolid package insert)
prescribing information. New York, Pharmacia & Upjohn Co., New
York, NY.
18. Wong E, Rab S. 2014. Tedizolid phosphate (Sivextro): a second genera-
tion oxazolidinone to treat acute bacterial skin and skin structure infec-
tions. P T 39:555579.
19. Drusano GL, Liu W, Kulawy R, Louie A. 2011. Impact of granulocytes on
the antimicrobial effect of tedizolid in a mouse thigh infection model.
Antimicrob Agents Chemother 55:5300 5305. http://dx.doi.org/10.1128
/AAC.00502-11.
20. Barman TK, Pandya M, Mathura T, Bhadauriya T, Rao M, Khan S,
Singhal S, Bhateja P, Sood R, Malhotra S, Das B, Paliwal J, Bhatnagar
PK, Upadhyay DJ. 2009. Novel biaryl oxazolidinones: in vitro and in vivo
activities with pharmacokinetics in an animal model. Int J Antimicrob
Agents 33:280 284. http://dx.doi.org/10.1016/j.ijantimicag.2008.08.025.
21. Mathur T, Kumar M, Barman TK, Kumar GR, Kalia V, Singhal S, Raj
VS, Upadhyay DJ, Das B, Bhatnagar PK. 2011. Activity of RBx 11760, a
novel biaryl oxazolidinone, against Clostridium difficile. J Antimicrob
Chemother 66:10871095. http://dx.doi.org/10.1093/jac/dkr033.
22. Clinical and Laboratory Standards Institute. 2012. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically. Ap-
proved standard M7-A09. Clinical and Laboratory Standards Institute,
Wayne, PA.
23. Lorian V. 2005. Antibiotics in laboratory medicine, 5th ed. Lippincott
Williams and Wilkins, Philadelphia, PA.
24. Hoellman DB, Lin G, Ednie LM, Rattan A, Jacobs MR, Appelbaum PC.
2003. Antipneumococcal and antistaphylococcal activities of ranbezolid
(RBX 7644) a new oxazolidinone, compared to those of other agents.
Antimicrob Agents Chemother 47:1148 1150. http://dx.doi.org/10.1128
/AAC.47.3.1148-1150.2003.
25. Mathur T, Bhateja P, Pandya M, Fatma T, Rattan A. 2004. In vitro
activity of RBx 7644 (ranbezolid) on biofilm producing bacteria. Int J
Antimicrob Agent 24:369 373. http://dx.doi.org/10.1016/j.ijantimicag
.2004.04.012.
26. Kalia V, Miglani R, Purnapatre KP, Mathur T, Singhal S, Khan S, Voleti
SR, Upadhyay DJ, Saini KS, Rattan A, Raj VS. 2009. Mode of action of
ranbezolid against Staphylococci and structural modeling studies of its
interaction with ribosomes. Antimicrob Agents Chemother 53:1427
1433. http://dx.doi.org/10.1128/AAC.00887-08.
27. Kadurugamuwa JL, Sin L, Albert E, Yu J, Francis K, DeBoer M, Rubin
M, Bellinger-Kawahara C, Parr TR, Jr, Contag PR. 2003. Direct contin-
uous method for monitoring biofilm infection in a mouse model. Infect
Immun 71:882 890. http://dx.doi.org/10.1128/IAI.71.2.882-890.2003.
28. Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D. 1999. Character-
ization of the importance of polysaccharide intercellular adhesin/
hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biom-
aterial-based infection in a mouse foreign body infection model. Infect
Immun 67:26272632.
29. Jonathan A, Meulbroek Oleksijew A, Tanaka SK, Alder JD. 1996.
Efficacy of ABT-719, a 2-pyridone antimicrobial, against enterococci,
Escherichia coli, and Pseudomonas aeruginosa in experimental murine
pyelonephritis. J Antimicrob Chemother 38:641 653. http://dx.doi.org
/10.1093/jac/38.4.641.
30. Andes DR, van Ogtrop ML, Peng J, Craig WA. 2002. In vivo pharma-
codynamics of a new oxazolidinone (linezolid). Antimicrob Agents Che-
mother 46:3484 3489. http://dx.doi.org/10.1128/AAC.46.11.3484-3489
.2002.
31. Ippolito JA, Kanyo ZF, Wang D, Franceschi FJ, Moore PB, Steitz TA,
Duffy EM. 2008. Crystal structure of the oxazolidinone antibiotic lin-
ezolid bound to the 50S ribosomal subunit. J Med Chem 51:33533356.
http://dx.doi.org/10.1021/jm800379d.
32. Miller JR, Waldrop GL. 2010. Discovery of novel antibacterials. Expert Opin
Drug Discov 5:145154. http://dx.doi.org/10.1517/17460440903493449.
33. Piddock LJV. 2012. The crisis of no new antibioticswhat is the way
forward? Lancet Infect Dis 12:249 253. http://dx.doi.org/10.1016/S1473
-3099(11)70316-4.
December 2016 Volume 60 Number 12
 Barman et al.
34. Sharma KK, Sangraula H, Mediratta PK. 2002. Some new concepts in
antibacterial drug therapy. Ind J Pharmacol 34:390 396.
35. Craig WA. 1993. Post antibiotic effects in experimental infection models:
relationship to in vitro phenomena and to treatment of infections in man.
J Antimicrob Chemother 31:149 158. http://dx.doi.org/10.1093/jac/31
.suppl_D.149.
36. Jones RN, Fritsche TR, Sader HS, Ross JE. 2007. LEADER surveillance
program results for 2006: an activity and spectrum analysis of linezolid using
clinical isolates from the United States (50 medical centers). Diagn Microbiol
Infect Dis 59:309 317. http://dx.doi.org/10.1016/j.diagmicrobio.2007.06
.004.
37. Jones RN, Ross JE, Castanheira M, Mendes RE. 2008. United States
resistance surveillance results for linezolid (LEADER Program for 2007).
Diagn Microbiol Infect Dis 62:416 426. http://dx.doi.org/10.1016/j
.diagmicrobio.2008.10.010.
38. Christensen GD, Simpson WA, Bisno AL, Beachey EH. 1982. Adherence
of biofilm-producing strains of Staphylococcus epidermidis to smooth sur-
faces. Infect Immun 37:318 3126.
39. Costerton JW, Stewart PS, Greenberg EP. 1999. Bacterial biofilms: a
common cause of persistent infections. Science 284:1318 1322. http://dx
.doi.org/10.1126/science.284.5418.1318.
40. Louie A, Liu W, Kulawy R, Drusano GL. 2011. In vivo pharmacodynam-
ics of torezolid phosphate (TR-701), a new oxazolidinone antibiotic,
against methicillin-susceptible and methicillin-resistant Staphylococcus
aureus strains in a mouse thigh infection model. Antimicrob Agents Che-
mother 55:34533460. http://dx.doi.org/10.1128/AAC.01565-10.
December 2016 Volume 60 Number 12 Antimicrobial Agents and Chemotherapy
41. Locke JB, Hilgers M, Shaw KJ. 2009. Novel ribosomal mutations in
Staphylococcus aureus strains identified through selection with the oxazo-
lidinones linezolid and torezolid (TR-700). Antimicrob Agents Che-
mother 53:52655274. http://dx.doi.org/10.1128/AAC.00871-09.
42. Rayner CR, Forrest A, Meagher AK, Birmingham MC, Schentag JJ.
2003. Clinical pharmacodynamics of linezolid in seriously ill patients
treated in a compassionate use program. Clin Pharmacokinet 42:1411
1423. http://dx.doi.org/10.2165/00003088-200342150-00007.
43. Ambrose PG, Bhavnani SM, Rubino CM, Louie A, Gumbo T, Forrest A,
Drusano GL. 2007. Pharmacokinetics-pharmacodynamics of antimicro-
bial therapy: its not just for mice anymore. Clin Infect Dis 44:79 86.
http://dx.doi.org/10.1086/510079.
44. Barros M, Branquinho R, Grosso F, Peixe L, Novais C. 2014. Linezolid-
resistant Staphylococcus epidermidis, Portugal, 2012. Emerg Infect Dis 20:
903905. http://dx.doi.org/10.3201/eid2005.130783.
45. Wagenlehner FME, Wydra S, Onda H, Kinzig-Schippers M, Srgel F,
Naber KG. 2003. Concentrations in plasma, urinary excretion, and bac-
tericidal activity of linezolid (600 milligrams) versus those of ciprofloxacin
(500 milligrams) in healthy volunteers receiving a single oral dose. Anti-
microb Agents Chemother 47:3789 3794. http://dx.doi.org/10.1128
/AAC.47.12.3789-3794.2003.
46. Matsukawa MY, Kunishima S, Takahashi K, Takeyama K, Tsukamoto
T. 2001. Staphylococcus aureus bacteriuria and surgical site infections by
methicillin-resistant Staphylococcus aureus. Int J Antimicrob Agents 17:
3273229. http://dx.doi.org/10.1016/S0924-8579(00)00358-7.
aac.asm.org 7145