In Vitro and In Vivo Characterization of NOSO-502, a Novel Inhibitor of Bacterial Translation

Antibacterial activity screening of a collection of Xenorhabdus strains led to the discovery of the odilorhabdins, a new antibiotic class with broad-spectrum activity against Gram-positive and Gram-negative pathogens. Odilorhabdins inhibit bacterial translation by a new mechanism of action on ribosomes.

Antibacterial activity screening of a collection of Xenorhabdus strains led to the discovery of the odilorhabdins (ODLs), a new antibiotic class with broad-spectrum activity against Gram-positive and Gram-negative pathogens (3). Odilorhabdins inhibit bacterial translation by a new mechanism of action on ribosomes (3). Their chemical tractability made them suitable for a lead optimization program by medicinal chemistry that led to the preclinical candidate NOSO-502 (Fig. 1).
We report here the in vitro and in vivo characterization of NOSO-502. The data demonstrate that NOSO-502 is active against a panel of Gram-positive and Gramnegative bacteria, including carbapenem-resistant and polymyxin-resistant strains, and exhibits promising in vivo activity in various murine infection models, a favorable in vitro safety profile, and a low potential for resistance development.

RESULTS
NOSO-502 exhibits potent antibacterial activity. The antibacterial activity spectrum of NOSO-502 was assessed by testing a panel of Gram-positive and Gram-negative wild-type strains. The compound was active against Gram-negative pathogens of the Enterobacteriaceae family, such as E. coli or K. pneumoniae, with MIC values between 0.5 and 4 g/ml, as well as Stenotrophomonas maltophilia. In comparison, the MIC values of NOSO-502 against Pseudomonas aeruginosa and Acinetobacter baumannii were Ͼ64 g/ml. For Gram-positive species, NOSO-502 was more active against staphylococci than Enterococcus or Streptococcus strains (Table 1).  The compound was also tested against a recent panel of Enterobacteriaceae clinical isolates. MIC 90 values were between 2 and 8 g/ml against E. coli, K. pneumoniae, Enterobacter cloacae, and Citrobacter freundii. The MIC range of NOSO-502 was narrow against C. freundii and E. cloacae (1 to 4 g/ml) but wider against E. coli (1 to 32 g/ml) and K. pneumoniae (0.5 to 16 g/ml). Nevertheless, only 3 isolates of E. coli and K. pneumoniae out of 101 and 57 tested, respectively, exhibited MIC values slightly higher than the MIC 90 (E. coli, 2 isolates at 16 g/ml and 1 at 32 g/ml; K. pneumoniae, 1 isolate at 4 g/ml, 1 at 8 g/ml, and 1 at 16 g/ml). The antibacterial activity of NOSO-502 was conserved against fluoroquinolone-, aminoglycoside-, and polymyxin B-resistant strains in the panel (Table 2).
MIC values of NOSO-502 were determined against selected CRE and colistin-resistant isolates. The CRE strains tested produce KPC enzymes (Ambler class A carbapenemaseproducing strains), metallo-␤-lactamases, such as NDM, VIM, or IMP (Ambler class B carbapenemase-producing strains), AmpC (Ambler class C carbapenem-resistant strains), and OXA-48 enzymes (Ambler class D carbapenemase-producing strains). NOSO-502 exhibited potent activity against all carbapenemase-producing Enterobacteriaceae strains (Table 3) and overcame multiple mechanisms of colistin acquired resistance (chromosomeencoded mutations or deletions of pmrA, pmrB, mgrB, or phoQ or expression of mcr-1, mcr-2, or mcr-3), except mechanisms involving mutations of the crrB gene (Table 4). The crrB gene belongs to a two-component system named crrAB. Mutations in this gene are responsible for the acquisition of colistin resistance via a lipopolysaccharide (LPS) modification and an upregulation of an RND-type efflux pump (4). The crrAB locus is variably present in K. pneumoniae genomes and absent in E. coli (5). NOSO-502 had rapid bactericidal activity against E. coli ATCC 25922 and K. pneumoniae ATCC 43816, causing a 3-log decrease in CFU per milliliter at 1 h (4ϫ and 8ϫ MIC) (Fig. 2). We observed regrowth of E. coli at 4ϫ MIC. Such regrowth at 24 h is not uncommon and has previously been reported for bactericidal antimicrobials, such as ciprofloxacin against E. coli (6).
The propensity of bacteria to develop resistance to NOSO-502 was assessed by determining the spontaneous frequency of resistance (FoR) to the compound with E. coli ATCC 25922 and K. pneumoniae ATCC 43816. Mutants of E. coli resistant to 4ϫ MIC (16 g/ml) or 8ϫ MIC (32 g/ml) of NOSO-502 were isolated at frequencies of 3.0 ϫ 10 Ϫ9 and Ͻ5.0 ϫ 10 Ϫ10 , respectively. The frequencies of resistance of K. pneumoniae were 2.4 ϫ 10 Ϫ9 at 4ϫ MIC (4 g/ml) and Ͻ7 ϫ 10 Ϫ10 at 8ϫ MIC (8 g/ml).
NOSO-502 has a good in vitro safety profile. The potential nephrotoxicity of NOSO-502 was assessed in cells derived directly from human kidney tissue, human renal proximal tubular epithelial cells (HRPTEpiC) and HK-2 cells. A multiplexed assay with HRPTEpiC was used to assess cellular stress induced in vitro by NOSO-502. Three parameters were measured: a decrease in cell viability, the expression of heat shock protein 27 (HSP27), and the level of kidney injury molecule 1 (KIM-1). A decrease in cell viability is a very sensitive marker to detect general toxicity but is not sufficient to predict nephrotoxicity, whereas increases in the level of biomarkers, such as KIM-1 or HSP27, are well correlated with dose levels of known nephrotoxic compounds (7,8). HSP27 is expressed in response to cellular stress to block the apoptotic pathway. KIM-1 is a well-accepted marker of renal proximal tubule injury. NOSO-502 showed no cytotoxicity to HRPTEpiC and the molecule did not significantly increase (5-fold) KIM-1 or HSP27 levels at concentrations up to 100 M (0.1-fold and 1.5-fold increases at 100 M in KIM-1 and HSP27 signals, respectively). Polymyxin B and gentamicin, used as comparators in this study, showed different toxicity profiles. Polymyxin B was cytotoxic at low concentrations (50% inhibitory concentration [IC 50 ] ϭ 11.8 M) and induced a The cardiotoxic effect of NOSO-502 was evaluated using the automated patch clamp human ether-a-go-go related gene (hERG) potassium channel assay. This test is now accepted as an early predictor of potential cardiotoxicity and is used routinely at an early stage in the drug discovery process. NOSO-502 did not significantly inhibit hERG currents at concentrations up to 512 M (2.6% inhibition at 256 M and 1.9% inhibition at 512 M). We also measured the effect of the compound on the voltage-gated cardiac sodium ion channel Nav 1.5. This channel is a key component for the initiation and transmission of the electrical signal throughout the heart. The IC 50 of NOSO-502 in the patch clamp Nav 1.5 sodium channel assay was higher than 512 M.
The genotoxic potential of NOSO-502 was investigated using the micronucleus (MN) assay. This test detects both aneugenic (whole chromosome) and clastogenic (chromosome breakage) damage in interphase cells (9). There was no significant increase of micronuclei in cells treated with 512 M NOSO-502 versus an S9 medium negative control (0.61% cells with micronuclei for NOSO-502 versus 0.7% for S9 medium).
NOSO-502 had no cytotoxic effect against mammalian HepG2 (human hepatocellular carcinoma) cells at concentrations up to 512 M (0% inhibition from 16 to 256 M and 4.2% inhibition at 512 M) and did not show any hemolytic activity at 100 M. The compound (10 M) had no significant activity against any of the 55 cell surface receptors or enzymes tested in a broad-based screen.
NOSO-502 is resistant to biotransformation by hepatocytes and microsomes. NOSO-502 was resistant to biotransformation when incubated in mouse, rat, dog, monkey, and human liver microsomes and hepatocytes during the in vitro study conducted to evaluate metabolic stability. After 45 min, 70.5 to 78.6% of NOSO-502 remained after incubation with microsomes of the different species. The half-lives of NOSO-502 were 116, 129, 101, 147, and 145 min in mouse, rat, dog, monkey, and human liver microsomes, respectively. After 60 min, 79.5 to 91.9% of NOSO-502 remained after incubation with hepatocytes of the different species. The half-lives of NOSO-502 in hepatocytes were 192, 194, 483, 698, and 329 min for mouse, rat, dog, monkey, and human microsomes, respectively.
NOSO-502 shows variable stability in plasma of different species. NOSO-502 showed variable stability to biotransformation when incubated in mouse, rat, dog, monkey, and human plasma; 10.1 to 61.2% of NOSO-502 remained after incubation with plasma of the different species over the 120-min test period. The half-lives of NOSO-502 were 54, 36, 158, 96, and 79 min in mouse, rat, dog, monkey, and human plasma, respectively.
Pharmacokinetics. The pharmacokinetics of NOSO-502 were evaluated in normal female CD-1 mice or normal female Sprague-Dawley rats. NOSO-502 was administered intravenously at 30 mg/kg of body weight to mice and 15 mg/kg to rats. The concentration-versus-time curves and the results of the pharmacokinetic analysis are summarized in Fig. 3. In mice, NOSO-502 displayed moderate clearance (1.13 liters/h/ kg), a moderate volume of distribution (0.66 liters/kg), and a half-life of 25 min. The pharmacokinetics of NOSO-502 in rats showed a longer half-life (90 min) but were consistent with the results in mice, with a plasma clearance of 1.92 liters/h/kg and a volume of distribution of 0.94 liter/kg. NOSO-502 showed moderate plasma protein binding, with 19.8, 20.5, 17.6, and 18.7% unbound in mouse, rat, dog, and human plasma, respectively.
NOSO-502 shows efficacy in several murine infection models. The efficacy of NOSO-502 was evaluated in murine infection models to determine whether NOSO-502 has potential as a clinical therapy. In vivo efficacy studies were conducted by administering NOSO-502 subcutaneously (s.c.). The efficacy of NOSO-502 was first assessed in a neutropenic murine sepsis infection model. This model, with E. coli EN122 (extendedspectrum ␤-lactamase [ESBL], clinical isolate; MIC of NOSO-502 ϭ 4 g/ml), was established in female NMRI mice. NOSO-502 was administered subcutaneously 1 h postinoculation at set concentrations of 1.3, 2.5, 5, 10, 20, and 40 mg/kg, whereas colistin was administered by the same route at 5 mg/kg. Five hours postchallenge, blood samples were collected, and the mice were euthanized. Blood was serially plated and colonies enumerated to determine the CFU per milliliter of blood. NOSO-502 was highly effective, achieving a 50% effective dose (ED 50 ) of 3.5 mg/kg and 1-, 2-, and 3-log reductions in blood burden at 2.6, 3.8, and 5.9 mg/kg, respectively (Fig. 4).
A mouse E. coli UTI89 (MIC of NOSO-502 ϭ 4 g/ml) upper urinary tract infection model was established in female C3H/HeN mice. Administration of 24 mg/kg of NOSO-502 once daily resulted in a statistically significant reduction in urine, bladder, and kidney burdens relative to vehicle control animals. At 4 days postinfection, NOSO-502 reduced the urine burden by 2.39 log 10 CFU/ml (P Յ 0.0001), the bladder burden by 1.96 log 10 CFU/ml (P ϭ 0.0012), and the kidney burden by 1.36 log 10 CFU/ml (P ϭ 0.0123) relative to vehicle (Fig. 5).
A neutropenic mouse E. coli ATCC BAA-2469 (NDM-1; MIC of NOSO-502 ϭ 2 g/ml) intraperitoneal (i.p.) sepsis infection model was established in male CD-1/ICR mice. Ninety percent of the vehicle-treated mice succumbed to infection prior to the end of the study. All NOSO-502-treated mice (4, 12, and 24 mg/kg) survived up to the end of  Antimicrobial Agents and Chemotherapy the study at 24 h (P ϭ 0.0009 relative to vehicle). The vehicle group had mean and median survival times of 19.8 h and 20.2 h, respectively. One subcutaneous administration of NOSO-502 resulted in statistically significant dose-dependent reductions in blood and i.p. wash burdens relative to vehicle control animals at all doses. Treatment with 4 mg/kg of NOSO-502 reduced the blood and i.p. wash burdens by 1.48 log 10 CFU/ml (P ϭ 0.0081) and 0.68 log 10 CFU/ml (P ϭ 0.0145), respectively. Treatment with 12 mg/kg reduced the blood burden by 2.14 log 10 CFU/ml (P Ͻ 0.0001) and the i.p. wash burden by 2.07 log 10 CFU/ml (P Յ 0.0001), and treatment with 24 mg/kg reduced the blood burden by 2.37 log 10 CFU/ml (P Յ 0.0001) and the i.p. wash burden by 2.74 log 10 CFU/ml (P Յ 0.0001) (Fig. 6). A neutropenic mouse K. pneumoniae NCTC 13442 (OXA-48; MIC of NOSO-502 ϭ 1 g/ml) lung infection model was established in male CD-1/ICR mice. NOSO-502 was administered subcutaneously 2 h, 8 h, 14 h, and 20 h postinoculation at set concentrations of 2, 6, and 20 mg/kg (equivalent to 8, 24, and 80 mg/kg/day), whereas tigecycline was administered by the same route at 40 mg/kg (equivalent to 160 mg/kg/day). NOSO-502 was also administered once 2 h postinoculation at 80 mg/kg. Twenty-six hours postchallenge, mice were euthanized, and the lungs were collected. Administration of NOSO-502 resulted in statistically significant reductions in lung burdens relative to vehicle control animals at all doses. Treatment with 8, 24, and 80 mg/kg/day of NOSO-502 reduced the lung burden by 2.69, 3.99, and 4.07 log 10 CFU/gram of lung tissue, respectively (P Յ 0.0001). Treatment with 80 mg/kg once reduced the lung burden by 3.98 log 10 CFU/gram of lung tissue (P Յ 0.0001), and treatment with 160 mg/kg/day of tigecycline reduced the lung burden by 3.14 log 10 CFU/gram of lung tissue (P Յ 0.0001) (Fig. 7).

DISCUSSION
The urgent need to discover new antibiotics active against Gram-negative bacteria with a novel mechanism of action to counter the threat of drug-resistant infection is widely recognized. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). ODLs are cationic peptides that inhibit bacterial translation by a novel mechanism of action. ODLs bind to the small subunit of bacterial ribosomes at a site not exploited by any known ribosome-targeting antibiotic. When bound to the ribosome, ODLs make contacts with both the rRNA and tRNA and kill bacteria by interfering with the decoding of genetic information and inhibiting ribosome progression along the mRNA in a context-specific manner (3).
NOSO-502 is active against Enterobacteriaceae, including CRE belonging to all classes of the Ambler classification and resistant to gentamicin, polymyxin B, or tigecycline. This is crucial, because these antibiotics, classically used for the treatment of such infections, are associated with high levels of resistance, ranging from 9.7 to 51.3% (mean, 22.6%) for colistin, 5.6 to 85.4% (mean, 43.5%) for gentamicin, and 0 to 33% (mean, 15.2%) for tigecycline (10,11,12,13,14,15,16,17,18,19). Current options    (24,25,26,27,28). These infections have been associated with mortality rates exceeding 50% in some reports (29,30,31,32). NOSO-502 can overcome multiple mechanisms of colistin-resistant strains. Furthermore, the compound demonstrated rapid bactericidal activity and a low potential for the development of resistance. NOSO-502 is effective in mouse models of serious hospital-acquired infections. It provided significant protection against the Gram-negative pathogens E. coli and K. pneumoniae, the highest-incidence pathogens in complicated intra-abdominal and urinary tract infections, in septicemia following peritoneal challenge, and in acute pyelonephritis. NOSO-502 was active in mouse infection models against E. coli strains expressing the metallo-␤-lactamase NDM-1 and resistant to other major antibiotic classes, including fluoroquinolones, macrolides, aminoglycosides, ␤-lactams, cephalosporins, and carbapenems. These results are encouraging and show the strong potential for in vivo efficacy of NOSO-502. Effective doses will be optimized after the best dosing schedule is defined during a pharmacokinetic-pharmacodynamic (PKPD) study.
NOSO-502 showed a good safety profile, with no in vitro nephrotoxicity, cardiotoxicity, genotoxicity, or cytotoxicity at concentrations up to 512 M. Nephrotoxicity is a serious side effect of many drugs, including cationic antibiotics aminoglycosides and polymyxins (33,34,35). Polymyxins accumulate extensively within proximal tubular cells (PTCs) of the kidneys, where they induce damage, which may lead to acute kidney injury (AKI) in patients (36). AKI is the major dose-limiting adverse effect of this class of antibiotics and affects 50 to 60% of patients receiving them (35,37). Aminoglycosides are filtered across the glomerulus and then excreted, with 5 to 10% of a parenteral dose being taken up and sequestered by the PTCs, in which the aminoglycoside can achieve high concentrations (38). AKI due to acute tubular necrosis is a relatively common complication of aminoglycoside therapy and affects 10 to 20% of patients (33,34). The results of NOSO-502 on HRPTEpiC and HK-2 cells are promising but must be confirmed by histopathological examination of kidney cells following in vivo administration to animals, the standard assay for studying nephrotoxicity effects.
Cardiotoxicity issues are associated with many antibiotics, including macrolides, ketolides, and fluoroquinolones. These classes have been associated with prolongation of cardiac repolarization. All these agents produce a blockage of the hERG channeldependent potassium current in myocyte membranes, resulting in a prolonged QTc interval, which may give rise to ventricular fibrillation or tachycardia (39). Nav 1.5 is another channel involved in cardiotoxicity issues. Its activation induces depolarization of the cell membrane. Failure of the Nav 1.5 sodium channel to adequately conduct the electrical current across the cell membrane can result in a potentially fatal disorder. NOSO-502 did not show any effects on hERG or Nav 1.5 channels at high concentrations.
In this study, we confirmed that NOSO-502, like many other therapeutic peptides, is safe and highly selective. NOSO-502 interacts strongly with a specific site on the 30S subunit of bacterial ribosomes but has no significant activity against any of the 55 cell surface receptors, transporters, or ion channels tested. There is increasing interest in peptides in pharmaceutical research and development (R&D), and approximately 140 are currently being evaluated in clinical trials and more than 500 are in preclinical development (40,41). The main limitation of peptides is their predisposition to enzymatic degradation. Thus, most do not circulate in blood for more than a few minutes, preventing their usefulness as therapeutic agents. However, NOSO-502 showed good stability in plasma, microsomes, and hepatocytes, probably due to the presence in its structure of three nonstandard amino acid residues: ␣,␥-diamino-␤-hydroxybutyric acid [Dab(␤OH)] at position 2 (N terminus), ␣,␤-dehydroarginine (Dha) at position 9 (C terminus), and D-ornithine at position 5. This translates into relatively long half-lives in mice and rats.
NOSO-502 represents a new class of very promising bacterial ribosomal inhibitors to combat bacterial multidrug resistance. Multiplexed HRPTEpiC cytotoxicity assay. The multiplexed cytotoxicity assay on human renal proximal tubule epithelial cells (HRPTEpiC) was conducted by Eurofins Panlabs (Eurofins Panlabs, Inc., St. Charles, MO) by using an image-based high content analysis (HCA) technique where cells were fixed and stained with nuclear dye to visualize nuclei and fluorescently labeled antibodies to detect drug-induced cellular injury and cellular stress arising from oxidative and chemical stress. Cells were seeded into 384-well plates and grown in RPMI 1640, 10% fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 1 mM sodium pyruvate in a humidified atmosphere of 5% CO 2 at 37°C. NOSO-502, gentamicin, and polymyxin B were added 24 h after cell seeding. Compounds were serially diluted 3.16-fold and assayed over 10 concentrations in a final assay concentration of 0.5% dimethyl sulfoxide (DMSO) from 100 M to 3.7 nM. At the same time, a time zero untreated cell plate was generated. After a 48-h incubation period, cells were fixed and stained with fluorescently labeled antibodies and nuclear dyed to allow visualization of nuclei, injured cells, and stressed cells. Injured cells were detected using a KIM-1 (kidney injury molecule 1) antibody. Stressed cells were detected using an anti-HSP27 (heat shock protein 27) antibody. Cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation assay output was referred to as the relative cell count. To determine the cell proliferation endpoint, the cell proliferation data output was transformed to percentage of control (POC) using the following formula: POC ϭ relative cell count in compound wells/relative cell count in vehicle wells ϫ 100. The signal intensity of the incorporated cellular stress and injury measurements were normalized with the relative cell count from each well. Automated fluorescence microscopy was carried out using a Molecular Devices ImageXpress micro-imager, and images were collected with a 4ϫ objective.

MATERIALS AND METHODS
Cytotoxicity testing. HK-2 and HepG2 cell cytotoxicity assays were run by Eurofins-Cerep (Cerep cytotoxicity profile; Eurofins-Cerep SA, Poitiers, France) as described in reference 44. Cell viability was measured using a luciferase-coupled ATP-quantitation assay (CellTiter-Glo; Promega, Madison, WI). In this assay, the luminescent signal is proportional to the amount of ATP and thus to the number of metabolically competent cells; cell injury and death result in a marked decrease in intracellular ATP levels. HK-2 and HepG2 cells were dispensed at 6,000 to 3,000 cells/5 l/well in white tissue-culture treated 96-well solid-bottom assay plates and incubated at 37°C for 16 h to allow cell attachment, followed by the addition of NOSO-502 at 16, 32, 64, 128, 256, and 512 M. After compound addition, plates were incubated for 48 h at 37°C. At the end of the incubation period, 5 l of CellTiter-Glo reagent was added, the plates were incubated at room temperature for 30 min, and the luminescence intensity of each well was determined. Each experiment was carried out in duplicate.
hERG tail current inhibition. Inhibition of the human ether-a-go-go-related gene (hERG) cardiac potassium ion channel was determined by Eurofins Panlabs (Eurofins Panlabs, Inc., St. Charles, MO) in CHO-K1 (Chinese hamster ovary) cells stably transfected with human hERG cDNA using QPatch automated whole-cell patch clamp electrophysiology as described in reference 45. NOSO-502 was tested at 64, 256, and 512 M. In this method, the extracellular solution (control) is applied first and the cell is stabilized in the solution for 5 min. Then the test compound is applied from low to high concentrations sequentially on the same cell, with 5 min for each test concentration, at room temperature. Nav 1.5 peak current inhibition. Inhibition of the Nav 1.5 human sodium ion channel was determined by Eurofins Panlabs in HEK-293 cells stably transfected with human Nav1.5 cDNA (type V voltage-gated sodium channel alpha subunit, GenBank accession number NM_000335) using IonWorks Quattro automated whole-cell patch clamp electrophysiology. NOSO-502 was tested at 4, 8, 16, 32, 64, 128, 256, and 512 M. In this method, the voltage protocol is applied prior to compound addition (Pre), the compounds are added and incubated for 600 s at room temperature, and then the voltage protocol is applied a final time (Post) on the IonWorks Quattro.
In vitro micronucleus assay. The test was conducted by Eurofins Panlabs. CHO-K1 cells were preloaded with a cell dye that stains the cytoplasm, after which the cells were treated with NOSO-502 at 32, 64, 128, 256, and 512 M for 24 h. At the end of the incubation period the cells were fixed, and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescence microscope coupled with proprietary automated image analysis software (46). The percent micronucleated cells was calculated. A marginally positive result ("Ϫ/ϩ") is defined as a value significantly higher than controls (t test, P Ͻ 0.05) and at least 2-fold higher than controls. A positive result ("ϩ") is defined as a value significantly higher than controls (t test, P Ͻ 0.05) and at least 3-fold higher than controls.
Hemolytic activity. Mouse red blood cells were washed with phosphate-buffered saline (PBS) and resuspended in PBS to 10% (vol/vol). NOSO-502 was tested at a final concentration of 100 M. PBS and deionized water were used as 0 and 100% hemolytic controls, respectively. Assays were incubated at 35°C for 45 min. The release of hemoglobin in the supernatant was monitored by absorbance at 540 nm. Experiments were performed in triplicate.
Hepatocyte stability. The hepatocyte metabolic stability assays were performed by Cyprotex Discovery Ltd. (Macclesfield, UK). This assay utilizes cryopreserved pooled hepatocytes from different species (mouse, rat, dog, monkey, and human), stored in liquid nitrogen prior to use. Williams E media supplemented with 2 mM L-glutamine, 25 mM HEPES, and NOSO-502 (NOSO-502 final substrate concentration of 1 M, test compound prepared in water; control compound final substrate concentration of 3 M, final DMSO concentration of 0.25%) were preincubated at 37°C prior to the addition of a suspension of cryopreserved hepatocytes (final cell density of 0.5 ϫ 10 6 viable cells/ml in Williams E media supplemented with 2 mM L-glutamine and 25 mM HEPES) to initiate the reaction. The final incubation volume was 500 l. Two control compounds were included with each species alongside an appropriate vehicle control. The reactions were stopped by transferring an aliquot of the mixture to 40% trichloroacetic acid (TCA) in water, containing an internal standard for the test compounds (NOSO-95216; 1 M final concentration) or methanol for the control compounds, at various time points (0, 5, 10, 20, 40, and 60 min). The termination plates were centrifuged at 2,500 rpm at 4°C for 30 min to precipitate the protein. Following protein precipitation, the test compound sample supernatants were diluted with analytical-grade water, whereas the control compounds were diluted with an internal standard (metoprolol) in water. The test compound samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Microsome stability. The microsome metabolic stability assays were performed by Cyprotex Discovery Ltd. (Macclesfield, UK). Pooled microsomes from different species (mouse, rat, dog, monkey, and human) were stored at Ϫ80°C prior to use. Microsomes (final protein concentration of 0.5 mg/ml), 0.1 M phosphate buffer (pH 7.4), and NOSO-502 (test compound final substrate concentration of 1 M, test compound prepared in water; control compound final substrate concentration of 3 M, final DMSO concentration of 0.25%) were preincubated at 37°C prior to the addition of NADPH (final concentration of 1 mM) to initiate the reaction. The final incubation volume was 500 l. A minus cofactor control incubation was included for each compound tested, in which 0.1 M phosphate buffer (pH 7.4) was added instead of NADPH (minus NADPH). Two control compounds were included with each species. Each compound was incubated for 0, 5, 15, 30, and 45 min. The control (minus NADPH) was incubated for 45 min only. The reactions were stopped by transferring an aliquot of the mixture to 40% TCA in water containing an internal standard (NOSO-95216, 1 M final concentration) for the test compounds, or methanol for the control compounds, at the desired time points. The termination plates were centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the protein. Following protein precipitation, the test compound sample supernatants were diluted with analytical-grade water, whereas the control compounds were diluted with an internal standard (metoprolol) in water. The test compound samples were analyzed by LC-MS/MS. Plasma stability. The plasma stability assays were performed by Cyprotex Discovery Ltd. Speciesspecific plasma (heparin anticoagulant) was adjusted to pH 7.4 at 37°C, and NOSO-502 or control compound (test compound final substrate concentration of 10 M, test compound prepared in water; control compound final substrate concentration of 1 M, final DMSO concentration of 2.5%) was added to initiate the reaction. The final incubation volume was 200 l. All incubations were performed singularly for each compound at each time point. A vehicle control incubation was included using either water or DMSO, along with a control compound known to be metabolized specifically by each species. Each compound was incubated for 0, 15, 30, 60, and 120 min at 37°C. The reactions were stopped by the 4, 10, 20, or 40 mg/kg NOSO-502, a vehicle (PBS [pH 7.4]), or 5 mg/kg of colistin (Sigma-Aldrich; no. 4461) subcutaneously as a single dose in 0.2 ml. Four hours after treatment, mice were anesthetized and blood was collected by axillary cutdown. Blood samples were serially diluted and plated on blood agar plates (SSI Diagnostica, Hillerød, Denmark), with subsequent counting of colonies after incubation overnight at 35°C in ambient air. The mice were observed during the study for clinical signs of infection, such as lack of curiosity, social withdrawal, changes in body position and patterns of movement, distress, or pain.
Mouse UTI model. All animal studies were performed under UK Home Office license P2BC7D240 with local ethical committee clearance. All studies were performed by technicians who completed parts A, B, and C of the UK Home Office personal license course and hold current personal licenses. All experiments were performed in dedicated biohazard 2 facilities (this site holds a certificate of designation).
NOSO-502 was tested against E. coli UTI89 (MIC ϭ 4 g/ml) in a mouse UTI model by Evotec (Manchester, UK). Female C3H/HeN mice, 18 to 22 g (Janvier Laboratories, UK), were allowed to acclimatize for 7 days. Following acclimatization, drinking water was replaced with water containing 5% glucose from 5 days preinfection. Previously prepared frozen stocks of E. coli UTI89 were diluted to 1 ϫ 10 10 CFU/ml immediately prior to infection. Mice were infected by directly administering a 0.05-ml inoculum (5 ϫ 10 8 CFU/mouse) via the urethra into the bladder under parenteral anesthesia (90 mg/kg of ketamine and 9 mg/kg of xylazine). Bladders were emptied prior to infection, and once infected, infection catheters were left in the urinary tract for 10 min to reduce the risk of the organism flowing back out. Following catheter removal, mice were allowed to fully recover in warmed humidified cages. Dose formulations of NOSO-502 were prepared in 25 mM PBS. Treatment with 4, 12, and 24 mg/kg of NOSO-502 was initiated 24 h postinfection and was administered once daily (q24h) by subcutaneous injection or intravenously (ciprofloxacin) for 3 days. Mice were euthanized 96 h postinfection (three doses administered). Ciprofloxacin (Bayer; lot BXHEFTI), administered at 10 mg/kg/dose intravenously twice a day (BID), was included as a comparator (six doses administered), and 25 mM PBS was used as a vehicle. Urine was collected 24 h postinfection from all animals and used to assess the infection level of each mouse prior to initiation of treatment; all mice were successfully infected. In addition, five mice were euthanized by pentobarbitone overdose to provide a 24-h pretreatment control group. The clinical condition and body weight of all remaining animals were assessed and urine samples were collected 96 h postinfection. Animals were then euthanized by pentobarbitone overdose and the kidneys and bladders removed and weighed. Tissue samples were homogenized using a Precellys 24 dual-bead beater in 2 ml of ice-cold sterile PBS. Homogenates and urine samples were quantitatively cultured onto MacConkey's agar plates and incubated at 37°C for 24 h before colonies were counted. The data from the culture burdens were analyzed using appropriate nonparametric statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons between groups) with StatsDirect software v. 2.7.8 and compared to pretreatment and vehicle controls.
Mouse neutropenic i.p. sepsis model. All animal studies were performed under UK Home Office license P2BC7D240 with local ethical committee clearance. All studies were performed by technicians who completed parts A, B, and C of the UK Home Office personal license course and hold current personal licenses. All experiments were performed in dedicated biohazard 2 facilities (this site holds a certificate of designation).
NOSO-502 was tested against E. coli ATCC BAA-2469 (MIC ϭ 2 g/ml) in an i.p. sepsis model by Evotec (Manchester, UK). Male CD1/ICR mice, 25 to 30 g (Charles River, UK), were allowed to acclimatize for 11 days. Mice were rendered neutropenic with two intraperitoneal injections of 150 mg/kg of cyclophosphamide 4 days before infection and 100 mg/kg 1 day before infection. Previously prepared frozen stocks of E. coli ATCC BAA-2469 were diluted immediately prior to infection to 6.8 ϫ 10 7 CFU/ml. Mice were infected by directly administering a 0.5-ml inoculum (3.4 ϫ 10 7 CFU/mouse) via intraperitoneal injection. Dose formulations of NOSO-502 were prepared in 25 mM PBS. Treatment was initiated 1 h postinfection, and NOSO-502 doses (4, 12, and 24 mg/kg) were administered once by subcutaneous injection. Tigecycline (MIC ϭ 0.5 g/ml), administered at 40 mg/kg/dose subcutaneously BID, was included as a comparator, and two doses were administered. Animals from the pretreatment groups were euthanized 1 h postinfection, and all remaining mice were euthanized 25 h postinfection. The clinical condition and body weight of all remaining animals were assessed 25 h postinfection or when animals reached the ethical severity endpoint (whichever came first). Mice were anesthetized using 2.5% isoflurane-97.5% oxygen, followed by a pentobarbitone overdose. When mice were deeply unconscious, blood was collected from all animals under terminal cardiac puncture into EDTA blood tubes. In addition, an intraperitoneal wash with sterile PBS (2 ml i.p. injected and 1 ml removed for culture) was collected. Five mice were also euthanized by pentobarbitone overdose to provide a 1-h pretreatment control group. Blood and i.p. wash samples were quantitatively cultured onto cystine lactose electrolyte-deficient (CLED) agar plates and incubated at 37°C for 24 h before colonies were counted. The data from the culture burdens were analyzed using appropriate nonparametric statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons between groups) with StatsDirect software v.2.7.8 and compared to pretreatment and vehicle controls. Mouse lung infection model. All animal experiments were performed under UK Home Office license 40/3644, and with local ethical committee clearance (The University of Manchester AWERB). All experiments were performed by technicians who had completed at least parts 1 to 3 of the Home Office personal license course and held current personal licenses.
NOSO-502 was tested against K. pneumoniae NCTC 13442 (expresses OXA-48 carbapenemase, MIC ϭ 1 g/ml) in a neutropenic mouse pulmonary infection model by Evotec (Manchester, UK). Male CD-1/ICR mice, 6 to 8 weeks old (Charles River UK), were allowed to acclimatize for 7 days and then rendered neutropenic by i.p. injection of cyclophosphamide (200 mg/kg on day 4 and 150 mg/kg on day 1 before infection). Mice were infected by the intranasal route (ϳ4 ϫ 10 6 CFU/mouse) under parenteral anesthesia. At 2 h, 8 h, 14 h, and 20 h postinfection, mice received treatments with NOSO-502 at 2, 6, or 20 mg/kg or with tigecycline at 40 mg/kg administered by the s.c. route in a volume of 10 ml/kg (8 mice per dose). At 2 h postinfection, NOSO-502 was delivered once by the s.c. route at 80 mg/kg in a volume of 10 ml/kg (8 mice). At 2 h postinfection, one infected group was humanely euthanized, and lungs were processed for pretreatment quantitative culture to determine Klebsiella burdens. At 26 h postinfection, all remaining mice were humanely euthanized. Lungs were aseptically removed, homogenized, serially diluted, and plated on CLED agar for CFU titers.