Protein synthesis; bacterial 50S ribosomal subunit

The activities of the oxazolidinone antibacterial agents Eperezolid (PNU-100592) and linezolid (PNU-100766) were compared with that of vancomycin against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus (n = 200), coagulase-negative staphylococci (n = 100), and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium (n = 50). Eperezolid and linezolid demonstrated good in vitro inhibitory activity, regardless of methicillin susceptibility for staphylococci (MIC at which 90% of the isolates are inhibited [MIC90] range, 1 to 4 microg/ml) or vancomycin susceptibility for enterococci (MIC90 range, 1 to 4 microg/ml). In time-kill studies, eperezolid and linezolid were bacteriostatic in action. A postantibiotic effect of 0.8+/-0.5 h was demonstrated for both eperezolid and linezolid against S. aureus, S. epidermidis, E. faecalis, and E. faecium.[1]

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The oxazolidinones are a novel class of antibiotics that act by inhibiting protein synthesis. It as been reported that the drug exerts its primary activity on the initiation phase of translation. In order to study the possibility of direct interaction between the drug and the ribosome, we have developed a binding assay using 14C-labelled Eperezolid (PNU-100592; formerly U-100592). Eperezolid binds specifically to the 50S ribosomal subunit of Escherichia coli. The specific binding of eperezolid is dose dependent and is proportional to the ribosome concentrations. Scatchard analysis of the binding data reveals that the dissociation constant (Kd) is about 20 microM. The binding of eperezolid to the ribosome is competitively inhibited by chloramphenicol and lincomycin. However, unlike chloramphenicol and lincomycin, Eperezolid does not inhibit the puromycin reaction, indicating that the oxazolidinones have no effect on peptidyl transferase. In addition, whereas lincomycin and, to some extent, chloramphenicol inhibit translation termination, eperezolid has no effect. Therefore, we conclude that the oxazolidinones inhibit protein synthesis by binding to the 50S ribosomal subunit at a site close to the site(s) to which chloramphenicol and lincomycin bind but that the oxazolidinones are mechanistically distinct from these two antibiotics [3].

The in vivo effectiveness of oxazolidinones Eperezolid (U-100592) and linezolid (U-100766) against one strain each of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium was examined in a rat model of intra-abdominal abscess. MICs of both drugs were 2 microg/ml for each strain. At doses of 25 mg/kg of body weight twice daily intravenously or orally, linezolid produced small but statistically significant reductions in abscess bacterial density for E. faecalis. The reduction in viable cells observed would not likely be clinically relevant. Eperezolid was ineffective at this dose. At a dosage of 100 mg/kg/day, linezolid treatment led to an approximately 100-fold reduction in viable cells per gram of abscess. Against E. faecium infections, intravenous eperezolid and oral linezolid were effective, reducing densities approximately 2 log(10) CFU/g. Both oxazolidinones demonstrated activity against enterococci in this model. However, results were modest with the dosing regimens employed.[2]

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Oxazolidinone treatment for E. faecalis. [2]
For Eperezolid, the q12h intravenous regimen studied showed no activity against E. faecalis, despite mean peak levels of approximately 10 times the MIC (Table 3). Administration of the same total daily dose (50 mg/kg) by continuous 24-h infusion intravenously or by q12h oral dosing provided no advantage. Levels of linezolid in plasma were approximately twice those of eperezolid when drugs were administered at 25 mg/kg q12h intravenously and approximately three times greater when these compounds were given orally. Both routes of administration at this dosage resulted in small but statistically significant decreases in viable bacteria after 4.5 days of therapy with linezolid. Doubling the total daily dose (100 mg/kg) with more-frequent administration resulted in a 2-log10 CFU/g decrease in bacterial counts. No animal had sterile abscess contents.

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Oxazolidinone treatment for E. faecium. [2]
Against this organism, intravenous and oral Eperezolid, but not intravenous linezolid, reduced viable cell densities significantly. However, linezolid demonstrated activity when administered orally. Almost 2-log10 CFU/g reductions in residual bacteria were noted with oral linezolid and intravenous eperezolid.

Binding assay. [3]
Radiolabelled compounds, [14C]Eperezolid (59.32 mCi/mg, 23.4 mCi/mmol) and [14C]PNU-96499 (142 mCi/mg, 55 mCi/mmol), were synthesized. D-threo-[Dichloroacetyl-1-14C]chloramphenicol (166 mCi/mg, 54 mCi/mmol) was used. The binding studies were performed in microcentrifuge tubes that contained a total of 100 ml of reaction mixture which included 0.3 to 2.0 nmol of ribosomes, 1 to 100 mM radiolabelled compound with either 1 ml of dimethyl sulfoxide or an excess amount (100- to 1,000-fold) of unlabelled compound, 50 mM Tris-HCl (pH 7.5), 5 mM Mg(CH3COO)2, and 200 mM KCl. All other ingredients were mixed together before the addition of ribosome. The reaction mixture was allowed to incubate at 25°C for 10 min, and the reaction was terminated by the addition of 50 ml of 100% ice-cold ethanol to precipitate the ribosomes and bound drug. After incubation at 4°C for 30 to 60 min, the suspension was centrifuged at full speed in an Eppendorf microcentrifuge for 20 min. The supernatant was then carefully removed, and the radioactivity in the pellet was measured. All datum points represent the mean 6 standard error of the mean (SEM) of at least three independent determinations. For the measurement of specific binding of a compound, the total and nonspecific binding must be measured. Total binding was measured directly by adding a high concentration of radiolabelled ligand. It is assumed that at these high concentrations a very high proportion of the binding is entirely nonspecific. The specific binding sites would be completely saturated at lower concentrations, so the amount of specific binding would be small. Nonspecific binding is determined by the addition of unlabelled compound at 1,000 times the concentration of the radiolabelled compound. Virtually all of the high-affinity binding to the specific binding site will be displaced, but the nonspecific binding will not. Nonspecific binding is defined as the amount of radiolabelled compound remaining bound in the presence of an excess amount of unlabelled compound. The specific binding of the compounds is determined by subtracting the nonspecific binding from the total binding. NaCl at 1.5 M displaced the binding of the oxazolidinones, indicating that the binding was not covalent.

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Puromycin reaction. [3]
For f[3 H]Met-tRNA-AUG-ribosome complex formation, a reaction mixture containing 0.65 nmol of total ribosomes, 0.2 nmol of f[3 H] Met-tRNA (32.92 mCi/mg, 9.7 Ci/mmol), and 2.5 nmol of AUG in binding buffer (20 mM Tris-HCl [pH 7.4], 10 mM MgCl2, 150 mM NH4Cl) in a volume of 50 ml was incubated at 30°C for 30 min. A reaction mixture containing 3 ml of the complex described above, 3 mM puromycin, 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 150 mM NH4Cl in a volume of 50 ml, in the presence or absence of chloramphenicol, lincomycin, linezolid, or Eperezolid, was incubated at 30°C for 30 min. The reaction was terminated by the addition of 250 ml of 100 mM potassium phosphate buffer (pH 6), and then the f[3 H]Met-puromycin product was extracted into ethyl acetate. The radioactivity in the ethyl acetate layer was measured by liquid scintillation spectrometry.

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Termination reaction. [3]
A reaction mixture containing 5 ml of the complex described above, 50 mM Tris-HCl (pH 7.4), 30 mM MgCl2, 75 mM NH4Cl, 80 mM UAA, and 3.5 mg of termination factors in a volume of 50 ml, in the presence or absence of chloramphenicol, lincomycin, Eperezolid, or linezolid, was incubated at 22°C for 30 min. Termination factors were isolated as described by Ganoza et al. Concentrated termination factors from the phosphocellulose column step were used in the assay. The reaction was halted by the addition of 250 ml of 0.1 N HCl and extraction of the f[3 H]Met product into ethyl acetate. The radioactivity in the ethyl acetate layer was measured by liquid scintillation spectrometry. If either the termination triplet codon (UAA) or the termination factors were absent, there was no release of f[3 H]Met.

Clinical isolates of Staphylococcus aureus, S. epidermidis, various other coagulase-negative staphylococci, E. faecalis, and E. faecium were collected over a 6-month period from hospitalized patients at Detroit Receiving Hospital and University Health Center, Detroit, Mich. Methicillin susceptibility was determined by the oxacillin disk method.[1]

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MICs were determined by a microdilution method with Mueller-Hinton broth supplemented (SMHB) with calcium (25 mg/liter) and magnesium (12.5 mg/liter). Susceptibility testing for each drug was performed according to the guidelines of the National Committee for Clinical Laboratory Standards.[1]

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The bactericidal activities of Eperezolid and linezolid were compared to that of vancomycin by use of time-kill analyses. Four representative clinical isolates (methicillin-resistant S. aureus R323, methicillin-susceptible S. epidermidis R264, vancomycin-resistant E. faecalis R581, and vancomycin-resistant E. faecium R20) were evaluated. The test strains were grown overnight at 35°C in SMHB and diluted to yield a starting inoculum of 106 CFU/ml. Sufficient stock antibiotic solution was added to achieve a desired concentration of four times the respective MICs. Growth controls were prepared in a similar fashion with substitution of the appropriate medium in place of the stock antibiotic solution. All tubes were incubated at 35°C with constant rotation for 24 h. Samples (0.1 ml) were removed at 0, 4, 8, and 24 h; diluted at least 250-fold with 0.9% sodium chloride to reduce antibiotic carryover; and plated on tryptic soy agar. The limit of detection for this method is 30 CFU/plate, corresponding to 300 CFU/ml (11). At time points at which bacterial counts were expected to be below limits of detection, 0.1-ml samples were placed in 10 ml of cold 0.9% sodium chloride and filtered by a 0.45-μm-pore-diameter filter (Millipore, Bedford, Mass.). Filters were placed aseptically on tryptic soy agar and incubated for 24 h. The limit of detection for this method is 10 CFU/plate, corresponding to 100 CFU/ml. All time-kill-curve experiments were performed in duplicate.[1]

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The presence of a postantibiotic effect (PAE) was determined for Eperezolid, linezolid, and vancomycin for representatives of each group of organisms by the method described by Craig and Gudmundsson. An overnight growth of S. aureus, S. epidermidis, E. faecalis, or E. faecium was diluted into fresh SMHB to 106 CFU/ml and then incubated on a rotor at 37°C for 3 to 4 h until the logarithmic growth phase was achieved. At the end of this period, the inoculum size was determined, and each tube containing the test organisms was then exposed to eperezolid, linezolid, or vancomycin at the MIC and at four times the MIC for 1 h at 37°C on a rotor. One test tube of each organism was also used as a growth control and was subjected to the same procedures as described above but was not exposed to the antibiotic. Following incubation with the antibiotic, the cultures, including the growth controls, were diluted 1:1,000 into 10 ml of fresh prewarmed SMHB and reincubated at 37°C. Samples were removed in duplicate every 1.0 h and plated onto tryptic soy agar to determine the PAE. Each PAE experiment was performed in duplicate. The duration of the PAE was calculated by the equation PAE = T − C, where T is the time required for the CFU count in the culture exposed to antibiotic to increase 1 log10 unit above the count observed immediately after antibiotic removal and C is the time required for the CFU count in the control to increase 1 log10 unit above the count observed immediately after the same procedure used on the test culture for the antibiotic removal.[1]

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The activities of Eperezolid and linezolid compared to that of vancomycin are shown in Table 1. For S. aureus, vancomycin was one- to twofold more active than eperezolid and linezolid. The oxazolidinones were equipotent to vancomycin against all coagulase-negative staphylococci tested. Compound eperezolid was at least one- to twofold more active than linezolid against coagulase-negative staphylococci. In time-kill studies, eperezolid and linezolid displayed bacteriostatic action against all isolates tested. As expected, vancomycin displayed bactericidal activity against S. aureus and S. epidermidis but not E. faecalis or E. faecium (Fig. 1)[1].

Animal model. [2]
Enterococcal intra-abdominal abscesses were created in male Sprague-Dawley rats weighing 175 to 250 g, by using a model which has been described previously. For all surgical procedures rats were anaesthetized with ketamine and xylazine. Suspensions of 0.5 ml of E. faecalis 1310 or E. faecium A1221 at a density of 106 CFU/ml in BHI broth, along with heat-killed Bacillus fragilis ATCC 23745 and sterilized rat cecal contents (in ratios of 1:1:2 parts, respectively) and barium sulfate (10% [wt/vol]) were put into double-gelatin capsules which were then implanted intraperitoneally through a 1-cm midline abdominal incision.

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Antimicrobial therapy. [2]
Treatment was started 4 h after implantation of the inoculum and was given either intravenously or by peroral gavage. For intravenous administration, oxazolidinones were dissolved in dimethyl sulfoxide which was diluted to 5% (vol/vol) in sterile saline. The doses used in these experiments were based on those which appeared to be active in previously published mouse protection studies. Antimicrobials were administered at desired intervals as 15- to 20-min infusions or by continuous infusion over 24 h via a central catheter, which was surgically implanted through the left internal jugular vein into the superior vena cava the day before the infection as previously described. For peroral administration, oxazolidinones were suspended in 2 ml of saline and the slurry was given by gavage every 12 h (q12h). As a positive (effective) control, ampicillin was administered by continuous intravenous infusion at a dosage of 400 mg/kg of body weight/24 h, which has previously been shown to achieve a mean steady-state serum concentration of approximately 15 μg/ml. Continuous-infusion therapy was administered for 4.5 days. For twice-daily regimens a total of nine doses were given over 4.5 days. Untreated control rats were included in each experiment.

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Monitoring of therapy and outcome. [2]
For determination of concentrations of oxazolidinones in plasma, blood was obtained by retro-orbital puncture 0 to 5 min after completion of intermittent infusion or 45 to 60 min after gavage for peak levels and just prior to the next scheduled dose for trough levels. Samples were treated with 25 μl of 2% disodium EDTA, and plasma was separated, frozen, and sent to Pharmacia & Upjohn, where oxazolidinone concentrations were measured by high-performance liquid chromatography by Guy Padbury. Details of the assay have been published elsewhere. The technique has been validated to concentrations as low as 0.01 μg/ml. Coefficients of variation for intra-assay and interassay samples by this method are <10%.

Rats were sacrificed 12 h after the last dose of antimicrobials. The abdomen was opened under aseptic conditions, and the abscess contents were harvested, weighed, and homogenized in 2 ml of sterile saline and then serially diluted. Because of the potential for the occasional translocation of noninoculated bacteria into intra-abdominal abscess contents in the rat model of fecal peritonitis, suspensions were plated both on blood agar plates in duplicate for colony counts and on Enterococcosel agar plates, as a selective medium for enterococci. Plating either organism on this medium resulted in counts identical to those on blood agar plates (data not shown). Results from the selective plates were used only on those few occasions in which a question of contaminating bacteria arose. Statistical analysis was carried out by the two-tailed Mann-Whitney test. P values < 0.05 were considered statistically significant.

[1]. Comparative in vitro activities and postantibiotic effects of the oxazolidinone compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus vancomycin against Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Antimicrob Agents Chemother. 1998 Mar;42(3):721-4.
[2]. Activities of the oxazolidinones linezolid and eperezolid in experimental intra-abdominal abscess due to Enterococcus faecalis or vancomycin-resistant Enterococcus faecium. Antimicrob Agents Chemother. 1999 Dec;43(12):2873-6.
[3]. The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob Agents Chemother. 1997 Oct;41(10):2127-31.

However, there are two lines of reasoning to suggest that the percentage of time above MIC in plasma may not be a particularly accurate predictor of response in this model. First, in our infected rats receiving multiple doses of linezolid at 25 mg/kg q12h, it would appear from examination of an admittedly small number of data points that the terminal half-life may have been closer to 1.75 h. In that case, the percentage of time above MIC would have been closer to 60% for both linezolid and eperezolid. Second, although measurements of plasma concentrations were not available for rats treated with a continuous infusion of eperezolid, based on concentrations achieved with q12h dosing we estimated plasma levels of approximately twice the MIC for the entire dosing interval. It would thus appear that plasma concentrations substantially in excess of the MIC for a significant percentage of the dosing interval are required for demonstration of significant reduction in bacterial densities by these bacteriostatic agents in our model. We have confirmed previous observations documenting the in vivo activity of the oxazolidinones eperezolid and linezolid in experimental enterococcal infection. Nevertheless, effects were modest and therefore of uncertain clinical relevance. Further studies using optimized dosing based on comparative pharmacokinetics in humans and experimental animals and on the results of safety and tolerability profiles from clinical trials with the oxazolidinones may be appropriate. [2]

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The results of this study demonstrate rather convincingly that the oxazolidinones have no effect on peptidyl transferase or translation termination. If, in fact, the oxazolidinones act to block translation initiation, as has been suggested, it is tempting to postulate that the oxazolidinones bind to a site on the 50S subunit closely related to the chloramphenicol and lincomycin binding site and near the interface with the 30S subunit. The resulting distorted site may prevent the correct positioning of the 30S initiation complex from forming the 70S initiation complex and hence inhibit translation initiation. [3]

C18H20FN4O5

391.3794

394.165

C, 55.24; H, 5.15; F, 4.85; N, 14.32; O, 20.44

165800-04-4

73214

White to off-white Solid powder

1.37g/cm3

701.2ºC at 760 mmHg

377.9ºC

1.22E-20mmHg at 25°C

1.579

0.386

2

7

5

28

599

1

FC1C([H])=C(C([H])=C([H])C=1N1C([H])([H])C([H])([H])N(C(C([H])([H])O[H])=O)C([H])([H])C1([H])[H])N1C(=O)O[C@@]([H])(C([H])([H])N([H])C(C([H])([H])[H])=O)C1([H])[H]

SIMWTRCFFSTNMG-AWEZNQCLSA-N

InChI=1S/C18H23FN4O5/c1-12(25)20-9-14-10-23(18(27)28-14)13-2-3-16(15(19)8-13)21-4-6-22(7-5-21)17(26)11-24/h2-3,8,14,24H,4-7,9-11H2,1H3,(H,20,25)/t14-/m0/s1

N-[[(5S)-3-[3-fluoro-4-[4-(2-hydroxyacetyl)piperazin-1-yl]phenyl]-2-oxo-1,3-oxazolidin-5-yl]methyl]acetamide

Eperezolid; U 100592; Eperezolid; 165800-04-4; U-100592; N-[[(5S)-3-[3-fluoro-4-[4-(2-hydroxyacetyl)piperazin-1-yl]phenyl]-2-oxo-1,3-oxazolidin-5-yl]methyl]acetamide; C460ZSU1OW; PNU-100,592; PNU 100,592; U-100,592; PNU 100592;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~44 mg/mL (~111.56 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)