Nano-E Antibacterial Emulsion Poster 49th ICAAC Annual Meeting


AU-FQ antibacterial compounds: activity against Gram+ organisms and development of an emulsion formulation.

Sofya Dvoskin, D.V.M.,1 Andrew X. Chen, Ph.D.,2 HauChen, Ph.D.,2 and George E. Wright, Ph.D.1
1GLSynthesisInc, Worcester, MA 2LATITUDE Pharmaceuticals Inc., San Diego, CA

GLSynthesis Inc.
One Innovation Drive
Worcester, MA 01605
Ph 508 7546700
FAX 508 7547075


  • Background: “AU-FQ” hybrid antibacterials-6-anilinouracils connected via the 3 position to the 7-piperazinyl group of fluoroquinolones-are potent antibacterials in vitro and in vivo, and show promise as parenteraltreatments for multiantibioticresistant Gram+ infections. The preclinical candidate 259C, aka MBX500 (see structure), has posed delivery challenges because of its high dose and its pH-dependent solution stability. The product profile requires a dosage form that is easy to use parenterallyand stable under standard storage conditions. These challenges have been addressedby the development of F-20,a stable, pH-neutral, detergent-free, and lyophilizableformulation.
  • Methods: 259C wassynthesized by a proprietary method. Screening against Gram+ clinical isolates was done by MicromyxLLC, Kalamazoo, MI. Animal studies employed Swiss-Webster mice and Fischer rats, and analyses of parent compound and metabolite in plasma were done by LC-MS. Intravenous emulsions were rationally developed using a unique Nano-E™ emulsion technology, and analyzed by laser light scattering, microscopic evaluation, pH measurements, and HPLC.
  • Results: 259C was an effective antibacterial with MIC90values of 0.12-4 μg/mLagainst multiple clinical isolates of 18 Gram+ strains. Experimental emulsions of 259C up to 8 mg/mLwere prepared and analyzed for particle size and stability. The best emulsion “F-20” was stable for at least 6 months in liquid form at 2-8 oCand under accelerated conditions as lyophilized powder (30oC/70% RH). Analysis of plasma of IV-dosed rats revealed complex elimination of the parent compound and conversion to a single major metabolite, likely the carboxyl glucuronide.
  • Conclusions: Successful development of the F-20 emulsion formulation for 259C has enabled GLP preclinical toxicity studies in rats to be initiated. The emulsion does not adversely affect efficacy, pharmacokinetics or metabolism of 259C in the mouse or rat. The liquid emulsionswere stable for at least 6 months at 2-8oC, and the lyophilized versions, after reconstitution, were stable up to 6 months.



259C/MBX500 is the lead preclinical candidate from a group of hybrid antibacterials, consisting of a DNA polymerase IIIC inhibitory “EMAU” moiety (left hand) covalently bound to a “fluoroquinolone, FQ” antibacterial moiety via the 7-cyclic amine function (right hand). Members of this family of antibacterialsare potent inhibitors of DNA polymerase IIIC from Gram+ bacteria, and weak inhibitors of topoisomerase/gyrase. It is believed that the compounds owe their potent antibacterial activity to facilitation of uptake in sensitive organisms by the FQ moiety, rather than to dual target activity for example, the hybrids are equally active against cipro-resistant and sensitive organisms (Table 1).

Antibacterial activity

Gram-positive spectrum: 259C/MBX500 is a potent and selective inhibitor of Gram-positive aerobic bacteria. Table 1 summarizes the MIC50and MIC90values of the compound against 306 clinical isolates, and the effect of comparators vancomycin, linezolid, daptomycin, ciprofloxacin and oxacillinagainst the same organisms. MIC50values of 259C varied from 0.06-2 μg/mLand MIC90values varied from 0.12-4 ?g/mL.Strains that were resistant to comparators were uniformly sensitive to 259C.

Table 1. MICs of 259C and comparators vs. Gram+ clinical isolates
      MIC50, MIC90 (μg/ml)
Organism Phenotype # isolates 259C Vanco Linezolid Dapto Cipro Oxacillin
S. aureus MSSA 25 0.5,1 1,1 4,4 1,1 0.5,1 0.2,0.5
" MRSA 25 2,4 1,1 2,4 1,1 >32,
" CA-MRSA* 25 0.5,2 1,1 2,2 1,2 0.5,
S. epidermidis MSSE 25 1,2 2,2 1,2 0.5,1 0.25,
" MRSE 25 2,4 2,2 1,2 0.5,1 >32,
E. faecium VSE 15 2,4 1,1 2,2 8,8 >32,
" VRE 10 2,4 >64,>64 2,2 4,8 >32,
E. faecalis/faecium LRE† 11 2,4 >64,>64 16,32   >32,
S. pneumoniae PSSP 10 1,2 0.5,0.5 0.5,1 0.25,0.5 1,2 ≤0.06,0.25
" PISP 10 0.5,1 0.25,0.25 0.5,1 0.12,0.25 0.5,1 2,4
" PRSP 10 0.5,1 0.25,0.5 0.5,1 0.12,0.25 0.5,2 8,8
S. pyogenes   25 0.06,
0.5,0.5 1,1 0.06,0.12 0.12,
S. agalactiae   25 1,1 0.5,0.5 1,1 0.25,0.5 1,1 0.5,0.5
H. influenzae   20 1,1 >64,>64 8,16   ≤0.008,
M. catarrhalis   20 0.5,1 64,>64 4,4   ≤0.03,
*community acquired MRSA. †linezolid resistant Enterococci.

Emulsion development

A stable intravenous nanoemulsionformulation for 259C (F-20, Table 2) was developed using proprietary Nano-E™ technology, which is especially suitable for formulating highly insoluble small molecules, peptides and proteins. All components of the Nano-E™ system are GRAS listed and of compendialgrade. A Nano-E™ emulsion is a pH neutral, semi-transparent and lyophilizableemulsion with an average droplet size below 200 nm allowing for sterilization by filtration.

Table 2. Compositions of F-20 emulsions.
Components Vehicle 6 mg/g 8 mg/g
% (w/w) % (w/w) % (w/w)
259C 0 0.6 0.8
Soybean oil, super-refined 7 7 7
Miglyol 812 (medium chain triglycerides) 7 7 7
Phospholipon 90G (soy lecithin) 7 7 7
Sodium oleate* 0.3 0.3 0.3
Sucrose 15 15 15
NaOH and HCl (for pH adjustments) 8.0-8.2 8.0-8.2 8.0-8.2
Water-for-Injection QS QS QS
Total 100 100 100

F-20 has achieved a high drug load to about 8 mg/mLfor the highly insoluble 259C. Both liquid and lyophilized forms have been prepared (see Figures). The lyophilized form can be reconstituted with water for injection.

Liquid and lyophilized emulsions

Emulsion stability

F-20 stability parameters include appearance (visual and microscopic), pH, droplet size (see Table 3), assay (see Table 4) and chromatographic purity. No changes were observed in appearance and pH after 6 months of the liquid emulsion at 2-8°C or of the lyophilized emulsion up to 25°C.

Table 3. Average droplet size by laser light scattering (RH, relative humidity).
Sample Storage condition Initial 1 week 2 weeks 3 months 6 months
Liquid F-20 2-8°C ambient RH 71.3 73.2 93.5 76.9 77.1
25°C 60% RH 79.2 83.3 90.8 105
Lyophilized F-20 2-8°C ambient RH 168 127 120 138 170
25°C 60% RH 125 121 141 163

Table 4. 259C concentration by HPLC.
Sample Storage condition Initial 1 week 2 weeks 3 months 6 months
Liquid F-20 2-8°C ambient RH 8.0 7.8 8.0 7.8 7.9
25°C 60% RH 7.7 7.7 7.2 7.1
Lyophilized F-20 2-8°C ambient RH 7.8 8.2 8.2 7.9 7.9
25°C 60% RH 8.0 8.1 7.9 7.6

Efficacy and toxicity in mice

The F-20 emulsion formulation enabled delivery of 259C via the tail vein to Swiss-Webster mice to determine antibacterial efficacy against IP infection with S. aureus (Smith) and to determine acute toxicity. Efficacy and toxicity were similar to those obtained with the artificial vehicle “DCP” (Table 5), but without the severe thrombophelebitisobserved at the site of injection with the latter vehicle.

Table 5. Table 5. Efficacy and toxicity of 259C in mice*.
Vehicle ED50† mg/kg LD50 mg/kg
DCP 16 220
F-20 18 200
* via tail vein. † Sa(Smith) IP infection. DCP is 10% dimethylacetamide, 10% Cremophorin PBS, pH ca. 9.5.

Pharmacokinetics and metabolism in rats

The 259C emulsion was given as a bolus injection of 40 mg/kg in the tail vein of Sprague-Dawleyrats. Blood samples were removed at the indicated times from groups of 6 rats. The plot shows the average plasma concentration of 259C obtained from LCMS analysis vs. time. A major metabolite, the glucuronide259C_G, independently confirmed by MS, is seen with peak plasma concentration at ca. 120 min.

Averaged MS Data

PK parameters were derived from WinNonLinusinga one compartment model (Table 6). Results were similar to those from dosing of 259C in DCP or in 5% DMSO in saline, pH 10.2, via jugular vein catheters (not shown).

Table 6. Rat PK parameters for IV 259C in F-20
Parameter Value StdError CV%
t½ β 40.9 min 21.2 52
Cl 0.0273L/min 0.0096 35.3
Vss 1.61L/kg 0.53 33.4
PK parameters for 259C_G
Cmax 25.0 μM 2.49 10.0
Tmax 120 min


  • 259C is a broad, Gram+ spectrum antibacterial with no cross-resistance to current antibiotics.
  • The F-20 emulsion formulation enables IV dosing of 259C without irritation at the SOI.
  • The F-20 emulsion does not adversely affect the efficacy of 259C in mice.
  • The F-20 emulsion does not alter the PK properties of 259C in the rat.

GLP preclinical tox/path and safety pharmacology studies are underway with 259C in F-20 emulsion at 8 mg/ml.

Acknowledgements of support

Technical: Ivan Yanachkov, MilkaYanachkova
Financial: SBIR grant AI068349
Development: Microbiotix Inc.