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BERGEON ET AL.
the potential of charged glycolipids to form ion-pairs
with a model drug like piperacillin.9,10
liposaccharide derivatives were mixed with the
commercial salt and dissolved in pure glacial acetic
acid; the excess acid was removed under vacuo and
the mixture was then lyophilised; (ii) with piperacillin
acid, the liposaccharide derivatives and the drug were
dissolved in water/acetonitrile (1:1) and lyophilised.
ESI-MS and MS/MS analyses were carried out on a
PE Sciex API3000 triple quadrupole mass spectro-
meter, using a mixture of solvent A (1% formic acid in
water) and B (1% formic acid in 9:1 acetonitrile/water)
In an earlier study, we reported that the in vitro
permeability of piperacillin across Caco-2 monolayers
could be notably improved by associating this anionic
b-lactam antibiotic to a positively charged liposac-
charide containing a D-glucose moiety coupled to a 12-
carbon long lipoamino acid (LAA) (a-amino acid with
lipophilic alkyl side-chain).10 Positive results were
also observed when varying the lipid chain length.
LAAs are versatile molecules with the unique ability
to be linked through either their amino or carboxyl
extremities. They can be readily obtained from alkyl
bromide precursors in a few, short and easy synthetic
steps. LAAs combined with carbohydrates offer
certain advantages versus lipids alone; for example,
it allows for a modulation of the degree of lipophilicity
brought by the lipid chain via the hydroxyl residues
present on the carbohydrate moiety, hence preserving
the water solubility characteristics of the modified
drug while still increasing its lipophilicity.
at 0.1 mL/min. For LC–MS/MS experiments,
Phenomenex luna C18 column (5 mm, 50 mm Â
2.0 mm) equipped with C18 guard column
a
a
(4 mm  3.0 mm) was attached to the mass spectro-
meter and a 30–100% gradient of B in A over 4 min
was used for elution at a flow rate of 0.5 mL/min with
a 1:10 splitter upstream from the ionisation source
(Shimadzu LC-10AT system).10 Data were acquired
with Analyst 1.4 software (Applied Biosystems/MDS
Sciex, Toronto, Canada).
The amine function of the LAAs is subject to acido-
basic equilibrium and therefore is not constantly
protonated, which can be problematic as this residual
charge is the key element of the interaction between
the drugs of interest and counter ionic liposacchar-
ides. Quaternisation of the terminal amine was
therefore considered to create a constantly, positively
charged residue. Also, as the sugar moiety introduced
a significant degree of hydrophilicity, additional
lipophilic components were considered in the design
of new charged liposaccharides for a better, adjus-
table balance of lipophilicity versus hydrophilicity.
Isothermal Titration Microcalorimetry (ITC)
ITC measurements were performed on a MicroCal
VP-ITC microcalorimeter (Northampton, MA), with
Origin 5.0 and VP viewer 2000 software. Experiments
were performed at 378C in purified water (MiiliQ
Gradient A10, Millipore, North Ryde, NSW, Austra-
lia) with degassed, sonicated samples. The reference
cell was loaded with water while the sample cell
(1.4395 mL) was charged with piperacillin acid (0.05–
0.5 mM) and rotated at 300 rpm. The liposaccharide
derivatives (4 mM) were injected into the sample cell
using a microsyringe at a rate of 3–10 mL every 6 min
(total injection volume 300 mL).
MATERIALS AND METHODS
Chemistry and Analytical Characterisation
Antimicrobial Assays (MIC)
All solvents and reagents were obtained at the
highest available purity from Sigma–Aldrich (Castle
Hill, NSW, Australia) and used directly without
further purification. The liposaccharide derivatives
presented in this publication were prepared according
to the methodology described by Abdelrahim et al.11
Commercially available piperacillin sodium was
converted into piperacillin acid using an IR-120-
[Hþ] cationic-exchange resin (Rohm Haas, Philadel-
phia, PA). The salt was dissolved in water and
vigorously stirred with the resin for 30 min, until pH
stabilised at 3 and piperacillin acid precipitated. The
precipitate was then dissolved in acetonitrile and the
resin was filtered off, washed with acetonitrile, and
the filtrate was then lyophilised on an Alpha 2-4/LSC
(Martin Chris, Osterode am Harz, Germany) at
À808C, <150 psi. Two methodologies for lyophilisa-
tion were used; (i) with piperacillin sodium, the
Minimum inhibitory concentration (MIC) experi-
ments were carried using a broth dilution method.
Two strains of bacteria were used, Escherichia coli
(MC4100) and Pseudomonas aeruginosa (A01). Bac-
teria were cultured in Luria-Bertani (LB) broth
medium (1%, w/v NaCl, 1%, w/v tryptone, 0.5%, w/v
yeast extract, adjusted to pH 7 and autoclaved at
1218C for 20 min before use; reagents purchased from
Sigma–Aldrich). Bacterial growth was monitored by
optical density (OD) measurements, performed on an
Ultrospec 2000 (Pharmacia Biotech, Uppsala, Swe-
den). Bacteria were grown to an OD ꢀ 1 then diluted
with LB broth and subcultured again to reach an OD
of 0.3–0.8. They were then diluted to a count of
1 Â 106 cfu/100 mL (OD 0.1) and plated in 96-well
round bottom plates (TPP, Zurich, Switzerland).
Lyophilised preparations of piperacillin and lipo-
saccharides were dissolved in 5% dimethylsulfoxide
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
DOI 10.1002/jps