In vitro and in vivo evaluation of positively charged liposaccharide derivatives as oral absorpti

For Peer Review

In vitro and in vivo evaluation of positively charged liposaccharide derivatives as oral absorption enhancers for

the delivery of anionic drugs

Journal: Journal of Pharmaceutical Sciences

Manuscript ID: 09-326

Wiley - Manuscript type: Research Article

Date Submitted by the Author:

22-May-2009

Complete List of Authors: Bergeon, Julie; The University of Queensland, School of Chemistry

and Molecular Biosciences Ziora, Zita; The University of Queensland, Centre for Integrated Preclinical Drug Development (CIPDD) Abdelrahim, Adel; The University of Queensland, School of Chemistry and Molecular Biosciences Pernevi, Niklas; The University of Queensland, School of Chemistry and Molecular Biosciences Moss, Anne; The University of Queensland, School of Chemistry and Molecular Biosciences Toth, Istvan; The University of Queensland, Centre for Integrated Preclinical Drug Development (CIPDD); The University of

Queensland, School of Chemistry and Molecular Biosciences

Keywords: Oral drug delivery, Absorption enhancer, Bioavailability, Caco-2 cells, Calorimetry (ITC), Pharmacokinetics/pharmacodynamics, Cationic lipids

John Wiley & Sons, Inc.

Journal of Pharmaceutical Sciences

For Peer Review

In vitro and in vivo evaluation of positively charged liposaccharide derivatives as oral absorption

enhancers for the delivery of anionic drugs

Julie A. Bergeon1, Zyta M. Ziora

2, Adel S. Abdelrahim

1, Niklas U. Pernevi, Anne R. Moss

1, Istvan Toth

1,2

1 The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072,

Australia 2 The University of Queensland, Centre for Integrated Preclinical Drug Development (CIPDD),

Brisbane, Qld 4072, Australia

Keywords: charged liposaccharide, ion-pairing, Caco-2 cells, calorimetry (ITC), absorption enhancer,

piperacillin, oral drug delivery, bioavailability

ABSTRACT

Oral delivery of hydrophilic, ionisable drugs remains a major challenge in drug development and a

number of active pharmaceuticals fail to reach the market of oral drugs because of a lack of

absorption and/or stability issues. One possible approach to improving the bioavailability of such

drug candidates is to increase their lipophilicity, which is a key parameter in the permeation across

cell membranes. However, modifying the chemical structure of an active compound by adding lipid

residues often results in changes in activity. With ionised molecules, ion-pairing can be considered to

associate charged lipid moieties with the parent drug without altering its structure and therefore

activity. This study presents the results of in vitro and in vivo evaluation of a series of positively

charged liposaccharide derivatives combined with an anionic model drug, piperacillin. The

antimicrobial activity, plasma stability, permeability in Caco-2 cell monolayers and oral absorption of

the synthesised conjugates were assessed.

INTRODUCTION

Recent advances in drug development have shown the crucial need for developing new active drugs

to treat and/or prevent diseases which currently resist conventional therapies.

However, the promising therapeutical profiles of a large number of potential drug candidates

(including ionic/ionisable molecules, peptides and protein-like pharmaceuticals with a high

hydrophilic character) is often dampened by issues such as low oral bioavailability and poor in situ

stability.1,2

To overcome these difficulties, different strategies such as structural modifications by

adjunction of various protecting groups to improve stability, or use of surfactants and absorption

enhancers to increase membrane permeability, essentially by passive diffusion, have been

investigated. 3,4

Considering the lipidic nature of the bilayered cell membrane, the derivatisation of

drugs by lipidation has been extensively investigated and improvements in enzymatic stability and

membrane permeability have been reported. 5,6

Although increased lipophilicity can facilitate

absorption across membranes, it is also frequently associated with solubility difficulties in aqueous

media.

Another approach to overcome absorption problems revolves around the ionizability of polar

molecules. Hydrophilic drugs are commonly commercialised as salts (e.g. sodium, acetate, chloride

salts), more soluble and often more stable7 than the parent molecule, which means that the drug

itself can potentially exhibit charges depending on pH conditions (both in solution and in situ). This

offers ample possibilities to associate lipophilic counter-ionic moieties by ion-pairing in order to

enhance the overall lipophilicity of these hydrophilic drugs while preserving their biologically active

chemical structure. 8

Page 1 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

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 β-lactam antibiotic to a positively

charged liposaccharide containing a D-glucose moiety coupled to a twelve-carbon long lipoamino

acid (α-amino acid with lipophilic alkyl side-chain). 9 Positive results were also observed when

varying the lipid chain length. Lipoamino acids (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. Lipoamino acids combined with

carbohydrates offer several advantages versus lipids alone. Firstly, 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. Secondly, sugars can be selected to interact with specific

membrane receptors where available, offering the possibility of a mediated transport across

membranes as an alternative to passive diffusion.

The amine function of the lipoamino acids 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 liposaccharides. Quaternization 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, adjustable balance of

lipophilicity versus hydrophilicity.

MATERIALS AND METHODS

1. Chemistry and analytical characterisation

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. 10

. Commercially available piperacillin sodium was converted into piperacillin acid using an IR-

120-[H+] cationic exchange resin (Rohm Haas, Philadelphia, PA, USA). The salt was dissolved in water

and vigorously stirred with the resin for 30 mins, 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 -80°C, < 150 psi. Two methodologies for lyophilisation were used; (i) with

piperacillin sodium, the 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

spectrometer, using a mixture of solvent A (1% formic acid in water) and B (1% formic acid in 9:1

acetonitrile/water) at 0.1 mL/min. For LC–MS/MS experiments, a Phenomenex luna C18 column (5

µm, 50 mm × 2.0 mm) equipped with a C18 guard column (4 × 3.0 mm) was attached to the mass

spectrometer and a 30-100% gradient of B in A over 4 minutes 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). 9

Data were acquired with Analyst 1.4 software (Applied Biosystems/ MDS Sciex, Toronto, Canada).

2. Isothermal Titration Microcalorimetry (ITC)

ITC measurements were performed on a MicroCal VP-ITC microcalorimeter (Northampton, MA,

USA), with Origin 5.0 and VP viewer 2000 software. Experiments were performed at 37°C in purified

water (MiiliQ Gradient A10, Millipore, North Ryde, NSW, Australia) with degassed, sonicated

Page 2 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

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 µL every 6 minutes (total

injection volume 300 µL).

3. Antimicrobial assays (MIC)

MIC experiments were carried using a broth dilution method. Two strains of bacteria were used,

Escherichia coli (MC4100) and Pseudomonas aeruginosa (A01). Bacteria 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 121°C for 20 minutes before use; reagents purchased from Sigma-Aldrich, Castle

Hill, NSW, Australia). Bacterial growth was monitored by optical density (OD) measurements,

performed on an Ultrospec 2000 (Pharmacia Biotech, Uppsala, Sweden). Bacteria were grown to an

OD ≥ 1 then diluted with LB broth and sub-cultured again to reach an OD of 0.3-0.8. They were then

diluted to a count of 1 × 106 cfu / 100 µL (OD 0.1) and plated in 96-well round bottom plates (TPP,

Zurich, Switzerland).

Lyophilised preparations of piperacillin and liposaccharides were dissolved in 5% dimethylsulfoxide

(DMSO)/water and serially diluted (1:3) with LB broth to reach a final piperacillin concentration of 0-

25 mM for E. coli and 0-52 mM for P. aeruginosa. The bacteria (100 µL) were then mixed with the

drug solutions (100 µL) and incubated for 16-20 hrs at 37°C, after which bacterial growth was

assessed by OD measurements at 600 nm on a Spectramax 250 plate reader (Molecular Devices,

Sunnyvale, CA, USA). The MICs for each formulation was then determined by plotting the OD values

vs piperacillin concentrations.

4. Haemolytic assays

Haemolytic assays were undertaken with approval from the University of Queensland Ethics

Committee (approval # 2006000950). Full venous blood samples were taken from healthy adult

volunteers. Red blood cells (hRBCs) were isolated from the whole blood by centrifugation at 2576 G

for 15 min (Sigma 2-5 centrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany).

Plasma was removed and the cells were washed several times with PBS before being resuspended in

PBS (to original blood volume) and aliquoted (100 µL) in 96-well flat bottom plates (TPP, Zurich,

Switzerland).

Solutions of the tested compounds were prepared in PBS (0.2 – 20 µM) and dispensed in the wells

containing the hRBCs preparations. The plates were then incubated at 37°C for 60 mins on a

Heidolph ShakingIncubator & Titramax 1000 (Schwabach, Germany) at 400 rpm, after which they

were centrifuged at 2129 G for 15 min; 75 µL of the supernatant was then removed and placed into

new plates before absorbance was measured at 540 nm using a Spectramax 250 microplate reader.

PBS was used as a negative control; water and SDS were used as positive controls. The percentage of

haemolysis caused by each of the tested formulation was calculated by the following equation:

where min A540 represents the absorbance at 540 nm of PBS alone, and max A540 represents the

absorbance at 540 nm of SDS.

5. Caco-2 permeability assay

Cell culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA) except of Hank’s

balanced saline solution (HBSS) and 14

C-labelled mannitol which were supplied by Sigma–Aldrich

(Castle Hill, NSW, Australia) and Amersham Biosciences (Piscataway, NJ, USA), respectively. Tissue

culture flasks (75 cm2) were obtained from BD Bioscience (Franklin Lakes, NJ, USA).

Page 3 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Caco-2 cells from the American type culture collection (Rockville, MD, USA) were maintained in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS),

and 1% nonessential amino acids at 95% humidity and 37°C in an atmosphere of 5% CO2. The

medium was changed every second day. After reaching 80% confluence, the cells were subcultured

using 0.2% ethylenediaminetetraacetic acid (EDTA) and 0.25% trypsin. 100 µL of a solution

containing approx. 106 cells/mL (passage number 31-40) were seeded onto polycarbonate

Transwell® inserts supplied by Coastar (Cambridge, MA, USA; mean pore size = 0.45 µm, 6.5 mm

diameter) and cultivated in DMEM supplemented with 10% FBS, 1% non-essential amino acids, 100

U/mL penicillin and 100 mg/mL streptomycin. The cells were allowed to grow for 21–28 days and the

medium was changed every other day.

The tested compounds were dissolved in HBSS–HEPES (HBSS buffered with 25 mM 4-(2-

hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.4) to a final concentration of 200 µM.

Prior to the transport studies, the Caco-2 monolayers were washed three times with pre-warmed

HBSS–HEPES and the integrity of the monolayers was assessed by measuring the TEER values using a

Millicell-ERS system (Millipore Corp., Bedford, MA, USA); TEER were also measured after each

experiment. Typical values were in the range 0.8-1.6 kΩ.cm2. The drug solutions (100 µL) were added

to the donor side of the monolayers and the Transwell® plates were placed in a shaking incubator

set to 400 rpm and 37°C for the duration of the experiment. At pre-determined time points (30, 90,

120 and 150 min), samples (400 µL) were taken from the receiver chamber and replaced with the

same amount of HBSS–HEPES. All compounds were tested in two to three independent assays, using

four wells each time. Concentrations in piperacillin were determined by LC–MS/MS. The transport of 14

C-mannitol (200 µM), a marker of paracellular transport, was also measured in parallel. After

dilution of samples (400 µL) with 4 mL of Wallac OptiPhase HiSafe 3 liquid scintillation cocktail

(Montreal, Canada), radioactivity was measured using a Beckman Coulter LS650 Multipurpose liquid

scintillation spectrometer (Beckman Instruments, Fullerton, CA) and permeability values determined

by the following equation:

where dC/dt is the steady-state rate of change in the chemical in the receiver chamber (mM, or dpm

mL-1

), Vr is the volume of the receiver chamber (mL), A is the surface area of the cell monolayers

(cm2) and C0 is the initial concentration in the donor chamber (mM, or dpm.mL

-1).

6. Oral absorption studies

Animal studies were undertaken with approval from the University of Queensland Ethics Committee

(approval #SMMS/002/08/ARC). 8-week old male Sprague Dawley rats (300-350g, 4 rats/compound)

were administered formulations of the tested compounds in 5% DMSO/water by oral gavage, to a

level of 50 mg/kg of piperacillin for each rat. At regular time points (0, 5, 10, 15, 30, 60, 90, 120

minutes), blood samples (200 µL) were collected in heparinised tubes by tail vein bleeding. The rats

were euthanized immediately after the experiment by inhalation of a 1:1 O2/CO2 mixture; death was

further asserted by cervical dislocation.

Blood samples were centrifuged for 20 min at 1717 g and the plasma was collected, treated with

acetonitrile (1:1 v/v) to precipitate the proteins and centrifuged. The supernatant was then collected

for analyses by LC-MS/MS (or kept at -30°C if analyses could not be run immediately).

7. Statistical analysis

Results were expressed as mean values ± standard error mean (SEM). Where applicable, statistical

analysis of values was performed by one-way analysis of variance (ANOVA) of repeated measures to

a significance level of 0.05 (p < 0.05), followed by Tukey’s post hoc test (multiple pairwise

Page 4 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

comparison of means). Computations were realised using GraphPad Prism v5.01 (GraphPad

software, La Jolla, CA, USA).

RESULTS AND DISCUSSION

Design and preparation of a new series of charged liposaccharide derivative carriers

A novel series of liposaccharide derivatives was synthesised by modifying the amino extremity of the

liposaccharide. The resulting compounds comprised a D-glucose (G) moiety and a twelve carbon long

LAA (C12) scaffold, onto which additional amino residues, namely leucine, phenylalanine (two of the

most hydrophobic amino acids) 11

and another C12 were attached. The N-terminus amino group was

then converted into a quaternary ammonium salt using Amberlite 400 [HO-] as a base

10 and the

obtained liposaccharide derivatives were lyophilised together with the model drug piperacillin (Fig.

1).

Fig. 1 – Synthesised charged liposaccharide derivatives associated with model drug piperacillin

Initially, lyophilisation was attempted directly with the commercially available piperacillin sodium

salt under acidic conditions (glacial acetic acid); however, preliminary observations (binding studies

and nuclear magnetic resonance analyses, results not shown) suggested that limited interactions

occurred between the anionic drug and its cationic liposaccharide counterparts. It is believed that

this could be due to strong ionic binding between the piperacillin anion and the sodium cation,

which is smaller and has a greater charge density compared to the charged liposaccharide

derivatives (calculated Hückel charge of 0.788 (GC12C12)); as a consequence, the exchange between

the two cations was likely to be thermodynamically unfavourable. To facilitate this substitution,

piperacillin sodium was dissolved in water and stirred with a cation-exchange resin under controlled

pH to obtain piperacillin acid, which was then immediately lyophilised with different liposaccharide

derivatives from a 1:1 acetonitrile-water solution. The prepared conjugates were stored at -20°C and

their integrity was monitored by analytical examination by 1H-NMR.

Different ratios of drug/liposaccharide were considered in this study. Initial measurements of

binding by isothermal titration microcalorimetry (ITC) revealed that two concurrent processes were

taking place, binding (ion-pairing) and micellization. A 1:1 ratio did not seem to be sufficient to

observe optimum ionic interactions, possibly due to the presence of multiple interaction sites on the

model drug. Indeed, when examining interactions between piperacillin and GC12C12, a ratio close to

1:2 drug/liposaccharide was found to give optimal ion interactions (Fig. 2). The negative enthalpy

observed initially below a 1:2 ratio is indicative of favourable ion-pairing, while above 1:2, the

process becomes endothermic as a result of micellization occurring. As the ratio of

drug/liposaccharide was increased, the micellization of the lipidic cations was predominantly

observed over the binding process (results not shown) and therefore ion-pairing, if present, could

not be significantly assessed.

Figure 2. Isothermal titration microcalorimetry (ITC) binding assay between piperacillin (acid form,

0.2 mM) and liposaccharide derivative GC12C12 at 4 mM in purified water.

All liposaccharide derivatives were therefore tested in association with piperacillin in a 1:2

drug/counter-ion ratio. A 1:1 ratio was also examined to see if even a lower quantity of

Page 5 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

liposaccharide derivatives was sufficient to generate an increase in oral absorption (penetration

enhancing effect).

Antimicrobial activity

The antimicrobial activity of the drug conjugates was assessed in vitro in two Gram-negative

bacterial strains, Escherichia coli and Pseudomonas aeruginosa. Minimum inhibitory concentrations

(MICs) were determined by a broth dilution method 12

and compared to the MIC of piperacillin

alone. Previously synthesised 2-amino-N-(2’-α,D-gluco)dodecanamide (GC12) 9 was also tested in the

same 1:1 and 1:2 molar ratios.

After 16-20 hours incubation with bacterial cultures at 37 °C, resistance to piperacillin alone was

found to be in agreement with reference values of 2-4 µg/mL for E. Coli 13

and 3-6 µg/mL for most

strains of P. aeruginosa. 14,15

. When the drug was tested in association with the synthesised charged

liposaccharide derivatives, no significant change was observed in the MIC values for P. aeruginosa,

all averaging 7-12 µg/mL (Fig. 3). For E. coli, slight, however not significant, increases in the MICs

were noticed when piperacillin was formulated with GC12Phe (1:1 and 1:2) and GC12C12 (1:1).

The pairing of the liposaccharide derivatives to piperacillin therefore does not appear to have

negatively impacted the original antimicrobial activity against either bacterial strain. The absence of

significant variation in MICs (verified using a one-way Anova statistical comparison followed by

Tukey’s post-hoc test) when using different ratios of liposaccharides would indicate that the

modified liposaccharides do not have antimicrobial or antagonist properties of their own.

Figure 3. MICs of piperacillin alone and formulated with the synthesised liposaccharide derivatives

for two Gram-negative bacterial strains, E. coli and P. aeruginosa. Concentrations were

determined by optical density measurements at 600 nm after 24 hr incubation in bacterial culture

medium at 37°C. (Values given as mean ± SEM, n≥3)

Haemolytic activity

Although destined to oral drug delivery, the liposaccharide derivatives and their ionic complexes

with piperacillin were nonetheless tested for signs of haemolytic activity, which could cause

disruptions to the biological membranes and yield to toxicity.16,17

Haemolytic activity was assessed in

triplicate against human red blood cells (hRBCs) over 60 minutes at 6 different concentrations,

ranging from 0.1 µM to 10 mM; the tested solutions were formulated in phosphate buffered saline

(PBS) and compared to piperacillin alone. Sodium dodecylsulphate (SDS), a surfactant known to be

toxic to hRBCs at the concentrations used in this assay, was also included as a positive control.

Measurements were done by absorbance readings at 540 nm and the percentage of haemolysis was

determined by the following equation:

Initially, only the synthesised liposaccharide derivatives were considered; one of the complexes, Pip-

GC12C12, was additionally evaluated to examine any possible synergistic effect.

Figure 4. Haemolytic activity measured against hRBCs at 37°C over 1hr of piperacillin (alone and

with GC12C12) and the synthesised liposaccharide derivatives in PBS. The percentage of haemolysis

was calculated by absorbance readings at 540 nm (results given as mean ± SEM, n=3)

Page 6 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

As shown in Figure 4, the synthetic liposaccharide derivatives expectedly induced haemolysis of

hRBCs to some extent, yet the values remain significantly less than those of SDS in the low

concentration range. Interestingly, the comparison between GC12 and GC12C12 revealed a two-fold

increase on average in haemolytic activity, suggesting a direct correlation between the greater

lipophilicity brought by the second C12 moiety and the haemolytic properties of the liposaccharide.

At commonly used therapeutic concentrations however (i.e. < 1 mM when considering the

standardised intravenous administration of piperacillin sodium), most liposaccharide derivatives

tested showed less than 25 % haemolysis, which is considered to be the threshold for tolerable

toxicity to hRBCs. 18

However, piperacillin in association with GC12C12 in 1:1 and 1:2 ratios yielded

synergistic effects resulting in increased haemolysis, to levels comparable to SDS.

When examining the EC10 (drug concentration causing 10% haemolysis of hRBCs; see Table 1 – the

values were estimated from the graphical data presented in Fig. 4), GC12 and GC12Leu were found to

be moderately haemolytic (0.5 mM < EC10 < 5 mM) while the other two liposaccharide derivatives

GC12Phe and GC12C12 as well as the combined Pip-GC12C12 were classified as strongly haemolytic (0.5

mM < EC10), along with SDS. 17

Table 1. Estimated EC10 of drug, liposaccharide derivatives and combined entities.

These findings are of great importance when considering LAAs as penetration enhancer candidates

for general drug delivery. Although the concentrations used in this assay are far greater than those

used in vivo, the LAA-based molecules tested here showed noticeable signs of toxicity against hRBCs.

In vitro permeability assessment in Caco-2 cells

Prior to conducting oral absorption studies, the in vitro permeability of the synthesised compounds

was assessed in Caco-2 cells. This cell line originates from a human colorectal adenocarcinoma and is

commonly used as a model for predicting the transport of drugs across the intestinal epithelium.

When cultured, Caco-2 cells spontaneously form differentiated, polarised monolayers which express

most structural and functional characteristics of the small intestine, including enzymes and efflux

proteins. Artursson et al. demonstrated that the apparent permeability coefficients determined with

this model can be correlated to human oral drug absorption values with good respect. 19

.

200 µM solutions of piperacillin associated with the different liposaccharide derivatives (1:1 and 1:2

molar ratios) in Hank’s balanced salt solution (HBSS) were added to the apical side of the Caco-2

monolayers and permeability to the basolateral side of the monolayer was studied over 150 min,

using the paracellular marker 14

C-mannitol as a negative control (typical permeability values of 14

C-

mannitol are around 1-5 × 10-7

cm.s-1

19

). Apparent permeability coefficients (Fig. 5) were calculated

from concentrations of piperacillin determined by liquid chromatography coupled to mass

spectrometry (LC-MS/MS) against an 8 point standard curve while 14

C-mannitol was quantified by

liquid scintillation counting (β-emission) 9.

Piperacillin alone expectedly showed very low permeability across Caco-2 monolayers; the observed

values were higher than reported previously, 3.07(OH)-3.22(Na) × 10-7

cm.s-1

versus 3.24(Na) × 10-8

cm.s-1

9. A similar 10-fold variation was noticed for Pip-GC12 [1:1] and

14C-mannitol, this variability

could be explained by the difference in cell passage numbers, since the cells used in the present

study were much younger than in the previous examination. Reports in literature have shown that

the differentiation of the cells was affected by early passage numbers, mainly a lower cohesion of

Page 7 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

the monolayer and reduced tightness of the paracellular junctions. 20

This was also evidenced by

measurement of transepithelial electrical resistances (TEER), 21

which averaged 0.9-1.1 kΩ in this

study, slightly less than in previous experiments.

As shown in Figure 5, piperacillin demonstrated a marked increase in membrane permeability when

associated in a 1:1 ratio with GC12, which is in agreement with previously reported data. 9

Interestingly, when testing GC12C12 in that same ratio, the increase was slightly less. This could be

correlated to the ITC data which suggest that a 1:1 ratio was not sufficient in the case of GC12C12 to

obtain optimal binding between the parent drug and the charged liposaccharide derivatives. Indeed,

when assessing the drug in a 1:2 ratio with the liposaccharide derivative, the permeability was

greatly enhanced to reach a mean value of 1.27 × 10-5

cm.s-1

, nearly forty times higher than

piperacillin alone. Drugs with apparent permeability coefficients across Caco-2 monolayers of 10-6

cm.s-1

or greater usually present excellent oral bioavailability. 19

; the results obtained in this in vitro

assay therefore suggest that Pip-GC12C12 [1:2] might be a good candidate for oral absorption

evaluation.

Figure 5. Apparent permeability coefficients determined in 21-28 day old monolayers of Caco-2

cells (passage 31-40) over a 150 min period at 37°C (Results given +/- SEM, n=2-3; 4 wells/exp).

The major increase in permeability observed with Pip-GC12C12 [1:2] can also, to a certain extent, be

linked to its toxicity, as evidenced earlier in the haemolytic assay. The measurement of the TEER

values prior to, and after each experiment revealed a drop in resistance across the monolayer, sign

of loss of integrity of the monolayer. It is however unknown whether the loss was permanent or if

the monolayer would recover given time and optimum culture conditions. The other conjugates

tested did not yield to such marked variation in TEER. Surprisingly, the penetration of piperacillin

was not significantly improved when combined with GC12 in a 1:2 molar ratio. No satisfactory ITC

data could be obtained for GC12 so the ratio for optimal binding could not be assessed accurately. It

is also possible that alternative phenomena occurred that could decrease the permeability

enhancing effects of GC12 (e.g. efflux, aggregation, adhesion to membranes). The other

liposaccharide derivatives tested in formulation with piperacillin did not yield significant changes in

permeability, independent of the ratio used.

Oral absorption in rats

Based on the in vitro results and particularly the permeability assays, four of the tested formulations

were evaluated in vivo, namely Pip-GC12 [1:1], Pip-GC12 [1:2], Pip-GC12C12 [1:1] and Pip-GC12C12 [1:2].

Male Sprague-Dawley rats (300-350 g) were administered the formulations by oral gavage; blood

samples were taken from the tail vein over a two hour period and analysed by LC-MS/MS.

Piperacillin given both orally and intravenously (i.v.) was used as a control. The i.v. curve (Fig. 6a)

demonstrates a rapid distribution of piperacillin after injection with mean peak concentration of 70

µg.mL-1

at around 5 minutes. The antibiotic is then rapidly cleared from the circulatory system with

virtually no drug detected after 1h.

The oral uptake of piperacillin was calculated from the AUC120 (mean area under the curve, see Table

2) and was expectedly very low (0.5 %). When examining the absorption of the piperacillin

conjugates, the absorption curves were very similar to that of piperacillin alone, administered orally.

Surprisingly, the improvements in permeability noticed in vitro with the Caco-2 monolayer model

were not observed in vivo, either with GC12 (1:1) or GC12C12 (1:1 and 1:2). The determination of the

AUCs showed 0.14 % to 0.38 % absorption, which statistically is not significantly different from the

0.5 % absorption found for commercial piperacillin sodium (Fig. 6b).

Page 8 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 6a. In vivo pharmacokinetics of piperacillin sodium administered intravenously at 50 mg/kg

in saline (0.9 % NaCl) over a 2h period; piperacillin concentration in blood was determined by LC-

MS/MS (Results given +/- SEM, n=3-5).

Figure 6b. In vivo absorption and pharmacokinetics of oral formulations of piperacillin, alone or in

association with liposaccharide derivatives GC12 and GC12C12, in 1:1 and 1:2 ratios in water + 5%

DMSO, over a two hour period. A dose equivalent to 50 mg piperacillin was administered by oral

gavage to 300-350 g Sprague-Dawley rats and piperacillin concentration in blood determined by

LC-MS/MS (Results given +/- SEM, n=3-5).

Table 2. Area Under the Curve (AUC120) values for the in vivo pharmacokinetics profile of

piperacillin alone (oral and i.v.) and in association with liposaccharide derivatives GC12 and GC12C12.

Results are given as mean and relative values against intravenous piperacillin levels.

The literature abounds in reports correlating in vitro predictions of permeability obtained with the

Caco-2 model of intestinal epithelium and in vivo oral absorption. The variations observed in these

experiments are therefore more likely to be inherent to the nature of the compounds tested, as it

appears that piperacillin was most probably released from the liposaccharide derivatives post-

administration.

CONCLUSION

We report here the biological evaluation of a novel series of ionic liposaccharide derivatives

designed as potential oral penetration enhancers. The synthetic constructs were engineered to

combine in a single molecular entity a carbohydrate-based hydrophilic moiety coupled to lipophilic

residues, such as hydrophobic amino acids and twelve carbon long LAAs. Piperacillin, a potent β-

lactam antibiotic with known low oral bioavailability, was used as a model drug and associated in

various molar ratios with the liposaccharide derivatives by lyophilisation. ITC measurements were

performed to determine the critical micelle concentration and binding affinity of the amphiphilic

conjugates and led to two ratios being considered for further examinations, respectively 1:1 and 1:2

drug/liposaccharide.

Antimicrobial assays revealed that the minimum inhibitory concentration of drug was practically

unchanged when piperacillin was combined with the liposaccharide derivatives. When examining the

effect of the tested conjugates against human red blood cells, an increase of haemolytic activity to

levels similar to SDS was observed in the case of Pip-GC12C12, yet the levels of toxicity remained low

when piperacillin and the liposaccharide derivatives were tested alone; the values observed denoted

a direct correlation between the lipophilicity of the conjugates and their haemolytic activity. The in

vitro evaluation of the synthesised associates in Caco-2 monolayers showed that apparent

membrane permeability values were significantly improved with three formulations, Pip-GC12 (1:1),

Pip-GC12C12 (1:1) and Pip-GC12C12 (1:2). The latter displayed in vitro permeability coefficients nearly

forty times higher than the parent piperacillin drug alone. Unfortunately, these promising results

were not confirmed in vivo. Current research is now being focused on investigating and

characterising the nature of the interactions between the drug and its counter-ionic liposaccharide

derivative, as well as their stability at various pH values.

ACKNOWLEDGEMENTS

Page 9 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

This work was supported by an ARC project grant (DP0558334). The authors wish to thank Prof

Michael F. Jennings for his kind assistance in providing facilities and equipment to conduct the

microbiology experiments, and Mr Michael Moore for his help with handling animals.

REFERENCES

1. Trapani A, Garcia-Fuentes M, Alonso MJ. 2008. Novel drug nanocarriers combining hydrophilic

cyclodextrins and chitosan. Nanotechnology 19(18):185101/185101-185101/185110.

2. Cano-Cebrian MJ, Zornoza T, Granero L, Polache A. 2005. Intestinal absorption enhancement

via the paracellular route by fatty acids, chitosans and others: A target for drug delivery. Curr

Drug Deliv 2(1):9-22.

3. Shaji J, Patole V. 2008. Protein and Peptide Drug Delivery: Oral Approaches. Indian J Pharm Sci

70(3):269-277.

4. Touitou E, Barry BW. 2006. Enhancement in Drug Delivery. ed.: CRC Press. p 633.

5. Pouton CW, Porter CJH. 2008. Formulation of lipid-based delivery systems for oral

administration: Materials, methods and strategies. Adv Drug Deliv Rev 60(6):625-637.

6. Wong A, Toth I. 2001. Lipid, Sugar and Liposaccharide Based Delivery Systems. Curr Med Chem

8:1123-1136.

7. Bastin RJ, Bowker MJ, Slater BJ. 2000. Salt Selection and Optimisation Procedures for

Pharmaceutical New Chemical Entities. Organic Process Research & Development 4(5):427-

435.

8. Neubert R. 1989. Ion-pair transport across membranes. Pharm Res 6(9):743-747.

9. Violette A, Cortes DAF, Bergeon JA, Falconer RA, Toth I. 2008. Optimized LC-MS/MS

quantification method for the detection of piperacillin and application to the development of

charged liposaccharides as oral penetration enhancers. Int J Pharm 351(1-2):152-157.

10. Abdelrahim AS, Ziora ZM, Bergeon JA, Moss AR, Toth I. 2009. Synthesis and calorimetric

titration of new glucolipopeptides as potential cationic penetration enhancers. Tetrahedron

((submitted)).

11. Kyte J, Doolittle RF. 1982. A simple method for displaying the hydropathic character of a

protein. J Mol Biol 157(1):105-132.

12. Andrews JM. 2001. Determination of minimum inhibitory concentrations. J Antimicrob

Chemoth 48:5-16.

13. Nolting A, DallaCosta T, Rand KH, Derendorf H. 1996. Pharmacokinetic pharmacodynamic

modeling of the antibiotic effect of piperacillin in vitro. Pharm Res 13(1):91-96.

14. Hoogkampkorstanje JAA, Westerdaal NAC. 1979. Activity and synergy of piperacillin and

aminoglycosides against Pseudomonas-Aeruginiosa. A Van Leeuw J Microb 45(4):617-618.

15. Korvick JA, Yu VL. 1991. Antimicrobial agent therapy for Pseudomonas aeruginosa. Antimicrob

Agents Chemother 35(11):2167-2172.

16. Pignatello R, Noce C, Campisi A, Acquaviva R, Bucolo C, Puglisi G, Toth I. 2007. Evaluation of

Cell Tolerability of a Series of Lipoamino Acids Using Biological Membranes and a

Biomembrane Model. Curr Drug Deliv 4:109-121.

17. Ross BP, Braddy AC, McGeary RP, Blanchfield JT, Prokai L, Toth I. 2004. Micellar aggregation

and membrane partitioning of bile salts, fatty acids, sodium dodecyl sulfate, and sugar-

conjugated fatty acids: Correlation with hemolytic potency and implications for drug delivery.

Molecular Pharmaceutics 1(3):233-245.

18. Amin K, Dannenfelser RM. 2006. In vitro hemolysis: Guidance for the pharmaceutical scientist.

J Pharm Sci 95(6):1173-1176.

19. Artursson P, Palm K, Luthman K. 2001. Caco-2 monolayers in experimental and theoretical

predictions of drug transport. Adv Drug Deliv Rev 46(1-3):27-43.

Page 10 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20. BriskeAnderson MJ, Finley JW, Newman SM. 1997. The influence of culture time and passage

number on the morphological and physiological development of Caco-2 cells. Proceedings of

the Society for Experimental Biology and Medicine 214(3):248-257.

21. Donna AV. 2008. Variability in Caco-2 and MDCK cell-based intestinal permeability assays. J

Pharm Sci 97(2):712-725.

Page 11 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Table 1. Estimated EC10 of drug, liposaccharide derivatives and combined entities. 58x42mm (600 x 600 DPI)

Page 12 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Table 2. Area Under the Curve (AUC120) values for the in vivo pharmacokinetics profile of

piperacillin alone (oral and i.v.) and in association with liposaccharide derivatives GC12 and GC12C12. Results are given as mean and relative values against intravenous piperacillin levels.

123x61mm (150 x 150 DPI)

Page 13 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Fig. 1 – Synthesised charged liposaccharide derivatives associated with model drug piperacillin 78x36mm (600 x 600 DPI)

Page 14 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 2. Isothermal titration microcalorimetry (ITC) binding assay between piperacillin (acid form, 0.2 mM) and liposaccharide derivative GC12C12 at 4 mM in purified water.

96x135mm (600 x 600 DPI)

Page 15 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 3. MICs of piperacillin alone and formulated with the synthesised liposaccharide derivatives for two Gram-negative bacterial strains, E. coli and P. aeruginosa. Concentrations were determined by optical density measurements at 600 nm after 24 hr incubation in bacterial culture medium at

37°C. (Values given as mean ± SEM, n≥3) 254x190mm (150 x 150 DPI)

Page 16 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 4. Haemolytic activity measured against hRBCs at 37°C over 1hr of piperacillin (alone and with GC12C12) and the synthesised liposaccharide derivatives in PBS. The percentage of haemolysis

was calculated by absorbance readings at 540 nm (results given as mean ± SEM, n=3) 229x162mm (150 x 150 DPI)

Page 17 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 5. Apparent permeability coefficients determined in 21-28 day old monolayers of Caco-2 cells (passage 31-40) over a 150 min period at 37°C (Results given +/- SEM, n=2-3; 4 wells/exp).

254x190mm (150 x 150 DPI)

Page 18 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 6a. In vivo pharmacokinetics of piperacillin sodium administered intravenously at 50 mg/kg in saline (0.9 % NaCl) over a 2h period; piperacillin concentration in blood was determined by LC-

MS/MS (Results given +/- SEM, n=3-5). 297x210mm (150 x 150 DPI)

Page 19 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 6b. In vivo absorption and pharmacokinetics of oral formulations of piperacillin, alone or in association with liposaccharide derivatives GC12 and GC12C12, in 1:1 and 1:2 ratios in water + 5%

DMSO, over a two hour period. A dose equivalent to 50 mg piperacillin was administered by oral gavage to 300-350 g Sprague-Dawley rats and piperacillin concentration in blood determined by LC-

MS/MS (Results given +/- SEM, n=3-5). 297x210mm (150 x 150 DPI)

Page 20 of 20



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Documents

In vitro and in vivo evaluation of positively charged liposaccharide derivatives as oral absorpti