Amphiphilic ion pairs of tobramycin with lipoamino acids

Article abstract of DOI:10.1016/j.ejmech.2011.02.015

A series of amphiphilic ion pairs of the aminoglycoside antibiotic tobramycin (TOB) with lipoamino acids (LAA) bearing an alkyl side chain of 10-14 carbon atoms are described. TOB-LAA ion pairs were obtained by reduced pressure evaporation of an aqueous-e

Full text of DOI:10.1016/j.ejmech.2011.02.015

European Journal of Medicinal Chemistry 46 (2011) 1665e1671
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Amphiphilic ion pairs of tobramycin with lipoamino acids
,
b
a
c
b
Rosario Pignatello a , Annalisa Mangiafico , Livia Basile , Barbara Ruozi , Pio M. Furneri
*
a Department of Drug Sciences, University of Catania, Catania, Italy
b Department of Microbiological Sciences and Gynaecological Sciences, University of Catania, Catania, Italy
c Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, Modena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of amphiphilic ion pairs of the aminoglycoside antibiotic tobramycin (TOB) with lipoamino acids
(LAA) bearing an alkyl side chain of 10e14 carbon atoms are described. TOB-LAA ion pairs were obtained
by reduced pressure evaporation of an aqueous-ethanol co-solution of TOB and LAAs. A different degree
of substitution of TOB amine groups was obtained by using increasing the drug to LAA molar fractions
(1:1 to 1:5). FTIR analysis corroborated their structure, powder X-ray diffractometry (PXRD) and
differential scanning calorimetry (DSC) were used to identify the formation of new chemical species.
The prepared compounds were submitted to an in vitro microbiological assay against different
bacterial strains, both susceptible and resistant to aminoglycosides. The presence of only one LAA moiety
did not improve the in vitro antibacterial activity of TOB free base. Analogously, equimolar physical
mixtures (PhM) of TOB with LAA failed to exert a remarkable cell growth inhibitory activity.
Noteworthy, when three or all the five amine groups of TOB were salified with LAA residues, very
Received 9 October 2010
Received in revised form
11 January 2011
Accepted 11 February 2011
Available online 19 February 2011
Keywords:
Tobramycin
Lipoamino acids
Amphiphilicity
Hydrophobicity
Antimicrobial activity
Gram negative bacteria
active compounds were produced, showing MIC values lower than the detectable limit of 0.03 mg/ml.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Moreover, in the presence of lower drug concentrations the inac-
tivating activity of bacterial enzymes can be more successful.
As many antibacterial drugs, TOB has been submitted to
extensive investigations looking for more active derivatives, as well
as to preformulation studies aimed at obtaining more stable forms
and medicines with improved properties.
The sensitivity of some specific positions in the molecule of TOB
to chemical modifications has been well delineated, particularly for
the five amine groups. In some instances such changes resulted in
strong changes of the antibacterial activity or sensitivity to inacti-
vating enzymes [2e5].
Aminoglycoside antibiotics are generally converted to salt forms
during recovery and purification procedures. For example, TOB is
converted to the pentasulfate salt to obtain a water soluble
compound suitable for parenteral injection. Conversion to inor-
ganic salts can change the pharmacokinetics of the drug, but may
be unable to affect their interaction with and uptake by the
bacterial cells. Recently, the so-called hydrophobic ion pairing
approach (HIP) has been proposed as a chemical strategy to
reversibly modify the properties of drug molecules. By the HIP, in
drug compounds containing ionizable groups, polar counter ions
are stoichiometrically replaced with more hydrophobic ones. The
resulting ion pairs, being more hydrophobic than the parent
compounds, can improve drug permeability and transport through
biological membranes, ensure a better systemic adsorption, and
enhance the drug uptake by cells [6e8]. Furthermore, the
Aminoglycoside antibiotics are bactericide agents extensively
used in the clinical therapy of many infectious diseases. Among
them, tobramycin (TOB; Fig. 1) is broad-spectrum antibiotic,
effective against Gram negative bacteria, especially the Pseudo-
monas species. It represents a 10% component of the antibiotic
complex, nebramycin, produced by Streptomyces tenebrarius.
TOB exerts a bactericidal activity against many bacterial strains
involved in clinical infections. It is particularly indicated for the
treatment of septicemia, complicated and recurrent urinary tract
infections, lower respiratory infections, serious skin and soft tissue
infections including burns and peritonitis, ophthalmic and central
nervous system infections caused by organisms resistant to other
antibiotics, including other aminoglycosides.
Bacteria can show a natural resistance to aminoglycosides
because of the reduced penetration of the antibiotic inside cells,
and an acquired resistance due to a low affinity of the drug for the
bacterial ribosome, or drug deactivation by microbial cell enzymes
[1]. A reduced cellular permeability can result in an insufficient
(sub-active) concentration of the antibiotic in the target sites.
* Corresponding author. Dipartimento di Scienze del Farmaco, Città Universitaria,
viale A. Doria 6, I-95125 Catania, Italy. Tel.: þ39 0957384005; fax: þ39 095222239.
E-mail address: r.pignatello@unict.it (R. Pignatello).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.015

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R. Pignatello et al. / European Journal of Medicinal Chemistry 46 (2011) 1665e1671
of this antibiotic, especially against anaerobic Gram negative
bacteria. Therefore, this study was designed to assess whether the
amphiphilicity, induced by ion pairing TOB with the LAA moieties,
will ameliorate the antibacterial activity. Moreover, from a techno-
logical point of view it is conceivable that the increased lipophilic
character of TOB-LAA ion pairs would positively affect the drug
encapsulation and retention in lipid-based carrier systems, such as
liposomes and lipid nanoparticles; this aspect will be examined in
a separate research.
LAA moieties with a different length of the side alkyl chain (10,
12, or 14 carbon atoms) (Fig. 1) were used in the present study. The
Fig. 1. General structure of TOB and lipoamino acids. Their ion pairs were obtained
starting from a co-solution of the drug and LAA at a 1:1, 1:3, or 1:5 M ratio.
amphiphilic features of these complexes might be useful from
a technological point of view, for instance by enhancing the drug
dissolution in nonaqueous solvents and its encapsulation efficiency
and retention time in various drug delivery systems [9e11]. Until
very recently, some HIP studies also focused on antibiotics, to
improve their absorption and biological activity, or optimize their
loading in nanocarrier systems [12e15].
We have recently undertaken a series of studies concerning the
reversible modification of antibiotic molecules through the forma-
tion of organic amphiphilic ion pairs with lipoamino acids (LAA).
LAA have become increasingly important because of their chemical
simplicity and versatility, surface activity, aggregation properties,
broad biological activityand low toxicity profile [16,17]. Inparticular,
some of us have focused the attention upon some a-amino acids
bearing a saturated alkyl chain of different length in 2-position
(Fig. 1). Because of the contemporary presence of a lipophilic alkyl
chain and the polar amino acid head, LAA can impart amphiphilic
properties to drugs, the so-called membrane-like character [18].
Consequently, they have been proposed as useful promoieties to
modulate or enhance the interaction with and penetration through
cell membranes and biological barriers of compoundswhich possess
poor biopharmaceutical properties [19,20]. Toth and collaborators
have described some conjugates of b-lactam antibiotics covalently
linked to LAA [21], with the aim at enhancing their adsorption in the
gastro-intestinal tract. For instance, oral administration of an
ampicillin-LAA conjugate significantly improved the antibacterial
activity [22]. In a more recent study, a
D-glucuronic acid-LAA
conjugate has been ion paired with gentamicin resulting in a better
oral absorption in rodents [14].
In our lab LAA have been recently used to produce amphiphilic
ion pairs of erythromycin [23]. We observed that although an
electrostatic interaction between the LAA moieties and the drug
occurred, as suggested by spectrophotometric analyses, the growth
inhibitory activity profile of the ion pairs remained close to the
parent drug against different bacterial strains, both sensitive and
resistant to macrolides. However, the presence of the lipid modifier
is expected to modify the in vivo pharmacokinetics of erythromycin
and its penetration into bacterial cells.
The choice of LAA as counter ions mainly relies on their
amphiphilic properties. In contrast with other markedly hydro-
phobic counter ions, such as fatty acids, often used for salifying
drug molecules, a drug-LAA complex should retain the ability of the
LAA moiety to interact with the biological membranes and facilitate
the penetration of the active compound inside the target cells. We
have previously proved a similar occurrence through the covalent
linkage between drugs and LAA, but our aim is to verify the same
Fig. 2. DSC curves of TOB-LAA10 derivatives (endotherm down). The coevaporates
were obtained at different drug-LAA molar ratios (1:1, 1:3, and 1:5); the physical
mixture (PhM) was formed at a 1:1 M ratio. DSC curves of TOB (free base) of the
starting lipoamino acid were included for comparison. Each sample was analyzed from
20 to 300 ꢀC, at a heating rate of 5 ꢀC/min and a cooling rate of 10 ꢀC.
possibility by
a reversible ionic complexation of ionizable
compounds with LAA.
The presence of multiple amino and hydroxyl groups makes TOB
a hydrophilic polycation. Such a feature may affect the low activity

R. Pignatello et al. / European Journal of Medicinal Chemistry 46 (2011) 1665e1671
1667
ion pairs were prepared by reduced pressure evaporation of
a water/ethanol co-solution of the drug (as free base) and LAA. To
verify the effect of progressive ion pairing of the five amine groups
present in TOB molecule, different drug to LAA molar ratios were
examined (1:1, 1:3 or 1:5) labelled as TOB-LAAn-11, -13 or -15,
respectively.
Characterization by FTIR analysis was made to confirm the
structure of the prepared ion pairs, while differential scanning
calorimetry (DSC) and powder X-ray diffractometry (PXRD) were
used to evaluate the formation of a new saline species in respect to
the starting components. The experimental data were compared
with the corresponding equimolar physical mixtures (PhM) of TOB
and the different LAAs, obtained in the absence of any solvent by
simple mechanical mixing of the ingredients.
In a preliminary biological assessment, TOB-LAA ion pairs and
PhMs were tested in vitro against different bacterial strains to
assess the influence of the LAA moiety upon the antibacterial
activity of the drug.
2. Results and discussion
2.1. Chemistry
To investigate the interactions between TOB and LAA, their
coevaporates and PhMs were analyzed in the solid state by
conventional spectroscopic techniques (FTIR, PXRD, DSC).
In the DSC experiments with pure TOB (as the free base) (Fig. 2),
the first observed endothermic peak can be attributed to the
dehydration of the specimen, followed at 164 ꢀC by the fusion into
a metastable form. The latter then recrystallized into the stable
form, as suggested by an endothermic peak at 197.5 ꢀC, and finally
melted at 217 ꢀC [24]. Pure LAA samples showed strong endo-
thermic peaks at the specific melting temperatures, typically
around 220 and 260 ꢀC.
The calorimetric analysis of TOB-LAA ion pairs suggested the
formation of new chemical complexes. In their DSC curves the
endothermic peaks of the two starting compounds were not visible
and were replaced by broad signals, at variable temperature peaks,
which can be assigned to the ionic domains formed by TOB amino
cations and LAA carboxylic anions in the originated ion pairs (Figs. 2
and 3).
The physical mixing of TOB and the different LAA instead gave
more complex DSC profiles (Fig. 2), in which the endothermic
signals related to the melting of pure TOB (both the metastable and
stable forms) and LAA were essentially superimposed.
Fig. 3. DSC curves of TOB-LAA12 and LAA14 derivatives (at 1:1 M ratio). Samples were
analyzed from 20 to 300 ꢀC, at a heating rate of 5 ꢀC/min and a cooling rate of 10 ꢀC.
Endotherm down.
Finally, the DSC curves obtained for the TOB-LAA10 ion pairs
with a 1:3 and 1:5 M ratios were nearly analogous to those
Table 1
IR data (KBr) of TOB base and TOB-LAA coevaporates and physical mixtures.
Attributiona
TOB
TOB-LAA10ꢁ11 PhM10
TOB-LAA12ꢁ11 PhM12
TOB-LAA12ꢁ13 TOB-LAA12ꢁ15 TOB-LAA14ꢁ11 PhM14
NH stretching (s)
OH stretching (s)
Aliphatic CH
stretching (m)
COOH stretching
(w) (LAA)
3450e3200 3550e3250
3345e3240 3420e3364
3350
2922
1685
3348e3275
3349e3271
3489e3240
3346e3282
2910
2843
1690
2844
1685
2923
1654
2911
2912
2851
2930
e
1655
1655
1690
1685
LAA internal lactam (s)
NH bending (s)
CH2 scissoring (m)
In-plane OH bending
e
e
1605
1574
1463
e
1606
1592
1462
1640, 1600 (w) 1600 (w)
e
1610
1580
1416
1377e1345
(m)
1588
1461
1574
1505
1583
1461
1574
1463
1574
1463
1575
1470
1393e1335
(w)
1380, 1349 1346e1336
(m)
1348e1336 1394, 1384
(m)
1394, 1387, 1349 1346, 1337
(m)
1340, 1335
(m)
(w)
(w)
(m)
CN and CO
1032
1011
1028
1015
1034
1019
1020
1012
1030
stretching (s)
a
s: strong; m: medium: w: weak intensity band.

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R. Pignatello et al. / European Journal of Medicinal Chemistry 46 (2011) 1665e1671
registered for the corresponding equimolar ion pair. All showed an
endothermic peak due to the melting of the stable form of TOB and
a broad endothermic signal around 240e250 ꢀC, ascribed to the
melting of ion pair crystals (Fig. 2). Similar results were collected for
TOB-LAA12-13 and TOB-LAA12-15 ion pairs, with respect to the
corresponding equimolar compound (not shown). Such an infor-
mation suggests that, independently on the number of amino
groups that have been salified in TOB molecule, the crystalline
structure and the relative thermotropic behavior of the different
ion pairs were comparable, despite of the strong differences
observed in the microbiological activity (vide infra).
The FTIR analysis of pure TOB gave strong bands around 3450-
3200 cmꢁ1, at 2910 cmꢁ1 and at 1588 cmꢁ1, respectively attributed
to the stretching of amine and hydroxyl groups, the stretching of
aliphatic CH and the bending of NH groups (Table 1) [25]. The
spectra of pure LAA were mainly characterized by the presence of
a complex of three sharp peaks, in the range around 1580 and
1670 cmꢁ1, due to the carboxyl group and the formation of an
internal lactam with the free amine group (not shown).
ingredients. They mainly concerned the signals of the internal
lactam, visible in the PhM spectra at about 1600 cmꢁ1, and the
stretching of CeN and CeO bonds, that was obtained around
1230 cmꢁ1 in the PhMs, similarly to pure TOB, but fell at higher
fields (around 1010 cmꢁ1) for the corresponding ion pairs. Although
small, such differences were observed in all the samples and
confirm the formation of new chemical species between the drug
and the lipophilic moieties. Noteworthy, the signal due to the lac-
tam group was detected in the IR spectra of 1:3 and 1:5 ion pairs
(Table 1). This would suggest that an aliquot of free LAA is present
in these compounds and it can be explained by considering the
different pka values associated with the five amine groups in TOB
molecule. Actually, the basicity decrease from C-1 and C-3 amine
groups (pka values roughly ranging from 6 to 7), to the C-20 and C-
300 groups (pka between 7.8 and 8.3), to the less basic C-60 amine
group, showing a pka value of 8.9 [26]. Therefore, it is plausible that
the last amine group was only partially salified by the LAAs in the
ion pairs produced at a 1:5 M ratio. Elsewhere in the literature has
been already observed that, in the presence of multiple ionizable
groups, the process of ion pairing can reach an equilibrium prior to
a complete stoichiometric substitution [27].
Additionally, the FTIR spectra of the 1:1 PhMs showed a super-
imposition of the signals of the starting molecules. It is noteworthy
that also in these spectra a stretching band around 1580e1590 cmꢁ1
was visible, confirming what observed in DSC experiments, that an
ionic interaction between the carboxyl function of the LAA and the
amine groups of TOB was formed, even during the mere mechanical
trituration step, at room temperature and in the absence of any
solvent, that was used for the production of PhMs. A critical point of
the above assumption might be related to the presence of moisture
in the TOB sample, as supported by the DSC analysis (Fig. 2). This
Table 1 reports the FTIR data of all the tested samples; as an
example, the spectra of the TOB-LAA12 derivatives are reported in
Fig. 4. The IR spectra of TOB-LAA ion pairs and PhMs in a 1:1 M ratio
showed few but relevant points of difference, compared to the pure
Fig. 4. FTIR spectroscopic analysis of TOB (as a free base) and TOB-LAA12 hydrophobic
Fig. 5. PXRD profiles of TOB (free base) and TOB-LAA10 ion pair and PhM, prepared at
ion pair and PhM performed at a 1:1 M ratio.
a 1:1 M ratio.

R. Pignatello et al. / European Journal of Medicinal Chemistry 46 (2011) 1665e1671
1669
Table 2
Solubility (mg/ml) of TOB-LAA ion pairs in different solvents, at room temperature, compared to TOB base.
Solvent
TOB base
TOB-LAA10ꢁ11
15
10
TOB-LAA12ꢁ11
15
15
TOB-LAA14ꢁ11
10
10
TOB-LAA10ꢁ13
10
7.5
TOB-LAA10ꢁ15
10
7.5
Water
pH 7.4 phosphate
buffer
>50
>50
Ethanol
Acetone
Dichloromethane
5
5
1
>30
15
20
>30
20
>30
25
20
>30
>30
>30
>30
>30
>30
>30
little amount of water could of course have a role in the formation of
electrostatic interactions between TOB and the LAA. To verify this
hypothesis, a batch of TOB/LAA10 physical mixture was prepared as
described in the experimental section, but starting from a specimen
of TOB free base that had been dried overnight at 70 ꢀC under high
vacuum. The resulting FTIR analysis showed that, even in the
absence of residual moisture (confirmed by DSC, not shown), TOB
and LAA10 were able to develop an ionic interaction, although the
signal was smaller that that one observed in the previous sample
(not shown). We then concluded that the ionic interaction of TOB
with the counter ion, at least inpart, occurred upon a simple physical
mixing of the drug with the LAA.
The PXRD analysis corroborated both the DSC and FTIR results.
These studies well evidenced the different nature of TOB-LAA ion
pairs (coevaporates) compared to the PhMs. In Fig. 5 the behavior of
LAA10 derivatives is illustrated; the other two series of compounds
with the LAA12 and LAA14 gave very similar results (data not
shown). All the TOB-LAA 1:1 ion pairs, independently on the LAA
present, showed a diffractometric profile typical of amorphous
materials, with the weak signals of the working matrix. Conversely,
the corresponding PhMs gave a diffractogram nearly identical to
that one of pure TOB [25].
seemed also to be proportional to the number of LAA residues
interacting with the drug (1:5 z 1:3 > 1:1 M ratio).
This information can be important in the view of encapsulation
of TOB-LAA ion pairs in polymeric or lipid-based colloidal drug
carriers, such as liposomes, micelles or lipid nanoparticles.
2.3. Microbiological assays
TOB sulfate shows an inhibitory activity in various bacterial
strains. Literature data indicate MIC values in the 0.25e1
mg/ml
range against ATCC strains of Staphylococcus aureus, Pseudomonas
aeruginosa and Escherichia coli, and of 8e32
faecalis [28].
mg/ml for Enterococcus
As the free base, in our experiments TOB had practically no
activity against many Gram negative and Gram positive bacteria,
with MIC values equal or higher than 4
mg/ml (Table 3). This
suggests that in the base (penta-amine) form this drug is not able to
penetrate into cultured bacterial cells and then inhibit their growth.
The TOB-LAA coevaporates in a 1:1 M ratio, along with the
corresponding PhMs were tested in a first set of experiments. In
accordance with the above analytical assessment, neither these
compounds showed a notable growth inhibitory activity against the
assayed strains (Table 3). The conclusion of these experiments
would then be that salifying only one amino group in TOB molecule
did not produce compounds able to penetrate and act into the
bacterial cells.
To confirm this hypothesis, further series of coevaporates were
prepared between TOB and LAA10 or LAA12, in both a 1:3 and 1:5 M
ratio, so that to achieve a greater level of complexation of the basic
groups in drug molecule. Interestingly, reduced MIC values were
obtained (Table 3), always lower than the measurable value of
0.03 mg/ml, and anyway at least three two-fold dilutions lower than
the target MICs, i.e., the expected inhibitory values for TOB sulfate
against the used bacterial strains [28].
A clear experimental evidence of the present study is that sali-
fying with LAA residues more than one amine functions in TOB
molecule, up to all the five amine groups present, enhanced the
growth inhibitory activity against different Gram negative bacterial
strains. This is most probably due to a more efficacious and rapid
Based on these data, it can be assumed that the simple mechanical
mixing of TOB with the LAA gave a powder with the same crystalline
properties of the starting drug, whereas in the coevaporates the
ingredients were intimately mixed, and ‘diluting’ each other
produced a new species with a concomitant loss of crystallinity.
2.2. TOB-LAA ion pairs solubility profile
Increasing the liposolubility is one of the main targets of the HIP
approach. As a polycation molecule, TOB free base is freely soluble
in water, slightly soluble in ethanol and insoluble in chloroform and
ether (United States Pharmacopoeia, USP 29). In our experimental
conditions, such solubility pattern was confirmed (Table 2). The
formation of the coevaporates with one or more LAA moieties
reduced the solubility of the drug in aqueous media, but exerted
a positive influence on the solubility in the tested organic solvents
(Table 2). Increase of solubility in dichloromethane and ethanol
Table 3
In vitro growth inhibitory activity (MIC, mg/ml) of TOB (as the free base or penta-sulfate salt), TOB-LAA coevaporates and physical mixtures. Incubation time: 18 h.
Bacterial strain TOB
TOB
TOB-
PhM10 TOB-
TOB-
TOB-
PhM12 TOB-
TOB-
TOB-
PhM14
(free base) penta-sulfatea LAA10ꢁ11
LAA10ꢁ13 LAA10ꢁ15 LAA12ꢁ11
LAA12ꢁ13 LAA12ꢁ15 LAA14ꢁ11
E. coli
ATCC 25922
E. coli
ATCC 35218
E. faecalis
ATCC 29212
S. aureus
ATCC 29213
16
16
32
16
16
0.25e1
0.25e1
8e32
0.5
1
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
2
1
<0.03
<0.03
0.03
<0.03
<0.03
0.03
1
1
>4
>4
>4
>4
>4
0.5
0.5
0.5
0.5
16
>4
32
>4
>4
1
0.25e1
0.25e1
2
1
1
0.5
0.5
1
<0.03
<0.03
<0.03
<0.03
P. aeruginosa
0.5
0.5
1
ATCC 27853
a
MIC values taken from Ref. [28].

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R. Pignatello et al. / European Journal of Medicinal Chemistry 46 (2011) 1665e1671
penetration of the drug inside the bacterial cells, compared to the
commonly used penta-sulfate TOB salt.
in absolute ethanol. The two solutions were mixed for about 4 h at
40 ꢀC and then at overnight room temperature. Ethanol and part of
the water were removed under high vacuum at an external
temperature of 40 ꢀC. Residual water was finally removed by
freeze-drying (Edward Modulyo). The resulting fluffy, white
powders were stored in tight closed glass vials at 4 ꢄ 1 ꢀC until use.
1:3 Molar Ratio. TOB-LAA10-13 and TOB-LAA12-13 were obtained
in a similar manner, starting from 0.3 mmol TOB and 0.9 mmol of
the chosen LAA.
No evident relationship between the in vitro microbiological
activity and the length of the side alkyl chain in the used LAA was
registered. This can be due to the relatively narrow range of lip-
ophilicity within the three used LAA residues (LAA10 to LAA14),
although in many other studies on drug-LAA conjugates relevant
differences have been observed using analogous substituents
[29,30]. Alternatively, it could be concluded that the substitution of
the free amine groups in the TOB molecule, regardless of the
structure of the counterion moiety, would led to the hypothesized
effect on drug uptake into bacterial cells and, hence, on their
growth inhibitory effect.
1:5 Molar Ratio. TOB-LAA10-15 and TOB-LAA12-15 were obtained
in a similar manner, starting from 0.2 mmol TOB and 1 mmol of the
chosen LAA.
4.2. Preparation of the physical mixtures
3. Conclusion
TOB-LAA equimolar PhMs (PhM10, PhM12, and PhM14) were
prepared by mixing the two components in a porcelain mortar for
30 min. The products were stored in a refrigerator in closed glass
vials until use.
The experimental findings seem to confirm the initial working
hypothesis, and reinforce what has been previously observed for
analogous erythromycin-LAA coevaporates: the complexation of
these antibiotics with amphiphilic moieties, while maintaining or
even improving the microbiological activity profile of parent drugs,
can be an easy strategy to enhance the physicoechemical, phar-
macokinetic and, ultimately, pharmacological properties of drugs.
For instance, a specific study is in progress to assess whether the
amphiphilicity of the prepared coevaporates, as demonstrated by
their solubility pattern, could also positively affect the oral
absorption of TOB.
Such amphiphilic character could significantly affect the pene-
tration of TOB through biological membranes. By considering the
diffuse clinical use of TOB in ocular pathologies, experiments are
thus in course in our lab to assess the diffusion of TOB-LAA ion pairs
through rabbit cornea, with the aim at achieving higher levels of
the antibiotic in the aqueous humor and inner eye tissues [31].
4.3. Solubility determination
The solubility profile of coevaporates in a range of pharmaceu-
tically related solvents (water; 0.13 M phosphate buffer solution,
pH 7.4; ethanol; acetone; and dichloromethane) was measured at
room temperature. To a known volume of each solvent (about 2 ml)
in capped glass tubes, small amounts of TOB or TOB-LAA ion pairs
were progressively added. The mixture was vortex-mixed and
submitted to turbidimetry analysis (Shimadzu UV-1601; Shimadzu
Italia, Milan, Italy). The early measurement of an absorbance at
650 nm was considered as the solubility threshold. Experimental
data are reported in Table 2.
4.4. Microbiology
4. Materials and methods
4.4.1. Strains
TOB free base (CAS 32986-56-4) and TOB sulfate were purchased
from Calbiochem (Merck KGaA, Darmstadt, Germany). HPLC-grade
water was purchased from Merck (Darmstadt, Germany); absolute
ethanol was purchased fromSigmaeAldrich Chimica srl, Milan, Italy.
The LAA were synthesized as described elsewhere [32].
E. coli ATCC 25922, E. coli ATCC 35218, E. faecalis ATCC 29212, S.
aureus ATCC 29213, and P. aeruginosa ATCC 27853, were investi-
gated in this study.
4.4.2. Susceptibility test procedure
IR spectra were registered in KBr tablets with an FTIR Per-
kineElmer 1600 spectrophotometer. PXRD data were collected on
a PW3710 (Philips, Eindhoven, The Netherlands) powder diffrac-
The antimicrobial activity of TOB-LAA coevaporates and PhMs
was determined in comparison with that of the free drug, with
MICs determined by using the standard broth microdilution assay
[33]. The sample suspension was added so that to obtain an
equivalent drug concentration with respect to that of the free drug
solution, thus allowing to evaluate the effectiveness of the counter
ion moiety by a direct comparison of the results. Cation-adjusted
Muller-Hinton broth (CAMHB) was used except for streptococci,
where CAMHB added of 2.5% lysed horse blood was used.
tometer using Cu K
a radiation and a graphite monochromator, over
the range 5ꢀ ꢂ 2v ꢃ 30ꢀ at a scanning rate of 0.005ꢀ/s. DSC exper-
iments were performed with a Mettler DSC12E calorimeter, con-
nected to a Lauda Ecoline RE 207 thermocryostat. A sample of pure
indium was used to calibrate the instrument. The detection con-
sisted of a Mettler Pt 100 sensor, with a thermometric sensitivity of
56
m
V/ꢀC, a calorimetric sensitivity of about 3
mV/mW and a noise
A stock solution of TOB base (320
mg/ml) was prepared in water;
less than 60 nV (<1 mV). Each DSC scan showed an accuracy of
the stock solutions of tested samples were obtained by dissolving
them in dimethylsulfoxide and then diluting to 10 ml with the
culture broth. The further dilutions were obtained as proposed by
the CLSI [33]. A total of 10 concentrations of each sample were
prepared. A suspension of microorganisms was added to each well
to have a bacterial concentration of 103 CFU/ml in each well.
A positive control (growth) consisting of organisms in broth,
a negative control (sterility) consisting of uninoculated broth, and
a drug control consisting of broth containing the highest concen-
trations of TOB, TOB-LAA ion pairs or PhMs (concentrations 1, 10,
and 100 times higher than those used in the experiments) were
included for each tested bacterial strain. Plates were sealed with
transparent acetate film and incubated at 37 ꢀC under atmospheric
conditions for up to 18 h. Each dilution was assayed six times and
ꢄ0.4 ꢀC and reproducibility and resolution of 0.1 ꢀC. Samples
(5e10 mg) were sealed in a 40-ml aluminum pan, using an empty
pan as reference. Each sample was analyzed from 20 to 300 ꢀC, at
a heating rate of 5 ꢀC/min and a cooling rate of 10 ꢀC.
4.1. TOB-LAA ion pair preparation
1:1 Molar Ratio. TOB-LAA10-11, TOB-LAA12-11, and TOB-LAA14
-
11, equimolar ion pairs between TOB and the LAA bearing a side
alkyl chain of 7, 9, or 11 carbon atoms, respectively (Fig. 1) were
prepared by co-evaporation of a co-solution of the two compo-
nents. TOB base (0.3 mmol) was dissolved in water, while the
appropriate LAA (0.3 mmol) was dissolved under magnetic stirring

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