Polyamidoamine (PAMAM) dendrimers as biocompatible carriers of quinolone antimicrobials: An in vitro study

Article abstract of DOI:10.1016/j.ejmech.2006.12.035

Quinolones, an expanding class of clinically established potent antibiotics, is not freely soluble in water which prevents the design of liquid dosage forms and restricts their use in topical applications. In the present study we investigated the potential of polyamidoamine (PAMAM) dendrimers as drug carriers of quinolones (nadifloxacin and prulifloxacin) by aqueous solubility and antibacterial activity studies. Results showed that the aqueous solubility of nadifloxacin and prulifloxacin was significantly increased by PAMAM dendrimers. Microbiology studies showed that nadifloxacin and prulifloxacin still exhibit their strong antimicrobial activities in the presence of dendrimers. These studies indicated that PAMAM dendrimers might be considered as biocompatible carriers of quinolones under suitable conditions.

Full text of DOI:10.1016/j.ejmech.2006.12.035

European Journal of Medicinal Chemistry 42 (2007) 1032e1038
http://www.elsevier.com/locate/ejmech
Short communication
Polyamidoamine (PAMAM) dendrimers as biocompatible
carriers of quinolone antimicrobials: An in vitro study
a,b,*
b
, Haiou Qu , Minglu Ma , Zhenhua Xu ,
b
b
Yiyun Cheng
b
a
a,
Peng Xu , Yujie Fang , Tongwen Xu **
a
Laboratory of Functional Membranes, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China
b
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science
and Technology of China, Hefei, Anhui 230027, China
Received 6 September 2006; received in revised form 20 December 2006; accepted 21 December 2006
Available online 20 January 2007
Abstract
Quinolones, an expanding class of clinically established potent antibiotics, is not freely soluble in water which prevents the design of liquid
dosage forms and restricts their use in topical applications. In the present study we investigated the potential of polyamidoamine (PAMAM)
dendrimers as drug carriers of quinolones (nadifloxacin and prulifloxacin) by aqueous solubility and antibacterial activity studies. Results
showed that the aqueous solubility of nadifloxacin and prulifloxacin was significantly increased by PAMAM dendrimers. Microbiology studies
showed that nadifloxacin and prulifloxacin still exhibit their strong antimicrobial activities in the presence of dendrimers. These studies indicated
that PAMAM dendrimers might be considered as biocompatible carriers of quinolones under suitable conditions.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Polyamidoamine dendrimers; PAMAM; Drug carrier; Antibacterial drugs; Quinolones; Nadifloxacin; Prulifloxacin
1
. Introduction
numerous diseases [4]. Quinolones cover a host of aerobic
gram-negative, gram-positive and even some anaerobic spe-
cies responsible for various infections (prostatitis, tuberculo-
sis, pneumonia, bronchitis and urinary tract, respiratory tract,
skin, gastrointestinal, bone, joint, soft tissue, abdominal infec-
tions, some sexually transmitted diseases and some infections
that affect people with AIDS). Also, quinolones have gained
popularity in the ophthalmology field since they have been
shown to be equivalent to combination therapy in the treat-
ment of many ocular infections [3e5].
Bacterial infections remain major causes of morbidity and
mortality in hospitals around the world [1]. A new report esti-
mated that Staphylococcus aureus (S. aureus) infections alone
resulted in 9.5 billion dollars of extra hospital charges and
nearly 12,000 inpatient deaths per year [2].
Quinolones, an expanding class of clinically established po-
tent antibiotics, whose accidental discovery occurred in the
early 1960s, were very important in addition to the antibiotics
that we had already developed [3]. They are well known
broad-spectrum antibacterials and widely used to treat
Quinolones are usually available as tablets or liquid suspen-
sion to be taken by mouth.
Although quinolones are well absorbed when orally given,
they can still cause side effects. The most common of which
involve mainly the digestive system (stomach pain or upset,
nausea, vomiting, and diarrhea) [4,5]. In some situations, qui-
nolones should be given by intravenous injection for more se-
rious infections. However, such compounds exist mainly in
their zwitterionic form owing to the acid/base interaction
*
Corresponding author. Laboratory of Functional Membranes, Department
of Chemistry, University of Science and Technology of China, Hefei, Anhui
30026, China.
2
** Corresponding author.
E-mail addresses: yycheng@mail.ustc.edu.cn (Y. Cheng), twxu@ustc.edu.
cn (T. Xu).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.035

Y. Cheng et al. / European Journal of Medicinal Chemistry 42 (2007) 1032e1038
1033
between the basic nitrogen of the piperazine and the carbox-
ylic acid group. Such interaction also determines the low aque-
ous solubility of these compounds at pH close to 7 [6e8]. This
is the main factor, which prevents the design of liquid dosage
forms and restricts their use in topical applications, such as
parenteral and ophthalmic, because the aqueous compatibility
of these drugs occurs at rather basic or acid pH [9]. Further-
more, poor solubility is generally related to a low bioavailabil-
ity, which presents a major challenge during drug formulation.
In order to improve the solubility of quinolones in water,
carbomer hydrogelequinolone complexes were prepared to
enhance dissolution and absorption rate [6]. Also, liposomes
were used to encapsulate quinolones [7]. More recently,
a new class of water-soluble quinolones was synthesized
internal cavities which are able to encapsulate hydrophobic
guest molecules in the macromolecule interior. Drugs or other
molecules can either be attached to dendrimers’ end groups or
encapsulated in the macromolecule interior [19]. These spe-
cific properties make dendrimers suitable for drug delivery
systems [15e18]. Drugs bound to dendrimers are at early
stages of development and data on them are limited. Several
authors reported on the encapsulation of non-steroidal anti-in-
flammatory drugs (NSAIDs) and anti-cancer drugs in den-
drimers [21,22]. In the previous study, we also studied the
solubilization of sulfonamides by dendrimers [23]. However,
to our knowledge there are no studies devoted to the solubili-
zation of quinolone antibacterial drugs in the presence of den-
drimers. Here, we focus on using ethylenediamine (EDA) core
PAMAM dendrimers as potential drug carriers, which are
emerging as a promising group of safer and perhaps more
effective alternatives to traditional quinolones as exemplified
by nadifloxacin and prulifloxacin.
[
10]. However, the aqueous solubility of carbomer hydrogels
or liposomes is insufficient to stabilize drugs at high doses.
Moreover, high synthesis costs limit the use of these new
quinolones.
Dendrimers are new artificial macromolecules topologi-
cally based on the structure of a tree. They are hyperbranched,
monodisperse, three-dimensional molecules, having defined
molecular weight and hosteguest entrapment properties.
Due to their special synthesis in a stepwise manner from
branched monomer units, they allow the precise control of
size, shape, dimensions, density, polarity, flexibility, solubility
and placement of functional groups by choosing of these
building units and functional group chemistry. As a result,
they combine typical characteristics of small organic mole-
cules and polymers that result in special physical and chemical
properties [11e14]. Up to now, dendrimers have already at-
tracted increasing attention for their applications in many
fields including model chemistry or combination chemistry,
electrochemistry and photochemistry, nanoparticle synthesis
template, water purification, dye decolorization, monomolecu-
lar membranes, curing agents in epoxy resin systems, cata-
lyzer in extensive areas, drug delivery systems and gene
transfection in biomedical fields. Among them the use of den-
drimers as drug carriers in delivery systems has been of great
interest.
Nadifloxacin, a fluorinated quinolone antimicrobial, is
widely used for the treatment of multiple inflamed acne le-
sions as a topical agent [23]. It has a potent bactericidal activ-
ity against P. acnes and other gram-positive and gram-negative
bacteria [24]. In addition to its bactericidal activity, nadiflox-
acin has also been suggested to have anti-inflammatory actions
which may have a beneficial effect on some aspects of inflam-
matory acne [23e25]. Furthermore, nadifloxacin does not
show cross-resistance with other new quinolones. Prulifloxa-
cin, the lipophilic prodrug of ulifloxacin, is also a broad-spec-
trum oral fluoroquinolone antibacterial agent [26]. After oral
administration, prulifloxacin is absorbed by the intestine and
rapidly metabolized by esterases to ulifloxacin, the active me-
tabolite of prulifloxacin [27e30].
The aim of the present work was (a) to investigate the po-
tential of PAMAM dendrimers as solubility enhancers of qui-
nolones as exemplified by nadifloxacin and prulifloxacin; (b)
to study the effect of concentration, generation and pH value
on the solubility of nadifloxacin and prulifloxacin and (c) to
investigate antibacterial activities of quinolones in the pres-
ence of PAMAM dendrimers.
Polyamidoamine (PAMAM) dendrimer with an ellipsoidal
Ò
or spheroidal shape is one of the most studied starburst mac-
2. Experiments
romolecules. Due to specific synthesis, PAMAM dendrimers
have some interesting properties, which distinguish them
from classical linear polymers, e.g. PAMAM has a much
higher amino group density comparing with the conventional
macromolecules, a third generation PAMAM prepared from
2.1. Materials
Nadifloxacin and prulifloxacin were purchased from Bei-
jing Maijing Pharmacy Factory (Beijing, China); ethylenedi-
amine, methyl acrylate, methanol, dimethyl sulfoxide (HPLC
grade) were obtained from Shanghai Chemical Co. (Shanghai,
China). For both solubility and antibacterial studies, distilled
water was used.
À4
ammonia core has 1.24 Â 10 amine moieties per unit vol-
À6
ume (cubic Angstrom units) in contrast to the 1.58 Â 10
amine moieties per unit volume of a conventional star polymer
[
14]. The high density of functional groups (eNH , eCOOH,
2
eOH) in PAMAM dendrimers may be expected to have poten-
tial applications in enhancing the solubility of low aqueous
solubility drugs and delivery systems for bioactive materials
2.2. Synthesis of PAMAM dendrimers
[
20]. Also, These functional groups on the outer shell are re-
PAMAM dendrimers were synthesized according to
Ref. [31]. Ethylenediamine (10.0 g, 0.166 mol) was dissolved
in 100 ml methanol in a 1-l round-bottomed flask. Methyl ac-
sponsible for high reactivity which means dendrimers can be
modified or conjugated with a list of interesting guest mole-
cules. Furthermore, PAMAM dendrimers possess empty
ꢀ
rylate (94.6 g, 0.751 mol) was added at 40 C and the system

1034
Y. Cheng et al. / European Journal of Medicinal Chemistry 42 (2007) 1032e1038
was stirred for 24 h under nitrogen. Excess methyl acrylate
was removed under vacuum at room temperature. A Michael
addition between the amine and the acrylate yielded a product
bearing four terminal methyl ester groups, defined as the G0.5
PAMAM. Subsequently, ethylenediamine (120 g, 2.00 mol)
was dissolved in methanol and added to the G0.5 PAMAM
and, after stirring for 48 h under nitrogen and removing excess
reactants by vacuum distillation, a product bearing four termi-
nal amino groups was obtained, defined as the G1 PAMAM.
By repeating the above cycle, higher generation PAMAM den-
drimers (up to G5) were synthesized. Purity of the synthesized
at the same drug concentration. After that, they were diluted
in 100 ml of distilled water to reach a final concentration of
5 mg/ml (nadifloxacin or prulifloxacin concentration). Den-
drimer and DMSO were also evaluated in the absence of qui-
nolones. When conducting the antibacterial activity studies,
50 ml of LuriaeBertani liquid medium was first distributed
from the second to the twelfth well of a 96-well plate. Then,
100 ml of test solutions prepared as above were added to the
first test well of each line, and 50 ml of scalar dilution was
transferred from the second to the eleventh well, the twelfth
well was added without any drug. Finally, 50 ml of a microbial
1
13
6
PAMAM dendrimers was characterized via FT-IR, H and
NMR and element analysis [14].
C
suspension (w10 colony forming units, CFU/ml), obtained
ꢀ
from an overnight growth at 37 C, was added to each well
of the plate. The final concentration of these samples used to
evaluate the antibacterial activity was from 2.5 mg/ml (second
well) to 4.9 ng/ml (eleventh well). The plate was incubated for
24 h at 37 C and examined by measuring the optical density
in a spectrophotometer (630 nm).
2
.3. Solubility test
The solubility of nadifloxacin or prulifloxacin was deter-
ꢀ
mined using the equilibrium solubility method [32]. Excess
drugs were added to 500 ml of each test solution to ensure
the drug solution reaching saturation. The solution was me-
3. Results and discussion
ꢀ
chanically shaken for 24 h at 37 C and then the solutions
were centrifuged at 10,000 rpm for 3 min. Three repeats
were conducted for each sample. The effect of initial pH con-
dition on solubility of nadifloxacin and prulifloxacin was ex-
amined over the range 7e10, which was achieved by
addition of 0.1 M HCl in 10 mg/ml G4 PAMAM dendrimer
solutions. The obtained dendrimer solutions with different
pH values (7e10) were used for nadifloxacin and prulifloxacin
solubility studies as mentioned above.
3
.1. UVevis spectroscopy studies about the interactions
between dendrimers and quinolones
The interactions between quinolones with dendrimer mole-
cules were determined by UVevis spectroscopy. The UV ab-
sorption peak of the free nadifloxacin appeared at 297 nm
(
Fig. 1). After the addition of G4 PAMAM dendrimers, blue
shifts were observed (the peak shifted about 8 nm from
97 nm). In general, blue shift means that drug molecules
2
2
.4. UVevis spectroscopy
move to hydrophobic environment, which is an indication of
hydrophobic encapsulation of nadifloxacin by the hydrophobic
cavities of PAMAM dendrimers. In addition, electrostatic in-
teraction between the carboxyl group of nadifloxacin and the
amine groups of PAMAM dendrimers can also explain this
blue shift. From the calibration curve of free nadifloxacin in
the absence/presence of PAMAM dendrimers, the absorbance
of the samples at 297/289 nm showed a linear relationship
Quinolones in ethanol solution or in distilled water give
maximum absorbance in UV region at their characteristic
wavelengths (297 nm for nadifloxacin and 281 nm for pruli-
floxacin). PerkineElmer UVevis spectrometer was used to es-
timate the amount of drug incorporated in the dendrimer. The
saturated solutions obtained from the solubility studies were
diluted to a proper concentration (500Â, distilled water). Since
the dendrimers in the diluted solutions give extremely low
absorbance between 250 and 700 nm, the absorbance obtained
from quinolones dendrimer solution would be solely from na-
difloxacin or prulifloxacin. The absorbance of nadifloxacin
and prulifloxacin at their characteristic wavelengths was re-
lated with the amount/solubility of quinolones in the den-
drimer solutions or distilled water.
1
1
1
.4
.2
.0
10 mg/ml dendrimer
8 mg/ml dendrimer
6 mg/ml dendrimer
4
mg/ml dendrimer
2 mg/ml dendrimer
without dendrimer
0.8
0
0
0
0
.6
.4
.2
.0
2
.5. Antibacterial activity test
The compounds were tested against Escherichia coli (E.
coli) for their antibacterial activities, using a common Lur-
iaeBertani liquid medium microdilution method as described
in Ref. [33]. Before the antibacterial tests, the drug formations
in the presence/absence of dendrimer were prepared as fol-
lows: nadifloxacin and prulifloxacin were dissolved in
Nadifloxacin
2
00
300
400
Wavelength (nm)
500
600
700
1
0
0 mg/ml G4 PAMAM dendrimer to a concentration of
.5 mg/ml, while pure quinolones were dissolved in DMSO
Fig. 1. UVevis spectrum of nadifloxacin at different G4 PAMAM dendrimer
concentrations.

Y. Cheng et al. / European Journal of Medicinal Chemistry 42 (2007) 1032e1038
1035
with the concentration of nadifloxacin (data not shown). The
absorbance of the test solutions at 289 nm obtained from sol-
ubility studies increased significantly with the concentration of
PAMAM dendrimers. Since the PAMAM dendrimers in the di-
luted solutions give nearly no absorbance at 289 nm at the
studied concentration, the absorbance obtained from samples
would be solely from the drug, which indicated that the solu-
bility of nadifloxacin increased significantly with dendrimer
concentration. Similar results were obtained when prulifloxa-
cin were investigated, as shown in Fig. 2, the UV absorption
peak of the dendrimereprulifloxacin mixture appeared at
small guest molecules inside and make dendrimers suitable
for enhancing the solubility of hydrophobic drug molecules
such as nadifloxacin and prulifloxacin in aqueous solutions
[36,37]. Furthermore, there are tertiary amines in these inter-
nal cavities, which could interact with the atoms of the quino-
lone molecules by hydrogen bond formation [37]. Therefore,
PAMAM dendrimers possess open and internal cavities and
many terminal functional groups which are responsible for
high solubility and reactivity. These specific properties make
PAMAM series dendrimers suitable for solubility enhancers
of quinolones.
2
(
76 nm, shifting 7 nm from the peak of free prulifloxacin
281 nm). These results concluded that quinolone molecules
3.3. Effect of initial pH condition of dendrimers on
solubility of quinolones
could interact with PAMAM dendrimers by electrostatic inter-
action as well as hydrophobic encapsulation.
To ascertain the most effective initial pH condition on sol-
ubility of quinolones using PAMAM dendrimers, dendrimer
solutions were produced at a range of pH values (7e10), the
concentration of G4 PAMAM dendrimer being constant. The
results are shown in Figs. 3 and 4. As can be seen, the process
was highly pH-dependent. The solubility of nadifloxacin and
prulifloxacin in PAMAM dendrimer solutions was highest at
pH 10, less at pH 9 and 8, and least at pH 7. As discussed
in Section 3.2, the solubility enhancement of quinolones is
due to (a) electrostatical interaction between the surface amine
groups of dendrimer molecule and the carboxyl group of qui-
nolones, (b) hydrophobic and open cavities in PAMAM den-
drimers and (c) hydrogen bond formations between tertiary
amines in internal cavities of dendrimers and the atoms of
guest molecules. At lower pH conditions, there is no signifi-
cant increase of solubility of nadifloxacin in dendrimer solu-
tion compared to that at higher pH conditions. This is
because the nadifloxacin or prulifloxacin, exist mainly in their
zwitterionic form owing to the acid/base interaction between
the basic nitrogen of the piperazine and the carboxylic acid
group, is unionized at pH conditions close to 7 and hence can-
not interact electrostatically with the surface amine groups of
dendrimer molecule. Furthermore, earlier studies have shown
3
solubility of quinolones
.2. Effect of PAMAM dendrimer concentration on
The effect of PAMAM dendrimer on solubility of nadiflox-
acin was carried out using G4 PAMAM dendrimer of molec-
ular weight 6900 Da and 32 amine groups in the outer shell,
and the results are shown in Fig. 1. It was observed that the
solubility of nadifloxacin increased significantly with PA-
MAM concentrations. In the presence of G4 PAMAM den-
drimer, the solubility of nadifloxacin in the dendrimer
solutions increased in an approximately linear manner with
an increase in dendrimer concentration. Similar results were
obtained with prulifloxacin (Fig. 2). These results were pre-
sumably due to the increase in the number of surface amines
and internal cavities that are available to interact with or
encapsulate nadifloxacin molecules. Firstly, PAMAM den-
drimers have large numbers of primary amines on their sur-
face, which could interact electrostatically with the carboxyl
group in the quinolone molecules [21,34]. Secondly, PAMAM
dendrimers possess empty internal cavities and an open struc-
ture (low generation PAMAM dendrimers) [35], due to these
specific and interesting properties of PAMAM dendrimers,
the hydrophobic cavities in PAMAM dendrimers can keep
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
A - pH 10
B - pH 9
C - pH 8
D - pH 7
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
1
8
6
4
0 mg/ml dendrimer
mg/ml dendrimer
mg/ml dendrimer
mg/ml dendrimer
2 mg/ml dendrimer
without dendrimer
A
B
C
Nadifloxacin
Prulifloxacin
500
D
2
00
300
400
Wavelength (nm)
600
200
300
400 500
Wavelength (nm)
600
700
Fig. 2. UVevis spectrum of prulifloxacin at different G4 PAMAM dendrimer
concentrations.
Fig. 3. UVevis spectrum of nadifloxacin at different pH conditions (G4 den-
drimer concentration being constant, 10 mg/ml).

1036
Y. Cheng et al. / European Journal of Medicinal Chemistry 42 (2007) 1032e1038
0
0
0
0
0
.8
.6
.4
.2
.0
1.2
A - pH 10
A - G5 dendrimer
B - G4 dendrimer
C - G3 dendrimer
B - pH
C - pH
D - pH
9
8
7
1
0
0
0
.0
.8
.6
.4
A
B
A
C
D
Prulifloxacin
600
B
C
0.2
2
00
300
400 500
Wavelength (nm)
700
Prulifloxacin
600
0
.0
2
00
300
400 500
Wavelength (nm)
700
Fig. 4. UVevis spectrum of prulifloxacin at different pH conditions (G4 den-
drimer concentration being constant, 10 mg/ml).
Fig. 6. UVevis spectrum of prulifloxacin at different PAMAM dendrimer gen-
erations (dendrimer mole concentration being constant, 7 Â 10À4 M).
that the cavity environment inside the PAMAM dendrimer is
more hydrophobic than that of the water phase outside [38].
Therefore, in PAMAM dendrimer solutions, quinolone mole-
cules could be solubilized in the cavities of the PAMAM den-
drimer at the site of low polarity [37]. However, at low pH
conditions, protonation of tertiary amines in the cavities of
PAMAM dendrimers enhances the polar level of environment
inside the dendrimer, which causes no significant increase in
the solubility of nadifloxacin and prulifloxacin as observed
in the experiment. As a result, the solubility of nadifloxacin
in PAMAM dendrimer solutions was lowest at pH 7.
dendrimer solutions likely depends on the dendrimer genera-
tion (size) [39]. Since the number of primary and tertiary
amines in the dendrimer increases with generation size, at
a given pH condition, higher generation dendrimer has a ten-
dency to entrap more hydrophobic compound inside than lower
ones. Also, the solubility of nadifloxacin in PAMAM solutions
depends on the surface area and primary amino groups of PA-
MAM particles, which cause the higher generation PAMAM
particles to have a higher ability to absorb and interact with
the nadifloxacin molecule. In this way, we could explain why
higher generation dendrimers could enhance the solubility of
nadifloxacin more efficiently than lower ones. Similar results
were obtained when prulifloxacin were studied.
3.4. Effect of PAMAM generation on solubility
of quinolones
The effect of various generations of PAMAM dendrimers
G3eG5) in the process was investigated. The results are shown
(
3.5. Antibacterial activities
in Figs. 5 and 6, from which it is clear that the solubility of na-
difloxacin and prulifloxacin were affected by the generation of
PAMAM dendrimer. The solubility of nadifloxacin in higher
generation PAMAM solution was in fact higher than those in
lower ones. The solubility of hydrophobic compounds in
To check whether the quinolones still exhibit their antibac-
terial activities in the presence of PAMAM dendrimers, the
antibacterial activity of quinolones (nadifloxacin and pruliflox-
acin), dendrimer and quinoloneedendrimer solution were
investigated. The results presented in Figs. 7 and 8 indicate
1
1
0
0
0
.2
.0
.8
.6
.4
A - G5 dendrimer
B - G4 dendrimer
C - G3 dendrimer
1.0
0.8
0.6
0.4
0.2
0.0
A
B
N-G4 dendrimer
N-DMSO
0.2
G4 dendrimer
DMSO
C
Nadifloxacin
600
Control
0.0
2
00
300
400
500
Wavelength (nm)
700
2
4
6
Dilution times
8
10
Fig. 5. UVevis spectrum of nadifloxacin at different PAMAM dendrimer gen-
erations (dendrimer mole concentration being constant, 7 Â 10À4 M).
Fig. 7. Antibacterial activity of different formulations of nadifloxacin.

Y. Cheng et al. / European Journal of Medicinal Chemistry 42 (2007) 1032e1038
1037
We are in the process of conducting pre-clinical testing to
evaluate the potential of dendrimers as carrier for nadifloxa-
cin/prulifloxacin and other antibacterial drugs. Although toxic-
ity problems may exist, modification of the structure of
dendrimers should resolve this issue.
0
0
0
0
0
.8
.6
.4
.2
.0
Acknowledgements
P-G4 dendrimer
P-DMSO
Financial supports from the Innovation Foundation of
Graduate Student in University of Science and Technology
of China (KD2004035) was highly appreciated.
G4 dendrimer
DMSO
Control
2
4
6
Dilution times
8
10
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Fig. 8. Antibacterial activity of different formulations of prulifloxacin.
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1
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