Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20

The effect of ferrous ions, calcium ions and citric

acid on absorption of ciprofloxacin across caco-2
cells: practical and structural approach

Dina El-Sabawi, Rana Abu-Dahab, Waleed A. Zalloum, Fadia Ijbara & Imad I.
Hamdan

To cite this article: Dina El-Sabawi, Rana Abu-Dahab, Waleed A. Zalloum, Fadia Ijbara &
Imad I. Hamdan (2018): The effect of ferrous ions, calcium ions and citric acid on absorption
of ciprofloxacin across caco-2 cells: practical and structural approach, Drug Development and
Industrial Pharmacy

To link to this article: https://doi.org/10.1080/03639045.2018.1539495

Accepted author version posted online: 23

Oct 2018.

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The effect of ferrous ions, calcium ions and citric acid on absorption of ciprofloxacin

across caco-2 cells: practical and structural approach

Dina El-Sabawia, Rana Abu-Dahaba, Waleed A. Zalloumb, Fadia Ijbaraa, Imad I.

Hamdana*

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a
School of Pharmacy, The University of Jordan, Amman 11942, Jordan.

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b
Department of pharmacy, Faculty of health science, American University of Madaba,

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P.O. Box: 2882, Amman 11821, Jordan
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*Corresponding author:
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Faculty of Pharmacy, The University of Jordan.

Queen Rania Street, Amman 11942, Jordan.

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Tel.: + 962 6 5355000 ext. 23302

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Fax: + 962 6 5300250

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Email: I.hamdan@ju.edu.jo
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Abstract

Objective: To study the potential influence of selected metal ions on absorption (and

hence oral bioavailability of ciprofloxacin (Cipro) in presence and absence of a

competing ligand. Significance: The presence of metal ions together with Cipro results

in complexes exhibiting a decreased bioavailability. Attempts were made to better

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understand the mechanism of decreased Cipro bioavailability in the presence of metals

such as calcium and ferrous ions, and a small-sized ligand citric acid (CitA). Methods:

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Effect of complex size or other potential factors was studied using diffusion through

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synthetic membrane, permeation studies across Caco-2 cells and capillary
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electrophoresis. A molecular dynamics (MD) simulation study was conducted to find the

arrangement and the nature of the interactions between Cipro molecules and ferrous ions.
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Results: Cipro was shown to form complexes with metals and CitA. The presence of

CitA improved permeation of Cipro through the synthetic membrane but this was not as
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obvious in case of Caco-2 cells. Capillary electrophoresis suggested the existence of

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large molecular aggregates of Cipro: metal complexes. MD simulations offered clear

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evidence of large size aggregates in line with the experimental findings. CitA alone

significantly improved permeation of Cipro through Caco-2 cells. Conclusions: The size
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of the formed complexes, rather than the decrease in the solubility of formed complexes,

plays a significant role in permeation (absorption) of Cipro. CitA might ameliorate the

effect of co-administered metal ions on the bioavailability of Cipro.

Keywords: Ciprofloxacin, metal ions, permeability, Caco-2, bioavailability, citric acid,

molecular dynamics simulation.

Introduction

Ciprofloxacin (Cipro, Fig. 1) is a broad-spectrum fluoroquinolone (FQ) antibiotic that is

widely used for treating a variety of infections, particularly urinary tract infections [1].

Cipro exists mainly as a zwitterion at physiological pH which imparts substantial

hydrophilicity to the compound. Despite this, Cipro exhibits good oral bioavailability

(60-80%) which been attributed to the drug being actively transported utilizing different

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types of organic anion transporters [2,3]. Cipro has also been shown to be actively

secreted from the serosal side to luminal side by an active saturable mechanism [4]; thus

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other organic compounds are likely to interfere with the absorption and pharmacokinetics

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of Cipro.
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Cipro and FQs in general are known to form chelate complexes with metal ions leading
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to a decrease in their oral bioavailability when administered with metal-containing

pharmaceuticals such as antacids or mineral supplements [5-9]. It is estimated that this

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chelation interaction might be found in 22 -76% of all patients prescribed FQs, with up to
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90% reduction in bioavailability [10]. Almost all divalent and trivalent metal ions have
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been shown to have this effect including Al3+, Fe3+, Mg2+, Zn+2, Ca2+, Fe2+ and Cu2+ [11,

12]. The magnitude of the effect was shown to decrease in the order Al3+>Fe2+> Zn+2>
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Mg2+> Ca2+, which might reasonably correlate with the estimated formation constants of

the complexes [6, 13].

While it is now generally accepted that chelate complexes with metal ions are largely

behind the observed decrease in bioavailability of FQs, the exact mechanism has long

been a controversial issue [12, 14-16]. Early hypotheses to explain this effect primarily
suggested a decrease in the solubility of the FQs as they form complexes with metals [5]

and were later supported by some relevant studies [7, 11, 12, 17].

However, other studies report conflicting findings, with metal complexation shown to

increase the solubility of FQs [14, 18-20]. Interestingly, one report showed differential

behavior amongst the FQs where ofloxacin was found to form highly soluble carboxylate

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salts with metals while Cipro did not [21]. Other reports suggest that Cipro-metal

complexes may adsorb on the precipitated metal hydroxides [17, 22]. One report even

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suggested that the decreased Cipro bioavailability with concomitant ingestion of milk

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was a result of its adsorption on the protein casein rather than binding to calcium ions
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[23], but that was disputed later by others [8, 15]. Finally, few researchers suggested the

possibility that the formed complexes might not be absorbed due to other factors such as
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size, geometry and ability to interact with drug transporters [8, 15, 16, 24].
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Zakelj et al. [24] reported no effect of calcium and magnesium ions on the permeability
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of Cipro, using side-by-side diffusion chambers with saturated solutions of drug and
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metal cations in the donor compartment. Using similar methodology, the same group

confirmed later that a formed Cipro-aluminum complex does not permeate through the
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intestinal mucosal membrane [8, 15]. To the best of our knowledge there has been only

one study that employed human carcinoma cell culture monolayer (Caco- 2) to assess the

effect of metal ions on the permeation of FQs, which reported a decrease in Cipro

permeability up to 30% and 60% in presence of Al3+ and Mg2+ ions, respectively [25].

The authors concluded that neither adsorption of FQs on the solid metal precipitate,
change in solubility, nor the decrease in permeability of the formed complex could fully

explain the investigated interaction and called for further investigations.

In this study we hypothesize that the expected large size of the formed Cipro-metal

complexes might play a major role in the observed overall decrease in Cipro permeability

when co-administered with metal ions. In order to test the hypothesis, the complexation

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behavior of Cipro with ferrous (Fe2+) and calcium (Ca2+) ions was also studied in

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presence of a selected small-sized chelating agent (ligand) with the expectation that either

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smaller size ternary complexes of Cipro would be formed (hypothesis 1), or a

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competition between the added new ligand and Cipro might take place depending on
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complex formation constant and concentration of the ligand, leading to increased free

absorbable Cipro (hypothesis 2).

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Several authors have reported ternary complexes formed between Cipro, metals and some
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other auxiliary ligands including histidine, bibyridyl and phenanthroline [7, 26, 27]. In
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this study citric acid (CitA) with its well-known calcium-chelating properties, was
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selected as a hydrophilic ligand. In addition to common techniques, molecular dynamics

(MD) simulations were employed to gain insight about the structure of Cipro:iron
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complexes.
Experimental

Materials

Ciprofloxacin hydrochloride was a kind gift of Dar Al Dawa (Amman, Jordan).

Potassium dihydrogen phosphate was obtained from Sigma-Aldrich (Germany). HPLC

grade acetonitrile and ethanol were obtained from Tedia Company (Tedia INC, USA).

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Trypan blue (MTT) cell proliferation assay kits were from iNtRON Biotechnology, INC.,

Korea. All other chemicals were procured from Sigma-Aldrich (Germany).

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Preparation and characterization of Cipro complexes
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Cipro:metal complexes were prepared to contain an estimated theoretical ratio of

Cipro:metal of 1:1 as follows; to prepare Cipro:Fe2+ complex 1.67 g of ferrous sulfate

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(FeSO4) were dissolved in 500 mL of distilled water (solution A); 0.333 g of Cipro were

dissolved in 50 mL distilled water and mixed with 75 mL of solution A, then the mixture
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was left in the fumehood until a precipitate was obtained. For preparation of Cipro:Fe2+
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complex in presence of the selected ligand CitA, the adequate weight of CitA to form a
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Cipro:CitA complex of 1:1 ratio was dissolved in the minimum volume of distilled water

and then mixed with a mixture of Cipro and ferrous sulfate as described above.
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Cipro:Ca2+ complex was prepared in a similar manner using 0.672 g of calcium chloride

(CaCl2). Complexes of CitA with the investigated metals were also similarly prepared

but in absence of Cipro (Blank complexes). The formed complexes were characterized

by melting point, HPLC, FTIR using standard KBr method and UV spectroscopy. Proton

NMR spectra were obtained in D2O using Bruker® NMR spectrometer (500 MHz).
FTIR spectra were recorded using Shimadzu FTIR spectrophotometer (Japan). A Cary

UV double beam spectrophotometer was employed to obtain all UV measurements. The

chromatographic system consisted of a Merck Hitachi HPLC unit equipped with a UV

detector, water bath to control temperature, and a computer with Claritylight® software

for data handling. The HPLC method employed a phenyl column (phenomenex 150mm

x 4.6 i.d.) and a mobile phase consisting of 10% acetonitrile in 50 mM phosphate buffer

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(pH 3) and containing 50 mg% CitA. Peaks were monitored using a UV detector set at

277 nm with column temperature maintained at 47 ºC and flow rate of 1.5 mL/min. For

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determination of CitA in the relevant complexes, 195 nm was employed for detection.

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The method was validated properly for sensitivity, selectivity, linearity, precision and
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accuracy with satisfactory outcome. Linearity was established in the range 0.25 -50

g/mL with a correlation coefficient > 0.995 and a typical calibration equation: Peak area
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= 350.8 x (x is concentration in g/mL). Precision of the method was determined at the
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lowest, medium and highest concentration and in all cases RSD values of < 4.2 % were
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obtained. Therefore, the lowest limit of quantification was taken as 0.25 g/mL.
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Accuracy was also determined at three concentration levels and the percentage error was
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< 1.1%. Selectivity was established by demonstrating that none of the background

samples (tissue culture medium/ buffer) exhibited any measurable response at the
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retention time of interest.

UV spectroscopic titrations

All solutions were prepared in 20mM Tris buffer of pH 7. Cipro was titrated with FeSO4

by adding increasing volumes of FeSO4 solution (75 mg%) to 5 mL of Cipro solution

(100 mg%) in separate tubes and completing the volumes to 20 mL with the buffer.

Solutions were vortex mixed, left to stand for 1 h then scanned in the range of 200-550

nm. For titrations involving CitA, a presumed Cipro:Fe2+ complex was prepared in situ

at an equimolar ratio by mixing 50 mL of Cipro solution (200 mg%) with 50 mL of Fe2+

solution (100 mg%). To 5 mL aliquots of the obtained mixture, increasing volumes (0-10

mL) of CitA solution (2.6 mM) were added before the volumes were made up to 20 mL

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with the buffer. Solutions were vortex mixed, left to stand for 1 h and then scanned in the

range of 200-550 nm.

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Capillary electrophoretic studies
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Capillary electrophoresis (CE) experiments were carried out using Agilent 1000 CE

equipment (Germany) equipped with a PDA detector. Fused silica capillaries (75 µm
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I.D.) with effective length of 40 cm were employed. Tris buffer at a concentration of 50

mM and at a physiological pH of 7 was employed as a background electrolyte, along with

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a voltage of 18 KV, and temperature set at 37 ºC in all capillary electrophoresis (CE)

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experiments. Electropherograms (e-gram) were monitored at 277 nm although spectra

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were recorded over the range 200-350 nm. Solutions containing a fixed concentration of

Cipro (0.26 mg/mL) were mixed with increasing amounts of the relevant metal ions using
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stock solutions of FeSO4 (0.48 mg/mL) or CaCl2 (0.19 mg/mL) to prepare six tubes

having an increasing ratio of Cipro to metal in the range 10-0.25, including a solution of

Cipro alone. A similar series of solutions was prepared for each metal but in presence of

the auxiliary ligand CitA at equimolar concentration with Cipro. All solutions were

successively injected onto CE system, using hydrodynamic injection mode (25 mbar x 20
s) starting with the solutions that contained no metals and ending with those containing

highest concentrations of the metal.

Solubility and partition coefficient

Solubility of Cipro and the potential complexes was determined by adding excess amount

of the solid material to 2 mL of Tris buffer (pH 7) and left on a shaker water bath

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maintained at 37 ºC for 24 h. Samples were then filtered and properly diluted before

being injected onto HPLC and determined using the described HPLC method. Cipro

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concentration representing solubility was determined using a properly constructed

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calibration curve for Cipro in Tris buffer. Apparent partition coefficients were
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determined for Cipro alone and for the presumed complexes which were formed in situ

i.e. 50 mg of Cipro alone or in addition to the weight of equimolar amounts of either

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CaCl2 or FeSO4 plus the equimolar amount of CitA were dissolved in 200 mL Tris buffer

(pH 7). From the obtained clear solutions, 2 mL aliquots were mixed with 2 mL butanol
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and left on shaker water bath (37 ºC) for 24 h, the aqueous layer was diluted as
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appropriate before injection. From the organic layer (butanol), 100 µL were mixed well
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with 5 mL methanol and 100 µL Tris buffer before being injected onto HPLC system and

concentrations were determined using calibration curves as described earlier.

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Diffusion through cellulose membrane

For the diffusion experiment, a locally designed glass Franz diffusion cell was employed

with a radius of 1 cm. The employed semi-permeable cellulose membrane had a

molecular weight cut-off of 12000 Dalton. 10 mL of Cipro solution in 50 mM Tris buffer

(pH 7) were placed in the donor compartment. In cases where metal ions and/or CitA

had to be examined they were dissolved in the same solution containing Cipro at the

required concentration. The receiver compartment was filled with 40 mL Tris buffer

solution and samples (0.5 mL) were withdrawn from the receiver compartment at

intervals of 0.5, 1, 2, 3, and 24 h with buffer replacements. The withdrawn samples were

centrifuged and analyzed by HPLC (as described above) to determine the concentrations

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of diffused Cipro.

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Permeation through Caco-2 cells

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Caco-2 cells with passage number (4) were thawed and maintained as an attached
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monolayer cell culture, supplemented with 10% heat-inactivated fetal bovine serum

(FBS), 1% penicillin-streptomycin and 0.14% gentamicin. When the attached cells on

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the flask surface became 80% confluent (observed by inverted light microscope), the

media was aspirated and the attached cells were washed out with 5 mL phosphate
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buffered saline(PBS). Then 2 mL of trypsin-EDTA were added to the flask, and

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incubated at 37 ºC for 10-15 min until detachment of cells was observed under the
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inverted light microscope. Thereafter, 5 mL media were added to deactivate the trypsin

and the resulted detached cells suspension was aspirated and transferred to a conical tube.
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Finally, cells were diluted to the required seeding density and used for seeding, or were

re-suspended into fresh media and grown in a new culture flask.

For counting viable cells, an aliquot of trypsinized cells was diluted with trypan blue dye

in a ratio of (1:4) and was used to completely fill the chamber of the heamocytometer and

then placed under the microscope. Finally, the total number of cells per milliliter was
found, and appropriate volumes of the cell suspensions withdrawn and used for seeding

of the 96-well and trans-well plates at the target density.

In order to determine the maximum non-toxic concentration of the test drugs that could

be used on Caco-2 cells, an MTT proliferation assay was performed. In summary, Caco-

2 cells at a density of 1×104 cells per well were seeded on 96-well plates, and incubated

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for 10 days until confluent monolayers were obtained. On the day of experiment, cell

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culture media was removed, and monolayers were washed three times, and incubated

with PBS for 2 h at 37 ºC. Then PBS was aspirated and solutions of Cipro and CitA in

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PBS were added separately to each well (6 serial dilutions at 1:2 ratio were used starting
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from maximum soluble concentration), the cells were then incubated for 1 h. Following

incubation, cells were washed by PBS, then 100 µL of culture media and 15 µL of MTT
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reagent were added to the wells, and the cells were incubated for an additional 4 h. After
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4 h incubation, the produced formazan crystals were dissolved by the addition of

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dimethyl sulfoxide (DMSO) to each well, and the absorbance was measured at 570 nm
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using a 96-well plate reader. All of the experiments were conducted in quadruplicate.
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Caco-2 cells with an average cell density of 1.5×105 cells/cm2 were seeded onto 12-well

trans-well plates, and were allowed to grow for 21 days until a confluent monolayer was
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obtained. Monolayer integrity was assured by recording the trans-epithelial electrical

resistance (TEER) during culture. Total TEER values were corrected for background, to

obtain the TEERfinal value. Membranes were used in the experiments when the TEERfinal

value reached was more than1000 Ω.cm2. To confirm monolayer integrity, the lucifer
yellow rejection assay was completed on the day before the transport study and those

inserts showing more than 99% rejection were used.

On the day of experiment culture media was aspirated, and both sides of the monolayers

were washed three times with Hank's balanced salt solution(HBSS) and pre-incubated

with the transport buffer (HBSS pH 7.4) for 60 min at 37 ºC. Subsequently, 500 µL of

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Cipro solutions alone (15g/ml) or in presence of equimolar amounts of Fe2+, Ca2+ and/or

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CitA at molar ratio of CitA to Cipro = 1or 5 were added to the apical compartment (for A

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→ B transport). The receiver (basolateral) chambers were filled with 1500 µL of fresh

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buffer (all experiments were carried out in triplicate). The transport study was initiated
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by placing the trans-well plate on microplate thermo-shaker incubator at 37 ºC and 250

oscillations per min. Samples (150 µL) were withdrawn from the receiver compartments
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at time points 30, 60, 120 and 240 min, and were replaced with fresh HBSS to maintain
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sink conditions. At the last time point (240 min), samples were taken from the donor
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compartments as well in order to confirm mass balance. Samples were stored at -20 ºC
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until HPLC analysis for Cipro content.

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Molecular dynamics simulation

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Preparation of the initial configuration

Ciprofloxacin molecules ionization state was predicted using the Marvin suite from

ChemAxon® [28], which showed that Cipro molecules are predominantly present in their

zwitterionic form at physiological pH (positively charged piperazine ring and negatively

charged carboxylic acid group). Therefore, the input structure of Cipro was set to the
zwitterion form during the molecular dynamics (MD) simulation. Then, the initial

configuration of Cipro molecules was created by the Packmol module [29], where 10

molecules of Cipro were created randomly (initial configuration). Atomic point charges

for Cipro molecules were derived from a AM1-BCC charge model with

ANTECHAMBER in AMBER14 [30]. The initial configuration was then exported to the

xleap module of Amber14 to build the topology and coordinate files, which were

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prepared using 12-6-4 LJ-type non-bonded model for parameters of Fe2+ ions and GAFF

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force field for the rest of parameters [30]. The MD simulation was run using an explicit

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water SPCE octahedral solvent box model with 8Å cut. Twenty Fe2+ ions were added to

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the initial configuration, and then the whole system was neutralized by Cl- ions.
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Minimization and MD simulation
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Water was minimized for 10000 cycles using steepest descent followed by conjugate

gradient algorithms. The whole system was then minimized for 2500 cycles using
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steepest descent and then a conjugate gradient algorithm. Then, water was equilibrated
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for 20 ps at constant volume with periodic boundaries, where weak positional restraints

with a force constant of 5 Å was applied on Cipro molecules and Fe2+ ions during the
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equilibrium stage. The whole system was then equilibrated for 100 ps using constant
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pressure periodic boundaries with no restraints. After the system minimization and

equilibration steps, it was subjected to unrestrained molecular dynamics simulation at

constant temperature of 300 K and constant pressure of 1 atm for 200 ns. Cut off

distance for the calculation of non-bonded forces was set to 8 Å, and SHAKE bond

length constraint involving hydrogen atoms was turned on.

Clustering

The 200 ns MD simulation trajectories were imaged and then water molecules were

stripped off. All trajectories were aligned against the first frame of the MD using heavy

atoms only excluding Fe2+ ions. All frames were then clustered by DBSCAN algorithm

implemented in cpptraj of AMBER14 on Fe2+ ions using minimum points of 4 and

epsilon of 30 with no frame orientation (no fit), since they are previously aligned against

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the first frame [30-32]. This clustered all frames according to the Fe2+ ions, which could

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show the arrangement of Cipro molecules and Fe2+ ions in space. The most two

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populated clusters were chosen to represent the complex formation between Cipro

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molecules and Fe2+ ions.
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Results and discussion
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Characterization of complexes
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Precipitates were obtained out of aqueous solutions upon standing in a fume cabinet. The
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obtained precipitates were characterized by melting point, UV spectra, FTIR and HPLC.
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A summary of the obtained melting points and HPLC analysis of Cipro content within the

precipitates is shown in (Table 1). The purpose of this characterization step was to assess
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the possibility of ternary complex formation between Cipro, metals and CitA. While the

measured melting pointof Cipro:Ca2+ complex was almost close to that of free Cipro, the

measured melting point for Cipro:Fe2+ complex was clearly lower (~30 ºC). Potential

complexes involving the auxiliary ligand (CitA) exhibited even much lower melting

points than that of Cipro alone (~100 ºC for Cipro:Ca2+:CitA and 40 ºC for
Cipro:Fe2+:CitA) and indicating, firstly the formation of a new chemical entity which is

most likely a complex involving Cipro, CitA and the metal (Fe2+ and Ca2+). Secondly the

formed species seem to have adisturbed molecular arrangement and intermolecular

interactions which were reflected as a significant decrease in melting point.

Further support of ternary complex formation was obtained through HPLC analysis of the

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percentage content of Cipro and CitA in each product (shown in Table 1). According to

the obtained percentages, theoretical calculations were performed to predict possible

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formulae that match the practically found values. With the exception of Cipro:CitA case,

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other potential complexes clearly fitted a formulae of 1:1 Cipro:CitA and/or metal (Table

1).
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UV spectra for the obtained precipitates showed only slight shifts in the two max values
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for the free Cipro (272 -275 nm and 319-329 nm), with the most obvious shifts observed
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for complexes containing iron. FTIR spectra were obtained for the different precipitated
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materials; sample spectra for Cipro, Cipro:Ca2+ and Cipro:CitA were presented in Figure
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2. It was interesting to find that Cipro appeared to form adducts with CitA e.g. ion pairs

even in absence of metals. Many absorption bands in the FTIR spectrum of Cipro were
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shifted to a higher or a lower frequency, new peaks emerged, overlapped or even almost

completely vanished (see arrows in Fig. 2). The most prominent absorption bands of

Cipro were shown at 3529-3373 cm-1, 2503-2692 cm-1, 1709 cm-1and 1624 cm-1 which

accords well with previous reports. The broad band above 3370 cm-1 with a clearly

defined maximum at 3529 cm-1corresponds to different formats of N-H and carboxylic O-

H groups stretching and may include those of water molecules. Further reflections for

the dimeric carboxyl O-H stretch, and a reflection for the protonated amino group are

seen as more than one band in the range of 2503-2692 cm-1 [33]. The strong bands at

1707 cm-1 and 1624 cm-1 correspond to the carbonyl groups of the carboxylic acid and

epicyclic keton groups respectively [34, 35]. In the FTIR spectra (Fig. 2) for Cipro

complexes with Ca2+, the most obvious changes were: (1) the N-H and O-H stretch

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regions where a very broad band replaced the distinct sharper ones in free Cipro spectrum

suggesting deprotonation of the carboxylic group, involvement of N-H bond in new

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hydrogen bonds, and potential presence of hydrogen bonded water molecules, (2) the

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obvious decrease in the intensity of the multiple absorption bands in the region of 2503-
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2692 cm-1 suggested disruption of Cipro dimeric species upon complexation, and (3)

significant decrease in the intensity of carboxyl band at 1707 cm-1 with increase in the
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relative intensity of the band at 1629 cm-1 (which also became slightly broader) which

suggests involvement of both carbonyl groups (carboxylic acid and epicylcic ketone) in
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the process of complex formation in accordance with most of the published reports about
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Cipro metal complexes [20, 33-38]. However, in some of the reported studies the band at
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1707 cm-1 vanished almost completely upon complexation, although other studies

indicated only a significant decrease in intensity which is similar to results reported in our
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study [21, 29, 38, 39].

The FTIR spectra for Cipro:metal complexes (Cipro:M) in presence of CitA differed

from either Cipro, Cipro:M or free CitA itself. The obtained FTIR spectrum for

Cipro:Fe2+:CitA was obviously different from Cipro, Cipro:Fe2+ or Cipro:CitA and thus,
the formation of a ternary complex was evident. For the potential ternary complex

(Cipro:Ca2+:CitA) the obtained spectrum was similar to that obtained for Cipro:CitA

complex, thus it could not be confirmed by FTIR if ternary complexes involving Cipro,

calcium and CitA were formed; although there was a significant difference in the melting

points of the two products ~ 45 ºC (Table 1).

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Percentage content of Cipro, as determined by HPLC, in each precipitate and the likely

stoichiometric ratios of the potential complexes are presented in Table 1. The obtained

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binary complexes Cipro:Ca2+ and Cipro:Fe2+ were most likely of 1:1 stoichiometry, being

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consistent with the FTIR data, where the frequency for both carbonyl carbons of ketone
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and carboxylic acid groups were shifted as in many previous reports [6, 36, 39-41].

HPLC analysis demonstrated the presence of both of CitA and Cipro in the relevant
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complexes. According to calculated percentages of Cipro and CitA, ternary complexes

involving calcium appeared to join one molecule of the auxiliary ligand CitA to one
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molecule of Cipro together with one atom of calcium. For binary complexes between
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Cipro and CitA (without calcium) the obtained ratio was clearly two molecules of Cipro
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joined to one molecule of CitA, in accordance with salt formation between the

tricarboxylic acid CitA and the monobasic Cipro.

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For CitA-iron ternary complexes with Cipro, the estimated ratios allowed for two

stoichiometric ratios: either 1 or 2 molecules of Cipro attached to one molecule of CitA

and one atom of iron.

Solubility and partition coefficient

The solubility data and butanol/water partition coefficients values for Cipro and its

various presumed complexes are shown in Table2. Cipro exhibits very high aqueous

solubility at pH 7 of about 28 mg/mL which is consistent with previously reported values

[41]. Complex formation with either Fe2+ or Ca2+significantly decreased its aqueous

solubility to < 2 mg/mL, which is still regarded as a highly soluble chemical species

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according to FDA Biopharmaceutics Classification System (BCS) [42]. Thus the

observed decrease in solubility of Cipro:M (~ 1-1.5 mg/mL) complexes as compared to

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Cipro(~29 mg/mL) cannot be the main reason behind the impaired absorption and

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bioavailability of Cipro in the presence of metal ions. CitA appeared to have a
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differential effect on the metal complexes i.e. it almost did not alter the solubility of

Cipro:Ca2+ complex, but significantly improved that of Cipro:Fe2+. These observations

M
may accord with the analysis of the components of the complexes where the

Cipro:Fe2+:CitA complex contained two molecules of ionized Cipro instead of one and
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thus its solubility might be higher than Cipro:Fe2+.

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Initially, attempts were made to determine apparent partition coefficients in an

octanol/water system, but the highly hydrophilic Cipro displayed minimal partitioning in
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that system, so a butanol/water system was employed (Table 2). Even in the significantly

more polar butanol, Cipro was found to still favor the aqueous phase (obtained logP =

0.8). Cipro complexes with Fe2+and Ca2+ exhibited even a lower apparent logP value

(0.46) compared to free Cipro. It was interesting to observe that despite the observed

lower aqueous solubility of Cipro:M complexes, they did not exhibit higher logP values
which suggests that the change induced by complexation is not simply a change in

hydrophilicity but possibly a generation of large molecular structures that are held by

extended intermolecular interactions. Such extended polymeric molecular structures of

large size have been previously reported for Cipro:M complexes using X-Ray

crystallography and some have been shown to have extended helical arrangements [43-

45].

t
ip
CitA raised the measured logP value in presence of Ca2+or Fe2+ to 0.65, thus approaching

cr
that of native Cipro. The obtained logP values for both metal ternary complexes

us
involving CitA were intermediate between that of their binary complexes values and free
an
Cipro. CitA appeared to shift both solubility and logP values towards those of native

Cipro and thus was a promising agent in counteracting the effect of metals on
M
bioavailability of Cipro. Overall, solubility and apparent logP values tend to support the

large molecular aggregations hypothesis for explaining the in vivo decreased absorption
e d

of Cipro when co-administered with metal ions such as Ca2+and Fe2+,rather than a simple
pt

decrease in hydrophilicity.
ce

UV titration of Cipro and Cipro:Fe2+ with CitA

Ac

The complex formed between Fe2+ and Cipro (Cipro:Fe2+) was characterized by a

yellowish color that is a result of increased absorption of light at ~ 450nm. No change in

absorptivity was observed for Fe2+ complexes with CitA due to limited chromophore.

This difference was exploited to examine the effect of CitA on Cipro:Fe2+ complexes by

spectrophotometric titration of an equimolar aliquot of Cipro:Fe2+ with CitA. CitA was

shown to significantly decrease the absorbance at 450 nm (Fig. 3). The decrease in

absorbance at about 450 nm confirms its ability to effectively compete with Cipro to form

Fe2+ complexes. Moreover, the CitA titration curve suggests the formation of a ternary

complex involving Cipro:Fe2+:CitA rather than liberating Cipro form its iron complex to

form a Cipro:CitA complex. That was inferred from the observed break point (ratio) in

the curve which was closer to 0.5, rather than 1 or more.

t
ip
Capillary electrophoresis studies

cr
Samples containing a fixed amount of Cipro and increasing concentrations of the metals

us
(Fe2+ or Ca2+) were injected onto CE whilst employing Tris buffer (pH 7). Cipro was
an
shown to migrate so early before electroosmotic flow (EOF) indicating it was positively

charged under the experimental pH. As more iron was added to the Cipro sample, a new
M
peak started to appear at a slightly longer migration time (tm) than Cipro peak while the

latter decreased with only a very small shoulder to the left (Fig. 4). With larger
e d

concentrations of iron (i.e. lower Cipro to metal ratio), the small shoulder (peak) started
pt

to increase in size on the left side of Cipro peak (shorter tm) while that on the right
ce

continued to become larger and broader on account of the Cipro peak. The obtained e-

grams indicated that Cipro:Fe2+ complexes were so strong so that they did not dissociate
Ac

completely during the high voltage of electrophoresis which is rather uncommon. In

most similar cases metals dissociate from ligands under the high voltage applied [46, 47].

E-grams (Fig.4) not only provide a direct evidence of complexes of Cipro:Fe2+ that are

sufficiently stable to withstand the electrophoretic conditions, but also tend to shed some

light on the chemical nature of the formed complexes. It was interesting to observe that
the complexes formed at higher Cipro:Fe2+ ratios (~ 4 and 2) tend to have longer

migration times than the positively charged free Cipro i.e. closer to the migration time of

EOF. The positive charge on Cipro can only be increased as a result of complexation

with the cations because of the positive charge on the metal, and if so the expected

migration time should be rather lower than that of Cipro under the operating CE

conditions. Having the complex migrating in the opposite direction suggests a

t
ip
significantly larger species so that it migrates more slowly towards the cathode, which

accords with our suggestion that the size of Cipro:M complexes is significantly large.

cr
However, the observed small peak of the complexed species that started to appear below

us
a Cipro:M ratio of approximately 1 suggests that it belongs to a 1:1 Cipro:M complex
an
which has different properties (namely size) from 2:1 or higher order complexes. The

envisaged 2:1 complexes in this study might be in fact composed of several Cipro
M
molecules with several Fe2+ atoms, but with ratio 2:1. The so obtained large-sized

complexes can explain the observed unexpected shift in the migration time of Cipro:Fe2+
e d

2:1 complexes. That might help explaining the rather conflicting findings in literature
pt

pertaining the physicochemical properties of Cipro:M complexes compared to free Cipro

ce

i.e. the obtained data for 2:1 complexes may significantly differ from the 1:1 complexes.

To the best of our knowledge this is the first experimental evidence that the 1:1 Cipro:M
Ac

complexed species differ significantly in physicochemical behavior than their 2:1

counterparts. When the experiment was repeated in presence of equimolar CitA, the

faster migrating complex (1:1) started to form earlier i.e. at higher ratios of Cipro:M (~

4:1- 2:1) and the apparently larger complexes appeared when the ratio of Cipro:M

approached 1:1(Fig. 4b). The obtained data could be explained as CitA competes with
Cipro, leading to formation of ternary complexes rather than expelling free Cipro and

when excess iron is available then both ligands (Cipro and CitA) form independent

complexes so that the larger-sized Cipro:M complexes with higher migration times

reappeared.

On the other hand, Cipro:Ca2+ complexes appeared to be significantly weaker so that they

could not withstand the electrophoretic conditions as there was no evidence of complexed

t
ip
species i.e no change in peak shape or emergence of new peaks. Presence of CitA did not

change the picture apart from making the peak of Cipro (even when present alone)

cr
sharper, indicating strong intermolecular association of Cipro with CitA rather than with

us
calcium ions.
an
Diffusion through cellulose membrane
M
The total diffused amounts (eflux) of Cipro through cellulose membranes with molecular

weight cut-off of 12000 Dalton were determined in the presence and absence of metals
e d

and/or CitA (Fig.5). Figure 5 clearly shows a significant decrease (~ 50%) in the
pt

diffused amount of Cipro when Fe2+or Ca2+ ions were present as compared to free Cipro.
ce

Although no precipitates were observed in the donor or the receiver compartments, still

the complexes formed by metal ions and Cipro led to a decrease in the diffused amount of
Ac

Cipro, which supported the hypothesis that the size of the formed complex, rather than its

solubility, was a major determinant of its permeation through the membrane. The

presence of CitA (without a metal) also decreased permeation of Cipro, although to a

lesser extent than metals did.

Permeation experiments in the manner described above have been previously

documented as evidence of large self-associated aggregates for non-polymeric molecules

e.g. cyclodextrins and their guest molecules [48, 49]. Therefore, the observed decrease in

permeation of Cipro across the cellulose membrane in presence of metal ions suggests the

formation of large-sized aggregates of self-associated Cipro: metal complexes. Previous

reports have shown the unique ability of Cipro complexes in particular to form such large

t
ip
molecular aggregates in elongated helical shapes that are maintained in the solid state

[45], which is in line with the suggested hypothesis and experimental findings.

cr
us
The presence of CitA in the donor compartment together with Fe2+ and Cipro
an
significantly enhanced the total efflux of Cipro even to a slightly higher level than that of

free Cipro. The counteracting effect of CitA to Fe2+ ions could be explained as CitA
M
preferentially contributing to complex formation with Fe2+not necessarily as a single

CitA:Fe2+ complex, but rather ternary complexes involving Cipro, CitA and Fe2+.
e d

Regarding calcium ions, CitA appeared to only marginally enhance the efflux of Cipro,
pt

suggesting large sizes of the relevant complexes.

ce

Permeation through Caco-2 cells

Ac

The permeability of Cipro across Caco-2 cells was measured in HBSS at pH 7.4. The

results as the cumulative percent transported were plotted versus time, all transport

experiments were performed from the apical to the basolateral side (Fig.6).

Notably, the diffused amount of free Cipro increased linearly at earlier time points of the

experiment but became almost constant after 2 h which is most likely due to its cellular
efflux. The diffused amount of Cipro alone was generally low, mounting to not more

than 38% after 4 h of incubation, being consistent with the reported active efflux of the

drug from receiver to donor compartment [4, 24, 50]. The mass balance calculations

revealed around 85% recovery with about 15% of the drug trapped in the cellular

monolayer, possibly due to interaction of Cipro with intracellular or membrane protein

components. Presence of Ca2+ led to a linear permeation curve with obvious reduction in

t
ip
the total permeated amount of Cipro. After 4 h incubation, the recovery of the drug in

presence of calcium ions reached about 90%. In comparison, the reduction of the

cr
permeated amount brought about by presence of Fe2+ ions was modest, reaching nearly

us
20% at maximum but associated with obvious reduction in recovery (~70%). The
an
observed higher percentage of lost drug across the cellular layer, in presence of Fe2+,

accords with some previous reports where magnesium was shown to encourage
M
interaction of FQs with bacterial membrane components [51].
e d

As characterization experiments indicated formation of ion pair salt between CitA and
pt

Cipro, permeability experiments of Cipro were also performed in presence of equimolar

ce

amounts of CitA. The results indicated a significant increase in the permeation of Cipro

at later time points of incubation (Fig. 6a). The total amount of transported Cipro was
Ac

almost doubled in presence of CitA. This could be attributed to the differing properties

of the formed ion pair (Cipro:CitA) which, unlike Cipro, might not be a substrate to the

active efflux pump. It is worth to mention that CitA, when present with Cipro, caused a

decrease in the efflux of Cipro through the synthetic membrane i.e. opposite to its effect
in case of Caco-2 cells, which indicates that the observed effects in the cellular system

could have been due to interaction with some cellular targets rather than a size issue.

The presence of CitA as a competing ligand exhibited variable effects on the two

Cipro:metal complexes. In the case of calcium, the presence of CitA at equimolar

concentration of Cipro, decreased Cipro permeability dramatically to a level even lower

t
ip
than that observed in its absence. At concentrations 5 times that of Cipro it improved the

permeability significantly to the extent that it became close to that of free Cipro (Fig. 6b).

cr
The observed concentration dependent effect of CitA on permeation of Cipro accords

us
with a model where at lower concentrations of CitA, the competition of CitA with the
an
metal (Ca2+ in particular) can only result in the formation of ternary complexes involving

Ca2+, Cipro and CitA, but the chance of liberating Cipro from its calcium complex is
M
quite low. Ternary complexes involving Cipro, Ca2+ and CitA appear to be even worse in

their ability to cross through the cellular monolayer than Cipro either due to their higher
e d

ability to form large molecular aggregates, or a three-dimensional shape and functionality

pt

which facilitates interaction with cellular components (proteins), thus hindering

ce

permeation.

Understandably higher ratios of CitA to Cipro (5 times) may shift the equilibrium
Ac

towards the formation of binary CitA:Ca2+ complexes leading to liberation of Cipro

which consequently would travel cross the Caco-2 cellular layer in the same mechanism

and rate of free Cipro.

On the other hand, when CitA was added to Cipro:Fe2+at an equimolar concentration to

Cipro, or even five folds higher,the permeation profile did not change significantly (Fig.

6a).

A possible explanation to the observed differential effect of the two metals is that the

Cipro:Fe2+ complexes were significantly stronger than Cipro:Ca2+ complexes, as

experimentally observed through CE studies.

t
ip
NMR study of Cipro:Ca2+:CitA complexes

cr
Since the effect of CitA on permeation of Cipro in presence of Ca2+ was shown to be

us
concentration dependent, attempts were made to obtain further insight about the potential
an
complexes. NMR spectra were obtained for Cipro alone and in presence of Ca2+ and/or

CitA (at equimolar and 5 fold levels) in distilled water. Presence of calcium ions resulted
M
in obvious shifts in the frequency of the aromatic protons of Cipro in particular. The

most obvious change was that of the aromatic protons at positions 2 and 3(Fig. 7) which
e d

appeared as one overlapped signal at ~ 7.2 ppm for Cipro alone [52]. In the presence of
pt

Ca2+, that peak was split into two peaks, each of them appearing as a doublet, which
ce

obviously suggests serious changes in Cipro molecular arrangements e.g. stacking that

allows coupling of protons on positions 2 and 3 with each other leading to appearance of
Ac

each as doublets. Because protons 2 and 3 are para to each other, their coupling would

not be observed in most cases (as was seen for native Cipro), thus the most plausible

explanation is through space coupling (scalar coupling) which might be enabled upon

complexation where protons 2 and 3 from two different molecules that may come close

enough to each other [53].

Addition of CitA (at two concentrations) to the presumed Cipro:Ca2+ complex showed

only slight changes in the NMR spectra, thus being consistent with FTIR data which also

could not show a clear evidence of ternary complex formation (Fig. 7). Presence of CitA

with Cipro resulted in a similar change (almost identical to that obtained for Cipro:Ca2+),

which supports the suggestion that the most dramatic effect on Cipro’s structure was

changes in the pattern of molecular stacking.

t
ip
Although results of the two permeation experiments (synthetic membrane and Caco-2

cr
cells) indicated generally similar results in the sense that metals decreased permeation of

us
Cipro, which can be alleviated to some extent by CitA, some differences could be
an
highlighted. These findings suggest that size, while a crucial determinant, is not the only

factor that controls permeation through GIT epithelial cells. Rather a combination of
M
complex size, molecular shape and functionality that allows or hinders interaction with

bio-molecules on cell surface (receptors) or proteins whilst crossing the cellular

e d

monolayer. A hydrophilic ligand such as CitA at high concentrations appears to offer

pt

means by which the negative effect of some metals on absorption of Cipro might be
ce

counteracted.
Ac

Molecular dynamics simulation

Molecular dynamics (MD) simulation has been proved to be a useful method to detect

complex formation [54]. In this study, MD simulation has been used to explore the

nature of the interaction between Fe2+ ions and Cipro molecules as well as their spatial

orientation upon complex formation. Figure 8 shows the root mean square deviation
(RMSD) of Cipro molecules from the initial configuration. This indicates that Cipro

molecules have deviated from the initial configuration, which is the dispersed molecules,

and rearranged themselves to a more energy favored spatial arrangement with Fe2+ions.

Furthermore, the RMSD pattern shows that Cipro molecules are present in different

clusters that have different space arrangement from the initial configuration, however

close to each other.

t
ip
The clustering procedure produced two closely related major clusters, where Cipro

cr
molecules are arranged in three molecules per complex (Fig.9a and Fig. 9c). Closer

us
inspection of these clusters showed that Cipro molecules are arranged so that all
an
carboxylic acid groups are aligned on top of each other with a narrow angle between

them of ~30º (Fig. 9b and Fig. 9d). Two Fe2+ ions are coordinated to the carboxylic acid
M
groups of three Cipro molecules. This arrangement could be created by the formation of

coordination bonds between lone pairs of carboxylic acid groups and Fe2+ ions, which
e d

allow the approach of the negatively charged carboxylic acid groups. This arrangement
pt

puts the protons of the aromatic rings in a more shielded position, thus it is predicted that
ce

the NMR spectrum of the complex is different from that of the Cipro molecules in their

dispersed form. Indeed, the obtained NMR spectrum for the Cipro:Ca2+complex, which
Ac

is predicted to form a similar complex, was different from that for separated Cipro

molecules (Fig. 7), and in accordance with MD simulation prediction. The anticipated

Cipro: metal complex according to MD simulation is clearly several times larger in size

than the isolated monomers, which supports that lower permeation of the complex is

attributed to size effect rather than solubility issues.

Finally, to inspect the strength of the complex formation, we extracted frames at 0 ns, 50

ns, 100 ns and 200 ns of the MD simulation (Fig. 10). It is clear from this figure that the

complex between Cipro molecules and Fe2+ ions has been formed early from the

beginning of the MD simulation. This demonstrates that this complex is strong and its

formation is highly favored, since it lasted till the end of simulation, which is in

accordance to our findings using capillary electrophoresis. A graphic representation of

t
ip
the hypothesis and overall findings is presented in Fig. 11.

cr
Conclusion

us
Complexes of Cipro with iron and/or calcium were shown to form in aqueous medium.
an
Some ternary complexes have also been shown to form involving Cipro, iron and CitA.

The solubility and partition coefficients of the so obtained complexes, although changed
M
negatively, they were not likely to be the reason behind the usually observed decrease of

oral bioavailability of Cipro when co-administered with metal ions. Diffusion studies
e d

through synthetic cellulose membrane provided good evidence that size of the obtained
pt

Cipro: metal complexes might be a key element in the decreased absorption of Cipro.
ce

MD simulation studies provided excellent evidence on the most likely three-dimensional

structure of the anticipated Cipro complexes with iron, demonstrating the significantly
Ac

increased molecular size as compared to the free Cipro monomers.

Presence of the auxiliary ligand CitA appeared to counteract the effect of iron ions only

on the permeation of the drug through the synthetic membrane; most likely through

involvement of ternary complexes which might be of smaller sizes than binary Cipro:

metal complexes.
Using Caco-2 cells, we confirmed that calcium in particular decreases the permeability of

Cipro across the monolayer even at low concentrations (15 g/ml), which precludes the

possibility of solubility being the reason behind the decrease in permeation of Cipro in

presence of metals. CitA as an auxiliary ligand had a concentration dependent effect on

Cipro permeability, and it was found to minimize the negative effect of metals on

t
ip
absorption of Cipro when available at 5 fold molar concentration of Cipro. Thus in

urgent cases where metals have to be given along with the drug for the purpose of

cr
maximizing clinical benefits, a formula comprising CitA might offer a good option.

us
Consumption of food rich in CitA e.g. citrus fruits might have to be revised when Cipro
an
is prescribed. Most interesting perhaps was the observation that CitA might form an ion

pair with Cipro leading to almost two fold increase in bioavailability, which might be
M
very promising in developing more clinically effective forms of Cipro.
e d

Acknowledgment
pt

The authors wish to thank the Deanship of Academic Research at The University of
ce

Jordan for financial support (Grant recommendation No. 27/2014-2015). Sincere thanks

to Dr Matthew D Jones from the Department of Pharmacy and Pharmacology,University

Ac

of Bath, for manuscript proofreading.

Conflict of interest

The authors declare no conflict of interest.

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52- Turcu I, Bogdan M. Size dependence of molecular self-assembling in stacked
aggregates. 1. NMR investigation of ciprofloxacin self-association. J Phys Chem B.
2012;116:6488-6498.
M
53- Bifulco G, Mangoni A. 1H-1H scalar coupling across two stacked aromatic rings: DFT
d

calculations and experimental proof. Magnetic Res Chem.2008;46:199-201.

54- Ghattas MA, Bryce RA, Al Rawashdah S, et al. Comparative molecular dynamics
e

simulation of aggregating and non‐aggregating inhibitor solutions: understanding the

pt

molecular basis of promiscuity. Chem Med Chem. 2018;13:500-506.

ce
Ac
Figure 1

t
ip
cr
Fig. 1: Structure of ciprofloxacin (Cipro).

us
an
M
e d
pt
ce
Ac
Figure 2

t
ip
cr
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e d
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ce
Ac

Fig. 2: FTIR spectra of Cipro (A, red), Cipro:Ca2+ (B, blue ) and Cipro:Ca2+:CitA (C,
black). Colors are visible on the net version.
Figure 3
Cipro with Fe Cipro:Fe with CitA

0.7

0.6
Absorbance at 450nm

0.5

0.4

t
0.3

ip
0.2

cr
0.1

us
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Molar ratio
an
Fig. 3: Titration of Cipro with Fe2+ ions in Tris buffer (■), in comparison to titration of
M
equimolar mixture of Cipro and Fe2+ with CitA (○) .
e d
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ce
Ac
Figure 4
A B

r= ∞

r= 4

t
ip
cr
r= 0.5

us
an
Fig. 4: E-grams of Cipro in presence of decreasing ratios of Cipro to Fe2+ (r) in absence
(A) and presence (B) of CitA.
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e d
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Ac
Figure 5
5000

4500

4000

3500
Flux of Cipro over 5 hrs

3000

t
2500

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2000

cr
1500

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1000

500
an
0
Cipro Cipro:Fe Cipro:Ca Cipro:CitA Cipro:Fe:1xCitA Cipro:Ca:1xCitA
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Fig. 5: Summary of the total efflux of Cipro through synthetic membrane calculated at
5 hours, alone and in presence of metals and/or CitA.
e d
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ce
Ac
Figure 6
Cipro alone Cipro+Fe Cipro:Fe:1xCitA

Cipro:Fe:5x CitA Cipro: CitA

80
70
60
50
% diffused

40

t
30

ip
20 A

cr
10
0

us
0 1 2 3 4 5
Time (hr)

Cipro alone
Cipro:Ca:1x CitA
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Cipro+Ca
Cip:Ca: 5x CitA
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45
40
35
d

30
e
% diffused

25
pt

20
15
ce

10 B
5
Ac

0
0 1 2 3 4 5
Time (hr)

Fig. 6: Plot of percentage diffused Cipro against time, in presence and absence of
metals (Fe2+ (A) and Ca2+ (B)); details for each case are shown on the plates.
Figure 7

Cipr
o

t
ip
Cipro:Ca2
+

cr
us
an Cipro:Ca2+:Cit
A
M
Cipro:Ca2+:5x
d

CitA
e
pt
ce

Fig. 7: Proton NMR spectra in the aromatic region, for Cipro alone and with calcium
Ac

and/or CitA. Colors are visible on the net version.

Figure 8

t
ip
cr
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Fig. 8: Root mean square deviation (RMSD) of Cipro molecules during molecular
dynamics (MD) simulation referenced to the first frame.
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Ac
Figure 9
A B

t
ip
C D

cr
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Fig. 9: First (A) and second (B) clusters of Cipro:Fe2+ complex. Expanded view of first
(C) and second (D) clusters. The large spheres represent Fe+2 ions.
e d
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ce
Ac
Figure 10

t
ip
0 ns 50 ns

cr
us
an
M
100
ns 200 ns
d

Fig. 10: Snapshots of the production MD simulation of Cipro molecules and Fe2+ ions at
e

0 ns, 50 ns, 100 ns and 200 ns. Cipro molecules are represented as surfaces
while Fe2+ ions are represented by large spheres.
pt
ce
Ac
Figure 11

t
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cr
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e d
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ce
Ac

Fig. 11: A graphical representation of the experimental findings, (a) reasonable

permeation of ciprofloxacin, (b) presence of metals results in large size complexes that
lead to low permeation, (c) presence of citric acid interferes with the metal-ciprofloxacin
complexes and to some extent counteracts the effect of metals, and (d) presence of citric
acid alone results in significant improvement of permeation, likely through ion pair
formation.
Table 1: Summary of the physicochemical parameters measured for the obtained
complexes.

Compound Melting point % Cipro % CitA Conclusion / Predicted

(ºC) formula
Cipro 317-319 NA NA NA
Cipro:Ca2+ 324* 71.5 NA Ratio of Cipro:Ca2+ must be <
2:1 i.e.1:1 e.g.
[Cipro:Ca2+].6H2O
Cipro:Ca2+:CitA 228-232 63.4 33.4 Ratio of Cipro:Ca2+ must be <
2:1 i.e.1:1 e.g.

t
Cipro:Ca2+:CitA

ip
Cipro:Fe2+ 290* 62.7 NA Ratio of Cipro:Ca2+ must be <
2:1 i.e.1:1 e.g.

cr
[Cipro:Fe2+].6H2O
Cipro:Fe2+:CitA 278* 65.4 21.3% Allows for both ratios 2:1 and

us
1:1 (more likely)
[(Cipro)2:Fe2+:CitA] or
an [Cipro: Fe2+:CitA]

Cipro:CitA 265 82.2 20.1 (Cipro)2:CitA

M
* Indicates degradation
e d
pt
ce
Ac
 Table 2: Summary of solubility in Tris buffer pH 7 and partition coefficient
(butanol/Tris buffer pH 7) for Cipro and its complexes with Ca2+ and Fe2+ in
presence and absence of CitA ligand (n=3, RSD < 7%).

Partition coefficient logP in presence of: Solubility (µg/mL) when complexed with:
Compound
Ca2+ Fe2+ none Ca2+ Fe2+ none
Cipro alone 0.46 0.46 0.8 > 1500 1165 27953
Cipro:CitA 0.65 0.65 1406 5266

t
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cr
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e d
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Ac