Biopharmaceutical Characterization of Ciprofloxacin HCl–Ferrous Sulfate Interaction

Biopharmaceutical Characterization of Ciprofloxacin HCl–Ferrous Sulfate Interaction

PHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY Biopharmaceutical Characterization of Ciprofloxacin HCl–Ferrous Sulfate Interaction ˇ C, ´ 1 ALEKSANDR...

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Faculty of Pharmacy, University of Belgrade, Serbia


School of Pharmacy and Pharmaceutical Sciences, Trinity College, University of Dublin, Ireland

Received 29 January 2011; accepted 24 June 2011 Published online 24 July 2011 in Wiley Online Library ( DOI 10.1002/jps.22707 ABSTRACT: The ciprofloxacin–iron interaction, resulting in a lower bioavailability, is well documented in vivo; however, a mechanistic explanation supported by experimental data of this interaction is missing. In the present study, ciprofloxacin hydrochloride (HCl) and ferrous sulfate interaction was simulated in vitro by performing solubility and dissolution studies in the reactive media containing ferrous sulfate. Characterization of the precipitate formed indicated its probable chemical structure as Fe(SO4 2− )2 (Cl− )2 (ciprofloxacin)2 × (H2 O)n , where n is up to 12 molecules of water. The solubility of this complex in water was estimated to be approximately 2 mg/mL, being about 20-fold lower than the solubility of ciprofloxacin HCl. The solubility of the complex was used as input parameter for an in silico modeling by GastroPlusTM and the resulting predicted plasma time curves were in good agreement with the in vivo data. These results strongly indicate that ciprofloxacin–iron interaction in vivo is caused by the formation of a low soluble complex. This interaction was also simulated by in vitro dissolution, in which a mini scale apparatus provided more biorelevant results than the standard dissolution apparatus, probably because the drug concentrations in the mini apparatus were higher and, thus, closer to the conditions encountered in vivo. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5174–5184, 2011 Keywords: bioavailability; Biopharmaceutics Classification System (BCS); ciprofloxacin HCl; dissolution; drug interaction; in silico modeling; ferrous sulfate; oral absorption; solubility

INTRODUCTION Drug absorption from the gastrointestinal tract is a complex and dynamic process governed by a number of factors including the drug and the dosage form properties, as well as the physiological conditions present. With the introduction of Biopharmaceutics Classification System (BCS)1 and the increased understanding of gastrointestinal physiology gained over the last decade, there is an increased confidence in in vitro dissolution testing as a biorelevant and predictive methodology.2–5 Mechanistic modeling and simulation of drug dissolution and absorption have been sought as a base for in silico bioequivalence Additional Supporting Information may be found in the online version of this article. Supporting Information Correspondence to: Jelena Parojˇci´c (Telephone: + 381(0)113951363; Fax: + 381(0)113972840; E-mail: jelena.parojcic Journal of Pharmaceutical Sciences, Vol. 100, 5174–5184 (2011) © 2011 Wiley-Liss, Inc. and the American Pharmacists Association


studies6 and there is an increased interest for in vitro and in silico investigation of drug–drug and food– drug interactions.7–9 It has been shown in a previous study that in vitro solubility/dissolution studies can be used to predict physicochemical interactions that are likely to influence drug absorption rate in vitro.10 The ciprofloxacin–iron interaction is well known and documented in a number of in vivo studies.11–16 The observed, reduced, ciprofloxacin absorption may result in plasma concentrations lower than its minimal inhibitory concentration and thus compromise its antibiotic effect.11 Formation of a nonabsorbable complex has been postulated as the interaction mechanism,12,17–20 although some authors commented that other physicochemical factors, such as solubility, may also play a role.14,16 Published data related to ciprofloxacin complexation and solubility in the presence of iron and/or other metal cations are somewhat contradictory. Although ciprofloxacin tablet dissolution has been shown to be retarded,19,21 some solubility studies indicate an increased



solubility or no effect in the presence of divalent or trivalent cations.17,18,22 Increased solubility in the presence of metal cations was reported for ciprofloxacin base.22 Numerous ciprofloxacin— iron complexes have been isolated and described in the literature.23–26 Although most of the in vitro studies performed were related to the interaction of ciprofloxacin base and Fe (II) and/or Fe (III) ion, biopharmaceutical relevance of such studies is questionable because the therapeutically relevant form of ciprofloxacin for oral administration as immediate release (IR) tablets is its hydrochloride (HCL) salt and iron is orally administered in the form of Fe (II) salts. Ciprofloxacin HCl is, generally, classified as the BCS class IV drug because of its low solubility at pH 6.8 and limited bioavailability. Yet, in a recent review by Olivera et al.,27 it was emphasized that therapeutic inequivalence between brand name drug products and US Food and Drug Administration-approved generic drug products has not been reported and that risk of bioinequivalence caused by an excipient and/ or manufacturing variables for IR ciprofloxacin HCl solid oral dosage forms is low. In the present study, ciprofloxacin–iron interaction was simulated in vitro by performing solubility and dissolution studies of ciprofloxacin HCl in the reactive media containing ferrous sulfate. The precipitate formed was investigated for its quantitative composition and solid state properties. In silico simulations using the GastroPlusTM software were used to asses the biopharmaceutical impact of the low soluble complex formed, investigate the potential mechanism of this drug interaction, and propose dissolution methodology that would reflect the in vivo situation.

Experimental In Vivo Data A detailed survey of the literature data available on ciprofloxacin–iron interaction has been performed. The data collected have been carefully reviewed. The set of in vivo data on ciprofloxacin plasma concentrations following oral administration of 500 mg ciprofloxacin (in the form of HCl salt) with/without the ferrous sulfate preparation reported by Kara et al.13 and the intravenous (i.v.) data reported by Ljungberg and Nilson-Ehle28 were used for further evaluation applying both gastrointestinal simulation and numerical deconvolution. Solubility Study Solubility studies in water and the reactive media containing 7.2, 36, and 72 mM ferrous sulfate were performed using ciprofloxacin HCl salt and ciprofloxacin base. The investigated samples were continuously shaken on a laboratory shaker (Unimax DOI 10.1002/jps


1010, Heidolph, Schwabach, Germany) for 6 h and, after centrifugation (where necessary) and filtration, appropriately diluted and assayed UV spectrophotometrically at 276 nm (UV-Vis spectrophotometer (Evolution 300 spectrophotometer, Thermo Fisher Scientific, Madison, Wisconsin). In order to investigate the potential for complex formation, ciprofloxacin HCl and ferrous sulfate solutions (0.05 and 0.1 M, respectively) were mixed in different ratios (9:1, 7:3, 5:5, 3:7, and 1:9) and examined for precipitate formation. The precipitate formed was collected and, after drying at room temperature, examined using powder X-ray diffraction (XRD), differential scanning calorimetry/ thermogravimetric analysis (DSC/TGA), and Fourier transform infrared (FTIR) analysis. The quantities of ciprofloxacin, iron, chloride, and sulfate in the solid phase were determined, as well as the water content.

In Silico Absorption Simulation In silico absorption simulation was performed using the commercially available software GastroPlusTM version 6.0.1004 (Simulations Plus Inc., Lancaster, CA) based on the advanced compartmental absorption and transit model.29 Model optimization was performed based on the set of input parameters describing drug and dosage form characteristics, which were determined experimentally and/or taken from the literature. Summary of input parameters used is given in Table 1. Experimentally determined solubility of ciprofloxacin HCl and its complex with iron were used as a reference. Permeability value (Peff ) was estimated from ciprofloxacin bioavailability data using the exponential relationship established by Amidon et al.1 The relevant pharmacokinetic data were calculated from the in vivo data following ciprofloxacin i.v. administration.28 Human physiology fasted mode was used for simulation. Default absorption scale factor values were modified to reflect the in vivo data indicating rapid drug absorption in the proximal segments of the gastrointestinal tract. Consequently, they were scaled to zero below the “jejunum 2” compartment. The pH value in “duodenum” compartment was adjusted to 4.04 in accordance with the ciprofloxacin HCl experimental data. The model developed was used to simulate the influence of solubility on ciprofloxacin HCl plasma concentration profile and to estimate drug absorption with and without ferrous sulfate coadministration. Hypothetical in vivo absorption profiles calculated by numerical deconvolution were used as a reference. The relevant percent prediction error (PE%) values between the in vivo observed and in silico predicted pharmacokinetic parameters were calculated as follows: PE% =

PKpredicted − PKobserved × 100 PKobserved




Table 1. GastroPlus summary of ciprofloxacin HCl input parameters Parameters Molecular weight log P pK a1 pK a2 Dose Solubility at pH 4.04 Solubility of ciprofloxacin–iron complex Diffusion coefficient Drug particle density Peff (human jejunal permeability) Body weight Blood/plasma concentration ratio Unbound percent in plasma Clearance (CL) Volume of distribution (Vc ) Peripheral volume (V2 ) Elimination half-life (T1/2 ) (h) Distribution rate constants k12 k21

Value 385.8 g/mol 1.32a 8.62b 6.16b 500 mg 42 mg/mLc 2.2 mg/mLc 0.75 × 105 cm2 /s 1.2 g/mLd 1.57 × 10−4 cm/se 70 kg 1f 70%g 26 L/hh 0.56 L/kgh 1.347 L/kgf 4.08f 2.3753 L/hh 0.98752 L/hh

a See

Ref. 30. Ref. 31. c Experimental value. d GastroPlus default values. e See text. f In silico predicted (ADMETPredictorTM module). g See Ref. 32. h Calculated by Kinetica program from Ref. 28. b See

where PK denotes the relevant pharmacokinetic parameter (i.e., maximum plasma concentration, Cmax or area under the plasma concentration time curve, AUC). Numerical Deconvolution In order to estimate the relevant absorption kinetics, numerical deconvolution was performed using the selected set of data on ciprofloxacin plasma concentrations following oral administration without (control study) and with ferrous sulfate reported by Kara et al.13 The pharmacokinetic profile observed after i.v. administration28 was employed as the weighting function. Commercially available software Thermo Kinetica version 5.0 (Thermo Fisher Scientific) was used for numerical deconvolution. Dissolution Studies Dissolution studies of commercially available ciprofloxacin HCl tablets (Marocen, Hemofarm STADA, Serbia) were performed under the compendial conditions, using water as the dissolution medium and the paddle rotating speed of 50 rpm (British Pharmacopoeia 2010). Standard paddle dissolution apparatus, as well as the mini paddle (Erweka DT 700, Heusenstamm, Germany) were used in order to investigate the effect of media volume on drug dissolution. The experiments were performed in 900, 250, 150, and 50 mL of dissolution media with JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011

and without ferrous sulfate added. Dissolution media samples were withdrawn at the predetermined time intervals, appropriately diluted and assayed for ciprofloxacin UV spectrophotometrically at 276 nm. The solid phase from the dissolution vessel was collected and dried at room temperature for further analysis. Solid Phase Characterization

Differential Scanning Calorimetry Differential scanning calorimetry experiments were performed using a Mettler Toledo DSC 821e with a refrigerated cooling system LabPlant RP-100. Nitrogen was used as the purge gas. Aluminum sample holders were sealed with a lid and pierced to provide three vent holes. Sample volume was sufficient to provide proper contact between the powder and the bottom of the pan, and sample weight was 5 mg or more. DSC measurements were carried out at a heating/cooling rate of 10◦ C/min. The DSC system was controlled by Mettler Toledo STARe software (version 6.10) working on a Windows NT operating system. The unit was calibrated with indium and zinc standards.

Thermogravimetric Analysis Thermogravimetric analysis was performed using a Mettler TG 50 module linked to a Mettler MT5 balance. Samples (5–12 mg) were placed into open aluminum pans. A heating rate of 10◦ C/min was implemented in all measurements. Analysis was carried out in the furnace under nitrogen purge and monitored by Mettler Toledo STARe software (version 6.10) with a Windows NT operating system.

Powder X-ray Diffraction Powder XRD analysis was conducted using a Miniflex II Desktop X-ray diffractometer Rigaku with Ilaskris cooling unit. The tube output voltage used was 30 kV and tube output current was 15 mA. A Cu-tube with Ni-filter suppressing K$ radiation was used. Measurements were taken at ambient conditions from 5 to 40 on the 2θ scale at a step size of 0.05◦ per second in each case.

Fourier Transform Infrared Analysis Infrared spectra were recorded on a Nicolet Magna IR 560 E.S.P. spectrophotometer equipped with MCT/A detector, working under Omnic software version 4.1. A spectral range of 650–4000 cm−1 , resolution 2 cm−1 , and accumulation of 64 scans were used in order to obtain good quality spectra. A KBr disk method was used with a 1% sample loading. KBr disks were prepared by direct compression under 8 bar pressure for 1 min. DOI 10.1002/jps


Table 2.


Summary of the in vivo data on ciprofloxacin absolute bioavailability

Dose (mg)

ka (h−1 )

Cmax (mg/L)

tmax (h)

50 100 750

0.28 0.49 2.65

0.58 0.825 1.16

100 250 500 1000

0.73 1.59 2.77 5.57

1 1.25 1.54 1.75

3.633 3.367 3.120 2.433

100 250/fasting 250/fed 500 750

0.37 1.04 0.83 1.51 1.97

1.16 1 1.34 1.18 1.26

1.782 2.034 2.142 1.476 1.842




200 750

1.18 2.97

0.69 1.38

500 750

2.7 3.8


AUC (mgh/L)

Fa (%)

Number of Volunteers

1.0 1.9 12.2

77 63 54


Hoffken et al.34

2.10 5.28 9.61 22.84

82.7 83.1 75.7 89.9


Bergan et al.35

1.77 4.23 3.58 6.78 8.77


12 10 10 10 12

Borner et al.36



Drusano et al.37

4.18 15.3

69.0 69.1


Plaisance et al.38

10.7 16.8

75 78


Lettieri et al.39

0.545 0.441 0.283 0.302

8.58 1.48

Inductive-Coupled Plasma Mass Spectrometry



LCK 311 test. The test is based on the photometric determination (8 = 468 nm, Dr. Lange Lasa 100 spectrophotometer; Dr. Bruno Lange GmbH) of Fe (III) thiocynate concentration formed by thiocynate ions released from mercury thiocynate reacting with the chloride ions.

The samples were first digested with 69% HNO3 and H2 O2 and then made up to a final volume of 50 mL with deionized water. This digest was then analyzed by inductive-coupled plasma mass spectrometry (ICPMS) Varian 820 to measure the level of iron. Certified calibration standards were used to calibrate the ICPMS and the samples were diluted where necessary to bring them into the range of the calibration curve.

RESULTS AND DISCUSSION In Vivo Bioavailability

Quantification of Chloride and Sulfate Anions

Ciprofloxacin HCl absorption from tablets is, generally, described as well and rapid, with absolute bioavailability of approximately 70%.33 Biopharmaceutical characteristics of ciprofloxacin HCl have been recently reviewed by Olivera et al.27 Summary of the literature data on ciprofloxacin absolute bioavailability is given in Table 2. The in vivo data indicate rapid and somewhat variable drug absorption with

Concentration of the sulfate ions was determined using a Dr. Lange LCK 153 test based on the principle of barium sulfate formation. The resulting turbidity was measured photometrically (Dr. Lange Lasa 100 spectrophotometer; Dr. Bruno Lange GmbH, Berlin, Germany) at a wavelength of 430 nm. Concentration of the chloride ions was performed with a Dr. Lange Table 3.

Summary of the in vivo data on ciprofloxacin HCI -iron interaction following 500 mg ciprofloxacin oral administration

Study Description Control 1 Control 2 Iron (II) Sulfate Control Iron (II) fumarate Control Iron (II) sulfate Iron (II) gluconate Control study Iron (II) sulfate slow release Iron (II) gluconate

Iron Dose (mg) Cmax (mg/L) tmax (h) AUC (mgh/L) – – 65 (t.i.d.) – 70 – 60 69.6 – 100

2.8 3.2 0.7 2.1 0.6 3.0 2.0 1.3 2.15 0.95 2.4 1.0

1.5 1.5 1.2 0.9 1.1 1.3 1.0

14.66 15.71 5.37 13.3 4.0 16.2 9.4 5.8 12.2 5.0 18.3 10.1

Fa (%) (relative Number of to control) Volunteers NA NA 36 (19–61) NA 30.1 NA 58.0 35.8 NA 41



Polk et al.11


Brouwers et al.12


Kara et al.13


Lehto et al.14 Pazzucconi et al.15


NA, data not available. t.i.d., three times a day.

DOI 10.1002/jps




the absolute bioavailability values ranging from 52% to 90%. Rapid drug absorption is documented by the relatively low tmax values in the range of 0.58–1.75 h and relatively high absorption rate constants with the values reported being mostly within the range of 1.5–3.6 h−1 . Both pharmacokinetic analysis and remote control capsule study revealed that ciprofloxacin is predominantly absorbed in duodenum and upper jejunum, with as much as 78% of dose absorbed from this gastrointestinal segment.40 Such data are indicative of the existence of a narrow absorption window in the proximal intestine. Ciprofloxacin HCl coadministration with iron salts results in substantially diminished bioavailability. An overview of the literature data related to ciprofloxacin–iron in vivo studies is given in Table 3. Although available data are relatively variable, a general trend exhibiting 40%–70% reduced ciprofloxacin bioavailability in the presence of different iron salts has been documented. The observed, limited ciprofloxacin absorption, might result from, either, limited solubility or limited permeability of the ciprofloxacin–iron interaction product. In a reˇ cent report, Zakelj et al.20 suggested that the effect observed in vivo resulted from the decreased permeability of ciprofloxacin–iron complex. They investigated permeability of ciprofloxacin in the presence of different metal cations using the isolated rat jejunum in a side-by-side diffusion chambers. Donor suspensions with high concentrations of metal cations and fluoroquinolones were applied and it was concluded “that the fluoroquinolone–metal cation complexes do not permeate through the intestinal mucosal membrane at all.”20 Interestingly, significant/intensive precipitation in the donor compartment was reported. Solubility Aqueous solubility of ciprofloxacin HCl salt was determined to be 42 mg/mL, that is, 109 mM (final pH 4.04). The presence of ferrous sulfate substantially decreased drug solubility. A more than threefold decrease of ciprofloxacin HCl concentration in solution was observed in media containing 72 mM ferrous sulfate. Ciprofloxacin base solubility in water was 0.097 mg/mL (pH 6.98), and it increased significantly in media containing ferrous sulfate, consistent with ˇ the findings of Zakelj et al.22 The concentration of ciprofloxacin in solution (with pH values) determined in the reactive media containing different amounts of ferrous sulfate is presented in Figure 1. Theoretical pH solubility profile of ciprofloxacin (based on pKa values 8.62 and 6.16) is presented as an inset in Figure 1, as well as the experimental values obtained for ciprofloxacin HCl salt (open circle), ciprofloxacin base (closed circle), and ciprofloxacin–iron complex (closed diamond) in water. The results obtained indicate that decreased ciprofloxacin concentration in reactive media is not just a pH related effect. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011

When clear solutions of 0.05 M ciprofloxacin HCl and 0.1 M ferrous sulfate were mixed in different volumetric ratios, immediate coloration was observed followed by some precipitation, with the most abundant precipitation in the mixture containing 25 mM ciprofloxacin HCl and 50 mM ferrous sulfate. Ciprofloxacin concentration in the solution above the precipitate was found to be 2.2 mg/mL (pH 2.8). Solubility of this complex was therefore almost a 20-fold lower than that of ciprofloxacin HCl salt. Although ciprofloxacin classification as a BCS class IV drug27 is based on the lowest solubility in the physiologically relevant pH range, with the relevant dose/solubility ratio approximately 5000 (dose number 20) for a 500 mg dose, the dose/solubility ratio of ciprofloxacin HCl salt in water and acidic media is approximately 14 (dose number 0.05) and approximately 230 for ciprofloxacin–iron complex (dose number 0.9). Incomplete ciprofloxacin absorption in the presence of iron, in spite of the relatively low dose/solubility ratio of ciprofloxacin–iron complex, could implicate a limited fluid volume available at the site of absorption, limiting its dissolution. Relevant drug concentrations that could be expected in vivo, taking into account the intestinal fluid volume range of 10–350 mL,41–43 could be as high as 150 mM. Transport to regions with increased pH values would cause ciprofloxacin to precipitate, thus limiting its absorption to a relatively short segment of the intestine. It can be postulated that, following concomitant drug administration, excess iron is present in the gastrointestinal lumen, causing less soluble complex formation and thus limiting ciprofloxacin absorption and its bioavailability. In order to test the above hypothesis, GastroPlusTM simulations and dissolution study in different media volumes were performed. Gastrointestinal Simulation The results obtained in vitro and literature in vivo data were combined in the GastroPlusTM simulation in order to investigate the impact of solubility change on ciprofloxacin bioavailability following oral administration with/without iron. Drug solubility was varied within the range starting at 1 mg/mL up to the experimentally observed 42 mg/mL (with the actual inputs being 1, 3, 5, 10, 20, 30, and 42 mg/mL). The simulated ciprofloxacin plasma levels are presented in Figure 2, together with the mean plasma profiles for ciprofloxacin administration with and without ferrous sulfate reported by Kara et al.13 The best fit of the actual in vivo data observed when ciprofloxacin tablets were given without ferrous sulfate (i.e., control study) was obtained with the input solubility of 42 mg/ mL, referring to the experimentally obtained aqueous solubility of ciprofloxacin HCl. The relevant PE% values were 5.57 for Cmax and 1.05 for AUC. Reduced DOI 10.1002/jps



Figure 1. Concentration of ciprofloxacin hydrochloride (HCl) dissolved in media containing different concentrations of ferrous sulfate. Inset: theoretical pH solubility profile of ciprofloxacin with the data points representing experimentally obtained ciprofloxacin solubility in water (full circle refers to ciprofloxacin base, open circle to ciprofloxacin HCl, and closed diamond to ciprofloxacin–iron complex).

ciprofloxacin absorption observed in the presence of iron was best described when ciprofloxacin solubility was reduced to approximately 3 mg/mL (PE% 0.23 for Cmax and 4.57 for AUC). This finding corresponds well with the solubility of ciprofloxacin–iron complex obtained in the current study. The gastrointestinal absorption models developed were used to estimate the relevant drug absorption kinetics and regional absorption. Ciprofloxacin absorption profiles without and with coadministered ferrous sulfate generated by GastroPlusTM were plotted together with the results obtained by numerical deconvolution (Fig. 3). The in vivo input profiles obtained suggest that after oral administration, either with or without iron, ciprofloxacin absorption kinetics exhibit initial rapid absorption in the first 2 h after administration. The absorption rate constants in this period calculated from the deconvoluted in vivo profiles were 0.76 and 0.80 h−1 for the control and drug interaction study, respectively. The amounts of drug absorbed from different regions of the gastrointestinal tract with/without ferrous sulfate coadministration are given in Figure 4. The regional absorption profiles indicate predominant ciprofloxacin absorption in the proximal part of the gastrointestinal tract and are consistent with data reported from the in vivo remote control capsule study.40 The simulations suggest that drug absorption when coadministered with iron was not impaired in “duodenum,” whereas it was substantially decreased in “jejunum 1 and 2.”

DOI 10.1002/jps

Dissolution Study Ciprofloxacin tablet dissolution profiles obtained in different volumes of media with/without ferrous sulfate added are presented in Figure 5. Taking into account the in vitro data suggesting that ciprofloxacin– iron interaction occurs at relatively high drug concentrations, three ciprofloxacin tablets and an equivalent of three doses of ferrous sulfate were used in the dissolution experiments. The relevant ciprofloxacin/iron molar ratio was 1.2 with the actual concentrations of ciprofloxacin HCl and ferrous sulfate being 5 and 6 mM, 18 and 21 mM, and 30 and 36 mM in dissolution media volume of 900 , 250, and 150 mL, respectively. Although drug interaction was not evident from the standard (900 mL volume) dissolution vessel, in the mini scale apparatus, with the media volume of 150 mL, decreased ciprofloxacin dissolution was observed, which was in a rank order with the in vivo input estimated by numerical deconvolution and/or GastroPlusTM simulations. A dissolution study was also conducted with single tablets in 50 mL media. The extent of ciprofloxacin dissolution was reduced to 43.5%, which was somewhat lower than the data obtained with three doses added to 150 mL media volume. The results obtained indicate that small volume dissolution experiments may be useful for performing biorelevant dissolution tests predictive of certain physicochemically based drug interactions occurring in the gastrointestinal lumen. Such results are in accordance with recent findings reported by Takano et al.44 emphasizing the importance of mini scale




Figure 2. In vivo observed (symbols) and in silico generated (lines) ciprofloxacin plasma profiles using various solubility input values: (a) 42 mg/mL, (b) 30 mg/mL, (c) 20 mg/mL, (d) 10 mg/mL, (e) 5 mg/mL, (f) 3 mg/mL, and (g) 1 mg/mL.

dissolution studies in biopharmaceutical drug characterization. Solid-State Characterization The solid phases generated from the interaction mixture of ciprofloxacin HCl solution and ferrous sulfate solution as well as that collected from the dissolution study in reactive media were examined by powder XRD, DSC, TGA, and FTIR.

X-ray diffraction scan of the precipitate formed from the mixture of ciprofloxacin HCl and ferrous sulfate solutions showed a very prominent peak at 9.40 2θ and the diffractogram was different than those of ciprofloxacin base and the HCl salt (Fig. 6). The sample obtained from the ciprofloxacin tablet dissolution study was crystalline, although the peak intensities did not exceed 1500 cps in contrast to the precipitate obtained from ciprofloxacin HCl and ferrous sulfate

Figure 3. Ciprofloxacin absorption profiles calculated by numerical deconvolution and estimated by GastroPlusTM simulation from the in vivo data set reported by Kara et al.13 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011

DOI 10.1002/jps



Figure 4. Regional absorption of ciprofloxacin with/without ferrous sulfate coadministration (estimated by GastroPlusTM simulation).

solutions interaction (Fig. 6). The decrease in peak intensities may be due to a reduction in the crystalline fraction of the precipitate caused by the presence of tablet excipients. The precipitate formed from ciprofloxacin HCl and ferrous sulfate solution exhibited a 19.5% mass loss (by TGA) in the temperature range corresponding to a broad endothermic peak seen by DSC with a peak at approximately 115◦ C (data not shown). This was followed by a broad and weak endotherm peaking at approximately 227◦ C associated with a mass loss by TGA, indicating thermal decomposition. In contrast, thermogravimetry of ciprofloxacin HCl showed approximately 6.9% mass loss corresponding to crystalline and adsorbed water removal. A melting endotherm for ciprofloxacin HCl was seen in DSC at approximately 309◦ C, whereas that of base appeared at approximately 270◦ C (data not shown). No thermal events of ciprofloxacin HCl or base were observed in the thermogram of the precipitate. Thermal analysis of the solid phase collected from ciprofloxacin tablet dissolution in the presence of ferrous sulfate showed a major endothermic event peaking at approximately 110◦ C preceded by a minor endotherm with an onset at approximately 54◦ C (data not shown). These two processes were accompanied by a mass loss of approximately 16.6% and attributed to adsorbed/crystalline water evaporation. Another, broad but weak, endotherm appeared at approximately 214◦ C (peak temperature) and it was associated with a further continuous mass loss, indicating thermal decomposition. The DSC trace of powdered ciprofloxacin tablet was comparable to the thermogram of the pure drug. The peaks of ciprofloxacin HCl DOI 10.1002/jps

monohydrate were shifted to lower temperatures due to the presence of other tablet ingredients. The precipitate formed from ciprofloxacin HCl and ferrous sulfate solution exhibited a broad IR absorption band at 3416 cm−1 , most likely of OH stretch vibrations of water molecules, and two peaks at 1701 and 1630 cm−1 ascribed to the carboxylic and ketone C O groups, respectively,45 vibrations, as observed for ciprofloxacin HCl (1709 and 1624 cm−1 , respectively) (data not shown). It was observed earlier that fluorochinolones are able to coordinate to metals, for example, copper ions through the carbonyl group attached to the ring and oxygen of the ionized carboxylic group.46 This type of direct coordination type is however not supported by the FTIR analysis and the carboxylic peak is still visible. Therefore, it is likely that ciprofloxacin complexes to the ferrous ion as a cation formed by protonation of the piperazine ring and forms an outer-sphere complex. Ionization of the piperazinic nitrogen is confirmed by the presence of NH2 + stretching vibrations appearing between 2600 and 2400 cm−1 . Another interesting and characteristic band is a broad and strong peak at 1103 cm−1 assigned as the SO4 2− vibrations. The FTIR spectrum of the solid phase collected from ciprofloxacin tablet/dissolution in the reactive media was very similar to the precipitate formed from ciprofloxacin HCl and ferrous sulfate solution and was characterized by a broad and irregular in shape peak with a maximum at 3415 cm−1 (data not shown). Because of its broadness, it may be described as a hydrogen-bonded OH stretch, most likely from the water present in the precipitate. The two carbonyl bands, at 1705 and 1630 cm−1 , are slightly shifted in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011



Figure 5. Ciprofloxacin tablet dissolution in different volumes of dissolution media (a) without and (b) with ferrous sulfate added.

comparison to the hydrochloric salt and the precipitate formed from ciprofloxacin HCl and ferrous sulfate solution, and still clearly discernible in the spectrum. Ciprofloxacin tablet powdered material gave a very similar FTIR spectrum to that of pure ciprofloxacin HCl. Quantitative estimation of the precipitate formed from the ciprofloxacin HCl and ferrous sulfate solution mixture gave ciprofloxacin content of 58.7% (w/ w), 5% (w/w) iron, and approximately 19.5% (w/w) water. The relevant amount of the sulfate was 15.4% (w/w) and that of the chloride ion was 5.3% (w/w). On the basis of the results of quantitative analysis presented above, the probable chemical structure of the ciprofloxacin–iron complex obtained in this study is Fe(SO4 2− )2 (Cl− )2 (ciprofloxacin)2 × (H2 O)n , where n is up to 12 molecules of water. It was found that JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011

solid phase collected from ciprofloxacin tablet dissolution study in media containing ferrous sulfate contained approximately 60% (w/w) ciprofloxacin, 5.4% (w/w) iron, and 16.6% (w/w) water. Thus, the similarity of the solid adducts obtained from the mixture of ciprofloxacin HCl and ferrous sulfate solutions and from ciprofloxacin tablet mini scale dissolution study in media containing ferrous sulfate was confirmed by quantitative analysis, thermal behavior, FTIR, and XRD patterns.

CONCLUSIONS Biopharmaceutical characterization of the ciprofloxacin–iron interaction based on literature in vivo data, in vitro drug solubility and dissolution, and in silico simulations indicated that DOI 10.1002/jps



Figure 6. XRD scans of ciprofloxacin base (a), ciprofloxacin HCl monohydrate (b), powdered ciprofloxacin tablets (c), solid phase from the mixture of ciprofloxacin HCl/ferrous sulfate solution (d), and solid phase collected from ciprofloxacin tablet dissolution study in media containing ferrous sulfate (e).

the observed drug interaction, involving formation of a poorly soluble complex of ciprofloxacin, with ferrous ion, chloride, and sulfate is sufficient to explain the reduced absorption observed in vivo. Ciprofloxacin HCl solubility, as well as its dissolution from tablets, was clearly reduced in media containing ferrous sulfate. The extent of the reduction observed varied depending on the concentration ratio of the interacting species. Interaction mixtures containing relatively high drug concentrations, in the range of around 30 mM or higher, for both ciprofloxacin HCl salt and ferrous sulfate resulted in the formation of a new solid phase. Complex formation occurs in mixtures containing relatively high drug concentrations, which would be encountered in vivo under the conditions of relatively low fluid volume available. Such observations are supported by the results of recent studies indicating that available gastrointestinal fluid volume is relatively limited and emphasize the importance of mini scale dissolution testing equipment for biorelevant drug interaction studies. The approach presented could be applied to other fluorquinolone–metal interactions showing reduced absorption in vivo.

ACKNOWLEDGMENTS Aleksandra Stojkovi´c would like to acknowledge the PhD grant from the Ministry of Science and Technological Development, Republic of Serbia. Part of the work was performed under the project TR-34007 supported by the Ministry of Science and TechnologDOI 10.1002/jps

ical Development, Republic of Serbia. Lidia Tajber, Krzysztof J. Paluch, and Owen Corrigan wish to acknowledge support for this research from the Irish Research Council for Science and Engineering Technology and the Solid State Pharmaceutical Cluster, supported by Science Foundation Ireland under grant number 07/SRC/B1158.

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DOI 10.1002/jps