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Original Research
Investigation of b-cyclodextrin–norfloxacin inclusion complexes. Part 2. Inclusion mode and stability studies Expert Rev. Anti Infect. Ther. 13(1), 131–140 (2015)
Cassiana Mendes*1, Aline Buttchevitz1, Andersson Barison2, Fernanda Maria Marins Ocampos2, Larissa Sakis Bernardi3, Paulo Renato Oliveira3 and Marcos Antoˆnio Segatto Silva1 1 Post graduation Program in Pharmaceutical Sciences, Quality Control Laboratory, Federal University of Santa Catarina (UFSC), Floriano´polis, SC, Brazil 2 NMR center, Federal University of Parana´ (UFPR), Curitiba, PR, Brazil 3 Post graduation Program in Pharmaceutical Sciences, Universidade Estadual do Centro Oeste/UNICENTRO, 85040-080, Guarapuava, PR, Brazil *Author for correspondence: Tel.: +55 483 721 4585 Fax: +55 483 721 9350 [email protected]
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Introduction: Norfloxacin (NFX) is a broad spectrum antibiotic with low solubility and permeability, which is unstable on exposure to light and humidity. Objective: In this study, the mode of NFX inclusion into b-cyclodextrin complexes was evaluated and a complete physical, chemical and microbiological stability study of the inclusion complexes was carried out. Methods: Potentiometric titrations were performed to evaluate changes in the pKa of the NFX molecule due to the formation of an inclusion complex and NMR analysis demonstrated that the NFX molecule is included in the b-cyclodextrin cavity. Results: Inclusion complexes obtained by kneading followed by freeze-drying showed improved NFX stability compared with the isolated drug or the physical mixture. This method was effective in terms of protecting the drug from photodegradation and also avoiding hydrolysis. Differences between NFX and the complexes could be evidenced by thermal analysis, infrared spectroscopy and x-ray powder diffraction as well as by determining the solubility and drug content. The antimicrobial potency was also preserved on applying the promising method of kneading. Conclusion: The satisfactory stability indicates that the NFX/b-cyclodextrin complexes could be useful as an alternative to the existing NFX drug formulation. KEYWORDS: b-cyclodextrin • inclusion complexes • inclusion mode • norfloxacin • stability studies
Several drugs are unstable and can degrade when exposed to light, elevated temperatures and humidity. Therefore, the best conditions for storage should be investigated to guarantee the product stability and efficiency. A welldesigned stability study takes into account all of the factors that the product could be exposed to and it is an increasingly important issue in the development of any drug delivery system [1,2]. Norfloxacin (NFX) is a broad spectrum fluorquinolone antibiotic that has been extensively used for decades to treat mainly urinary infections, as well as urethritis, pharyngitis and proctitis, due to its effectiveness against Gram positive and Gram negative bacteria. NFX is a hygroscopic and photosensitive drug and its physical–chemical properties are affected by 10.1586/14787210.2015.982092
exposure to visible and UV light, affecting its bioavailability. Furthermore, prolonged exposure to heat and acidic solutions can lead to its hydrolysis, resulting in a carboxyl product which is responsible for the microbiological activity [3,4]. According to the Biopharmaceutical Classification System [5,6], NFX is a class IV drug (low solubility and low permeability) with low absorption (around 30–40%) associated with oral administration. This problem can be solved by the formation of inclusion complexes, mainly with cyclodextrin (CD), which usually increases the stability, solubility, dissolution rate and bioavailability [7,8]. In this regard, b-cyclodextrin (bCD) is a torus-shaped oligosaccharide containing seven glucose units, with a hydrophilic outer surface and a
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131
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Original Research
Mendes, Buttchevitz, Barison et al.
lipophilic inner cavity which can accommodate a wide variety of lipophilic drugs, forming the so-called inclusion complexes. The stability of inclusion complexes can be provided by noncovalent interactions, such as hydrophobic interactions, electronic effects, steric factors and van der Walls forces. During the inclusion complex formation, the water molecules from the inner bCD cavity are replaced by appropriate lipophilic guests, which are energetically favored. Therefore, the presence of water plays an important role in the formation and stability of guest/CD inclusion complexes. The formation of complexes usually increases the stability, solubility, dissolution rate and bioavailability [7,8]. Moreover, in inclusion complexes the incorporation of drugs into the lipophilic cavity of bCD can protect them from light, humidity and high temperatures, with a consequent improvement in their stability [9,10]. However, it should be highlighted that pharmaceutical forms should contain as little CD as possible, because an excess can cause some problems associated with the formulation bulk or can be toxic, besides reducing the drug bioavailability and efficacy [11]. In the recent literature, much attention has been focused on stability studies. The inclusion complexes with NFX in the hydrophobic cavity of bCD can even protect the drug from light, moisture and temperature, thus improving the drug stability [9,10]. There are, however, few reports that explore the protection provided by the inclusion complex formation. In this context, to investigate the properties of inclusion complexes this study reports: the NMR and pKa results to verify the inclusion and show that the NFX is included in the bCD cavity and a complete study on the physical, chemical and microbiological stability. Experimental Materials
NFX was obtained from Galena, the CDs were kindly donated by Roquette Corporate (Labonathus, Sa˜o Paulo, Brazil) and all other chemicals were of analytical grade. Preparation of inclusion complexes
Three different methods were employed to produce the inclusion complexes with equimolar amounts of NFX in bCD: co-evaporation and kneading followed by freeze-drying or spraydrying. For the control, physical mixtures of NFX in bCD were prepared by blending equimolar amounts of NFX and bCD in a mortar until a homogeneous mixture was obtained. Co-evaporation
Equimolar amounts of NFX and bCD were dissolved in an EtOH/H2O solution (1:1 v/v) and the pH adjusted to 3.0. The solution was then stirred for 96 h at room temperature. Finally, the solvent was removed under reduced pressure at 50oC. Kneading
Stoichiometric amounts of NFX and bCD (1:1) were mixed for 20 min, 1 ml of water was then added and mixing applied again for 5 min. This procedure was then repeated. Next, 50 ml of water (40˚C) was added to the paste formed, the pH 132
was adjusted to 3.0 and the solution was stirred for 20 min. The resulting solution was then filtered and freeze-dried or spray-dried to dry the complexes formed. For the freeze-drying the solution was reduced to -40˚C and freeze-dried for 24 h, while for the spray-drying the solution was placed in a Buchi B-290 mini spray-dryer with an inlet temperature of 180˚C, outlet temperature of 62˚C, air flow rate of 30 ml/min and air pressure of 1.5 bar. Potentiometric titrations
Potentiometric investigations were carried out with a Corning350 pH meter fitted with blue-glass and Ag/AgCl reference electrodes calibrated to read -log [H+] directly. The electrode was calibrated with standard 0.1 M solutions of HCl and KOH. The ionic strength of the HCl solution was maintained at 0.1 M with the addition of KCl. Measurements were carried out in a thermostated cell containing 50 ml of a NFX solution (0.05 mm) with ionic strength adjusted to 0.1 M with the addition of KCl at 25 ± 0.05˚C. The experiments were performed under argon to eliminate the presence of atmospheric CO2. All experimental solutions were prepared in acetonitrile/water (1:1, v/v), owing to the low solubility of the drug in water. The pKw of the acetonitrile/water containing 0.1 M of KCl was 15.40 [12]. Computations were carried out with the BEST7 program. NMR analysis
The formation of a complex between NFX and bCD was also investigated by means of NMR spectroscopy analysis. For this, 1 H as well as 1D and 2D rotational frame nuclear overhauser effect spectroscopy (ROESY) NMR experiments were performed at 303 K in D2O on a Bruker AVANCE III HD 600 NMR spectrometer operating at 14.1 T, observing 1H at 600.13 MHz. The spectrometer was equipped with a 5 mm quadrinuclear inverse detection probe with a z-gradient. The 1 H NMR spectra were acquired with a spectral width of 6330 Hz (~10.5 ppm) and 64K data points, providing a digital resolution of 0.19 Hz, recycle delay of 1.0 s and 8 transients. The 1D ROESY NMR experiments were obtained with a selective 180˚ excitation pulse on the signals at 3.84 and 3.74 ppm, corresponding to H-3 and H-5 in the bCD moiety and selective refocusing with shaped pulses using the Bruker gradient selrogp pulse sequence, with a mixing time of 400 ms for ROESY spinlock, recycle delay of 2.0 s, 32K data points, 4K transients and the same spectral width used to obtain the 1H NMR spectra. The 1H and 1D ROESY NMR spectra were processed by applying a Fourier transform with zero-filling to 128K data points and by applying an exponential multiplication of the FIDs by a factor of 0.3 and 1 Hz for 1H and 1D ROESY NMR, respectively. All 1H NMR chemical shifts are given in ppm in relation to the TMSP-d4 signal at 0.00 ppm as the internal reference, and all pulse programs were supplied by Bruker. Stability evaluation
Stability assays were performed according to published guidelines on stability tests [13]. The physical mixture and inclusion Expert Rev. Anti Infect. Ther. 13(1), (2015)
Original Research
Investigation of b-cyclodextrin–norfloxacin inclusion complexes
pKa1 O
+
HN 2
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+ H2N
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Figure 1. Schematic representation of norfloxacin and norfloxacin/b-cyclodextrin inclusion complex in protonated and unprotonated forms, in different pH values.
complexes were monitored for up to 6 months in stability chambers at 40˚C and 75% relative humidity. The same samples were stored in photostability chambers with exposure to 200 Wh/m2 UV and 1,200,000 lux h visible. After 6 months, the samples were removed and submitted to differential scanning calorimetry (DSC), thermogravimetry (TG), diffuse Fourier transform infrared (FT-IR) spectroscopy, x-ray powder diffraction (XRPD), solubility assays, drug content determination and antimicrobial assay.
injection volume of 20 mL. The decarboxylated derivate was produced using a method in which 250 mg of NFX was refluxed with 70 ml of hydrochloric acid (2 mM) for 48 h at 150˚C, protected from light [15]. Subsequently, the solution was cooled and the pH was adjusted to 7.5 with sodium hydroxide (2 mM). The solution was then evaporated under vacuum to dryness and the residue extracted by EtOH and filtered through a 0.45 mm pore size filter. The final product was evaluated by HPLC. Differential scanning calorimetry & thermogravimetric analysis
Drug content determination
Drug content was determined by the ‘n-hexane wash’ method [14]. This method is based on the fact that bCD and its complexes are insoluble in n-hexane, while the free drug is soluble. In this procedure, 5 mg samples of each product were extracted with 5 ml of n-hexane containing 2% of acetic acid. The top-phase (n-hexane) was dried under reduced pressure at 40˚C. The residue was dissolved in 2 ml of mobile phase, filtered (0.45 mm pore size) and quantified by high performance liquid chromatography (HPLC), as described below. The drug content included in the bCD cavity was determined by dissolving the previously washed complex with a small amount of dimethyl sulfoxide (400 ml) and diluting it with water containing 2% of acetic acid. After 12 h the solution was centrifuged for 10 min at 3000 rpm and the top phase was filtered (0.45 mm pore size) and quantified by HPLC. The HPLC analysis was performed on a Shimadzu liquid chromatograph consisting of a LC-10A VP quaternary pump and a fluorescence detector (l excitation at 330 nm and l emission at 445 nm). The chromatograph was equipped with a Phenomenex C18 reversed-phase column chromatograph (150 4.0 mm; 5 mm particle size) conditioned in a SPD-10AVP column oven. The column was eluted with an isocratic mobile phase consisting of phosphate buffer (25 mM, pH 3.0) and acetonitrile (86:14 v/v) at a flow rate of 1.0 ml/min and informahealthcare.com
DSC curves for the NFX, bCD, NFX/bCD physical mixtures and inclusion complexes were obtained on a Shimadzu DSC-60 using aluminum crucibles with around 2.0 mg of
A
B
C
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
Figure 2. 1H nuclear magnetic resonance spectra. (A) NFX/bCD inclusion complex, (B) 1D ROESY spectra of NFX/bCD obtained by selective irradiation of 3.84 and (C) 3.74 ppm signals from bCD H-3 and H-5, respectively.
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HN
and an average of over 32 scans, at a spectral resolution of 4 cm-1. A background spectrum was obtained for each experimental condition.
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H2C
CH2 CH2
X-ray powder diffraction N H
H3
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The crystallinity of the samples was evaluated by X-ray powder diffractometry. XRPD patterns were recorded on an X’Pert Philips diffractometer, with a CuKa tube, operating at 40 kV and 40 mA, in the range of 5–40 (2) with a pass time of 5 s.
F
H2C
H
N
Solubility of NFX/bCD complexes
To evaluate the solubility of the samples, an excess of the solid powder was added to 10 ml of a simulated intestinal fluid (0.05 M potassium phosphate - pH 6.8, without enzyme) in amber glass flasks which were sealed and magnetically stirred at human body temperature (37˚C) for 24 h. The samples were filtered through a 0.45-mm pore size filter and the NFX concentration determined by HPLC. The solubility of the inclusion complexes was evaluated in triplicate.
O H 5
H5 H O
HO
Figure 3. Schematic representation of norfloxacin/ b-cyclodextrin inclusion complex, showing norfloxacin structure partially inserted into the b-cyclodextrin cavity.
Antimicrobial assay
sample in a dynamic N2 atmosphere (50 ml/min) with a heating rate of 10˚C/min, in the temperature range of 30–300˚C. TG experiments were conducted on a Shimadzu TGA-50 thermobalance using a platinum cell containing approximately 4 mg of sample, in a dynamic N2 atmosphere (50 ml/min) with a heating rate of 10˚C/min, in the temperature range of 30–600˚C. Diffuse reflectance Fourier transform infrared spectroscopy
The FT-IR spectra were acquired on a PerkinElmer spectrophotometer (Frontier), within a scan range of 600–4000 cm-1 O
O
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N
The antimicrobial activity of the NFX/bCD inclusion complexes was evaluated by the microdilution method, following the procedure outlined in the Manual of Clinical Microbiology [16]. Fresh cultures of Staphylococcus epidermidis (ATCC 12228), cultivated on Muller-Hinton agar were suspended in 0.9% NaCl. The suspension obtained was adjusted to the McFarland turbidity standard (0.5) and diluted to obtain an inoculum of 106 colony forming units per ml. The NFX, physical mixture and inclusion complexes were dissolved in 0.1 M phosphate buffer (pH 8) and then diluted using Muller-Hinton Broth [17,18]. Twofold serial concentrations of the compounds
F
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H3C Norfloxacin
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OH NH
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NH2
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Ethylenediamine derivative
Figure 4. Structures of norfloxacin and its degradants products, according to Alnajjar et al. [4].
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Investigation of b-cyclodextrin–norfloxacin inclusion complexes
Original Research
Table 1. Norfloxacin content, minimal inhibitory concentration, solubility and antibacterial potency are listed below for norfloxacin alone, physical mixture and complexes obtained by co-evaporation, kneading followed by freeze-drying and kneading followed by spray-drying at the initial time, after 6 months in the stability chamber (75% U.R., 40˚C), after ultraviolet exposition and visible exposition. Formulation
Drug content (%)
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Initial
Solubility† (mg/ml)
MIC (mg/ml)
Antibacterial potency‡ (%)
SC
UV
VIS
Initial
SC
UV
VIS
Initial
SC
UV
VIS
Initial
SC
UV
VIS
NFX
100
87.7
74.7
74.1
1
2
1
1
58.9
49.5
41.1
41.5
100.0
92.4
98.7
99.3
PM
50
44.5
37.2
37.1
1
2
1
1
59.1
48.1
39.5
40.2
99.7
90.9
99.9
98.8
COE
97
77.5
60.5
67.6
1
2
1
1
62.8
53.9
30.2
35.6
83.2
72.4
82.6
84.1
KFD
86
85.9
84.6
85.8
1
1
1
1
137.9
135.7
136.5
135.2
123.3
120.4
126.1
119.5
KSD
90
90
78.2
86.3
1
1
1
1
97.5
98.7
86.7
93.6
110.4
108.4
107.2
108.6
†
Drug solubility in simulated intestinal fluid pH 6.8 (37ºC). ‡ Growth inhibition zone is proportional to log of concentration, and are calculated by Hewitt equations. COE: Complexes obtained by co-evaporation; KFD: Kneading followed by spray-drying; MIC: Minimal inhibitory concentration; MIC: Minimal inhibitory concentration; NFX: Norfloxacin; PM: Physical mixture; SC: Stability chamber; UV: Ultraviolet exposition; VIS: Visible exposition.
were employed to determine the MIC, which ranged from verified by ANOVA (p = 0.05). Composition of Medium 1: 128.00 to 0.25 mg/ml. The MIC was defined, in triplicate, as peptone (6.0 g), pancreatic digest of casein (4.0 g), yeast extract the lowest concentration inhibiting visible microbial growth (3.0 g), beef extract (1.5 g), dextrose (1.0 g), agar (15.0 g), after incubation at 37˚C for 18 h. The antimicrobial activity of the NFX/ A bCD inclusion complexes was evaluated B by agar diffusion assay, as described by C Froehlich and Schapoval (1990). Fresh M cultures of Staphylococcus epidermidis N ATCC 12228 cultivated on Antibiotic D O Medium 1 were suspended in 0.9% NaCl and the suspension obtained was P adjusted to 25 ± 2% of transmittance at E 580 nm, to obtain the inoculum. To F form the base layer, 20 ml aliquots of G Antibiotic Medium 11 were placed in six Petri dishes. After its solidification, a H 5 ml portion of Antibiotic Medium 11, Q to which was added the inoculum R (0.7%), was poured onto the base layer. After solidification, stainless steel cylinS I ders (8 6 10 mm) were placed over J T the surface of the inoculated agar on each K dish. To each cylinder, 150 ml of NFX standard solution or inclusion complex L was added, at presumed concentrations of 4000 3500 3000 2500 2000 1500 1000 600 10, 20 and 40 mg/ml. After incubation Wavenumber (cm–1) (32–35˚C for 16–18 h), the cylinders were removed and the diameters of the 4000 3500 3000 2500 2000 1500 1000 600 Wavenumber (cm–1) inhibition zones were measured with the aid of an electronic digital caliper Figure 5. Fourier transform infrared spectroscopy spectra. (A) Norfloxacin NFX, (Starret). Assays were performed in (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after duplicate, using six plates for each one. 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PMThe antimicrobial potency of the NFX VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COEcomplexes was calculated according to VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by the Hewitt equation [19]. The validity spray-drying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC. of the parallel-line model chosen was informahealthcare.com
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B C
D
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E F G H
I J K
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M N O P Q
R S T 100
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Figure 6. Differential scanning calorimetry curves. (A) Norfloxacin NFX, (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PM-VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COE-VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by spray-drying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC.
water (1000 ml), pH after sterilization 6.6 ± 0.1; and composition of Medium 11: same as Medium 1, except that the final pH after sterilization is 8.3 ± 0.1 [18,20]. 136
Results & discussion
Potentiometric investigations were carried out to determine the pKa (protonation–deprotonation equilibrium) for NFX when it is inserted in the lipophilic cavity of the bCD. NFX shows four different pKa values (3.1, 6.1, 8.6 and 11.3), which hinders its solubility at physiological pH (6.8). On the other hand, in the NFX/bCD system, the pKa was increased to 9.9 and 12.5, respectively. These basic pKa values suggest that the less protonated NFX is inserted in the bCD cavity. The formation of an inclusion complex is well known to improve the solubility of the guest, and the pKa value is an essential parameter to predict the behavior in biological fluids. The results revealed that at physiological pH (6.8) all NFX molecules (>99%) are present in the protonated form and, as a consequence, complexed NFX has improved solubility when compared with the free drug, due to the pKa value, in addition to the inclusion complex contribution. FIGURE 1: The formation of inclusion complexes obtained by kneading followed by freeze-drying can be evidenced by changes, including broadening, in the 1H NMR signals for the drug, when inside the lipophilic cavity of the bCD, compared to signals of the drug without the presence of bCD recorded under the same NMR conditions [21,22]. Therefore, the first approach was to acquire 1H NMR spectra of both the NFX and NFX/bCD, in D2O. However, only the NFX/bCD system was soluble in D2O and its 1H NMR spectrum showed NFX signals, evidencing the formation of a NFX/bCD inclusion complex. Moreover, the 1H NMR spectra showed a 7:1 area ratio for the bCD to NFX signals, revealing a 1:1 stoichiometry. The formation of the NFX/bCD inclusion complex was supported by the one-dimensional rotating frame nuclear Overhauser effect (1D ROESY) NMR experiments. The 1D ROESY NMR analysis carried out by selective excitation of the hydrogens H-3 and H-5 at 3.84 and 3.74 ppm in bCD caused an NOE enhancement in the 8.60, 7.64, 4.37 and 1.42 ppm NFX signals (FIGURE 2). A high NOE intensification of the NFX signals was observed only when the H-3 resonance frequency at 3.84 ppm was selectively irradiated (FIGURE 2). However, the selective irradiation of the H-5 resonance frequency at 3.74 showed a higher intensification of the NFX signal at 8.60 ppm, indicating that this NFX hydrogen is closer to the bCD H-5 than to the other bCD hydrogens. The intensification of the NFX signals in the 1D ROESY NMR spectra, after selective excitation of the resonance frequency of H-3 and/or H-5 bCD hydrogens, revealed that a NFX/bCD inclusion complex was formed, with the NFX structure partially inserted in the bCD cavity, as represented in FIGURE 3. Physcochemical stability Photostability studies
Physicochemical stability studies provide information required for the development of the preformulation, highlighting particular requirements regarding unstable drugs, such as NFX. Expert Rev. Anti Infect. Ther. 13(1), (2015)
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Investigation of b-cyclodextrin–norfloxacin inclusion complexes
According to Alnajjar and coworkers, the three photodegradation products of NFX are formylpiperazine, amine and ethylenediamine (FIGURE 4) [23]. NFX degrades around 26% (assay 74%) when exposed to UV and visible light (TABLE 1). The FT-IR analysis (FIGURE 5A) shows the difference in the region of 950–900 cm-1 for NFX before and after light exposure. This result provides evidence of the presence of photodegradation products, because this region corresponds to the imine moiety. From the data in TABLE 1, the same decomposition of the physical mixture can be noted, indicating that without the formation of an inclusion complex the presence of bCD does not guarantee NFX light protection. There was no protection provided by the formation of the inclusion complex obtained applying the co-evaporation method and FIGURE 6 shows the change in the imine region after photoexposure. However, very low NFX photodegradation was observed for the inclusion complexes obtained through kneading followed by freeze-drying. In the case of the spray-drying method, a low degree of photodegradation was observed on exposure to visible light (assay 86.3%) and more accentuated degradation in the presence of UV light (assay 78.2%). Although the inclusion complex does not contain the piperazine ring moiety, it can be inferred that the complex obtained by kneading followed by freeze-drying prevented the photodegradation of this part of the NFX molecule. In this case, bCD could provide some spatial protection from the reactive species or some particle aggregation of these amorphous complexes could occur in contrast to the crystalline form obtained by co-evaporation technique. The DSC analysis of NFX showed an endothermic event at 217˚C (DHfusion = -170.93 J/g) assigned to the fusion point typical of crystalline substances, while bCD showed a water loss at 93˚C (DHfusion = -245.22 J/g) (FIGURE 6). On the other hand, the inclusion complex obtained by co-evaporation presented an event at 140˚C (FIGURE 6I), while those obtained by kneading followed by freeze-drying or spray-drying exhibited an endothermic event at 160˚C (FIGURE 6M & 6K). NFX, the guest molecule, entered the bCD cavity and some of the cavity water was lost, therefore the energy of the remaining water was altered, which may explain the slight shift in the endothermic events to higher temperature observed for the inclusion complexes (160˚C). This finding suggests that the NFX/bCD samples obtained by kneading followed by freezedrying or by spray-drying are more stable than those produced by co-evaporation. After photoexposure, other events were observed for the inclusion complexes obtained by coevaporation as well as by kneading followed by spray-drying probably due to the presence of photodegradation products. However, in the inclusion complex obtained by kneading followed by freeze-drying no changes in the endothermic event were observed (FIGURE 6M–6P). The inclusion complexes obtained by this technique could maintain the thermal stability indicated initially by the DSC analysis. The XRPD patterns for NFX showed characteristic crystalline peaks at 7.86, 9.90, 10.70, 11.99, 14.92, 16.09, 18.76, informahealthcare.com
Original Research
A B C D E F G
H
I J
K L
M N
O P Q R S T 5
10
15
20 25 2q
30
35 40
Figure 7. X-ray powder diffraction patterns. (A) Norfloxacin NFX, (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PM-VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COE-VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by spraydrying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC.
20.68, 22.76 and 25.02 (FIGURE 7). This XRPD pattern presents excellent agreement with the well-defined polymorph A, defined as the commercial form [24]. After photoexposure, peaks which differed from those of the NFX polymorph A appeared, probably due to NFX photodegradation products. On the other hand, the NFX/bCD system obtained by co-evaporation 137
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Mendes, Buttchevitz, Barison et al.
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0 50
50
solubility of the NFX, free or in the presence of bCD, reduced considerably. Therefore, the presence of the polymer did not affect this parameter. The coevaporated complex did not maintain its solubility after photoexposure. On the other hand, the systems obtained by kneading followed by freeze-drying or spray-drying presented no significant decrease in solubility. The most relevant observation to emerge from this investigation is that the ability of these inclusion complexes to protect the NFX molecule against degradation and improve its solubility is maintained. Temperature & humidity
–1
–1
After 6 months of storage at 40˚C and 75% humidity, the NFX underwent –2 –2 hydrolysis to its decarboxyl derivative, as –0 200 400 600 –0 200 400 600 evidenced by the absence of stretching at Temperature (°C) Temperature (°C) 3500–3550 and 1716 cm-1, related to E F OH and carbonyl C=O groups, DrTGA TGA DrTGA TGA respectively (FIGURE 5), in the FT-IR analy% % sis. This degradation product was also 2 0.5 100 100 detected in the HPLC analysis (data not 0 0 shown). On the other hand, according to 50 50 the FT-IR, DSC and XRPD analysis, no –20 –0.5 NFX degradation was observed in the –0 –0 case of the NFX/bCD systems obtained –1 –4 by kneading followed by freeze-drying or –0 200 400 600 –0 200 400 600 spray-drying (FIGURES 5–7). In contrast, the Temperature (°C) Temperature (°C) DSC analysis showed other endothermic events in the NFX/bCD system obtained Figure 8. Thermogravimetric and derivative thermogravimetric curves. (A) NFX, (B) b-cyclodextrin, (C) physical mixture and complexes obtained by (D) co-evaporation, by co-evaporation, which produced an (E) kneading followed by freeze-drying or (F) spray-drying. inclusion complex in the crystal form. Moreover, the FT-IR analysis showed the showed unique crystalline peaks at 2 angles of 11.7, 13.5, absence of COOH stretching, indicating a decarboxylated deg14.6, 15.4 and 17.6, identifying the formation of a new crystal. radation product. The XRPD analysis of the complex obtained After photoexposure, the characteristic intense peaks of NFX by co-evaporation shows intense NFX peaks at 2 angles of (11.99 and 20.68) and bCD (15.19 and 16.91) changed, indi- 12.07 and 20.7, as observed after exposure to light, indicating cating destabilization of the complex. On the other hand, the that this inclusion complex is sensitive to light and humidity. NFX/bCD complexes obtained by kneading followed by In other words, the inclusion complex obtained by cofreeze-drying or spray-drying showed an XRPD pattern typical evaporation generated low stability complexes. of amorphous compounds. Even after light exposure the amorAccording to the TG analysis (FIGURE 8), the physical mixture phous pattern remained the same, indicating that there was and NFX/bCD systems obtained by co-evaporated had higher no degradation. water contents (10.4 and 10.9%, respectively). However, the The solubility assays performed under simulated intestinal NFX/bCD systems obtained by kneading followed by freezeconditions with the NFX/bCD system obtained by kneading drying or spray-drying showed lower water contents (7.5% for followed by freeze-drying or spray-drying showed improvement both). These results indicate that in the kneading followed by in terms of the solubility, while no improvement was observed freeze-drying or spray-drying procedures NFX replaces more for the samples obtained from the physical mixture and the water molecules than in that of the other approaches. NFX can NFX/bCD system obtained by co-evaporation (TABLE 1). These absorb water and form a hydrate, which shows an endothermic findings indicate the formation of a NFX/bCD inclusion com- event at 136.4˚C. However, the TG analysis shows events at plex applying the kneading procedure. After photoexposure, the 61.6 and 64˚C for the NFX/bCD systems obtained by –0
138
–0
Expert Rev. Anti Infect. Ther. 13(1), (2015)
Investigation of b-cyclodextrin–norfloxacin inclusion complexes
kneading followed by freeze-drying or spray-drying, respectively, related to the absorption of natural humidity by the amorphous solid, leading to the low temperatures observed on the DSC endotherm (FIGURE 6). Therefore, these results provided further support for the hypothesis that NFX instability can be overcome through the production of an inclusion complex by kneading followed by freeze-drying.
Original Research
reflects directly in the antibacterial activity, because the carboxyl ring is responsible for the NFX activity. On the other hand, in the NFX/bCD system obtained by kneading followed by freeze-drying or spray-drying the NFX molecule remained inside the bCD cavity. Therefore, these preparation methods preserved the antibacterial potency because the drug was protected from the hydrolysis process. Conclusions
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Microbiological stability
The physical mixture presented the same inhibitory activity as the free NFX, whilst the isolated bCD showed no antibacterial effect. Furthermore, the microbiological study showed that the inclusion complexes also had the same MIC as the free NFX, indicating that all of the preparation methods allowed the drug stability to be maintained (TABLE 1). The MIC obtained for NFX in the isolated form or in inclusion complexes was 1 mg/ml (TABLE 1), according to a previous study [25]. To evaluate microbiological stability, the antibacterial potency of NFX alone and as inclusion complexes after the stability studies was determined. After photoexposure there was no statistical difference in the antimicrobial activity. This result can be attributed to the fact that all of the photoproducts keep the carboxyl moiety and the fluor atom, which are responsible for the antimicrobial activity. However, after exposure to humidity and heat, an important difference between the isolated NFX and some of the complexes was observed (TABLE 1). After 6 months exposure it was noted that the NFX lost its initial antibacterial properties and the antibacterial potency decreased to 92.4%. The same was observed for the physical mixture and the co-evaporated NFX/bCD system, as a consequence of the NFX degradation. After exposure to humidity and heat, NFX degrades to its decarboxylated derivative, which
The results of this study provided an insight into the mode of NFX inclusion in the b-CD cavity, based on NMR analysis and the change in the pKa values due to the inclusion formation. Kneading followed by freeze-drying was found to be the most appropriate method for the production of the inclusion complexes because it provided chemical, physical and microbiological stability. This method prevented degradation on exposure to UV and visible light, besides inhibiting the hydrolysis of the NFX. Preserving the antibacterial activity with the use of a simple, low cost method is an important issue in the obtainment of inclusion complexes which can be applied on an industrial scale. The NFX/bCD complexes obtained by kneading followed by freeze-drying represent a potential alternative to the existing NFX drug formulation. Financial & competing interests disclosure
This study was supported by the National Counsel of Technological and Scientific Development (CNPq) and Coordination for Enhancement of Higher Education Personnel (CAPES). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Key issues • NMR analysis demonstrated the mode of the norfloxacin (NFX) molecule inclusion in the b-cyclodextrin (bCD) cavity. • The pKa determination provided information on the ionization of NFX when inserted in the bCD cavity. • Without the formation of an inclusion complex the presence of bCD does not guarantee NFX light protection. • The improvement of the solubility could be maintained by systems obtained by kneading followed by freeze-drying or spray-drying. • The thermal stability of the inclusion complexes obtained by kneading followed by freeze-drying was maintained after photoexposure. • After exposure to high humidity and temperature no NFX degradation was observed in the case of the NFX/bCD systems obtained by kneading followed by freeze-drying or spray-drying. • After photoexposure the antibacterial potency of NFX in the inclusion complexes formed by kneading followed by freeze-drying or spray-drying was preserved. • NFX degradation into its decarboxylated derivative (without pharmacological activity) could be prevented through the formation of inclusion complexes by kneading followed by freeze-drying or spray-drying.
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composite matrix for expanded bed adsorption: preparation and characterization of physical properties. Carbohydr Polym 2010;80(4):1085-90
References 1.
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Nyu Medical Center on 02/17/15 For personal use only.
2.
3.
4.
5.
6.
7.
8.
Yang L-J, Chen W, MA S-X, et al. Host-guest system of taxifolin and native cyclodextrin or its derivative: preparation, characterization, inclusion mode, and solubilization. Carbohydr Polym 2011; 85(3):629-37 Zeng J, Ren Y, Zhou C, et al. Preparation and physicochemical characteristics of the complex of edaravone with hydroxypropyl-b-cyclodextrin. Carbohydr Polym 2011;83(3):1101-5 El Khateeb SZ, Abdel Razek SA, Amer M. Stability-indicating methods for the spectrophotometric determination of norfloxacin. J Pharm Biomed Anal 1998; 17(4):829-40 Alnajjar A, Idris AM, AbuSeada HH. Development of a stability-indicating capillary electrophoresis method for norfloxacin and its inactive decarboxylated degradant. Microchem J 2007;87(1):35-40
18.
Brazilian Pharmacopoeia. Brası´lia, Brazil: 5th ed 2010)
19.
Hewitt W. Microbiological assay for pharmaceutical analysis: a rational approach. CRC Press, Boca Raton, FL; 2013)
20.
The United States Pharmacopoeia, 37nd ed. (USP 37). United States Pharmacopeial Convention; Rockville, MD: 2013
21.
Asbahr ACC, Franco L, Barison A, et al. Binary and ternary inclusion complexes of finasteride in HPbCD and polymers: Preparation and characterization. Bioorg Med Chem 2009;17(7):2718-23
22.
Grebogi IH, Tibola APO, Barison A, et al. Binary and ternary inclusion complexes of dapsone in cyclodextrins and polymers: preparation, characterization and evaluation. J Inclusion Phenom Macrocyclic Chem 2012;73(1-4):467-74
23.
Hussein K, Turk M, Wahl MA. Comparative evaluation of ibuprofen/betacyclodextrin complexes obtained by supercritical carbon dioxide and other conventional methods. Pharm Res 2007; 24(3):585-92
Alnajjar A, AbuSeada HH, Idris AM. Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization. Talanta 2007;72(2):842-6
24.
Oliveira PR, Bernardi LS, Mendes C, et al. Liquid chromatographic determination of norfloxacin in extended-release tablets. J Chromatogr Sci 2009;47(9):739-44
Barbas R, Martı´ F, Prohens R, et al. Polymorphism of norfloxacin: evidence of the enantiotropic relationship between polymorphs A and B. Cryst Growth Des 2006;6(6):1463-7
25.
Norrby SR. Useful agents in the management of urinary tract infections. Int J Antimicrob Agents 1994;4(2):129-34
10.
Gould S, Scott RC. 2-Hydroxypropyl-bcyclodextrin (HP-b-CD): a toxicology review. Food Chem Toxicol 2005;43(10): 1451-9 Kiss T, Fenyvesi F, Ba´cskay I, et al. Evaluation of the cytotoxicity of b-cyclodextrin derivatives: evidence for the role of cholesterol extraction. Eur J Pharm Sci 2010;40(4):376-80
12.
Herrador M, Gonza´lez AG. Potentiometric titrations in acetonitrile–water mixtures: evaluation of aqueous ionisation constant of ketoprofen. Talanta 2002;56(4):769-75
13.
Food and Drug Administration. Guidance for industry Q1A (R2) stability testing of new drug substances and products. Food and Drug Administration. Rockville, MD, 2003. Available from: www.fda.gov/ downloads/RegulatoryInformation/ Guidances/ucm128204.pdf [Last accessed 19 March 2013]
Breda SA, Jimenez-Kairuz AF, Manzo RH, et al. Solubility behavior and biopharmaceutical classification of novel high-solubility ciprofloxacin and norfloxacin pharmaceutical derivatives. Int J Pharm 2009;371(1):106-13
14.
Tommasini S, Raneri D, Ficarra R, et al. Improvement in solubility and dissolution rate of flavonoids by complexation with b-cyclodextrin. J Pharm Biomed Anal 2004; 35(2):379-87
15.
140
Froehlich PE, Schapoval EES. Doseamento microbiolo´gico do norfloxacino, me´todo da difusa˜o em a´gar (cilindro em placas). Braz J Pharm Sci 1990;12:161-5
Brewster ME, Loftsson T. Cyclodextrins as pharmaceutical solubilizers. Adv Drug Deliv Rev 2007;59(7):645-66
Amidon GL, Lennernas H, Shah VP, et al. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 1995;12(3): 413-20
Zhao J, Lin D-Q, Wang Y-C, et al. A novel b-cyclodextrin polymer/tungsten carbide
17.
9.
11.
16.
Clinical and Laboratory Standards Institute; Wayne, PA: 2009)
CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standards: M7-A7.
Expert Rev. Anti Infect. Ther. 13(1), (2015)
Original Research
Investigation of b-cyclodextrin–norfloxacin inclusion complexes. Part 2. Inclusion mode and stability studies Expert Rev. Anti Infect. Ther. 13(1), 131–140 (2015)
Cassiana Mendes*1, Aline Buttchevitz1, Andersson Barison2, Fernanda Maria Marins Ocampos2, Larissa Sakis Bernardi3, Paulo Renato Oliveira3 and Marcos Antoˆnio Segatto Silva1 1 Post graduation Program in Pharmaceutical Sciences, Quality Control Laboratory, Federal University of Santa Catarina (UFSC), Floriano´polis, SC, Brazil 2 NMR center, Federal University of Parana´ (UFPR), Curitiba, PR, Brazil 3 Post graduation Program in Pharmaceutical Sciences, Universidade Estadual do Centro Oeste/UNICENTRO, 85040-080, Guarapuava, PR, Brazil *Author for correspondence: Tel.: +55 483 721 4585 Fax: +55 483 721 9350 [email protected]
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Introduction: Norfloxacin (NFX) is a broad spectrum antibiotic with low solubility and permeability, which is unstable on exposure to light and humidity. Objective: In this study, the mode of NFX inclusion into b-cyclodextrin complexes was evaluated and a complete physical, chemical and microbiological stability study of the inclusion complexes was carried out. Methods: Potentiometric titrations were performed to evaluate changes in the pKa of the NFX molecule due to the formation of an inclusion complex and NMR analysis demonstrated that the NFX molecule is included in the b-cyclodextrin cavity. Results: Inclusion complexes obtained by kneading followed by freeze-drying showed improved NFX stability compared with the isolated drug or the physical mixture. This method was effective in terms of protecting the drug from photodegradation and also avoiding hydrolysis. Differences between NFX and the complexes could be evidenced by thermal analysis, infrared spectroscopy and x-ray powder diffraction as well as by determining the solubility and drug content. The antimicrobial potency was also preserved on applying the promising method of kneading. Conclusion: The satisfactory stability indicates that the NFX/b-cyclodextrin complexes could be useful as an alternative to the existing NFX drug formulation. KEYWORDS: b-cyclodextrin • inclusion complexes • inclusion mode • norfloxacin • stability studies
Several drugs are unstable and can degrade when exposed to light, elevated temperatures and humidity. Therefore, the best conditions for storage should be investigated to guarantee the product stability and efficiency. A welldesigned stability study takes into account all of the factors that the product could be exposed to and it is an increasingly important issue in the development of any drug delivery system [1,2]. Norfloxacin (NFX) is a broad spectrum fluorquinolone antibiotic that has been extensively used for decades to treat mainly urinary infections, as well as urethritis, pharyngitis and proctitis, due to its effectiveness against Gram positive and Gram negative bacteria. NFX is a hygroscopic and photosensitive drug and its physical–chemical properties are affected by 10.1586/14787210.2015.982092
exposure to visible and UV light, affecting its bioavailability. Furthermore, prolonged exposure to heat and acidic solutions can lead to its hydrolysis, resulting in a carboxyl product which is responsible for the microbiological activity [3,4]. According to the Biopharmaceutical Classification System [5,6], NFX is a class IV drug (low solubility and low permeability) with low absorption (around 30–40%) associated with oral administration. This problem can be solved by the formation of inclusion complexes, mainly with cyclodextrin (CD), which usually increases the stability, solubility, dissolution rate and bioavailability [7,8]. In this regard, b-cyclodextrin (bCD) is a torus-shaped oligosaccharide containing seven glucose units, with a hydrophilic outer surface and a
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Original Research
Mendes, Buttchevitz, Barison et al.
lipophilic inner cavity which can accommodate a wide variety of lipophilic drugs, forming the so-called inclusion complexes. The stability of inclusion complexes can be provided by noncovalent interactions, such as hydrophobic interactions, electronic effects, steric factors and van der Walls forces. During the inclusion complex formation, the water molecules from the inner bCD cavity are replaced by appropriate lipophilic guests, which are energetically favored. Therefore, the presence of water plays an important role in the formation and stability of guest/CD inclusion complexes. The formation of complexes usually increases the stability, solubility, dissolution rate and bioavailability [7,8]. Moreover, in inclusion complexes the incorporation of drugs into the lipophilic cavity of bCD can protect them from light, humidity and high temperatures, with a consequent improvement in their stability [9,10]. However, it should be highlighted that pharmaceutical forms should contain as little CD as possible, because an excess can cause some problems associated with the formulation bulk or can be toxic, besides reducing the drug bioavailability and efficacy [11]. In the recent literature, much attention has been focused on stability studies. The inclusion complexes with NFX in the hydrophobic cavity of bCD can even protect the drug from light, moisture and temperature, thus improving the drug stability [9,10]. There are, however, few reports that explore the protection provided by the inclusion complex formation. In this context, to investigate the properties of inclusion complexes this study reports: the NMR and pKa results to verify the inclusion and show that the NFX is included in the bCD cavity and a complete study on the physical, chemical and microbiological stability. Experimental Materials
NFX was obtained from Galena, the CDs were kindly donated by Roquette Corporate (Labonathus, Sa˜o Paulo, Brazil) and all other chemicals were of analytical grade. Preparation of inclusion complexes
Three different methods were employed to produce the inclusion complexes with equimolar amounts of NFX in bCD: co-evaporation and kneading followed by freeze-drying or spraydrying. For the control, physical mixtures of NFX in bCD were prepared by blending equimolar amounts of NFX and bCD in a mortar until a homogeneous mixture was obtained. Co-evaporation
Equimolar amounts of NFX and bCD were dissolved in an EtOH/H2O solution (1:1 v/v) and the pH adjusted to 3.0. The solution was then stirred for 96 h at room temperature. Finally, the solvent was removed under reduced pressure at 50oC. Kneading
Stoichiometric amounts of NFX and bCD (1:1) were mixed for 20 min, 1 ml of water was then added and mixing applied again for 5 min. This procedure was then repeated. Next, 50 ml of water (40˚C) was added to the paste formed, the pH 132
was adjusted to 3.0 and the solution was stirred for 20 min. The resulting solution was then filtered and freeze-dried or spray-dried to dry the complexes formed. For the freeze-drying the solution was reduced to -40˚C and freeze-dried for 24 h, while for the spray-drying the solution was placed in a Buchi B-290 mini spray-dryer with an inlet temperature of 180˚C, outlet temperature of 62˚C, air flow rate of 30 ml/min and air pressure of 1.5 bar. Potentiometric titrations
Potentiometric investigations were carried out with a Corning350 pH meter fitted with blue-glass and Ag/AgCl reference electrodes calibrated to read -log [H+] directly. The electrode was calibrated with standard 0.1 M solutions of HCl and KOH. The ionic strength of the HCl solution was maintained at 0.1 M with the addition of KCl. Measurements were carried out in a thermostated cell containing 50 ml of a NFX solution (0.05 mm) with ionic strength adjusted to 0.1 M with the addition of KCl at 25 ± 0.05˚C. The experiments were performed under argon to eliminate the presence of atmospheric CO2. All experimental solutions were prepared in acetonitrile/water (1:1, v/v), owing to the low solubility of the drug in water. The pKw of the acetonitrile/water containing 0.1 M of KCl was 15.40 [12]. Computations were carried out with the BEST7 program. NMR analysis
The formation of a complex between NFX and bCD was also investigated by means of NMR spectroscopy analysis. For this, 1 H as well as 1D and 2D rotational frame nuclear overhauser effect spectroscopy (ROESY) NMR experiments were performed at 303 K in D2O on a Bruker AVANCE III HD 600 NMR spectrometer operating at 14.1 T, observing 1H at 600.13 MHz. The spectrometer was equipped with a 5 mm quadrinuclear inverse detection probe with a z-gradient. The 1 H NMR spectra were acquired with a spectral width of 6330 Hz (~10.5 ppm) and 64K data points, providing a digital resolution of 0.19 Hz, recycle delay of 1.0 s and 8 transients. The 1D ROESY NMR experiments were obtained with a selective 180˚ excitation pulse on the signals at 3.84 and 3.74 ppm, corresponding to H-3 and H-5 in the bCD moiety and selective refocusing with shaped pulses using the Bruker gradient selrogp pulse sequence, with a mixing time of 400 ms for ROESY spinlock, recycle delay of 2.0 s, 32K data points, 4K transients and the same spectral width used to obtain the 1H NMR spectra. The 1H and 1D ROESY NMR spectra were processed by applying a Fourier transform with zero-filling to 128K data points and by applying an exponential multiplication of the FIDs by a factor of 0.3 and 1 Hz for 1H and 1D ROESY NMR, respectively. All 1H NMR chemical shifts are given in ppm in relation to the TMSP-d4 signal at 0.00 ppm as the internal reference, and all pulse programs were supplied by Bruker. Stability evaluation
Stability assays were performed according to published guidelines on stability tests [13]. The physical mixture and inclusion Expert Rev. Anti Infect. Ther. 13(1), (2015)
Original Research
Investigation of b-cyclodextrin–norfloxacin inclusion complexes
pKa1 O
+
HN 2
O
O
F
F
OH
N+ H
pKa2
N+ H
+ H2N
H3C
N+ H H3C
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1
2
3
H2N+
O N H3 C
5
6
O
N
8
H2+ N HN
H3C
N
H3C
9.9 10
H2+ N
11
H3C
O
12
12.5
13 H N
N
O
HO
N H3C
F
N
–
O
11.3 9
+
O
HN
8.6 7
O F
–
O
N H2N+
6.1 4
O F
–
N+ H
pKa4
O
F
O
3.1 pH
O
O –
N+ H
pKa3
N
F
N
H3 C
O
pKa1
O HO
F
N
O
pKa2
O HO
Figure 1. Schematic representation of norfloxacin and norfloxacin/b-cyclodextrin inclusion complex in protonated and unprotonated forms, in different pH values.
complexes were monitored for up to 6 months in stability chambers at 40˚C and 75% relative humidity. The same samples were stored in photostability chambers with exposure to 200 Wh/m2 UV and 1,200,000 lux h visible. After 6 months, the samples were removed and submitted to differential scanning calorimetry (DSC), thermogravimetry (TG), diffuse Fourier transform infrared (FT-IR) spectroscopy, x-ray powder diffraction (XRPD), solubility assays, drug content determination and antimicrobial assay.
injection volume of 20 mL. The decarboxylated derivate was produced using a method in which 250 mg of NFX was refluxed with 70 ml of hydrochloric acid (2 mM) for 48 h at 150˚C, protected from light [15]. Subsequently, the solution was cooled and the pH was adjusted to 7.5 with sodium hydroxide (2 mM). The solution was then evaporated under vacuum to dryness and the residue extracted by EtOH and filtered through a 0.45 mm pore size filter. The final product was evaluated by HPLC. Differential scanning calorimetry & thermogravimetric analysis
Drug content determination
Drug content was determined by the ‘n-hexane wash’ method [14]. This method is based on the fact that bCD and its complexes are insoluble in n-hexane, while the free drug is soluble. In this procedure, 5 mg samples of each product were extracted with 5 ml of n-hexane containing 2% of acetic acid. The top-phase (n-hexane) was dried under reduced pressure at 40˚C. The residue was dissolved in 2 ml of mobile phase, filtered (0.45 mm pore size) and quantified by high performance liquid chromatography (HPLC), as described below. The drug content included in the bCD cavity was determined by dissolving the previously washed complex with a small amount of dimethyl sulfoxide (400 ml) and diluting it with water containing 2% of acetic acid. After 12 h the solution was centrifuged for 10 min at 3000 rpm and the top phase was filtered (0.45 mm pore size) and quantified by HPLC. The HPLC analysis was performed on a Shimadzu liquid chromatograph consisting of a LC-10A VP quaternary pump and a fluorescence detector (l excitation at 330 nm and l emission at 445 nm). The chromatograph was equipped with a Phenomenex C18 reversed-phase column chromatograph (150 4.0 mm; 5 mm particle size) conditioned in a SPD-10AVP column oven. The column was eluted with an isocratic mobile phase consisting of phosphate buffer (25 mM, pH 3.0) and acetonitrile (86:14 v/v) at a flow rate of 1.0 ml/min and informahealthcare.com
DSC curves for the NFX, bCD, NFX/bCD physical mixtures and inclusion complexes were obtained on a Shimadzu DSC-60 using aluminum crucibles with around 2.0 mg of
A
B
C
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
Figure 2. 1H nuclear magnetic resonance spectra. (A) NFX/bCD inclusion complex, (B) 1D ROESY spectra of NFX/bCD obtained by selective irradiation of 3.84 and (C) 3.74 ppm signals from bCD H-3 and H-5, respectively.
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HN
and an average of over 32 scans, at a spectral resolution of 4 cm-1. A background spectrum was obtained for each experimental condition.
H2C
H2C
CH2 CH2
X-ray powder diffraction N H
H3
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The crystallinity of the samples was evaluated by X-ray powder diffractometry. XRPD patterns were recorded on an X’Pert Philips diffractometer, with a CuKa tube, operating at 40 kV and 40 mA, in the range of 5–40 (2) with a pass time of 5 s.
F
H2C
H
N
Solubility of NFX/bCD complexes
To evaluate the solubility of the samples, an excess of the solid powder was added to 10 ml of a simulated intestinal fluid (0.05 M potassium phosphate - pH 6.8, without enzyme) in amber glass flasks which were sealed and magnetically stirred at human body temperature (37˚C) for 24 h. The samples were filtered through a 0.45-mm pore size filter and the NFX concentration determined by HPLC. The solubility of the inclusion complexes was evaluated in triplicate.
O H 5
H5 H O
HO
Figure 3. Schematic representation of norfloxacin/ b-cyclodextrin inclusion complex, showing norfloxacin structure partially inserted into the b-cyclodextrin cavity.
Antimicrobial assay
sample in a dynamic N2 atmosphere (50 ml/min) with a heating rate of 10˚C/min, in the temperature range of 30–300˚C. TG experiments were conducted on a Shimadzu TGA-50 thermobalance using a platinum cell containing approximately 4 mg of sample, in a dynamic N2 atmosphere (50 ml/min) with a heating rate of 10˚C/min, in the temperature range of 30–600˚C. Diffuse reflectance Fourier transform infrared spectroscopy
The FT-IR spectra were acquired on a PerkinElmer spectrophotometer (Frontier), within a scan range of 600–4000 cm-1 O
O
F
N
The antimicrobial activity of the NFX/bCD inclusion complexes was evaluated by the microdilution method, following the procedure outlined in the Manual of Clinical Microbiology [16]. Fresh cultures of Staphylococcus epidermidis (ATCC 12228), cultivated on Muller-Hinton agar were suspended in 0.9% NaCl. The suspension obtained was adjusted to the McFarland turbidity standard (0.5) and diluted to obtain an inoculum of 106 colony forming units per ml. The NFX, physical mixture and inclusion complexes were dissolved in 0.1 M phosphate buffer (pH 8) and then diluted using Muller-Hinton Broth [17,18]. Twofold serial concentrations of the compounds
F
N
O
F
OH
N
HN
O
O
H2N
N
OH N
HN
H3C Descarboxylated derivative
H3C Norfloxacin
O
H3C Amino derivative
O
O
O
F
F OH N
OH NH
N
N H3C O Formylpiperazine derivative
NH2
N H3C
Ethylenediamine derivative
Figure 4. Structures of norfloxacin and its degradants products, according to Alnajjar et al. [4].
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Investigation of b-cyclodextrin–norfloxacin inclusion complexes
Original Research
Table 1. Norfloxacin content, minimal inhibitory concentration, solubility and antibacterial potency are listed below for norfloxacin alone, physical mixture and complexes obtained by co-evaporation, kneading followed by freeze-drying and kneading followed by spray-drying at the initial time, after 6 months in the stability chamber (75% U.R., 40˚C), after ultraviolet exposition and visible exposition. Formulation
Drug content (%)
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Initial
Solubility† (mg/ml)
MIC (mg/ml)
Antibacterial potency‡ (%)
SC
UV
VIS
Initial
SC
UV
VIS
Initial
SC
UV
VIS
Initial
SC
UV
VIS
NFX
100
87.7
74.7
74.1
1
2
1
1
58.9
49.5
41.1
41.5
100.0
92.4
98.7
99.3
PM
50
44.5
37.2
37.1
1
2
1
1
59.1
48.1
39.5
40.2
99.7
90.9
99.9
98.8
COE
97
77.5
60.5
67.6
1
2
1
1
62.8
53.9
30.2
35.6
83.2
72.4
82.6
84.1
KFD
86
85.9
84.6
85.8
1
1
1
1
137.9
135.7
136.5
135.2
123.3
120.4
126.1
119.5
KSD
90
90
78.2
86.3
1
1
1
1
97.5
98.7
86.7
93.6
110.4
108.4
107.2
108.6
†
Drug solubility in simulated intestinal fluid pH 6.8 (37ºC). ‡ Growth inhibition zone is proportional to log of concentration, and are calculated by Hewitt equations. COE: Complexes obtained by co-evaporation; KFD: Kneading followed by spray-drying; MIC: Minimal inhibitory concentration; MIC: Minimal inhibitory concentration; NFX: Norfloxacin; PM: Physical mixture; SC: Stability chamber; UV: Ultraviolet exposition; VIS: Visible exposition.
were employed to determine the MIC, which ranged from verified by ANOVA (p = 0.05). Composition of Medium 1: 128.00 to 0.25 mg/ml. The MIC was defined, in triplicate, as peptone (6.0 g), pancreatic digest of casein (4.0 g), yeast extract the lowest concentration inhibiting visible microbial growth (3.0 g), beef extract (1.5 g), dextrose (1.0 g), agar (15.0 g), after incubation at 37˚C for 18 h. The antimicrobial activity of the NFX/ A bCD inclusion complexes was evaluated B by agar diffusion assay, as described by C Froehlich and Schapoval (1990). Fresh M cultures of Staphylococcus epidermidis N ATCC 12228 cultivated on Antibiotic D O Medium 1 were suspended in 0.9% NaCl and the suspension obtained was P adjusted to 25 ± 2% of transmittance at E 580 nm, to obtain the inoculum. To F form the base layer, 20 ml aliquots of G Antibiotic Medium 11 were placed in six Petri dishes. After its solidification, a H 5 ml portion of Antibiotic Medium 11, Q to which was added the inoculum R (0.7%), was poured onto the base layer. After solidification, stainless steel cylinS I ders (8 6 10 mm) were placed over J T the surface of the inoculated agar on each K dish. To each cylinder, 150 ml of NFX standard solution or inclusion complex L was added, at presumed concentrations of 4000 3500 3000 2500 2000 1500 1000 600 10, 20 and 40 mg/ml. After incubation Wavenumber (cm–1) (32–35˚C for 16–18 h), the cylinders were removed and the diameters of the 4000 3500 3000 2500 2000 1500 1000 600 Wavenumber (cm–1) inhibition zones were measured with the aid of an electronic digital caliper Figure 5. Fourier transform infrared spectroscopy spectra. (A) Norfloxacin NFX, (Starret). Assays were performed in (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after duplicate, using six plates for each one. 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PMThe antimicrobial potency of the NFX VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COEcomplexes was calculated according to VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by the Hewitt equation [19]. The validity spray-drying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC. of the parallel-line model chosen was informahealthcare.com
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E F G H
I J K
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M N O P Q
R S T 100
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Figure 6. Differential scanning calorimetry curves. (A) Norfloxacin NFX, (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PM-VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COE-VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by spray-drying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC.
water (1000 ml), pH after sterilization 6.6 ± 0.1; and composition of Medium 11: same as Medium 1, except that the final pH after sterilization is 8.3 ± 0.1 [18,20]. 136
Results & discussion
Potentiometric investigations were carried out to determine the pKa (protonation–deprotonation equilibrium) for NFX when it is inserted in the lipophilic cavity of the bCD. NFX shows four different pKa values (3.1, 6.1, 8.6 and 11.3), which hinders its solubility at physiological pH (6.8). On the other hand, in the NFX/bCD system, the pKa was increased to 9.9 and 12.5, respectively. These basic pKa values suggest that the less protonated NFX is inserted in the bCD cavity. The formation of an inclusion complex is well known to improve the solubility of the guest, and the pKa value is an essential parameter to predict the behavior in biological fluids. The results revealed that at physiological pH (6.8) all NFX molecules (>99%) are present in the protonated form and, as a consequence, complexed NFX has improved solubility when compared with the free drug, due to the pKa value, in addition to the inclusion complex contribution. FIGURE 1: The formation of inclusion complexes obtained by kneading followed by freeze-drying can be evidenced by changes, including broadening, in the 1H NMR signals for the drug, when inside the lipophilic cavity of the bCD, compared to signals of the drug without the presence of bCD recorded under the same NMR conditions [21,22]. Therefore, the first approach was to acquire 1H NMR spectra of both the NFX and NFX/bCD, in D2O. However, only the NFX/bCD system was soluble in D2O and its 1H NMR spectrum showed NFX signals, evidencing the formation of a NFX/bCD inclusion complex. Moreover, the 1H NMR spectra showed a 7:1 area ratio for the bCD to NFX signals, revealing a 1:1 stoichiometry. The formation of the NFX/bCD inclusion complex was supported by the one-dimensional rotating frame nuclear Overhauser effect (1D ROESY) NMR experiments. The 1D ROESY NMR analysis carried out by selective excitation of the hydrogens H-3 and H-5 at 3.84 and 3.74 ppm in bCD caused an NOE enhancement in the 8.60, 7.64, 4.37 and 1.42 ppm NFX signals (FIGURE 2). A high NOE intensification of the NFX signals was observed only when the H-3 resonance frequency at 3.84 ppm was selectively irradiated (FIGURE 2). However, the selective irradiation of the H-5 resonance frequency at 3.74 showed a higher intensification of the NFX signal at 8.60 ppm, indicating that this NFX hydrogen is closer to the bCD H-5 than to the other bCD hydrogens. The intensification of the NFX signals in the 1D ROESY NMR spectra, after selective excitation of the resonance frequency of H-3 and/or H-5 bCD hydrogens, revealed that a NFX/bCD inclusion complex was formed, with the NFX structure partially inserted in the bCD cavity, as represented in FIGURE 3. Physcochemical stability Photostability studies
Physicochemical stability studies provide information required for the development of the preformulation, highlighting particular requirements regarding unstable drugs, such as NFX. Expert Rev. Anti Infect. Ther. 13(1), (2015)
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Investigation of b-cyclodextrin–norfloxacin inclusion complexes
According to Alnajjar and coworkers, the three photodegradation products of NFX are formylpiperazine, amine and ethylenediamine (FIGURE 4) [23]. NFX degrades around 26% (assay 74%) when exposed to UV and visible light (TABLE 1). The FT-IR analysis (FIGURE 5A) shows the difference in the region of 950–900 cm-1 for NFX before and after light exposure. This result provides evidence of the presence of photodegradation products, because this region corresponds to the imine moiety. From the data in TABLE 1, the same decomposition of the physical mixture can be noted, indicating that without the formation of an inclusion complex the presence of bCD does not guarantee NFX light protection. There was no protection provided by the formation of the inclusion complex obtained applying the co-evaporation method and FIGURE 6 shows the change in the imine region after photoexposure. However, very low NFX photodegradation was observed for the inclusion complexes obtained through kneading followed by freeze-drying. In the case of the spray-drying method, a low degree of photodegradation was observed on exposure to visible light (assay 86.3%) and more accentuated degradation in the presence of UV light (assay 78.2%). Although the inclusion complex does not contain the piperazine ring moiety, it can be inferred that the complex obtained by kneading followed by freeze-drying prevented the photodegradation of this part of the NFX molecule. In this case, bCD could provide some spatial protection from the reactive species or some particle aggregation of these amorphous complexes could occur in contrast to the crystalline form obtained by co-evaporation technique. The DSC analysis of NFX showed an endothermic event at 217˚C (DHfusion = -170.93 J/g) assigned to the fusion point typical of crystalline substances, while bCD showed a water loss at 93˚C (DHfusion = -245.22 J/g) (FIGURE 6). On the other hand, the inclusion complex obtained by co-evaporation presented an event at 140˚C (FIGURE 6I), while those obtained by kneading followed by freeze-drying or spray-drying exhibited an endothermic event at 160˚C (FIGURE 6M & 6K). NFX, the guest molecule, entered the bCD cavity and some of the cavity water was lost, therefore the energy of the remaining water was altered, which may explain the slight shift in the endothermic events to higher temperature observed for the inclusion complexes (160˚C). This finding suggests that the NFX/bCD samples obtained by kneading followed by freezedrying or by spray-drying are more stable than those produced by co-evaporation. After photoexposure, other events were observed for the inclusion complexes obtained by coevaporation as well as by kneading followed by spray-drying probably due to the presence of photodegradation products. However, in the inclusion complex obtained by kneading followed by freeze-drying no changes in the endothermic event were observed (FIGURE 6M–6P). The inclusion complexes obtained by this technique could maintain the thermal stability indicated initially by the DSC analysis. The XRPD patterns for NFX showed characteristic crystalline peaks at 7.86, 9.90, 10.70, 11.99, 14.92, 16.09, 18.76, informahealthcare.com
Original Research
A B C D E F G
H
I J
K L
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20 25 2q
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35 40
Figure 7. X-ray powder diffraction patterns. (A) Norfloxacin NFX, (B) after ultraviolet exposition NFX-UV, (C) after visible exposition NFX-VIS, (D) after 6 months in the stability chamber NFX-SC; (E) physical mixture PM, (F) PM-UV, (G) PM-VIS, (H) PM-SC; (I) complexes obtained by co-evaporation COE, (J) COE-UV, (K) COE-VIS, (L) COE-SC; (M) complexes obtained by kneading followed by freeze-drying KFD, (N) KFD-UV, (O) KFD-VIS, (P) KFD-SC; (Q) complexes obtained by kneading followed by spraydrying KSD, (R) KSD-UV, (S) KSD-VIS, (T) KFD-SC.
20.68, 22.76 and 25.02 (FIGURE 7). This XRPD pattern presents excellent agreement with the well-defined polymorph A, defined as the commercial form [24]. After photoexposure, peaks which differed from those of the NFX polymorph A appeared, probably due to NFX photodegradation products. On the other hand, the NFX/bCD system obtained by co-evaporation 137
Original Research A
Mendes, Buttchevitz, Barison et al.
DrTGA mg/min
TGA % 100
2
B
DrTGA mg/min
TGA % 100
0
1 50
–2 50
0 –0
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DrTGA 1 01
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solubility of the NFX, free or in the presence of bCD, reduced considerably. Therefore, the presence of the polymer did not affect this parameter. The coevaporated complex did not maintain its solubility after photoexposure. On the other hand, the systems obtained by kneading followed by freeze-drying or spray-drying presented no significant decrease in solubility. The most relevant observation to emerge from this investigation is that the ability of these inclusion complexes to protect the NFX molecule against degradation and improve its solubility is maintained. Temperature & humidity
–1
–1
After 6 months of storage at 40˚C and 75% humidity, the NFX underwent –2 –2 hydrolysis to its decarboxyl derivative, as –0 200 400 600 –0 200 400 600 evidenced by the absence of stretching at Temperature (°C) Temperature (°C) 3500–3550 and 1716 cm-1, related to E F OH and carbonyl C=O groups, DrTGA TGA DrTGA TGA respectively (FIGURE 5), in the FT-IR analy% % sis. This degradation product was also 2 0.5 100 100 detected in the HPLC analysis (data not 0 0 shown). On the other hand, according to 50 50 the FT-IR, DSC and XRPD analysis, no –20 –0.5 NFX degradation was observed in the –0 –0 case of the NFX/bCD systems obtained –1 –4 by kneading followed by freeze-drying or –0 200 400 600 –0 200 400 600 spray-drying (FIGURES 5–7). In contrast, the Temperature (°C) Temperature (°C) DSC analysis showed other endothermic events in the NFX/bCD system obtained Figure 8. Thermogravimetric and derivative thermogravimetric curves. (A) NFX, (B) b-cyclodextrin, (C) physical mixture and complexes obtained by (D) co-evaporation, by co-evaporation, which produced an (E) kneading followed by freeze-drying or (F) spray-drying. inclusion complex in the crystal form. Moreover, the FT-IR analysis showed the showed unique crystalline peaks at 2 angles of 11.7, 13.5, absence of COOH stretching, indicating a decarboxylated deg14.6, 15.4 and 17.6, identifying the formation of a new crystal. radation product. The XRPD analysis of the complex obtained After photoexposure, the characteristic intense peaks of NFX by co-evaporation shows intense NFX peaks at 2 angles of (11.99 and 20.68) and bCD (15.19 and 16.91) changed, indi- 12.07 and 20.7, as observed after exposure to light, indicating cating destabilization of the complex. On the other hand, the that this inclusion complex is sensitive to light and humidity. NFX/bCD complexes obtained by kneading followed by In other words, the inclusion complex obtained by cofreeze-drying or spray-drying showed an XRPD pattern typical evaporation generated low stability complexes. of amorphous compounds. Even after light exposure the amorAccording to the TG analysis (FIGURE 8), the physical mixture phous pattern remained the same, indicating that there was and NFX/bCD systems obtained by co-evaporated had higher no degradation. water contents (10.4 and 10.9%, respectively). However, the The solubility assays performed under simulated intestinal NFX/bCD systems obtained by kneading followed by freezeconditions with the NFX/bCD system obtained by kneading drying or spray-drying showed lower water contents (7.5% for followed by freeze-drying or spray-drying showed improvement both). These results indicate that in the kneading followed by in terms of the solubility, while no improvement was observed freeze-drying or spray-drying procedures NFX replaces more for the samples obtained from the physical mixture and the water molecules than in that of the other approaches. NFX can NFX/bCD system obtained by co-evaporation (TABLE 1). These absorb water and form a hydrate, which shows an endothermic findings indicate the formation of a NFX/bCD inclusion com- event at 136.4˚C. However, the TG analysis shows events at plex applying the kneading procedure. After photoexposure, the 61.6 and 64˚C for the NFX/bCD systems obtained by –0
138
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Expert Rev. Anti Infect. Ther. 13(1), (2015)
Investigation of b-cyclodextrin–norfloxacin inclusion complexes
kneading followed by freeze-drying or spray-drying, respectively, related to the absorption of natural humidity by the amorphous solid, leading to the low temperatures observed on the DSC endotherm (FIGURE 6). Therefore, these results provided further support for the hypothesis that NFX instability can be overcome through the production of an inclusion complex by kneading followed by freeze-drying.
Original Research
reflects directly in the antibacterial activity, because the carboxyl ring is responsible for the NFX activity. On the other hand, in the NFX/bCD system obtained by kneading followed by freeze-drying or spray-drying the NFX molecule remained inside the bCD cavity. Therefore, these preparation methods preserved the antibacterial potency because the drug was protected from the hydrolysis process. Conclusions
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Microbiological stability
The physical mixture presented the same inhibitory activity as the free NFX, whilst the isolated bCD showed no antibacterial effect. Furthermore, the microbiological study showed that the inclusion complexes also had the same MIC as the free NFX, indicating that all of the preparation methods allowed the drug stability to be maintained (TABLE 1). The MIC obtained for NFX in the isolated form or in inclusion complexes was 1 mg/ml (TABLE 1), according to a previous study [25]. To evaluate microbiological stability, the antibacterial potency of NFX alone and as inclusion complexes after the stability studies was determined. After photoexposure there was no statistical difference in the antimicrobial activity. This result can be attributed to the fact that all of the photoproducts keep the carboxyl moiety and the fluor atom, which are responsible for the antimicrobial activity. However, after exposure to humidity and heat, an important difference between the isolated NFX and some of the complexes was observed (TABLE 1). After 6 months exposure it was noted that the NFX lost its initial antibacterial properties and the antibacterial potency decreased to 92.4%. The same was observed for the physical mixture and the co-evaporated NFX/bCD system, as a consequence of the NFX degradation. After exposure to humidity and heat, NFX degrades to its decarboxylated derivative, which
The results of this study provided an insight into the mode of NFX inclusion in the b-CD cavity, based on NMR analysis and the change in the pKa values due to the inclusion formation. Kneading followed by freeze-drying was found to be the most appropriate method for the production of the inclusion complexes because it provided chemical, physical and microbiological stability. This method prevented degradation on exposure to UV and visible light, besides inhibiting the hydrolysis of the NFX. Preserving the antibacterial activity with the use of a simple, low cost method is an important issue in the obtainment of inclusion complexes which can be applied on an industrial scale. The NFX/bCD complexes obtained by kneading followed by freeze-drying represent a potential alternative to the existing NFX drug formulation. Financial & competing interests disclosure
This study was supported by the National Counsel of Technological and Scientific Development (CNPq) and Coordination for Enhancement of Higher Education Personnel (CAPES). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Key issues • NMR analysis demonstrated the mode of the norfloxacin (NFX) molecule inclusion in the b-cyclodextrin (bCD) cavity. • The pKa determination provided information on the ionization of NFX when inserted in the bCD cavity. • Without the formation of an inclusion complex the presence of bCD does not guarantee NFX light protection. • The improvement of the solubility could be maintained by systems obtained by kneading followed by freeze-drying or spray-drying. • The thermal stability of the inclusion complexes obtained by kneading followed by freeze-drying was maintained after photoexposure. • After exposure to high humidity and temperature no NFX degradation was observed in the case of the NFX/bCD systems obtained by kneading followed by freeze-drying or spray-drying. • After photoexposure the antibacterial potency of NFX in the inclusion complexes formed by kneading followed by freeze-drying or spray-drying was preserved. • NFX degradation into its decarboxylated derivative (without pharmacological activity) could be prevented through the formation of inclusion complexes by kneading followed by freeze-drying or spray-drying.
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composite matrix for expanded bed adsorption: preparation and characterization of physical properties. Carbohydr Polym 2010;80(4):1085-90
References 1.
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by Nyu Medical Center on 02/17/15 For personal use only.
2.
3.
4.
5.
6.
7.
8.
Yang L-J, Chen W, MA S-X, et al. Host-guest system of taxifolin and native cyclodextrin or its derivative: preparation, characterization, inclusion mode, and solubilization. Carbohydr Polym 2011; 85(3):629-37 Zeng J, Ren Y, Zhou C, et al. Preparation and physicochemical characteristics of the complex of edaravone with hydroxypropyl-b-cyclodextrin. Carbohydr Polym 2011;83(3):1101-5 El Khateeb SZ, Abdel Razek SA, Amer M. Stability-indicating methods for the spectrophotometric determination of norfloxacin. J Pharm Biomed Anal 1998; 17(4):829-40 Alnajjar A, Idris AM, AbuSeada HH. Development of a stability-indicating capillary electrophoresis method for norfloxacin and its inactive decarboxylated degradant. Microchem J 2007;87(1):35-40
18.
Brazilian Pharmacopoeia. Brası´lia, Brazil: 5th ed 2010)
19.
Hewitt W. Microbiological assay for pharmaceutical analysis: a rational approach. CRC Press, Boca Raton, FL; 2013)
20.
The United States Pharmacopoeia, 37nd ed. (USP 37). United States Pharmacopeial Convention; Rockville, MD: 2013
21.
Asbahr ACC, Franco L, Barison A, et al. Binary and ternary inclusion complexes of finasteride in HPbCD and polymers: Preparation and characterization. Bioorg Med Chem 2009;17(7):2718-23
22.
Grebogi IH, Tibola APO, Barison A, et al. Binary and ternary inclusion complexes of dapsone in cyclodextrins and polymers: preparation, characterization and evaluation. J Inclusion Phenom Macrocyclic Chem 2012;73(1-4):467-74
23.
Hussein K, Turk M, Wahl MA. Comparative evaluation of ibuprofen/betacyclodextrin complexes obtained by supercritical carbon dioxide and other conventional methods. Pharm Res 2007; 24(3):585-92
Alnajjar A, AbuSeada HH, Idris AM. Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization. Talanta 2007;72(2):842-6
24.
Oliveira PR, Bernardi LS, Mendes C, et al. Liquid chromatographic determination of norfloxacin in extended-release tablets. J Chromatogr Sci 2009;47(9):739-44
Barbas R, Martı´ F, Prohens R, et al. Polymorphism of norfloxacin: evidence of the enantiotropic relationship between polymorphs A and B. Cryst Growth Des 2006;6(6):1463-7
25.
Norrby SR. Useful agents in the management of urinary tract infections. Int J Antimicrob Agents 1994;4(2):129-34
10.
Gould S, Scott RC. 2-Hydroxypropyl-bcyclodextrin (HP-b-CD): a toxicology review. Food Chem Toxicol 2005;43(10): 1451-9 Kiss T, Fenyvesi F, Ba´cskay I, et al. Evaluation of the cytotoxicity of b-cyclodextrin derivatives: evidence for the role of cholesterol extraction. Eur J Pharm Sci 2010;40(4):376-80
12.
Herrador M, Gonza´lez AG. Potentiometric titrations in acetonitrile–water mixtures: evaluation of aqueous ionisation constant of ketoprofen. Talanta 2002;56(4):769-75
13.
Food and Drug Administration. Guidance for industry Q1A (R2) stability testing of new drug substances and products. Food and Drug Administration. Rockville, MD, 2003. Available from: www.fda.gov/ downloads/RegulatoryInformation/ Guidances/ucm128204.pdf [Last accessed 19 March 2013]
Breda SA, Jimenez-Kairuz AF, Manzo RH, et al. Solubility behavior and biopharmaceutical classification of novel high-solubility ciprofloxacin and norfloxacin pharmaceutical derivatives. Int J Pharm 2009;371(1):106-13
14.
Tommasini S, Raneri D, Ficarra R, et al. Improvement in solubility and dissolution rate of flavonoids by complexation with b-cyclodextrin. J Pharm Biomed Anal 2004; 35(2):379-87
15.
140
Froehlich PE, Schapoval EES. Doseamento microbiolo´gico do norfloxacino, me´todo da difusa˜o em a´gar (cilindro em placas). Braz J Pharm Sci 1990;12:161-5
Brewster ME, Loftsson T. Cyclodextrins as pharmaceutical solubilizers. Adv Drug Deliv Rev 2007;59(7):645-66
Amidon GL, Lennernas H, Shah VP, et al. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 1995;12(3): 413-20
Zhao J, Lin D-Q, Wang Y-C, et al. A novel b-cyclodextrin polymer/tungsten carbide
17.
9.
11.
16.
Clinical and Laboratory Standards Institute; Wayne, PA: 2009)
CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standards: M7-A7.
Expert Rev. Anti Infect. Ther. 13(1), (2015)
