Jpp-d-08-00422 1.6

Preparation, physicochemical characterization and biological Received August 26, 2008Accepted March 18, 2009 evaluation of cefodizime metal ion complexes Sayed H. Audaa,b, Y. Mrestanic, Dietrich H. Niesd, Cornelia Großed Institutes of aPharmacy, cApplied Dermatopharmacy and dBiology, Martin-Luther-University Halle-Wittenberg,Halle (Saale), Germany, and bDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy,Al-Azhar University, Assiut Branch, Assiut, Egypt Objectives Cefodizime is a broad spectrum cephalosporin belonging to the thirdgeneration agents. In this study, attention has been paid to the preparation, physicochemicalcharacterization and biological evaluation of new Cu2+, Zn2+, Fe3+, Co2+ and Al3+complexes of cefodizime.
Methods The stoichiometrics and the mode of bonding of the complexes were deducedfrom their elemental and metal analysis, electrical conductivity measurements, UV-vis, IRand Raman spectroscopic investigations. Study of the stoichiometry of these complexesreferred to the formation of 1 : 1 ratios of metal to ligand. Antimicrobial activity of thecomplexes was determined using two strains of Gram-positive (Bacillus subtilis andProteus vulgaris) and two strains of Gram-negative (Escherichia coli W3110 andPseudomonas putida) bacteria. The minimal inhibitory concentration was determined asthe lowest concentration inhibiting bacterial growth on solid Luria Bertani medium.
Key findings The spectra gave evidence as to the position of binding. In addition, theaqueous solubility of cefodizime was strongly reduced by complexation.
Conclusions The antibacterial activity of cefodizime was not affected by complexationwith Al3+ but it was reduced by complexation with the other tested metal ions against thebacteria under study.
Keywords antibacterial activity; cefodizime; metal complexes; physicochemicalcharacterization The cephalosporin antibiotics are semisynthetic antibacterials derived from cephalosporin C,a natural antibiotic produced by the mould Cephalosporium acremonium.[1] The most widelyused system of classification of cephalosporins is by generations. According to theirantimicrobial spectrum of activity, they are classified into four generations. The firstgeneration cephalosporins are very active against Gram-positive cocci. They have limitedactivity against Gram-negative bacteria.[2] The second generation cephalosporins havesomewhat increased activity against Gam-negative microorganisms but are much less activethan the third and fourth generation agents.[3,4] Cefodizime is a broad-spectrum, third-generation, parenteral cephalosporin which possesses a prolonged elimination half-life, >3.5 h.[5,6] The chemical structure ofcefodizime disodium salt is shown in Figure 1.
Cephalosporin antibiotics have long been known to behave as relatively efficient chelating agents.[7] A list of clinically used chelating agents may be found in mostpharmacopoeia, while new chelating agents continue to be sought.[8,9] The medicinal uses of metal complexes are of increasing clinical and commercial Correspondence: Reinhard H. H.
Neubert, Institute of Pharmacy, importance. Fluorouracil-oxaliplatin complex is used in Europe and the USA for treatment of colorectal cancer.[10,11] Ranitidine-bismuth citrate complex is marketed in the USA as ranitidine bismutrex for the management of peptic ulcer and ulcers associated with Helicobacter pylori.[12] Gold and ruthenium complexes of chloroquine and clotrimazole have been investigated for their antiparasitic activity.[13,14] Furthermore, it was found that some chloroquine complexes are useful even in chloroquine-resistant cases. Iqbal et al.[15] reported that copper–cephalexin complex exhibited a good anti-inflammatory activity and their colours the other complexes were not suitable for the An HP 8452 A (Hewlett-Packard, Waldbronn, Germany) was used to determine the UV–vis spectra of cefodizime and its metal complexes in phosphate buffer (pH 7.4).
The chemical structure of cefodizime disodium.
C, H, N and S contents were analysed using an elemen-tal analyser, CHN -932, Leco Corporation (St Joseph, had more antibacterial effect than the free cephalexin. The effect of metal ions on drug activity was confirmed byseveral studies.[16,17] The aim of this work was the preparation and physico- Metal contents were determined by direct titration against chemical characterization of cefodizime metal complexes as standard EDTA (for the Zn2+ complex) or by a back titration well as the investigation of their antibacterial activity.
technique using standard Zn solution (for the remainingcomplexes) after complete decomposition of the complexes achieved by boiling with concentrated nitric acid for 10 min.
Water content determinationWater content in the prepared complexes was determined All chemicals were of reagent grade and were used without using the Karl-Fischer method using the Karl-Fischer- any further purification. Cefodizime disodium was obtai- Titrator AQUA 40.00 instrument, Elektrochemie Halle ned from Hoechst (Frankfurt, Germany). CuSO4 and ZnSO4.7H2O were purchased from Sigma-Aldrich GmbH(Seelza, Germany). FeCl3.6H2O was supplied by Roanal (Budapest, Hungary). Co(NO3)2.6H2O was obtained from Quantitative solubility of the complexes was determined Gruessing (Filsum, Germany). NiCl3.7H2O and AlCl3 were spectrophotometrically in phosphate buffer (pH 7.4) by the purchased from Germed (Dresden, Germany). Dimethyl equilibrium solubility method, which employs a saturated sulfoxide (DMSO) was obtained from Carl Roth GmbH & solution of the material, obtained by stirring an excess of the Co. (Karlsruhe, Germany). Ethylene diamine tetraacetate material in the solvent for a prolonged period until equilibrium (EDTA), nitric acid, dipotassium hydrogen phosphate and is achieved. At 270 nm and room temperature the extinction potassium dihydrogen phosphate were supplied by E. Merck coefficient was between 6.51 and 6.70 l/mmol/cm.
(Darmstadt, Germany). Methanol, ethanol, diethyl ether,acetonitrile, acetone and dimethylformamide were obtained from Riedel-de Haen AG (Seelze, Germany).
The buffer solution was prepared by dissolving 1.237 gdipotassium hydrogen phosphate and 0.394 g potassium dihydrogen phosphate in 600 ml distilled water, and then Cefodizime disodium (2 mmol) was dissolved in 20 ml adding distilled water to reach a volume of 1000 ml. The pH methanol. Metal salts, CuSO4, ZnSO4.7H2O, FeCl3.6H2O, of the buffer was measured at 25oC using a microprocessor Co(NO3)2.6H2O and AlCl3 (1 mmol) were separately pH meter obtained from Testo GmbH and Co. (Lenzkirch, dissolved in 10 ml methanol. The two solutions were mixed while stirring for 30 min. Coloured products pre-cipitated and were isolated by filtration. The products were washed with water, acetone and dimethyl ether, and dried in Saturated solutions of cefodizime and its complexes were prepared by adding an excess mass of powder to a constantvolume (2 ml) of phosphate buffer, pH 7.4. Saturated solutions of cefodizime and its complexes were kept on a magnetic stirrer in closed glass tubes for 24 h at 25oC. Before The FTIR spectra of cefodizime and its metal complexes the analysis all samples were filtered through 0.45-mm were recorded using a FTIR spectrometer Vertex 70 by Millipore PTFE filters (Millipore Corp., Bedford, MA, USA).
Bruker Optics (Ettlingen, Germany). The samples were diluted with an adequate amount of KBr and compressed topellets. The pellets were measured in the range from 370 to Antimicrobial activity of the complexes was determined using two strains of Gram-positive (Bacillus subtilis andProteus vulgaris) and two strains of Gram-negative (Escher- ichia coli W3110 and Pseudomonas putida) bacteria. The The FT-Raman measurements of cefodizime and its Zn2+ and minimal inhibitory concentration (MIC) was determined as Al3+ complexes were acquired by using the RFS 100/S the lowest concentration inhibiting bacterial growth on solid spectrometer (Bruker Optics, Karlsruhe, Germany). Due to Luria Bertani medium (DifcoTM Lennox; Becton Dickinson, Germany). A preculture for each bacterium was incubated at 30∞C, 250 rev/min, for 17 h. This was then diluted 1 : 400 in Evidence for complex formation was obtained by comparing fresh medium and incubated for 2 h at 30∞C, 250 rev/min.
the most characteristic infrared (IR) spectral bands of the free This 2 h-culture was used for streaking onto plates contain- cefodizime and its complexes. In general, cephalosporins ing the complexes (dissolved in DMSO) in different have three characteristic C=O absorptions for the stretching concentrations. The plates were incubated at 30∞C for 17 h.
vibrations of the b-lactam ring, the carboxylate and amide I.
Using the paper disc diffusion method on solid Luria Cephalosporins have a zwitterionic character. Thus, their Bertani medium another technique was performed. The spectra of free ligand show bands of antisymmetric (nas) and complexes were tested at a concentration of 3 mg/ml in symmetric (ns) vibrations of the carboxylate group. Disap- DMSO. As a control, DMSO alone was applied to the paper pearance of one or more of such bands may indicate the discs. A preculture and a 2 h-culture (as described above) participation of it or them in metal coordination. The were done. A 500 ml sample of the 2 h-culture were plated important IR frequencies of cefodizime and its metal onto nutrient agar (Carl Roth, Germany), dried and paper complexes along with their assignments are given in Table 2.
discs with 10 ml of the complex solution were applied. After17 h at 30∞C the inhibition zone was measured.
The main Raman spectra of cefodizime and its Zn2+ and Al3+ The one-way analysis of variance post-hoc test was used for complexes are listed in Table 3. Due to their colours the rest the determination of significant differences in the study of the complexes were not suitable for Raman investigation.
(OriginPro 7.5). The analysis of variance post-hoc Tukey’stest was used to compare all the samples with one other (antibacterial activity of cefodizime and its complexes Five-point standard calibration curves of cefodizime and its complexes in phosphate buffer, pH 7.4, gave linearitycorrelation coefficients ranging from 0.996 to 0.999. The compounds were determined spectrophotometrically at lmax = 270 nm using the same solvent medium as the The Fe3+ and Co2+ complexes were brown and rose,respectively. The Zn2+ and Al3+ complexes were white, while the Cu2+ complex was green. Microanalytical and The results of the MIC test are shown in Table 5. Cefodizime complexometric titration data (Table 1) confirmed the interferes with cell-wall synthesis of bacteria, leading to lysis formation of 1 : 1 metal to ligand ratio. Water content of the infectious microorganisms. In-vitro antibacterial determined by the Karl-Fischer method showed that the Cu2+ activity of cefodizime and its complexes were tested using complex was tetrahydrated, the Al3+ complex was penta- the MIC and the paper disc diffusion method using two hydrated, whilst the Zn2+, Fe3+, and Co2+ complexes were strains of Gram-positive (B. subtilis and P. vulgaris) and two hexahydrated. UV-vis spectra showed no significant differ- strains of Gram-negative (E. coli W3110 and P. putida) ence between cefodizime and its metal complexes.
bacteria. Antibacterial activity of cephalosporin metal ion Elemental analysis data of cefodizime metal complexes n = 4 (contents in % ± SE). Found (calcd.).
The main FTIR spectra of cefodizime and its metal complexes The main FT-Raman spectra of cefodizime and its Zn2+ and Al3+ complexes The aqueous solubility of cefodizime and its complexes in either as a monodentate or a bidentate ligand, giving changes in the relative positions of the antisymmetric and symmetricstretching vibrations.[4] The FT-IR spectra of the complexes gave a separation value of >200 cm-1 suggesting mono-dentate bonding for the carboxylate group.[20] of cefodizime at 1041 cm-1 which showed no significant changes in any of the complexes, suggesting that there was no coordination through this group to the metal ion.[21] In addition, IR spectra of cefodizime exhibited a band at Phosphate buffer was pH 7.4. SE, standard error.
1356 cm-1 due to n(C–N) of the b-lactam and thiazole ringnitrogen atom.[22] This band appeared in all studiedcomplexes without further change, indicating that the complexes depends mainly on the type of cephalosporin b-lactam and thiazole ring nitrogen atom were not participat- used, the type of metal ion and the type of microorganism ing in the bonding. Bands at 3191 and 3310 cm-1 in the spectra of cefodizime were due to antisymmetric andsymmetric NH stretching of the carbamate NH2 group. These bands appeared relatively at the same position in the spectraof all the complexes with the exception of the Co2+ complex.
In the FT-IR spectra (Table 2) of cefodizime, a characteristic This may provide evidence to the participation of this group band arising from stretching vibrations of the carbonyl group in the coordination only in the case of the Co2+ complex and of the b-lactam ring appeared at 1777 cm-1. This band its inertness towards coordination in the other complexes.
appeared in all studied complexes almost at the same wave Similar to the IR spectra, Raman spectra of cefodizime number. This may suggest that the carbonyl oxygen atom showed a characteristic band arising from stretching vibra- from the b-lactam ring was not engaged in metal binding.
tions of the carbonyl group of the b-lactam ring at 1764 cm-1 Furthermore, the FT-IR spectra of cefodizime revealed a (Table 3). This band appeared in all the studied complexes band at 1659 cm-1 due to stretching vibrations of the amide almost at the same wave number. It may indicate that the carbonyl group. This band either vanished or appeared as a carbonyl oxygen atom from the b-lactam ring was not shoulder in metal complexes suggesting the coordination of engaged in metal binding. Raman spectra of cefodizime revealed a band at 1623 cm-1 due to stretching vibrations of The bands of antisymmetric (as) and symmetric (s) the amide carbonyl group. This band was significantly vibrations of the carboxylate group arose at 1588 and shifted in metal complexes suggesting the coordination of 1375 cm-1, respectively. In spectra of metal complexes, metals through this group. The bands of antisymmetric (as) these two bands were shifted towards the higher wave and symmetric (s) vibrations of carboxylate groups of number. This suggested interaction between the metal ions cefodizime arose at 1585 and 1397 cm-1, respectively.
and the carboxylate group of cefodizime.[19] On the other Although the shift of these bands in the Raman spectra of hand, a carboxylate ligand could bind to the metal atom metal complexes was small, they gave a significant shift in Antibacterial activity of cefodizime and its complexes against different bacteria* *Bacteria were incubated (30∞C) in solid Luria Bertani medium containing increasing concentrations of cefodizime or its complexes. Minimalinhibitory concentration, the lowest concentration that inhibited the formation of single colonies. The experiment was performed in triplicate.
the FT-IR spectra. This may suggest interaction between exhibited significant differences at the 0.05 level only for metal ions and the carboxylate group of cefodizime.
B. subtilis, B. vulgaris and E. coli with P. putida. The The solubility values of complexes that were calculated population means were significantly different (one-way from these determinations showed significant decrease when compared with the solubility of cefodizime. These valuesare listed in Table 4. The solubility of cefodizime in the phosphate buffer (pH 7.4) was 164.41 mg/ml while thesolubility of complexes ranged from 1.963 to 2.58 mg/ml.
Cefodizime formed complexes with different metal ions. The The decreased solubility of metal complexes may have been stoichiometric ratio of these complexes was 1 : 1 metal to attributed to the decrease in their hydrophilicity compared ligand. Furthermore, the coordination of ligand with metal with cefodizime.[23] According to this study we could arrange ions occurred through carboxylate and amide carbonyl groups.
the solubility of complexes in phosphate buffer (pH 7.4) in the In addition, the aqueous solubility of the cefodizime was following order, with the highest solubility for the cefodizime- strongly affected by complexation. With the exception of the Co2+ complex and the least solubility for the cefodizime–Fe3+ cefodizime-Al complex, all tested metal ion complexes were complex: cefodizime-Co2+ > cefodizime-Zn2+ > cefodizime- less active than cefodizime against the bacteria under study.
Al3+ > cefodizime-Cu2+> cefodizime-Fe3+.
Except for the cefodizime-Al3+ complex, all tested metal ion complexes were shown to be less active than cefodizime against the bacteria investigated. Cefodizime-Al3+ complex exhibited an antibacterial activity similar to the parent drugagainst B. subtilis, P. vulgaris and E. coli and 1.16-times less The Author(s) declare(s) that they have no conflicts of than cefodizime against P. putida. The antimicrobial activity of the other complexes ranged from 1.3 to 5.8-times less active than the pure antibiotic. Cefodizime-Zn2+ andcefodizime-Fe3+ complexes had a middling activity, while This research received no specific grant from any funding the cefodizime-Cu2+ and cefodizime-Co2+ complexes had the agency in the public, commercial or not-for-profit sectors.
On the other hand, the inhibition zone of cefodizime and its metal ion complexes against the same bacteria was measuredusing the paper disc diffusion method on solid Luria Bertani 1. Parfitt K. Martindale. The complete drug reference, 33rd edn.
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Q1: Please check whether it is  (upsilon) or n (nu) symbol to be used for symmetric vibrations throughout the article.


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Gabriel Maislos, DPM, FACFAS BIOGRAPHICAL DATA: Date of Birth: June 24, 1974 Place of Birth: Detroit, MI HIGH SCHOOL EDUCATION Kinkaid High School Houston, TX 1988-1992 UNDERGRADUATE EDUCATION: Tufts University Medford, MA Bachelor of Science, Biology 1992-1996 MEDICAL EDUCATION: Dr. William M. Scholl College of Podiatric Chicago, IL Medicine

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