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P 55 Preformulation Studies for Incorporating of a New Ruthenium (III) Complex with Enrofloxacin in Topical Fomulations
B. S. Velescu1, V. Anuta1, C. Dinu-Prvu1,2, V. Uivaroi1
University of Medicine and Pharmacy, Carol Davila, Faculty of Pharmacy, Bucharest, Romania 2 University Politehnica of Bucharest, Bucharest, Romania.
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Introduction Modulation and optimization of pharmaceutical formulation, regardless the route of administration, is based on the preformulation studies. for many metal complexes, their low solubility in water or other biocompatible vehicles is the critical factor in formulation. So far, different solubilization techniques were studied and used for medicinal substances poorly soluble in water. in this study we analyzed several ways to increase the solubility of a new complex with antitumor action, with general formula RuCl3(enrofloxacin)2(DM SO)0.5(H2O) (RuE) by means of different co-solvent systems. We studied the influence of co-solvent type, the influence of pH on solubility, the stability of test solutions obtained with appropriate co-solvent mixtures. the optimum solubilization models were used for designing biocompatible solvent systems, for allowing the complex to be incorporated in topical hydrogel formulations. The influence of the solubilization system on the release kinetics of RuE from the hydrogels was studied. Materials and Methods Materials: Dioxan, DMSO, PEG 400, all of all of analytical grade, were purchased from Merck. The HPLC grade ethanol was obtained from Sigma Aldrich. Methods: For determination of the RuE complex solubility, the shake-flask method was applied: 10 ml of each co-solvent system to an excess of drug substance, using 10 ml volumetric flasks (all determinations were performed in triplicate). The vials were vigorously mixed for 30 seconds, and maintained under sonication for 30 minutes, at 30 C, by means of a SonoSwiss thermostated ultrasonic bath. The samples

were placed for 24 hour on a Heidolph Vibramax 100 stirer. The samples were furthemore centrifuged and the supernatant was filtered throgh a celulose-esther membrane with the average pore size 0,45 m. Spectrophotometric methods were implemented for quantitative evaluations of drug dissolved, with calibration probes prepared in methanol and processed blank media as reference. Quantitative determinations were made at = 280 nm. Mechanism of co-solvent solubilisation is interpreted by the high capacity of these fluids to form multiple intermolecular hydrogen bonds between molecules of the same kind and with molecules of different nature. in practice, to approximate solubility of compounds in co-solvent is used the theory to take account of dielectric requirements. According to this theory, any solute has a maximum solubility in a given solvent mixture, with one or more specific dielectric constant. Solubility of RuE in different water:dioxane mixtures was evaluated and the dielectric constant for the optimum solubilizing mixture was established. Different biocompatible solvent systems (PEG 400, propylene glycol, ethanol, water) with the same dielectric constant were prepared and used for preparing hydrogels with 1% or 2% CMCNa. The release kinetics of RuE from the hydrogels was studied on a Hanson Microette system (Hanson Research Inc., USA) with 12 ml vertical diffusion cells, by using water:propylene glycol 1:1 v:v, receptor media

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Results and Discutions The results are presented in the next tables and figures: Table 1. Solubility of RuE in different water:dioxane mixtures H2O:dioxane (v:v) ap Solubility (mg/mL) 3 1 73.27 0.59 2 2 64.61 0.96 1 3 48.43 0.97 1 9 28.12 0.71 9 1 76.36 0.08 In fig 1 is represented the variation of solubility with the value of ap the solubility of the studied complex was evaluated. It was found that the solubility depends on pH, having the higher values in basic medium, due to the formation of sodium salt. Although the value of solubility in basic medium is suitable for pharmaceutical formulation process should still verify the maintaining of a therapeutic effect comparable to that of neutral complex (often forming a salt reduces / cancels therapeutic effect). Various co-solvent biocompatible systems used for solubilization of RuE are presented in tables 3-5 Table 3. Water: propylene glycol systems used for solubilization of RuE H2O (mL) 3.8 1.4 Propylene glycol (mL) 6.2 9.6 64.61 48.43 S (mg/mL) 0.89 0.91

Table 4 Water: PEG 400 system used for solubilization of RuE H2O (mL) 1.5 Fig 1 the variation of solubility with the value of ap Table.5 Water: propylene glycol: ethanol system used for The pH and experimental conditions influence on RuE solubility is presented in table 2 and fig 2. Table 2 pH influence on RuE solubility pH 1.46 5.00 7.08 8.12 10.40 11.92 S (mg/ml) 0.017 0.016 0.019 0.400 0.700 1.210 The results shown that the solubility is almost identical to that in water:dioxane of the same dielectric constant. A representative result following the release kinetics of RuE from 1% CMCNa hydrogel is presented in figure 3. solubilization of RuE H2O (mL) 5 Propylene glycol (mL) 10 Ethanol (mL) 10 51.76 S (mg/mL) 0.84 PEG 400 (mL) 23.5 48.43 S (mg/mL) 0.99

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Fig. 2. Experimental conditions influence on RuE solubility The influence of certain experimental parameters on
th

Fig. 3. Higuchi kinetic profile pethe diffusion of RuE in the receptor media

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The in vitro drug release rates range is highly dependent on viscosity, and increasing almost linearly with the concentration of cellulose derivatives. Conclusions Studies have shown a significant improvement in solubility of Ru(III) enrofloxacin complex (approximately 30 times using three different co-solvent systems) with co-solvent solubilization method, which allowed us to select a biocompatible system useful for further pharmaceutical formulation studies. The influence of certain experimental parameters on the solubility of the studied complex was evaluated. It was found that the solubility depends on pH, having the higher values in basic medium, due to the formation of sodium salt. Although the value of solubility in basic medium is suitable for pharmaceutical formulation process should still verify the maintaining of a therapeutic effect comparable to that of neutral complex (often forming a salt reduces / cancels therapeutic effect). The solubility value is influenced by sonication, sonication temperature, type and time of stirring. The complex has proved to be stable in the biocompatible systems proposed, providing real benefits in future formulation processes (regardless the route of administration chosen). Acknowledgements This work was supported by the Grant Development of systems with improved solubility for new ruthenium (lll) complexes with quinolone antibiotics and testing their anticancer activity of the Romanian Ministry of Education and Research, Capacities Programme, Module III Consulted references: 1. Dal Pozzo A., Liggeri E., Delucca C., Calabrese G. Prediction of skin permeation of highly lipophilic compounds; in vitro model with a modified receptor phase, Int. J. Pharm., 1991, 70 (3), pag. 219-223 2. Shah V.P., Elkins J.S., Williams R.L. Evaluation of the test system used for in vitro release of drugs for topical dermatological drug products, Pharm. Dev. Technol., 1999, 4 (3), pag. 377-385 3. Singh S.K., Durrani M.J., Reddy I.K., Khan M.A. Effect of permeation enhancers on the release of ketoprofen through transdermal drug delivery systems, Pharmazie, 1996, 51 (10), pag. 741-744 4. Yener G., Gnll ., ner M., Araman A. Effect of vehicles and penetration enhancers on the in vitro percutaneous absorption of celecoxib through human skin, Pharmazie, 2003, 58 (5), pag. 330-333

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