Binary Inclusion Complexes of Enrofloxacin in 2-Hydroxypropyl-β-Cyclodextrin: Preparation and Characterization
V.F. Zadra1; C.C. Custodio2; M.A.S. Carvalho3; L.N. Rodrigues-Carpentieri4
Cyclodextrins (CDs) are cyclic torus-shaped molecules with a hydrophilic outer surface and a lipophilic central cavity, which can accommodate a variety of lipophilic drugs (Loftsson & Duchêne 2007). Formation of complexes usually results in favorable changes in many physicochemical properties, such as solubility, dissolution rate, stability and bioavailability. Enrofloxacin (HERX) is a typical second-generation quinolone antimicrobial drug with a broad spectrum of activity against a wide range of gram-negative and gram-positive bacteria, including those resistant to ?-lactam (editor's note: the symbol for ?-lactam wasn't readable in the original) antibiotics and sulfonamides. It is used in veterinary medicine in cattle, pigs, poultry, dog and cat (Martinez et al. 2006). HERX is a poorly water-soluble base-type drug and it is usually available as tablet, subcutaneous injection, oral solution and topical (ear drops) (Lizondo et al. 1997). The purpose of this study was to investigate the effects of 2-HPβCD on solubility of enrofloxacin.
Materials and Methods
In order to evaluate the influence of HPβCD on the HERX solubility, the interactions in aqueous solution were studied by phase-solubility analysis. The influence of HPβCD on the enrofloxacin physicochemical properties was evaluated by preparing solid systems with equimolar quantities of HPβCD and HERX by coevaporation. Differential scanning calorimetry (DSC) and powder X-ray diffractometry (XRD) analysis were used to characterize the binary system, comparing them with the physical mixtures prepared in the same molar ratio.
The solubility of HERX increased linearly as a function of HPβCD concentration, giving AL-type phase solubility diagram and indicating the formation of a complex where the complexing agent (HPβCD) is present in first order degree with respect the drug. As the slope of these solubility diagram were less then 1 it was possible to asses a 1:1 stoichiometry and calculate the apparent stability constants (KC) of the binary complexes using the equation of Higuchi & Connors (1965). The estimated values of S0, slopes of phase-solubility diagrams and KC were 8.60 mM, 0.33768 and 59.3 M-1, respectively. Loftsson, Hreinsdóttir and Másson (2007) proposed another method to evaluate the solubilizing effects of CDs, the determination of complexation efficiency (CE). Based in this value, the drug: CD ratio can be determined. Complexation efficiency (CE) value of 0.0345 and molar ratio of 1:3 were found what means that about one out every 3 CD molecules in solution are forming a water-soluble complex with HERX. DSC was used to characterize HERX complexes in the solid state and to obtain further supporting evidence on complex formation. The thermal curve of pure HERX was typical of a crystalline anhydrous substance with a sharp endothermic peak at 227.44°C corresponding to the melting point of the drug. A broader endothermal effect, associated with water loss, was instead recorded for HPβCD. In physical mixtures (PM) the characteristic thermal profile of HERX was shifted to lower temperatures at around 188.6°C. In the binary system, the fusion endotherm of HERX was totally absent, indicating the formation of amorphous entities and/or inclusion complexes. The XRD pattern of HERX revealed high-intensity reflections corresponding to the diffraction peaks 7.09°; 9.83° and 14.80°, which were indicative of its crystalline character, while a hollow pattern was recorded for HPβCD, demonstrating their amorphous state. Some diffraction peaks attributable to HERZ crystals are still detectable in physical mixtures, whereas they are absent in the binary systems, indicating the formation of a true inclusion complex.
Studies by several different research groups have shown that cyclodextrins form both inclusion and non-inclusion complexes and that several complexes can coexist in aqueous solution. According to Rama and cols (2005), only complex presenting Kc between 100 e 1000 M-1 may have industrial application. Stability constants lower than 100 M-1 characterizes instable drug-CD systems and complexes with Kc higher than 1000 M-1 could prejudice drug absorption. Kc is determinate from the slope and the intrinsic solubility (S0) of the drug in the aqueous complexation medium. Theoretically the intercept (Sint) of the phase solubility diagram should be identical to S0. For poorly soluble drugs with aqueous solubility < 0.1 mM, S0 is in general much larger the Sint. This can lead to erroneous KC values. A more accurate method for determination of solubilizing efficiency of cyclodextrins is to determine their complexation efficiency (CE) i.e., the concentration ratio between cyclodextrin in a complex and free cyclodextrin. CE is calculated from the slope of the phase-solubility diagrams and is independent of both S0 and Sint (Loftsson et al. 2007). Thermal methods are widely employed in the assessment of the solid phase aiming essentially to determine significant differences between traces obtained from scanning the untreated mixture (PM) and those from the interacted mixture (generally indicated as an "inclusion complexes) (Ribeiro et al 2003). In this study, DSC was used to characterize HERX complexes in the solid state and to obtain further supporting evidence on complex formation. Powder XRD is a useful method for the detection of CD complexation in powder or microcrystalline states. The diffraction pattern of the complex should be clearly distinct from those of the superposition of each component if a true inclusion complex exists (Veiga et al 1996). Complete drug amorphousness was detected in the diffraction patterns of the HERX/HPβCD binary system.
The results obtained indicated the formation of HERX/HPβCD binary complex in solution and in the solid state. HERX solubility increased in 32.5% in binary system. Based on these results, we believe that the interaction of HERX with HP,CD, through the formation of inclusion complexes, can lead to important modifications in the physicochemical and biological properties of the guest molecule, which might eventually have relevant pharmaceutical potential.
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