Fabiana L. Alovero

Engineering the Specificity of Antibacterial Fluoroquinolones: Benzenesulfonamide Modifications at C-7 of Ciprofloxacin Change Its Primary Target in Streptococcus pneumoniae from Topoisomerase IV to Gyrase

ABSTRACT We have examined the antipneumococcal mechanisms of a series of novel fluoroquinolones that are identical to ciprofloxacin except for the addition of a benzenesulfonylamido group to the C-7 piperazinyl ring. A number of these derivatives displayed enhanced activity againstStreptococcus pneumoniae strain 7785, including compound NSFQ-105, bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at C-7, which exhibited an MIC of 0.06 to 0.125 μg/ml compared with a ciprofloxacin MIC of 1 μg/ml. Several complementary approaches established that unlike the case for ciprofloxacin (which targets topoisomerase IV), the increased potency of NSFQ-105 was associated with a target preference for gyrase: (i) parC mutants of strain 7785 that were resistant to ciprofloxacin remained susceptible to NSFQ-105, whereas by contrast, mutants bearing a quinolone resistance mutation in gyrA were four- to eightfold more resistant to NSFQ-105 (MIC of 0.5 μg/ml) but susceptible to ciprofloxacin; (ii) NSFQ-105 selected first-step gyrAmutants (MICs of 0.5 μg/ml) encoding Ser-81-to-Phe or -Tyr mutations, whereas ciprofloxacin selects parC mutants; and (iii) NSFQ-105 was at least eightfold more effective than ciprofloxacin at inhibiting DNA supercoiling by S. pneumoniae gyrase in vitro but was fourfold less active against topoisomerase IV. These data show unequivocally that the C-7 substituent determines not only the potency but also the target preference of fluoroquinolones. The importance of the C-7 substituent in drug-enzyme contacts demonstrated here supports one key postulate of the Shen model of quinolone action.

Release kinetics and up-take studies of model fluoroquinolones from carbomer hydrogels

Hydrogels of carbomer (C) loaded with model slightly soluble fluoroquinolone antimicrobials (AMFQ), norfloxacin (I) and ciprofloxacin (II) were prepared to evaluate their physical and delivery properties. Thus, dispersions of 0.25% of C loaded with 0.2-0.5 mol equivalents of AMFQ and 0.2-0.5 mol equivalents of NaOH yielded pseudoplastic hydrogels with a high negative electrokinetic potential and good physical stability. Concentration of AMFQ in the hydrogels was, respectively, 7.2 and 34 times higher than I and II aqueous solubility, indicating a high increase in aqueous compatibility. Release of AMFQ in bicompartimetal Franz type cell occurred by zero order kinetics. Delivery rate constant (k(0)) was five to six times higher as water was replaced by NaCl solution as receptor medium. Release in agar dishes revealed that, even under high dilution, delivery remains modulated. Intestinal absorption flux coefficient in everted rat intestine (k(U)) were measured with reference solutions (RS) of free AMFQ (k(U)(RS) II>k(U)(RS) I) and with hydrogels (H), in which the pattern was reversed since k(U)(H) I>k(U)(H) II. As expected k(U)(H) II was 0.55 times lower than k(U)(RS) II. However, k(U)(H) I was 1.37 times higher than its reference, which cannot be explained from the analysis of k(0) and k(U)(RS) alone. Hydrogels C-AMFQ behave as a reservoir of AMFQ able to deliver it at a constant rate and would be useful to design topical and or systemic dosage forms.

Small-Colony Mutants of Staphylococcus aureus Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones

ABSTRACT Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of Staphylococcus aureus RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the gyrA, gyrB, grlA, or grlB gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of S. aureus that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had gyrA(S84L) alterations; those obtained with sparfloxacin carried a gyrA(D83A) mutation or a novel gyrB deletion (ΔRKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of S. aureus GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type grlB-grlA gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant gyrA or gyrB alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.

Formulation of a neutral solution of ciprofloxacin upon complexation with aluminum.

Clear solutions of 0.5 and 1.0% ciprofloxacin (CF) of pH 7.2 were prepared by the addition of aluminum chloride hexahydrate (AlCl3.6H2O) in the molar proportion CF:AlCl3.6H2O (3:1). Minimum inhibitory concentrations (MIC) of these solutions were the same as an equimolar solution of CF.HCl. Solutions exhibited good physical, chemical and microbiological stability and satisfactorily overcame an ocular irritation test on rabbits.

A new class of fluoroquinolones : benzenesulfonamidefluoroquinolones (BSFQs), antibacterial activity and SAR studies

Abstract N-Sulfanilylnorfloxacin was chosen as lead compound to develop a new series of benzenesulfonamidefluoroquinolones (BSFQs). Eight new compounds with different p -substituents on the phenyl group were prepared and their in vitro antibacterial activities were evaluated. The presence of the benzenesulfonylamido groups (BS) bound to the piperazinyl ring shifted the activity of classic antimicrobial fluoroquinolones from being more active against Gram negative to Gram positive strains. Analogs with p -NHCH 3 , p -H or p -NO 2 were more active than norfloxacin. QSAR studies through Hansch analysis showed a linear correlation of the activity with electronic distribution (empirical descriptors) along with Sterimol parameters. Small electron–donor groups with hydrophilic properties increase the in vitro activity against Gram positive bacteria.

In vitro pharmacodynamic properties of a fluoroquinolone pharmaceutical derivative: hydrochloride of ciprofloxacin/aluminium complex

Some in vitro pharmacodynamic properties of a new aqueous soluble ciprofloxacin (CIPX) derivative, the hydrochloride of its aluminum complex: (HCl.CIPX)(3)Al, are reported. Although (HCl.CIPX)(3)Al had the same MIC as CIPX, the minimum bactericidal activity against Escherichia coli was 2-fold higher than that of CIPX and the rate of killing was slightly delayed compared with time-kill curves obtained with CIPX. (HCl.CIPX)(3)Al showed a longer post-antibiotic effect (PAE). As pharmacodynamic properties of CIPX are not drastically affected by being complexed with aluminium, the increased aqueous compatibility of the complex remains as the main formulation factor for liquid dosage forms.

Azithromycin loaded on hydrogels of carbomer: Chemical stability and delivery properties

Hydrogels of carbomer (C) and azithromycin (AZI) were prepared by neutralizing with AZI 50% of the carboxylic groups of 0.25% C(974) and C(934) dispersions. The hydrogels exhibit pH close to 8 and are physically stable. Titration with NaCl revealed a high degree of counterion condensation C-AZI. The release of AZI in a Franz cell was almost negligible when the receptor compartment was filled with water but was increased about 20 times as water is replaced by NaCl solution. Two analytical methods were used to evaluate the effect of the counterionic condensation on the chemical stability of AZI, a microbial assay and an HPLC method. Degradation of AZI in buffered aqueous solution was used as reference. The stability of AZI was significantly improved in the hydrogels retaining more than 75% of the initial concentration along a period of 18-20 months evaluated and the self life (t(90)) of the drug was increased 27 and 20 times over the reference. The improvement of AZI stability could be attributed to the high degree of counterion condensation in which drug molecules remain associated to the macromolecular phase having a high negative electrokinetic potential and higher viscosity and lower kinetic energy than those in the fluid phase.

A novel gel based on an ionic complex from a dendronized polymer and ciprofloxacin: Evaluation of its use for controlled topical drug release

The development and characterization of a novel, gel-type material based on a dendronized polymer (DP) loaded with ciprofloxacin (CIP), and the evaluation of its possible use for controlled drug release, are presented in this work. DP showed biocompatible and non-toxic behaviors in cultured cells, both of which are considered optimal properties for the design of a final material for biomedical applications. These results were encouraging for the use of the polymer loaded with CIP (as a drug model), under gel form, in the development of a new controlled-release system to be evaluated for topical administration. First, DP-CIP ionic complexes were obtained by an acid-base reaction using the high density of carboxylic acid groups of the DP and the amine groups of the CIP. The complexes obtained in the solid state were broadly characterized using FTIR spectroscopy, XRP diffraction, DSC-TG analysis and optical microscopy techniques. Gels based on the DP-CIP complexes were easily prepared and presented excellent mechanical behaviors. In addition, optimal properties for application on mucosal membranes and skin were achieved due to their high biocompatibility and acute skin non-irritation. Slow and sustained release of CIP toward simulated physiological fluids was observed in the assays (in vitro), attributed to ion exchange phenomenon and to the drug reservoir effect. An in vitro bacterial growth inhibition assay showed significant CIP activity, corresponding to 38 and 58% of that exhibited by a CIP hydrochloride solution at similar CIP concentrations, against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. However, CIP delivery was appropriate, both in terms of magnitude and velocity to allow for a bactericidal effect. In conclusion, the final product showed promising behavior, which could be exploited for the treatment of topical and mucosal opportunistic infections in human or veterinary applications.

Bioadhesive and biocompatible films as wound dressing materials based on a novel dendronized chitosan loaded with ciprofloxacin

The bioadhesive polymeric films as topical drug delivery systems are interesting alternatives to improve the pharmacotherapy and patient compliances. New derivate biomaterials based on weisocyanate- dendronized PVP- crosslinked chitosan and loaded with ciprofloxacin (CIP), as model drug, were used to prepare bioadhesive films. Relevant in vitro/in vivo attributes to define main physicochemical and biopharmaceutical characteristics for topical wound-healing applications were evaluated. A high proportion of CIP, uniformly dispersed along throughout the film, was loaded. An extended release of CIP and different behaviors of release profiles, depending on the presence of dendron, were observed. The films loaded with CIP were effective in inhibiting the growth of both Gram positive and Gram negative bacteria. In addition, biocompatibility and bioadhesion into conjuntival-sacs of the rabbits suggests that these films have good properties to be applied over skin wounds for topical applications, allowing a reduction of the frequency of administration and improving the residence time of the films.

Polyelectrolyte-drug ionic complexes as nanostructured drug carriers to design solid and liquid oral delivery systems

Aqueous dispersions of acid or basic polyelectrolytes (PE) loaded with ionizable drugs (D) of opposite charge are characterized by a high degree of counterionic condensation. In solid state, these PE-D complexes are stable amorphous materials that, in many cases, quickly swell reverting to the original dispersion in the presence of aqueous fluids. Such systems exhibit a set of unique properties. Among them, the reversibility of the PE-D association modulates the release of D when dispersions are in contact with biological fluids. Besides, some of them exhibit interesting bioadhesive properties. By compaction of solid complexes swellable PE-D matrices (SPDM) are obtained. Kinetics and rate of D delivery from SPDM can be modulated by changes in their composition to satisfy the design of extended delivery systems. Among the factors that control the delivery of D, the rate of swelling, complex dissociation of ionic pairs and further diffusion of D, as well as hydrogel layer erosion, are mainly involved. PE-D complexes formulated as matrices, multiparticulate or dispersed systems play an important role in the design of drug delivery systems.