Ruben H. Manzo

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.

Solubility behavior and biopharmaceutical classification of novel high-solubility ciprofloxacin and norfloxacin pharmaceutical derivatives

The hydrochlorides of the 1:3 aluminum:norfloxacin and aluminum:ciprofloxacin complexes were characterized according to the Biopharmaceutics Classification System (BCS) premises in comparison with their parent compounds. The pH-solubility profiles of the complexes were experimentally determined at 25 and 37 degrees C in the range of pH 1-8 and compared to that of uncomplexed norfloxacin and ciprofloxacin. Both complexes are clearly more soluble than the antibiotics themselves, even at the lowest solubility pHs. The increase in solubility was ascribed to the species controlling solubility, which were analyzed in the solid phases at equilibrium at selected pHs. Additionally, permeability was set as low, based on data reported in the scientific literature regarding oral bioavailability, intestinal and cell cultures permeabilities and also considering the influence of stoichiometric amounts of aluminum. The complexes fulfill the BCS criterion to be classified as class 3 compounds (high solubility/low permeability). Instead, the active pharmaceutical ingredients (APIs) currently used in solid dosage forms, norfloxacin and ciprofloxacin hydrochloride, proved to be BCS class 4 (low solubility/low permeability). The solubility improvement turns the complexes as potential biowaiver candidates from the scientific point of view and may be a good way for developing more dose-efficient formulations. An immediate release tablet showing very rapid dissolution was obtained. Its dissolution profile was compared to that of the commercial ciprofloxacin hydrochloride tablets allowing to dissolution of the complete dose at a critical pH such as 6.8.

Double-layered mucoadhesive tablets containing nystatin

The objective of this work was to design a mucoadhesive tablet with a potential use in the treatment of oral candidosis. A 2-layered tablet containing nystain was formulated. Lactose CD (direct compression), carbomer (CB), and hydroxypropylmethylcellulose (HPMC) were used as excipients. Tablets were obtained through direct compression. Properties such as in vitro mucoadhesion, water uptake, front movements, and drug release were evaluated. The immediate release layer was made of lactose CD (100 mg) and nystatin (30 mg). The CB:HPMC 9∶1 mixture showed the best mucoadhesion properties and was selected as excipient for the mucoadhesive polymeric layer (200 mg). The incorporation of nystatin (33.3 mg) in this layer affected the water uptake, which, in turn, modified the erosion front behavior. Nystatin showed a first-order release. The polymeric layer presented an anomalous kinetic (n=0.82) when this layer layer was individually evaluated. The mucoadhesive tablet formulated in this work releases nystatin quickly from the lactose layer and then in a sustained way, during approximately 6 hours. from the polymeric layer. The mixture CB:HPMC 9∶1 showed good in vitro mucoadhesion. A swelling-diffusion process modulates the release of nystatin from this layer. A non-Fickian (anomalous) kinetic was observed.

Biowaiver Monographs for Immediate Release Solid Oral Dosage Forms: Ciprofloxacin Hydrochloride

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of new multisource and reformulated immediate release (IR) solid oral dosage forms containing ciprofloxacin hydrochloride as the only active pharmaceutical ingredient (API) are reviewed. Ciprofloxacin hydrochlorides solubility and permeability, its therapeutic use and index, pharmacokinetics, excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. Solubility and BA data indicate that ciprofloxacin hydrochloride is a BCS Class IV drug. Therefore, a biowaiver based approval of ciprofloxacin hydrochloride containing IR solid oral dosage forms cannot be recommended for either new multisource drug products or for major scale-up and postapproval changes (variations) to existing drug products.

Equilibrium properties and mechanism of kinetic release of metoclopramide from carbomer hydrogels

Equilibrium properties and kinetics of metoclopramide release of carbomer-metoclopramide (C-M) hydrogels are reported. A set of (C-M)(X) (x=moles percent of M=50, 75, 100) that covers a pH range between 6.49 and 8.40 was used. Hydrogels exhibited a high negative electrokinetic potential (zeta). Concentrations of ion pair [R-COO(-)MH(+)] and free species [M] and [MH(+)] were determined by the selective extraction of M with 1,2-dichloroethane (DCE) together with pH measurements. The system (C-M) is characterized by a high proportion of drug present in the form of ion pairs and a negative zeta potential that attracts MH(+) and H(+) and repeals OH(-), providing a microenvironment of higher acidity than the bulk medium. Delivery rates of M were measured in a Franz type bi-compartmental device using water and NaCl 0.9% solution as receptor media. (C-M) hydrogels behave as a reservoir that releases the drug at a slow rate to water; the rate increases 14 times as water is replaced by NaCl solution. The pH effect on delivery rate suggests that, under the main conditions assayed, the rate of dissociation of R-COO(-)MH(+) together with the low change of pH in the polyelectrolyte environment are the factors that control releasing rates.

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.

Drugs solubilization in ascorbyl–decanoate micellar solutions

Abstract Alkanoyl-6- O -ascorbic acid esters are obtained upon esterification of ascorbic acids primary hydroxyl groups with fatty acids. Being more hydrophobic than vitamin C, they dissolve in lipophilic media, and behave as surfactants in water, where they produce micellar aggregates or spreading monolayers, depending on the side chain length. These amphiphiles keep the same antioxidant activity of vitamin C, and can be used for solubilization and protection of hydrophobic species from oxidative degradation. In this paper we report a study on the micellar aggregates formed by decanoyl-6- O -ascorbic acid in water, through surface tension, conductivity, and solubility experiments, and on its coagels produced at low temperatures, by means of differential scanning calorimetry and scanning electron microscopy. Solubilization of some hydrophobic molecules (phenacetin, danthron, and griseofulvin) in ascorbyl–decanoate (ASC10) micelles confirms that the supramolecular assemblies significantly enhance the solubility of these drugs in the liquid phase.

Characterization of the solubility and solid‐state properties of saccharin salts of fluoroquinolones

Saccharinates salts of the fluoroquinolone antibiotics norfloxacin, ciprofloxacin, ofloxacin, and enrofloxacin were obtained as pure crystalline anhydrous solids with sweet taste. The products were characterized by one- ((13)C) and two-dimensional ((1)H-(13)C) dimensions solid state Nuclear Magnetic Resonance and infrared spectroscopy showing ionic interactions between the saccharine amide and the fluoroquinolone piperazine. Several intermolecular bindings were also identified. Thermal behavior and powder X-ray diffraction provided complementary evidences of salt formation. The series of products showed improved properties with respect to water solubility. A solubility model was developed. These salts would be a good way forward to developing more suitable formulations of these APIs.

Evaluation of the surfactant properties of ascorbyl palmitate sodium salt

In this paper we report on the physicochemical surface properties of ascorbyl palmitate (Asc16) and of its sodium salt (Asc16Na) with a view to their use as surfactants. Asc16Na was synthesized from ascorbyl palmitate by neutralizing the -OH groups in position 3 of the ascorbyl ring. The acid-base properties, thermal analysis and stability of Asc16Na monomers were determined. Self-assembling parameters of micellar aggregates in aqueous dispersions through critical micellar concentration (CMC) and critical micellar temperature (CMT) were measured. Asc16Na micellar dispersions efficiently solubilize poorly soluble drugs such as phenacetin and griseofulvin, and enhance their apparent solubility in aqueous environments. Stability tests showed that Asc16Na is more unstable than ascorbyl palmitate. Ascorbyl palmitate and its sodium salt are insoluble at room temperature in water, but their solubilities strongly depend on temperature, and largely increase above the CMT. Although Asc16Na is insoluble at room temperature, it is more soluble than Asc16, and its CMT significantly lowers in the undissociated acidic form. The apparent solubilities of phenacetin and griseofulvin are increased in Asc16Na aqueous solutions. The Asc16Na potential use as surfactant is restricted by its low stability in water, therefore the addition of some antioxidant species is necessary.