Development and validation of a reversed-phase high-performance liquid chromatographic method with solid-phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies.

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed-phase liquid chromatography (RP-HPLC) method coupled with UV detection (RP-HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP-HPLC with UV detection required a sample preparation step by solid-phase extraction. The method was validated in the concentration range of 8.05 × 10-7 to 3.22 × 10-5 m for HCT. Chromatographic parameters, namely carry-over, lower limit of quantification (1.4491 × 10-7 m), limit of detection (3.8325 × 10-8 m), selectivity, inter- and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.

Permeability Profiles and Intestinal Toxicity Assessment of Hydrochlorothiazide and Its Inclusion Complex with ß-Cyclodextrin Loaded into Chitosan Nanoparticles.

Here, a novel drug delivery system was developed for the hydrochlorothiazide (HCT):ß-cyclodextrin (ßCD) inclusion complex loaded into chitosan (CS) nanoparticles (NPs) [CS/HCT:ßCD NPs]. It was found, for the first time, that exposure of the intestinal mucosa to free HCT resulted in an increased and abnormal intestinal permeability associated with several injuries to the intestinal epithelium. Nevertheless, the HCT delivery system obtained ameliorated the damage of the intestinal epithelium induced by HCT. Furthermore, we found that the corresponding permeability profiles for both the free HCT and the CS/HCT:ßCD NPs were exponential and lineal, respectively. We propose that the increased intestinal uptake and severe tissue injury of HCT to the intestinal epithelium could be directly related to possible effects of this drug on the ionoregulatory Na+/K+-ATPase channel. Thus, it is postulated that the CS/HCT:ßCD NPs may increase the gastrointestinal retention of the HCT, which would provide increased adherence to the mucus barrier that lines the intestinal epithelium; consequently, this would act as a slow HCT release delivery system and maintain lower drug levels of luminal gut in comparison with the administration of free HCT, leading to less severe local injury.

Biowaiver monographs for immediate release solid oral dosage forms: furosemide.

Literature and new experimental data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing furosemide are reviewed. The available data on solubility, oral absorption, and permeability are sufficiently conclusive to classify furosemide into Class IV of the Biopharmaceutics Classification System (BCS). Furosemide's therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems are also taken into consideration. In view of the data available, it is concluded that the biowaiver procedure cannot be justified for either the registration of new multisource drug products or major postapproval changes (variations) to existing drug products.

Biowaiver monographs for immediate release solid oral dosage forms: acetazolamide.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing acetazolamide are reviewed. Acetazolamide's solubility and permeability characteristics according to the Biopharmaceutics Classification System (BCS), as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems are taken into consideration. The available data on solubility, on oral absorption and permeability are not sufficiently conclusive to classify acetazolamide with certainty. Taking a conservative approach, no biowaiver is considered justified for the registration of new multisource drug products. However, SUPAC level 1 and level 2 postapproval changes and most EU Type I variations can be approved waiving in vivo BE studies.

Effects of hydroxypropyl-beta-cyclodextrin on the chemical stability of a naphthoquinone in aqueous solutions.

2-Hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (1), an antibacterial agent, was shown to form inclusion complexes with HP-beta-CD in aqueous solution. In the present work the kinetics of 1 degradation in aqueous buffer solution was investigated as a function of pH (2.34-3.95), HP-beta-CD concentration (0%-28% (w/v)) and temperature (60-90 degrees C). A second-order derivative spectroscopic methodology was developed for the kinetic investigations. The degradation showed to follow pseudo-first-order kinetics. Also, an specific acid catalysis was found and the introduction of up to 28% (w/v) HP-beta-CD to the reaction medium did not change this kinetic behaviour. The obtained results indicated that HP-beta-CD stabilises 1 against degradation in aqueous solutions.

Effect of hydroxypropyl-beta-cyclodextrin on the solubility of an antibacterial isoxazolyl-naphthoquinone.

The complexation of 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I) with a highly soluble cyclodextrin, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was studied in aqueous media by solubility methods. I is an antibacterial and trypanocidal agent that is undergoing preclinical testing. Unfortunately, I exhibits low water solubility, and it is therefore difficult to prepare the solutions for biological tests. I inclusion took place with 1:1 stoichiometry. The stability constants of the I complexes calculated from the slope and the intercept of the phase solubility diagrams are larger in the less ionized form, whereas greater overall solubility is obtained in basic media.

Isoxazoles. 8. Preformulation studies of an isoxazolylnaphthoquinone derivative.

The degradation kinetics of 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone 4-imine (1) in a 25% solution of ethyl alcohol in water has been studied. The rate constants were observed to follow pseudo-first-order kinetics in all cases. The pH-rate profile indicated a negligible decomposition at pH values higher than its pKa2 value [5.4 +/- 0.14 (*n = 6)]. Un-ionized 1 was subject to specific acid catalysis. The ionic strength did not affect the stability of the drug. These data can be used to develop a stable oral liquid dosage form of the drug.

Isoxazoles. VII: Hydrolysis of 4-methyl-5-isoxazolylnaphthoquinone derivatives in aqueous solutions.

The kinetics for the degradation of 2-(4-methyl-5-isoxazolylamine)-N-(4-methyl-5-isoxazolyl)-1,4 -naphthoquinone-4- imine (1) in solution were investigated at 70 degrees C and at a constant ionic strength of 0.5 over a pH range of 1.75 to 12.85. The degradation rates were determined by absorption and second-derivative UV spectrometry. Two degradation products were identified in acidic and neutral pHs; they are 4-N-(4-methyl-5-isoxazolyl)-1,2-naphthoquinone (2) and 2-methyl-cyanoacetamide (5), respectively. In alkaline pH, two degradation products, 2-hydroxy-N-(4-methyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (3) and 5-amino-4-methylisoxazole (4), were isolated. The pathway for degradation of 1 in acidic and neutral pH followed consecutive first-order kinetics since 2 undergoes hydrolysis giving 2-hydroxy-1,4-napthoquinone (6) and 2-methylcyanoacetamide (5). No appreciable buffer effect on the degradation of 1 and 2 was observed for any of the buffer species in this study. The pH-rate profiles exhibited specific acid and specific basic catalysis for 1 and specific acid catalysis for 2. The maximum stability for 1 and 2 occurred in the neutral pH region.

Isoxazoles. VI: Aspects of the chemical stability of a new naphthoquinone-amine in acidic aqueous solution.

Some aspects of the chemical degradation of N-(3,4-dimethyl-5-isoxazolyl)-4-amino-1,2-naphthoquinone were investigated as a function of pH and temperature. In acid and neutral pH, four main degradation products were identified: 2-hydroxy-1,4-naphthoquinone, 2-butanone, ammonia, and hydroxylamine. No significant buffer effects were observed for the buffer species used in this study. The pH-rate profile exhibited a specific acid catalysis which is important at pH values less than 3.5, and an inflection point at pH 1.10 corresponding to a pKa value. From Arrhenius plots, the activation energy was found to be 17.8 +/- 0.3 kcal/mol.

Isoxazoles V: chemical stability of diisoxazolylnaphthoquinone in aqueous solution.

The hydrolytic degradation of 2-(3,4-dimethyl-5-isoxazolylamine)-N-(3,4-dimethyl-5-isoxazolyl )-1,4- naphthoquinone-4-imine (1) was investigated over a wide range of pH values and at different temperatures. The degradation rates were determined by reversed-phase HPLC and were observed to follow pseudo-first-order kinetics with respect to the concentration of 1. The pH-rate profile was linear with slopes -1 and +1 in acid and alkaline pH, respectively, becoming pH independent in the region of maximum stability from pH 4.5 to 10.0. Neither primary salt effects nor buffer catalysis was observed due to the buffer species employed.