Leandro Tasso

Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery.

Spray-dried powders for lung delivery of sodium alendronate (SA) were prepared from hydroalcoholic solutions. Formulations display geometric particle size below to 12 μm and spherical shape associated to a hollow structure. The addition of leucine and ammonium bicarbonate leads to porous particles with rough surfaces. The tapped density ranges from 0.016 to 0.062 g/cm(3), decreasing with the increase of the leucine concentration. For all formulations, the calculated aerodynamic diameters are lower than 5 μm. The in vitro aerodynamic evaluation shows that all powders present a high emitted fraction of 100%, a fine particle fraction ranging from 34.4% to 62.0% and an alveolar fraction ranging from to 23.7% to 42.6%. An optimized sample was evaluated regarding sodium alendronate acute pulmonary toxicity and lung bioavailability. The bronchoalveolar lavage study shows that the intratracheal administration of sodium alendronate dry powder and sodium alendronate aqueous solution do not induce significant increases of lung toxicity indicators as compared with the positive control. Moreover, the intratracheal administration of sodium alendronate dry powder results in a 6.23 ± 0.83% bioavailability, a 3.5-fold increase as compared to oral bioavailability. Finally, these results suggest that sodium alendronate pulmonary delivery could be a new and promising administration route.

Evaluation of gatifloxacin penetration into skeletal muscle and lung by microdialysis in rats

This study aimed to investigate gatifloxacin distribution into skeletal muscle and lung interstitial fluid by microdialysis and to correlate free tissue and free plasma levels of the drug. Microdialysis recoveries were determined in vitro by extraction efficiency and retrodialysis at 80, 160 and 400 ng/ml resulting in 33.5+/-1.3%, 33.1+/-1.2%, 31.8+/-2.7% and 31.4+/-2.6%, 33.1+/-2.2%, 30.6+/-3.3%, respectively. In vivo recovery by retrodialysis in Wistar rats skeletal muscle and lung were 29.1+/-1.0% and 30.7+/-1.4%, respectively. The recovery was constant and independent on the method or media used. Gatifloxacin tissue penetration was investigated after intravenous dosing of 6 mg/kg to Wistar rats. Free skeletal muscle, lung and plasma profiles were virtually superimposable resulting in similar area under the curve (AUC(0-9)) of 3888+/-734 ng h/ml, 4138+/-1071 ng h/ml and 3805+/-577 ng h/ml, respectively (alpha=0.05). The tissue distribution factors were 1.02 and 1.08 for muscle and lung relative to plasma. In conclusion, free plasma levels are a good surrogate for gatifloxacin active levels at the infection site.

Validation of an efficient LC-microdialysis method for gemifloxacin quantitation in lung, kidney and liver of rats

A liquid chromatography method has been established for the reliable determination of unbound gemifloxacin concentrations in kidney, lung and liver microdialysates of rats. Microdialysis probes were inserted into tissues of rats, and then dialysates were collected at regular time intervals after intravenous administration of gemifloxacin (40 mg kg(-1)). A pilot study was performed to assess gemifloxacin penetration in lung, kidney and liver of rats. Gemifloxacin was separated on a C(18) column eluted using triethylamine solution (0.5%, v/v), adjusted to pH 3.0±0.1 with 85% phosphoric acid, methanol and acetonitrile (71:15:14, v/v/v) as mobile phase at a flow rate of 1.1 mL min(-1). The fluorescence detector was set at excitation and emission wavelengths of 344 nm and 399 nm, respectively. The limit of quantitation was found to be 50 ng mL(-1). Linearity was found to be over a concentration range of 50-2000 ng mL(-1). The intra-assay and inter-assay precision and accuracy values were determined from the analysis of six quality control samples. The results obtained at three concentration levels showed R.S.D. values lower than 6.06% and 4.10% for repeatability and intermediate precision, respectively. The accuracy (R.E.%) ranged from 90.0 to 106.5%. The chromatographic run time of each sample was performed in 9 min. Drug stability in microdialysates was shown at room temperature for 8h, after three freeze-thaw cycles, in freezer at -80 °C for 14 days, and in the autosampler after processing for 8h. The relative recoveries determined by extraction efficiency (EE) and retrodialysis (RD) in vitro employing a flow rate of 1.5 μL min(-1) were 29.24±3.67% and 23.67±3.31%, respectively. In vivo recoveries determined by RD in Wistar rats kidney, lung and liver were 27.69±2.09%, 23.12±3.79% and 17.38±0.68%, respectively. The method was successfully applied to investigate tissue penetration of unbound gemifloxacin into the kidney, lung and liver of rats.

Reversed phase liquid chromatography method with fluorescence detection of gemifloxacin in rat plasma and its application to the pharmacokinetic study.

A simple, accurate and precise high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin (GEM) in rat plasma using furosemide as internal standard (I.S.). Plasma samples were pretreated by direct deproteinization and all samples and standard solutions were chromatographed at 45°C using triethylamine solution (0.5%, v/v, pH 3.0±0.1), methanol and acetonitrile (63:30:7, v/v/v) as the mobile phase. Chromatographic resolution was achieved using a RP-C(18) column (Atlantis, Waters, 150 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL min(-1) and an injection volume of 30 μL. The analytes were measured by fluorescence detection with excitation and emission wavelengths of 344 nm and 399 nm, respectively. The retention times for GEM and I.S. were approximately 7.5 and 12.6 min, respectively. The lower limit of quantitation (LLOQ) was 20 ng mL(-1) and the calibration curves were linear over a concentration range of 20-5000 ng mL(-1). The intra- and inter-day precisions, expressed by relative standard deviation (R.S.D.) were lower than 6.24% and 4.49%, respectively. The accuracy ranged from 91.3% to 112% and from 98.8% to 106% for the lower and upper limit of quantitation of the calibration curve, respectively. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. No interferences from endogenous substances were found. The recovery of GEM and I.S. from plasma was greater than 90%. Drug stability in plasma was shown at room temperature for 4h, after three freeze-thaw cycles for 24h, in freezer at -80°C for 60 days, and in the autosampler after processing for 12h. The utility of the assay was confirmed by the successful analysis of plasma samples from GEM pharmacokinetics studies in the rats after intravenous administration.

Effect of hyperbaric oxygen therapy on tooth extraction sites in rats subjected to bisphosphonate therapy-histomorphometric and immunohistochemical analysis.

ObjectiveThis study aimed to investigate the effect of hyperbaric oxygen therapy (HBOT) on tooth extraction sites in rats treated with bisphosphonate.Materials and methodsRats were treated with zoledronic acid, subjected to tooth extractions and allocated into groups: (1) 7xa0days of HBOT, (2) 14xa0days of HBOT, (3) 7-day control, and (4) 14-day control. The site of tooth extractions was analyzed by histomorphometry and immunohistochemistry.ResultsOn macroscopic analysis, HBOT did not significantly affect bone exposure volume either at 7 or 14xa0days. On hematoxylin and eosin (H&E) analysis, the 14-day HBOT group showed less non-vital bone compared to both controls and 7-day HBOT group. HBOT significantly lowered expression of vascular endothelial growth factor (VEGF), receptor activator NF-kB ligand (RANKL), bone morphogenetic protein-2 (BMP-2), and osteoprotegerin (OPG) at 7xa0days, compared to control, whereas at 14xa0days, there was no significant difference for these variables.ConclusionHBOT can reduce the amounts of non-vital bone microscopically detected in tooth extraction sites of rats subjected to bisphosphonate therapy. The effect seems to occur in a dose-dependent mode. Further studies are required to clarify the mechanisms accounting for this effect.Clinical relevanceTreatment of bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been a challenging task, where the effectiveness of HBOT is controversial. This study reports important effects of HBOT on the maxillae of rats subjected to bisphosphonate treatment, making an important contribution to the knowledge about the applicability of HBOT in BRONJ.

HPLC Determination of Gemifloxacin in Different Tissues of Rats Under Normobaric and Hyperbaric Exposure

The aim of this paper is to develop and validate an HPLC method to investigate the tissue distribution of gemifloxacin (GEM) in rats following intravenous administration and compare the penetration of this drug in different tissues undergoing normobaric and hyperbaric oxygen (HBO) exposure. The chromatographic conditions consisted of a C18 column and mobile phase composition of triethylamine (0.5% v/v, pH 3.0), methanol and acetonitrile (65:28:7, v/v/v) at a flow rate of 1.0xa0mLxa0min−1. The calibration curves were linear from 0.2 to 30xa0μgxa0mL−1 for GEM, with correlation coefficient rxa0≥xa00.99. Retention times of GEM and furosemide (internal standard) were approximately 8 and 16xa0min, respectively. The intra-day variation was less than 5.56, 8.94 and 5.74% for lung, kidney and liver, respectively, and the inter-day variation was less than 1.58, 3.71 and 3.45% for lung, kidney and liver, respectively. The accuracy ranged from 89.0 to 112.9%. Recoveries of GEM and furosemide were up to 90%. Short-term, freeze–thaw, long-term and autosampler stability were demonstrated. The present study showed that the highest tissue concentration of GEM under normobaric exposure was obtained in the kidney (51.22xa0μgxa0g−1), followed by liver (32.78xa0μgxa0g−1) and lung (28.49xa0μgxa0g−1). The t1/2β obtained was 6.35, 2.83 and 2.41xa0h for lung, kidney and liver, respectively. Statistical difference (pxa0<xa00.05) was observed in rat tissues—lung, liver and kidney, when the rats were exposed to normobaric and HBO (2.5xa0h at 1.7 ATA).

Development and validation of a sensitive and selective LC—MS/MS method for the determination of an antimalarial drug candidate in rat plasma, and its application to a preclinical pharmacokinetic study

An accurate and reliable LC—MS/MS assay was firstly developed and validated for quantitative determination of a new antimalarial prototype drug, 3β-hydroxyurs-12-en-28-oic acid (LAFIS 01), in rat plasma. Dexamethasone was employed as internal standard. Simple protein precipitation by acetonitrile for the sample preparation was used. Effective separation was achieved with Phenomenex Luna C18 (50 × 2 mm, 5 μm) column. The mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.1% acetic acid, delivered by gradient elution. The column temperature was maintained at 40 °C. The LAFIS 01 was monitored by electrospray ionization interface, operating in the negative mode (ESI−) in multiple reactions monitoring (MRM), checking the transitions 455 > 455 for LAFIS 01 and 451 > 361 for the IS. Once LAFIS 01 demonstrated low fragmentation by collision-induced dissociation (CID) nonpresenting abundant high-intensity fragments to meet the desired concentration levels quantification, only pseudomolecul...

Galenic approaches in troubleshooting of glibenclamide tablet adhesion in compression machine punches

This study aimed to examine the adhesion of glibenclamide 5xa0mg tablets to the tools of compression machines. This problem is not commonly reported in the literature, since it is considered as tacit knowledge. The starting point was the implementation of three technical alternatives: changing the parameters of compression, evaluating the humidity of the powder blend and the manufacturer of the lubricant magnesium stearate. The adhesion was directly related to the characteristics of magnesium stearate from different manufacturers, and the feasibility of evaluating powder flow characteristics by different techniques that are not routinely followed in various pharmaceutical companies. In vitro dissolution tests showed that the magnesium stearate manufacturer can influence on the dissolution profile of glibenclamide tablets. This study presented various aspects of tablet adhesion to compression machine punches. Troubleshooting approaches can be, most of times, conducted based on previous experience, or an experimental research needs to be implemented in order to have confident results.

Efavirenz dissolution enhancement III: Colloid milling, pharmacokinetics and electronic tongue evaluation

Abstract Efavirenz (EFV), a non‐nucleoside reverse transcriptase inhibitor (NNRTI), is part of first‐line therapy for the treatment of human immunodeficiency virus type 1 infection (HIV‐1/AIDS). This drug shows relatively low oral absorption and bioavailability, as well as high intra‐ and inter‐subject variability. Several studies have shown that treatment failure and adverse effects are associated with low and high EFV plasma concentrations, respectively. Some studies suggest different EFV formulations to minimize inter‐patient variability and improve its solubility and dissolution; however, all of these formulations are complex, using for instance, cyclodextrins, dendrimers and polymeric nanoparticles, rendering them inviable industrially. The aim of this work was to prepare simple and low‐cost suspensions of EFV for improvement of solubility and dissolution rate by using colloid mill, spray or freeze‐drying, and characterization of the powders obtained. The results demonstrated an increase in the dissolution rate of EFV, using 0.2% of sodium lauryl sulfate (SLS) and 0.2% of hydroxypropylcellulose (HPC) or hydroxypropylmetilcellulose (HPMC) in both freeze and spray dried powders. The pharmacokinetic studies demonstrated improved pharmacokinetic parameters for the formulation containing SLS and HPC. The powders obtained, which present enhanced dissolution properties, can be incorporated in a solid dosage form for treatment of AIDS in paediatric patients with promising results.

Stability-indicating RP-HPLC method for determination of beclomethasone dipropionate in nanocapsule suspensions

A simple stability-indicating RP-HPLC/UV method was validated for determination of beclomethasone dipropionate (BD) in nanocapsule suspensions. Chromatographic conditions consisted of a RP C18column (250 mm x 4.60 mm, 5 µm, 110 A), using methanol and water (85:15 v/v) as mobile phase at 1.0 mL/min with UV detection at 254 nm. The calibration curve was found to be linear in the concentration range of 5.0-25.0 µg/mL with a correlation coefficient > 0.999. Precision was demonstrated by a relative standard deviation lower than 2.0%. Accuracy was assessed by the recovery test of BD from nanocapsules (98.03% to 100.35%). Specificity showed no interference from the components of nanocapsules or from the degradation products derived from acid, basic and photolytic conditions. In conclusion, the method is suitable to be applied to assay BD in bulk drug and in nanocapsules, and it can be employed to study stability and degradation kinetics.