Bruna Gaelzer Silva Torres

Comparison of Fluconazole Renal Penetration Levels in Healthy and Candida albicans-Infected Wistar Rats

ABSTRACT The aims of this study were to evaluate free levels of fluconazole (FCZ) in the kidneys of healthy and Candida albicans-infected Wistar rats using microdialysis and to establish the relationship between free renal and total plasma levels under both conditions. Microdialysis recovery rates were determined in vitro by dialysis, and retrodialysis recovery rates were determined in vivo by retrodialysis. The recovery rate was around 50%, independent of the method, drug concentration, or condition (in vitro or in vivo) used. FCZ kidney penetration in healthy and infected rats was investigated after the administration of 10 mg/kg of body weight intravenously (i.v.) or 50 mg/kg orally (n = 6/group) and blood and microdialysate sample harvesting at predetermined time points up to 24 and 18 h, respectively. There were no statistical differences between the area under the free concentration-time curve (AUC0–∞) values in plasma and in tissue for either healthy or infected groups for the same dose regimen investigated. The antifungal tissue penetrations were similar for both doses and under all conditions investigated (ranging from 0.77 to 0.84). The unbound fraction of FCZ was concentration independent (86.0% ± 2.0%), allowing the prediction of free renal levels using pharmacokinetic parameters obtained from total plasma fitting. The results showed that free renal and free plasma levels are similar in healthy and systemically C. albicans-infected rats. Therefore, free plasma levels are a good surrogate to estimate free FCZ renal concentrations in systemic candidiasis and can be used to optimize dosing regimens for this drug.

Validation of a sensitive HPLC/fluorescence method for assessment of ciprofloxacin levels in plasma and prostate microdialysate samples from rats

Chronic bacterial prostatitis treatment consists of broad-spectrum antibiotic therapy for long periods of time. Drug penetration into the prostate makes the treatment a challenged. Ciprofloxacin is one of the most prescribed drugs for this treatment. A liquid chromatography with fluorescence detection method was developed and validated for determining ciprofloxacin concentrations in two different matrices: plasma and prostate microdialysate. Ciprofloxacin was separated on a C18 column eluted with a mobile phase constituted of a mixture of 0.4% aqueous triethylamine:methanol:acetonitrile (75:15:10, v/v/v) and 0.4% aqueous triethylamine:acetonitrile (88:12, v/v) for microdialysate and plasma samples, respectively. Linearity was obtained over a concentration range of 5-1000 ng/mL (microdialysate) and 10-2000 ng/mL (plasma), with coefficients of determination ≥0.9956. Precision was determined from the analysis of six quality control samples and showed RSD values <11.1 and 7.4% for intra and inter-assay precision, respectively. The accuracy ranged from 85.6 to 114.3%. The method was applied to a preliminary pharmacokinetic study to investigate ciprofloxacin concentrations in prostate, sampled by microdialysis, and plasma after a 7 mg/kg intravenous dose to Wistar rats. The method showed high sensitivity using only protein precipitation as plasma sample clean-up and was successfully applied to investigate ciprofloxacin prostate penetration.

HIGHLY SENSITIVE LC-MS/MS METHOD FOR THE DETERMINATION OF CLOZAPINE IN RAT PLASMA: APPLICATION TO A PRECLINICAL PHARMACOKINETIC STUDY

A highly sensitive, rapid assay method has been developed and validated for the quantification of clozapine in rat plasma using LC-MS/MS. The extraction process involved plasma protein precipitation with acetonitrile and elution with a mobile phase constituted by acetonitrile and acidic water (80/20), at 0.8 mL/min flow rate (split 1:3). Chromatographic separation was performed by C18 Symmetry column (4.6 × 75 mm, 3.5 µm). The lower limit of quantification was 1 ng/mL and the linearity was observed between 1–1000 ng/mL with a determination coefficient >0.9810. The intra- and inter-day precisions were less or equal to 8.38 and 4.36%, respectively. The method showed sensitivity, linearity, precision, accuracy, and specificity necessary for quantification of plasma clozapine in preclinical pharmacokinetics studies.

Pre-clinical pharmacokinetics and acute toxicological evaluation of a monastrol derivative anticancer candidate LaSOM 65 in rats.

Abstract 1. The present work investigated the pharmacokinetic and tissue distribution as well as acute toxicity of a new chemical entity (NCE), the anticancer candidate LaSOM 65 in Wistar rats. 2. LaSOM 65 pharmacokinetics was investigated after intravenous (i.v., 1 mg/kg) and oral (p.o., 10 and 30 mg/kg) dosing. Tissue distribution was assessed after i.v. bolus dose. Acute toxicity was evaluated after i.v. (1, 2.5 and 5 mg/kg) and p.o. (50, 100 and 150 mg/kg) administration. 3. Short half-life (1.75 ± 0.71 h), a clearance of 0.85 ± 0.18 L/h/kg and a volume of distribution of 1.76 ± 0.24 L/kg were observed after i.v. dosing. The compound showed good bioavailability and linear pharmacokinetics after oral doses. The NCE distributes consistently in lung and fatty tissues, with penetration ratios of 2.7 and 1.4, respectively. The other tissues investigated presented smaller penetration ratios. Adverse clinical symptoms were observed only after i.v. administration, and regressed 3 h after dosing. Compared with controls, no statistical differences were found for serum analysis, body weight and relative organ weight, indicating no acute toxicological effects. 4. Overall, LaSOM 65 showed good pharmacokinetic characteristics and no signs of acute toxicity, indicating that it is a promising anticancer candidate.

Bioanalytical method for the quantification of the monastrol derivative anticancer candidate lasom 65 in pre-clinical pharmacokinetic investigations

A simple HPLC/UV method was developed for the determination of the anticancer candidate LaSOM 65 in rat plasma. Samples were cleaned by protein precipitation with acetonitrile (recovery > 95%), after which they were subjected to chromatography under the isocratic elution of an acetonitrile:water (45:55, v/v) solution with detection at 303 nm. The method was linear (r2 > 0.98) over the concentration range (0.05–2 µg mL−1) with intra- and inter-day precision ranging from 9.6% to 13.6% and 4.3% to 5.4%, respectively. The accuracy of the method ranged from 85% to 113.6%, and it showed sufficient sensitivity to determine pharmacokinetic parameters of LaSOM 65 after intravenous administration to Wistar rats.

Pre-clinical pharmacokinetics of the acridine antitumour candidate AC04 and its 1-oxo-metabolite plasma profile

This work aimed to investigate plasma pharmacokinetics and tissue distribution of a new acridine derivative 5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione (AC04) and its 1-oxo-AC04 metabolite disposition in Wistar rats. After a single AC04 1.5 mg/kg intravenous (i.v.) bolus dose, blood samples were taken up to 120 h. Plasma samples were deproteinization, and AC04 and metabolite were quantified by validated liquid chromatography in tandem with mass spectrometry method. Protein binding was determined by ultrafiltration. AC04 tissue disposition was evaluated after i.v. bolus dose. Individual AC04 concentration–time profiles were best fitted by a two-compartment model showing CLtot of 3.4 ± 3.4 L/h/kg, VdSS of 137.9 ± 91.4 L/kg, AUC0–∞ of 788 ± 483 ng·h/mL and a t1/2 of 45.5 ± 31.5 h. Protein binding was 98.1 ± 1.6%. AC04 showed higher penetration into the lung, spleen and liver, with AUC0–96 of 798,443, 263,211 and 303,722 ng·h/mL, respectively. The 1-oxo-AC04 metabolite represented 10% of AC04 plasma concentration, showing a t1/2 of 23.2 ± 10.4 h. These results suggest that, despite the small free plasma fraction, AC04 penetrates extensively reaching high concentrations in most tissues residing for a long time, which is important for its activity on solid tumours. All results combined indicate that AC04 is potentially a good antitumour candidate.

Lapachol, a compound targeting pyrimidine metabolism, ameliorates experimental autoimmune arthritis

BackgroundThe inhibition of pyrimidine biosynthesis by blocking the dihydroorotate dehydrogenase (DHODH) activity, the prime target of leflunomide (LEF), has been proven to be an effective strategy for rheumatoid arthritis (RA) treatment. However, a considerable proportion of RA patients are refractory to LEF. Here, we investigated lapachol (LAP), a natural naphthoquinone, as a potential DHODH inhibitor and addressed its immunosuppressive properties.MethodsMolecular flexible docking studies and bioactivity assays were performed to determine the ability of LAP to interact and inhibit DHODH. In vitro studies were conducted to assess the antiproliferative effect of LAP using isolated lymphocytes. Finally, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA) models were employed to address the anti-arthritic effects of LAP.ResultsWe found that LAP is a potent DHODH inhibitor which had a remarkable ability to inhibit both human and murine lymphocyte proliferation in vitro. Importantly, uridine supplementation abrogated the antiproliferative effect of LAP, supporting that the pyrimidine metabolic pathway is the target of LAP. In vivo, LAP treatment markedly reduced CIA and AIA progression as evidenced by the reduction in clinical score, articular tissue damage, and inflammation.ConclusionsOur findings propose a binding model of interaction and support the ability of LAP to inhibit DHODH, decreasing lymphocyte proliferation and attenuating the severity of experimental autoimmune arthritis. Therefore, LAP could be considered as a potential immunosuppressive lead candidate with potential therapeutic implications for RA.

Population Pharmacokinetic Modeling as a Tool To Characterize the Decrease in Ciprofloxacin Free Interstitial Levels Caused by Pseudomonas aeruginosa Biofilm Lung Infection in Wistar Rats

ABSTRACT Biofilm formation plays an important role in the persistence of pulmonary infections, for example, in cystic fibrosis patients. So far, little is known about the antimicrobial lung disposition in biofilm-associated pneumonia. This study aimed to evaluate, by microdialysis, ciprofloxacin (CIP) penetration into the lungs of healthy and Pseudomonas aeruginosa biofilm-infected rats and to develop a comprehensive model to describe the CIP disposition under both conditions. P. aeruginosa was immobilized into alginate beads and intratracheally inoculated 14 days before CIP administration (20 mg/kg of body weight). Plasma and microdialysate were sampled from different animal groups, and the observations were evaluated by noncompartmental analysis (NCA) and population pharmacokinetic (popPK) analysis. The final model that successfully described all data consisted of an arterial and a venous central compartment and two peripheral distribution compartments, and the disposition in the lung was modeled as a two-compartment model structure linked to the venous compartment. Plasma clearance was approximately 32% lower in infected animals, leading to a significantly higher level of plasma CIP exposure (area under the concentration-time curve from time zero to infinity, 27.3 ± 12.1 μg · h/ml and 13.3 ± 3.5 μg · h/ml in infected and healthy rats, respectively). Despite the plasma exposure, infected animals showed a four times lower tissue concentration/plasma concentration ratio (lung penetration factor = 0.44 and 1.69 in infected and healthy rats, respectively), and lung clearance (CLlung) was added to the model for these animals (CLlung = 0.643 liters/h/kg) to explain the lower tissue concentrations. Our results indicate that P. aeruginosa biofilm infection reduces the CIP free interstitial lung concentrations and increases plasma exposure, suggesting that plasma concentrations alone are not a good surrogate of lung concentrations.