Maiara Cássia Pigatto

Induction of lipid peroxidation and decrease of antioxidant defenses in symptomatic and asymptomatic patients with X-linked adrenoleukodystrophy.

Patients affected by X‐linked adrenoleukodystrophy (X‐ALD) present a progressive brain and peripheral demyelination and adrenal cortex insufficiency, associated with accumulation of the very long chain fatty acids (VLCFA) hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and biological fluids. X‐ALD is characterized by heterogeneous clinical phenotypes. Seven clinical variants have been described for this genetic disorder, being the childhood cerebral form (CCER), adrenomyeloneuropathy (AMN) and asymptomatic the most common clinical forms. In a previous work, we showed evidence that oxidative stress is involved in the pathophysiology of X‐ALD symptomatic patients. In the present study, we compared oxidative stress parameters, namely thiobarbituric acid reactive substances (TBA‐RS) and total antioxidant status (TAS), in plasma from patients with CCER, AMN and in asymptomatic X‐ALD patients. It was observed that symptomatic and asymptomatic X‐ALD patients presented a significant increase of plasma TBA‐RS measurement, indicating a stimulation of lipid peroxidation. Furthermore, lipid peroxidation was higher in AMN, as compared to CCER and asymptomatic patients. We also observed that the total antioxidant defenses (TAS) were decreased in symptomatic but not in asymptomatic X‐ALD patients. Therefore, it may be presumed that asymptomatic patients seem to be protected against oxidative stress because of their normal antioxidant defenses and that other factors besides oxidative damage may be responsible for the severity of the symptoms in X‐ALD and need to be investigated.

Hexacosanoic and docosanoic acids plasma levels in patients with cerebral childhood and asymptomatic X-linked adrenoleukodystrophy: Lorenzo's oil effect.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal metabolism, biochemically characterized by deficient β-oxidation of saturated very long chain fatty acids (VLCFA). The consequent accumulation of these fatty acids in different tissues and in biological fluids is associated with a progressive central and peripheral demyelination, as well as with adrenocortical insufficiency and hypogonadism. Seven variants of this disease have been described, being cerebral childhood the most frequent. The recommended therapy consists of the use of the glyceroltrioleate/glyceroltrierucate mixture known as Lorenzo’s Oil (LO), combined with a VLCFA-poor diet, but only in asymptomatic patients will this treatment prevent the progression of the symptomatology. In the present study we evaluated the biochemical course of patients with cerebral childhood (CCER) and asymptomatic clinical forms of X-ALD treated with LO associated with a VLCFA-restricted diet. We observed that hexacosanoic acid plasma concentrations and hexacosanoic/docosanoic ratio were significantly reduced in CCER patients during treatment when compared with diagnosis. Hexacosanoic acid plasma level was significantly reduced when compared with that at diagnosis and achieved the normal levels only in asymptomatic patients under LO treatment. In asymptomatic patients the magnitude of hexacosanoic acid decrease was higher than that of the CCER patients. These results show the good biochemical response of LO treatment in asymptomatic X-ALD patients. It is possible to suppose that this could be correlated with the prevention of the appearance of neurological signals in this group of patients treated with LO.

Metabolism evaluation of the anticancer candidate AC04 by biomimetic oxidative model and rat liver microsomes

Jacobsen reagents, in the presence of monooxygen donors, appear as an alternative to produce metabolites from biological active compounds. This reaction may mimic the oxidation and oxygenation reactions of cytochrome P450 (CYP450) enzymes upon various drugs and biologically active compounds. Acridines represent a well-known group of polyaromatic compounds capable of acting as DNA intercalating agents. Viewing to search for new anticancer agents, one promising new acridine, the 5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione (AC04) (2), has been studied by our group and the in vitro metabolism was investigated in this work, aiming to advance in the pre-clinical pharmacokinetic investigation. A systematic investigation of the gas-phase reaction, supported by computational chemistry, of the AC04 (2) was studied to help the structure elucidation of possible in vivo metabolites. To confirm the methodology, the oxidized product was obtained in large scale for NMR analysis and the data confirmed the structure. In addition, AC04 (2) was submitted to an in vitro metabolism assay employing rat liver microsomes and also, a pilot study was conducted in rats after AC04 intravenous (i.v.) dosing of 1.5 mg/kg. A single oxidized product was obtained from microsomal metabolism and detected in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis corresponding to the same product formed by Jacobsen-catalyzed reaction. These results indicate that Jacobsen oxidation reactions, combined with in vitro metabolism assays employing isolated microsomes, might replace some in vivo metabolism studies, thus reducing the use of animals in new chemical entities pre-clinical investigation.

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.

HPLC-UV method for quantifying etoposide in plasma and tumor interstitial fluid by microdialysis: application to pharmacokinetic studies

A simple and sensitive bioanalytical method was developed and validated for determination of etoposide in plasma and microdialysis samples of Walker-256 tumor-bearing rats. A microdialysis probe was implanted in the center of a subcutaneous tumor and Ringers solution was used as perfusion medium. Chromatographic separation was conducted on a Shimadzu CLC-C8 column using a mobile phase consisting of water-acetonitrile (70:30; v/v) adjusted to pH 4.0 ± 0.1 with formic acid at a gradient flow rate of 1.0-0.6 mL/min, an injection volume of 30 μL and UV detection at 210 nm. Microdialysate samples were analyzed without processing and plasma samples (100 μL) were spiked with phenytoin as internal standard (IS) (1 µg/mL) followed by extraction with tert-butyl methyl ether. The organic layer was evaporated and reconstituted with 100 μL of mobile phase before injection. The methods for plasma and microdialysate were linear in the ranges of 25-10,000 ng/mL and of 10-1500 ng/mL, respectively. All the validation parameters such as intra- and inter-day precision and accuracy and stability were within the limits established by international guidelines. The present method was successfully applied in the investigation of etoposide pharmacokinetics in rat plasma and microdialysate tumor samples following a single 15 mg/kg intravenous dose.

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.

DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE PRE-CLINICAL PHARMACOKINETIC INVESTIGATION OF THE ANTICANCER CANDIDATE AC04 IN RODENTS

A liquid chromatography-tandem mass spectrometry method was developed for accurate quantification of the acridinic anticancer candidate AC04 (5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione) in rat plasma viewing the drugs pre-clinical pharmacokinetic investigation. The sample purification was performed by protein precipitation technique with acetonitrile, and terbinafine was used as the internal standard (IS). The chromatographic separation was achieved using an isocratic mobile phase consisting of a mixture of acetonitrile and 0.1% formic acid, (95:5, v/v) flowing through an ACE® C18 column (5 µm, 2.1 × 50 mm) at a flow rate of 0.45 mL/min. Mass spectrometer with electrospray ionization (ESI) monitored, in positive mode, the transitions m/z 411.1 > 104.8 and 292.4 > 142 for AC04 and internal standard, respectively. An extensive method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method showed linearity in the concentration range of 2.5–500.0 ng/mL with correlation coefficient 0.993. Intra- and inter-day precision were less than 8.5 and 3.7%, respectively. The applicability of the method for pharmacokinetic investigation was determined in a pilot investigation of AC04 plasma profiles after a 1.5 mg/kg intravenous administration to rats.

Pharmacokinetic/pharmacodynamic modeling of etoposide tumor growth inhibitory effect in Walker-256 tumor-bearing rat model using free intratumoral drug concentrations

&NA; The purpose of this study was to establish a population pharmacokinetic/pharmacodynamic (PK/PD) model linking etoposide free tumor and total plasma concentrations to the inhibition of solid tumor growth in rats. Walker‐256 tumor cells were inoculated subcutaneously in the right flank of Wistar rats, which were randomly divided in control and two treated groups that received etoposide 5 or 10 mg/kg i.v. bolus every day for 8 and 4 days, respectively, and tumor volume was monitored daily for 30 days. The plasma and intratumoral concentrations‐time profiles were obtained from a previous study and were modeled by a four‐compartment population pharmacokinetic (popPK) model. PK/PD analysis was conducted using MONOLIX v.4.3.3 on average data and by mean of a nonlinear mixed‐effect model. PK/PD data were analyzed using a modification of Simeoni Tumor Growth Inhibition (TGI) model by introduction of an Emax function to take into account the concentration dependency of k2variable parameter (variable potency). The Simeoni TGI‐Emax model was capable to fit schedule‐dependent antitumor effects using the tumor growth curves from the control and two different administered schedules. The PK/PD model was capable of describing the tumor growth inhibition using total plasma or free tumor concentrations, resulting in higher k2max (maximal potency) for free concentrations (25.8 mL·&mgr;g− 1·day− 1 ‐ intratumoral vs. 12.6 mL·&mgr;g− 1·day− 1 total plasma). These findings indicate that the plasma concentration may not be a good surrogate for pharmacologically active free tumor concentrations, emphasizing the importance of knowing drug tumor penetration to choose the best antitumor therapy. Graphical abstract Figure. No caption available.

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.

Validation of LC‐MS/MS method applied to evaluation of free tissue concentrations of vildagliptin in diabetic rats by microdialysis

A novel LC-MS/MS method was developed for the quantification of vildagliptin in an aqueous matrix. The method was successfully validated, meeting all the requisites of US Food and Drug Administration guide for a bioanalytical method. The developed method presented a limit of quantification of 10 ng/mL and the range of concentration achieved was 10-1875 ng/mL. The injection volume necessary was only 10 μL, and retention time was 4.60 min. The mobile phase employed was methanol-ammonium acetate 5 mm (95:5). The stability of the drug was evaluated in the different conditions through which the samples passed. A pharmacokinetic experiment was conducted with diabetic male Wistar rats, and the concentration of drug in liver was evaluated through a microdialysis technique. The perfusion fluid employed was ultrapure water. The dose administrated was 50 mg/kg and the method allowed the quantification of vildagliptin for more than three half lives, successfully characterizing the pharmacokinetic profile when the developed method was applied. This is the first report on the tissue pharmacokinetics of a DPP-4 inhibitor and could contribute to drug dosage optimization in the future.