Randomized, phase 1/2, double-blind pioglitazone repositioning trial combined with antifungals for the treatment of cryptococcal meningitis - PIO study.

BACKGROUND: Cryptococcosis affects more than 220,000 patients/year, with high mortality even when the standard treatment [amphotericin B (AMB), 5-flucytosin (5-FC) and fluconazole] is used. AMB presents high toxicity and 5-FC is not currently available in Brazil. In a pre-clinical study, pioglitazone (PIO - an antidiabetic drug) decreased AMB toxicity and lead to an increased mice survival, reduced morbidity and fungal burden in brain and lungs. The aim of this trial is to evaluate the efficacy and safety of PIO combined with standard antifungal treatment for human cryptococcosis. METHODS: A phase 1/2, randomized, double blind, placebo-controlled trial will be performed with patients from Belo Horizonte, Brazil. They will be divided into three groups (placebo, PIO 15 mg/day or PIO 45 mg/day) and will receive an additional pill during the induction phase of cryptococcosis' treatment. Our hypothesis is that treated patients will have increased survival, so the primary outcome will be the mortality rate. Patients will be monitored for survival, side effects, fungal burden and inflammatory mediators in blood and cerebrospinal fluid. The follow up will occur for up 60 days. CONCLUSIONS: We expect that PIO will be an adequate adjuvant to the standard cryptococcosis' treatment. TRIAL REGISTRATION: ICTRP/WHO (and International Clinical Trial Registry Plataform (ICTRP/WHO) (http://apps.who.int/trialsearch/Trial2.aspx?TrialID=RBR-9fv3f4), RBR-9fv3f4 (http://www.ensaiosclinicos.gov.br/rg/RBR-9fv3f4). UTN Number: U1111-1226-1535. Ethical approvement number: CAAE 17377019.0.0000.5149.

Validation of a HPLC method for quantification of midazolam in rat plasma: Application during a Maytenus ilicifolia-drug interaction study.

Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC-UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol-isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC-UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb-drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol-water (1:1). The analyte was eluted throughout a C18 column using sodium acetate buffer (10 mm, pH 7.4)-methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from -19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

Evaluation of the Effects of Maytenus ilicifolia on the Activities of Cytochrome P450 3A and P-glycoprotein.

BACKGROUND: Maytenus ilicifolia is a Brazilian popular medicine commonly used to treat ulcer and gastritis. Despite the absence of toxicity regarding its consumption, possible interactions when co-administrated with conventional drugs, are unknown. OBJECTIVE: This study aimed to evaluate the effects of M. ilicifolia extracts on Cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) activities. METHODS: The extracts were obtained by infusion (MI) or turbo-extraction using hydro-acetonic solvent (MT70). The content of polyphenols in each extract was determined. To assess the modulation of M. ilicifolia on P-gp activity, the uptake of fexofenadine (FEX) by Caco-2 cells was investigated in the absence or presence of MI or MT70. The effect on CYP3A activity was evaluated by the co-administration of midazolam (MDZ) with each extract in male Wistar rats. The pharmacokinetic parameters of the drug were determined and compared with those from the control group. The content of total phenolic compounds, tannins, and flavonoids on MT70 extract was about double of that found in MI. RESULTS: In the presence of the extracts, the uptake of the P-gp marker (FEX) by Caco-2 cells increased from 1.7 ± 0.4 ng.mg-1 protein (control) to 3.5 ± 0.2 ng.mg-1 protein (MI) and 4.4 ± 0.5 ng.mg-1 protein (MT70), respectively. When orally co-administrated with MDZ (substrate of CYP3A), the extracts augmented the AUC(0-∞) (Control: 911.7 ± 215.7 ng.h.mL-1; MI: 1947 ± 554.3 ng.h.mL-1; MT70: 2219.0 ± 506.3 ng.h.mL-1) and the Cmax (Control: 407.7 ± 90.4 ng.mL-1; MI: 1770.5 ± 764.5 ng.mL-1; MT70: 1987.2 ± 544.9 ng.mL-1) of the drug in rats indicating a 50% reduction of the oral Cl. No effect was observed when midazolam was given intravenously. CONCLUSION: The results suggest that M. ilicifolia can inhibit the intestinal metabolism and transport of drugs mediated by CYP3A and P-gp, respectively, however, the involvement of other transporters and the clinical relevance of such interaction still need to be clarified.

Toxicity and in vivo release profile of sirolimus from implants into the vitreous of rabbits' eyes.

PURPOSE: To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. METHODS: PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. RESULTS: After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. CONCLUSIONS: Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.

Influence of PEG coating on the biodistribution and tumor accumulation of pH-sensitive liposomes.

Liposomes are lipid vesicles widely used as nanocarriers in targeted drug delivery systems for therapeutic and/or diagnostic purposes. A strategy to prolong the blood circulation time of the liposomes includes the addition of a hydrophilic polymer polyethylene glycol (PEG) moiety onto the surface of the vesicle. Several studies claim that liposome PEGylation by a single chain length or a combination of PEG with different chain lengths may alter the liposomes' pharmacokinetic properties. Therefore, the purpose of this study was to evaluate the influence of PEG on the biodistribution of pH-sensitive liposomes in a tumor-bearing animal model. Three liposomal formulations (PEGylated or not) were prepared and validated to have a similar mean diameter, monodisperse distribution, and neutral zeta potential. The pharmacokinetic properties of each liposome were evaluated in healthy animals, while the biodistribution and scintigraphic images were evaluated in tumor-bearing mice. High tumor-to-muscle ratios were not statistically different between the PEGylated and non-PEGylated liposomes. While PEGylation is a well-established strategy for increasing the blood circulation of nanostructures, in our study, the use of polymer coating did not result in a better in vivo profile. Further studies must be carried out to confirm the feasibility of the non-PEGylated pH-sensitive liposomes for tumor treatment.

Molecularly imprinted polymer for determination of lumefantrine in human plasma through chemometric-assisted solid-phase extraction and liquid chromatography.

Lumefantrine is the first-choice treatment of Falciparum uncomplicated malaria. Recent findings of resistance to lumefantrine has brought attention for the importance of therapeutic monitoring, since exposure to subtherapeutic doses of antimalarials after administration is a major cause of selection of resistant parasites. Therefore, this study focused on the development of innovative, selective, less expensive and stable molecularly imprinted polymers (MIPs) for solid-phase extraction (SPE) of lumefantrine from human plasma to be used in drug monitoring. Polymers were synthesized by precipitation polymerization and chemometric tools (Box-Behnken design and surface response methodology) were employed for rational optimization of synthetic parameters. Optimum conditions were achieved with 2-vinylpyridine as monomer, ethylene glycol dimethacrylate as crosslinker and toluene as porogen, at molar ratio of 1:6:30 of template/monomer/crosslinker and azo-bisisobutyronitrile as initiator at 65 °C. The MIP obtained was characterized and exhibited high thermal stability, adequate surface morphology and porosity characteristics and high binding properties, with high affinity (adsorption capacity of 977.83 µg g-1) and selectivity (imprinting factor of 2.44; and selectivity factor of 1.48 and selectivity constant of 1.44 compared with halofantrine). Doehlert matrix and fractional designs were satisfactorily used for development and optimization of a MISPE-HPLC-UV method for determination of lumefantrine. The method fulfilled all validation parameters, with recoveries ranging from 83.68% to 85.42%, and was applied for quantitation of the drug in plasma from two healthy volunteers, with results of 1407.89 and 1271.35 ng mL-1, respectively. Therefore, the MISPE-HPLC-UV method optimized through chemometrics provided a rapid, highly selective, less expensive and reproducible approach for lumefantrine drug monitoring.

Comparison of HPLC, UV spectrophotometry and potentiometric titration methods for the determination of lumefantrine in pharmaceutical products.

This paper describes the development and evaluation of a HPLC, UV spectrophotometry and potentiometric titration methods to quantify lumefantrine in raw materials and tablets. HPLC analyses were carried out using a Symmetry C(18) column and a mobile phase composed of methanol and 0.05% trifluoroacetic acid (80:20), with a flow rate of 1.0ml/min and UV detection at 335nm. For the spectrophotometric analyses, methanol was used as solvent and the wavelength of 335nm was selected for the detection. Non-aqueous titration of lumefantrine was carried out using perchloric acid as titrant and glacial acetic acid/acetic anhydride as solvent. The end point was potentiometrically determined. The three evaluated methods showed to be adequate to quantify lumefantrine in raw materials, while HPLC and UV methods presented the most reliable results for the analyses of tablets.