Polymorphisms of leptin, beta-lactoglobulin and pituitary transcription factor have no effect on milk characteristics in crossbred cows / Polimorfismos da leptina, beta-lactoglobulina e fator de transcrição da hipófise não afetam as características do leite em vacas mestiças

O objetivo deste trabalho foi identificar polimorfismos genéticos de leptina, ß-lactoglobulina e fator de transcrição pituitária (PIT1) e avaliar seus efeitos na composição química e na contagem de células somáticas de leite de vacas leiteiras mestiças que vivem em um clima quente. Um total de 291 vacas leiteiras mestiças foram investigadas. Foram coletadas amostras de sangue para extração de DNA e amostras de leite. As amostras foram classificadas em três grupos genéticos: 12/ (42), 34/ (83) e 78/ (166) Holandês x Guzerá. As frequências de alelos e genótipos foram determinadas e o equilíbrio Hardy-Weinberg foi avaliado. Foram realizadas análises da composição do leite (gordura, proteína, lactose e extracto seco desengordurado), contagem de células somáticas e rendimento leiteiro. Os grupos genéticos e os polimorfismos genéticos para cada gene foram utilizados como efeitos fixos na análise. O único polimorfismo encontrado em equilíbrio de Hardy-Weinberg foi para o genótipo da ß-lactoglobulina. No presente estudo, era esperado que a maioria das variáveis de composição variasse entre os genótipos. Já se sabe que os cruzamentos dão origem a animais com características fenotípicas e genotípicas. No entanto, os polimorfismos não influenciaram a composição e a qualidade do leite nas vacas 12/ , 34/ e 78/ Holstein x Guzerá mantidas em um clima quente.(AU)

Development and validation of a bioanalytical method by LC-MS/MS for the quantification of the LAFIS 10 - an antimalarial candidate - and its pharmacokinetics first evaluation.

A rapid and highly sensitive method by LC-MS/MS was developed and validated for the quantification of an antimalarial candidate (LAFIS10) in rat plasma using dexamethasone as internal standard (IS). The chromatographic separation was performed with a Poroshell 120 EC-C18 column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 10 m m of ammonium formate and 0.1% formic acid, delivered in the form of elution gradient. The LAFIS10 was monitored using an electrospray ionization interface operating in the positive mode in multiple reaction monitoring mode, monitoring the transitions 681.47 → 538.2 for LAFIS10 and 393.20 → 355.30 for the IS. The flow rate was 500 µL/min. The column temperature was kept at 40 °C and the injection volume was 2 µL. The lower limit of quantification was of 10 ng/mL and linearity between 10 and 1000 ng/mL was observed, with an R(2) > 0.99. The accuracy of the method was >90%. The relative standard deviations intra- and interday were <8.80 and <6.37%, respectively. The method showed sensitivity, linearity, precision, accuracy and selectivity required to quantify LAFIS 10 in preclinical pharmacokinetic studies according to criteria established by the US Food and Drug Administration and European Medicines Agency.

Development and characterization of parenteral nanoemulsions containing thalidomide.

This study reports the development of nanoemulsions intended for intravenous administration of thalidomide (THD). The formulations were prepared by spontaneous emulsification method and optimized with respect to thalidomide (0.01-0.05%, w/w), and hydrophilic emulsifier (polysorbate 80; 0.5-4.0%, w/w) content. The formulations were evaluated concerning physical appearance and drug crystallization; droplet size; zeta potential and drug assay. Only the formulation containing 0.01% THD and 0.5% polysorbate kept its properties in a satisfactory range over the evaluated period (60 days), i.e. droplet size around 200nm, drug content around 95% and zeta potential around -30mV. The transmission electron microscopy revealed emulsion droplets almost spherical in shape confirming the results obtained by photon correlation spectroscopy. Drug crystallization observed for higher content (THD 0.05%, w/w) nanoemulsions was investigated. The crystals observed at optical microscopy presented a different crystal habit compared to that of the raw material used. It was speculated whether the kind of THD polymorph employed could influence nanoemulsion formulation. Formulations were prepared with either one of THD polymorphs (ß- or α-) and crystals were characterized by fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). It was observed that regardless of the polymorph employed (ß- or α-), drug crystallization occurs in the α-form. THD solubility in oils was not influenced by the polymorphic form. In addition, the in vitro dissolution profile of the selected formulation (THD 0.01%, w/w; polysorbate 0.5%, w/w) was assessed by bulk-equilibrium reverse dialysis sac technique and demonstrated a release profile similar to that of a THD acetonitrile solution, with around 95% THD being dissolved within 4h. Finally, a pharmacokinetic simulation of an intravenous infusion of 250mL of the selected nanoemulsion suggests that the parenteral administration of a dose as low as 25mg might lead to therapeutic plasma concentrations of thalidomide.

Microdialysis as a tool to determine free kidney levels of voriconazole in rodents: a model to study the technique feasibility for a moderately lipophilic drug.

Microdialysis has been employed for the in vivo measurement of endogenous compounds and a variety of drugs in different tissues. The applicability of this technique can be limited by drug lipophilicity which can impair the diffusion through dialysis membrane. The objective of this study was to evaluate the feasibility of using microdialysis to study kidney penetration of voriconazole, a moderately lipophilic antifungal triazolic agent (LogD7.4=1.8). Microdialysis probe recoveries were investigated in vitro by dialysis and retrodialysis using four different drug concentrations (0.1-2 microg/mL) at five flow rates (1-5 microL/min). Recoveries were dependent on the method used for the determination as well as on the flow rate, but independent of drug concentration. The average apparent recoveries determined by dialysis and retrodialysis, at flow rate of 2 microL/min, were 21.1+/-1.5% and 28.7+/-2.0%, respectively. Recovery by retrodialysis was bigger than the recovery by dialysis. The average apparent dialysis/retrodialysis recovery ratio in vitro was 0.73 for all concentrations investigated. The differences between retrodialysis and dialysis recoveries were attributed to the drug's binding to the plastic tubing before and after the dialysis membrane which was experimentally evaluated and mathematically modeled. The in vivo apparent recovery determined by retrodialysis in healthy Wistar rats' kidney was 38.5+/-3.5%, similar to that observed in vitro using the same method (28.7+/-2.0%). The in vivo apparent recovery after correcting for plastic tubing binding (25.1+/-2.8%) was successfully used for determining free kidney levels of voriconazole in rats following 40 and 60mg/kg oral dosing. The results confirmed that microdialysis can be used as sampling technique to determine free tissue levels of moderately lipophilic drugs once the contribution of tubing binding and membrane diffusion on the apparent recovery are disentangled.

Validation of rapid and simple LC-MS/MS method for determination of voriconazole in rat plasma.

A rapid, simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of voriconazole (VRC) in rat plasma, using ketoconazole as internal standard (IS). Analysis was performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water-formic acid (60:40:0.05, v/v/v), at a flow of 1.0 mL/min (split ratio 1:5), and a mass spectrometer Micromass, equipped with a double quadrupole and an electrospray ionization interface, operated in a positive mode. Plasma samples were deproteinized with methanol (1:2) and 30 microL of the supernatant was injected into the system. The retention times of VRC and IS were approximately 3.3 and 2.7 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 50-2500 ng/mL with determination coefficient >0.98. The lower limit of quantification was 50 ng/mL. The accuracy of the method was within 5%. Intra- and inter-day relative standard deviations were less or equal to 12.5 and 7.7%, respectively. The applicability of the LC-MS-MS method for pharmacokinetic studies was tested using plasma samples obtained after intravenous administration of VRC to male Wistar rats. The reported method provided the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of VRC in pre-clinical pharmacokinetic studies.