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- Combinatorial delivery of Crizotinib–Palbociclib–Sildenafil using TPGS-PLA micelles for improved cancer treatmentPublication . Diogo, Duarte Miguel de Melo; Gaspar, Vítor Manuel Abreu; Costa, Elisabete C.; Moreira, André; Oppolzer, David; Gallardo, Eugenia; Correia, Ilídio Joaquim SobreiraThe co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3 nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib–Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.
- Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesisPublication . Pedro, Augusto; Oppolzer, David; Bonifácio, M J; Maia, C J; Queiroz, João; Passarinha, L ACatechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.
- Hair analysis for alcohol biomarkers: assessing excessive consumption in a student populationPublication . Oppolzer, David Jerónimo; Gallardo Alba, Maria Eugénia; Barroso, Mário Jorge Dinis; Passarinha, Luís António PaulinoAlcohol consumption within the student population has become an increasing health concern in several countries. The use of alcohol has deleterious consequences on intellectual performance of students, and can be associated with a variety of risky situations, as injuries, unplanned and unsafe sex, sexual and non-sexual violence, property damage and reckless driving. Monitoring alcohol consumption in the student population is therefore important to identify the degree of the problem and to assess consumption trends, allowing also for the implementation of adequate policies, as well as verifying their effectiveness. Alcohol consumption can be monitored using statistics, such as survey studies performed on the population by means of questionnaires. These approaches have relative efficiency and are widely used; however, considerable limitations are associated to their use, making them unreliable for clinical and/or forensic purposes. Alcohol biomarkers are measurable substances in a biological sample, whose presence indicates some form of exposure to alcohol, and their use to assess alcohol exposure in clinical and forensic scenarios has been applied in a variety of biological specimens in the past years. One of the most notable of these specimens is hair, which presents several advantages, including non-invasive collection, ease of availability, long windows of detection and low chance of adulteration. Hair testing for alcohol exposure relies mainly on the analysis of two biomarkers, ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). These markers have been widely applied in several studies, and were shown to present good sensitivity and specificity. Therefore, the objective of the present work was to evaluate the alcohol consumption on a university student population using two main approaches, one based on questionnaire analysis, and the second on the analytical determination of alcohol biomarkers in hair. A total of 1192 hair samples, and respective self-completion questionnaire were collected, 975 samples were analysed thereof. Through analysis of the questionnaires it was found that alcohol consumption started mostly at the age of 15, and that most of the students considered their consumption as moderate, while almost one-third consumed alcohol excessively at least once in the last month. Beer was reported to be the most consumed beverage, and drinking was widely preferred accompanied. Places for alcohol consumption are mostly public places, such as coffee bars. Higher ingested quantities of alcohol per drinking occasion were associated with the male genre, but both genres presented mean alcohol volumes indicating risky and excessive drinking. The use of other substances was also assessed, including tobacco, which was a habit in one-third of the students. The incidence of the use of illicit substances was similar, and cannabis was the most consumed substance, but mainly on rare occasions. The use of illicit substances was however associated with drinking events in more than 50% of the students. For alcohol biomarker analysis in hair, a method was developed for the analysis of EtG. The compound was extracted from the hair matrix by ultra-sonication with water, clean-up using mixed anion-exchange solid-phase extraction (SPE) and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A second method was developed for the analysis of four FAEEs (ethyl myristate, palmitate, oleate and stearate). The compounds were extracted from the hair matrix by incubation with heptane, extracts were cleaned-up with aminopropyl SPE and analysed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Both methods were validated according to guidelines from the Food and Drug Administration (FDA) and International Conference on Harmonization (ICH). Assessed parameters included sensitivity, limits, linearity, precision and accuracy, recovery, stability and matrix effect. The methods were found to be selective, with a limit of quantification of 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Linearity was obtained in the ranges of 3 – 500 pg/mg for EtG and 30 – 5000 pg/mg for FAEEs, and precision and accuracy were found acceptable according to the guidelines. Overall recovery ranged from 74.79% and 97.90%, and processed EtG samples were stable for at least 96h, and FAEEs samples for at least 24h. Matrix effects were evaluated for EtG analysis and were not significant. Higher concentrations of both biomarkers were obtained for the male genre, and at the universities of Beira Interior and Coimbra. Based on hair sample results, individuals were categorized as abstinent, moderate or excessive drinkers, according to cut-off concentrations proposed by the Society of Hair Testing (SoHT) for both biomarkers. These values are 7 pg/mg of EtG, and 0.2 ng/mg (for 0-3 cm segments) or 0.4 ng/mg (for 0-6 cm segments) of FAEEs, to distinguish abstinence from moderate drinking. To distinguish moderate from excessive drinking, a value of 30 pg/mg of EtG, and 0.5 ng/mg (for 0-3 cm segments) or 1.0 ng/mg (for 0-6 cm segments) of FAEEs was used. At the proposed cut-off values, EtG presented good sensitivity (60-81.6%) and specificity (56.3–90.3%), while for FAEEs good specificity was obtained (87.5–100%), but sensitivity varied between 30.5% and 100%. In order to verify if the currently proposed cut-off values are adequate for the studied population, receiver operating characteristic (ROC) analysis was performed, to determine the optimal cut-offs based on the results. Optimal EtG cut-off concentrations were determined to be 7.30 and 29.85 pg/mg, respectively for abstinence and excessive drinking, which are similar to those proposed by the SoHT. For FAEEs, optimal concentrations were 0.185 and 0.378 ng/mg for abstinence, at 0–3 cm and 0–6 cm hair segments, respectively, both similar to the proposed values. At the cut-off for excessive drinking, similar cut-off values were obtained for both lengths (0.817 and 0.889 ng/mg), but these are not similar to the proposed values. However, they confirm that harmonization of the excessive drinking cut-off value for FAEEs can be possible, at a concentration close to 0.8 ng/mg, regardless the length of the hair segment. The use of hair washing products, such as hair conditioner and mask, was found to be associated with lower concentrations of EtG in hair. Cosmetic treatments as bleaching and/or dyeing were associated with the same effect. No effect was associated with the use of hairspray, gel or wax, and for the FAEEs there was no observable effect associated with any of the mentioned products. This demonstrates the importance of documenting the use of hair washing products and cosmetic treatments during sample collection, and the high care that must be taken during result interpretation, considering this information when positivity or negativity for a hair sample is to be given. Both biomarkers correlated with the self-reported consumption habit, while only EtG correlated with the ingested quantities of alcohol per occasion. Inconclusive cases were obtained during combined interpretation of EtG and FAEEs. These cases could mostly be explained by the use of hair products and cosmetic treatments; however, in a number of cases where no simple explanation could be presented, the results of EtG analysis were in agreement with the questionnaire data. This indicates that EtG should be regarded as the first choice in alcohol consumption assessment, while FAEEs should be used to confirm the results from EtG analysis. Only 56.3% of the self-reported abstinent cases could be confirmed by combined analysis of EtG and FAEEs; for moderate drinkers this percentage was 71.6% and for excessive drinkers 60%. Alcohol consumption underestimation or overestimation are assumed as major reasons for this percentages. Combined alcohol biomarker analysis showed that the student population is majorly composed of moderate drinkers (69.39%), followed by abstinent (18.82%) and excessive drinkers (11.69%). Overall, hair analysis for alcohol biomarkers proved to be a powerful tool for the assessment of alcohol consumption in a student population, with considerable advantages. Hair is a sample easy to collect through non-evasive manners, easily stored and the chances of adulteration are low. Therefore, it has good potential to be applied in population studies, especially because it can complete and confirm a great part of the information obtained by questionnaire analysis with added reliability, promoting as such sound and defensible results.
- Detection of biogenic amines in urine and plasma by liquid chromatography coupled to electrochemical detection HPLC-ED using microextraction in packed syringe MEPSPublication . Oppolzer, David Jerónimo; Alba, Maria Eugénia Gallardo; Passarinha, Luís António PaulinoBiogenic amines are neurotransmitters involved in several physiological processes. Changes and disturbs in their function are associated to several neurological disorders or diseases, as well as consumption of psychotherapeutic or psychotropic drugs. The detection of these compounds in biological fluids is therefore of clinical and neurochemical interest. The goal of this work was to develop and validate and analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in both urine and plasma samples. The internal standard used was 3,4-dihydroxybenzylamine (DHBA). The MEPS extraction procedure was optimized using the design of experiments (DOE) tool, and the final conditions of 8 strokes, no washing, and two elutions of 100 FL methanol were chosen. The method was fully validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50-1000 ng/mL for 5-HT and between 5-1000 ng/mL for DA and NE, with determination coefficients (R2) higher than 0.99 for all compounds. The limits of detection and quantification were respectively 20 and 50 ng/mL for 5-HT, and 2 and 5 ng/mL for DA and NE. Intra- and interday precision ranged from 1 to 10 %, while accuracy was within a ±15% interval for all compounds. Authentic urine and plasma samples were analysed by the validated method, and the three compounds were detectable and quantifiable in urine, while only 5-HT was detected and quantified in plasma. This is the first time that a commercially available MEPS column was used for the simultaneous detection of biogenic amines in urine, and also the first time that 5-HT was detected and quantified in plasma using MEPS. MEPS proved to be fast to perform with the use of less solvent volumes, saving time and money and being less laborious. The validated method is useful for the determination of biogenic amines in laboratorial routine.
- Co-delivery of Sildenafil (Viagra®) and Crizotinib for Synergistic and Improved Anti-tumoral TherapyPublication . Marques, João Filipe Gonçalves; Gaspar, Vítor Manuel Abreu; Oppolzer, David; Costa, Elisabete C.; Gallardo, Eugenia; Correia, Ilídio Joaquim SobreiraPurpose Cancer multi-drug resistance is a major issue associated with current anti-tumoral therapeutics. In this work, Crizotinib an anti-tumoral drug approved for the treatment of non-small lung cancer in humans, and Sildenafil (Viagra®), were loaded into micellar carriers to evaluate the establishment of a possible synergistic anti-tumoral effect in breast cancer cells. Methods Micellar carriers comprised by PEG-PLA block co-polymers were formulated by the solvent displacement method in which the simultaneous encapsulation of Crizotinib and Sildenafil was promoted. Encapsulation efficiency was analyzed by a new UPLC method validated for this combination of compounds. Micelle physicochemical characterization and cellular uptake were characterized by light scattering and confocal microscopy. The bio- and hemocompatibility of the carriers was also evaluated. MCF-7 breast cancer cells were used to investigate the synergistic anti-tumoral effect. Results Our results demonstrate that this particular combination induces massive apoptosis of breast cancer cells. The co-delivery of Crizotinib and Sildenafil was only possible due to the high encapsulation efficiency of the micellar systems (>70%). The micelles with size ranging between 93 and 127 nm were internalized by breast cancer cells and subsequently released their payload in the intracellular compartment. The results obtained demonstrated that the delivery of both drugs by micellar carriers led to a 2.7 fold increase in the anti-tumoral effect, when using only half of the concentration that is required when free drugs are administered. Conclusions Altogether, co-delivery promoted a synergistic effect and demonstrated for the first time the potential of PEG-PLA-Crizotinib-Sildenafil combination for application in cancer therapy.