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Background: Current drug for the treatment of endometriosis is not able of completely cure and significant side effects hinder the continuation of treatment. So, the search for new drug candidates is necessary, and the use of plants extracts is highlighted. Babassu oil and copaiba oilresin have several therapeutic properties. We investigated the in vitro effects of two nanoemulsions containing babassu oil (Orbignya speciosa) (SNEDDS-18) and/or copaiba oilresin (Copaifera langsdorffii) (SNEDDS-18/COPA) on cultured human eutopic endometrium stromal cells from endometrial biopsies of patients without (CESC) and with (EuESC) endometriosis, and human stromal cells from biopsies of endometriotic lesions (EctESC). Methods: CESC, EuESC and EctESC were established and treated with SNEDDS-18 and SNEDDS-18/COPA to evaluate its effects on cytotoxicity, cell morphology, proliferation, and signaling pathways. Results: After 48 hours incubation with SNEDDS-18 and SNEDDS-18/COPA, cell viability and proliferation were inhibited, especially in EctESC. The lowest concentration of both nanoemulsions reduced cell viability and proliferation and breakdown the cytoskeleton in EctESCs. After 24h treatment it was observed a decrease in IL-1, TNF-α, and MCP-1, and an increase in IL-10 production. Conclusions: Both nanoemulsions can affect the endometriotic stromal cell behaviors, thus revealing two potential candidates for new phytotherapeutic agents on the management of endometriosis.

Background: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans. Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to high resolution mass spectrometry. Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies with rats. Conclusion: Its extensive characterization and commercial availability suggests that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites.

The third generation of antiepileptic drugs that have been approved by international regulatory agencies between 2007 and 2018 include rufinamide, stiripentol, eslicarbazepine acetate, lacosamide, perampanel, brivaracetam and everolimus. As part of demonstrating their safety profile, stability indicating methods are developed to monitor these drugs and their impurities. In this context, this review describe some characteristics, impurities and the stability indicating methods used for the determination of these drugs and the presence of their related substances. Through a search in official compendia and scientific articles, fifty-six analytical methodologies were identified up to October 2020. The methodologies were developed using techniques of HPLC, UPLC, HPTLC, GC and UV/Vis spectrophotometry. A majority of the methods (∼70%) employed HPLC-UV. A number of these antiepileptic drugs were found to have had a small number of studies related to their stability and for the detection of impurities. The presentation of the current level of research on third generation antiepileptic drugs highlights the need for new stability and safety studies that are necessary to develop new pharmaceutical products containing these drugs.

Background: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 μM for promastigotes and an IC50 of 70 μM for amastigotes. Objective: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems Method: NPs of poly (D,L-lactic-co-glycolic acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for encapsulation efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by transmission electron microscopy (TEM). Results: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite selectivity index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). Conclusion: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

The human oral cavity is colonized by a diversity of microorganisms living harmoniously. When an imbalance occurs in this population, also called dysbiosis, opportunistic microorganisms lead to oral infections. Nystatin, a traditional antifungal, and red propolis, a resinous mixture collected by honeybees from plant source, can be used to control oral infections. Using β-cyclodextrin (βCD) inclusion complexes with nystatin and red propolis may improve the solubility, stability and antimicrobial activity of these substances, making it possible to include these complexes in pharmaceutical formulations. This study aimed to obtain these inclusion complexes by solubilization and characterize and evaluate their antimicrobial activity against microorganisms from the oral cavity. The inclusion complexes showed loss of crystallinity and the infrared spectra showed interactions between the substances. The content of total polyphenol ranged from 4.32-4.62 mg/g, while the content of flavonoids was between 3.05-3.83 mg/g. The red propolis (Prop):βCD(1:1 and 2:1) complexes were the most active against S.aureus, S.epidermidis, S.mutans and C.albicans with Minimum Inhibitory Concentration (MIC) ranging between 32-256 µg/mL. The inclusion complex of nystatin (Nys):βCD(1:1) effectively inhibited C.albicans growth (MIC=4µg/mL). Interestingly, the association between Nys:βCD(1:1) and Prop:βCD(1:1 or 2:1) showed a synergic effect, improving antifungal activity when compared to red propolis complex alone.

Abstract: Prediction of pulmonary metabolites following inhalation of a locally acting pulmonary drug is essential to the successful development of novel inhaled medicines. The lungs present metabolic enzymes, therefore they influence drug disposal and toxicity. The present review provides an overview of alternative methods to evaluate the pulmonary metabolism for the safety and efficacy of pulmonary delivery systems. In vitro approaches for investigating pulmonary drug metabolism were described, including subcellular fractions, cell culture models and lung slices as the main available in vitro methods. In addition, in silico studies are promising alternatives that use specific software to predict pulmonary drug metabolism, determine whether a molecule will react with a metabolic enzyme, the site of metabolism (SoM) and the result of this interaction. They can be used in an integrated approach to delineate the major cytochrome P450 (CYP) isoforms to rationalize the use of in vivo methods. A case study about a combination of experimental and computational approaches was done using fluticasone propionate as an example. The results of three tested software, RSWebPredictor, SMARTCyp and XenoSite, demonstrated greater probability of the fluticasone propionate being metabolized by CYPs 3A4 at the S1 atom of 5-S-fluoromethyl carbothioate group. As the in vitro studies were not able to directly detect pulmonary metabolites, those alternatives in silico methods may reduce animal testing efforts, following the principle of 3Rs (Replacement, Reduction and Refinement), and contribute to the evaluation of pharmacological efficacy and safety profiles of new drugs in development.

In Brazil, health products are subject to health technology and are marketed only after they are registered by regulated companies. It is important to monitor the performance of these products in the market during the post-marketing phase, in an effort to prevent, intervene, and act in response to complaints and adverse events. Objectives: Evaluate and perform a functional benchmarking to identify best practices in health technology monitoring of health product companies, and determine critical points regarding the execution of health technology assessment programs. Methods: The sampling of the target population was non-probabilistic and the investigation was conducted with the collection of different kinds of information related to technical complaint (TC) and adverse event (AE) procedures performed by companies that register health products. Results: A pilot study was performed. After the preparation of a questionnaire, it was applied in a functional benchmarking in 22 medium/large companies that follow-up with consumers of health products. Conclusions: The questionnaire developed throughout the study proved to be a useful tool for the diagnosis of the degree of implementation of health products monitoring procedures. Company “A” appeared to be meeting what Brazilian legislation requires as a standard procedure for health technology monitoring.

Lopinavir/ritonavir is a potent coformulation of protease inhibitors used against HIV infection. Lopinavir is the main responsible for viral load suppression, whereas ritonavir is a pharmacokinetic enhancer. Both of them have recently gained relevance as candidate drugs against severe coronavirus disease (COVID-19). However, significant beneficial effects were not observed in randomized clinical trials. This review summarizes the main physical-chemical, pharmacodynamic, and pharmacokinetic properties of ritonavir and lopinavir, along with the analytical methodologies applied for biological matrices, pharmaceutical formulations, and stability studies. The work also aimed to provide a comprehensive impurity profile for the combined formulation. Several analytical methods in four different pharmacopeias and 37 articles in literature were evaluated and summarized. Chromatographic methods for these drugs frequently use C8 or C18 stationary phases with acetonitrile and phosphate buffer (with ultraviolet detection) or acetate buffer (with tandem mass spectrometry detection) as the mobile phase. Official compendia methods show disadvantages as extended total run time and complex mobile phases. HPLC tandem-mass spectrometry provided high sensitivity in methodologies applied for human plasma and serum samples, supporting the therapeutic drug monitoring in HIV patients. Ritonavir and lopinavir major degradation products arise in alkaline and acidic environments, respectively. Other non-chromatographic methods were also summarized. Establishing the impurity profile for the combined formulation is challenging due to a large number of impurities reported. Easier and faster analytical methods for impurity assessment are still needed.

Central nervous system tuberculosis (CNS-TB) is the most severe form of the disease especially due to the inability of therapeutics to cross the blood-brain barrier (BBB). Clofazimine (CFZ) stands out for presenting high in vitro activity against multi-drug resistant strains of Mycobacterium tuberculosis, however, CFZ physicochemical and pharmacokinetics properties limit drug penetration into the CNS and, consequently, its clinical use. The aim of this work was to develop polymeric nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) loaded with CFZ and functionalized with a transferrin receptor (TfR)-binding peptide, aiming brain drug delivery for CNS-TB treatment by the intravenous route. The poor water solubility and high lipophilicity of CFZ was overcome through its entrapment into PLGA-PEG NPs manufactured by both conventional and microfluidic techniques using the nanoprecipitation principle. In vitro studies in brain endothelial hCMEC/D3 cells demonstrated that CFZ incorporation into the NPs was advantageous to reduce drug cytotoxicity. The TfR-binding peptide-functionalized NPs showed superior cell interaction and higher CFZ permeability across hCMEC/D3 cell monolayers compared to the non-functionalized NP control, thus indicating the efficacy of the functionalization strategy on providing CFZ transport through the BBB in vitro. The functionalized NPs demonstrate suitability for CFZ biological administration, suggested with low plasma protein binding, off-target biodistribution and precise delivery of CFZ towards the brain parenchyma.

Background Rotigotine is a dopaminergic agonist developed for the treatment of Parkinson’s disease and restless leg syndrome. The pure levorotatory enantiomer is marketed in several countries as a transdermal patch. Reports of oxidation and instability in a previous formulation indicate the need to evaluate impurities in both the raw material and pharmaceutical dosage forms of rotigotine to ensure product quality. Objective This review examines the main analytical methods for analyzing rotigotine in raw material and its transdermal patches with the aim of assisting the development of new pharmaceutical formulations and stability studies. Methods Analytical methods based on high-performance liquid chromatography for rotigotine from pharmacopoeias and literature were evaluated. A comparison was made between the methods found in the literature and official rotigotine monographs described by the United States, European, and British Pharmacopoeias, including a discussion of their acceptance limits for impurities related to the drug. The different impurities from the synthesis processes and degradation studies of rotigotine were also evaluated, as well as the main articles that describe methods for assessing their chiral purity. Results Qualified and unofficial official impurities found in forced degradation studies were verified. The methods presented show adequate specificity and selectivity in determining the drug in the presence of its impurities. Conclusions The approached methods are promising, but more detailed studies on the stability of rotigotine are still lacking, mainly in the pharmacokinetic and toxicological characterization of its impurities.

The objective of this study was to develop a dissolution test in order to establish an in vitro–in vivo correlation (IVIVC) model for desvenlafaxine succinate monohydrate (DVSM) extended release (ER) tablets. The in vitro release characteristics of the drug were determined using USP apparatus 1 at 75 rpm, with volume of HCl pH 1.2, acetate buffer solution (ABS) pH 4.5, or phosphate buffer solution (PBS) pH 6.8. In vivo plasma concentrations and pharmacokinetic parameters in healthy volunteers were obtained from a bioequivalence study. The similarity factors f1 and f2 were used to compare the dissolution data. The IVIVC model was developed using fraction dissolved and fraction absorbed of the reference product. For predictability, the results showed that the percentage prediction error (%PE) value of Cmax was 7.63%. The observed low prediction error for Cmax demonstrated that the IVIVC model was valid for this parameter.

In this study we aimed to develop a roflumilast (R) and formoterol fumarate (F) dry powder inhaler formulation (DPI) incorporating HPβCD by spray drying and evaluated if it attenuates the inflammatory process and improves lung function in a murine model of ovalbumin induced allergic asthma. The DPI was characterized by powder X-ray diffraction, thermal analysis, scanning electron microscopy, particle size, density, specific surface area and dynamic vapor sorption analyses. In vitro deposition studies were performed using a NGI, while transepithelial permeability and in vivo effects on lung mechanics and inflammation in a model of allergic asthma were also assessed. The R:F formulation was amorphous with high glass transition temperatures, comprised of wrinkled particles, had low bulk and tapped densities, high surface area, suitable particle size for pulmonary delivery and exhibited no recrystallization even at high relative humidities. MMAD were statistically similar of 4.22 ± 0.19 and 4.32 ± 0.13 µm for F and R, respectively. Fine particle fractions (5 µm) were of more than 50% of the emitted dose. The R:F formulation led to reduced eosinophil infiltration and airway collagen fiber content, yielding decreased airway hyperresponsiveness. In the current asthma model, the R:F formulation combination decreased inflammation and remodeling, thus improving lung mechanics.

Uncaria tomentosa (UT) extracts have been shown to have promising anti-tumor activity. We hypothesized that its incorporation into nanostructured systems could improve the anticancer properties. Here, poly-e-caprolactone (PCL) and poly-d,l-lactide-co-glycolide (PLGA) were employed to generate nanoparticles loaded with UT extract in a single emulsion solvent evaporation method. The nanoparticles were characterized by particle size, zeta potential, morphology and entrapment efficiency along with stability and release profiles. The nanoparticles presented entrapment efficiencies above 60% and a mean diameter below 300nm. UT-PCL nanoparticles presented higher entrapment efficiency and mean particle size as well as a slow release rate. The UT-PLGA nanoparticles showed higher drug loading. Two prostate cancer cell-lines, LNCaP and DU145 that were derived from metastatic sites, served as model systems to assess cytotoxicity and anti-cancer activity. In vitro, both formulations reduced the viability of DU145 and LNCaP cells. Yet, the UT-PLGA nanoparticles showed higher cytotoxicity towards DU145 cells while the UTPCL against LNCaP cells. The results confirm that the incorporation of UT into nanoparticles could enhance its anti-cancer activities that can offer a viable alternative for the treatment of prostrate canner and highlights the potential of nanostructured systems to provide a promising methodology to enhance the activity of natural extracts.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.

Zebrafish has become a popular model organism in several lines of biological research sharing physiological, morphological and histological similarities with mammals. In fact, many human cytochrome P450 (CYP) enzymes have direct orthologs in zebrafish, suggesting that zebrafish xenobiotic metabolic profiles may be similar to those in mammals. The focus of the review is to analyse the studies that have evaluated the metabolite production in zebrafish over the years, either of the drugs themselves or xenobiotics in general (environmental pollutants, natural products, etc.), bringing a vision of how these works were performed and comparing, where possible, with human metabolism. Early studies that observed metabolic production by zebrafish focused on environmental toxicology, and in recent years the main focus has been on toxicity screening of pharmaceuticals and drug candidates. Nevertheless, there is still a lack of standardization of the model and the knowledge of the extent of similarity with human metabolism. Zebrafish screenings are performed at different life stages, typically being carried out in adult fish through in vivo assays, followed by early larval stages and embryos. Studies comparing metabolism at the different zebrafish life stages are also common. As with any non-human model, the zebrafish presents similarities and differences in relation to the profile of generated metabolites compared to that observed in humans. Although more studies are still needed to assess the degree to which zebrafish metabolism can be compared to human metabolism, the facts presented indicate that the zebrafish is an excellent potential model for assessing xenobiotic metabolism.

Benign Prostatic Hyperplasia (BPH) is a common progressive condition that affects the quality of life of older men. Since current pharmacological interventions can cause severe side effects, this study aimed to develop, characterize and evaluate a natural alternative based on the use of babassu oil (BBS) and copaiba oil-resin (COP) incorporated into self-emulsifying drug delivery systems (SEDDS). Initially, the major components of each of the natural oils were analyzed by gas chromatography/mass spectrometry to provide a means to track their incorporation into nanoformulations. An effective analytical methodology was developed to detect lauric acid and β-caryophyllene as chemical tracers for BBS and COP, respectively. The physical characteristics of the resulting nanoemulsions were next analyzed to identify preparations suitable for in vitro toxicity and in vivo tests. The two most promising SEDDS formulations, containing BBS with and without COP, were thermodynamically stable with a zeta potential less than −20 mv and droplet size around 30 nm. In addition, both were non-cytotoxic and genotoxic. These formulations, in rat model of BPH, showed promising potential to reduce the onset of this pathological process. In conclusion, SEDDS containing BBS and COP were successfully developed that could serve as natural alternatives for the prevention of BPH.

The aim of this study was to develop roflumilast dry powder inhaler (DPI) formulations by spray drying using hydroxypropyl-β-cyclodextrin (HPβCD) and to determine their suitability for pulmonary delivery. Different feed solution concentrations, solvent systems and spray drying parameters were used to obtain the formulations which were characterized using X-ray powder diffraction, thermal analysis, scanning electron microscopy, particle size distribution, bulk and tapped density, specific surface area, dynamic vapour sorption, in vitro deposition properties using a Next Generation Impactor (NGI) and transepithelial permeability. Microparticles spray dried from ethanol were wrinkled and amorphous, exhibiting high glass transition temperatures while those from methanol:n-butyl acetate consisted of irregularly shaped porous particles partially crystalline. All formulations presented low density, particle size and residual solvent content exhibiting high depositon in the lower stages of the NGI. Mass median aerodynamic diameters (MMADs) were in the range of 3.32-4.49 μm, with high fine particle fractions (FPF 5 μm). Stability studies demonstrated no significant modifications in the solid-state nature and in the aerolisation performance of the selected formulation which presented a P

In this study, the formation of caffeine/dapsone (CAF/DAP) cocrystals by scalable production methods, such as liquid-assisted grinding (LAG) and spray drying, was investigated in the context of the potential use of processed cocrystal powder for pulmonary delivery. A CAF/DAP cocrystal (1:1 M ratio) was successfully prepared by slow evaporation from both acetone and ethyl acetate. Acetone, ethyl acetate, and ethanol were all successfully used to prepare cocrystals by LAG and spray drying. The powders obtained were characterized by X-ray diffractometry (XRD), differential scanning calorimetry (DSC), thermogravimetry (TGA), and Fourier transform infrared spectroscopy (FTIR). Laser diffraction analysis indicated a median particle size (D50) for spray-dried powders prepared from acetone, ethanol, and ethyl acetate of 5.4 ± 0.7, 5.2 ± 0.1, and 5.1 ± 0.0 μm respectively, which are appropriate sizes for pulmonary delivery by means of a dry powder inhaler. The solubility of the CAF/DAP cocrystal in phosphate buffer pH 7.4, prepared by spray drying using acetone, was 506.5 ± 31.5 μg/mL, while pure crystalline DAP had a measured solubility of 217.1 ± 7.8 μg/mL. In vitro cytotoxicity studies using Calu-3 cells indicated that the cocrystals were not toxic at concentrations of 0.1 and of 1 mM of DAP, while an in vitro permeability study suggested caffeine may contribute to the permeation of DAP by hindering the efflux effect. The results obtained indicate that the CAF/DAP cocrystal, particularly when prepared by the spray drying method, represents a possible suitable approach for inhalation formulations with applications in pulmonary pathologies.

Gliclazide (GLZ) is a second generation hypoglycemic drug used for the treatment of Type 2 diabetes mellitus. The low solubility of GLZ has been described as the rate limiting step for drug dissolution and absorption, thus a prediction of its in vivo behavior based on a discriminative dissolution test should lead to a relevant in vitro-in vivo correlation (IVIVC). The aim of this study was to develop a dissolution method for GLZ modified-release (MR) tablets using an United States Pharmacopeia (USP) apparatus 3 through its evaluation by an IVIVC analysis. Various dissolution parameters were evaluated to establish an in vitro method for GLZ tablets. The final dissolution conditions, referred to as method 3, utilized a 400 µm mesh and 30 dips per minute over a total period of 10 h that included 1h in HCl media (pH 1.2), 2h in acetate buffer solution (pH 4.5), 1 h in phosphate buffer solution (PBS; pH 5.8), 5h in PBS (pH 6.8) and finally 1h in PBS (pH 7.2). The calculated point-to-point IVIVC (R2=0.9970) was significantly greater than other methods. The robustness of method 3 suggests it could be applied to pharmaceutical equivalence studies and for quality control analyses of GLZ.

Roflumilast is a phosphodiesterase type 4 inhibitor that is administered orally as a long-term, in the clinical treatment of chronic obstructive pulmonary disease and asthma. Launched in 2010 for the European market, it currently does not have an official monograph. Here, a reproducible gradient RP-HPLC method was developed and validated for the separation and determination of Roflumilast in the presence of its six major degradation products. Separation was performed on a C18 analytical column (250 × 4.6 mm, 5 µm) with a mobile phase-A of ACN and a phase-B of ammonium acetate buffer (5 mM, pH 4.2) containing triethylamine (0.5% v/v). The most effective RP-HPLC gradient program was determined to be 0/80, 35/10, 36/80, 40/80 (time in minutes/% mobile phase-B). The flow rate was 1.0 ml/min and the column temperature was 25°C. The success of separation of the degradation products with different chemical characteristics was obtained by extending the time of the gradient, changing the proportion of the ...

We have developed a microemulsion (ME)-based hydrogel, containing propylene glycol, Azone®, Labrasol®, isobutanol and water (20:3:18:3:56), for the transdermal delivery of rivaroxaban (RVX). Formulation ME-1:RVX, which was loaded with 0.3 mg/g of RVX, presented as a clear, homogenous fluid with a droplet size of 82.01 ± 6.32 nm and a PdI of 0.207 ± 0.01. To provide gelation properties, 20 % (w/w) of Pluronic® F-127 was added to ME-1:RVX to generate formulation PME-1a. An added benefit was an increased capacity for RVX to 0.4 mg/g (formulation PME-1b). PME-1b displayed spherical droplets with a nanoscale diameter as observed by Transmission Electron Microscopy. The release of RVX from PME-1b was 20.71 ± 0.76 μg/cm2 with a permeation through pig epidermis of 18.32 ± 8.87 μg/cm2 as measured in a Franz Cell for 24 h. PME-1b presented a pseudoplastic behavior, pH value compatible with the skin and good stability over 60 days at room and elevated temperatures. The prothrombin time was assessed for each concentration of RVX obtained in the permeation assay and each demonstrated a relevant anticoagulant activity. PME-1b also presented no cytotoxicity against HaCaT cells. Utilizing GastroPlus® software, an in silico analysis was performed to simulate the delivery of PME-1b through a transdermal system that suggested a minimum dose of RVX for the treatment and prevention of venous thromboembolism could be achieved with an 8 h administration regimen. These results suggest that PME-1b is a promising transdermal formulation for the effective delivery of RVX that could be a viable alternative for the treatment and prevention of venous thromboembolism.

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd.

The preparation of new biocompatible polymer systems could accelerate processes underlying guided bone tissue regeneration (GBR) by acting as adjuvants. Here, different reaction conditions were investigated to engineer nanocomposites based on chitosan and sodium montimorilonite (Na-Mt) that can be used to generate biocompatible membranes. The goal was a balanced combination of flexibility and tensile strength for use in orthopedic and periodontal surgical procedures for improved GBR. The execution of different experimental protocols aimed to control the polymer intercalation process inside the Na-Mt lamellae, and to avoid their exfoliation, by varying its concentration while applying, at same time, ultrasonic energy as a pretreatment. To increase the therapeutic efficacy of the membranes formed, sodium alendronate was included in the compositions as well as plasticizers to optimize their mechanical and physical-chemical properties. All membranes were analyzed by powder x-ray diffraction, nuclear magnetic resonance, differential scanning calorimetry and thermogravimetric analysis, as well as high performance chromatography to characterize the sodium alendronate content. Surface roughness, water absorption (swelling) and tensile strength were evaluated by scanning electron microscope and profilometry. As a model for human osteoblasts, the proliferation of Saos-2 cells was measured by colorimetric assays and microscopy to assess the potential performance of the membranes in GBR. Membranes with improved mechanical and physical-chemical properties along with excellent biocompatibility could be generated from 33% sodium montmorillonite (w/w) intercalated with chitosan and containing sodium alendronate, glycerol (20% [w/w]) and Labrasol® (3% [w/w]) that also supported the induction of Saos-2 cell proliferation and differentiation, exhibiting potential to develop a novel biomaterial device for GBR

The aim of this study was to develop and validate a discriminating in vitro release test to evaluate rivastigmine transdermal patches. The Exelon® Patch was chosen as a model transdermal product. The studies of in vitro release were designed to determine the impact of the official apparatus chosen (USP apparatus 5 and USP apparatus 6), the rotation speed, and the dissolution medium characteristics on the rivastigmine release profile from transdermal patches. Patches with different drug release profiles were tested in order to evaluate the discriminating power of the in vitro release test developed and validated. Variables such as the apparatus type, the dissolution medium, and the rotation speed have a significant influence on the drug release characteristics from a transdermal patch. The in vitro release methodologies using the USP apparatus 5 at 50 rpm and USP apparatus 6 at 25 rpm using the medium phosphate-buffered saline pH 7.4 were considered discriminative and adequate to characterize the rivastigmine (RV) release from a commercial transdermal patch, Exelon® Patch.

Objective: Titanium tetrafluoride (TiF4) is a topical agent used in the control of dental caries; however, it is highly acidic. To minimize this effect, cyclodextrins (CDs) are used. This study evaluated the in vitro potential of TiF4 and β-CD on remineralization. Methods: Forty bovine enamel blocks were selected by microhardness and randomly assigned to four groups (n = 10 per group): control (distilled and deionized water), 1% β-CD solution, 1% TiF4 solution, and TiF4: β-CD solution. The blocks were subjected to a pH cycling regimen for 8 days. After that, samples were evaluated by cross-sectional microhardness (CSMH), scanning electron microscopy (SEM), and energy dispersive spectrometry (EDS). Data were assessed for normality and analyzed using ANOVA and Tukey's tests (α = 0.05). Results: Regarding CSMH, TiF4: β-CD was statistically superior to the control (P = 0.033), β-CD (P = 0.022), and TiF4 (P = 0.006). SEM photomicrography revealed the titanium dioxide coating on slabs treated with TiF4 and TiF4: β-CD. EDS assessment demonstrated the presence of titanium on the surface of slabs treated with TiF4 and TiF4: β-CD. Conclusion: The solution containing the inclusion nanocomplex formed of TiF4 and β-CD was able to reharden the enamel subsurface.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with development of parasite resistance. In order to overcome these problems, efforts have been made to search for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate the leishmanicidal effect of N,N'-disubstituted thioureas against Leishmania amazonensis, with evaluation of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC50 21.48-189.10 µM), with low cytotoxicity on mice peritoneal macrophages (CC50>200 µM), except for thiourea 3e (CC50=49.22 µM). After that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC50 ranging from 70 to 150 µM against L. amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These data indicate that thiourea derivatives are good candidates as leading compounds for the development of new leishmanicidal drugs.

Sildenafil is a potent selective inhibitor of phosphosdiesterase-5 previously used in erectile dysfunction and subsequently approved in 2005 for pulmonary arterial hypertension treatment. Since oral administration of sildenafil shows pharmacokinetic problems with mean absolute bioavailability of 41%, the goal of this work was to develop a novel sildenafil self-emulsifying drug delivery system (SEDDS) for oral absorption improvement and management of dosage. One pharmaceutical solution and four SEDDS containing sildenafil were successfully obtained and SEDDS formed O/W nanoemulsion with droplet size less than 300 nm. The stability studies evidenced that the SEDDS containing 3.3% w/w of sildenafil yielded the best results. The safety of 2-pyrrolidone/isobutanol in oral formulations was assessed in mice and no lethality was achieved in the placebo groups with LD50 of 490 mg/Kg for SEDDS II-3.3, suggesting it as a safe excipient for humans. Therewithal, in silico studies using PBPK models provided the pharmacokinetic profile of sildenafil SEDDS. Subsequently, in silico evaluation indicated that the sildenafil SEDDS droplet size influenced its bioavailability, enhancing absorption, assuring a good pharmacokinetic profile. These findings suggest that the formulations developed here presented the potential to enhance drug oral absorption with the possibility to control drug dosage as they are liquid pharmaceutical formulations.

Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor-positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We carried out a post-marketing surveillance of the generic tamoxifen formulation in current use at INCA, by comparing plasma concentrations of the parent drug and metabolites obtained with the generic vs the reference formulation. Thirty patients participated in an open-label, bracketed protocol, comprising 3 successive phases of 30-32 days each: the generic formulation was used in phases 1 and 3 and the reference formulation in phase 2. Two blood samples were collected in the last 4 days of each phase, for LC-MS/MS quantification of tamoxifen and metabolites in plasma. The median plasma concentrations (ng/mL) for the reference formulation were as follows: tamoxifen, 135.0 (CI 95% 114.2-155.8); endoxifen, 35.3 (30.0-40.8); and 4-hydroxytamoxifen, 4.8 (4.2-5.4). The endoxifen/tamoxifen plasma concentration ratio was 0.27 (0.21-0.25). ANOVA detected no statistically significant difference in plasma concentrations of tamoxifen, metabolites or the endoxifen/tamoxifen ratio among the three phases. The genetic component (rGC) of the CYP2D6-mediated conversion of tamoxifen into endoxifen, estimated using the repeated drug administration procedure across the three phases, was 0.87, pointing to an important component of genetic variability. In conclusion, this first post-marketing surveillance trial of oncologic generic drugs carried out in Brazilian patients verified the switchability between the reference and the generic tamoxifen formulation currently used at our institution. The adopted bracketed protocol adds confidence to this conclusion and may serve as a frame for future trials of post-marketing assessment of other generic drug products.

Injectable solutions containing epinephrine (EPI) and norepinephrine (NE) are not stable, and their degradation is favored mainly by the oxidation of catechol moiety. As studies of these drugs under forced degradation conditions are scarce, herein, we report the identification of their degradation products (DP) in anesthetic formulations by the development of stability-indicating HPLC method. Finally, the risk assessment of the major degradation products was evaluated using in silico toxicity approach. HPLC method was developed to obtain a higher selectivity allowing adequate elution for both drugs and their DPs. The optimized conditions were developed using a C18 HPLC column, sodium 1-octanesulfonate, and methanol (80:20, v/v) as mobile phase, with a flow rate of 1.5 mL/min, UV detection at 199 nm. The analysis of standard solutions with these modifications resulted in greater retention time for EPI and NE, which allow the separation of these drugs from their respective DPs. Then, five DPs were identified and analyzed by in silico studies. Most of the DPs showed important alerts as hepatotoxicity and mutagenicity. To the best of our acknowledgment, this is the first report of a stability-indicating HPLC method that can be used with formulations containing catecholamines.

Leishmaniasis is a neglected tropical disease caused by protozoa of the genusTo develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems.Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM).NPs with thiourea 1 showed an improvedThe results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by

The primary course of melanoma treatment involves surgery in association with chemotherapy. One potential agent is vinorelbine (VNR). While it displays anti-proliferative activity against melanoma cell lines, it presents a low oral bioavailability and intravenous administration often causes irritation, phlebitis and pain at the injection site. The aim of this study was to develop Pluronic® F-127 (PF-127) hydrogels containing VNR for the transdermal route of administration. Hydrogels composed of either 15% (G15), 20% (G20), 23% (G23) or 25% (G25) PF-127 aqueous solutions were loaded with a VNR propylene glycol solution. Their release and permeation profiles for VNR were evaluated in a Franz diffusion cell. In addition, their rheological behavior, visual aspect during storage and the in silico prediction of VNR absorption using GastroPlus® were evaluated. The G20 and G23 formulations showed similar release and permeation profiles with the G20 demonstrating a higher permeation of VNR at 8h (p

Methyldopa is a centrally acting antihypertensive agent, whose pharmacological activity is attributed to the S-α-methyldopa enantiomer. The assessment of enantiomeric purity is an essential quality control test for chiral drugs, as the presence of certain impurities in a drug can interfere with the desired pharmacological effect. The present study was undertaken to evaluate different chiral stationary phases composed of macrocyclic glycopeptides (vancomycin, teicoplanin and aglycone teicoplanin) and the use of superficially porous particles for the efficient separation of methyldopa enantiomers. The reversed-phase and polar ionic elution modes were evaluated, with different proportions of solvents and additives. The influence of the flow rate and temperature on the analysis were also evaluated. Different stationary phases in different elution modes exhibited suitable selectivity and fast analysis. Total separation of the enantiomers were obtained with the teicoplanin and teicoplanin aglycone stationary phases. The teicoplanin aglycone column produced excellent resolution values and very fast separation analyzes. The use of the teicoplanin aglycone chiral stationary phase for the analysis of MDopa enantiomers is innovative and has not yet been explored in the literature. The best method was obtained and validated with teicoplanin aglycone in the reversed-phase mode using 20 mM ammonium acetate buffer pH 4.0/MeOH (20:80), column temperature of 45 °C and flow rate of 1.0 mL/min, which presented a total run time less than 5 min, a resolution of 5.05 and good peak symmetry. The method can be used in pharmaceutical quality control laboratories and support the stereospecific synthesis of the drug.

Tobramycin (TBM) is an aminoglycoside antibiotic mostly used for infections caused by gram-positive although it can be indicated for some gram-negative organisms. Due to the absence of chromophore groups in TBM and its basicity, several different analytical approaches have been described in the literature for its quantification using HPLC that address specific aspects of its physical–chemical properties. Some methods are based on derivatization with chromophores and fluorescence molecules. Others apply techniques for detection such as mass spectrometry, electrochemical pulse detector, amperometric pulse detection, refractive index and evaporative light scattering. There are also approaches to the separation that include capillary and micellar electrophoresis. The applicability of different types of detectors in the quantification of TBM, along with its degradation products, are discussed to present advantages and disadvantages as well as the use of ion pairs and different stationary phases.

Lutein is a carotenoid with antioxidant activity that is present in various dosage forms. The bioavailability of carotenoid from oral dosage formulations depends on their release, dissolution and its permeability through the gastrointestinal tract. Here, a dissolution test was developed for evaluating formulations and the bioavailability was assessed. The test utilized a USP-apparatus II with rotations of 50, 75 and 100rpm in water with P80 at 1, 2 and 5% (w/v). A non-everted rat intestinal sac model was used in conjunction to assess the intestinal permeability. The most discriminative conditions were 100rpm in water with 2% polysorbate 80, which showed profile differences between two formulations. The intestinal permeation studies showed a lag-time and apparent permeability coefficient that were characteristic of highly permeable drugs. We suggest that a dissolution test can be an essential quality control tool for formulations containing compounds as lutein, although not mandatory by the regulation agencies.

In the present study, in vitro permeation experiments in a Franz diffusion cell were performed using different synthetic polymeric membranes and pig ear skin to evaluate a rivastigmine (RV) transdermal drug delivery system. In vitro-in vivo correlations (IVIVC) were examined to determine the best model membrane. In vitro permeation studies across different synthetic membranes and skin were performed for the Exelon(®) Patch (which contains RV), and the results were compared. Deconvolution of bioavailability data using the Wagner-Nelson method enabled the fraction of RV absorbed to be determined and a point-to-point IVIVC to be established. The synthetic membrane, Strat-M™, showed a RV permeation profile similar to that obtained with pig ear skin (R(2)=0.920). Studies with Strat-M™ resulted in a good and linear IVIVC (R(2)=0.991) when compared with other synthetic membranes that showed R(2) values less than 0.90. The R(2) for pig ear skin was 0.982. Strat-M™ membrane was the only synthetic membrane that adequately simulated skin barrier performance and therefore it can be considered to be a suitable alternative to human or animal skin in evaluating transdermal drug transport, potentially reducing the number of studies requiring human or animal samples.

Introduction:One of the most frequent parents’ concerns is that oral antibiotic formulations induce dental damage in their children’s. This study aimed to assess the cariogenic and erosive potentials of 29 pediatric antibiotics.Materials and Methods:Replicates of each antibiotic were analyzed for the concentration of sugars (sucrose, glucose and fructose) and sorbitol by high performance liquid chromatography (HPLC). The pH was determined by digital pHmeter. Titratable acidity was determined in triplicate using the same pHmeter by gradual addition of 0.1N sodium hydroxide (NaOH) until pH 7.0. Viscosity measurements were carried out using a viscosimeter. In order to rank the relative performance of each medicine, the DEA (Data Envelopment Analysis) methodology was used.Results:Sucrose was present in most samples (n=24) with concentrations ranging from 26 to ≈ 100g% (w/w). Only one antibiotic contained sorbitol (66.9g%). Twenty seven antibiotics presented pH values ranging from 4.1 to 6.9 and most of them (n=15) showed the pH below the critical value for dissolution of hydroxyapatite. The values of titratable acidity and viscosity ranged from 0.26 to 40.48 ml and from 20 to 1780cP, respectively. DEA methodology showed that two medicines were distant from the performance frontier (Klaricid®50mg and Zinnat®250mg), which means that these medicines showed the worst performance and, therefore, greater potential for dissolution of dental enamel.Conclusion:Many antibiotics presented high concentration of sugars, high titratable acidity, pH below the critical value and high viscosity which can be considered risk factors for dental caries and erosion, when consumed frequently.

In this work, newly developed nanocomposites based upon lamellar silicates are evaluated to determine their potential in controlling endometriosis. The preparation of the new nanocarriers is detailed, properties characterized and in vitro pharmacological evaluation performed. The nanocomposites in this study were obtained from the reaction of copaiba oil-resin (COPA) with the polymer polyvinylpyrrolidone (PVP K-30). COPA was selected due to its antiinflammatory and anticancer activities along with the organophilic derivatives of sodium montmorillonite, Viscogel B8, S7 and S4. The results indicated that it was feasible to obtain a good yield of a COPA nanocomposite using a simple process. Intercalation was confirmed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). In vitro release experiments demonstrated that COPA was released from the nanocomposite in a delayed fashion. Whereas, in vitro pharmacological studies showed a reduction in viability and proliferation of endometriotic cell cultures upon COPA nanocomposite treatment, suggesting that the system developed here can be a promising alternative therapy for the oral treatment of endometriosis.

The purpose of this study was to prepare engineered particles of rivastigmine hydrogen tartrate (RHT) and to characterize the physicochemical and aerodynamic properties, in comparison to a lactose carrier formulation (LCF). Microparticles were prepared from ethanol/water solutions containing RHT with and without the incorporation of L-leucine (Leu), using a spray dryer. Dry powder inhaler formulations prepared were characterized by scanning electron microscopy, powder X-ray diffraction, laser diffraction particle sizing, ATR-FTIR, differential scanning calorimetry, bulk and tapped density, dynamic vapour sorption and in vitro aerosol deposition behaviour using a next generation impactor. The smooth-surfaced spherical morphology of the spray dried microparticles was altered by adding Leu, resulting in particles becoming increasingly wrinkled with increasing Leu. Powders presented low densities. The glass transition temperature was sufficiently high (>90 °C) to suggest good stability at room temperature. As Leu content increased, spray dried powders presented lower residual solvent content, lower particle size, higher fine particle fraction (FPF

The aim of this work was the development and characterization of nisin-loaded nanoparticles and the evaluation of its potential antifungal activity. Candidiasis is a fungal infection caused by Candida sp. considered as one of the major public health problem currently. The discovery of antifungal agents that present a reduced or null resistance of Candida sp. and the development of more efficient drug release mechanisms are necessary for the improvement of candidiasis treatment. Nisin, a bacteriocin commercially available for more than 50 years, exhibits antibacterial action in food products with potential antifungal activity. Among several alternatives used to modulate antifungal activity of bacteriocins, polymeric nanoparticles have received great attention due to an effective drug release control and reduction of therapeutic dose, besides the minimization of adverse effects by the preferential accumulation in specific tissues. The nisin nanoparticles were prepared by double emulsification and solvent evaporation methods. Nanoparticles were characterized by dynamic light scattering, zeta potential, Fourier transform infrared, X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Antifungal activity was accessed by pour plate method and cell counting using Candida albicans strains. The in vitro release profile and in vitro permeation studies were performed using dialysis bag method and pig vaginal mucosa in Franz diffusion cell, respectively. The results revealed nisin nanoparticles (300 nm) with spherical shape and high loading efficiency (93.88 ± 3.26%). In vitro test results suggest a promising application of these nanosystems as a prophylactic agent in recurrent vulvovaginal candidiasis and other gynecological diseases.

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose.