Preclinical validation of a new hybrid molecule loaded in liposomes for melanoma management.

The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma.

Topical Allopurinol-Loaded Nanostructured Lipid Carriers: A Novel Approach for Wound Healing Management.

Nanostructured lipid carriers (NLC) have been widely studied as delivery systems for a variety of routes, including the skin. Their composition results in an imperfect lipid matrix, allowing increased drug encapsulation. Allopurinol (AP), a xanthine oxidase inhibitor, is characterized by low water solubility and high melting point, which has hampered its use through the topical route. In this work, AP was incorporated in a NLC formulation to enhance drug-carrier association and skin delivery as a topical approach to treat wounds. AP-NLC system was characterized in terms of size, charge, rheological behavior, and in vitro skin permeation. The in vitro cytotoxicity was evaluated using HaCaT cells. The wound healing efficacy of the AP-NLC formulation on animal skin lesions was evaluated in male Wistar rats. The AP-NLC presented a mean size of 193 ± 15 nm with a PdI of 0.240 ± 0.02, zeta potential values around -49.6 mV, and an encapsulation efficiency of 52.2%. The AP-NLC formulation presented an adequate profile to be used topically, since epidermal and dermal drug retention were achieved. No reduction in HaCaT cells viability was observed at the tested concentrations (AP < 10 µg/mL). The in vivo application of the AP-NLC formulation resulted in the regeneration of skin lesions when compared with non-treated controls.

Development and Mechanistic Insight into the Enhanced Cytotoxic Potential of Parvifloron D Albumin Nanoparticles in EGFR-Overexpressing Pancreatic Cancer Cells.

Pancreatic cancer is one of the most lethal cancers, with an extremely poor prognosis. The development of more effective therapies is thus imperative. Natural origin compounds isolated from Plectranthus genus, such as parvifloron D (PvD), have cytotoxic and antiproliferative activity against human tumour cells. However, PvD is a very low water-soluble compound, being nanotechnology a promising alternative strategy to solve this problem. Therefore, the aim of this study was to optimize a nanosystem for preferential delivery of PvD to pancreatic tumour cells. Albumin nanoparticles (BSA NPs) were produced through a desolvation method. Glucose cross-linking and bioactive functionalization profiles of BSA platform were elucidated and analysed using static lattice atomistic simulations in vacuum. Using the optimized methodology, PvD was encapsulated (yield higher than 80%) while NPs were characterized in terms of size (100-400 nm) and morphology. Importantly, to achieve a preferential targeting to pancreatic cancer cells, erlotinib and cetuximab were attached to the PvD-loaded nanoparticle surface, and their antiproliferative effects were evaluated in BxPC3 and Panc-1 cell lines. Erlotinib conjugated NPs presented the highest antiproliferative effect toward pancreatic tumour cells. Accordingly, cell cycle analysis of the BxPC3 cell line showed marked accumulation of tumour cells in G1-phase and cell cycle arrest promoted by NPs. As a result, erlotinib conjugated PvD-loaded BSA NPs must be considered a suitable and promising carrier to deliver PvD at the tumour site, improving the treatment of pancreatic cancer.

Pharmaceutical Benefits of Fluticasone Propionate Association to Delivery Systems: In Vitro and In Vivo Evaluation.

The objective of the present work was to characterize the ability of liposomes and cyclodextrin (CyD) complexes to modulate the in vivo profile of fluticasone (FTZ). In vitro cell compatibility tests were performed, exposing A549 cells to FTZ in the free form and FTZ associated to liposomes and complexed with CyD. The in vivo fate of a selected FTZ liposomal formulation and of several FTZ CyD complexes was achieved following intranasal instillation or pulmonary administration in BALB/c mice, respectively. For pulmonary administration, an inhalation chamber was constructed to enable the simultaneously pulmonary administration to six mice. Thirty minutes and 3 h after administration, mice were sacrificed, their blood, lungs, livers, and spleens were removed, and FTZ level was determined by HPLC using an extraction procedure. The in vitro tests revealed no toxic effects of FTZ formulations, as cellular viability was always superior to 90% for FTZ concentrations ranging from 5 to 60 µM 72 h after incubation. The in vivo biodistribution results showed that FTZ incorporated in liposomes resulted in 20 and 30 times higher accumulation in the lungs in comparison with free FTZ, at 0.5 and 3 h after i.n. administration, respectively. FTZ associated to Hydroxypropyl-γ-cyclodextrin (HP-CyD) was the complex that permitted the higher accumulation of FTZ in the lungs in comparison with the respective free form. The results also suggest that the inhalation chamber apparatus can effectively facilitate the evaluation of in vivo inhalation. The establishment of an animal model of asthma allows us to further study the therapeutic efficacy of the developed FTZ formulations.

Development of Parvifloron D-loaded Smart Nanoparticles to Target Pancreatic Cancer.

Pancreatic cancer is the eighth leading cause of cancer death worldwide. For this reason, the development of more effective therapies is a major concern for the scientific community. Accordingly, plants belonging to Plectranthus genus and their isolated compounds, such as Parvifloron D, were found to have cytotoxic and antiproliferative activities. However, Parvifloron D is a very low water-soluble compound. Thus, nanotechnology can be a promising delivery system to enhance drug solubility and targeted delivery. The extraction of Parvifloron D from P. ecklonii was optimized through an acetone ultrasound-assisted method and isolated by Flash-Dry Column Chromatography. Then, its antiproliferative effect was selectivity evaluated against different tumor cell lines (IC50 of 0.15 ± 0.05 µM, 11.9 ± 0.7 µM, 21.6 ± 0.5, 34.3 ± 4.1 µM, 35.1 ± 2.2 µM and 32.1 ± 4.3 µM for BxPC3, PANC-1, Ins1-E, MCF-7, HaCat and Caco-2, respectively). To obtain an optimized stable Parvifloron D pharmaceutical dosage form, albumin nanoparticles were produced through a desolvation method (yield of encapsulation of 91.2%) and characterized in terms of size (165 nm; PI 0.11), zeta potential (-7.88 mV) and morphology. In conclusion, Parvifloron D can be efficiently obtained from P. ecklonii and it has shown selective cytotoxicity to pancreatic cell lines. Parvifloron D nanoencapsulation can be considered as a possible efficient alternative approach in the treatment of pancreatic cancer.

Microencapsulated Solid Lipid Nanoparticles as a Hybrid Platform for Pulmonary Antibiotic Delivery.

Solid lipid nanoparticles (SLN) containing rifabutin (RFB), with pulmonary administration purposes, were developed through a technique that avoids the use of organic solvents or sonication. To facilitate their pulmonary delivery, the RFB-loaded SLN were included in microspheres of appropriate size using suitable excipients (mannitol and trehalose) through a spray-drying technique. Confocal analysis microscopy showed that microspheres are spherical and that SLN are efficiently microencapsulated and homogeneously distributed throughout the microsphere matrices. The aerodynamic diameters observed an optimal distribution for reaching the alveolar region. The dry powder's performance during aerosolization and the in vitro drug deposition were tested using a twin-impinger approach, which confirmed that the microspheres can reach the deep lung. Isothermal titration calorimetry revealed that SLN have higher affinity for mannitol than for trehalose. Upon microsphere dissolution in aqueous media, SLN were readily recovered, maintaining their physicochemical properties. When these dry powders reach the deep lung, microspheres are expected to readily dissolve, delivering the SLN which, in turn, will release RFB. The in vivo biodistribution of microencapsulated RFB-SLN demonstrated that the antibiotic achieved the tested organs 15 and 30 min post pulmonary administration. Their antimycobacterial activity was also evaluated in a murine model of infection with a Mycobacterium tuberculosis strain H37Rv resulting in an enhancement of activity against M. tuberculosis infection compared to nontreated animals. These results suggest that RFB-SLN microencapsulation is a promising approach for the treatment of tuberculosis.

Ethosomes for enhanced skin delivery of griseofulvin.

Griseofulvin (GRF) is an important antifungal drug with low bioavailability and, for this reason, a topical formulation with a targeted action and minimal systemic effects, appears to be a preferable solution. GRF poor solubility has limited the development of topical formulations and their release to the market. The aim of this work was to prepare a new GRF formulation for topical application using lipid-based nanosystems; to study its permeation and penetration, cell viability and to evaluate its therapeutic action. Ethosomal systems composed of soy bean phosphatidylcholine, ethanol and water were prepared for incorporating GRF. After the characterization of the vesicles in terms of size, charge and penetrability, permeation through newborn pig using Franz diffusion cells was conducted. Cell viability at different concentrations of the chosen formulation was determined. At last, skin adapted agar diffusion test was performed to assess the therapeutic efficacy of the formulation. GRF vesicles had mean size of 130nm. Permeation and penetration assays revealed that GRF-loaded ethosomes have an adequate profile to be used in a topical formulation since drug retention in the stratum corneum was achieved. Cell viability tests proved this formulation presented no cytotoxicity to HaCaT cells for concentrations below 50µg/mL. The skin diffusion test evidenced the potential of developed formulation to target skin dermatophytes. The results obtained in this study contribute to a new perspective in topical treatment of fungal infections.

Lipid-based nanoformulations of trifluralin analogs in the management of Leishmania infantum infections.

AIM: To improve the potential of trifluralin (TFL) in the management of Leishmania infantum infections through the synthesis of analogs (TFLA) and incorporation in nanoparticulate drug delivery systems (NanoDDS), liposomes and solid lipid nanoparticles, for selective targeting to leishmania infection sites. MATERIAL & METHODS: In vitro screening of 18 TFLA was performed by flow cytometry. NanoDDS were loaded with active TFLA and evaluated for antileishmanial efficacy in mice through determination of parasite burden in liver and spleen. RESULTS: The in vitro testing revealed the most active and nontoxic TFLAs, which were selected for the in vivo studies based on high incorporation in liposomes and lipid nanoparticles (>90%). Selected TFLA nanoformulations showed superior antileishmanial activity in mice (parasite burden >80%), over free TFLA and Glucantime. CONCLUSION: The modification of TFL structure to obtain active TFLA, together with their incorporation in NanoDDS, improved their in vivo performance against L. infantum infection.

Hemisynthetic trifluralin analogues incorporated in liposomes for the treatment of leishmanial infections.

Leishmaniasis, a vector-borne parasitic disease caused by Leishmania protozoa, is one of the most neglected tropical diseases in terms of drug discovery and development. Current treatment is based on a limited number of chemotherapeutic agents all of which present either/or resistance issues, severe toxicities and adverse reactions associated with extended treatment regimens, and high cost of therapy. Dinitroanilines are a new class of drugs with proven in vitro antileishmanial activity. In previous work a liposomal formulation of one dinitroaniline (TFL) was found to be active against Leishmania parasites in a murine model of visceral leishmaniasis (VL) and in the treatment of experimental canine leishmaniasis. In this study we have investigated the use of dinitroaniline analogues (TFL-A) associated to liposomes, as means to further improve TFL antileishmanial activity. The potential of the liposomal formulations was assessed in vitro against Leishmania infantum promastigotes and intracellular amastigotes and in vivo in a murine model of zoonotic VL. Free and liposomal TFL-A were active in vitro against Leishmania parasites, and they also exhibited reduced cytotoxicity and haemolytic activity. Treatment of infected mice with liposomal TFL-A reduced the amastigote loads in the spleen up to 97%, compared with the loads for untreated controls. These findings illustrate that chemical synthesis of new molecules associated with the use of Nano Drug Delivery Systems that naturally target the diseased organs could be a promising strategy for effective management of VL.

Lycopene from tomatoes: vesicular nanocarrier formulations for dermal delivery.

This experimental work aimed to develop a simple, fast, economic, and environmentally friendly process for the extraction of lycopene from tomato and incorporate this lycopene-rich extract into ultradeformable vesicular nanocarriers suitable for topical application. Lycopene extraction was conducted without a cosolvent for 30 min. The extracts were analyzed and incorporated in transfersomes and ethosomes. These formulations were characterized, and the cellular uptake was observed by confocal microscopy. Dermal delivery of lycopene formulations was tested under in vitro and in vivo conditions. Lycopene extraction proved to be quite safe and selective. The vesicular formulation was taken up by the cells, being more concentrated around the nucleus. Epicutaneous application of lycopene formulations decreased the level of anthralin-induced ear swelling by 97 and 87%, in a manner nonstatistically different from the positive control. These results support the idea that the lycopene-rich extract may be a good alternative to the expensive commercial lycopene for incorporation into advanced topical delivery systems.

Formulation of oryzalin (ORZ) liposomes: in vitro studies and in vivo fate.

Oryzalin (ORZ) is a dinitroaniline that has attracted increasing interest for the treatment of leishmaniasis. The possible use of ORZ as an antiparasitic agent is limited by low water solubility associated with an in vivo rapid clearance. The aim of this work was to overcome these unfavorable pharmaceutical limitations potentiating ORZ antileishmanial activity allowing a future clinical use. This was attained by incorporating ORZ in appropriate liposomes that act simultaneously as drug solvent and carrier delivering ORZ to the sites of Leishmania infection. The developed ORZ liposomal formulations efficiently incorporated and stabilised ORZ increasing its concentration in aqueous suspensions at least 150 times without the need of toxic solvents. The incorporation of ORZ in liposomes reduced the in vitro haemolytic activity and cytotoxicity observed for the free drug, while ORZ exhibits a stable association with liposomes during the first 24h after parenteral administration, significantly reducing ORZ blood clearance and elimination from the body. Simultaneously, an increased ORZ delivery was observed in the main organs of leishmanial infection with a 9-13-fold higher accumulation as compared to the free ORZ. These results support the idea that ORZ performance was strongly improved by the incorporation in liposomes. Moreover, ORZ liposomal formulations can be administrated in vivo in aqueous suspensions without the need of toxic solvents. It is expected an improvement in the therapeutic activity of liposomal ORZ that will be tested in future work.

Synthesis, stability, biochemical and pharmacokinetic properties of a new potent and selective 4-oxo-ß-lactam inhibitor of human leukocyte elastase.

The 4-oxo-ß-lactams (azetidine-2,4-diones) are potent acylating agents of the human leukocyte elastase (HLE), a neutrophil serine protease that plays a key role in several inflammatory diseases. A novel 4-oxo-ß-lactam containing a N-(4-(phenylsulphonylmethyl)phenyl) group, 3, was designed as a potential mechanism-based inhibitor capable of undergoing elimination of phenylsulphinate upon Ser-195 acylation. Compound 3 was found to be a potent slow-tight binding inhibitor of HLE, presenting a remarkable second-order rate constant of 1.46 x 106 M⁻¹s⁻¹ and displaying selectivity over the proteinase 3 and cathepsin G. However, liberation of phenylsulphinate was not observed in the hydrolysis of 3 in both pH 7.4 phosphate buffer and human plasma. The C(max) values of 1207 µg/total blood, 179 µg/g spleen and 106 µg/g lung were determined by HPLC, following a single 30 mg/kg dose of 3 given intraperitoneally to NMRI mice, suggesting that the inhibitor distributes well into tissues. Although being a powerful selective inhibitor of HLE, 4-oxo-ß-lactam 3 has a limited stability, being susceptible to off-target reactions (plasma and liver enzymes).

Trifluralin liposomal formulations active against Leishmania donovani infections.

The purpose of this study was to increase the therapeutic index of the antiparasitic drug, trifluralin (TFL), to allow its parenteral administration without the need of toxic solvents. This was achieved by incorporating TFL in liposomes with high loading capacity. These formulations were stable in freeze-dried form during at least one year and in frozen form during at least three months. Therapeutic activity, assessed on a visceral model of infection, showed that TFL liposomes reduced the number of parasites by up to one third or one half as compared to negative control and to free TFL, respectively.

Biochemical changes in arthritic rats: dehydroascorbic and ascorbic acid levels.

The objective of this work was to evaluate the use of vitamin C as a biomarker in the inflammatory phase of the rat adjuvant arthritis and to correlate it with other parameters used for disease evaluation. Paw swelling was used for physical evaluation and the levels of ascorbate and dehydroascorbate in the serum of male rats, before and after adjuvant arthritis induction, were quantified by a high-performance liquid chromatography method (HPLC). The optimised HPLC assay enabled the quantification of both forms of the vitamin in rat sera, with the same extraction method and using different detectors, instead of obtaining dehydroascorbate by subtraction of the total ascorbate measurement. This method was used to follow the severity of adjuvant arthritis and the results were correlated with other already established disease activity parameters. A decrease of ascorbic acid and dehydroascorbic acid was observed with the increase of right paw circumference during the course of adjuvant arthritis. The disease associated changes in the serum concentrations of ascorbic acid, from biosynthesis and from recycling, can be evaluated by the direct quantification of dehydroascorbic acid. This provides some evidence for the potential of the quantification of these biomarkers to study the disease activity, and as a tool for the establishment of therapeutic protocols, to evaluate the anti-inflammatory effect of new drugs or formulations.