Nanostructured lipid carriers containing benznidazole: physicochemical, biopharmaceutical and cellular in vitro studies.

Chagas disease is a neglected endemic disease prevalent in Latin American countries, affecting around 8 million people. The first-line treatment, benznidazole (BNZ), is effective in the acute stage of the disease but has limited efficacy in the chronic stage, possibly because current treatment regimens do not eradicate transiently dormant Trypanosoma cruzi amastigotes. Nanostructured lipid carriers (NLC) appear to be a promising approach for delivering pharmaceutical active ingredients as they can have a positive impact on bioavailability by modifying the absorption, distribution, and elimination of the drug. In this study, BNZ was successfully loaded into nanocarriers composed of myristyl myristate/Crodamol oil/poloxamer 188 prepared by ultrasonication. A stable NLC formulation was obtained, with ≈80% encapsulation efficiency (%EE) and a biphasic drug release profile with an initial burst release followed by a prolonged phase. The hydrodynamic average diameter and zeta potential of NLC obtained by dynamic light scattering were approximately 150 nm and -13 mV, respectively, while spherical and well-distributed nanoparticles were observed by transmission electron microscopy. Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and small-angle X-ray scattering analyses of the nanoparticles indicated that BNZ might be dispersed in the nanoparticle matrix in an amorphous state. The mean size, zeta potential, polydispersity index, and %EE of the formulation remained stable for at least six months. The hemolytic effect of the nanoparticles was insignificant compared to that of the positive lysis control. The nanoparticle formulation exhibited similar performance in vitro against T. cruzi compared to free BNZ. No formulation-related cytotoxic effects were observed on either Vero or CHO cells. Moreover, BNZ showed a 50% reduction in CHO cell viability at 125 µg/mL, whereas NLC-BNZ and non-loaded NLC did not exert a significant effect on cell viability at the same concentration. These results show potential for the development of new nanomedicines against T. cruzi.

Novel Phenobarbital-Loaded Nanostructured Lipid Carriers for Epilepsy Treatment: From QbD to In Vivo Evaluation.

Pharmacological treatments of central nervous system diseases are always challenging due to the restrictions imposed by the blood-brain barrier: while some drugs can effectively cross it, many others, some antiepileptic drugs among them, display permeability issues to reach the site of action and exert their pharmacological effects. The development of last-generation therapeutic nanosystems capable of enhancing drug biodistribution has gained ground in the past few years. Lipid-based nanoparticles are promising systems aimed to improve or facilitate the passage of drugs through biological barriers, which have demonstrated their effectiveness in various therapeutic fields, without signs of associated toxicity. In the present work, nanostructured lipid carriers (NLCs) containing the antiepileptic drug phenobarbital were designed and optimized by a quality by design approach (QbD). The optimized formulation was characterized by its entrapment efficiency, particle size, polydispersity index, and Z potential. Thermal properties were analyzed by DSC and TGA, and morphology and crystal properties were analyzed by AFM, TEM, and XRD. Drug localization and possible interactions between the drug and the formulation components were evaluated using FTIR. In vitro release kinetic, cytotoxicity on non-tumoral mouse fibroblasts L929, and in vivo anticonvulsant activity in an animal model of acute seizures were studied as well. The optimized formulation resulted in spherical particles with a mean size of ca. 178 nm and 98.2% of entrapment efficiency, physically stable for more than a month. Results obtained from the physicochemical and in vitro release characterization suggested that the drug was incorporated into the lipid matrix losing its crystalline structure after the synthesis process and was then released following a slower kinetic in comparison with the conventional immediate-release formulation. The NLC was non-toxic against the selected cell line and capable of delivering the drug to the site of action in an adequate amount and time for therapeutic effects, with no appreciable neurotoxicity. Therefore, the developed system represents a promising alternative for the treatment of one of the most prevalent neurological diseases, epilepsy.

Nebulization of a polyelectrolyte-drug system for systemic hypertension treatment.

Hypertension is a chronic pathology where blood pressure levels are continuously high, causing cardiac, renal, cerebral, and vascular damage leading to early morbi-mortality. This illness is the main risk factor for cardiovascular diseases and the main cause of atrial fibrillation. Atenolol (AT) is a ß-1 blocker drug useful for antihypertension and antiarrhythmic treatments. However, this drug possesses low oral bioavailability associated to its low permeability and extensive hepatic first-pass metabolism. To solve the conventional AT-administration problems, oral controlled-release and transdermal delivery have been reported. In this work, an alternative AT inhalatory system administered by nebulization is presented. This system is based on an ionic complex between acidic groups of alginic acid and cationic groups of AT (AA-AT), which was obtained by spray-drying. Pharmaceutical and biopharmaceutical properties for AA-AT inhalatory administration using a jet nebulizer were investigated. The aerodynamic performance (assayed at different cup-nebulizer loadings) of the nebulized system demonstrated that around 40% of the formulation would deposit in the respiratory membrane, with mass median aerodynamic diameters of 3.4-3.6 µm. The AT carried in the AA-AT system was released adequately by ionic exchange in saline solution by permeation through a cellulose membrane. The presence of AA as polyelectrolyte conferred mucoadhesive properties to the ionic complex. Even at high relative AA-AT concentrations, no cytotoxic effect was detected in A-549 cell line. Finally, the preliminary pharmacokinetic assay in the in vivo model confirmed that AT was absorbed from the lung to the systemic circulation, with a greater plasmatic AUC compared to the pure drug (around 50% higher). Then, the system and the nebulization administration demonstrated potential for drug cardiac targeting.

Parabens inhibit hNaV 1.2 channels.

Propylparaben, a commonly used antimicrobial preservative, has been reported as an anticonvulsant agent targeting neuronal Na+ channels (NaV). However, the specific features of the NaV channel inhibition by this agent have so far not been extensively studied. Moreover, it is still unclear if it shares this pharmacological activity with other parabens. Here, we fully characterized the mechanism of action of the inhibitory effect that propylparaben and benzylparaben induce on human NaV 1.2 channel isoform (hNaV1.2). We established a first approach to know the parabens structural determinants for this channel inhibition. The parabens effects on hNaV1.2 channel mediated currents were recorded using the patch-clamp whole-cell configuration on hNaV1.2 stably transfected HEK293 cells. Propylparaben induced a typical state-dependent inhibition on hNaV1.2 channel carried current, characterized by a left-shift in the steady-state inactivation curve, a prolongation in the time needed for recovery from fast inactivation and a frequency-dependent blocking behavior. The state-dependent inhibition is increased for butylparaben and benzylparaben and diminished for methylparaben, ethylparaben and p-hydroxybenzoic acid (the major metabolite of parabens hydrolysis). Particularly, butylparaben and benzylparaben shift the steady-state inactivation curve 2- and 3-times more than propylparaben, respectively. Parabens are blockers of hNaV1.2 channels, sharing the mechanism of action of most of sodium channel blocking antiseizure drugs. The potency of this inhibition increases with the size of the lipophilic alcoholic residue of the ester group. These results provide a basis for rational drug design directed to generate new potential anticonvulsant agents.

Trypanosoma cruzi Virulence Factors for the Diagnosis of Chagas' Disease.

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.

Hybrid Compounds as Anti-infective Agents.

Hybrid drugs are multi-target chimeric chemicals combining two or more drugs or pharmacophores covalently linked in a single molecule. In the field of anti-infective agents, they have been proposed as a possible solution to drug resistance issues, presumably having a broader spectrum of activity and less probability of eliciting high level resistance linked to single gene product. Although less frequently explored, they could also be useful in the treatment of frequently occurring co-infections. Here, we overview recent advances in the field of hybrid antimicrobials. Furthermore, we discuss some cutting-edge approaches to face the development of designed multi-target agents in the era of omics and big data, namely analysis of gene signatures and multitask QSAR models.

Neglected Tropical Protozoan Diseases: Drug Repositioning as a Rational Option.

Neglected tropical diseases represent a major sanitary problem and a huge economic burden to endemic countries, and are currently expanding to non-endemic countries owing to migration currents. Though long abandoned in the past, recent research on novel therapeutics has already started to show results. Drug repositioning is one of the prominent, more successful strategies to approach the development of new treatments for these diseases. Here we present an overview on the limitations of the current available medications to treat African trypanosomiasis, Chagas disease and Leishmaniasis, along with a review on drug candidates presently undergoing clinical trials and drug candidates identified through drug repositioning initiatives.

Saxagliptin affects long-bone microarchitecture and decreases the osteogenic potential of bone marrow stromal cells.

Diabetes mellitus is associated with a decrease in bone quality and an increase in fracture incidence. Additionally, treatment with anti-diabetic drugs can either adversely or positively affect bone metabolism. In this study we evaluated: the effect of a 3-week oral treatment with saxagliptin on femoral microarchitecture in young male non-type-2-diabetic Sprague Dawley rats; and the in vitro effect of saxagliptin and/or fetal bovine serum (FBS), insulin or insulin-like growth factor-1 (IGF1), on the proliferation, differentiation (Runx2 and PPAR-gamma expression, type-1 collagen production, osteocalcin expression, mineralization) and extracellular-regulated kinase (ERK) activation, in bone marrow stromal cells (MSC) obtained from control (untreated) rats and in MC3T3E1 osteoblast-like cells. In vivo, oral saxagliptin treatment induced a significant decrease in the femoral osteocytic and osteoblastic density of metaphyseal trabecular bone and in the average height of the proximal cartilage growth plate; and an increase in osteoclastic tartrate-resistant acid phosphatase (TRAP) activity of the primary spongiosa. In vitro, saxagliptin inhibited FBS-, insulin- and IGF1-induced ERK phosphorylation and cell proliferation, in both MSC and MC3T3E1 preosteoblasts. In the absence of growth factors, saxagliptin had no effect on ERK activation or cell proliferation. In both MSC and MC3T3E1 cells, saxagliptin in the presence of FBS inhibited Runx2 and osteocalcin expression, type-1 collagen production and mineralization, while increasing PPAR-gamma expression. In conclusion, orally administered saxagliptin induced alterations in long-bone microarchitecture that could be related to its in vitro down-regulation of the ERK signaling pathway for insulin and IGF1 in MSC, thus decreasing the osteogenic potential of these cells.

Metformin prevents anti-osteogenic in vivo and ex vivo effects of rosiglitazone in rats.

Long-term treatment with the insulin-sensitizer rosiglitazone reduces bone mass and increases fracture risk. We have recently shown that orally administered metformin stimulates bone reossification and increases the osteogenic potential of bone marrow progenitor cells (BMPC). In the present study we investigated the effect of a 2-week metformin and/or rosiglitazone treatment on bone repair, trabecular bone microarchitecture and BMPC osteogenic potential, in young male Sprague-Dawley rats. Compared to untreated controls, rosiglitazone monotherapy decreased bone regeneration, femoral metaphysis trabecular area, osteoblastic and osteocytic density, and TRAP activity associated with epiphyseal growth plates. It also decreased the ex vivo osteogenic commitment of BMPC, inducing an increase in PPARγ expression, and a decrease in Runx2/Cbfa1 expression, in AMP-kinase phosphorylation, and in osteoblastic differentiation and mineralization. After monotherapy with metformin, with the exception of PPARγ expression which was blunted, all of the above parameters were significantly increased (compared to untreated controls). Metformin/rosiglitazone co-treatment prevented all the in vivo and ex vivo anti-osteogenic effects of rosiglitazone monotherapy, with a reversion back to control levels of PPARγ, Runx2/Cbfa1 and AMP-kinase phosphorylation of BMPC. In vitro co-incubation of BMPC with metformin and compound C-an inhibitor of AMPK phosphorylation-abrogated the metformin-induced increase in type-1 collagen production, a marker of osteoblastic differentiation. In conclusion, in rodent models metformin not only induces direct osteogenic in vivo and ex vivo actions, but when it is administered orally in combination with rosiglitazone it can prevent several of the adverse effects that this thiazolidenedione shows on bone tissue.