Can amphotericin B and itraconazole be co-delivered orally? Tailoring oral fixed-dose combination coated granules for systemic mycoses.

The incidence and prevalence of invasive fungal infections have increased significantly over the last few years, leading to a global health problem due to the lack of effective treatments. Amphotericin B (AmB) and itraconazole (ITR) are two antifungal drugs with different mechanisms of action. In this work, AmB and ITR have been formulated within granules to elicit an enhanced pharmacological effect, while enhancing the oral bioavailability of AmB. A Quality by Design (QbD) approach was utilised to prepare fixed-dose combination (FDC) granules consisting of a core containing AmB with functional excipients, such as inulin, microcrystalline cellulose (MCC), chitosan, sodium deoxycholate (NaDC) and Soluplus® and polyvinyl pyrrolidone (PVP), coated with a polymeric layer containing ITR with Soluplus® or a combination of Poloxamer 188 and hydroxypropyl methyl cellulose-acetyl succinate (HPMCAS). A Taguchi design of experiments (DoE) with 7 factors and 2 levels was carried out to understand the key factors impacting on the physicochemical properties of the formulation followed by a Box-Behnken design with 3 factors in 3 levels chosen to optimise the formulation parameters. The core of the FDC granules was obtained by wet granulation and later coated using a fluidized bed. In vitro antifungal efficacy was demonstrated by measuring the inhibition halo against different species of Candida spp., including C. albicans (24.19-30.48 mm), C. parapsilosis (26.38-27.84 mm) and C. krusei (11.48-17.92 mm). AmB release was prolonged from 3 to 24 h when the AmB granules were coated. In vivo in CD-1 male mice studies showed that these granules were more selective towards liver, spleen and lung compared to kidney (up to 5-fold more selective in liver, with an accumulation of 8.07 µg AmB/g liver after twice-daily 5 days administration of granules coated with soluplus-ITR), resulting in an excellent oral administration option in the treatment of invasive mycosis. Nevertheless, some biochemical alterations were found, including a decrease in blood urea nitrogen (∼17 g/dl) and alanine aminotransferase (<30 U/l) and an increase in the levels of bilirubin (∼0.2 mg/dl) and alkaline phosphatase (<80 U/l), which could be indicative of a liver failure. Once-daily regimen for 10 days can be a promising therapy.

PET Imaging and Neurohistochemistry Reveal that Curcumin Attenuates Brain Hypometabolism and Hippocampal Damage Induced by Status Epilepticus in Rats.

Numerous preclinical studies provide evidence that curcumin, a polyphenolic phytochemical extracted from Curcuma longa (turmeric) has neuroprotective, anti-inflammatory and antioxidant properties against various neurological disorders. Curcumin neuroprotective effects have been reported in different animal models of epilepsy, but its potential effect attenuating brain glucose hypometabolism, considered as an early marker of epileptogenesis that occurs during the silent period following status epilepticus (SE), still has not been addressed. To this end, we used the lithium-pilocarpine rat model to induce SE. Curcumin was administered orally (300 mg/kg/day, for 17 days). Brain glucose metabolism was evaluated in vivo by 2-deoxy-2-[18F]Fluoro-D-Glucose ([18F]FDG) positron emission tomography (PET). In addition, hippocampal integrity, neurodegeneration, microglia-mediated neuroinflammation, and reactive astrogliosis were evaluated as markers of brain damage. SE resulted in brain glucose hypometabolism accompanied by body weight (BW) loss, hippocampal neuronal damage, and neuroinflammation. Curcumin did not reduce the latency time to the SE onset, nor the mortality rate associated with SE. Nevertheless, it reduced the number of seizures, and in the surviving rats, curcumin protected BW and attenuated the short-term glucose brain hypometabolism as well as the signs of neuronal damage and neuroinflammation induced by the SE. Overall, our results support the potential adaptogen-like effects of curcumin attenuating key features of SE-induced brain damage.

Micro- and Nano-Systems Developed for Tolcapone in Parkinson's Disease.

To date there is no cure for Parkinson's disease (PD), a devastating neurodegenerative disorder with levodopa being the cornerstone of its treatment. In early PD, levodopa provides a smooth clinical response, but after long-term therapy many patients develop motor complications. Tolcapone (TC) is an effective adjunct in the treatment of PD but has a short elimination half-life. In our work, two new controlled delivery systems of TC consisting of biodegradable PLGA 502 (poly (D,L-lactide-co-glycolide acid) microparticles (MPs) and nanoparticles (NPs) were developed and characterized. Formulations MP-TC4 and NP-TC3 were selected for animal testing. Formulation MP-TC4, prepared with 120 mg TC and 400 mg PLGA 502, exhibited a mean encapsulation efficiency (EE) of 85.13%, and zero-order in vitro release of TC for 30 days, with around 95% of the drug released at this time. Formulation NP-TC3, prepared with 10 mg of TC and 50 mg of PLGA 502, exhibited mean EE of 56.69%, particle size of 182 nm, and controlled the release of TC for 8 days. Daily i.p. (intraperitoneal) doses of rotenone (RT, 2 mg/kg) were given to Wistar rats to induce neurodegeneration. Once established, animals received TC in saline (3 mg/kg/day) or encapsulated within formulations MP-TC4 (amount of MPs equivalent to 3 mg/kg/day TC every 14 days) and NP-TC3 (amount of NPs equivalent to 3 mg/kg/day TC every 3 days). Brain analyses of Nissl-staining, GFAP (glial fibrillary acidic protein), and TH (tyrosine hydroxylase) immunohistochemistry as well as behavioral testing (catalepsy, akinesia, swim test) showed that the best formulation was NP-TC3, which was able to revert PD-like symptoms of neurodegeneration in the animal model assayed.

Wound healing, anti-inflammatory and anti-melanogenic activities of ursane-type triterpenes from Semialarium mexicanum (Miers) Mennega.

ETHNO-PHARMACOLOGICAL RELEVANCE: The bark of Semialarium mexicanum commonly known as 'Cancerina' is used as an infusion in Central America and Mexico to treat various wound infections, as well as skin and vaginal ulcers. AIM OF THE STUDY: This study aimed to determine the wound healing, anti-inflammatory and anti-melanogenic activities of the aqueous extract of Semialarium mexicanum and to identify the compounds related to these activities. MATERIALS AND METHODS: A bio-guided isolation of the active compounds of Semialarium mexicanum was carried out, selecting the sub-extracts and fractions depending on their wound healing, anti-inflammatory and anti-melanogenic activities in the RAW 264.7, NIH/3T3 and B16-F10 cells. RESULTS: Three compounds were obtained and characterised by nuclear magnetic resonance and mass spectrometry. These compounds are (3ß)-3-Hydroxy-urs-12-en-28-oic acid (1), (3ß)-Urs-12-ene-3,28-diol (2) and (2α, 19α)-2,19-Dihydroxy-3-oxo-urs-12-en-28-oic acid (3). Regarding the anti-inflammatory activity, the three compounds inhibited the production of NF-κB and NO, however, compound 3 was the most active with IC50 values of 8.15-8.19 µM and 8.94-9.14 µM, respectively, in all cell lines. The anti-melanogenic activity of these compounds was evaluated by the inhibition of tyrosinase and melanin in the B16-F10 cell line. The three compounds showed anti-melanogenic activity, however, compound 3 was the most active with an IC50 of 8.03 µM for the inhibition of tyrosinase production, and an IC50 of 8.53 µM for the inhibition of melanin production. Finally, concerning the wound healing activity, the three compounds presented proliferative activity in all the tested cell lines, however, compound 3 showed higher cell proliferation percentages than compounds 1 and 2 (88.89-89.60% compared to 64.92-65.71% and 71.53-71.99%, respectively). CONCLUSION: The wound healing, anti-inflammatory and anti-melanogenic activity of the aqueous extract of Semialarium mexicanum was tested and analysed in the present study, after having isolated three ursane-type triterpenes.

Controlled Release of Highly Hydrophilic Drugs from Novel Poly(Magnesium Acrylate) Matrix Tablets.

The potential of a new poly(magnesium acrylate) hydrogel (PAMgA) as a pharmaceutical excipient for the elaboration of matrix tablets for the extended release of highly hydrophilic drugs was evaluated. The polymer was synthetized with two different crosslinking degrees that were characterized by FTIR and DSC. Their acute oral toxicity was determined in a mouse model, showing no toxicity at doses up to 10 g/kg. Matrix tablets were prepared using metformin hydrochloride as a model drug and the mechanisms involved in drug release (swelling and/or erosion) were investigated using biorrelevant media. This new hydrogel effectively controlled the release of small and highly hydrophilic molecules as metformin, when formulated in matrix tablets for oral administration. The rate of metformin release from PAMgA matrices was mainly controlled by its diffusion through the gel layer (Fickian diffusion). The swelling capacity and the erosion of the matrix tablets influenced the metformin release rate, that was slower at pH 6.8, where polymer swelling is more intensive, than in gastric medium, where matrix erosion is slightly more rapid. The crosslinking degree of the polymer significantly influenced its swelling capacity in acid pH, where swelling is moderate, but not in intestinal fluid, where swelling is more intense.

Cannabidiol Enhances the Passage of Lipid Nanocapsules across the Blood-Brain Barrier Both in Vitro and in Vivo.

Diseases affecting the central nervous system (CNS) should be regarded as a major health challenge due to the current lack of effective treatments given the hindrance to brain drug delivery imposed by the blood-brain barrier (BBB). Since efficient brain drug delivery should not solely rely on passive targeting, active targeting of nanomedicines into the CNS is being explored. The present study is devoted to the development of lipid nanocapsules (LNCs) decorated with nonpsychotropic cannabinoids as pioneering nonimmunogenic brain-targeting molecules and to the evaluation of their brain-targeting ability both in vitro and in vivo. Noticeably, both the permeability experiments across the hCMEC/D3 cell-based in vitro BBB model and the biodistribution experiments in mice consistently demonstrated that the highest brain-targeting ability was achieved with the smallest-sized cannabinoid-decorated LNCs. Importantly, the enhancement in brain targeting achieved with the conjugation of cannabidiol to LNCs outperformed by 6-fold the enhancement observed for the G-Technology (the main brain active strategy that has already entered clinical trials for the treatment of CNS diseases). As the transport efficiency across the BBB certainly determines the efficacy of the treatments for brain disorders, small cannabinoid-decorated LNCs represent auspicious platforms for the design and development of novel therapies for CNS diseases.

Nanotechnology-based drug delivery of ropinirole for Parkinson's disease.

A new drug delivery system is developed for ropinirole (RP) for the treatment of Parkinson's disease (PD) consisting of biodegradable poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The formulation selected was prepared with 8 mg RP and 50 mg PLGA 502. This formulation exhibited mean encapsulation efficiency of 74.8 ± 8.2%, mean particle size lower than 155 nm, the zeta potential of -14.25 ± 0.43 mV and zero-order in vitro release of RP (14.13 ± 0.17 µg/h/10 mg NPs) for 5 d. Daily doses of the neurotoxin rotenone (2 mg/kg) given i.p. to male Wistar rats induced neuronal and behavioral changes similar to those of PD. Once neurodegeneration was established (15 d) animals received RP in saline (1 mg/kg/d for 35 d) or encapsulated within PLGA NPs (amount of NPs equivalent to 1 mg/kg/d RP every 3 d for 35 d). Brain histology and immunochemistry (Nissl-staining, glial fibrillary acidic protein and tyrosine hydroxylase immunohistochemistry) and behavioral testing (catalepsy, akinesia, rotarod and swim test) showed that RP-loaded PLGA NPs were able to revert PD-like symptoms of neurodegeneration in the animal model assayed.

Analgesic and anti-inflammatory controlled-released injectable microemulsion: Pseudo-ternary phase diagrams, in vitro, ex vivo and in vivo evaluation.

PURPOSE: Development of analgesic and anti-inflammatory controlled-released injectable microemulsions utilising lysine clonixinate (LC) as model drug and generally regarded as safe (GRAS) excipients. METHODS: Different microemulsions were optimised through pseudo-ternary phase diagrams and characterised measuring droplet size, viscosity, ex vivo haemolytic activity and in vitro drug release. The anti-inflammatory and analgesic activity was tested in mice (Hot plate test) and rats (Carrageenan-induced paw edema test) respectively and their activity was compared to an aqueous solution of LC salt. RESULTS: The aqueous solution showed a faster and shorter response whereas the optimised microemulsion increased significantly (p<0.01) the potency and duration of the analgesic and anti-inflammatory activity after deep intramuscular injection. The droplet size and the viscosity were key factors to control the drug release from the systems and enhance the effect of the formulations. CONCLUSIONS: The microemulsion consisting of Labrafil®/Lauroglycol®/Polysorbate 80/water with LC (56.25/18.75/15/10, w/w) could be a promising formulation after buccal surgery due to its ability to control the drug release and significantly achieve greater analgesic and anti-inflammatory effect over 24h.

Efficacy of Ropinirole-Loaded PLGA Microspheres for the Reversion of Rotenone- Induced Parkinsonism.

A new controlled delivery system has been developed for ropinirole (RP) for the treatment of Parkinson´s Disease (PD) consisting in PLGA microparticles (MPs) which exhibited in vitro constant release of RP (78.23 µg/day/10 mg MPs) for 19 days. The neuroprotective effects of RP released from MPs were evaluated in SKN-AS cells after exposure to rotenone (20 µM). Cell apoptosis was significantly reduced by RP (100-120 µM). Daily doses of rotenone (2 mg/kg) given i.p. to rats induced neuronal and behavioral changes similar to those of PD. After 15 days, animals received RP in saline (1 mg/kg/day for 45 days) or as MPs at two dose levels (amount of MPs equivalent to 7.5 mg/kg or 15 mg/kg RP given on days 15 and 30). Brain immunochemistry (Nisslstaining, GFAP and TH immunohistochemistry) and behavioral testing (catalepsy, akinesia, rotarod and swim test) showed that animals receiving RP either in solution or encapsulated within the MPs reverted the PD symptoms with the best results obtained in animals receiving RP microspheres at the highest dose assayed, thereby confirming the potential therapeutic interest of the new RP delivery system.

An effective novel delivery strategy of rasagiline for Parkinson's disease.

This is the first report on the efficacy of a new controlled release system developed for rasagiline mesylate (RM) in a rotenone-induced rat model of Parkinson's disease (PD). PLGA microspheres in vitro released RM at a constant rate of 62.3 µg/day for two weeks. Intraperitoneal injection of rotenone (2 mg/kg/day) to Wistar rats produced typical PD symptoms. Catalepsy, akinesia and swim tests outcomes in animals receiving RM either in solution or within microspheres showed a reversal in descent latency when compared to rotenone-treated animals, being this reversal specially pronounced in animals receiving RM microspheres (dose equivalent to 1 mg/kg/day RM injected i.p. every 15 days). Nissl-staining of brain sections showed selective degeneration of the substantia nigra (SNc) dopaminergic neurons in rotenone-treated animals which was markedly reverted by RM microspheres. PET/CT with (18)F-DG resulted in mean increases of accumulation of radiotracer in striatum and SNc of around 40% in animals treated with RM microspheres which also had significant beneficial effects on Bcl-2, Bax, TNF-α mRNA and SOD2 levels as detected by real-time RT-PCR. Our results confirm the robust effect achieved by the new controlled release system developed for RM which exhibited better in vivo efficacy than RM given in solution.

Cytotoxicity and biocompatibility evaluation of a poly(magnesium acrylate) hydrogel synthesized for drug delivery.

We report the synthesis and characterization as well as cytotoxicity and biocompatibility studies of a poly(magnesium acrylate) hydrogel (PAMgA) developed for drug delivery applications. Two hydrogels with different mesh sizes, large and short, were synthesized (L-C PAMgA and S-C PAMgA). The hydrogels were characterized through swelling, FT-IR and DSC. Cytotoxicity in vitro was evaluated on cell line NIH-3T3 fibroblasts via direct contact and two indirect contact methods (MTT and flow citometry). Both PAMgA hydrogels exhibited low cytotoxicity with survival rates higher than 90%. To select their administration route, biocompatibility was evaluated after intraperitoneal, subcutaneous, and oral administration to mice of both hydrogels at different dose ranges. Swelling percentages obtained were 33.3 ± 4.2% and 166.7 ± 8.3% for L-C PAMgA and S-C PAMgA respectively, showing a great difference in both hydrogels. Among the administration routes assayed, the hydrogels were well tolerated after oral administration of a wide dose range (10-500 mg/kg), thereby indicating that both PAMgA hydrogels are excellent candidates for oral administration due to their in vitro biocompatibility and oral non-toxicity. These results together with the fact that their synthesis is simple and inexpensive make them good candidates for the design of oral drug delivery devices.

Relationship between vasodilation capacity and phenolic content of Spanish wines.

We aimed to determine: 1) the concentration of polyphenols in Spanish red wines, 2) the vasodilatory properties of those wines in relation with their polyphenol concentrations and 3) the vasodilation induced by some of these polyphenols in rat aortic rings. In the wines studied the concentration of rutin and kaempferol was high compared with other polyphenols. All wines relaxed precontracted rat aortic rings and this effect was directly related with the concentration of myricetin and kaempferol in the wines. Kaempferol and rutin also induced endothelium-dependent and independent relaxation, kaempferol was more potent. This relaxation was not inhibited by the estrogen receptor alpha antagonist ICI 182,760. Kaempferol also potentiated the endothelium-dependent relaxation induced by acetylcholine, which was reversed by Nw-nitro-l-arginine methyl ester (l-NAME). These findings show a good correlation between the concentration of polyphenols (especially kaempferol) of Spanish red wines and the vasodilatory effect, which may confer on them unique features in the prevention of cardiovascular disease.

Resveratrol treatment controls microbial flora, prolongs shelf life, and preserves nutritional quality of fruit.

Resveratrol is known as a grapevine secondary metabolite with fungicide activity. Its exogenous application on harvested grapes resulted in the reduction of microbial flora growth, and consequently, prolonged shelf life, without affecting the nutritional quality of the fruit. Resveratrol treatment also resulted in being effective on fruit that normally does not accumulate such metabolites as, for example, tomatoes, apples, avocado pears, and peppers. As a result, all treated fruits maintained their post-harvest quality and health longer than the untreated ones. This study demonstrates the potential use of resveratrol as a natural pesticide to reduce post-harvest fungi development on a broad spectrum of fruit types.