Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Application of Accelerated Predictive Stability Studies in Extemporaneously Compounded Formulations of Chlorhexidine to Assess the Shelf Life.

Industrially fabricated medicines have a well-defined shelf life supported by rigorous studies before their approval for commercialization. However, the shelf life of extemporaneous compounding topical formulations prepared at hospitals tends to be shorter, especially when no data are available to prove a longer stability period. Also, the storage conditions are unknown in many circumstances. Accelerated Predictive Stability (APS) studies have been shown to be a useful tool to predict in a faster and more accurate manner the chemical stability of extemporaneously compounded formulations requiring a minimum amount of formulation, thereby reducing the chemical drug waste per study. Shelf life will be allocated based on scientific data without compromising drug efficacy or safety. In this work, the APS approach was applied to the commercially available Cristalmina® (CR) and an extemporaneously compounded formulation of chlorhexidine (DCHX). A different degradation kinetic was found between DCHX and CR (Avrami vs. zero-order kinetics, respectively). This can explain the different shelf life described by the International Council for Harmonisation of Technical Requirements Registration Pharmaceuticals Human Use (ICH) conditions between both formulations. A predicted stability for the DCHX solution was obtained from the extrapolation of the degradation rate in long-term conditions from the Arrhenius equation. The estimated degradation from the Arrhenius equation for DCHX at 5 °C, 25 °C, and 30 °C at 365 days was 3.1%, 17.4%, and 25.9%, respectively. The predicted shelf life, in which the DCHX content was above 90%, was 26.67 months under refrigerated conditions and 5.75 and 2.24 months at 25 and 30 °C, respectively. Currently, the Spanish National Formulary recommends a shelf life of no longer than 3 months at room temperature for DCHX solution. Based on the predicted APS and confirmed by experimental long-term studies, we have demonstrated that the shelf life of DCHX extemporaneously compounded formulations could be prolonged by up to 6 months.

Effect of enantiomerism on the bioequivalence of a new ibuprofen 600-mg tablet formulation obtained by roller compaction.

The bioequivalence of a new ibuprofen 600-mg film-coated tablet obtained by roller compaction was studied in a crossover study with 22 healthy volunteers. Bioequivalence was analyzed based on (a) the S-enantiomer, (b) the R-enantiomer, and (c) the sum of both enantiomers (representing the results of an achiral assay). The bioequivalence conclusion for ibuprofen products should be based not only on AUC and Cmax but also on tmax since tmax is related to the onset of action. However, it is not possible to ensure if bioequivalence has been demonstrated for tmax as regulators have not defined the acceptance range for the difference between medians of tmax in those cases, where tmax is clinically relevant. In this study, it was possible to conclude bioequivalence for tmax based on S-ibuprofen, though this conclusion might be questioned if the decision is based on R-ibuprofen or the achiral method.

Evaluating the Potential of Ursolic Acid as Bioproduct for Cutaneous and Visceral Leishmaniasis.

Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo efficacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial effects on different Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the effect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.

Efficacy of a poly-aggregated formulation of amphotericin B in treating systemic sporotrichosis caused by Sporothrix brasiliensis.

In severe cases of sporotrichosis, it is recommended to use amphotericin B deoxycholate (D-AMB) or its lipid formulations and/or in association with itraconazole (ITC). Our aim was to evaluate the antifungal efficacy of a poly-aggregated amphotericin B (P-AMB), a nonlipid formulation, compared with D-AMB on systemic sporotrichosis caused by Sporothrix brasiliensis. In vitro assays showed that Sporothrix schenckii sensu stricto and S. brasiliensis yeast clinical isolates were susceptible to low concentrations of P-AMB and D-AMB. Although P-AMB presented a higher minimal inhibitory concentration (MIC) compared to D-AMB, its cytotoxic effect on renal cells and erythrocytes was lower. For the in vivo assays, male BALB/c mice were intravenously infected with S. brasiliensis yeasts, and P-AMB or D-AMB was administered 3 days post-infection. The efficacy of five therapeutic regimens was tested: intravenous monotherapy with P-AMB or D-AMB, intravenous pulsed-therapy with P-AMB or D-AMB, and intravenous therapy with P-AMB, followed by oral ITC. These treatments increased murine survival and controlled the fungal burden in the liver, spleen, lungs, and kidneys. However, only D-AMB monotherapy or the pulsed-therapies with D-AMB or P-AMB led to 100% survival of the mice 45 days post-infection; only pulsed administration of D-AMB was able to control the fungal load in all organs 45 days post-infection. Accordingly, the histopathological findings showed reductions in the fungal burden and inflammatory reactions in these treatment regimens. Together, our results suggest that the P-AMB formulation could be considered as an alternative drug to D-AMB for treating disseminated sporotrichosis.

Permeability Characteristics of a New Antifungal Topical Amphotericin B Formulation with γ-Cyclodextrins.

Amphotericin B is a low soluble broad-spectrum antifungal agent. Cyclodextrins can be added to amphotericin formulations to enhance both their solubility and antifungal properties. Semisolid amphotericin formulations containing gamma cyclodextrin (AGCD) were prepared and compared with two reference formulations-one of them without any solubility enhancer (A) and the other with DMSO (ADMSO). Rheological, the permeability through hairless mouse skin and antifungal characteristics of the different formulations were evaluated. All three semisolid formulations show low thixotropy characteristics. ADMSO was the formulation with the least consistency, lowest viscosity, and greatest extensibility. The AGCD formulation had the opposite behavior and had both the greatest consistency and viscosity and the lowest extensibility. The lowest permeability was obtained with the reference A formulation while both AGCD and ADMSO had a similar permeability enhancement. According to the antimicrobial in vitro efficacy trials, the AGCD formulation showed 45⁻60% more activity than the reference A formulation. It can be concluded that γ-cyclodextrin is a useful excipient to improve the solubility, permeability, and antifungal activity of amphotericin B in semisolid topical formulations.

Agitation Rate and Time for Complete Dissolution in BCS Biowaivers Based on Investigation of a BCS Biowaiver for Dexketoprofen Tablets.

The objective of the present work is to investigate the validity of the existing requirements for BCS biowaivers of immediate release products containing a class I drug in relation to the agitation rate (50 or 75 rpm in the paddle apparatus) and the time limit for complete dissolution (30 min) in the current biowaivers in vitro dissolution tests. Further, the possibility of extensions will be examined since it has been proposed that the time limit for complete dissolution should be revised to 60 min, and also, if cone formation occurs with apparatus 2 at 50 rpm, then a higher agitation rate is acceptable to eliminate it. The development of four generic dexketoprofen immediate release tablets is described. Dexketoprofen is the eutomer of ketoprofen. According to the BCS, dexketoprofen is a class I drug. Three out of the four products failed to show bioequivalence for Cmax in the initial bioequivalence study conducted with the product despite similar but nonrapid dissolution profiles at 50 rpm in the paddle apparatus, or similar and very rapid dissolution profiles at 75 rpm. In conclusion, these data indicate that BCS biowaivers for class I drugs should be granted only when dissolution with the paddle apparatus is complete in 30 min at 50 rpm. The time limit for complete dissolution should not be extended to 60 min. Furthermore, the agitation rate should not be increased to 75 rpm, even in the case of a coning effect.

Oral particle uptake and organ targeting drives the activity of amphotericin B nanoparticles.

There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within N-palmitoyl-N-methyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index.

Unraveling the Impact of the Oil Phase on the Physicochemical Stability and Skin Permeability of Melatonin Gel Formulations.

Melatonin's antioxidant properties make it a valuable component in anti-aging semisolid topical products. This study explores the role of Pemulen®, an acrylic-based viscosifying agent, in stabilizing cream-gel formulations. Remarkably, even at low concentrations (0.4%), Pemulen® successfully produced physicochemical stable topical formulations. In this work, the impact of the ratio of the oily phase-comprising olive oil and isopropyl myristate from 0 to 20%-was investigated to understand the internal microstructure effect on skin permeability, rheological properties, and stability. The formulations exhibited pseudoplastic behavior, with a significant positive correlation (p-value < 0.1) between the oily phase ratio, viscosity, spreadability, skin adhesiveness, and permeability. Formulations without the oil phase exhibited greater skin permeability. However, higher oily phase content enhanced viscosity, spreadability, and skin adhesion. Given that melatonin primarily degrades through oxidation, incorporating antioxidant excipients in semisolid formulations is crucial for maintaining its chemical stability. A quality by design (QbD) approach was used to assess the impact of four excipients-(a) DL-α-tocopheryl acetate (0.05%), (b) ascorbic acid (0.1%), (c) ethylene diamine tetraacetic acid (0.1%), and (d) sodium metabisulphite (0.5%)-on melatonin's stability. Our findings indicate that maintaining the physical stability of the formulation with a 20% oil phase is more critical for protecting melatonin from oxidation than merely adding antioxidant excipients.

Leveraging 3D-printed microfluidic micromixers for the continuous manufacture of melatonin loaded SNEDDS with enhanced antioxidant activity and skin permeability.

Vesicants are chemical warfare agents (CWAs) capable of causing severe skin damage and systemic toxicity. Melatonin, known for its anti-inflammatory and antioxidant properties, can mitigate the effects of these agents. Self-nano-emulsifying drug delivery systems (SNEDDS) containing a high melatonin concentration (5 %, 50 mg/g) were optimized using a quality-by-design approach from biocompatible, non-irritant excipients with a particle size of about 100 nm. The melatonin-loaded SNEDDS showed a 43-fold greater permeability than a conventional melatonin cream. Chemical stability at ambient temperature (25 °C) was maintained for one year. The preparation of optimised melatonin-loaded SNEDDS using a simple mixing method was compared to microfluidic micromixers. Mixing was successfully achieved using a 3D-printed (fused deposition modeling or stereolithography) T-shaped toroidal microfluidic chip (with a channel geometry optimized by computational fluid dynamics), resulting in a scalable, continuous process for the first time with a substantial reduction in preparation time compared to other conventional mixing approaches. No statistically significant differences were observed in the key quality attributes, such as particle size and melatonin loading, between mixing method till kinetic equilibrium solubility is reached and mixing using the 3D-printed micromixers. This scalable, continuous, cost-effective approach improves the overall efficiency of SNEDDS production, reduces the cost of quality control for multiple batches, and demonstrates the potential of continuous microfluidic manufacture with readily customizable 3D-printed micromixers at points of care, such as military bases.

Guiding Clinical Prescription of Topical Extemporaneous Formulations of Sodium Cromoglycate Based on Pharmaceutical Performance.

Cromoglycate (SCG) is widely used for allergy processes, and inflammatory states acting as a mast cell membrane stabilizer that inhibits the histamine and mediator release. Currently, SCG topical extemporaneous compounding formulations are prepared in hospitals and community pharmacies, as no industrial fabricated medicines are available in Spain. The stability of these formulations is unknown. Additionally, there are no clear guidelines on which concentration and vehicle are more suitable to enhance permeation across the skin. In this work, the stability of commonly prescribed topical SCG formulations in clinical practice was evaluated. Different vehicles commonly employed by pharmacists daily for formulating topical SCG were investigated (Eucerinum, Acofar Creamgel, and Beeler's base) at different concentrations, ranging from 0.2 to 2%. The stability of topical extemporaneous compounded SCG formulations can be extended for up to three months at room temperature (25 °C). Creamgel 2% formulations significantly improved the topical permeation of SCG across the skin, being 4.5-fold higher than formulations prepared with Beeler's base. The reason attributed to this performance can be related to the lower droplet size formed upon dilution in aqueous media combined with a lower viscosity, which facilitates its application and extensibility on the skin. The higher the SCG concentration in Creamgel formulations, the higher the permeability across both synthetic membranes and pig skin (p-value < 0.05). These preliminary results can be used as a guide to prompt a rational prescription of topical SCG formulations.

Melatonin as Modulator for Sulfur and Nitrogen Mustard-Induced Inflammation, Oxidative Stress and DNA Damage: Molecular Therapeutics.

Sulfur and nitrogen mustards, bis(2-chloroethyl)sulfide and tertiary bis(2-chloroethyl) amines, respectively, are vesicant warfare agents with alkylating activity. Moreover, oxidative/nitrosative stress, inflammatory response induction, metalloproteinases activation, DNA damage or calcium disruption are some of the toxicological mechanisms of sulfur and nitrogen mustard-induced injury that affects the cell integrity and function. In this review, we not only propose melatonin as a therapeutic option in order to counteract and modulate several pathways involved in physiopathological mechanisms activated after exposure to mustards, but also for the first time, we predict whether metabolites of melatonin, cyclic-3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, and N1-acetyl-5-methoxykynuramine could be capable of exerting a scavenger action and neutralize the toxic damage induced by these blister agents. NLRP3 inflammasome is activated in response to a wide variety of infectious stimuli or cellular stressors, however, although the precise mechanisms leading to activation are not known, mustards are postulated as activators. In this regard, melatonin, through its anti-inflammatory action and NLRP3 inflammasome modulation could exert a protective effect in the pathophysiology and management of sulfur and nitrogen mustard-induced injury. The ability of melatonin to attenuate sulfur and nitrogen mustard-induced toxicity and its high safety profile make melatonin a suitable molecule to be a part of medical countermeasures against blister agents poisoning in the near future.

Exploring flubendazole formulations for use in sheep. Pharmacokinetic evaluation of a cyclodextrin-based solution.

BACKGROUND: Flubendazole (FLBZ) is a poor water solubility broad-spectrum BZD methylcarbamate anthelmintic compound. Cyclodextrins (CDs) are usually used to increase aqueous solubility of poor hydrosoluble compounds. The comparative in vitro aqueous solubility of FLBZ and other BZD anthelmintics in the presence of hydroxypropyl-ß-cyclodextrin (HPßCD) was evaluated in the current work. Additionally, the comparative pharmacokinetic behaviour of FLBZ (and its metabolites) administered by the intraruminal (i.r.) or intraabomasal (i.a.) routes to sheep as either an aqueous CDs-based solution or a conventional carboximethylcellulose (CMC) suspension was assessed. Drug solubility studies involving albendazole, mebendazole, oxfendazole and FLBZ were performed in an aqueous solution (pH 1.2 or 7.4) with or without HPßCD (10%, w/v). The pharmacokinetic study involved two experiments. Experiment 1: In a crossover study, sheep received either a FLBZ-CDs solution (n = 3) or a FLBZ-CMC suspension (n = 3) by the i.r. route (3.8 mg/kg). The treatment Groups were reversed after a 21-days washout period. Experiment 2: sheep (n = 4) were treated by the i.a. route with the FLBZ-CDs solution (3.8 mg/kg). Plasma and abomasal fluid samples were collected between 0 and 72 h post-treatment. Samples were analysed by HPLC. RESULTS: Improvement of FLBZ aqueous solubility due to CDs resulted markedly higher than that observed for mebendazole and albendazole. However, oppositely to what was expected, the absorption-related pharmacokinetic parameters did not show any marked formulation-dependant effect. After the i.a. administration of FLBZ, the AUC and the Tmax of the parent compound were significantly (P < 0.05) reduced, which is consistent with ruminal bypass. CONCLUSION: The administration of FLBZ as a CDs-based solution, does not seem to achieve great practical relevance for parasite control in sheep.

In Vitro and In Vivo Characteristics of Olive Oil as Excipient for Topical Administration.

Oily excipients are vital components of dermatological products. In this study, the in vitro and in vivo characteristics of Wild Olive Oil (WOO) were compared with two other types of olive oils: Extra Virgin Olive Oil (EVOO) and Virgin Olive Oil (VOO). This work has also included Liquid Paraffin (LP) and Rosehip Oil (RO) as reference oils. Melatonin was used in the study as a model drug to demonstrate the antioxidant capacity of the oils. The melatonin carrier capacity and antioxidant performance was related to the degree of unsaturation of the oils and was highest for RO and WOO and lowest for LP. However, the most stable oil to oxidation was LP. The in vivo performance of the oils in the skin of eight healthy volunteers was investigated with a dermoanalyser. The highest increment of oil and hydration in the skin was obtained with RO. The lowest perception of oiliness was described for WOO, which produced the highest increase in elasticity of the skin area where it was applied. An in vitro-in vivo correlation was therefore performed through multivariable analysis (MVA).

Tailoring Rational Manufacturing of Extemporaneous Compounding Oral Dosage Formulations with a Low Dose of Minoxidil.

Low amounts of minoxidil in oral dosage forms are commonly prescribed as anti-alopecic pharmacological treatments. Side effects are usually related to individual susceptibility. However, poor drug content and mass uniformity can lead to a potential risk of overdosing, and higher chances to experience side effects. The impacts of four formulation variables on drug content and mass pharmaceutical quality attributes were studied with an experimental design at two levels. The first variable (A) was the particle size of the direct compression microcrystalline cellulose (MCC) used as a diluent (Avicel® PH 101 vs. LP 200). The second variable (B) was the type of production process (direct filling vs. wet granulation). The third variable (C) was the particle size of riboflavin added as a color mixture indicator agent (granular vs. milled). The fourth variable (D) was the type of oral solid dosage form (capsule vs. tablet). In half of the formulations, the mean minoxidil content and minoxidil uniformity were out of the specification limits of the Pharmacopoeia, demonstrating the importance of carefully selecting the excipients as well as the utilized process when manufacturing low oral dosage minoxidil formulations. The best minoxidil content uniformity was achieved when using MCC LP 200, wet granulation, granular riboflavin, and capsules. However, tablets are the recommended dosage form when utilizing Avicel® PH 101 or direct filling. Meeting these criteria, the content and mass uniformity are more likely to meet the specification limits of the Pharmacopeia. Techniques such as NIR spectroscopy should be implemented to control the quality of extemporaneous compounding formulations with a low dose of active ingredient.

Antileishmanial Drug Discovery and Development: Time to Reset the Model?

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania species. The disease affects humans and animals, particularly dogs, provoking cutaneous, mucocutaneous, or visceral processes depending on the Leishmania sp. and the host immune response. No vaccine for humans is available, and the control relies mainly on chemotherapy. However, currently used drugs are old, some are toxic, and the safer presentations are largely unaffordable by the most severely affected human populations. Moreover, its efficacy has shortcomings, and it has been challenged by the growing reports of resistance and therapeutic failure. This manuscript presents an overview of the currently used drugs, the prevailing model to develop new antileishmanial drugs and its low efficiency, and the impact of deconstruction of the drug pipeline on the high failure rate of potential drugs. To improve the predictive value of preclinical research in the chemotherapy of leishmaniasis, several proposals are presented to circumvent critical hurdles-namely, lack of common goals of collaborative research, particularly in public-private partnership; fragmented efforts; use of inadequate surrogate models, especially for in vivo trials; shortcomings of target product profile (TPP) guides.

One and Two-Step In Vitro-In Vivo Correlations Based on USP IV Dynamic Dissolution Applied to Four Sodium Montelukast Products.

Montelukast is a weak acid drug characterized by its low solubility in the range of pH 1.2 to 4.5, which may lead to dissolution-limited absorption. The aim of this paper is to develop an in vivo predictive dissolution method for montelukast and to check its performance by establishing a level-A in vitro-in vivo correlation (IVIVC). During the development of a generic film-coated tablet formulation, two clinical trials were done with three different experimental formulations to achieve a similar formulation to the reference one. A dissolution test procedure with a flow-through cell (USP IV) was used to predict the in vivo absorption behavior. The method proposed is based on a flow rate of 5 mL/min and changes of pH mediums from 1.2 to 4.5 and then to 6.8 with standard pharmacopoeia buffers. In order to improve the dissolution of montelukast, sodium dodecyl sulfate was added to the 4.5 and 6.8 pH mediums. Dissolution profiles in from the new method were used to develop a level-A IVIVC. One-step level-A IVIVC was developed from dissolution profiles and fractions absorbed obtained by the Loo-Riegelman method. Time scaling with Levy's plot was necessary to achieve a linear IVIVC. One-step differential equation-based IVIVC was also developed with a time-scaling function. The developed method showed similar results to a previously proposed biopredictive method for montelukast, and the added value showed the ability to discriminate among different release rates in vitro, matching the in vivo clinical bioequivalence results.

Toxicology of Blister Agents: Is Melatonin a Potential Therapeutic Option?

Blister or vesicant chemical warfare agents (CWAs) have been widely used in different military conflicts, including World War I and the Iran-Iraq War. However, their mechanism of action is not fully understood. Sulfur and nitrogen mustard exert toxic effects not only through the alkylation of thiol-bearing macromolecules, such as DNA and proteins, but also produce free radicals that can develop direct toxic effects in target organs such as the eyes, skin, and respiratory system. The lack of effective treatments against vesicant CWAs-induced injury makes us consider, in this complex scenario, the use and development of melatonin-based therapeutic strategies. This multifunctional indoleamine could facilitate neutralization of the oxidative stress, modulate the inflammatory response, and prevent the DNA damage, as well as the long-term health consequences mediated by vesicant CWAs-induced epigenetic mechanisms. In this context, it would be essential to develop new galenic formulations for the use of orally and/or topically applied melatonin for the prophylaxis against vesicant CWAs, as well as the development of post-exposure treatments in the near future.

Increased Efficacy of Oral Fixed-Dose Combination of Amphotericin B and AHCC® Natural Adjuvant against Aspergillosis.

Invasive pulmonary aspergillosis represents one of the most serious fungal infections among immunocompromised patients. In this study, we aimed to analyze the in vivo efficacy of prophylactic oral amphotericin B (AMB) encapsulated in modified chitosan-nanoparticles (Nanomerics' Molecular Envelope Technology (MET)) supplemented with a standardized extract of cultured Lentinula edodes mycelia (AHCC®) in a murine model of pulmonary aspergillosis. We determined fungal burden and survival of mice and additionally, we carried out a cytokine analysis in an attempt to understand the immunomodulation of the extract. Our results evidenced equivalent efficacy between orally administered AMB-MET and the intravenous liposomal AMB marketed formulation. Addition of the AHCC® supplement significantly improved efficacy in terms of burden reduction and survival increase of both oral and intravenous AMB therapies compared to the untreated control group. Moreover, a protective effect of the extract was observed in terms of weight loss. Regarding the cytokine profiles, the Th1 immune response was stimulated in treated animals when compared to the control group. This response was marked by an enhancement in the MCP-1, GM-CSF, VEGF, RANTES and IL-17 levels and a decrease in the IL-6, a biomarker related to the severity of the infection.

Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis.

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.