Piperine, quercetin, and curcumin identified as promising natural products for topical treatment of cutaneous leishmaniasis.

Leishmania braziliensis (L. braziliensis) causes cutaneous leishmaniasis (CL) in the New World. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administer. Topical treatment would be the ideal option for the treatment of CL. This underscores the urgent need for affordable and effective treatments, with natural compounds being explored as potential solutions. The alkaloid piperine (PIP), the polyphenol curcumin (CUR), and the flavonoid quercetin (QUE), known for their diverse biological properties, are promising candidates to address these parasitic diseases. Initially, the in vitro cytotoxicity activity of the compounds was evaluated using U-937 cells, followed by the assessment of the leishmanicidal activity of these compounds against amastigotes of L. braziliensis. Subsequently, a golden hamster model with stationary-phase L. braziliensis promastigote infections was employed. Once the ulcer appeared, hamsters were treated with QUE, PIP, or CUR formulations and compared to the control group treated with meglumine antimoniate administered intralesionally. We observed that the three organic compounds showed high in vitro leishmanicidal activity with effective concentrations of less than 50 mM, with PIP having the highest activity at a concentration of 8 mM. None of the compounds showed cytotoxic activity for U937 macrophages with values between 500 and 700 mM. In vivo, topical treatment with QUE daily for 15 days produced cured in 100% of hamsters while the effectiveness of CUR and PIP was 83% and 67%, respectively. No failures were observed with QUE. Collectively, our data suggest that topical formulations mainly for QUE but also for CUR and PIP could be a promising topical treatment for CL. Not only the ease of obtaining or synthesizing the organic compounds evaluated in this work but also their commercial availability eliminates one of the most important barriers or bottlenecks in drug development, thus facilitating the roadmap for the development of a topical drug for the management of CL caused by L. braziliensis.

3-styrylcoumarin scaffold-based derivatives as a new approach for leishmaniasis intervention: biological and molecular modeling studies.

Seven 3-styrylcoumarins were tested for antileishmanial activity against Leishmania (Viannia) panamensis amastigotes. Cytotoxic activity was also evaluated against mammalian U-937 cells. The 3-methoxy-4-hydroxy coumarin derivative 6 was the most active with an IC50 of 40.5 µM, and did not reveal any conspicuous toxicity toward mammalian U-937 cells. Therefore, it may have potential to be considered as candidate for antileishmanial drug development. Further, among several druggable Leishmania targets, molecular docking studies revealed that compound 6 had docking preference by the N-myristoyltransferase (Lp-NMT) of Leishmania panamensis, showing a higher docking score of - 10.1 kcal mol-1 than positive controls and making this protein as a presumably druggable target for this compound. On the other hand, molecular dynamics simulations affirm the docking hypothesis, showing a conformational stability of the 6/Lp-NMT complex throughout 100 ns simulation. Moreover, the molecular mechanics/Poisson-Boltzmann surface area method also support the docking findings, revealing a total free energy of binding of - 47.26 ± 0.08 kcal mol-1, and identifying through energy decomposition analysis that those key aminoacids are contributing strongly to ligand binding. Finally, an optimal pharmacokinetic profile was also estimated for 6. Altogether, coumarin 6 could be addressed as starting point for further pharmacological studies concerning the therapeutic leishmaniasis intervention.

Screening of the antileishmanial and antiplasmodial potential of synthetic 2-arylquinoline analogs.

In this study, six analogs of 2-arylquinoline were synthesized and evaluated for their in vitro and in vivo antiplasmodial and leishmanicidal activity. At a later stage, hemolytic activity and druggability were tested in vitro and in silico, respectively, observing as a result: firstly, compounds showed half-maximal effective concentration (EC50) values between 3.6 and 19.3 µM. Likewise, a treatment using the compounds 4a-f caused improvement in most of the treated hamsters and cured some of them. Regarding the antiplasmodial activity, the compounds showed moderate to high activity, although they did not show hemolytic activity. Furthermore, 4e and 4f compounds were not able to control P. berghei infection when administered to animal models. Molecular dynamic simulations, molecular docking and ligand binding affinity indicate good characteristics of the studied compounds, which are expected to be active. And lastly, the compounds are absorbable at the hematoencephalic barrier but not in the gastrointestinal tract. In summary, ADMET properties suggest that these molecules may be used as a safe treatment against Leishmania.

Biological response of degradation products of PEO-modified magnesium on vascular tissue cells, hemocompatibility and its influence on the inflammatory response.

Cardiovascular stenting is the most widely used therapy to treat coronary artery disease caused by partial or total obstruction of the artery due to atherosclerotic plaque formation, with potentially fatal effects. There are different types of stents: bare metal stents, drug-eluting stents, bioabsorbable stents and dual therapy stents. However, they can lead to long-term complications, such as in-stent restenosis and late thrombosis. To reduce these adverse effects, research has focused on biodegradable metallic stents, since they retain the mechanical properties necessary to contain the injured artery while it is being repaired and, once their function has been fulfilled, the stent degrades without altering the system or compromising the patient's health. In this work we have evaluated the biological response of the degradation products of a bare Mg based biomaterial surface-modified by the plasma electrolytic oxidation (PEO) method on vascular tissue cells, hemocompatibility and inflammatory response. The results obtained are compatible with a biosafe material for future use as a cardiovascular implant, but it is necessary to continue with in vivo and mechanical properties tests to ensure and guarantee its use. SIGNIFICANCE STATEMENT: The development of fully bioresorbable stents is a promising alternative for the management of coronary artery disease without causing long-term problems at the implantation site. In this work, the hematological and immunological biocompatibility of bare Mg modified superficially by plasma electrolytic oxidation (PEO-Mg) was evaluated by in vitro and ex vivo assays. PEO-Mg was found to be compatible with blood and immune components surrounding the implantation site with no signs of toxicity to endothelial cells, macrophages, and arterial tissue. In addition, degradation products of PEO-Mg are eliminated by phagocytosis. However, an in-depth study of the physical and mechanical properties and in vivo biocompatibility must be carried out for its future use as a biomedical implant.

Reversing the biofilm-inducing effect of two xanthones from Garcinia mangostana by 3-methyl-2(5H)-furanone and N-butyryl-D-L homoserine lactone.

BACKGROUND: According to the WHO, 12 bacteria cause numerous human infections, including Enterobacteriaceae Klebsiella pneumoniae, and thus represent a public health problem. Microbial resistance is associated with biofilm formation; therefore, it is critical to know the biofilm-inducing potential of various compounds of everyday life. Likewise, the reversibility of biofilms and the modulation of persister cells are important for controlling microbial pathogens. In this work, we investigated the biofilm-inducing effects of xanthones from Garcinia mangostana on Klebsiella pneumoniae. Furthermore, we investigated the reversal effect of 3-methyl-2(5H)-furanone and the formation of persister cells induced by xanthones and their role in modulating the biofilm to the antibiotic gentamicin. METHODS: To analyze the biofilm-inducing role of xanthones from Garcinia mangostana, cultures of K. pneumoniae containing duodenal probe pieces were treated with 0.1-0.001 µM α- and γ-mangostin, and the biofilm levels were measured using spectrophotometry. To determine biofilm reversion, cultures treated with xanthones, or gentamicin were mixed with 3-methyl-2(5H)-furanone or N-butyryl-DL-homoserine lactone. The presence of K. pneumoniae persister cells was determined by applying the compounds to the mature biofilm, and the number of colony-forming units was counted. RESULTS: The xanthones α- and γ-mangostin increased K. pneumoniae biofilm production by 40% with duodenal probes. However, 3-methyl-2(5H)-furanone at 0.001 µΜ reversed biofilm formation by up to 60%. Moreover, adding the same to a culture treated with gentamicin reduced the biofilm by 80.5%. This effect was highlighted when 3-methyl-2(5H)-furanone was administered 6 h later than xanthones. At high concentrations of α-mangostin, persister K. pneumoniae cells in the biofilm were about 5 - 10 times more abundant than cells, whereas, with γ-mangostin, they were about 100 times more. CONCLUSION: Two xanthones, α- and γ-mangostin from G. mangostana, induced biofilm formation in K. pneumoniae and promoted persister cells. However, the biofilm formation was reversed by adding 3-methyl-2(5H)-furanone, and even this effect was achieved with gentamicin. In addition, this compound controlled the persister K. pneumoniae cells promoted by α-mangostin. Thus, synthetic, and natural biofilm-inducing compounds could harm human health. Therefore, avoiding these substances and looking for biofilm inhibitors would be a strategy to overcome microbial resistance and recover antibiotics that are no longer used.

Neglected Zoonotic Diseases: Advances in the Development of Cell-Penetrating and Antimicrobial Peptides against Leishmaniosis and Chagas Disease.

In 2020, the WHO established the road map for neglected tropical diseases 2021-2030, which aims to control and eradicate 20 diseases, including leishmaniosis and Chagas disease. In addition, since 2015, the WHO has been developing a Global Action Plan on Antimicrobial Resistance. In this context, the achievement of innovative strategies as an alternative to replace conventional therapies is a first-order socio-sanitary priority, especially regarding endemic zoonoses in poor regions, such as those caused by Trypanosoma cruzi and Leishmania spp. infections. In this scenario, it is worth highlighting a group of natural peptide molecules (AMPs and CPPs) that are promising strategies for improving therapeutic efficacy against these neglected zoonoses, as they avoid the development of toxicity and resistance of conventional treatments. This review presents the novelties of these peptide molecules and their ability to cross a whole system of cell membranes as well as stimulate host immune defenses or even serve as vectors of molecules. The efforts of the biotechnological sector will make it possible to overcome the limitations of antimicrobial peptides through encapsulation and functionalization methods to obtain approval for these treatments to be used in clinical programs for the eradication of leishmaniosis and Chagas disease.

Temporal Evaluation of the Surface Area of Treated Skin Ulcers Caused by Cutaneous Leishmaniasis and Relation with Optical Parameters in an Animal Model: A Proof of Concept.

Cutaneous leishmaniasis (CL) is a neglected disease caused by an intracellular parasite of the Leishmania genus. CL lacks tools that allow its understanding and treatment follow-up. This article presents the use of metrical and optical tools for the analysis of the temporal evolution of treated skin ulcers caused by CL in an animal model. Leishmania braziliensis and L. panamensis were experimentally inoculated in golden hamsters, which were treated with experimental and commercial drugs. The temporal evolution was monitored by means of ulcers' surface areas, as well as absorption and scattering optical parameters. Ulcers' surface areas were obtained via photogrammetry, which is a procedure that allowed for 3D modeling of the ulcer using specialized software. Optical parameters were obtained from a spectroscopy study, representing the cutaneous tissue's biological components. A one-way ANOVA analysis was conducted to identify relationships between both the ulcers' areas and optical parameters. As a result, ulcers' surface areas were found to be related to the following optical parameters: epidermis thickness, collagen, keratinocytes, volume-fraction of blood, and oxygen saturation. This study is a proof of concept that shows that optical parameters could be associated with metrical ones, giving a more reliable concept during the assessment of a skin ulcer's healing.

Nanoemulsions for increased penetrability and sustained release of leishmanicidal compounds.

In the last decade, the World Health Organization has driven the development of drugs for topical use in patients with cutaneous leishmaniasis (CL), the most prevalent clinical form of leishmaniasis, a neglected tropical disease. The chemicals C6 I, TC1, and TC2 were reported as promising antileishmanial drugs. We aimed to develop a topical nanoformulation that enhances the advantageous effect of C6 I, TC1, and TC2, guaranteeing higher stability and bioavailability of the pharmacologically active components through the topical route. Nanoemulsions were prepared by ultrasonication based on oleic acid (0.5 g). A relation of Tween®-80/ethanol (1:3) and water was obtained; physicochemical characterization of all formulations was performed, and the preliminary stability and transdermal penetration of these nanoemulsions were also investigated. Newtonian-type fluids with high load capacity, 147-273 nm globule size, and -15 to -18 mV zeta potential were obtained with differential permeability rates in the first pig ear skin assay, first-order kinetics-release model for C6 I, and Weibull for TC1 and TC2. The nanoemulsion showed good stability, high encapsulation efficiency, and higher leishmanicidal activity against Leishmania braziliensis with lower cytotoxicity in U937 macrophages. In conclusion, nanoemulsions of ethanol-oleic acid/Tween®-80 increase the activity of compounds with leishmanicidal activity by increasing their penetration and sustained release.

Antileishmanial Activity and In Silico Molecular Docking Studies of Malachra alceifolia Jacq. Fractions against Leishmania mexicana Amastigotes.

Malachra alceifolia Jacq. (family Malvaceae), known as "malva," is a medicinal plant used as a traditional therapy in many regions of America, Africa and Asia. Traditionally, this plant is used in the form of extracts, powder and paste by populations for treating fever, stomachache, inflammation, and parasites. However, the ethnopharmacological validation of M. alceifolia has been scarcely researched. This study showed that the chloroform fraction (MA-IC) and subfraction (MA-24F) of the leaves of M. alceifolia exhibited a potential antileishmanial activity against axenic amastigotes of Leishmania mexicana pifanoi (MHOM/VE/60/Ltrod) and had high and moderate cytotoxic effects on the viability and morphology of macrophages RAW 264.7. This study reports, for the first time, possible terpenoid metabolites and derivatives present in M. alceifolia with activity against some biosynthetic pathways in L. mexicana amastigotes. The compounds from the subfractions MA-24F were highly active and were analyzed by gas chromatography-mass spectrometry (GC-MS) and by a molecular docking study in L. mexicana target protein. This study demonstrates the potential modes of interaction and the theoretical affinity energy of the metabolites episwertenol, α-amyrin and methyl commate A, which are present in the active fraction MA-24F, at allosteric sites of the pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, aldolase, phosphoglucose isomerase, transketolase, arginase and cysteine peptidases A, target proteins in some vital biosynthetic pathways were responsible for the survival of L. mexicana. Some phytoconstituents of M. alceifolia can be used for the search for potential new drugs and molecular targets for treating leishmaniases and infectious diseases. Furthermore, contributions to research and the validation and conservation of traditional knowledge of medicinal plants are needed globally.

Evaluation of Pharmacokinetic and Toxicological Parameters of Arnica Tincture after Dermal Application In Vivo.

Cutaneous leishmaniasis (CL) is classified as a neglected tropical disease by the World Health Organization. As the standard drugs for the treatment of this disease suffer from severe unwanted effects, new effective and safe therapeutic options are required. In our previous work, Arnica tincture showed promising antileishmanial effects in vitro and in vivo. For the potential treatment of human CL patients with Arnica tincture, data on the pharmacokinetic properties of the bioactive, antileishmanial compounds (the sesquiterpene lactone (STL) helenalin and its derivatives) are needed. Therefore, we studied the in vivo absorption of the bioactive compounds after the dermal application of Arnica tincture in rats. Moreover, we analyzed the blood plasma, urine, and feces of the animals by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Although the majority (84%) of the applied STLs (1.0 mg) were absorbed, the concentrations in the plasma, urine, and feces were below the limit of detection (0.3 ng/mL) in the samples for UHPLC-HRMS analysis. This result may be explained by extensive metabolism and slow permeation accompanied by the accumulation of STLs in the skin, as described in our previous work. Accordingly, the plasma concentration of STLs after the topical application of Arnica tincture was very far from a dose where toxicity could be expected. Additionally, tests for corrosive or irritant activity as well as acute and repeated-dose dermal toxicity did not show any positive results after the administration of the amounts of Arnica tincture that would be needed for the treatment of CL. Consequently, in the treatment of CL patients with Arnica tincture, no toxic effects are expected, other than the known sensitization potential of the STLs.

Effect of the variation in the extracellular concentration of l-arginine in the physiology of Leishmania (Viannia) braziliensis and its susceptibility to some antileishmanial drugs.

The knowledge about amino acid metabolism in trypanosomatids is a valuable source of new therapeutic targets. l-arginine is an essential amino acid for Leishmania parasites, and it participates in the synthesis of polyamines, a group of essential nutrients used for nucleic acids, proteins biosynthesis, and redox modulation necessary for proliferation. In the present study, we evaluated the effect of changes in the availability of this amino acid on promastigotes and intracellular amastigotes on U937 macrophages and showed that the absence of l-arginine in culture medium negatively influences the growth and infectivity of Leishmania (Viannia) braziliensis, causing a decrease in the percentage of the infected cells and parasite load tested through light microscopy. In addition, the absence of l-arginine resulted in the parasite's inability to regulate its reactive oxygen species (ROS) production, which persisted for up to 24 h by flow cytometry following the probe H2DCF-DA dye. Moreover, the differentiation of promastigote to amastigote in axenic culture was more significant at low concentrations of l-arginine suggesting that this depletion induces a stress environment to increase this transformation under axenic conditions. No association was established between the availability of l-arginine and the effectiveness of antileishmanial drugs. All these results confirm the importance of l-arginine in L. braziliensis life cycle vital processes, such as its replication and infectivity, as documented in other Leishmania species. Based on these results, we proposed that the l-arginine uptake/metabolism route is possible in exploring new antileishmanial drugs.

Therapeutic Efficacy of Arnica in Hamsters with Cutaneous Leishmaniasis Caused by Leishmania braziliensis and L. tropica.

Leishmaniasis may occur in three different clinical forms, namely, visceral, mucocutaneous and cutaneous, which are caused by different species of trypanosomatid protozoans of the genus Leishmania. Pentavalent antimonials are the leading treatment for cutaneous leishmaniasis despite the hepatic, renal, and cardiac toxicity. In addition, the response of some Leishmania species to pentavalent antimonials is increasingly poorer, and therefore new and more potent therapeutic alternatives are needed. Arnica montana L., Asteraceae, is a traditional medicinal plant of Europe and preparations of its flowers are commonly used externally to treat disorders of the musculoskeletal system as well as superficial inflammatory conditions. Previous studies have shown that Arnica tincture (AT), an ethanolic extract prepared from the flowerheads of Arnica montana as well as isolated Arnica sesquiterpene lactones (STLs) have antileishmanial activity in vitro against L. donovani and L. infantum, as well as in vivo against L. braziliensis. In this work, we studied the in vitro cytotoxicity and antileishmanial activity of AT and STLs against both L. braziliensis and L. tropica. The in vivo therapeutic effect of AT was studied in hamsters with cutaneous Leishmaniasis (CL) caused by experimental infection with L. braziliensis and L. tropica. Furthermore, various semisolid Arnica preparations were also evaluated against L. braziliensis. The STLs and the AT possess a very high in vitro activity against both Leishmania species with median effective concentrations (EC50) ranging from 1.9 to 5.9 µg/mL. The AT was not cytotoxic for human tissue macrophages, skin fibroblasts, and hepatic cells. The therapeutic response of hamsters infected with L. braziliensis to the topical treatment with AT was 87.5% at a dose of 19.2 µg STL/2× day/60 d, 72.7% at doses of 19.2 µg STL/1× d/60 d and 67% at a dose of 38.4 µg STL/2× d/60 d. In turn, the therapeutic response in hamsters infected with L. tropica was 100% when treated at a dose of 19.2 µg STL/2× day/60 d and 71% at a dose of 38.4 µg STL/2× d/60 d. On the other hand, the effectiveness of treatment with glucantime administered intralesionally at a dose of 200 mg/every three days for 30 days was 62.5% for L. braziliensis and 37.5% for L. tropica infection. These results are promising and encourage the implementation of clinical trials with AT in CL patients as a first step to using AT as a drug against CL.

In Silico, In Vitro, and Pharmacokinetic Studies of UBMC-4, a Potential Novel Compound for Treating against Trypanosoma cruzi.

The lack of therapeutic alternatives for the treatment of Chagas disease, a neglected disease, drives the discovery of new drugs with trypanocidal activity. Consequently, we conducted in vitro studies using UBMC-4, a potential Trypanosoma cruzi AKT-like pleckstrin homology (PH) domain inhibitory compound found using bioinformatics tools. The half effective concentration (EC50) on intracellular amastigotes was determined at 1.85 ± 1 µM showing low cytotoxicity (LC50) > 40 µM on human cell lines tested. In order to study the lethal effect caused by the compound on epimastigotes, morphological changes were assessed by scanning and transmission electron microscopy. Progressive alterations such as flagellum inactivation, cell size reduction, nuclear structure alteration, condensation of chromatin towards the nuclear periphery, vacuole formation, and mitochondrial swelling with kinetoplast integrity loss were evidenced. In addition, apoptosis-like markers in T. cruzi were assessed by flow cytometry, demonstrating that the effect of UBMC-4 on T. cruzi AKT-like kinase reduced the tolerance to nutritional stress-triggered, apoptosis-like events, including DNA fragmentation, mitochondrial damage, and loss of plasma membrane integrity. After this, UBMC-4 was formulated for oral administration and pharmacokinetics were analyzed in a mouse model. Finally, upon oral administration of 200 mg/kg in mice, we found that a UBMC-4 plasma concentration remaining in circulation beyond 24 h after administration is well described by the two-compartment model. We conclude that UBMC-4 has an effective trypanocidal activity in vitro at low concentrations and this effect is evident in T. cruzi cell structures. In mice, UBMC-4 was well absorbed and reached plasma concentrations higher than the EC50, showing features that would aid in developing a new drug to treat Chagas disease.

Menthol carbonates as potent antiparasitic agents: synthesis and in vitro studies along with computer-aided approaches.

INTRODUCTION: Despite the number of deaths and the significant economic and social costs associated with Chagas, Leishmaniasis and Malaria diseases worldwide, available drugs are limited and have serious side effects and high toxicity for the patient. Therefore, there is an urgent need for safe, low-cost, and effective treatments. Natural products are an important source of bioactive compounds and there is current interest in finding natural bioactive molecules that can be used for treating these parasitic diseases. In the present study we proposed to evaluate the in vitro antiparasitic activity of new menthol derivatives against Trypanosoma cruzi, Leishmania braziliensis and Plasmodium falciparum; moreover, we propose to explore their mode of action through in silico approaches. MATERIAL AND METHODS: A series of carbonate prodrugs (1-9) were synthesized from menthol with different aliphatic alcohols. Spectroscopic techniques were used to confirm the structures of the synthesized compounds. The cytotoxicity of the compounds was assessed using U-937 cells. In vitro trypanocidal, leishmanicidal and antiplasmodial activity were evaluated using a T. cruzi, L. braziliensis and P. falciparum organism, respectively. In addition, in silico studies were also performed through molecular dynamics simulations and MM-PBSA analysis. RESULTS: The assay revealed that most of the compounds were highly active against intracellular amastigotes of T. cruzi and L. braziliensis, and had moderate activity against the total forms of P. falciparum. Compound 2 was one of the drugs that showed a high selectivity index (SI) for the three organisms evaluated. The prediction of the ADME properties suggests that all the compounds have drug-like molecular properties and the probability to be lead candidates. Finally, molecular dynamics simulations, and MM-PBSA studies indicate that menthol at the substrate binding site of TcDHODH, LbDHODH and PfDHODH is structurally stable in the same order as the natural substrate; also, interactions of menthol with residues involved in the inhibition of TcDHODH and PfDHODH proteins were predicted. CONCLUSIONS: The present study demonstrates that menthol prodrugs are promising antiparasitic agents; however, the mechanisms of action proposed in this study need to be experimentally verified by future enzymatic assays.

Dermal Absorption of Sesquiterpene Lactones from Arnica Tincture.

Arnica tincture is a traditional herbal medicine used to treat blunt injuries, e.g., bruises and squeezes. In addition, a potential new use in the treatment of cutaneous leishmaniasis is currently under investigation. Therefore, detailed information about the dermal absorption of the tincture and especially its bioactive constituents, sesquiterpene lactones (STLs) of the helenalin- and 11α,13-dihydrohelenalin type, is mandatory. Consequently, this article reports on dermal absorption studies of Arnica tincture using diffusion cells and porcine skin as well as two human skin samples with different permeability. The amounts of STLs on the skin surfaces, in skin extracts and in the receptor fluids were quantified by ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS). It was found that Arnica STLs permeated into the receptor fluid already 4 h after the application, but the amount was rather low. Within 48 h, a maximum of 8.4%, 14.6% and 36.4% of STLs permeated through porcine skin, human skin A (trans-epidermal water loss (TEWL) = 11.518 g·m-2·h-1) and the more permeable human skin B (TEWL = 17.271 g·m-2·h-1), respectively. The majority of STLs was absorbed (penetrated into the skin; 97.6%, 97.8% and 99.3%) after 48 h but a huge portion could not be extracted from skin and is expected to be irreversibly bound to skin proteins. To better visualize the analytes in different skin layers, a fluorescence-labeled STL, helenalin 3,4-dimethoxycinnamate, was synthesized. Fluorescence microscopic images depict an accumulation of the fluorescent derivative in the epidermis. For the treatment of local, cutaneous complaints, an enrichment of the bioactive substances in the skin may be considered beneficial.

In Vitro Metabolism of Helenalin Acetate and 11α,13-Dihydrohelenalin Acetate: Natural Sesquiterpene Lactones from Arnica.

Arnica tincture is a herbal medicinal preparation with anti-inflammatory activity which is used traditionally for the topical treatment of blunt injuries as well as rheumatic muscle and joint complaints. Its main bioactive constituents are sesquiterpene lactones (STLs) of the helenalin and 11α,13-dihydrohelenalin types. Besides the mentioned activity, the tincture and its isolated STLs have antileishmanial activity. In a recent in vivo study, a treatment with Arnica tincture cured cutaneous Leishmaniasis (CL) in a golden hamster model. CL is a neglected tropical disease affecting more than two million people every year, for which new treatments are urgently needed. In order to use Arnica tincture on open CL lesions of human patients, it is important to know how the constituents are metabolized. Therefore, in vitro metabolism experiments with liver microsomes of different species (rat, pig and human) were performed with the Arnica STLs helenalin acetate and 11α,13-dihydrohelenalin acetate. Phase I and phase II metabolism experiments were performed, as well as a combination of both. Glutathione conjugation plays a major role in the metabolism of these STLs, as could be expected based on previous reports on their reactivity. Besides glutathione conjugates, several other metabolites were formed, e.g., water conjugates and hydroxides. Our results show for the first time a detailed picture of the metabolism of Arnica STLs. The fast and extensive formation of glutathione conjugates makes it unlikely that low absorbed levels of these compounds, as expected after dermal absorption from Arnica tincture, could be of toxicological concern.

Eugenol carbonate activity against Plasmodium falciparum, Leishmania braziliensis, and Trypanosoma cruzi.

Neglected tropical diseases are a major health problem throughout the world, and there are few effective and safe drugs. In this study, we report the design and synthesis of a novel series of carbonates of eugenol using different aliphatic alcohols and N,N-carbonyldiimidazole. Spectroscopic techniques, including 1 H nuclear magnetic resonance (NMR), 13 C NMR, Fourier transform infrared, and high-resolution mass spectrometry, were used to confirm the structures of the synthesized compounds. In vitro and in silico studies of prodrugs of eugenol were performed to determine their antiplasmodial, trypanocidal, and leishmanicidal activities, and also their cytotoxicity. Compounds were highly active against Leishmania braziliensis and Plasmodium falciparum, whereas the activity shown for Trypanosoma cruzi was moderate. Molecular docking was used to determine a possible mode of action of eugenol against the dihydroorotate dehydrogenase of the three parasites (TcDHODH, LbDHODH, and PfDHODH). Notably, the docking results showed that eugenol not only has binding energy similar to that of the natural substrate (-7.2 and -7.1, respectively) but also has interactions with relevant biological residues of PfDHODH. This result indicates that eugenol could act as a substrate for PfDHODH in the pyrimidine biosynthesis pathway of P. falciparum. In conclusion, the combination of certain aliphatic alcohols and eugenol through a carbonate bond could significantly increase the antiparasitic activity of this class of compounds, which merits further studies.

Variation of intraocular pressure comparing rebound (TONOVET Plus®) and applanation (TONO-PEN VET®) tonometry in New Zealand rabbits treated with Amlodipine® / Variación de la presión intraocular comparando la tonometría de rebote (TONOVET Plus®) y aplanación (TONO-PEN VET®) en conejos de Nueva Zelanda tratados con Amlodipino® / Variação da pressão intraocular comparando a tonometria de rebote (TONOVET Plus®) e de aplanação (TONO-PEN VET®) em coelhos da Nova Zelândia tratados com Amlodipina®

Abstract Rabbits have played an important role in ophthalmological research processes, however in the environment there has been a deficiency of sensitive equipment in measurement of intraocular pressure for this species. To compare intraocular pressure (IOP) measured with rebound (TONOVET Plus®) and applanation (TONO-PEN VET®) tonometry in healthy New Zealand rabbits treated with Amlodipine. Twelve healthy male New Zealand white rabbits, 5 months old; two groups with 6 individuals were used in this study. The experimental group was given Amlodipine® at a dose of 5 mg/kg, once a day for a month. Blood pressure and intraocular pressure were measurement before treatment and 6 h after treatment in a period of one month. A trend is noted where the group of animals treated with amlodipine® presents lower values than the control group, both in systolic and diastolic pressure. The systolic pressure of the control group showed an average of 116.5 ± 7.9 mmHg and 117.4 ± 8.9 mmHg and the treatment group showed an average of 113.6 ± 7.9 mmHg and 111.8 ± 10.0 mmHg (pretreatment and posttreatment respectively). Tonometry: the values obtained using the TONOVET Plus® (19 ± 3 mmHg) are on average higher than the values obtained using the TONO-PEN VET® (17 ± 3 mmHg). It was possible to demonstrate the relationship between systemic blood pressure and intraocular pressure in New Zealand white rabbits. Rebound tonometry showed less variability in results than applanation tonometry. This study confirmed that both tonometers are reliable for measuring IOP in rabbits.

Resumen Los conejos han jugado un papel importante en los procesos de investigación oftalmológica, sin embargo, en el medio ha existido una deficiencia de equipos sensibles en la medición de la presión intraocular para esta especie. Para comparar la presión intraocular (PIO) medida con la tonometría de rebote (TONOVET Plus®) y aplanación (TONO-PEN VET®) en conejos Nueva Zelanda sanos y tratados con Amlodipino, fueron usados doce conejos Nueva Zelanda, machos, sanos, de 5 meses de edad y distribuidos en dos grupos con 6 individuos cada uno. El grupo experimental recibió Amlodipino® en una dosis de 5 mg/kg, una vez al día durante un mes. La presión arterial y la presión intraocular se midieron antes del tratamiento y 6 h después del tratamiento en un período de un mes. Se observa una tendencia donde el grupo de animales tratados con amlodipino® presenta valores más bajos que el grupo control, tanto en presión sistólica como diastólica. La presión sistólica del grupo control mostró un promedio de 116,5 ± 7,9 mmHg y 117,4 ± 8,9 mmHg y el grupo de tratamiento mostró un promedio de 113,6 ± 7,9 mmHg y 111,8 ± 10,0 mmHg (pretratamiento y postratamiento respectivamente). Tonometría: los valores obtenidos con el TONOVET Plus® (19 ± 3 mmHg) son en promedio superiores a los valores obtenidos con el TONO-PEN VET® (17 ± 3 mmHg). Fue posible demostrar la relación entre la presión arterial sistémica y la presión intraocular en conejos blancos Nueva Zelanda. La tonometría de rebote mostró menos variabilidad en los resultados que la tonometría de aplanamiento. Este estudio confirmó que ambos tonómetros son confiables para medir la PIO en conejos.

Resumo Os coelhos têm desempenhado um papel importante nos processos de pesquisa oftalmológica, porém no meio tem havido uma deficiência de equipamentos sensíveis na medida da pressão intraocular para esta espécie. Comparar a pressão intraocular (PIO) medida com tonometria de rebote (TONOVET Plus®) e de aplanação (TONO-PEN VET®) em coelhos saudáveis da Nova Zelândia tratados com Amlodipina®. Foram utilizados neste estudo doze coelhos brancos da Nova Zelândia, machos saudáveis, 5 meses de idade; distribuído em dois grupos com 6 indivíduos cada um. O grupo experimental recebeu Amlodipina® na dose de 5 mg / kg, uma vez ao dia durante um mês. A pressão arterial e a pressão intraocular foram medidas antes do tratamento e 6 horas após o tratamento em um período de um mês. Nota-se uma tendência onde o grupo de animais tratados com amlodipina® apresenta valores inferiores ao grupo controle, tanto na pressão sistólica quanto na diastólica. A pressão sistólica do grupo controle apresentou média de 116,5 ± 7,9 mmHg e 117,4 ± 8,9 mmHg e o grupo tratamento apresentou média de 113,6 ± 7,9 mmHg e 111,8 ± 10,0 mmHg (pré-tratamento e pós-tratamento, respectivamente). Tonometria: os valores obtidos com o TONOVET Plus® (19 ± 3 mmHg) são em média superiores aos valores obtidos com o TONO-PEN VET® (17 ± 3 mmHg). Foi possível demonstrar a relação entre a pressão arterial sistêmica e a pressão intraocular em coelhos brancos da Nova Zelândia. A tonometria de rebote mostrou menos variabilidade nos resultados do que a tonometria de aplanação. Este estudo confirmou que ambos os tonômetros são confiáveis para medir a PIO em coelhos.

In Vitro Evaluation of the Potential Pharmacological Activity and Molecular Targets of New Benzimidazole-Based Schiff Base Metal Complexes.

Metal-based drugs, including lanthanide complexes, have been extremely effective in clinical treatments against various diseases and have raised major interest in recent decades. Hence, in this work, a series of lanthanum (III) and cerium (III) complexes, including Schiff base ligands derived from (1H-benzimidazol-2-yl)aniline, salicylaldehyde, and 2,4-dihydroxybenzaldehyde were synthesized and characterized using different spectroscopic methods. Besides their cytotoxic activities, they were examined in human U-937 cells, primate kidney non-cancerous COS-7, and six other, different human tumor cell lines: U251, PC-3, K562, HCT-15, MCF-7, and SK-LU-1. In addition, the synthesized compounds were screened for in vitro antiparasitic activity against Leishmania braziliensis, Plasmodium falciparum, and Trypanosoma cruzi. Additionally, antibacterial activities were examined against two Gram-positive strains (S. aureus ATCC® 25923, L. monocytogenes ATCC® 19115) and two Gram-negative strains (E. coli ATCC® 25922, P. aeruginosa ATCC® 27583) using the microdilution method. The lanthanide complexes generally exhibited increased biological activity compared with the free Schiff base ligands. Interactions between the tested compounds and model membranes were examined using differential scanning calorimetry (DSC), and interactions with calf thymus DNA (CT-DNA) were investigated by ultraviolet (UV) absorption. Molecular docking studies were performed using leishmanin (1LML), cruzain (4PI3), P. falciparum alpha-tubulin (GenBank sequence CAA34101 [453 aa]), and S.aureus penicillin-binding protein 2a (PBP2A; 5M18) as the protein receptors. The results lead to the conclusion that the synthesized compounds exhibited a notable effect on model membranes imitating mammalian and bacterial membranes and rolled along DNA strands through groove interactions. Interactions between the compounds and studied receptors depended primarily on ligand structures in the molecular docking study.

Diffuse Reflectance Parameters of Treated Leishmaniasis Cutaneous Ulcers and Association with Histopathologies in an Animal Model: A Proof of Concept.

Cutaneous leishmaniasis (CL) is a parasitic disease that produces chronic skin ulcers. Although it has a worldwide presence, it is a neglected disease that still requires novel tools for its management. In order to study the use of optical tools in CL, this article presents a preliminary study of the correlation between CL histopathological and optical parameters. Optical parameters correspond to absorption and scattering coefficients obtained from diffuse reflectance spectra of treated CL in golden hamsters. Independently, histopathological data were collected from the same hamsters. As a result, after Spearman correlation and the Kruskal-Wallis test, inverse correlation was found between absorption/scattering optical parameters and inflammatory histopathological values, such as the scattering parameter related to the diameter of fibroblasts with the histopathological parameters of fibrosis, polymorphonuclear neutrophils, lymphocytes, plasmocytes, hyperplasia, and Leishmania, and the absorption parameter oxygen saturation showed a relation with the granulation tissue histopathological parameter. These correlations agree with the expected behavior of tissue composition during the healing process in CL. The results correspond to a proof of concept that shows that optical diffuse reflectance-based tools and methods could be considered as an alternative to assist in CL diagnosis and treatment follow-up.