Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions.

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

Evaluation of Silybin Nanoparticles against Liver Damage in Murine Schistosomiasis mansoni Infection.

Silybin (SIB) is a hepatoprotective drug known for its poor oral bioavailability, attributed to its classification as a class IV drug with significant metabolism during the first-pass effect. This study explored the potential of solid lipid nanoparticles with (SLN-SIB-U) or without (SLN-SIB) ursodeoxycholic acid and polymeric nanoparticles (PN-SIB) as delivery systems for SIB. The efficacy of these nanosystems was assessed through in vitro studies using the GRX and Caco-2 cell lines for permeability and proliferation assays, respectively, as well as in vivo experiments employing a murine model of Schistosomiasis mansoni infection in BALB/c mice. The mean diameter and encapsulation efficiency of the nanosystems were as follows: SLN-SIB (252.8 ± 4.4 nm, 90.28 ± 2.2%), SLN-SIB-U (252.9 ± 14.4 nm, 77.05 ± 2.8%), and PN-SIB (241.8 ± 4.1 nm, 98.0 ± 0.2%). In the proliferation assay with the GRX cell line, SLN-SIB and SLN-SIB-U exhibited inhibitory effects of 43.09 ± 5.74% and 38.78 ± 3.78%, respectively, compared to PN-SIB, which showed no inhibitory effect. Moreover, SLN-SIB-U demonstrated a greater apparent permeability coefficient (25.82 ± 2.2) than PN-SIB (20.76 ± 0.1), which was twice as high as that of SLN-SIB (11.32 ± 4.6) and pure SIB (11.28 ± 0.2). These findings suggest that solid lipid nanosystems hold promise for further in vivo investigations. In the murine model of acute-phase Schistosomiasis mansoni infection, both SLN-SIB and SLN-SIB-U displayed hepatoprotective effects, as evidenced by lower alanine amino transferase values (22.89 ± 1.6 and 23.93 ± 2.4 U/L, respectively) than those in control groups I (29.55 ± 0.7 U/L) and I+SIB (34.29 ± 0.3 U/L). Among the prepared nanosystems, SLN-SIB-U emerges as a promising candidate for enhancing the pharmacokinetic properties of SIB.

N, N'-disubstituted Ureas as Novel Antiplatelet Agents: Synthesis, Pharmacological Evaluation and In Silico Studies.

INTRODUCTION: Thrombotic disorders are among the leading causes of morbidity and mortality worldwide. Drugs used in the prevention and treatment of atherothrombosis have pharmacokinetic limitations and adverse effects such as hemorrhagic conditions, highlighting the importance of developing more effective antiplatelet agents. ethod: In this work, we synthesized N,N'-disubstituted ureas 3a-3j and evaluated their antiplatelet profiles through in vitro, ex vivo, and in silico studies. The synthesized derivatives exhibited a selective inhibitory profile against platelet aggregation induced by arachidonic acid (AA) in vitro, without significantly affecting other aspects of primary hemostasis and blood coagulation. The compounds that showed inhibition greater than 85% were submitted to the analysis of their potency by calculating the concentration required to inhibit 50% of platelet aggregation induced by AA (IC50). Urea derivative 3a was the most potent with IC50 of 1.45 µM. Interestingly, this derivative inhibited more than 90% of platelet aggregation induced by AA ex vivo, with a similar effect to acetylsalicylic acid. In the hemolysis assay, most of the urea derivatives presented values below 10% suggesting good hemocompatibility. Additionally, the compounds tested at 100 µM also showed no cytotoxic effects in HepG2 and Vero cells. RESULT: The in silico results suggested that compound 3a may bind to the key residue of COX-1 similar to AA and known COX-1 inhibitors, and the results are also in agreement with our SAR, which suggests that the inhibition of this enzyme is the most likely mechanism of antiplatelet activity. CONCLUSION: Therefore, these results demonstrated that N,N'-disubstituted ureas are promising candidates for the development of novel antiplatelet agents.

Use of In silico Methodologies to Predict the Bioavailability of Oral Suspensions: A Regulatory Approach.

BACKGROUND: Oral suspensions are heterogeneous disperse systems, and the particle size distribution, crystalline form of the dispersed solid, and composition of the formulation can be listed as parameters that control the drug dissolution rate and its bioavailability. OBJECTIVE: The aim of this work was to develop a discriminative dissolution test, which, in association with in silico methodologies, can make it possible to safely anticipate bioavailability problems. METHODS: Nimesulide and ibuprofen (BCS class II) and cephalexin (BCS class I) oral suspensions were studied. Previously, solid-state structure and particle size in active pharmaceutical ingredients were characterized and the impact of differences on solubility was evaluated for the choice of discriminative medium. Afterwards, particle size distribution (0.1 to 360 µm), dissolution profile, and in vitro permeability in Caco-2 cell of commercial suspensions, were determined. These parameters were used as input for the establishment of the in vitro-in vivo correlation (IVIVC) for the suspensions using the GastroPlus™ with Wagner-Nelson and Loo- Riegelmann deconvolution approach. RESULTS: The predicted/observed pharmacokinetic model showed good correlation coefficients (r) of 0.960, 0.950, and 0.901, respectively. The IVIVC was established for one nimesulide and two ibuprofen suspensions with r between 0.956 and 0.932, and the percent prediction error (%PE) did not exceed 15%. CONCLUSION: In this work, we have performed a complete study combining in vitro/in silico approaches with the aim of anticipating the safety and efficacy of oral pharmaceutical suspensions in order to provide a regulatory tool for this category of products in a faster and more economical way.

Molluscicidal and Cercaricidal Effects of Myrciaria floribunda Essential Oil Nanoemulsion.

Schistosomiasis is a tropical disease transmitted in an aqueous environment by cercariae from the Schistosoma genus. This disease affects 200 million people living in risk areas around the world. The control of schistosomiasis is realized by chemotherapy, wastewater sanitation, health education, and mollusk control using molluscicidal agents. This work evaluates the effects of a nanoemulsion containing essential oil from Myrciaria floribunda leaves as a molluscicidal and cercaricidal agent against Biomphalaria glabrata mollusks and Schistosoma mansoni cercariae. The Myrciaria floribunda essential oil from leaves showed nerolidol, ß-selinene, 1,8 cineol, and zonarene as major constituents. The formulation study suggested the F3 formulation as the most promising nanoemulsion with polysorbate 20 and sorbitan monooleate 80 (4:1) with 5% (w/w) essential oil as it showed a smaller droplet size of approximately 100 nm with a PDI lower than 0.3 and prominent bluish reflection. Furthermore, this nanoemulsion showed stability after 200 days under refrigeration. The Myrciaria floribunda nanoemulsion showed LC50 values of 48.11 µg/mL, 29.66 µg/mL, and 47.02 µg/mL in Biomphalaria glabrata embryos, juveniles, and adult mollusks, respectively, after 48 h and 83.88 µg/mL for Schistosoma mansoni cercariae after 2 h. In addition, a survival of 80% was observed in Danio rerio, and the in silico toxicity assay showed lower overall human toxicity potential to the major compounds in the essential oil compared to the reference molluscicide niclosamide. These results suggest that the nanoemulsion of Myrciaria floribunda leaves may be a promising alternative for schistosomiasis control.

In silico and pharmacological study of N,S-acetal juglone derivatives as inhibitors of the P2X7 receptor-promoted in vitro and in vivo inflammatory response.

Purinergic receptors are transmembrane proteins responsive to extracellular nucleotides and are expressed by several cell types throughout the human body. Among all identified subtypes, the P2×7 receptor has emerged as a relevant target for the treatment of inflammatory disease. Several clinical trials have been conducted to evaluate the effectiveness of P2×7R antagonists. However, to date, no selective antagonist has reached clinical use. In this work, we report the pharmacological evaluation of eleven N, S-acetal juglone derivatives as P2×7R inhibitors. Using in vitro assays and in vivo experimental models, we identified one derivative with promising inhibitory activity and low toxicity. Our in silico studies indicate that the 1,4-naphthoquinone moiety might be a valuable molecular scaffold for the development of novel P2×7R antagonists, as suggested by our previous studies.

Molluskicidal nanoemulsion of Neomitranthes obscura (DC.) N. Silveira for schistosomiasis control.

Schistosomiasis is caused by the parasite Schistosoma mansoni, which uses mollusks of the Biomphalaria genus as intermediate hosts. In 2020, approximately 241 million people worldwide underwent treatment for schistosomiasis. For this reason, the World Health Organization encourages research on alternative molluskicides based on plant species. The objective of this work was to investigate Neomitranthes obscura essential oil from leaf chemical composition and its essential oil nanoemulsion activity on intermediate hosts of schistosomiasis Biomphalaria glabrata control. The major chemical components of the Neomitranthes obscura essential oil were zonarene, seline-3,7(11)-diene, ß-selinene, and α-selinene. The nanoemulsion tested using 24-well plate methodology showed lethality and juvenile mollusks with LC90 values of 53.9 and 25.0 ppm after 48 h, respectively, and on their spawning with an LC90 of 66.2 ppm after 48 h. Additionally, the nanoemulsion exhibited an LC90 value against the infective form of the parasite Schistosoma mansoni of 11.5 ppm after 4 h. This pharmaceutical formulation acted inhibiting the acetylcholinesterase activity and was not toxic for Mellanoides sp. This result suggests the use of this nanoformulation as a promising alternative in the control of Biomphalaria glabrata and the transmission of schistosomiasis.

Aryl Hydrocarbon Receptor as a Therapeutical Target of Environmentally Induced Skin Conditions.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, expressed in several tissues and involved in the response to environmental stressors. Studies have already associated exposure to environmental factors, such as organic air pollutants, products of the skin microbiota, and solar radiation, with the development/worsening of skin conditions, mediated by AhR. On the other hand, recent studies have shown that synthetic and natural compounds are able to modulate the activation of some AhR signaling pathways, minimizing the harmful response of these environmental stressors in the skin. Thus, AhR constitutes a new therapeutic target for the prevention or treatment of skin conditions induced by the skin exposome. Herein, an overview of potential AhR ligands and their biologic effects in environmentally induced skin conditions are presented. The literature survey pointed out divergences in the mechanism of action from a therapeutic perspective. Although most studies point to the benefits of ligand downregulation of AhR signaling, counteracting the toxic effects of environmental factors on the skin, some studies suggest the AhR ligand activation as a therapeutical mechanism for some skin conditions. Furthermore, both agonist and antagonist profiles were identified in the AhR modulation by the synthetic and natural compounds raised. Despite that, this target is still little explored, and further studies are needed to elucidate the molecular mechanisms involved and identify new AhR ligands with therapeutic potential. SIGNIFICANCE STATEMENT: The aryl hydrocarbon receptor (AhR) is involved in different skin physiological and pathological processes, including toxic mechanisms of environmental factors. Synthetic and natural AhR ligands have demonstrated therapeutic potential for skin conditions induced by these agents. Thus, a comprehensive understanding of the skin toxicity mechanisms involving the AhR, as well as the use of AhR modulators from a therapeutic perspective, provides an alternative approach to the development of new treatments for skin disorders induced by the exposome.

Synthesis, Biological Evaluation and Molecular Modeling Studies of Naphthoquinone Sulfonamides and Sulfonate Ester Derivatives as P2X7 Inhibitors.

ATP acts in the extracellular environment as an important signal, activating a family of receptors called purinergic receptors. In recent years, interest in the potential therapeutics of purinergic components, including agonists and antagonists of receptors, has increased. Currently, many observations have indicated that ATP acts as an important mediator of inflammatory responses and, when found in high concentrations in the extracellular space, is related to the activation of the P2X7 purinergic receptor. In this sense, the search for new inhibitors for this receptor has attracted a great deal of attention in recent years. Sulfonamide derivatives have been reported to be potent inhibitors of P2X receptors. In this study, ten naphthoquinone sulfonamide derivatives and five naphthoquinone sulfonate ester derivatives were tested for their inhibitory activity on the P2X7 receptor expressed in peritoneal macrophages. Some compounds showed promising results, displaying IC50 values lower than that of A740003. Molecular docking and dynamic studies also indicated that the active compounds bind to an allosteric site on P2X7R. The binding free energy indicates that sulfonamides have an affinity for the P2X7 receptor similar to A740003. Therefore, the compounds studied herein present potential P2X7R inhibition.

Triazoles with inhibitory action on P2X7R impaired the acute inflammatory response in vivo and modulated the hemostatic balance in vitro and ex vivo.

OBJECTIVE: The present study aimed to investigate five triazole compounds as P2X7R inhibitors and evaluate their ability to reduce acute inflammation in vivo. MATERIAL: The synthetic compounds were labeled 5e, 8h, 9i, 11, and 12. TREATMENT: We administered 500 ng/kg triazole analogs in vivo, (1-10 µM) in vitro, and 1000 mg/kg for toxicological assays. METHODS: For this, we used in vitro experiments, such as platelet aggregation, in vivo experiments of paw edema and peritonitis in mice, and in silico experiments. RESULTS: The tested substances 5e, 8h, 9i, 11, and 12 produced a significant reduction in paw edema. Molecules 5e, 8h, 9i, 11, and 12 inhibited carrageenan-induced peritonitis. Substances 5e, 8h, 9i, 11, and 12 showed an anticoagulant effect, and 5e at a concentration of 10 µM acted as a procoagulant. All derivatives, except for 11, had pharmacokinetic, physicochemical, and toxicological properties suitable for substances that are candidates for new drugs. In addition, the ADMET risk assessment shows that derivatives 8h, 11, 5e, and 9i have high pharmacological potential. Finally, docking tests indicated that the derivatives have binding energies comparable to the reference antagonist with a competitive inhibition profile. CONCLUSIONS: Together, the results indicate that the molecules tested as antagonist drugs of P2X7R had anti-inflammatory action against the acute inflammatory response.

Alternative Methods for Pulmonary-Administered Drugs Metabolism: A Breath of Change.

Prediction of pulmonary metabolites following inhalation of a locally acting pulmonary drug is essential to the successful development of novel inhaled medicines. The lungs present metabolic enzymes, therefore they influence drug disposal and toxicity. The present review provides an overview of alternative methods to evaluate the pulmonary metabolism for the safety and efficacy of pulmonary delivery systems. In vitro approaches for investigating pulmonary drug metabolism were described, including subcellular fractions, cell culture models and lung slices as the main available in vitro methods. In addition, in silico studies are promising alternatives that use specific software to predict pulmonary drug metabolism, determine whether a molecule will react with a metabolic enzyme, the site of metabolism (SoM) and the result of this interaction. They can be used in an integrated approach to delineate the major cytochrome P450 (CYP) isoforms to rationalize the use of in vivo methods. A case study about a combination of experimental and computational approaches was done using fluticasone propionate as an example. The results of three tested software, RSWebPredictor, SMARTCyp and XenoSite, demonstrated greater probability of the fluticasone propionate being metabolized by CYPs 3A4 at the S1 atom of 5-S-fluoromethyl carbothioate group. As the in vitro studies were not able to directly detect pulmonary metabolites, those alternatives in silico methods may reduce animal testing efforts, following the principle of 3Rs (Replacement, Reduction and Refinement), and contribute to the evaluation of pharmacological efficacy and safety profiles of new drugs in development.

Nanoemulsion of Sideroxylon obtusifolium as an Alternative to Combat Schistosomiasis.

Schistosomiasis is caused by the intestinal parasite Schistosoma mansoni. Individuals are affected by schistosomiasis when they are exposed to aquatic environments contaminated with Schistosoma cercariae that emerged from the infected intermediate host mollusk of the genus Biomphalaria. The WHO recommends using molluscicidal products to reduce the snail population and disease transmission. The WHO encourages the search for alternative substances in schistosomiasis control. Natural products are seen as a promising alternative because they are abundant in countries where schistosomiasis is endemic and have many different substances in their extracts, impairing cases of resistance. Therefore, the nanoemulsion effect of a butanol-soluble fraction of Sideroxylon obtusifolium leaves was evaluated against three study points in the biological cycle of the disease, that is, adults and young Biomphalaria glabrata, spawning by the host mollusk, and infectious larvae of the parasite. Extract-SOB (butanol fraction) and nano-SOB (nanoemulsion) demonstrated promising activity in adult B. glabrata population control with an LC50 of 125.4 mg/L, an LC90 of 178.1 mg/L, an LC50 of 75.2 mg/L, and an LC90 of 97 mg/L. Nano-SOB presented greater potency against young B. glabrata, with an LC90 of 72.1 mg/L and an LC50 of 58.3 mg/L. Still, relevant activity against S. mansoni cercariae was eliminated in 4 h (LC90: 34.6 mg/L). Nano-SOB reduced viable spawning by approximately 30% at 178.1 and 97 mg/L. Referring to most substances in this extract, quercetin-3-rhamnosyl-(1-6)-galactoside and hyperoside may cause low environmental toxicity and human toxicity according to in silico analysis. Thus, nano-SOB is a promising agent to combat B. glabrata population growth and schistosomiasis transmission.

Eugenia sulcata (Myrtaceae) Nanoemulsion Enhances the Inhibitory Activity of the Essential Oil on P2X7R and Inflammatory Response In Vivo.

P2X7R is a purinergic receptor with broad expression throughout the body, especially in immune system cells. P2X7R activation causes inflammatory mediators to release, including interleukin-1ß (IL-1ß), the processing and release of which are critically dependent on this ion channel activation. P2X7R's therapeutic potential augments the discovery of new antagonistic compounds. Thus, we investigated whether the Eugenia sulcata essential oil could block P2X7R activity. The essential oil (ESO) dose-dependently inhibited ATP-promoted PI uptake and IL-1ß release with an IC50 of 113.3 ± 3.7 ng/mL and 274 ± 91 ng/mL, respectively, and the essential oil nanoemulsion (ESON) improved the ESO inhibitory effect with an IC50 of 81.4 ± 7.2 ng/mL and 62 ± 2 ng/mL, respectively. ESO and ESON reversed the carrageenan-activated peritonitis in mice, and ESON exhibited an efficacy higher than ESO. The majority substance from essential oil, ß-caryophyllene, impaired the ATP-evoked PI uptake and IL-1ß release with an IC50 value of 26 ± 0.007 ng/mL and 97 ± 0.012 ng/mL, respectively. Additionally, ß-caryophyllene reduced carrageenan-induced peritonitis, and the molecular modeling and computational simulation predicted the intermolecular interactions in the P2X7R situs. In silico, results indicated ß-caryophyllene as a potent allosteric P2X7R antagonist, although this substance may present toxic effects for humans. These data confirm the nanoemulsion of essential oil from E. sulcata as a promisor biotechnology strategy for impaired P2X7R functions and the inflammatory response.

Diterpenes isolated from Canistrocarpus cervicornis with virucidal activity against HIV-1: an in silico evaluation.

HIV is a public health problem, which makes necessary the development of new drugs. Natural products are known for their anti-HIV potential and a good strategy to suggest its mechanism of action is using in silico tools. Herein, diterpenes 1-3 had the binding mode evaluated in the HIV-1 glycoprotein; and properties ADMET in silico performed. In molecular docking important interactions between the hydrophobic cavity, and 1 and 2 were observed. In the molecular dynamics, 1 remained stable covering the entire hydrophobic cavity and performed hydrogen bond during all simulation. ADMET evaluation showed good properties for the diterpenes. Based on these findings, it was possible to suggest the potential from natural products as entry inhibitor and HIV-1 treatment.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis.

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure-activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1ß. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission.

KEY MESSAGE: NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. In this work, we used molecular modeling approach and site-directed mutagenesis to characterized the NADP-ME2 structural determinants necessary for allosteric regulation providing new insights for enzyme optimization. Structure-function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate's pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.

Development of rivaroxaban microemulsion-based hydrogel for transdermal treatment and prevention of venous thromboembolism.

We have developed a microemulsion (ME)-based hydrogel, containing propylene glycol, Azone®, Labrasol®, isobutanol and water (20:3:18:3:56), for the transdermal delivery of rivaroxaban (RVX). Formulation ME-1:RVX, which was loaded with 0.3 mg/g of RVX, presented as a clear, homogenous fluid with a droplet size of 82.01 ± 6.32 nm and a PdI of 0.207 ± 0.01. To provide gelation properties, 20 % (w/w) of Pluronic® F-127 was added to ME-1:RVX to generate formulation PME-1a. An added benefit was an increased capacity for RVX to 0.4 mg/g (formulation PME-1b). PME-1b displayed spherical droplets with a nanoscale diameter as observed by Transmission Electron Microscopy. The release of RVX from PME-1b was 20.71 ± 0.76 µg/cm2 with a permeation through pig epidermis of 18.32 ± 8.87 µg/cm2 as measured in a Franz Cell for 24 h. PME-1b presented a pseudoplastic behavior, pH value compatible with the skin and good stability over 60 days at room and elevated temperatures. The prothrombin time was assessed for each concentration of RVX obtained in the permeation assay and each demonstrated a relevant anticoagulant activity. PME-1b also presented no cytotoxicity against HaCaT cells. Utilizing GastroPlus® software, an in silico analysis was performed to simulate the delivery of PME-1b through a transdermal system that suggested a minimum dose of RVX for the treatment and prevention of venous thromboembolism could be achieved with an 8 h administration regimen. These results suggest that PME-1b is a promising transdermal formulation for the effective delivery of RVX that could be a viable alternative for the treatment and prevention of venous thromboembolism.

Clofazimine functionalized polymeric nanoparticles for brain delivery in the tuberculosis treatment.

Central nervous system tuberculosis (CNS-TB) is the most severe form of the disease especially due to the inability of therapeutics to cross the blood-brain barrier (BBB). Clofazimine (CFZ) stands out for presenting high in vitro activity against multi-drug resistant strains of Mycobacterium tuberculosis, however, CFZ physicochemical and pharmacokinetics properties limit drug penetration into the CNS and, consequently, its clinical use. The aim of this work was to develop polymeric nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) loaded with CFZ and functionalized with a transferrin receptor (TfR)-binding peptide, aiming brain drug delivery for CNS-TB treatment by the intravenous route. The poor water solubility and high lipophilicity of CFZ was overcome through its entrapment into PLGA-PEG NPs manufactured by both conventional and microfluidic techniques using the nanoprecipitation principle. In vitro studies in brain endothelial hCMEC/D3 cells demonstrated that CFZ incorporation into the NPs was advantageous to reduce drug cytotoxicity. The TfR-binding peptide-functionalized NPs showed superior cell interaction and higher CFZ permeability across hCMEC/D3 cell monolayers compared to the non-functionalized NP control, thus indicating the efficacy of the functionalization strategy on providing CFZ transport through the BBB in vitro. The functionalized NPs demonstrate suitability for CFZ biological administration, suggested with low plasma protein binding, off-target biodistribution and precise delivery of CFZ towards the brain parenchyma.

Inventive step assessment of top selling monoclonal antibodies in Brazil.

Introduction: The patent system is fundamental for the pharmaceutical industry development, providing a return on the large investment of time and financial resources. Among the patentability requirements, understanding how to comply with the inventive step is especially important for patent applicants. Regarding mAbs, due to the high affinity and specificity for their molecular therapeutic target, minimal structural changes can lead to unexpected properties, being a common issue among Patent Offices.Areas covered: The present research investigated the Brazilian patents covering top-selling mAbs.Expert Opinion: The more complete and detailed the mAb when the patent application is filed, the greater the chance of the patent being granted. It is necessary to disclose, at least, the six CDRs, the complete variable region, and/or the hybridoma. The Applicant shall specify faced obstacles during mAb generation, mainly if it is a common issue and resulted in improved properties. If it is possible, the Applicants shall compare the claimed mAbs to previous ones, focusing on the achieved unexpected or improved properties. After an objection by BRPTO, the Applicant shall submit data with quantitatively data about qualitative information disclosed at the Specification when filed. If applicable, show different epitope-binding and highlight clinical advantages of successful mAbs.

Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

ABSTRACT: Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and prothrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.

RESUMO: As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.