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.

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.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Assessment of predictivity of volatile organic compounds carcinogenicity and mutagenicity by freeware in silico models.

The application of in silico methods is increasing on toxicological risk prediction for human and environmental health. This work aimed to evaluate the performance of three in silico freeware models (OSIRIS v.2.0, LAZAR, and Toxtree) on the prediction of carcinogenicity and mutagenicity of thirty-eight volatile organic compounds (VOC) related to chemical risk assessment for occupational exposure. Theoretical data were compared with assessments available in international databases. Confusion matrices and ROC curves were used to evaluate the sensitivity, specificity, and accuracy of each model. All three models (OSIRIS, LAZAR and Toxtree) were able to identify VOC with a potential carcinogenicity or mutagenicity risk for humans, however presenting differences concerning the specificity, sensitivity, and accuracy. The best predictive performances were found for OSIRIS and LAZAR for carcinogenicity and OSIRIS for mutagenicity, as these softwares presented a combination of negative predictive power and lower risk of false positives (high specificity) for those endpoints. The heterogeneity of results found with different softwares reinforce the importance of using a combination of in silico models to occupational toxicological risk assessment.

Thieno[2,3-b]pyridine derivatives: a new class of antiviral drugs against Mayaro virus.

Mayaro virus (MAYV) is an arthropod-borne virus and a member of the family Togaviridae, genus Alphavirus. Its infection leads to an acute illness accompanied by long-lasting arthralgia. To date, there are no antiviral drugs or vaccines against infection with MAYV and resources for the prevention or treatment of other alphaviruses are very limited. MAYV has served as a model to study the antiviral potential of several substances on alphavirus replication. In this work we evaluated the antiviral effect of seven new derivatives of thieno[2,3-b]pyridine against MAYV replication in a mammalian cell line. All derivatives were able to reduce viral production effectively at concentrations that were non-toxic for Vero cells. Molecular modeling assays predicted low toxicity risk and good oral bioavailability of the substances in humans. One of the molecules, selected for further study, demonstrated a strong anti-MAYV effect at early stages of replication, as it protected pre-treated cells and also during the late stages, affecting virus morphogenesis. This study is the first to demonstrate the antiviral effect of thienopyridine derivatives on MAYV replication in vitro, suggesting the potential application of these substances as antiviral molecules against alphaviruses. Additional in vivo research will be needed to expand the putative therapeutic applications.

Identification, characterization and in silico ADMET prediction of Roflumilast degradation products.

The present study reports the degradation behavior of roflumilast (RFL), a new drug developed for the treatment of chronic obstructive pulmonary disease. The degradation of RFL was tested under various stress conditions as per the guidelines of the International Conference on Harmonization. The degradation products (DPs) of RFL were identified, characterized and in silico predictions were made of their pharmacokinetic properties, absorption, distribution, metabolism, excretion and toxicity (ADMET). RFL was subjected to various stress conditions including photodegradation, alkaline and acidic hydrolysis, oxidative and metallic degradation. After analysis by HPLC-DAD, the DPs were isolated by preparative TLC and characterized by high resolution mass spectrometry (HRMS), 1H NMR, 13C NMR and infrared (IR) spectroscopy. RFL tablets were prepared by the addition of solid stressing substances such as excipients and storage in an accelerated stability chamber (40°C; 75% r.h.) for sixteen months. Resulting DPs from the tablets were analyzed by UFLC-QTOF. The most drastic degradation conditions for RFL were 5M NaOH(aq), 6M HCl(aq), 7.5% v/v peracetic acid, which resulted in the isolation of four DPs. However, milder degradation conditions (1M NaOH(aq) and photolysis) generated six DPs (DP-1, 2, 3, 5, 7 and 8), and are more similar to the actual conditions the drug will be exposed. For tablets containing RFL exposed to an alkaline reagent, two DPs were formed: DP-1 and DP-11. Whereas RFL-containing tablets exposed to acid and oxidizing agents, formed one product DP-11. Forced degradation of RFL led to the formation of eleven DPs, seven of which have never been previously reported. RFL is stable under metallic stress and it is relatively stable during photodegradation testing. The UFLC-QTOF methodology detected a greater number of DPs that formed during the stress conditions tested when compared to the HPLC-DAD methodology. In silico prediction of the ADMET properties of the RFL degradation products and metabolites produced in this study are potentially hepatotoxic.

Antimycobacterial and anti-inflammatory activities of substituted chalcones focusing on an anti-tuberculosis dual treatment approach.

Tuberculosis (TB) remains a serious public health problem aggravated by the emergence of M. tuberculosis (Mtb) strains resistant to multiple drugs (MDR). Delay in TB treatment, common in the MDR-TB cases, can lead to deleterious life-threatening inflammation in susceptible hyper-reactive individuals, encouraging the discovery of new anti-Mtb drugs and the use of adjunctive therapy based on anti-inflammatory interventions. In this study, a series of forty synthetic chalcones was evaluated in vitro for their anti-inflammatory and antimycobacterial properties and in silico for pharmacokinetic parameters. Seven compounds strongly inhibited NO and PGE2 production by LPS-stimulated macrophages through the specific inhibition of iNOS and COX-2 expression, respectively, with compounds 4 and 5 standing out in this respect. Four of the seven most active compounds were able to inhibit production of TNF-α and IL-1ß. Chalcones that were not toxic to cultured macrophages were tested for antimycobacterial activity. Eight compounds were able to inhibit growth of the M. bovis BCG and Mtb H37Rv strains in bacterial cultures and in infected macrophages. Four of them, including compounds 4 and 5, were active against a hypervirulent clinical Mtb isolate as well. In silico analysis of ADMET properties showed that the evaluated chalcones displayed satisfactory pharmacokinetic parameters. In conclusion, the obtained data demonstrate that at least two of the studied chalcones, compounds 4 and 5, are promising antimycobacterial and anti-inflammatory agents, especially focusing on an anti-tuberculosis dual treatment approach.

Hologram QSAR models of 4-[(diethylamino)methyl]-phenol inhibitors of acetyl/butyrylcholinesterase enzymes as potential anti-Alzheimer agents.

Hologram QSAR models were developed for a series of 36 inhibitors (29 training set and seven test set compounds) of acetyl/butyrylcholinesterase (AChE/BChE) enzymes, an attractive molecular target for Alzheimer's disease (AD) treatment. The HQSAR models (N = 29) exhibited significant cross-validated (AChE, q2 = 0.787; BChE, q2 = 0. 904) and non-cross-validated (AChE, r2 = 0.965; BChE, r2= 0.952) correlation coefficients. The models were used to predict the inhibitory potencies of the test set compounds, and agreement between the experimental and predicted values was verified, exhibiting a powerful predictive capability. Contribution maps show that structural fragments containing aromatic moieties and long side chains increase potency. Both the HQSAR models and the contribution maps should be useful for the further design of novel, structurally related cholinesterase inhibitors.

Brown seaweed defensive chemicals: a structure-activity relationship approach for the marine environment.

The literature describes several diterpenes from brown seaweeds that act as defensive chemicals against natural enemies, such as competitors, epiphytes, pathogenic bacteria and herbivores. A structure-activity relationship is here presented using a new molecular modeling approach to identify structural and chemical features important to the defensive profile of four structurally related diterpenes (three dolastanes and one seco-dolastane) from Canistrocarpus cervicornis against the feeding process of the omnivorous sea urchin Lytechinus variegatus. Our experimental data revealed the herbivory inhibitory profile (HIE) for three of these evaluated compounds with (4R,7R, 14S)-4alpha,7alpha-diacetoxy-14-hydroxydolast-1(15),8-diene presenting the highest effect (HIE = 70%). Interestingly, the molecular modeling results infer that this biological activity seems to be related to several different structural features, including HOMO distribution, the molecular structure conformation, and the fulfillment of minimum requirements regarding molecular weight. These results reinforce the hypothesis about the intricate biological mechanism of these molecules due to the complexity of their chemical structures. Our work may help in the understanding of these defensive mechanisms and point to a new perspective of ecological and/or evolutionary evaluation in this area.