Synthesis and Antiallodynic Activity of Cannabidiol Analogue on Peripheral Neuropathy in Mice.

Cannabidiol (CBD) is a substance that exerts several therapeutic actions, including analgesia. CBD is generally administered orally, but its poor water solubility and metabolism impair its bioavailability. Thus, the development of molecules with better pharmacokinetic profile from cannabidiol becomes an interesting strategy for the design of novel analgesic drugs for the relief of painful conditions that are difficult to manage clinically, such as neuropathic pain. In the present study, an unprecedented analogue of CBD (1) was synthesized and some of its physicochemical properties were evaluated in silico as well as its stability in an acid medium. Additionally, its effect was investigated in a model of neuropathic pain induced by the chemotherapy drug paclitaxel in mice, in comparison with cannabidiol itself. Cannabidiol (20 mg/kg), pregabalin (30 mg/kg), or analogue 1 (5, 10, and 20 mg/kg), administered on the 14th day after the first administration of paclitaxel, attenuated the mechanical allodynia of the sensitized animals. The antinociceptive activity of analogue 1 was attenuated by previous administration of a cannabinoid CB1 receptor antagonist, AM 251, which indicates that its mechanism of action is related to the activation of CB1 receptors. In conclusion, the CBD analogue 1 developed in this study shows great potential to be used in the treatment of neuropathic pain.

Structure-based discovery of thiosemicarbazones as SARS-CoV-2 main protease inhibitors.

Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 µM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.

A clindamycin acetylated derivative with reduced antibacterial activity inhibits articular hyperalgesia and edema by attenuating neutrophil recruitment, NF-κB activation and tumor necrosis factor-α production.

We recently demonstrated that clindamycin exhibits activities in acute and chronic models of pain and inflammation. In the present study, we investigated the effects of clindamycin and a clindamycin acetylated derivative (CAD) in models of acute joint inflammation and in a microbiological assay. Joint inflammation was induced in mice by intraarticular (i.a.) injection of zymosan or lipopolysaccharide (LPS). Clindamycin or CAD were administered via the intraperitoneal route 1 h before zymosan or LPS. Paw withdrawal threshold, joint diameter, histological changes, neutrophil recruitment, tumor necrosis factor-α (TNF-α) production and phosphorylation of the IκBα and NF-κB/p65 were evaluated. In vitro assays were used to measure the antibacterial activity of clindamycin and CAD and also their effects on zymosan-induced TNF-α production by RAW264.7 macrophages. Clindamycin exhibited activity against Staphylococcus aureus and Salmonella Typhimurium ATCC® strains at much lower concentrations than CAD. Intraarticular injection of zymosan or LPS induced articular hyperalgesia, edema and neutrophil infiltration in the joints. Zymosan also induced histological changes, NF-κB activation and TNF-α production. Responses induced by zymosan and LPS were inhibited by clindamycin (200 and 400 mg/kg) or CAD (436 mg/kg). Both clindamycin and CAD inhibited in vitro TNF-α production by macrophages. In summary, we provided additional insights of the clindamycin immunomodulatory effects, whose mechanism was associated with NF-κB inhibition and reduced TNF-α production. Such effects were extended to a clindamycin derivative with reduced antibacterial activity, indicating that clindamycin derivatives should be investigated as candidates to drugs that could be useful in the management of inflammatory and painful conditions.

RI75, a curcumin analogue, inhibits tumor necrosis factor-α and interleukin-6 production and exhibits antiallodynic and antiedematogenic activities in mice.

Curcumin and its analogues exhibited anti-inflammatory activity in different experimental models. Recently, we synthesized (2E,3E)-3-buten-2-one-4-(4-hydroxy-3-methoxyphenyl)-2-(4-(4-methoxyphenyl)-2-thiazolyl)hydrazone (RI75), a curcumin analogue with a thiazolyl hydrazone moiety. In the present study, we investigated the effects induced by RI75 in different models of inflammation and pain in mice, as well as some underlying mechanisms. Pre-treatment with RI75 (40 mg/kg, intraperitoneal; i.p.) or curcumin (40 mg/kg, i.p.) reduced the mechanical allodynia and paw edema induced by intraplantar (i.pl) injection of carrageenan. RI75 antiallodynic activity was reduced by pre-treatment with naltrexone (5 and 10 mg/kg, i.p.) and cyproheptadine (10 mg/kg, i.p.), but not glibenclamide (20 and 40 mg/kg, i.p.). In a model of neuropathic pain, a single i.p. administration of RI75 (40 mg/kg) or curcumin (40 mg/kg) attenuated the ongoing mechanical allodynia induced by repeated administrations of paclitaxel. Pre-treatment with RI75 (40 mg/kg, i.p.) or curcumin (40 mg/kg, i.p.) also reduced tumor necrosis factor-α and interleukin-6 production and myeloperoxidase activity induced by carrageenan. The results of the present study demonstrate that RI75, a synthetic curcumin analogue, exhibits antiallodynic and antiedematogenic activities. Activation of opioidergic and serotonergic mechanisms and reduced production of inflammatory mediators and neutrophil recruitment may underlie RI75 activities.

Do Drug-likeness Rules Apply to Oral Prodrugs?

This paper describes a comparative analysis of the physicochemical and structural properties of prodrugs and their corresponding drugs with regard to drug-likeness rules. The dataset used in this work was obtained from the DrugBank. Sixty-five pairs of prodrugs/drugs were retrieved and divided into the following categories: carrier-linked to increase hydrophilic character, carrier-linked to increase absorption, and bioprecursors. We compared the physicochemical properties related to drug-likeness between prodrugs and drugs. Our results show that prodrugs do not always follow Lipinski's Rule of 5, especially as we observed 15 prodrugs with more than 10 hydrogen bond acceptors and 18 with a molecular weight greater than 500 Da. This fact highlights the importance of extending Lipinski's rules to encompass other parameters as both strategies (filtering of drug-like chemical libraries and prodrug design) aim to improve the bioavailability of compounds. Therefore, critical reasoning is fundamental to determine whether a structure has drug-like properties or could be considered a potential orally active compound in the drug-design pipeline.

Improving the solubility of an antifungal thiazolyl hydrazone derivative by cyclodextrin complexation.

RN104, named 2-[2-(cyclohexylmethylene)hydrazinyl)]-4-phenylthiazole, is a thiazolyl hydrazone derivative with promising antifungal activity. A HPLC-DAD method was carried out using C18 end-capped column (250 × 4.6 mm, 5 µm) and a mobile phase composed of water and acetonitrile (15:85 v/v) at a flow rate of 1.2 mL/min, injection volume 25 µL and DAD detection at 240 nm. The method showed to be selective, linear in the range of 20 to 240 µg/mL, precise, accurate and robust.Due to the low solubility of RN104 in water, the development of inclusion complexes using different cyclodextrins (ß-CD, γ-CD and 2-HP-ß-CD) was investigated, as well as the interaction mode between RN104 and cyclodextrins using molecular docking followed by semi-empirical calculations. Among tested cyclodextrins, the best results were obtained with 2-HP-ß-CD, which promoted an 18-fold increase in RN104's aqueous solubility.

Synthesis of quinoline derivatives as potential cysteine protease inhibitors.

Aim: Cysteine proteases are important molecular targets involved in the replication, virulence and survival of parasitic organisms, including Trypanosoma and Leishmania species. Methodology & results: Analogs of the 7-chloro-N-[3-(morpholin-4-yl)propyl]quinolin-4-amine were synthesized and their inhibitory activity against the enzymes cruzain and rhodesain as well as against promastigotes forms of Leishmania species and epimastigotes forms of Trypanosoma cruzi were evaluated. Five compounds showed activity against both enzymes with half maximal inhibitory concentration (IC50) values ranging from 23 to 123 µM. Among these, compounds 3 and 4 displayed leishmanicidal activity; compound 4 was the most promising with IC50 values <10 µM and no cytotoxicity for uninfected cells. Conclusion: The results obtained indicate that cysteine proteases are likely to be the molecular target of compounds 3 and 4.

Discovery and characterization of trypanocidal cysteine protease inhibitors from the 'malaria box'.

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 µM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 µM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs.

4-Methylbenzenecarbothioamide, a hydrogen sulfide donor, inhibits tumor necrosis factor-α and CXCL1 production and exhibits activity in models of pain and inflammation.

The gasotransmitter hydrogen sulfide (H2S) is known to regulate many pathophysiological processes. Preclinical assays have demonstrated that H2S donors exhibit anti-inflammatory and antinociceptive activities, characterized by reduction of inflammatory mediators production, leukocytes recruitment, edema and mechanical allodynia. In the present study, the effects induced by 4-methylbenzenecarbothioamide (4-MBC) in models of pain and inflammation in mice, the mechanisms mediating such effects and the H2S-releasing property of this compound were evaluated. 4-MBC spontaneously released H2S in vitro in the absence of organic thiols. Intraperitoneal (i.p.) administration of 4-MBC (100 or 150 mg/kg) reduced the second phase of the nociceptive response induced by formaldehyde and induced a long lasting inhibitory effect on carrageenan mechanical allodynia. 4-MBC antiallodynic effect was not affected by previous administration of naltrexone or glibenclamide. 4-MBC (50, 100 or 150 mg/kg, i.p.) induced a long lasting inhibitory effect on paw edema induced by carrageenan. The highest dose (150 mg/kg, i.p.) of 4-MBC inhibited tumor necrosis factor-α and CXCL1 production and myeloperoxidase activity induced by carrageenan. Mechanical allodynia and paw edema induced by carrageenan were not inhibited by the 4-MBC oxo analogue (p-toluamide). In summary, 4-MBC, an H2S releasing thiobenzamide, exhibits antinociceptive and anti-inflammatory activities. These activities may be due to reduced cytokine and chemokine production and neutrophil recruitment. The H2S releasing property is likely essential for 4-MBC activity. Our results indicate that 4-MBC may represent a useful pharmacological tool to investigate the biological roles of H2S.

Antifungal Activity Directed Toward the Cell Wall by 2-Cyclohexylidenhydrazo- 4-Phenyl-Thiazole Against Candida albicans.

BACKGROUND: The increasing incidence of invasive forms of candidiasis and resistance to antifungal therapy leads us to seek new and more effective antifungal compounds. OBJECTIVE: To investigate the antifungal activity and toxicity as well as to evaluate the potential targets of 2- cyclohexylidenhydrazo-4-phenyl-thiazole (CPT) in Candida albicans. METHODS: The antifungal activity of CPT against the survival of C. albicans was investigated in Caenorhabditis elegans. Additionally, we determined the effect of CPT on the inhibition of C. albicans adhesion capacity to buccal epithelial cells (BECs), the toxicity of CPT in mammalian cells, and the potential targets of CPT in C. albicans. RESULTS: CPT exhibited a minimum inhibitory concentration (MIC) value of 0.4-1.9 µg/mL. Furthermore, CPT at high concentrations (>60 x MIC) showed no or low toxicity in HepG2 cells and <1% haemolysis in human erythrocytes. In addition, CPT decreased the adhesion capacity of yeasts to the BECs and prolonged the survival of C. elegans infected with C. albicans. Analysis of CPT-treated cells showed that their cell wall was thinner than that of untreated cells, especially the glucan layer. We found that there was a significantly lower quantity of 1,3-ß-D-glucan present in CPT-treated cells than that in untreated cells. Assays performed on several mutant strains showed that the MIC value of CPT was high for its antifungal activity on yeasts with defective 1,3-ß-glucan synthase. CONCLUSION: In conclusion, CPT appears to target the cell wall of C. albicans, exhibits low toxicity in mammalian cells, and prolongs the survival of C. elegans infected with C. albicans.

A phenylthiazole derivative demonstrates efficacy on treatment of the cryptococcosis & candidiasis in animal models.

AIM: In this work we test 2-(2-(cyclohexylmethylene)hydrazinyl)-4-phenylthiazole (CHT) against Cryptococcus spp. and Candida albicans. METHODS: The ability of CHT to act in biofilm and also to interfere with C. albicans adhesion was evaluated, as well as the efficiency of the CHT in cryptococcosis and candidiasis invertebrate and murine models. RESULTS & CONCLUSION: In the present work we verified that CHT is found to inhibit Cryptococcus and C. albicans affecting biofilm in both and inhibited adhesion of Candida to human buccal cells. When we evaluated in vivo, CHT prolonged survival of Galleria mellonella after infections with Cryptococcusgattii, Cryptococcusneoformans or C. albicans and promoted a reduction in the fungal burden to the organs in the murine models. These results demonstrate CHT therapeutic potential.

Synthesis and evaluation of the antiparasitic activity of bis-(arylmethylidene) cycloalkanones.

A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promastigotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six compounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC50 values ranging from 7.0 to 249 µM. Besides these six compounds, eight other molecules exhibited significant leishmanicidal activity (IC50 values ranging from 0.6 to 110.4 µM). Two compounds can be considered as promising antiparasitic lead molecules because they showed IC50 values in the low-micromolar range (≤1.2 µM) with an adequate SI (≥19.9). To understand the mechanism of action of these compounds, two possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.

Development and validation of a simple and fast HPLC method for determination of lovastatin, pravastatin and simvastatin.

Statins are effective and often-prescribed drugs for the treatment of hypercholesterolemia. This study shows a simple and fast method validation by reversed-phase high-performance liquid chromatography in the linear range 28 to 52 µg/mL to quantify lovastatin, pravastatin sodium or simvastatin in bulk drug or dosage forms. Statins were determined using a C8 endcapped column (250 × 4 mm, 5 µm), isocratic mobile phase of acetonitrile and 0.1% phosphoric acid (65:35), 30°C, ultraviolet-diode array detection at λ 238 nm and 1.5 mL/min flow for lovastatin and simvastatin and 1.0 mL/min for pravastatin sodium. The developed method is fast, simple, reliable and shows appropriate linearity (r > 0.999), accuracy (98.8-101.6%), precision (relative standard deviation <2%) and selectivity toward placebo and/or degradation products in very similar chromatographic conditions for all statins.

Synthesis and evaluation of the anti parasitic activity of aromatic nitro compounds.

A series of nitroaromatic compounds was synthesized and evaluated as potential antileishmanial and trypanocidal agents. Five compounds exerted significant anti-leishmanial activity in vitro against promastigotes forms of Leishmania (L.) amazonensis, with IC(50) in the range of 23-59 µmol L(-1), but none were active against amastigotes intracellular forms of Trypanosoma cruzi. In vitro cytotoxicity on the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated with phytohemaglutinin (PHA) was also evaluated. Two compounds, 6 and 7, were found to present a promising anti-leishmanial activity with IC(50) values of 59.5 and 50.6 µM, respectively, without affecting the lymphocyte proliferation in PBMCs (selectivity index of 16.1 and 21.7, respectively), indicating low toxicity to human cells.

Preparation, characterization, and topical delivery of paromomycin ion pairing.

Topical chemotherapy with paromomycin (PA) has been used as an alternative for the treatment of cutaneous leishmaniasis; however, poor skin penetration of this drug limits the efficacy of formulations. The objective of this work was to study the ability of the PA free base to form ion pairing with organic acids, as well as evaluate the effect of these compounds on the topical delivery of PA. PA permeation across intact skin was low, while drug penetration into skin from PA ion pairing was the higher than that observed for the PA base. Data obtained on the stripped skin, a damaged skin model, clearly showed that the ion pairing presented a potential to improve PA skin permeation.

Determining subsampling effort for the development of a rapid bioassessment protocol using benthic macroinvertebrates in streams of Southeastern Brazil.

The purpose of this study was to establish a subsampling procedure for benthic macroinvertebrates to aid in the development of a multimetric index to assess the biological condition of streams. Data from six streams that are considered minimally impaired were used. Subsampling was done using a device divided into 24 quadrats. The sediment from each quadrat was sorted, and all organisms were removed and identified. Richness metrics were the most affected by subsample size. Relative-abundance metrics were the most stable, proving that the sample was well distributed throughout the tray and abundance proportions were maintained. The results showed that the macroinvertebrate assemblage present in the six quadrats was similar to that present in the full sample. These analyses indicated that six quadrats, randomly designated, with a minimum of 200 collected specimens could be used in place of the full sample. In routine water management, managing time and costs are a major challenge; therefore, this type of simplification is absolutely necessary to ensure that a biomonitoring tool is useful for practical applications.

Preparation and characterization of solid dispersion of simvastatin.

CONTEXT: Simvastatin (SIM), a widely used drug for the treatment of hypercholesterolemia, is a crystalline substance and practically insoluble in water. Its low dissolution rate leads to a poor absorption, distribution, and target organ delivery because the bioavailability of drugs with low aqueous solubility is limited by their dissolution rates. OBJECTIVE: The aim of this study was to prepare solid dispersions (SD) of SIM with inert carriers in an attempt to improve the release profile. METHODS: In this work, SIM SD with polyethylene glycol (PEG 6000) or polyvinylpyrrolidone (PVP K15) in 1:1, 1:2, 1: 3, 1:4, and 1:5 ratios were prepared and their stability and dissolution properties were investigated. SD were characterized by differential scanning calorimetry and X-ray powder diffraction. Tablets containing SD SIM : PEG 6000 were developed and their dissolution profile evaluated. RESULTS: Drug release from all SD was significantly improved when compared to their corresponding physical mixture or SIM alone. SD SIM:PVP showed drug degradation. The tablets gradually released SIM with a final quantity greater than 80% in 60 minutes. CONCLUSIONS: The preparation of SIM SD with PEG or PVP is a promising strategy to improve the bioavailability of the drug. SIM SD with PEG are more advantageous over the dispersions prepared with PVP because they do not show drug degradation during preparation.

Synthesis and antimalarial activity of semicarbazone and thiosemicarbazone derivatives.

Seventeen semicarbazone and thiosemicarbazone derivatives were prepared and tested in vitro against a chloroquine resistant strain of Plasmodium falciparum (W2) to evaluate their antiplasmodial potential. Three thiosemicarbazones were found to be active against the parasite and non-toxic to human peripheral blood mononuclear cells (PBMC). Among these, compound 5b presented the lowest IC50 value against P. falciparum (7.2 microM) and was the least toxic in the PBMC proliferation assay (IC50=73.5 microM). It was selected for in vivo tests on mice infected with Plasmodium berghei (strain NK-65). The thiosemicarbazone 5b was able to reduce the parasitaemia by 61% at 20 mg/kg on day 7 after infection without any sign of toxicity to the animals. In comparison, the standard drug chloroquine at 15 mg/kg showed a reduction around 95%. These in vitro and in vivo results make 5b an interesting lead for further development.

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors.

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).