Synthesis, in vitro and in vivo biological evaluation,COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives

The objective of this work was to obtain and evaluate anti-inflammatory in vitro, in vivo and in silico potential of novel indole-N-acylhydrazone derivatives. In total, 10 new compounds (3a–j) were synthesized in satisfactory yields, through a condensation reaction in a single synthesis step. In the lymphoproliferation assay, using mice splenocytes, 3a and 3b showed inhibition of lymphocyte proliferation of 62.7% (±3.5) and 50.7% (±2), respectively, while dexamethasone presented an inhibition of 74.6% (±2.4). Moreover, compound 3b induced higher Th2 cytokines production in mice splenocytes cultures. The results for COX inhibition assays showed that compound 3b is a selective COX-2 inhibitor, but with less potency when compared to celecoxib, and compound 3a not presented selectivity towards COX-2. The molecular docking results suggest compounds 3a and 3b interact with the active site of COX-2 in similar conformations, but not with the active site of COX-1, and this may be the main reason to the COX-2 selectivity of compound 3b. In vivo carrageenan-induced paw edema assays were adopted for the confirmation of the anti-inflammatory activity. Compound 3b showed better results in suppressing edema at all tested concentrations and was able to induce an edema inhibition of 100% after 5?h of carrageenan injection at the 30?mg?kg^?1 dosage, corroborating with the COX inhibition and lymphoproliferation results. I addition to our experimental results, in silico analysis suggest that compounds 3a and 3b present a well-balanced profile between pharmacodynamics and pharmacokinetics. Thus, our preliminary results revealed the potentiality of a new COX-2 selective derivative in the modulation of the inflammatory process.     

Antihypertensive effect of insect cells: In vitro and in vivo evaluation

In this study, we investigated the in vitro ACE inhibitory and in vivo antihypertensive effect of insect cell extracts. The IC₅₀ of three insect cell lines from different type and insect species origin: S2 (embryo, Drosophila melanogaster), Sf21 (ovary, Spodoptera frugiperda) and Bm5 (ovary, Bombyx mori), were evaluated. Most interesting results were that the IC₅₀ values ranged between 0.4 and 0.9 mg/ml, and that an extra hydrolysis with gastrointestinal enzymes did not increase the ACE inhibitory activity conspicuously. Finally, a single oral administration with a gavage of 150 mg cell extract/kg BW to spontaneous hypertensive rats (SHR) significantly decreased (p < 0.05) their systolic blood pressure (SBP) with 5-6% (9-12 mm Hg) compared to the controls at 6 h post-administration. Here the undigested and digested insect S2 cell extracts were equal in activity to lower the SBP. To the best of our knowledge, this is the first report of in vivo antihypertensive activity of insect cell extracts and this without an extra digestion requirement.     

Dinuclear copper(II) complexes containing 6-(benzylamino)purines as bridging ligands: Synthesis, characterization, and in vitro and in vivo antioxidant activities

A series of dinuclear copper(II) complexes involving 6-(benzylamino)purine derivatives, (HL_n), as bridging ligands were synthesized, characterized and tested for both their in vitro and in vivo antioxidant activities. Based on results of elemental analyses, temperature dependence of magnetic susceptibility measurements, UV-vis, FTIR, EPR, NMR and MALDI-TOF mass spectroscopy, conductivity measurements and thermal analyses, the complexes with general compositions of [Cu₂(μ-HL_n)₄Cl₂]Cl₂ · 2H₂O (1-4) and [Cu₂(μ-HL_n)₂(μ-Cl)₂Cl₂] (5-7) were prepared {where n = 1-4; HL₁ = 6-[(2-methoxybenzyl)amino]purine, HL₂ = 6-[(4-methoxybenzyl)amino]purine, HL₃ = 6-[(2,3-dimethoxybenzyl)amino]purine and HL₄ = 6-[(3,4-dimethoxybenzyl)amino]purine}. In the case of complexes 2, 3, 5 and 7, the antioxidant activities were studied by both in vitro {superoxide dismutase-mimic (SOD-mimic) activity} and in vivo {cytoprotective effect against the alloxan-induced diabetes (antidiabetic activity)} methods. The obtained IC₅₀ value of the SOD-mimic activity for the complex 5 (IC₅₀ = 0.253 μM) was shown to be even better than that of the native bovine Cu,Zn-SOD enzyme (IC₅₀ = 0.480 μM), used as a standard. As for the antidiabetic activity, the pretreatment of mice with complexes 3 and 7 led to the complete elimination of cytotoxic attack of alloxan and its free radical metabolites, used as a diabetogenic agent. The cytoprotective effect of these compounds was proved by the preservation of the initial blood glucose levels of the pretreated animals, as against the untreated control group.     

Combined chelation based on glycosyl-mono- and bis-hydroxypyridinones for aluminium mobilization: Solution and biodistribution studies

Taking into account the recognized interest of a poly-pharmacological strategy in chelation therapy, a study of aluminium combined chelation based on 3-hydroxy-4-pyridinone (3,4-HP) compounds with complementary properties, associated to different denticity, size and extrafunctionality, is presented herein. In particular, Al-chelation has been explored, using a tetradentate IDA bis-(3,4-HP) ligand, L, and two N-glycosyl mono-(3,4-HP) derivatives (A or B). Combined complexation studies with the tetradentate and the most promising bidentate ligand (A) evidenced the formation of ternary complexes with high thermodynamic stability (Al–L–A) being the predominant species at physiological pH. In vivo studies on the ability for radiotracer (⁶⁷Ga) removal from loaded mice, as a model of aluminium accumulation in body, have shown that the simultaneous administration to ⁶⁷Ga-loaded mice of a mono- and a bis-(3,4-HP) chelator (e.g. A and L) leads to a rapid metal elimination from main organs and whole animal model. This may be rationalized by coadjuvation and eventual synergistic effects, due to complementary accessibility of the chelators to different cellular compartments.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

Accuracy of in vivo and ex vivo ultrasonographic evaluation of ovarian follicles and corpora lutea in sows

Current study determined, in sows, the accuracy of ultrasonography for in vivo (n = 8) and ex vivo (n = 7) evaluation of corpora lutea (CLs) and follicles ≥1.5 mm in size, by comparison with macroscopic findings in sliced ovaries. The accuracy for ex vivo detection of follicles increased with follicle size (P < 0.05), being low for 1.5-1.9 mm follicles (65.9%) and higher for ≥6 mm follicles (93.3%); differences between ultrasonographic and macroscopic observations were significant only for follicles smaller than 3.9 mm (P < 0.05), due to underestimation. Ex vivo observation succeeded to detect presence or absence of CLs in all the ovaries; the efficiency for determining the exact number of CLs being 94.4%. The accuracy for in vivo detection of follicles also increased with follicle size (P < 0.05), dropping to values lower than 40% for 1.5-1.9 mm follicles; therefore, there were significant differences between ultrasonographic and macroscopic observations (P < 0.05). On the other hand, accuracy remained around 92% for ≥6 mm follicles. Ultrasonography was useful again for detecting presence of CLs in all the ovaries; the efficiency for determining CLs number reached 86.7%, due to underestimation in ovaries with higher number of CLs (P < 0.05). Overall, there were no significant differences when comparing the accuracy of ex vivo and in vivo scannings for determination neither of the number of follicles in each size-category larger than 1.9 mm nor of the presence of ovulations or of the CLs number in each ovary. In conclusion, the use of ultrasonography allows an accurate detection of the presence and number of CLs and follicles ≥2 mm of diameter in sows, without significant differences between in vivo and ex vivo observations.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

Enhancement of bone formation ex vivo and in vivo by a helioxanthin-derivative

To effectively treat serious bone defects using bone-regenerative medicine, a small chemical compound that potently induces bone formation must be developed. We previously reported on the osteogenic effect of 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin-derivative, in vitro. Here, we report on TH’s osteogenic effects ex vivo and in vivo. TH-induced new bone formation in both calvarial and metatarsal organ cultures. A novel monitoring system of osteoblastic differentiation using MC3T3-E1 cells revealed that TH was released from α-TCP bone cement and this release continued for more than one month. Lastly, the implantation of the α-TCP carrier containing TH into defects in mouse skull resulted in increased new bone areas within the defects after 4 weeks. A TH-containing scaffold may help establish a more efficient bone regeneration system.     

An efficient synthetic route for preparation of antimycobacterial wollamides and evaluation of their in vitro and in vivo efficacy

A convenient solid phase peptide synthetic (SPPS) route is reported for the preparation of antimycobacterial wollamides. The method is based on on-resin head-to-tail cyclization and is fast, efficient and amenable to automation. The in vitro antimycobacterial activities of the newly synthesized wollamides were evaluated against M. tuberculosis H37Rv (Mtb H37Rv). To assess their drug-likeness, in vitro pharmacokinetic (ADME) profiling was also performed. For wollamides with potent extracellular potency, intracellular activities and in vivo efficacy were determined. The results disclose the potent antimycobacterial (MIC_{Mtb H37Rv}?=?1.1?µM) and suitable drug-like properties of wollamide A (4b). Out of the synthesized wollamides, four compounds (4b–e) exhibited potent intracellular activities against Mtb H37Rv infected human macrophages (IC₅₀?=?0.2–1.3?µM). Results of in vivo blood exposure and efficacy assays for 4d and 4e are discussed.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Inhibition of c-Jun N-terminal kinase increases apoE expression in vitro and in vivo

Apolipoprotein E (apoE), a ligand for the low-density lipoprotein receptor family, has been implicated in modulating glial inflammatory responses and the risk of neurodegeneration associated with Alzheimer’s disease. Glial cells activated by lipopolysaccharide (LPS) have decreased apoE levels and we recently showed that apoE itself can modulate the inflammatory response by reducing c-Jun N-terminal kinase (JNK) activation. Reduced JNK phosphorylation is vital to overcome the LPS-induced decrease in apoE expression, suggesting that JNK inhibition may be an effective way to increase apoE protein and protract its anti-inflammatory properties. This study investigates the impact of JNK inhibition on apoE production using two JNK inhibitors. Our work in primary glia and in vivo in mice injected with JNK inhibitor demonstrates that inhibition of JNK may be an effective way to increase apoE expression.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin

Cantharidin is a natural poisonous compound secreted by male blister beetles. The effect of different doses of cantharidin on Leishmania major (MRHO/IR/75/ER) were investigated both in vitro (promastigote and amastigote viability) and in experimentally-infected BALB/c mice (skin lesions) using ointment or soluble cantharidin. In this study, cantharidin with concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg/ml inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30% and 80.04%, respectively. After 72 h, the mean number of amastigotes per macrophage in the culture using 2 μg/ml of cantharidin, (the 50% inhibitory concentration dose (IC₅₀)), was 1.2 while in the control group it was 2.7. In order to perform the inflammatory blister technique, 500 μg of cantharidin were solved in 25 μl of DMSO to show the formation of the blister which leads to treatment of cutaneous leishmaniasis. Using the blister technique, the small lesions (<5 mm) healed after one session. Two weeks of topical treatment with 0.1% cantharidin ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.     

Ornithine decarboxylase in Phycomyces: In vitro and in vivo properties

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5′-phosphate are 90 and 0.37 μm, respectively. Putrescine is a potent competitive inhibitor with a K_i of 75 μm. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.     

Formaldehyde induces hyperphosphorylation and polymerization of Tau protein both in vitro and in vivo

Background

Chronic formaldehyde exposure leads to memory impairment and abnormal elevation of endogenous formaldehyde has been found in the brains of Alzheimer's disease (AD) patients. Hyperphosphorylated Tau protein with subsequent aggregates as neurofibrillary tangles (NFTs) is one of the typical pathological characteristics in AD brains. The mechanism underlying abnormally elevated concentrations of endogenous formaldehyde that induce Tau hyperphosphorylation is unknown.

Methods

N2a cells and mice were treated with formaldehyde for different time points, then Western blotting and immunocytochemistry were utilized to determine the phosphorylation and polymerization of Tau protein. HPLC was used to detect the concentration of formaldehyde in cell media.

Results

Under formaldehyde stress, Tau became hyperphosphorylated, not only in the cytoplasm, but also in the nucleus of neuroblastoma (N2a) cells, and mouse brains. Polymers of cellular phospho-Tau were also detected. Significant accumulation of glycogen synthase kinase-3β (GSK-3β) in the nucleus of N2a and mouse brain cells, and elevation of its phosphorylation at Y216, was observed under formaldehyde stress. Formaldehyde-induced Tau hyperphosphorylation was blocked in the presence of LiCl and CT99021, inhibitors of GSK-3β, and by RNAi interference.

Conclusions

Formaldehyde, which may cause age-related memory loss, can act as a factor triggering Tau hyperphosphorylation via GSK-3β catalysis and induces polymerization of Tau.

General significance

Investigation of formaldehyde-induced Tau hyperphosphorylation may provide novel insights into mechanisms underlying tauopathies.