Amphiphilic chitosan-polyaminoxyls loaded with daunorubicin: Synthesis, antioxidant activity, and drug delivery capacity.

The binding of aminoxyls to polymers extends their potential use as antioxidants and EPR-reporting groups and opens up new horizons for tailoring new smart materials. In this work, we synthesized and characterized non-sulfated and N-sulfated water-soluble amphiphilic chitosans with a critical micelle concentration of 0.02-0.05 mg/mL that contain 13-18% of aminoglycosides bound with various aminoxyls. Chitosan-polyaminoxyls (CPAs) formed micelles with hydrodynamic radii Rh of ca. 100 nm. The EPR spectra of CPAs were found to depend on the rigidity of the aminoxyl-polymer bond and structural changes caused by sulfation. CPAs demonstrated antioxidant capacity/activity in three tests against reactive oxygen species (ROS) of various nature. The charge of micelles and structure of aminoxyls significantly affected their antioxidant properties. CPAs were low toxic against tumor (HepG2, HeLa, A-172) and non-cancerous (Vero) cells (IC50 > 0.8 mM of aminoglycosides). Sulfated CPAs showed better water solubility and the ability of binding and retaining the anti-tumor antibiotic daunorubicin (DAU). DAU-loaded micelles of CPAs (CPAs-DAU) demonstrated a 1.5-4-fold potentiation of DAU cytotoxicity against several cell lines. CPAs-DAU micelles were found to affect the cell cycle in a manner markedly different from that of free DAU. Our results demonstrated the ability of CPAs to act as bioactive drug delivery vehicles.

Preparation, Antioxidant Properties and Ability to Increase Intracellular NO of a New Pyridoxine Derivative B6NO.

In the case of various pathologies, an imbalance between ROS generation and the endogenous AOS can be observed, which leads to excessive ROS accumulation, intensification of LPO processes, and oxidative stress. For the prevention of diseases associated with oxidative stress, drugs with antioxidant activity can be used. The cytotoxic, antioxidant, and NO-donor properties of the new hybrid compound B6NO (di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) salt of 2-(nitrooxy)butanedioic acid) were studied. It was determined that B6NO chelates iron ions by 94%, which indicates B6NO's ability to block the Fenton reaction. The hybrid compound B6NO inhibits the process of initiated lipid peroxidation more effectively than pyridoxine. It was shown that B6NO exhibits antioxidant properties by decreasing ROS concentration in normal cells during the oxidative stress induction by tert-Butyl peroxide. At the same time, the B6NO antioxidant activity on tumor cells was significantly lower. B6NO significantly increases the intracellular nitrogen monoxide accumulation and showed low cytotoxicity for normal cells (IC50 > 4 mM). Thus, the results indicate a high potential of the B6NO as an antioxidant compound.

Novel potent bifunctional carboxylesterase inhibitors based on a polyfluoroalkyl-2-imino-1,3-dione scaffold.

An expanded series of alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (HOPs) 3 was obtained via Cu(OAc)2-catalyzed azo coupling. All were nanomolar inhibitors of carboxylesterase (CES), while moderate or weak inhibitors of acetylcholinesterase and butyrylcholinesterase. Steady-state kinetics studies showed that HOPs 3 are mixed type inhibitors of the three esterases. Molecular docking studies demonstrated that two functional groups in the structure of HOPs, trifluoromethyl ketone (TFK) and ester groups, bind to the CES active site suggesting subsequent reactions: formation of a tetrahedral adduct, and a slow hydrolysis reaction. The results of molecular modeling allowed us to explain some structure-activity relationships of CES inhibition by HOPs 3: their selectivity toward CES in comparison with cholinesterases and the high selectivity of pentafluoroethyl-substituted HOP 3p to hCES1 compared to hCES2. All compounds were predicted to have good intestinal absorption and blood-brain barrier permeability, low cardiac toxicity, good lipophilicity and aqueous solubility, and reasonable overall drug-likeness. HOPs with a TFK group and electron-donor substituents in the arylhydrazone moiety were potent antioxidants. All compounds possessed low cytotoxicity and low acute toxicity. Overall, a new promising type of bifunctional CES inhibitors has been found that are able to interact with the active site of the enzyme with the participation of two functional groups. The results indicate that HOPs have the potential to be good candidates as human CES inhibitors for biomedicinal applications.

Anatomy of the Bezymianny volcano merely before an explosive eruption on 20.12.2017.

Strong explosive eruptions of volcanoes throw out mixtures of gases and ash from high-pressure underground reservoirs. Investigating these subsurface reservoirs may help to forecast and characterize an eruption. In this study, we compare seismic tomography results with remote sensing and petrology data to identify deep and subaerial manifestations of pre-eruptive processes at Bezymianny volcano in Kamchatka shortly before its violent explosion on December 20, 2017. Based on camera networks we identify precursory rockfalls, and based on satellite radar data we find pre-eruptive summit inflation. Our seismic network recorded the P and S wave data from over 500 local earthquakes used to invert for a 3D seismic velocity distribution beneath Bezymianny illuminating its eruptive state days before the eruption. The derived tomography model, in conjunction with the presence of the high-temperature-stable SiO2 polymorph Tridymite in juvenile rock samples , allowed us to infer the coexistence of magma and gas reservoirs revealed as anomalies of low (1.5) and high (2.0) Vp/Vs ratios, respectively, located at depths of 2-3 km and only 2 km apart. The reservoirs both control the current eruptive activity: while the magma reservoir is responsible for episodic dome growth and lava flow emplacements, the spatially separated gas reservoir may control short but powerful explosive eruptions of Bezymianny.

Conjugates of tacrine and 1,2,4-thiadiazole derivatives as new potential multifunctional agents for Alzheimer's disease treatment: Synthesis, quantum-chemical characterization, molecular docking, and biological evaluation.

We synthesized conjugates of tacrine with 1,2,4-thiadiazole derivatives linked by two different spacers, pentylaminopropene (compounds 4) and pentylaminopropane (compounds 5), as potential drugs for the treatment of Alzheimer's disease (AD). The conjugates effectively inhibited cholinesterases with a predominant effect on butyrylcholinesterase (BChE). They were also effective at displacing propidium from the peripheral anionic site (PAS) of acetylcholinesterase (AChE), suggesting that they could block AChE-induced ß-amyloid aggregation. In addition, the compounds exhibited high radical-scavenging capacity. Conjugates 5 had higher anti-BChE activity and greater anti-aggregant potential as well relatively lower potency against carboxylesterase than compounds 4. Quantum-mechanical (QM) characterization agreed with NMR data to identify the most stable forms of conjugates for docking studies, which showed that the compounds bind to both CAS and PAS of AChE consistent with mixed reversible inhibition. Conjugates 4 were more potent radical scavengers, in agreement with HOMO localization in the enamine-thiadiazole system. Computational studies showed that all of the conjugates were expected to have good intestinal absorption, whereas conjugates 4 and 5 were predicted to have medium and high blood-brain barrier permeability, respectively. All conjugates were predicted to have medium cardiac toxicity risks. Overall, the results indicated that the conjugates are promising candidates for further development and optimization as multifunctional therapeutic agents for the treatment of AD.

Assessment of palmar subcutaneous tissue vascularization in patients with Dupuytren's contracture.

AIM: To investigate the structural and functional characteristics of palmar hypodermal tissue vascularization in Dupuytren's contracture patients of different age groups. METHODS: Eighty-seven Dupuytren's contracture patients underwent partial fasciectomy. Twenty-two of them were less than 55 years old (Y-group, n = 22); the others were 55 and older (O-group, n = 65). In surgically excised representative tissue samples, a histomorphometric analysis of the perforating arteries of the palmar aponeurosis and stereologic analysis of hypodermis vascularity were performed. The method of laser flowmetry estimated the microcirculation of the skin of the palm. RESULTS: Frequency of cases with rapid development of contracture (less than 5 years) was 13.6% in the Y-group and 40% in the O-group, P < 0.05. The external and luminal diameters of perforating arteries in palmar fascia were decreased more severely in Y. The thickness of intima increased three times compared with healthy control, and the intima/media relation also increased, especially in O. Increased numerical and volumetric micro-vessel densities in hypodermis, percentage of large vessels (more than 12 µm in diameter), and percentage of vessels with signs of periadventitial inflammatory infiltration were noted in Y. The percentage of vessels with adventitial fibrosis was greater in O than in Y. Base capillary flow in Y was increased compared to healthy control subjects and to O, and peak capillary flow was increased in comparison with control. CONCLUSION: Compared to the O-group, Y-group patients exhibited more severe constrictive remodeling of palmar fascia perforating arteries supplying hypodermis but more expressed compensatory changes of its capillarization.

Electron probe microanalysis оf experimentally stimulated osteoarthrosis in dogs.

AIM: To develop methods of articular cartilage preparation for X-ray-electron probe microanalysis and to study its elements content in experimental osteoarthrosis. METHODS: Twenty dogs aged 2-8 years were divided in research (aged 2 years, induction of osteoarthrosis - IOA) and intact group. Intact group included three subgroups (aged 2, 5 and 8 years). Samples of cartilage after araldite saturation and pouring were partially cut into semithin sections stained with methylene blue and with methylene blue-basic fuchsin. Their smooth surfaces were investigated by X-ray-electron probe microanalysis. Spatial distribution of sulfur, calcium and phosphorus and their concentrations (weight %) were investigated. RESULTS: X-ray electron probe microanalysis revealed non-uniform sulfur distribution in cartilage of intact animals: Its content increases from superficial zone to deep one, this regularity was preserved in animals with IOA. Differences of IOA with spontaneous chondropathy were revealed. Spontaneous aging was characterized by calcium and phosphorus storage in deep and calcified zones and compensatory increase of sulfated glycosaminoglycans in intermediate and deep cartilage zones as evidenced by the metachromatic reaction and microanalysis data. Unlike spontaneous chondropathy connected with aging in experimentally stimulated osteoarthrosis more intensive storage of calcium but minor phosphorus in intermediate zone were marked. In IOA the calcified cartilage thinning and osteoclastic resorption are apparent with few changes of elements composition; the only difference from control is minority phosphorus content. CONCLUSION: The obtained results demonstrate specific tricks of X-ray electron probe microanalysis and its possibility in the research of mechanisms of articular cartilage alterations in osteoarthrosis.

Benefits of Ilizarov automated bone distraction for nerves and articular cartilage in experimental leg lengthening.

AIM: To determine peculiarities of tissue responses to manual and automated Ilizarov bone distraction in nerves and articular cartilage. METHODS: Twenty-nine dogs were divided in two experimental groups: Group M - leg lengthening with manual distraction (1 mm/d in 4 steps), Group A - automated distraction (1 mm/d in 60 steps) and intact group. Animals were euthanized at the end of distraction, at 30th day of fixation in apparatus and 30 d after the fixator removal. M-responses in gastrocnemius and tibialis anterior muscles were recorded, numerical histology of peroneal and tibialis nerves and knee cartilage semi-thin sections, scanning electron microscopy and X-ray electron probe microanalysis were performed. RESULTS: Better restoration of M-response amplitudes in leg muscles was noted in A-group. Fibrosis of epineurium with adipocytes loss in peroneal nerve, subperineurial edema and fibrosis of endoneurium in some fascicles of both nerves were noted only in M-group, shares of nerve fibers with atrophic and degenerative changes were bigger in M-group than in A-group. At the end of experiment morphometric parameters of nerve fibers in peroneal nerve were comparable with intact nerve only in A-group. Quantitative parameters of articular cartilage (thickness, volumetric densities of chondrocytes, percentages of isogenic clusters and empty cellular lacunas, contents of sulfur and calcium) were badly changed in M-group and less changed in A-group. CONCLUSION: Automated Ilizarov distraction is more safe method of orthopedic leg lengthening than manual distraction in points of nervous fibers survival and articular cartilage arthrotic changes.

Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors.

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22-2.3µM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30µM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.

Histomorphometric analysis of knee synovial membrane in dogs undergoing leg lengthening by classic Ilizarov method and rapid automatic distraction.

PURPOSE: This pilot study aimed to test the theory that different lengthening methods affect the microscopic structure of knee joint synovium in diverse ways. METHODS: This was a descriptive and analytical cross-sectional study of synovium carried out in two experimental models of canine leg lengthening using the Ilizarov fixator. Group 1 (n = 6) used manual gradual distraction most commonly used in clinical practice at one millimetre/day divided into four equal increments, 0.25 mm at each increment. Group 2 (n = 7) used an increased rate of automatic distraction at three millimetres/day divided in 120 increments, 0.025 mm at each increment. At the end of distraction and after fixator removal, the animals were euthanised. Control was via intact dogs. The thickness of the synovial lining layer, number of synovial cell rows, and numerical density of subsynovial microvessels were assessed in digital images for semiautomated computerised analysis of semi-thin sections stained with toluidine blue and methylene blue-basic fuchsin. Comparison of synovitis manifestation was made with grading scale. The vascular and nerve changes in the subsynovial layer were also compared. RESULTS: Group 1 developed marked synovitis, synovium hypervascularisation, degeneration of the nerve fibres in subsynovial nerves with the tendency to regeneration. Group 2 had moderate to mild degree of synovitis with progressive degenerative changes in subsynovial vessels and nerves. CONCLUSION: Both methods used are unfavourable for the state of the joint synovium, but modify it in different ways.