The role of oxidative stress in the etiology of selected civilization diseases

The evolutionary ability to use oxygen in the process of respiration has provided living organisms with an efficient source of energy and made the development of life on Earth possible. However, oxygen and its derivatives can also pose a threat. During physiological processes, ROS (reactive oxygen species) are generated. Excessive amounts of these chemically active molecules may lead to the modification of biologically important macromolecules (proteins, nucleic acids), resulting in irreversible disturbances in the structure of key cell structures (cell nucleus, membranes). Another consequence of ROS activity is the disruption of signal transduction pathways in the cell, which leads to the development of many serious diseases (atherosclerosis, cancer). On the other hand, similarly to excess oxygen, its deficiency can be disastrous for cells. Hypoxia, i.e. a state of insufficient oxygen supply in relation to demand, is relevant not only in ischaemic heart disease and myocardial infarction but also in many other cardiovascular, neurodegenerative and even cancerous diseases. A major role in the response to hypoxia at the cellular level is played by hypoxiainducible factor (HIF), whose hyperactivation is associated with multidirectional disruption of intracellular pathways. There is a close link between hypoxia-related phenomena and ROS at the molecular level, which is based on bidirectional regulation. Therefore, both factors should be considered together in regards to the development of many pathologies. Despite the role of ROS and hypoxia in the development of lifestyle diseases, which has been discussed for years, it has still not been possible to introduce effective targeted therapy in this area. Notwithstanding encouraging initial data, many studies have provided inconclusive results on the efficacy of antioxidant therapy in neurodegenerative, cardiovascular and cancer diseases. In the latter case, however, the use of hypoxia-activated drugs and HIF-1 inhibitors seems to be a promising strategy. This demonstrates the need for a better understanding of the mechanisms involved in the action of the aforementioned factors and warrants further research in this area. The aim of this work is to present the mechanism of action of RFTs and hypoxia, their role in the pathophysiology of the most common human diseases and their potential use as targets for therapy

Studies of new purine derivatives with acetic acid moiety in human keratinocytes

Recently we described a group of purine derivatives based on theophylline structure with acetic acid moiety. Studies in a group of these compounds demonstrated their analgesic and anti-inflammatory properties. Taking into account wide spectrum of theophylline derivatives activity and searching for their new properties. the aim of the study was to evaluate safety of newly synthesized derivatives in human keratinocytes model. The effect of new purine derivatives with acetic acid moiety: 2-(8-methoxy-1,3-dimethyl-2,6-dioxo-purin-7-yl) acetic acid and 2-(1,3-dimethyl-2,6,8-trioxo-9H-purin-7-yl) acetic acid on proliferation rate and the ability of keratinocytes to migration was carried out. The results clearly demonstrate that purine derivatives with acetic acid moiety did not affect basic keratinocytes functions. Our compounds do not inhibit cells proliferation rate as well as their ability to migration. It can be therefore concluded that new purine derivatives with acetic acid moiety are safe versus normal cells. This observation opens up additional prospects in searching for their new applications

Lclet 4 enhances pro-apoptotic and anti-invasive effects of mitoxantrone on human prostate cancer cells : in vitro study

Triterpene saponosides are widely distributed plant secondary metabolites characterized by relatively low systemic cytotoxicity and a range of biological activities. These include anti-inflammatory, antimicrobial, vasoprotective and antitumor properties. In particular, the ability of saponins to enhance the cytotoxicity of chemotherapeutic drugs opened perspectives for their application in combined cancer chemotherapy. Here, we used human prostate cancer DU-145 cells as an in vitro model to elucidate the synergy of the interactions between biological activities of an oleanane type 13β,28-epoxy triterpene saponoside (Lclet 4) and mitoxantrone, which is a cytostatic drug commonly used in prostate cancer therapy. No cytotoxic or pro-apoptotic effect of Lclet 4 and mitoxantrone administered at the concentrations between 0.05 and 0.1 µg/ml could be seen. In contrast, cocktails of these agents exerted synergistic pro-apoptotic effects, accompanied by the activation of the caspase 3/7 system. This effect was paralleled by attenuating effects of Lclet 4/mitoxantrone cocktails on the invasive potential, metalloproteinase expression and motility of DU-145 cells. Multifaceted and additive effects of Lclet 4 and mitoxantrone on basic cellular traits crucial for prostate cancer progression indicate that the combined application of both agents at systemically neutral concentrations may provide the basis for new promising strategies of prostate cancer chemotherapy

Metabolic stability and its role in the discovery of new chemical entities

Determination of metabolic profiles of new chemical entities is a key step in the process of drug discovery, since it influences pharmacokinetic characteristics of therapeutic compounds. One of the main challenges of medicinal chemistry is not only to design compounds demonstrating beneficial activity, but also molecules exhibiting favourable pharmacokinetic parameters. Chemical compounds can be divided into those which are metabolized relatively fast and those which undergo slow biotransformation. Rapid biotransformation reduces exposure to the maternal compound and may lead to the generation of active, non-active or toxic metabolites. In contrast, high metabolic stability may promote interactions between drugs and lead to parent compound toxicity. In the present paper, issues of compound metabolic stability will be discussed, with special emphasis on its significance, in vitro metabolic stability testing, dilemmas regarding in vitro-in vivo extrapolation of the results and some aspects relating to different preclinical species used in in vitro metabolic stability assessment of compounds

Pan-Phosphodiesterase Inhibitors Attenuate TGF-β-Induced Pro-Fibrotic Phenotype in Alveolar Epithelial Type II Cells by Downregulating Smad-2 Phosphorylation

Airway remodeling is a pathological process that accompanies many chronic lung diseases. One of the important players in this process are epithelial cells, which under the influence of pro-inflammatory and pro-fibrotic factors present in the airway niche, actively participate in the remodeling process by increasing extracellular matrix secretion, acquiring migration properties, and overproducing pro-fibrotic transducers. Here, we investigated the effect of three new 8-arylalkylamino- and 8-alkoxy-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl-N-(5-(tert-butyl)-2-hydroxyphenyl)butanamides (1, 2, and 3), representing prominent pan-phosphodiesterase (pan-PDE) inhibitors on transforming growth factor type β (TGF-β)-induced alveolar epithelial type II cells (A549 cell line) of a pro-fibrotic phenotype. Our results demonstrate for the first time the strong activity of pan-PDE inhibitors in the prevention of TGF-β-induced mesenchymal markers’ expression and A549 cells’ migration. We also showed an increased p-CREB and decreased p-Smad-2 phosphorylation in TGF-β-induced A549 cells treated with 1, 2, and 3 derivatives, thereby confirming a pan-PDE inhibitor mesenchymal phenotype reducing effect in alveolar epithelial type II cells via suppression of the canonical Smad signaling pathway. Our observations confirmed that PDE inhibitors, and especially those active against various isoforms involved in the airway remodeling, constitute an interesting group of compounds modulating the pro-fibrotic response of epithelial cells

The application of in vitro models in a preclinical safety evaluation of new drug candidates

Modele in vitro to podstawowe narzędzie umożliwiające ustalenie profilu aktywności i sprawdzenie bezpieczeństwa kandydata na lek na etapie badań przedklinicznych. Dzięki nim możliwe jest wykonanie w krótkim czasie badań przesiewowych dla tysięcy aktywnych cząsteczek oraz przewidywanie ewentualnych działań niepożądanych. Pozwala to na wyeliminowanie potencjalnie niebezpiecznych związków na bardzo wczesnym etapie, jeszcze przed zastosowaniem modeli zwierzęcych. W niniejszej pracy zebrano najważniejsze informacje dotyczące wykorzystania metod in vitro, opierających się na modelach komórek prokariotycznych i eukariotycznych, w badaniu zarówno toksyczności narządowej (hepato-, neuro-, nefro-, kardiotoksyczności), jak i genotoksyczności związków aktywnych.In vitro models are an essential tool to determine the profile of activity and the safety of the drug candidate at the early stage of preclinical studies. Thanks to them it is possible to perform screening tests for thousands of active molecules in a short time and to predict their possible side effect. This allows to eliminate potentially dangerous compounds at a very early stage, before using animal models. This review presents the most important information about using in vitro methods, based on prokaryotic and eukaryotic cell models, in hepato-, neuro-, nephro-, cardio- and genotoxicity research of active compounds

S(+)-(2E)-N-(2-Hydroxypropyl)-3-Phenylprop-2-Enamide (KM-568) : a novel cinnamamide derivative with anticonvulsant activity in animal nodels of seizures and epilepsy

Epilepsy is one of the most frequent neurological disorders affecting about 1% of the world’s human population. Despite availability of multiple treatment options including antiseizure drugs, it is estimated that about 30% of seizures still remain resistant to pharmacotherapy. Searching for new antiseizure and antiepileptic agents constitutes an important issue within modern medicinal chemistry. Cinnamamide derivatives were identified in preclinical as well as clinical studies as important drug candidates for the treatment of epilepsy. The cinnamamide derivative presented here: S(+)-(2E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide (S(+)-N-(2-hydroxypropyl)cinnamamide, compound KM-568) showed anticonvulsant activity in several models of epilepsy and seizures in mice and rats. It was active in a genetic animal model of epilepsy (Frings audiogenic seizure-susceptible mouse model, ED50 = 13.21 mg/kg, i.p.), acute seizures induced electrically (maximal electroshock test ED50 = 44.46 mg/kg mice i.p., ED50 = 86.6 mg/kg mice p.o., ED50 = 27.58 mg/kg rats i.p., ED50 = 30.81 mg/kg rats p.o., 6-Hz psychomotor seizure model 32 mA ED50 = 71.55 mg/kg mice i.p., 44 mA ED50 = 114.4 mg/kg mice i.p.), chronic seizures induced electrically (corneal kindled mouse model ED50 = 79.17 mg/kg i.p., hippocampal kindled rat model ED50 = 24.21 mg/kg i.p., lamotrigine-resistant amygdala kindled seizure model in rats ED50 = 58.59 mg/kg i.p.), acute seizures induced chemically (subcutaneous metrazol seizure threshold test ED50 = 104.29 mg/kg mice i.p., ED50 = 107.27 mg/kg mice p.o., ED50 = 41.72 mg/kg rats i.p., seizures induced by picrotoxin in mice ED50 = 94.11 mg/kg i.p.) and the pilocarpine-induced status epilepticus model in rats (ED50 = 279.45 mg/kg i.p., ED97 = 498.2 mg/kg i.p.). The chemical structure of the compound including configuration of the chiral center was confirmed by NMR spectroscopy, LC/MS spectroscopy, elemental analysis, and crystallography. Compound KM-568 was identified as a moderately stable derivative in an in vitro mouse liver microsome system. According to the Ames microplate format mutagenicity assay performed, KM-568 was not a base substitution or frameshift mutagen. Cytotoxicity evaluation in two cell lines (HepG2 and H9c2) proved the safety of the compound in concentrations up to 100 µM. Based on the results of anticonvulsant activity and safety profile, S(+)-(2E)-N-(2-hydroxypropyl)-3-phenylprop-2-enamide could be proposed as a new lead compound for further preclinical studies on novel treatment options for epilepsy