The study of cellular cytotoxicity of argireline® - an anti-aging peptide

Argireline® is well know, innovative anti-aging product used in the cosmetic market. This short chain peptide is used as active ingredient in dermal ointment and creams. Argireline® prevents formation of skin lines and wrinkles in a very similar way to the botulinum toxin (Botox), inhibiting neurotransmitter release at the neuromuscular junction. Argireline® does not require under skin muscle injections and it is believed to be relatively safe. However, despite the fact that some toxicity data has been provided by the product manufacturer, there is an evident lack of reliable information about cytotoxicity of argireline® in the literature. The aim of the presented study was to estimate the antiproliferation effect of argireline® solution in several concentrations. The influence of argireline® on cellular proliferation was examined against: human embryonic kidney HEK-293 cell line, human neuroblastoma IMR-32 cell line, and human primary skin fibroblasts. Tests were performed using formazan-based cell proliferation assay: EZ4U, which allows to measure the efficiency of mitochondrial oxidative activity in living cells. The argireline® inhibitory concentration, IC50 values were calculated and the results were compared to the IC50 value of the reference compound: doxorubicin. In conclusion, the considered method resulted in dose-dependent argireline® anti-proliferation effects. However, the significant cytotoxicity of argireline® solution was observed under 18 to 10 000 fold higher concentrations (depending on cells that were examined) in comparison to doxorubicin

Are the hydantoin-1,3,5-triazine 5−HT6R5-HT_{6}R ligands a hope to a find new procognitive and anti-obesity drug? : considerations based on primary in vivo assays and ADME-Tox profile in vitro

$5-HT_{6}R$ Though the $5-HT_{6}R$ serotonin receptor is an important target giving both agonists and antagonists similar therapeutic potency in the treatment of topic CNS-diseases, no $5-HT_{6}R$ ligand has reached the pharmaceutical market yet due to the too narrow chemical space of the known $5-HT_{6}R$ agents and insuffcient "drugability." Recently, a new group of non-indole and non-sulfone hydantoin-triazine $5-HT_{6}R$ ligands was found, where 3-((4-amino-6-(4-methylpiperazin-1-yl)- 1,3,5-triazin-2-yl)methyl)-5-methyl-5-(naphthalen-2-yl)imidazolidine-2,4-dione (KMP-10) was the most active member. This study is focused on wider pharmacological and "druglikeness" characteristics for KMP-10. A computer-aided insight into molecular interactions with $5-HT_{6}R$ has been performed. "Druglikeness" was examined using an eight-test panel in vitro, i.e., a parallel artificial membrane permeability assay (PAMPA), and Caco-2 permeability-, P-glycoprotein (Pgp) affnity-, plasma protein binding-, metabolic stability- and drug–drug interaction-assays, as well as mutagenicity- and HepG2-hepatotoxicity risk tests. Behavioral studies in vivo, i.e., elevated plus-maze (EPM) and novel object recognition (NOR) tests, were performed. Extended studies on the influence of KMP-10 on rats' metabolism, including biochemical tests, were conducted in vivo. Results indicated significant anxiolytic and precognitive properties, as well as some anti-obesity properties in vivo, and it was found to satisfy the "druglikeness" profile in vitro for KMP-10. The compound seems to be a good lead-structure and candidate for wider pharmacological studies in search for new CNS-drugs acting via $5-HT_{6}R$

The 1,3,5-triazine derivatives as innovative chemical family of 5-HT6 serotonin receptor agents with therapeutic perspectives for cognitive impairment

Among serotonin receptors, the 5-HT6 subtype is the most controversial and the least known in the field of molecular mechanisms. The 5-HT6R ligands can be pivotal for innovative treatment of cognitive impairment, but none has reached pharmacological market, predominantly, due to insufficient “druglikeness” properties. Recently, 1,3,5-triazine-piperazine derivatives were identified as a new chemical family of potent 5-HT6R ligands. For the most active triazine 5-HT6R agents found (1-4), a wider binding profile and comprehensive in vitro evaluation of their drug-like parameters as well as behavioral studies and an influence on body mass in vivo were investigated within this work. Results indicated the most promising pharmacological/druglikeness profiles for 4-((1H-indol-3-yl)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (3) and 4-((2-isopropyl-5-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (4), which displayed a significant procognitive action and specific anxiolytic-like effects in the behavioral tests in vivo together with satisfied pharmaceutical and safety profiles in vitro. The thymol derivative (4) seems to be of higher importance as a new lead candidate, due to the innovative, non-indole and non-sulfone structure with the best 5-HT6R binding properties

Computer-Aided Studies for Novel Arylhydantoin 1,3,5-Triazine Derivatives as 5-HT6 Serotonin Receptor Ligands with Antidepressive-Like, Anxiolytic and Antiobesity Action In Vivo

This study focuses on the design, synthesis, biological evaluation, and computer-aided structure-activity relationship (SAR) analysis for a novel group of aromatic triazine-methylpiperazines, with an hydantoin spacer between 1,3,5-traizine and the aromatic fragment. New compounds were synthesized and their affinities for serotonin 5-HT6, 5-HT1A, 5-HT2A, 5-HT7, and dopamine D2 receptors were evaluated. The induced-fit docking (IFD) procedure was performed to explore the 5-HT6 receptor conformation space employing two lead structures. It resulted in a consistent binding mode with the activity data. For the most active compounds found in each modification line, anti-obesity and anti-depressive-like activity in vivo, as well as “druglikeness” in vitro, were examined. Two 2-naphthyl compounds (18 and 26) were identified as the most active 5-HT6R agents within each lead modification line, respectively. The 5-(2-naphthyl)hydantoin derivative 26, the most active one in the series (5-HT6R: Ki = 87 nM), displayed also significant selectivity towards competitive G-protein coupled receptors (6–197-fold). Docking studies indicated that the hydantoin ring is stabilized by hydrogen bonding, but due to its different orientation, the hydrogen bonds form with S5.44 and N6.55 or Q6.58 for 18 and 26, respectively. Compound 26 exerted anxiolytic-like and antidepressant-like activities. Importantly, it demonstrated anti-obesity properties in animals fed palatable feed, and did not show toxic effects in vitro

N-Benzyl-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) with hybrid structure as a candidate for a broad-spectrum antiepileptic drug

In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZinduced seizures inmice. Thus, AS-1may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 $\mu$M, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 $\mu$M)

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro

Are the Hydantoin-1,3,5-triazine 5-HT6R Ligands a Hope to a Find New Procognitive and Anti-Obesity Drug? Considerations Based on Primary In Vivo Assays and ADME-Tox Profile In Vitro

Though the 5-HT6 serotonin receptor is an important target giving both agonists and antagonists similar therapeutic potency in the treatment of topic CNS-diseases, no 5-HT6R ligand has reached the pharmaceutical market yet due to the too narrow chemical space of the known 5-HT6R agents and insufficient “drugability.” Recently, a new group of non-indole and non-sulfone hydantoin-triazine 5-HT6R ligands was found, where 3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-methyl-5-(naphthalen-2-yl)imidazolidine-2,4-dione (KMP-10) was the most active member. This study is focused on wider pharmacological and “druglikeness” characteristics for KMP-10. A computer-aided insight into molecular interactions with 5-HT6R has been performed. “Druglikeness” was examined using an eight-test panel in vitro, i.e., a parallel artificial membrane permeability assay (PAMPA), and Caco-2 permeability-, P-glycoprotein (Pgp) affinity-, plasma protein binding-, metabolic stability- and drug–drug interaction-assays, as well as mutagenicity- and HepG2-hepatotoxicity risk tests. Behavioral studies in vivo, i.e., elevated plus-maze (EPM) and novel object recognition (NOR) tests, were performed. Extended studies on the influence of KMP-10 on rats’ metabolism, including biochemical tests, were conducted in vivo. Results indicated significant anxiolytic and precognitive properties, as well as some anti-obesity properties in vivo, and it was found to satisfy the “druglikeness” profile in vitro for KMP-10. The compound seems to be a good lead-structure and candidate for wider pharmacological studies in search for new CNS-drugs acting via 5-HT6R

Evaluation influence acetyl hexapeptide-3 on the proliferation of eukaryotic cell lines selected in vitro

Przedmiotem pracy jest problematyka związana z procesami starzenia i tworzenia się zmarszczek. Skóra to największy organ w ludzkim organizmie, w którym następuje wiele procesów fizjologicznych. Narażona jest codziennie na wiele czynników ze środowiska zewnętrznego, w tym także na produkty kosmetyczne stosowane w codziennej pielęgnacji, które mogą stanowić dla niej zagrożenie. Formuły kosmetyków, czy ich komponenty również mogą stanowić potencjalne ryzyko wpływające nie tylko na skórę, ale cały organizm. W pracy omówiono wybrane metody alternatywne badań toksyczności kosmetyków, gdyż w związku z Dyrektywą Unii Europejskiej 76/768/EWG w Państwach Członkowskich zostało zakazane testowanie na zwierzętach całej formuły kosmetyku oraz poszczególnych składników. Jeden z tych testów został przeprowadzony i opisany na potrzeby napisania tejże rozprawy naukowej.Celem niniejszej pracy magisterskiej było przeprowadzenie i omówienie wyników badania cytotoksyczności komercyjnego wodnego roztworu argireliny oraz czystej substancji argireliny wobec następujących linii komórkowych: ludzkiej embrionalnej nerki HEK- 293, ludzkiej neuroblastomy IMR- 32 oraz ludzkich fibroblastów HSF. Żywotność komórek oceniano za pomocą testu EZ4U, który jest kolorymetrycznym testem opartym na formazanie. Dzięki eksperymentowi obliczono wartości IC50 argireliny wobec wcześniej wspomnianych linii komórkowych i zestawiono je z wartością IC50 wyliczoną dla doksorubicyny, która jest silnym cytostatykiem używanym w leczeniu nowotworów. Problematyka, która została poruszona w tej pracy ma ogromne znaczenie i pokazuje jak ważne dla przemysłu kosmetycznego jest przeprowadzenie badań toksyczności poszczególnych składników kosmetyków. Wyniki tych badań powinny być powszechnie udostępniane Konsumentowi, który korzystając z kosmetyków na co dzień, często darzy producenta bezkrytycznym zaufaniem i powierza mu swoje zdrowie, nie mając świadomości jakie mogą one mieć wpływ na skórę i organizm.The subject of this paper is the issues related to aging and wrinkle formation. The skin is the largest organ in the human body, in which there are many physiological processes. Skin is exposed daily to many factors from the external environment, including cosmetic products used in everyday care, which may be threat to it. Cosmetics formulas or its components are also a potential risk and may affect not only the skin, but the whole body. The paper discusses some alternative methods for toxicity testing, with respect to the EU Directive 76/768/EEC, which banned animal testing of cosmetic formulas and all individual components in the Member States. One of these tests was carried out and described for the purposes of that dissertation.The aim of this paper was to perform and discuss the results of cytotoxicity tests of commercial aqueous solution of argireline and the pure substance- argireline against following cell lines: human embryonic kidney HEK-293, human neuroblastoma IMR-32, human fibroblasts HSF. The cell viability was determined by a test EZ4U, which is a colorimetric test based on the formazan dye. Through the experiment, the IC50 of argireline was calculated against the mentioned cell lines, and in order to illustrate the anti-proliferative effect, the data were compared to the reference compound- doxorubicin, a potent chemotherapeutic agent, used in the treatment of tumors. The issue, which has been addressed in this work is of great importance and shows how it is important for the cosmetics industry to study the cytotoxicity of cosmetics components. The results of that research should be disseminated to Consumer, who use cosmetics every day and is no aware how it can affect the skin and body entrusting the manufacturer with his health