MM165 - A Small Hybrid Molecule Modulates the Kynurenine Pathway and Attenuates Lipopolysaccharide-Induced Memory Deficits and Inflammation.

Cognitive dysfunctions are now recognized as core symptoms of various psychiatric disorders e.g., major depressive disorder. Sustained immune activation may leads to cognitive dysfunctions. Proinflammatory cytokines shunt the metabolism of tryptophan towards kynurenine and quinolinic acid may accumulate at toxic concentrations. This acid triggers an increase in neuronal nitric oxide synthase function and promotes oxidative stress. The searching for small molecules that can regulate tryptophan metabolites produced in the kynurenic pathway has become an important goal in developing treatments for various central nervous system diseases with an inflammatory component. Previously we have identified a small hybrid molecule - MM165 which significantly reduces depressive-like symptoms caused by inflammation induced by lipopolysaccharide administration. In the present study, we investigated whether this compound would mitigate cognitive deficits induced by lipopolysaccharide administration and whether treatment with it would affect the plasma or brain levels of quinolinic acid and kynurenic acid. Neuroinflammation was induced in rats by administering lipopolysaccharide at a dose of 0.5 mg/kg body weight for 10 days. We conducted two tests: novel object recognition and object location, to assess the effect on memory impairment in animals previously treated with lipopolysaccharide. In plasma collected from rats, the concentrations of C-reactive protein and tumor necrosis factor alfa were determined. The concentrations of kynurenic acid and quinolinic acid were determined in plasma and homogenates obtained from the cerebral cortex of rats. Interleukin 6 in the cerebral cortex of rats was determined. Additionally, the body and spleen mass and spontaneous activity were measured in rats. Our study shows that MM165 may mitigate cognitive deficits induced by inflammation after administration of lipopolysaccharide and alter the concentrations of tryptophan metabolites in the brain. Compounds exhibiting a mechanism of action analogous to that of MM165 may serve as foundational structures for the development of a new class of antidepressants.

Chemometrics as a valuable tool for evaluating interactions between antiretroviral drugs and food.

AIMS: Clinically significant interactions with food occur for more than half of antiretroviral drugs. Different physiochemical properties deriving from the chemical structures of antiretroviral drugs may contribute to the variable food effect. Chemometric methods allow analysing a large number of interrelated variables concomitantly and visualizing correlations between them. We used a chemometric approach to determine the types of correlations among different features of antiretroviral drugs and food that may influence interactions. METHODS: Thirty-three antiretroviral drugs were analysed: ten nucleoside reverse transcriptase inhibitors, six non-nucleoside reverse transcriptase inhibitors, five integrase strand transfer inhibitors, ten protease inhibitors, one fusion inhibitor and one HIV maturation inhibitor. Input data for the analysis were collected from already published clinical studies, chemical records and calculations. We constructed a hierarchical partial least squares (PLS) model with three response parameters: postprandial change of time to reach maximum drug concentration (ΔTmax ), albumin binding (%) and logarithm of partition coefficient (logP). Predictor parameters were the first two principal components of principal component analysis (PCA) models for six groups of molecular descriptors. RESULTS: PCA models explained 64.4% to 83.4% of the variance of the original parameters (average: 76.9%), whereas the PLS model had four significant components and explained 86.2% and 71.4% of the variance in the sets of predictor and response parameters, respectively. We observed 58 significant correlations between ΔTmax , albumin binding (%), logP and constitutional, topological, hydrogen bonding and charge-based molecular descriptors. CONCLUSIONS: Chemometrics is a useful and valuable tool for analysing interactions between antiretroviral drugs and food.

Interactions of Antiretroviral Drugs with Food, Beverages, Dietary Supplements, and Alcohol: A Systematic Review and Meta-analyses.

Multiple factors may affect combined antiretroviral therapy (cART). We investigated the impact of food, beverages, dietary supplements, and alcohol on the pharmacokinetic and pharmacodynamic parameters of 33 antiretroviral drugs. Systematic review in adherence to PRISMA guidelines was performed, with 109 reports of 120 studies included. For each drug, meta-analyses or qualitative analyses were conducted. We have found clinically significant interactions with food for more than half of antiretroviral agents. The following drugs should be taken with or immediately after the meal: tenofovir disoproxil, etravirine, rilpivirine, dolutegravir, elvitegravir, atazanavir, darunavir, lopinavir, nelfinavir, ritonavir, saquinavir. Didanosine, zalcitabine, zidovudine, efavirenz, amprenavir, fosamprenavir, and indinavir should be taken on an empty stomach for maximum patient benefit. Antiretroviral agents not mentioned above can be administered regardless of food. There is insufficient evidence available to make recommendations about consuming juice or alcohol with antiretroviral drugs. Resolving drug-food interactions may contribute to maximized cART effectiveness and safety.

RESUMEN: Múltiples factores pueden afectar la terapia antirretroviral combinada (cART). Investigamos el impacto de los alimentos, las bebidas, los suplementos dietéticos y el alcohol en los parámetros farmacocinéticos y farmacodinámicos de 33 medicamentos antirretrovirales. Se realizó la revisión sistemática en apego a las guías PRISMA, con 109 reportes de 120 estudios incluidos. Para cada fármaco se realizaron metanálisis o análisis cualitativos. Hemos encontrado interacciones clínicamente significativas con alimentos para más de la mitad de los fármacos antirretrovirales. Los siguientes medicamentos deben tomarse durante o inmediatamente después de comer: tenofovir, disoproxil, etravirina, rilpivirine, dolutegravir, elvitegravir, atazanavir, darunavir, lopinavir, nelfinavir, ritonavir, saquinavir. Didanosina, zalcitabina, zidovudina, efavirenz, amprenavir, fosamprenavir e indinavir deben tomarse con el estómago vacío para obtener el máximo beneficio para el paciente. Los fármacos antirretrovirales no mencionados anteriormente se pueden administrar independientemente de los alimentos. No hay suficiente evidencia disponible para hacer recomendaciones sobre el consumo de jugo o alcohol con medicamentos antirretrovirales. Resolver las interacciones entre medicamentos y alimentos puede contribuir a maximizar la eficacia y la seguridad de cART.

Application of Sorbent-Based Extraction Techniques in Food Analysis.

This review presents an outline of the application of the most popular sorbent-based methods in food analysis. Solid-phase extraction (SPE) is discussed based on the analyses of lipids, mycotoxins, pesticide residues, processing contaminants and flavor compounds, whereas solid-phase microextraction (SPME) is discussed having volatile and flavor compounds but also processing contaminants in mind. Apart from these two most popular methods, other techniques, such as stir bar sorptive extraction (SBSE), molecularly imprinted polymers (MIPs), high-capacity sorbent extraction (HCSE), and needle-trap devices (NTD), are outlined. Additionally, novel forms of sorbent-based extraction methods such as thin-film solid-phase microextraction (TF-SPME) are presented. The utility and challenges related to these techniques are discussed in this review. Finally, the directions and need for future studies are addressed.

Relationships between Molecular Characteristics of Novel Organic Selenium Compounds and the Formation of Sulfur Compounds in Selenium Biofortified Kale Sprouts.

Due to problems with selenium deficiency in humans, the search for new organic molecules containing this element in plant biofortification process is highly required. Selenium organic esters evaluated in this study (E-NS-4, E-NS-17, E-NS-71, EDA-11, and EDA-117) are based mostly on benzoselenoate scaffolds, with some additional halogen atoms and various functional groups in the aliphatic side chain of different length, while one compound contains a phenylpiperazine moiety (WA-4b). In our previous study, the biofortification of kale sprouts with organoselenium compounds (at the concentrations of 15 mg/L in the culture fluid) strongly enhanced the synthesis of glucosinolates and isothiocyanates. Thus, the study aimed to discover the relationships between molecular characteristics of the organoselenium compounds used and the amount of sulfur phytochemicals in kale sprouts. The statistical partial least square model with eigenvalues equaled 3.98 and 1.03 for the first and second latent components, respectively, which explained 83.5% of variance in the predictive parameters, and 78.6% of response parameter variance was applied to reveal the existence of the correlation structure between molecular descriptors of selenium compounds as predictive parameters and biochemical features of studied sprouts as response parameters (correlation coefficients for parameters in PLS model in the range-0.521 ÷ 1.000). This study supported the conclusion that future biofortifiers composed of organic compounds should simultaneously contain nitryl groups, which may facilitate the production of plant-based sulfur compounds, as well as organoselenium moieties, which may influence the production of low molecular weight selenium metabolites. In the case of the new chemical compounds, environmental aspects should also be evaluated.

Self-Healable, Injectable Hydrogel with Enhanced Clotrimazole Solubilization as a Potential Therapeutic Platform for Gynecology.

Injectable, self-healing hydrogels with enhanced solubilization of hydrophobic drugs are urgently needed for antimicrobial intravaginal therapies. Here, we report the first hydrogel systems constructed of dynamic boronic esters cross-linking unimolecular micelles, which are a reservoir of antifungal hydrophobic drug molecules. The selective hydrophobization of hyperbranched polyglycidol with phenyl units in the core via ester or urethane bonds enabled the solubilization of clotrimazole, a water-insoluble drug of broad antifungal properties. The encapsulation efficiency of clotrimazole increases with the degree of the HbPGL core modification; however, the encapsulation is more favorable in the case of urethane derivatives. In addition, the rate of clotrimazole release was lower from HbPGL hydrophobized via urethane bonds than with ester linkages. In this work, we also revealed that the hydrophobization degree of HbPGL significantly influences the rheological properties of its hydrogels with poly(acrylamide-ran-2-acrylamidephenylboronic acid). The elastic strength of networks (GN) and the thermal stability of hydrogels increased along with the degree of HbPGL core hydrophobization. The degradation of the hydrogel constructed of the neat HbPGL was observed at approx. 40 °C, whereas the hydrogels constructed on HbPGL, where the monohydroxyl units were modified above 30 mol %, were stable above 50 °C. Moreover, the flow and self-healing ability of hydrogels were gradually decreased due to the reduced dynamics of macromolecules in the network as an effect of increased hydrophobicity. The changes in the rheological properties of hydrogels resulted from the engagement of phenyl units into the intermolecular hydrophobic interactions, which besides boronic esters constituted additional cross-links. This study demonstrates that the HbPGL core hydrophobized with phenyl units at 30 mol % degrees via urethane linkages is optimal in respect of the drug encapsulation efficiency and rheological properties including both self-healable and injectable behavior. This work is important because of a proper selection of a building component for the construction of a therapeutic hydrogel platform dedicated to the intravaginal delivery of hydrophobic drugs.

Potential of nafimidone derivatives against co-morbidities of epilepsy: In vitro, in vivo, and in silico investigations.

Nafimidone is known for its clinical antiepileptic effects and alcohol derivatives of nafimidone were reported be potent anticonvulsants. These compounds are structurally similar to miconazole, which is known to inhibit cholinesterases, protect neurons, and ameliorate cognitive decline. Herein, we aimed to reveal the potential of three nafimidone alcohol esters (5 g, 5i, and 5 k), which were previously reported for their anticonvulsant effects, against co-morbidities of epilepsy such as inflammatory and neuropathic pain, cognitive and behavioral deficits, and neuron death, and understand their roles in related pathways such as γ-butyric acid type A (GABAA ) receptor and cholinesterases using in vitro, in vivo and in silico methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for cytotoxicity evaluation, hippocampal slice culture assay for neuroprotection, formalin test for acute and inflammatory pain, sciatic ligation for neuropathic pain, Morris water maze and open field locomotor tasks for cognitive and behavioral deficits, radioligand binding for GABAA receptor affinity, spectrophotometric methods for cholinesterase inhibition in vitro, and molecular docking in silico. The compounds were non-toxic to fibroblast cells. 5 k was neuroprotective against kainic acid-induced neuron death. 5i reduced pain response of mice in both the acute and the inflammatory phases. 5i improved survival upon status epilepticus. The compounds showed no affinity to GABAA receptor but inhibited acetylcholinesterase, 5 k also inhibited butyrylcholinesterase. The compounds were predicted to interact mainly with the peripheric anionic site of cholinesterase enzymes. The title compounds showed neuroprotective, analgesic, and cholinesterase inhibitory effects, thus they bear promise against certain co-morbidities of epilepsy with neurological insults.

Role of Sulfur Compounds in Vegetable and Mushroom Aroma.

At the base of the food pyramid is vegetables, which should be consumed most often of all food products, especially in raw and unprocessed form. Vegetables and mushrooms are rich sources of bioactive compounds that can fulfill various functions in plants, starting from protection against herbivores and being natural insecticides to pro-health functions in human nutrition. Many of these compounds contain sulfur in their structure. From the point of view of food producers, it is extremely important to know that some of them have flavor properties. Volatile sulfur compounds are often potent odorants, and in many vegetables, belonging mainly to Brassicaeae and Allium (Amaryllidaceae), sulfur compounds determine their specific flavor. Interestingly, some of the pathways that form volatile sulfur compounds in vegetables are also found in selected edible mushrooms. The most important odor-active organosulfur compounds can be divided into isothiocyanates, nitriles, epithionitriles, thiols, sulfides, and polysulfides, as well as others, such as sulfur containing carbonyl compounds and esters, R-L-cysteine sulfoxides, and finally heterocyclic sulfur compounds found in shiitake mushrooms or truffles. This review paper summarizes their precursors and biosynthesis, as well as their sensory properties and changes in selected technological processes.

Star-Shaped Poly(furfuryl glycidyl ether)-Block-Poly(glyceryl glycerol ether) as an Efficient Agent for the Enhancement of Nifuratel Solubility and for the Formation of Injectable and Self-Healable Hydrogel Platforms for the Gynaecological Therapies.

In this paper, we present novel well-defined unimolecular micelles constructed a on poly(furfuryl glycidyl ether) core and highly hydrophilic poly(glyceryl glycerol ether) shell, PFGE-b-PGGE. The copolymer was synthesized via anionic ring-opening polymerization of furfuryl glycidyl ether and (1,2-isopropylidene glyceryl) glycidyl ether, respectively. MTT assay revealed that the copolymer is non-cytotoxic against human cervical cancer endothelial (HeLa) cells. The copolymer thanks to furan moieties in its core is capable of encapsulation of nifuratel, a hydrophobic nitrofuran derivative, which is a drug applied in the gynaecology therapies that shows a broad antimicroorganism spectrum. The study shows high loading capacity of the copolymer, i.e., 146 mg of nifuratel per 1 g of copolymer. The load unimolecular micelles were characterized using DLS and TEM microscopy and compared with the reference glyceryl glycerol ether homopolymer sample. The presence of numerous 1,2-diol moieties in the shell of PFGE-b-PGG macromolecules enabled the formation of reversible cross-links with 2-acrylamidephenylboronic acid-based polyacrylamide. The obtained hydrogels were both injectable and self-healable, which was confirmed with a rheological study.

The Slow-Releasing and Mitochondria-Targeted Hydrogen Sulfide (H2S) Delivery Molecule AP39 Induces Brain Tolerance to Ischemia.

Ischemic stroke is the third leading cause of death in the world, which accounts for almost 12% of the total deaths worldwide. Despite decades of research, the available and effective pharmacotherapy is limited. Some evidence underlines the beneficial properties of hydrogen sulfide (H2S) donors, such as NaSH, in an animal model of brain ischemia and in in vitro research; however, these data are ambiguous. This study was undertaken to verify the neuroprotective activity of AP39, a slow-releasing mitochondria-targeted H2S delivery molecule. We administered AP39 for 7 days prior to ischemia onset, and the potential to induce brain tolerance to ischemia was verified. To do this, we used the rat model of 90-min middle cerebral artery occlusion (MCAO) and used LC-MS/MS, RT-PCR, LuminexTM assays, Western blot and immunofluorescent double-staining to determine the absolute H2S levels, inflammatory markers, neurotrophic factor signaling pathways and apoptosis marker in the ipsilateral frontal cortex, hippocampus and in the dorsal striatum 24 h after ischemia onset. AP39 (50 nmol/kg) reduced the infarct volume, neurological deficit and reduced the microglia marker (Iba1) expression. AP39 also exerted prominent anti-inflammatory activity in reducing the release of Il-1ß, Il-6 and TNFα in brain areas particularly affected by ischemia. Furthermore, AP39 enhanced the pro-survival pathways of neurotrophic factors BDNF-TrkB and NGF-TrkA and reduced the proapoptotic proNGF-p75NTR-sortilin pathway activity. These changes corresponded with reduced levels of cleaved caspase 3. Altogether, AP39 treatment induced adaptative changes within the brain and, by that, developed brain tolerance to ischemia.

Novel Hydrogen Sulfide (H2S)-Releasing BW-HS-101 and Its Non-H2S Releasing Derivative in Modulation of Microscopic and Molecular Parameters of Gastric Mucosal Barrier.

Hydrogen sulfide (H2S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H2S-prodrug, BW-HS-101 with the ability to release H2S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5-50 µmol/kg) or its analogue without the ability to release H2S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE2 concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 µmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H2S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H2S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H2S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.

Determination of Volatile Terpenes in Coriander Cold Pressed Oil by Vacuum Assisted Sorbent Extraction (VASE).

Cold-pressed plant oils are of high interest to consumers due to their unique and interesting flavors. As they are usually only pressed at low temperatures and filtered, without further processing stages (as refining), they preserve their character that originates from the plant the oil was extracted from. Coriander cold pressed oil is gaining popularity as a novel product, obtained from its fruits/seeds; due to the high amount of terpenes, it has very characteristic flavor. A novel, vacuum-assisted sorbent extraction (VASE) method was used to extract terpenes from coriander cold pressed oil. Optimal parameters were determined. The profile of compounds extracted using VASE was compared with that of classic hydrodistillation method. Moreover, 17 monoterpene hydrocarbons and alcohols were identified with ß-linalool as the main compound, followed by α-pinene, γ-terpinene, camphor, sylvestrene, ß-pinene, and o-cymene. Differences were noted between profiles of terpenes after hydrodistillation and VASE extraction. For 8 out of 17 terpenes, VASE was used for their quantitative analysis. Regarding simplicity of the method, small sample requirement (200 mg) and short extraction time (5 min), VASE combined with GC/MS is well suited for characterization of terpenes in such matrix as plant oils.

In Search of a Phosphorus Dendrimer-Based Carrier of Rose Bengal: Tyramine Linker Limits Fluorescent and Phototoxic Properties of a Photosensitizer.

Photodynamic therapy (PDT) is a skin cancer treatment alternative to chemotherapy and radiotherapy. This method exploits three elements: a phototoxic compound (photosensitizer), light source and oxygen. Upon irradiation by light of a specific wavelength, the photosensitizer generates reactive oxygen species triggering the cascade of reactions leading to cell death. The positive therapeutic effect of PDT may be limited due to low solubility, low tumor specificity and inefficient cellular uptake of photosensitizers. A promising approach to overcome these obstacles involves the use of nanocarrier systems. The aim of this initial study was to determine the potential of the application of phosphorus dendrimers as carriers of a photosensitizer-rose bengal (RB). The primary goal involved the synthesis and in vitro studies of covalent drug-dendrimer conjugates. Our approach allowed us to obtain RB-dendrimer conjugates with the use of tyramine as an aromatic linker between the carrier and the drug. The compounds were characterized by FT-IR, 1H NMR, 13C NMR, 31P NMR, size and zeta potential measurements and spectrofluorimetric analysis. The dialysis to check the drug release from the conjugate, flow cytometry to specify intracellular uptake, and singlet oxygen generation assay were also applied. Finally, we used MTT assay to determine the biological activity of the tested compounds. The results of our experiments indicate that the conjugation of RB to phosphorus dendrimers via the tyramine linker decreases photodynamic activity of RB.

Inactivation of Thioglucosidase from Sinapis alba (White Mustard) Seed by Metal Salts.

The glucosinolates which are specialized plant metabolites of Brassica vegetables are prone to hydrolysis catalyzed by an endogenous enzyme myrosinase (thioglycoside hydrolase, thioglucosidase) that exists in Brassica plant tissue causing volatile isothiocyanates release. Currently existing literature data on the inactivation of myrosinase is insufficient in particular for use in the analysis of volatile and odor compounds in vegetables rich in glucosinolates. In this study, the impact of different metal salts in effective inactivation of enzyme activity was investigated by solid-phase microextraction (SPME) and GC/MS system in aqueous samples and kohlrabi matrix. A saturated solution of calcium chloride which is commonly used to stop enzyme activity in plant tissue inactivates the myrosinase-glucosinolate system. However, even without the participation of myrosinase, it changes the reaction pathway towards nitrile formation. The model experiment shows that optimum efficiency in inhibition of the enzyme system shows iron(III) ions, silver ions, and anhydride sodium sulfate resulting in no volatile products derived from glucosinolates. However, in the kohlrabi matrix, the strongest enzyme inhibition effect was observed for silver salt resulting in no volatile products, also both anhydrous Na2SO4 and saturated CaCl2 solution seem to be useful inhibitors in flavor studies.

Study on a Three-Step Rapid Assembly of Zolpidem and Its Fluorinated Analogues Employing Microwave-Assisted Chemistry.

We developed an efficient microwave-assisted three-step synthesis of zolpidem and its fluorinated analogues 1-3. The procedure relays on the utilization of easily accessible and inexpensive starting materials. Our protocol shows superior performance in terms of yield and purity of products, compared to conventional heating systems. Notably, the total time needed for reaction accomplishment is significantly lower comparing to oil bath heating systems. Finally, we have performed a detailed study on the preparation of zolpidem tartrate salt I, and we assessed its particle-sizes using a polarizing microscope. Our goal was to select the appropriate method that generates the acceptable particle-size, since the solid-size directly influences solubility in biological fluids and further bioavailability. We believe that the disclosed procedure will help to produce a lab-scale quantity of zolpidem and its fluorinated derivatives 1-3, as well as zolpidem tartrate salt I, with suitable fine-particle size for further biological experimentation.

Multicomponent Conjugates of Anticancer Drugs and Monoclonal Antibody with PAMAM Dendrimers to Increase Efficacy of HER-2 Positive Breast Cancer Therapy.

PURPOSE: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. METHODS: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. RESULTS: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. CONCLUSIONS: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.

Sugar Modification Enhances Cytotoxic Activity of PAMAM-Doxorubicin Conjugate in Glucose-Deprived MCF-7 Cells - Possible Role of GLUT1 Transporter.

PURPOSE: In order to overcome the obstacles and side effects of classical chemotherapy, numerous studies have been performed to develop the treatment based on targeted transport of active compounds directly to the site of action. Since tumor cells are featured with intensified glucose metabolism, we set out to develop innovative, glucose-modified PAMAM dendrimer for the delivery of doxorubicin to breast cancer cells. METHODS: PAMAM-dox-glc conjugate was synthesized and characterized by 1H NMR, FT-IR, size and zeta potential measurements. The drug release rate from conjugate was evaluated by dialysis under different pH conditions. The expression level of GLUT family receptors in cells cultured in full and glucose-deprived medium was evaluated by quantitative real-time RT-PCR and flow cytometry. The cytotoxicity of conjugate in presence or absence of GLUT1 inhibitors was determined by MTT assay. RESULTS: We showed that PAMAM-dox-glc conjugate exhibits pH-dependent drug release and increased cytotoxic activity compared to free drug in cells cultured in medium without glucose. Further, we proved that these cells overexpress transporters of GLUT family. The toxic effect of conjugate was eliminated by the application of specific GLUT1 inhibitors. CONCLUSION: Our findings revealed that the glucose moiety plays a crucial role in the recognition of cells with high expression of GLUT receptors. By selectively blocking GLUT1 transporter we showed its importance for the cytotoxic activity of PAMAM-dox-glc conjugate. These results suggest that PAMAM-glucose formulations may constitute an efficient platform for the specific delivery of anticancer drugs to tumor cells overexpressing transporters of GLUT family.

Anti-Alzheimer's multitarget-directed ligands with serotonin 5-HT6 antagonist, butyrylcholinesterase inhibitory, and antioxidant activity.

Serotonin 5-HT6 receptors, butyrylcholinesterase (BuChE) and oxidative stress are related to the pathophysiology of Alzheimer's disease. Inhibition of BuChE provides symptomatic treatment of the disease and the same effect was demonstrated for 5-HT 6 antagonists in clinical trials. Oxidative stress is regarded as a major and primary factor contributing to the development of Alzheimer's disease; therefore, antioxidant agents may provide a disease-modifying effect. Combining BuChE inhibition, 5-HT 6 antagonism, and antioxidant properties may result in multitarget-directed ligands providing cognition-enhancing properties with neuroprotective activity. On the basis of the screening of the library of 5-HT 6 antagonists against BuChE, we selected two compounds and designed their structural modifications that could lead to improved BuChE inhibitory activity. We synthesized two series of compounds and tested their affinity and functional activity at 5-HT 6 receptors, BuChE inhibitory activity and antioxidant properties. Compound 12 with K i and K b values against 5-HT 6 receptors of 41.8 and 74 nM, respectively, an IC 50 value of 5 µM against BuChE and antioxidant properties exceeding the activity of ascorbic acid is a promising lead structure for further development of anti-Alzheimer's agents.

Metabolic benefits of 1-(3-(4-(o-tolyl)piperazin-1-yl)propyl)pyrrolidin-2-one: a non-selective α-adrenoceptor antagonist.

PURPOSE: Previous studies have shown that several components of the metabolic syndrome, such as hypertension, obesity or imbalanced lipid and carbohydrate homeostasis, are associated with the sympathetic nervous system overactivity. Therefore, the inhibition of the adrenergic nervous system seems to be a reasonable and appropriate therapeutic approach for the treatment of metabolic disturbances. It has been suggested that non-selective adrenoceptor antagonists could be particularly beneficial, since α1-adrenoceptor antagonists can improve disrupted lipid and carbohydrate profiles, while the inhibition of the α2-adrenoceptor may contribute to body weight reduction. The aim of the present study was to investigate the metabolic benefits deriving from administration of a non-selective α-adrenoceptor antagonist from the group of pyrrolidin-2-one derivatives. The aim of the present study was to investigate the potential metabolic benefits deriving from chronic administration of a non-selective α-adrenoceptor antagonist, from the group of pyrrolidin-2-one derivatives. METHODS: The α1- and α2-adrenoreceptor affinities of the tested compound-1-(3-(4-(o-tolyl)piperazin-1-yl)propyl)pyrrolidin-2-one had been investigated previously by means of the radioligand binding assay. In the present study, we extended the pharmacological profile characteristics of the selected molecule by additional intrinistic activity assays. Next, we investigated the influence of the tested compound on body weight, hyperglycemia, hypertriglyceridemia, blood pressure in the animal model of obesity induced by a high-fat diet, and additionally we measured the spontaneous activity and body temperature. RESULTS: The intrinistic activity studies revealed that the tested compound is a potent, non-selective antagonist of α1B and α2A-adrenoceptors. After the chronic administration of the tested compound, we observed reduced level of triglycerides and glucose in the rat plasma. Interestingly, the tested did not reduce the body weight and did not influence the blood pressure in normotensive animals. Additionally, the administration of the tested compound did not change the animals' spontaneous activity and body temperature. CONCLUSION: Non-selective α-adrenoceptor antagonist seems to carry potential benefits in the improvement of the reduction of elevated glucose and triglyceride level. The lack of influence on blood pressure suggests that compounds with such a pharmacological profile may be particulary beneficial for the patients with disturbed lipid and carbohydrate profile, who do not suffer from hypertension. These results are particulary valuable, since currently there are no safe α2A-adrenoceptor antagonist drugs available in clinical use with the ability to modulate hyperglycemia that would not affect blood pressure.

Idalopirdine, a selective 5-HT6 receptor antagonist, reduces food intake and body weight in a model of excessive eating.

Obesity, from early childhood onwards, is a common societal problem. The overconsumption of sweet, salty and high-fat products are the main factors that cause excessive weight gain. It is therefore necessary to search for new drugs that affect satiety centers and reduce the sense of hunger and caloric intake. It has been suggested that the blockade of 5-HT6 receptors may reduce food intake, and since idalopirdine is a clinically tested, selective 5HT6 receptor antagonist, it was chosen to be examined in animal models of obesity. The activity of idalopirdine was measured in the rat model of excessive eating. Animals were on a high caloric diet that consisted of milk chocolate with nuts, cheese, salted peanuts and condensed milk. During a four-week experiment, the rats had constant access to standard feed and water ad libitum. Idalopirdine was administered intraperitoneally at a dose 5 mg/kg b.w./day. To establish whether idalopirdine would effectively suppress the rebound hyperphagia that accompanies refeeding, it was administered after a 20 h food deprivation period. Pica behavior was evaluated after the administration of idalopirdine to confirm that the suppression of food intake was not caused by visceral illness. The effect of the four-week treatment with idalopirdine on the amount of peritoneal adipose tissue, and on lipid and carbohydrate profiles in rats was also examined. The statistical significance was calculated using the one-way ANOVA post-hoc Tukey Multiple Comparison Test or the two-way ANOVA post-hoc Bonferroni Multiple Comparison Test. Idalopirdine significantly reduced caloric intake and prevented the development of obesity in tested animals. Rats, that received idalopirdine, had a smaller amount of adipose tissue in the peritoneum as well as lower glucose, triglyceride and cholesterol levels in comparison to the control group. Moreover, an anorectic action was not caused by abnormalities of the gastrointestinal tract, such as nausea. The obtained results indicate that idalopirdine reduces caloric intake and could be considered for further tests as a potential treatment of obesity.