Synthesis and antiageing properties of antioxidant pseudopeptides designed based on the bioisostere principle

Nineteen antioxidant pseudopeptides were designed and synthesized. They were confirmed as mild antioxidants, in which L1‐11 was the most active antioxidant with a cellular antioxidant activity (CAA) value of 5.65 ± 0.64 μmol QE/g, and L1‐12 was the second most active one (5.58 ± 0.66 μmol QE/g). The existence of nonnatural amino acids in L1‐12 increased its stability. Pretreatment with L1‐12 dose‐dependently extended the lifespan of Caenorhabditis elegans. L1‐12 improved resistance against UVB irradiation, oxidative stress induced by paraquat, and thermal shock. It decreased the reactive oxygen species level and upregulated the superoxide dismutase activity inside C. elegans. This pseudopeptide sensitively enhanced the expressions of the Cat‐1 and Nhr‐8 genes to reduce oxidative damage, leading to an extension of the lifespan. All the evidence support that L1‐12 may probably be a potential antiageing agent.     

Reorganizing metals: the use of chelating compounds as potential therapies for metal-related neurodegenerative disease

Metal ions, particularly copper, zinc and iron, are implicated in several amyloidogenic neurodegenerative disorders. In the brain, as elsewhere in the body, metal ion excess or deficiency can potentially inhibit protein function, interfere with correct protein folding or, in the case of iron or copper, promote oxidative stress. The involvement of metal ions in neurodegenerative disorders has made them an emerging target for therapeutic interventions. One approach has been to chelate and sequester the ions and thus limit their potential to interfere with protein folding or render them unable to undergo redox processes. Newer approaches suggest that redistributing metal ions has therapeutic benefits, and recent studies indicate that alleviating cellular copper deficiency may be a plausible way to limit neurodegeneration. In this review we discuss the role of metals in amyloidogenic, neurodegenerative disorders and highlight some mechanisms and compounds used in various therapeutic approaches.     

Alternative therapies to address the unmet medical needs of patients with phenylketonuria

Standard therapy for phenylketonuria (PKU), the most common inherited disorder in amino acid metabolism, is an onerous phenylalanine-restricted diet. Adherence to this stringent diet regimen decreases as patients get older, and this lack of adherence is directly associated with cognitive and executive dysfunction and psychiatric issues. These factors emphasize the need for alternative pharmacological therapies to help treat patients with PKU. Sapropterin dihydrochloride is a synthetic form of tetrahydrobiopterin, the cofactor of phenylalanine hydroxylase that in pharmacological doses can stabilize and increase residual enzyme activity in some patients with PKU. About one-third of all patients with PKU respond to oral sapropterin. Phenylalanine ammonia lyase (PAL) is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. Phase I and II trials have shown that injectable recombinant Anabaena variabilis PAL produced in Escherichia coli conjugated with PEG can reduce phenylalanine levels in subjects with PKU. The most frequently reported adverse events were injection-site reactions, dizziness and immune reactions. Additionally, oral administration of PAL and delivery of enzyme substitution therapies by encapsulation in erythrocytes are being investigated. Novel therapies for patients with PKU appear to be options to reduce phenylalanine levels, and may reduce the deleterious effects of this disorder.     

A scientific approach to anti-ageing therapies: state of the art

A lasting dream of human beings is to reverse or at least postpone ageing. During the last years, an increasing number of scientific meetings, articles, and books have been devoted to anti-ageing therapies. This subject, full of misleading, simplistic, or wrong ideas, is very popular among the general public, whose imagery has been fascinated by all possible tools to delay ageing, getting immortality. Here, we discuss anti-ageing strategies aimed not to rejuvenate but to slow ageing and delay the onset of age-related diseases. These approaches should be able to substantially slow down the ageing process, extending our productive, youthful lives.     

Anti-inflammatory,analgesic, and antioxidant activities of Pisonia aculeata: Folk medicinal use to scientific approach

Context: Pisonia aculeata leaves (Nyctagenaceae), a Folk medicinal plant used in the treatment of several inflammation, pain, and oxidative stress associated diseases.

Objective: To evaluate anti-inflammatory, analgesic, and antioxidant potential of crude methanol extract of P. aculeata leaves (MEPA).

Materials and methods: Analgesic and anti-inflammatory activities of MEPA (250 and 500?mg/kg) were evaluated using writhing, formalin, hot plate, tail flick, carrageenan-induced paw edema test, and membrane stabilizing activity. Free radical scavenging activity, total phenolic and flavonoid contents of MEPA were also determined using standard methods.

Results: Oral administration of MEPA showed significant (p < 0.001) inhibition of paw edema, pronounced at 4?h and 5?h after carrageenan injection, and at 200 µg/mL exerts 77.67 and 38.51% protective effect against hypotonic solution and heat induced hemolysis, respectively. MEPA (250 and 500?mg/kg) produced 35.21 and 79.14% inhibition of acetic acid-induced writhing. Furthermore, MEPA (500?mg/kg) inhibited 49.19% early and 73.14% late phase of formalin-induced hypernociception. In contrast, a lower dose of MEPA did not prevent hot plate induced nociception, while in the tail immersion method, pronounced analgesic activity was observed between 1 and 4?h postdosing. The extract possesses significant in vitro antioxidant activity and a lipid peroxidation inhibition effect. Total phenolic and total flavonoid content in MEPA were 87.99?±?0.87?mg GAE/g and 58.98?±?0.01?mg QE/g, respectively.

Discussion and conclusion: Our findings confirmed the analgesic, anti-inflammatory and antioxidant activities of Pisonia aculeata leaves. Contents of flavonoids and phenolic compounds in extract could be correlated with its observed biological activities.     

The mechanism of patulin's cytotoxicity and the antioxidant activity of indole tetramic acids

In LLC-PK1 cells exposed to patulin (50 microM), lipid peroxidation, abrupt calcium influx, extensive blebbing, and total LDH release appeared to be serially connected events with each representing a step in the loss of structural integrity of the plasma membrane. The aforementioned patulin-induced events were prevented by concurrent incubation with butylated hydroxytoluene, deferoxamine, and cyclopiazonic acid, a fungal metabolite. Patulin also caused depletion of nonprotein sulfhydryls, increased 86Rb+ efflux, dome collapse, and eventually the loss of cell viability. These events were not prevented by antioxidants, results consistent with the hypothesis that they were also serially connected but occurring parallel to those previously mentioned. The earliest events observed in patulin-treated cells were the decrease in nonprotein sulfhydryls and increase in 86Rb+ efflux (5 min) which occurred before statistically significant alterations in protein-bound sulfhydryls. The increased potassium efflux (86Rb+ efflux) occurred via a pathway distinct from BaCl2, quinine, or tetraethylammonium sensitive potassium channels. This is the first published report of the antioxidant activity of indole tetramic acids (cyclopiazonic acid and cyclopiazonic acid imine). The protective effect of tetramic acids in LLC-PK1 cells was restricted to indole tetramic acids, and their prevention of lipid peroxidation did not involve iron chelation. The results of this study demonstrate that cyclopiazonic acid is a potent inhibitor of azide-insensitive, ATP-dependent, a23187-sensitive calcium uptake by the lysate of LLC-PK1 cells. This result is consistent with the hypothesis that the endoplasmic reticulum calcium transport ATPase is a sensitive target for cyclopiazonic acid in LLC-PK1 cells. These findings raise the interesting possibility that the antioxidant activity of indole tetramic acids may involve multiple novel mechanisms: surface charge alterations on the cytoplasmic surface of plasma membranes, alterations in calcium permeability in the plasma and endoplasmic reticulum membrane, and inhibition of the calcium-dependent ATPase of the endoplasmic reticulum.     

Redox targeting of LY231617, an antioxidant with potential use in the treatment of brain damage

Several brain-targeting chemical delivery systems (CDS) based on a dihydropyridine ⇌ pyridinium salt-type targetor were synthesized and evaluated for LY231617 (1), a di-tert-butylated phenolic amine antioxidant with potential use in the treatment of brain injuries. The dihydropyridine moiety was chemically attached to the amine (by either amide or various substituted carbamate linkages) or to the phenolic hydroxyl (by carboxylic ester linkage) functionalities of LY231617. In vitro stability and in vivo tissue distribution studies (in the rat) were performed with the novel derivatives. The results indicated that a simple amide-type CDS demonstrated efficient delivery of LY231617-targetor conjugate to the CNS. This derivative which contains the intact pharmacophore might possess intrinsic pharmacological antioxidant activity. Favorable in vitro properties suggested that a substituted carbamate-type CDS might be a better delivery modality for LY231617.     

Factors associated with the use of solvents and cannabis by medical students

INTRODUCTION: The use of alcohol and other drugs among medical students has been a theme of growing interest and concern on the part of researchers, teaching institutions and medical associations since the decade of the 1960's. OBJECTIVE: Recent use of alcohol, tobacco, tranquillisers, amphetamines, cannabis, organic solvents, and cocaine among 456 medical students was surveyed. METHOD: Assessment was done by means of a self-report questionnaire according to World Health Organisation guidelines. RESULTS: Among medical students, after alcohol and tobacco, cannabis and solvents are the most frequently used psychoactive substances. As such, they were the most deeply analysed drugs in this study. Factors associated with the recent use of cannabis and solvents were established by logistic regression. Living with parents or a companion appeared as a protective factor for the use of cannabis. However, being male and regularly participating in the activities at the campus Sports Association showed as risk factors for the use of both cannabis and solvents. DISCUSSION: Concepts and misconceptions concerning protective and risk factors must be discussed in the light of cultural and circumstantial interferences. Harm reduction strategies should be seriously considered.     

The medical complications of alcohol use: understanding mechanisms to improve management

The use of alcohol in a dependent or even a regular heavy pattern predisposes the drinker to a range of adverse consequences. These include a risk of direct harm from alcohol, including organ damage, mental health disorders and a range of social and legal problems associated with behaviours due to alcohol's effects. The range of organ damage associated with regular heavy alcohol consumption is well described. Much new information on the mechanisms by which damage occurs is available and is reviewed in this paper. New knowledge can assist in the development of more appropriate management strategies for those affected by the medical complications of alcohol use. Genetic susceptibility to tissue injury is explored and the reasons why many heavy drinkers do not appear to experience organ damage are considered. Approaches to the management of certain alcohol-related disorders are outlined.     

Immunological properties of donkey's milk: its potential use in the prevention of atherosclerosis

Donkey's milk is the best substitute of human milk for its content in lactose, proteins, minerals, and omega-3 fatty acids. Here, we have evaluated the effects of colostrum and milk from donkeys (Martina Franca breed) on the function of human peripheral blood mononuclear cells (PBMCs) at different intervals from lactation. Colostrum induced more IgA responses, while milk induced predominantly more IgG responses. Both milk and colostrum induced expression of CD25 and CD69 on PBMCs. The ability to induce release of interleukins (IL) (IL-12, IL-1 beta and IL-10) and tumor necrosis factor-alpha was confined only to milk, while colostrum was devoid of this capacity. Finally, both colostrum and milk induced nitric oxide (NO) release from PBMCs but milk exhibited a greater capacity than colostrum in NO generation. Taken together, these immunological activities exerted by both colostrum and milk from donkeys may be useful in the treatment of human immune-related diseases. In particular, NO induction by donkey's milk may be very useful in the prevention of atherosclerosis, being a strong vasodilator and an effective antimicrobial agent since pathogens and/or their products may play a proatherogenic role.     

Focus on success: using a probabilistic approach to achieve an optimal balance of compound properties in drug discovery

The success of any drug will depend on how closely it achieves an ideal combination of potency, selectivity, pharmacokinetics and safety. The key to achieving this success efficiently is to consider the overall balance of molecular properties of compounds against the ideal profile for the therapeutic indication from the earliest stages of a drug discovery project. The use of in silico predictive models of absorption, distribution, metabolism and elimination (ADME) and physicochemical properties is a major aid in this exercise, as it enables virtual molecules to be assessed across a broad range of properties from initial library generation, through to candidate selection. Of course, no measurement, whether in silico, in vitro or in vivo, is perfect and the uncertainties in any data should be explicitly taken into account when basing conclusions on test results. In addition, in the early stages of drug discovery, when designing a library that is lead seeking or building compound structure-activity relationships, the quality of any set of molecules should also be balanced against the chemical diversity covered. Here, a scheme is presented for achieving these goals based on a suite of predictive ADME models, probabilistic scoring and multiobjective optimisation for library design. The use of this platform for applications in lead identification and optimisation is illustrated.     

Preliminary screening the antioxidant potential of in vitro-propagated Amsonia orientalis: An example to sustainable use of rare medicinal plants in pharmaceutical studies

Amsonia orientalis (European Bluestar) is a critically endangered plant species with medicinal and ornamental properties. The rare availability of the species in nature limits its potential to be used for various purposes. However, plant tissue culture is an effective method for the cultivation of such vulnerable species without damaging their natural populations, which are very limited in nature for scientific purposes. By taking advantage of plant tissue culture, this study aimed to measure the phenolic substance and flavonoid contents in leaf extracts of in vitro-propagated Amsonia orientalis, and to investigate their antioxidant potentials through phosphomolybdate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The crude extracts prepared in water, aqueous ethanol, methanol, and acetone were tested. The highest phenolic substance content was found in the ethanolic extracts, while statistically the same flavonoid contents were found in the ethanolic, methanolic, and acetone extracts. Although the water extract had lesser flavonoid content, it exhibited a notable antioxidant property. The ethanolic leaf extract gave the highest antioxidant and DPPH radical scavenging activity, especially when used at 1 mg mL^?1 concentration. Also, the TLC fingerprint profile validated the presence of valuable phytoconstituents in the leaves of the plant. This study indicated that ultrasound-assisted extraction of minimal amounts of dried leaf samples from in vitro-propagated plants might be adequate for the pre-screening of the antioxidant capacity of rare plant species.     

Involvement of cytotoxicity and variation of the mitochondrial membrane potential induced by hybrid agent

The pyrrolo[2,1‐c][1,4]benzodiazepine (PBD) derivative DC‐81 is a potent antitumor antibiotic produced by Streptomyces species. The marked cytotoxic potential of this drug may be the result of its interaction with DNA. Because DC‐81 only recognizes three DNA base pairs this has precluded its clinical utility. Combination of DC‐81 with an indole carboxylate moiety was a hybrid designed to have much higher sequence selectivity in DNA Interactivity. In this paper, the association between cytotoxicity and the changes of mitochondria membrane potential ΔΨ_mt after exposing human melanoma cell line A375 to the hybrid agent was examined using MTS cell proliferation assay and flow cytometry using the fluorochrome rhodamine 123. Our results indicated that the hybrid induced cytotoxity and a significant reduction in ΔΨ_mt of A375 cells. We suggest that the hybrid agent is a potent inducer of cell apoptosis in A375 cells. Drug Dev. Res. 61: 1–5, 2004. © 2004 Wiley‐Liss, Inc.     

Alcohol use disorders and current pharmacological therapies: the role of GABAA receptors

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABA_A receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABA_A receptors in the treatment of AUD and currently available and potentially novel pharmacological agents.     

Selective activation of mitomycin A by thiols to form DNA cross-links and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential

Mitomycin A (MA) but not mitomycin C (MC) cross-linked linearized (32)P-pBR322 DNA in the presence of dithiothreitol (DTT) or glutathione (GSH), as shown by a sensitive DNA cross-link assay. Incubation of calf-thymus DNA with MA and DTT or mercaptoethanol (MER) resulted in the formation of MA-DNA adducts, which were isolated from nuclease digests of the drug-DNA complexes by HPLC. The adducts were characterized by their UV absorption spectra, electrospray ionization mass spectrometry (ESIMS), and facile conversion from 7-methoxy- to 7-amino-substituted mitosene type adducts upon 10% NH(4)OH treatment, which were identical with known adducts of MC. Both DNA interstrand and intrastrand cross-link adducts, linking two deoxyguanosine residues at N(2), as well as several deoxyguanosine-N(2) monoadducts of MA, were identified. No DNA adducts were formed with MC under the same conditions. A specificity of DNA cross-link formation for the CpG sequence was observed using 12-mer synthetic oligodeoxyribonucleotides as substrates and as DNA sequence models, in analogy to the known CpG sequence specificity of MC-induced DNA cross-links. MA is known to be more cytotoxic by 2-3 orders of magnitude than MC, and this property correlates with redox potentials of MA (-0.19 V) and MA analogues that are higher than those of MC (-0.40 V) and its analogues. It is suggested that the biochemical basis for the higher cytotoxic potency of MA is MA's propensity to be reductively activated by cellular thiols while MC is resistant to thiol activation. This distinction is probably derived from the large difference between the quinone redox potentials of the two drugs.     

The application of pharmaceutical quality by design concepts to evaluate the antioxidant and antimicrobial properties of a preservative system including desferrioxamine

BackgroundThe purpose of the present study was to investigate the antioxidant and antimicrobial activities of a conventional preservative system containing desferrioxamine mesylate (DFO) and optimize the composition of the system through mathematical models.MethodsDifferent combinations of ethylenediaminetetraacetic acid (EDTA), sodium metabisulfite (SM), DFO and methylparaben (MP) were prepared using factorial design of experiments. The systems were added to ascorbic acid (AA) solution and the AA content over time, at room temperature and at 40 °C was determined by volumetric assay. The systems were also evaluated for antioxidant activity by a fluorescence-based assay. Antimicrobial activity was assessed by microdilution technique and photometric detection against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis. A multi-criteria decision approach was adopted to optimize all responses by desirability functions.ResultsDFO did not extend the stability of AA over time, but displayed a better ability than EDTA to block the pro-oxidant activity of iron. DFO had a positive interaction with MP in microbial growth inhibition. The mathematical models showed adequate capacity to predict the responses. Statistical optimization aiming to meet the quality specifications of the ascorbic acid solution indicated that the presence of DFO in the composition allows to decrease the concentrations of EDTA, SM and MP.ConclusionDFO was much more effective than EDTA in preventing iron-catalyzed oxidation. In addition, DFO improved the inhibitory response of most microorganisms tested. The Quality by Design concepts aided in predicting an optimized preservative system with reduced levels of conventional antioxidants and preservatives, suggesting DFO as a candidate for multifunctional excipient.Graphical abstractElectronic supplementary materialThe online version of this article (10.1007/s40199-020-00370-9) contains supplementary material, which is available to authorized users.     

A quantitative in vitro approach to study the intracellular fate of gold nanoparticles: from synthesis to cytotoxicity

Abstract

Due to their physico-chemical characteristics, gold nanoparticles (AuNPs) seem to be suitable for biomedical and therapeutic applications even if conflicting data on their toxicological profiles are present in literature. In order to better understand if AuNPs could be safe we must consider different biological endpoints such as cytotoxicity, genotoxicity, inflammation and biopersistence. Starting from these considerations, one of the first issues to be assessed is to better understand if AuNPs can be internalized by cells. In this work, we propose a methodological approach to radioactivate AuNPs by neutron activation and the quantification of their internalization by two in vitro cell systems such as MDCK and HepG2 after 24 h of exposure. Despite a dose-dependent internalization, no evidence of cytotoxicity, determined by two different standard in vitro methods such as Neutral Red Uptake and Colony Forming Efficiency, was observed.