Protection by extra virgin olive oil against oxidative stress in vitro and in vivo. Chemical and biological studies on the health benefits due to a major component of the Mediterranean diet.

We report the results of in vivo studies in Caenorhabditis elegans nematodes in which addition of extra virgin olive oil (EVOO) to their diet significantly increased their life span with respect to the control group. Furthermore, when nematodes were exposed to the pesticide paraquat, they started to die after two days, but after the addition of EVOO to their diet, both survival percentage and lifespans of paraquat-exposed nematodes increased. Since paraquat is associated with superoxide radical production, a test for scavenging this radical was performed using cyclovoltammetry and the EVOO efficiently scavenged the superoxide. Thus, a linear correlation (y = -0.0838x +19.73, regression factor = 0.99348) was observed for superoxide presence (y) in the voltaic cell as a function of aliquot (x) additions of EVOO, 10 µL each. The originally generated supoeroxide was approximately halved after 10 aliquots (100 µL total). The superoxide scavenging ability was analyzed, theoretically, using Density Functional Theory for tyrosol and hydroxytyrosol, two components of EVOO and was also confirmed experimentally for the galvinoxyl radical, using Electron Paramagnetic Resonance (EPR) spectroscopy. The galvinoxyl signal disappeared after adding 1 µL of EVOO to the EPR cell in 10 minutes. In addition, EVOO significantly decreased the proliferation of human leukemic THP-1 cells, while it kept the proliferation at about normal levels in rat L6 myoblasts, a non-tumoral skeletal muscle cell line. The protection due to EVOO was also assessed in L6 cells and THP-1 exposed to the radical generator cumene hydroperoxide, in which cell viability was reduced. Also in this case the oxidative stress was ameliorated by EVOO, in line with results obtained with tetrazolium dye reduction assays, cell cycle analysis and reactive oxygen species measurements. We ascribe these beneficial effects to EVOO antioxidant properties and our results are in agreement with a clear health benefit of EVOO use in the Mediterranean diet.

The isoflavonoid tectorigenin has better antiplatelet potential than acetylsalicylic acid.

BACKGROUND: One reason for the lower incidence of cardiovascular diseases in Asian countries may be the high intake of isoflavonoids and their antiplatelet effects may be an important factor. To date, there is limited comparison of a range of isoflavonoids and knowledge of their effects at different levels of platelet aggregation. PURPOSE: To screen the antiplatelet effects of a number of isoflavonoids on the arachidonic acid based aggregation pathway and investigate how the antiplatelet activity might occur. METHODS: The antiplatelet effects were first screened in whole human blood where platelet aggregation was induced by arachidonic acid. Further analysis was targeted at search of the mechanism of action. RESULTS: Thirteen of the eighteen tested isoflavonoids had significant inhibitory effect on platelet aggregation in whole human blood. Genistein had the same potency as clinically used acetylsalicylic acid (ASA) while tectorigenin was clearly stronger than ASA. Further analyses showed that the effect of tectorigenin was not based on inhibition of cyclooxygenase-1 in contrast to ASA or thromboxane synthase but by competitive antagonism at thromboxane receptors. CONCLUSION: Tectorigenin is a more potent antiplatelet compound than ASA and thus an interesting substance for further testing.

Intravenous rutin in rat exacerbates isoprenaline-induced cardiotoxicity likely due to intracellular oxidative stress.

OBJECTIVES: Rutin, quercetin-3-O-rutinoside, a natural flavonol glycoside, has shown various in vitro benefits with potential use treating human diseases, especially cardiovascular system disorders. Antioxidant properties are assumed to underlie the majority of these benefits. Yet rutin pro-oxidant properties have been reported as well. Our research group has recently shown aggravating effects on isoprenaline (ISO)-induced cardiotoxicity in Wistar:Han rats after 24 hours. METHODS: This study was designed to examine in more detail the reasons for the negative effects of rutin (11.5 and 46 mg/kg, i.v.) after administration of ISO (100 mg/kg, s.c.) in rats within 2 hours of continuous experiment and in the H9c2 cardiomyoblast-derived cell line. RESULTS: Like our previous findings, rutin did not (11.5 or 46 mg/kg, i.v.) reduce the ISO-induced mortality within 2 hours although the lower dose significantly reduced cardiac troponin T (cTnT) and partly improved the histological findings. In contrast, the higher dose increased the mortality in comparison with solvent (1.26% w/v sodium bicarbonate). This was not caused by any specific haemodynamic disturbances. It appears to be associated with oxidative stress as rutin enhanced intracellular reactive oxygen species formation in vitro and had the tendency to increase it in vivo. CONCLUSIONS: Rutin, likely due to its pro-oxidative effects, can exacerbate catecholamine cardiotoxicity depending on the dose used.

Protective Effects of D-Penicillamine on Catecholamine-Induced Myocardial Injury.

Iron and copper release participates in the myocardial injury under ischemic conditions and hence protection might be achieved by iron chelators. Data on copper chelation are, however, sparse. The effect of the clinically used copper chelator D-penicillamine in the catecholamine model of acute myocardial injury was tested in cardiomyoblast cell line H9c2 and in Wistar Han rats. D-Penicillamine had a protective effect against catecholamine-induced injury both in vitro and in vivo. It protected H9c2 cells against the catecholamine-induced viability loss in a dose-dependent manner. In animals, both intravenous D-penicillamine doses of 11 (low) and 44 mg/kg (high) decreased the mortality caused by s.c. isoprenaline (100 mg/kg) from 36% to 14% and 22%, respectively. However, whereas the low D-penicillamine dose decreased the release of cardiac troponin T (specific marker of myocardial injury), the high dose resulted in an increase. Interestingly, the high dose led to a marked elevation in plasma vitamin C. This might be related to potentiation of oxidative stress, as suggested by additional in vitro experiments with D-penicillamine (iron reduction and the Fenton reaction). In conclusion, D-penicillamine has protective potential against catecholamine-induced cardiotoxicity; however the optimal dose selection seems to be crucial for further application.

Antiplatelet Effects of Flavonoids Mediated by Inhibition of Arachidonic Acid Based Pathway.

Flavonoids, important components of human diet, have been claimed to possess a significant antiplatelet potential, in particular due to their effects on the arachidonic acid cascade. Due to variable and incomplete results, this study was aimed at delivering a detailed analysis of the effects of 29 structurally relevant, mainly natural flavonoids on three consecutive steps of the arachidonic acid cascade.Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygenase-1 inhibitory activity, which was higher than that of acetylsalicylic acid using the isolated ovine enzyme, and physiologically relevant, although lower than acetylsalicylic acid in human platelets. None of the tested flavonoids possesses an effect on thromboxane synthase in a clinically achievable concentration. Contrarily, many flavonoids, particularly those possessing an isolated 7-hydroxyl group and/or a 4'-hydroxyl group, acted as antagonists on thromboxane receptors. Interestingly, the substitution of the free 7-hydroxyl group by glucose might not abolish the activity.In conclusion, the consumption of few flavonoids in a diet, particularly of the isoflavonoids genistein and daidzein, may positively influence platelet aggregation.

Isoflavones Reduce Copper with Minimal Impact on Iron In Vitro.

Isoflavones are commonly consumed in many Asian countries and have potentially positive effects on human being. Only a few and rather controversial data on their interactions with copper and iron are available to date. 13 structurally related isoflavones were tested in the competitive manner for their Cu/Fe-chelating/reducing properties. Notwithstanding the 5-hydroxy-4-keto chelation site was associated with ferric, ferrous, and cupric chelation, the chelation potential of isoflavones was low and no cuprous chelation was observed. None of isoflavones was able to substantially reduce ferric ions, but the vast majority reduced cupric ions. The most important feature for cupric reduction was the presence of an unsubstituted 4'-hydroxyl; contrarily the presence of a free 5-hydroxyl decreased or abolished the reduction due to chelation of cupric ions. The results from this study may enable additional experiments which might clarify the effects of isoflavones on human being and/or mechanisms of copper absorption.

Antioxidant effects of coumarins include direct radical scavenging, metal chelation and inhibition of ROS-producing enzymes.

Coumarins represent a large group of 1,2-benzopyrone derivatives which have been identified in many natural sources and synthetized as well. Several studies have shown that their antioxidant capacity is not based only on direct scavenging of reactive oxygen and nitrogen species (RONS) but other mechanisms are also involved. These include: a) the chelation of transient metals iron and copper, which are known to catalyse the Fenton reaction; and b) the inhibition of RONS-producing enzymes (e.g. xanthine oxidase, myeloperoxidase and lipoxygenase), suggesting that mechanism(s) involved on cellular level are complex and synergistic. Moreover, many factors must be taken into account when analysing structure-antioxidant capacity relationships of coumarins due to different in vitro/in vivo methodological approaches. The structural features necessary for the direct RONS scavenging and metal chelation are apparently similar and the ideal structures are 6,7-dihydroxy- or 7,8-dihydroxycoumarins. However, the clinical outcome is unknown, because these coumarins are able to reduce copper and iron, and may thus paradoxically potentiate the Fenton chemistry. The similar structural features appear to be associated with inhibition of lipoxygenase, probably due to interference with iron in its active site. Contrarily, 6,7-dihydroxycoumarin seems to be the most active coumarin in the inhibition of xanthine oxidase while its derivative bearing the 4-methyl group or 7,8-dihydroxycoumarin are less active or inactive. In addition, coumarins may hinder the induction of inducible NO-synthase and cyclooxygenase- 2. Sparse data on inhibition of myeloperoxidase do not enable any clear conclusion, but some coumarins may block it.

Cardiovascular effects of coumarins besides their antioxidant activity.

Coumarins are a large group of substances, primarily of plant origin. Like their more intensively examined congeners flavonoids, many of them are antioxidants. Although such properties may be advantageous in cardiovascular diseases, it has been shown that coumarins exhibit direct effects on the cardiovascular system which are not based on antioxidant activity. The most common example is the well-known drug warfarin, a synthetic compound derived from natural dicoumarol. Moreover, other coumarins have been shown to possess antiplatelet and vasodilatory potential. Interestingly, the former effect may be mediated by the inhibition of various pathways leading to platelet aggregation, their differing effects on those pathways being due to structural differences between the various coumarins. Conversely, their vasodilatory potential is linked in the majority of cases to the inhibition of increases in intracellular calcium concentration in vascular smooth muscle cells, and in several coumarins also to NO-mediated vasodilatation. Available data on both activities are summarized in this review. At the end of this review, relevant data are provided from a few studies testing the in vivo effects of coumarins on major cardiovascular diseases; the clinical use of warfarin and other coumarin anticoagulants, as well as the limited data on the clinical use of coumarins in chronic venous insufficiency and the possible toxicological effects of coumarins.

LC-UV/MS methods for the analysis of prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone (BSIH) and its active chelator salicylaldehyde isonicotinoyl hydrazone (SIH).

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone-BSIH along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC-UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7-115 and 4-115 µM, respectively, and applied to BSIH activation experiments in vitro. An LC-MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06-23 and 0.24-23 µM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC-UV and LC-MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and its chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggests that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents.

Is a highly linear relationship between the dose of quercetin and the pharmacological effect possible? - a comment on Liu, et al. Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats. Molecules 2012, 17, 4281-4291.

We wish to offer some comments on the article by H. Liu et al. entitled "Evaluation of antioxidant and immunity activities of quercetin in isoproterenol-treated rats", published in Molecules in 2012 [1]. [...].

Oral administration of quercetin is unable to protect against isoproterenol cardiotoxicity.

Catecholamines are endogenous amines that participate in the maintenance of cardiovascular system homeostasis. However, excessive release or exogenous administration of catecholamines is cardiotoxic. The synthetic catecholamine, isoprenaline (isoproterenol, ISO), with non-selective ß-agonistic activity has been used as a viable model of acute myocardial toxicity for many years. Since the pathophysiology of ISO-cardiotoxicity is complex, the aim of this study was to elucidate the effect of oral quercetin pretreatment on myocardial ISO toxicity. Wistar-Han rats were randomly divided into four groups: solvent or quercetin administered orally by gavage in a dose of 10 mg kg(-1) daily for 7 days were followed by s.c. water for injection or ISO in a dose of 100 mg kg(-1). Haemodynamic, ECG and biochemical parameters were measured; effects on blood vessels and myocardial histology were assessed, and accompanying pharmacokinetic analysis was performed. Quercetin was unable to protect the cardiovascular system against acute ISO cardiotoxicity (stroke volume decrease, cardiac troponin T release, QRS-T junction elevation and histological impairment). The sole positive effect of quercetin on catecholamine-induced cardiotoxicity was the normalization of increased left ventricular end-diastolic pressure caused by ISO. Quercetin did not reverse the increased responsiveness of rat aorta to vasoconstriction in ISO-treated animals, but it decreased the same parameter in the control animals. Accompanying pharmacokinetic analysis showed absorption of quercetin and its metabolite 3-hydroxyphenylacetic acid formed by bacterial microflora. In conclusion, a daily oral dose of 10 mg kg(-1) of quercetin for 7 days did not ameliorate acute ISO-cardiovascular toxicity in rats despite minor positive cardiovascular effects.

Effect of novel 1-phenyl-3-methyl-4-acylpyrazolones on iron chelation and Fenton reaction.

Iron is an essential element in many physiological processes due to its ability to easily convert between two oxidation states Fe(III)/Fe(II). However, at a pathological state, unbound iron may promote the production of highly toxic hydroxyl radicals via Fenton reaction, particularly when it is present in the excess.Iron chelators forming tight complexes with iron may prevent this reaction. In this study, novel synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones were analyzed for their iron-chelating properties at four pathophysiologically relevant pH conditions (4.5-7.5) as well as for their effects on iron-based Fenton reaction. For the former competitive ferrozine spectrophotometric assay and for the latter HPLC method using salicylic acid as the indicator of hydroxyl radical production were used. All of the tested acylpyrazolones were efficient ferric chelators, however, their ferrous-chelating properties were clearly dependent on an acyl substitution. Interestingly, several acylpyrazolones had ferrous-chelating properties superior to those of the standard iron chelator - deferoxamine. Of particular interest is H2QpyQ, i.e. 2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine, whose ferrous-chelating properties were increasing while pH was decreasing. In spite of large differences in ferrous chelation, a majority of the tested acylpyrazolones were powerful inhibitors of Fenton reaction as deferoxamine. In conclusion, the novel 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones are efficient iron chelators and H2QpyQ may represent a prototype of specific iron chelators designed for chelation at acidic conditions in particular.

The relationship of oxidative stress markers and parameters of myocardial function in a rat model of cardiotoxicity.

Although a majority of studies related oxidative stress to cardiovascular diseases, the pathophysiological relevance has been remaining unknown. The aim of this study was to establish the relationship among different commonly used biomarkers of oxidative stress and cardiovascular dys/function in rats. A pathological state in many aspects similar to that of acute myocardial infarction was induced by administration of isoprenaline (100mg.kg(-1), s.c.) in Wistar:Han rats. Haemodynamic, biochemical and ECG parameters were measured in two sets of experiments: after 24hours and continuously during the first 2hours following the administration of isoprenaline. Serum cardiac troponin T (cTnT) correlated strongly with cardiac function, myocardial calcium levels, wet ventricles weight and relevant ECG parameters (T wave, R wave and J - junction - point amplitudes). However, only weak negative correlations were found for cTnT and total blood glutathione or serum vitamin C concentrations, while no significant associations were found with serum vitamin E and plasma TBARS. Although the oxidized form of glutathione correlated positively with heart rate, no correlation with the above-mentioned ECG parameters was found. However, correlations of in 8-isoprostane with both R wave and J-junction-point amplitudes were observed. Conclusively, more selective markers of oxidative stress may predict the functional status of the heart.

In vitro copper-chelating properties of flavonoids.

Copper is an indispensable trace element for human body and the association between a disruption of copper homeostasis and a series of pathological states has been well documented. Flavonoids influence the human health in a complex way and the chelation of transient metal ions indisputably contributes to their mechanism of the action, however, the information about their copper-chelating properties have been sparse. This in vitro study was thus aimed at the detailed examination of flavonoids-copper interactions by two spectrophotometric assays at four (patho)physiologically relevant pH conditions (4.5-7.5), with the emphasis on the structure-activity relationship. The tested flavonoids were compared with the clinically used copper chelator, trientine. Most of the 26 flavonoids chelated copper ions, however, in a variable extent. Only flavones and flavonols were able to form stable complexes with both cupric and cuprous ions. The 3-hydroxy-4-keto group and 5,6,7-trihydroxyl group represented the most efficient chelation sites in flavonols and flavones, respectively, and the 2,3-double bond was essential for the stable copper chelation. Interestingly, the 3´,4´-dihydroxyl (catechol) group was associated only with a weak activity. Although none of the tested flavonoids were more potent than trientine at physiological or slightly acidic conditions, 3-hydroxyflavone, kaempferol and partly baicalein surpassed trientine at acidic conditions. Conclusively, flavonoids containing appropriate structural features were efficient copper chelators and some of them were even more potent than trientine under acidic conditions.

Simultaneous determination of the novel thiosemicarbazone anti-cancer agent, Bp4eT, and its main phase I metabolites in plasma: application to a pilot pharmacokinetic study in rats.

Novel thiosemicarbazone metal chelators are extensively studied anti-cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC-MS/MS method for the simultaneous quantification of 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1-E and M1-Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C18 column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid-phase extraction on 96-well plates. This method was validated over the concentration range of 0.18-2.80 µM for Bp4eT, 0.02-0.37 µM for both M1-E and M1-Z, and 0.10-1.60 µM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration-time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti-cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class.

Mathematical calculations of iron complex stoichiometry by direct UV-Vis spectrophotometry.

The effects of iron-chelating agents on miscellaneous pathologies are currently largely tested. Due to various indications, different properties for chelators are required. A stoichiometry of the complex in relation to pH is one of the crucial factors. Moreover, the published data on the stoichiometry, especially concerning flavonoids, are equivocal. In this study, a new complementary approach was employed for the determination of stoichiometry in 10 iron-chelating agents, including clinically used drugs, by UV-Vis spectrophotometry at relevant pH conditions and compared with the standard Job's method. This study showed that the simple approach based on absorbance at the wavelength of complex absorption maximum was sufficient when the difference between absorption maximum of substance and complex was high. However, in majority of substances this difference was much lower (9-73 nm). The novel complementary approach was able to determine the stoichiometry in all tested cases. The major benefit of this method compared to the standard Job's approach seems to be its capability to reveal a reaction stoichiometry in chelators with moderate affinity to iron. In conclusion, using this complementary method may explain several previous contradictory data and lead to a better understanding of the underlying mechanisms of chelator's action.

Common biomarkers of oxidative stress do not reflect cardiovascular dys/function in rats.

BACKGROUND: Predicting cardiovascular events remains challenging despite the range of known biomarkers. AIM: To establish relationships between various biochemical and functional parameters of the cardiovascular system. METHOD: The relationship between cardiovascular dys/function and various biomarkers was examined in 145 experimental rats half of which received isoprenaline 100 mg/kg s.c. to induce cardiac impairment. RESULTS: Serum concentration of cardiac troponin T (cTnT), a known marker of cardiac derangement, correlated strongly with degree of myocardial injury (e.g. calcium overload, stroke volume) but correlations between cTnT and oxidative stress parameters were weak (for glutathione and vitamin C) or not found (for serum vitamin E and plasma thiobarbituric acid reactive substances levels). Relationships between cTnT and other parameters were exponential with the exception of myocardial calcium, where a power function was found. CONCLUSIONS: Commonly used biomarkers of oxidative stress cannot reliably predict cardiovascular dys/function in experimental rats.

Protection of cells against oxidative stress by nanomolar levels of hydroxyflavones indicates a new type of intracellular antioxidant mechanism.

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.

Novel method for rapid copper chelation assessment confirmed low affinity of D-penicillamine for copper in comparison with trientine and 8-hydroxyquinolines.

Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5-7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu(+)/Cu(2+) at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated.

Amino acid derivatives as transdermal permeation enhancers.

Transdermal permeation enhancers are compounds that temporarily decrease skin barrier properties to promote drug flux. In this study, we investigated enhancers with amino acids (proline, sarcosine, alanine, ß-alanine, and glycine) attached to hydrophobic chain(s) via a biodegradable ester link. The double-chain lipid-like substances displayed no enhancing effect, whereas single-chain substances significantly increased skin permeability. The proline derivative l-Pro2 reached enhancement ratios of up to 40 at 1% concentration, which is higher than that of the well-established and standard enhancers Azone, DDAIP, DDAK, and Transkarbam 12. No stereoselectivity was observed. l-Pro2 acted synergistically with propylene glycol. Infrared studies revealed that l-Pro2 forms a separate liquid ordered phase in the stratum corneum lipids and has no significant effect on proteins. l-Pro2 action was at least partially reversible as measured by skin electrical impedance. Toxicity in keratinocyte (HaCaT) and fibroblast (3T3) cell lines showed IC(50) values ranging from tens to hundreds of µM, which is comparable with standard enhancers. Furthermore, l-Pro2 was rapidly decomposed in plasma. In vivo transdermal absorption studies in rats confirmed the enhancing activity of l-Pro2 and suggested its negligible skin toxicity and minimal effect on transepidermal water loss. These properties make l-Pro2 a promising candidate for potential clinical use.