Anal Bioanal Chem (2010) 398:67–75 DOI 10.1007/s00216-010-3829-y
REVIEW
Recent advances in the assessment of the antioxidant capacity of pharmaceutical drugs: from in vitro to in vivo evidence Giangiacomo Beretta & Roberto Maffei Facino
Received: 11 March 2010 / Revised: 4 May 2010 / Accepted: 4 May 2010 / Published online: 25 May 2010 # Springer-Verlag 2010
Abstract In this review, some well-established assays and more recent markers developed for the understanding of the biological activity of pharmaceutical drugs belonging to different pharmacological classes (nonsteroidal antiinflammatory drugs, cardiovascular drugs, and centralnervous-system-acting drugs) are considered. The results of in vitro studies are reviewed and critically compared with those available from clinical trials, and their relevance for the elucidation of the mechanism of action of the drugs is discussed. Although from this examination a positive correlation between the in vitro and in vivo data seems to emerge, the small number of clinical trials available, their low number of patients enrolled, and sometimes the arbitrary or inappropriate choice of the biomarker(s) used highlight the need for (1) more standardized protocols to allow a reliable comparison of the results from different studies and (2) the development of new and more appropriate and specific biomarkers for the evaluation of oxidative stress before and after drug intervention. Keywords Pharmaceutical drugs . Free radicals . Antioxidant capacity . In vitro assays . Biomarkers
Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-3829-y) contains supplementary material, which is available to authorized users. G. Beretta (*) : R. M. Facino Department of Pharmaceutical Sciences “Pietro Pratesi”, Faculty of Pharmacy, University of Milan, via Mangiagalli 25, 20133 Milan, Italy e-mail:
[email protected]
Introduction Oxidative stress, often defined as an overall imbalance of oxidizing elements to reducing elements in the cell, has been recently better defined as disruption of the circuits constituted by redox elements (such as the pool of redox sensitive cysteine residues) controlled by glutathione (GSH), thioredoxins, and cysteine. These redox circuits control the signaling transduction pathways [1]. The adoption of such a definition could redirect research to identify perturbations of redox signaling and control, and lead to new treatments for oxidative-stress-related diseases. The formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been implicated in or associated with the pathogenesis of a plethora of human diseases, including atherosclerosis, type 2 diabetes, neurodegenerative diseases (Alzheimer, Parkinson, amyotrophic lateral sclerosis), infectious diseases, cardiac dysfunctions, and several type of cancers [2]. Hence, there is a need for simple, convenient, and reliable markers for the assessment both in vitro and in vivo of the metabolic/oxidative distress and of its modulation, if any, induced by the administration of pharmaceutical products. As will be outlined in this review, the data reported so far in the literature in the field of antioxidant activity of drugs limit the focus to the determination of the antioxidant properties evaluated in in vitro models, where a bulk of molecules is considered to be active and responsive as direct radical scavengers. Conversely, few clinical studies have reported the analytical profiles of some typical oxidation markers (in vivo or ex vivo), information that can provide a more reliable mechanistic explanation for the improvement of the disease and clarify whether the beneficial effects of drug intervention are really linked to an antioxidant action aimed at restoring the physiological
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redox signaling circuit disrupted by the pathological situation. Hence, the aim of the present review is to provide a brief panoramic view of the state of the art of the scientific knowledge on the antioxidant activity of pharmaceutical drugs, the methods used for its determination, the available biomarkers of oxidative stress, and their implications in health and disease. The identification of the existence of a correlation between in vitro and clinical findings will provide a solid background for the pharmacological and pharmaceutical mechanistic knowledge of a drug and stimulate scientific focus in this ever-growing research field.
Antioxidant assays and biomarkers The most popular techniques employed until now for the evaluation of the antioxidant activity of drugs can be basically classified in five different families of methods: 1. Direct detection of free radicals and other reactive oxidative species: electron spin resonance (ESR). This technique is based on the use of a spin trapper (the most common is 5,5-dimethyl-1-pyrrolidine-N-oxide; DMPO) and detection of hydroxyl radical (·OH) and superoxide (O2-) by the formation of the typical DMPO–OH spin adduct spectra (1:2:2:1 quartet with identical hyperfine splitting constants AH ¼ AN b ¼ 14:9) or DMPO–OOH (AN ¼ 14:3G; AH b ¼ 11:7G; AH g ¼ 1:25G), respectively [3, 4]. The scavenging activity of an antioxidant is measured on the basis of the decrease of the spectral signal height induced by the antioxidant with respect to the spectral signal height of a control sample. A simpler and less expensive method for the evaluation of anti-O2activity is based on the kinetic competition between the drug and the O2- acceptor nitrotetrazolium blue chloride (NBT), O2- being generated by the phenazine methosulfate/reduced β-nicotinamide adenine dinucleotide (NADPH) system. In this assay, first the rate of reduction of NBT by O2- is determined by spectrophotometrically recording the increase in absorbance at lmax =560 nm for 5 min at 25°C (controls) and then its inhibition induced by an O2- quenching drug [5]. The ESR technique has also been applied to monitor the entrapping of peroxyl radicals generated by the peroxidation of linoleic acid by the iron(II)/ascorbate system using α-(4-pyridyl-N-oxide)-tert-butylnitrone as a spin trapper (1:2:2:1; AN =16.0 G, AH b ¼ 2:6G) [6] and to directly detect nitric oxide (NO) radicals from NO donors using 2-(4-carboxyphenyl)-4,4,5,5tetramethyllimidazoline-1-oxyl-3-oxide as a spin trapper (1:2:3:2:1; AN =0.82 mT) [7].
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2. Thiobarbituric acid reactive substances (TBARS) assay. This assay, mainly used for the determination of lipid peroxidation products, is based on the antioxidantmediated inhibition of the increase in absorbance of the pink chromogen (lmax =535 nm, ε=155,000 mol-1) generated by the reaction of thiobarbituric acid with malondialdehyde (MDA) and structurally related species, which can be generated in vivo and ex vivo from different oxidizable biological substrates such as membrane lipids from liver microsomes, tissue homogenates, and plasma lipids [8]. A modification of this assay provides a suitable in test tube alternative to the ESR method for the evaluation of the anti-OH activity of a drug/antioxidant: exposure of the pentose sugar 2deoxyribose to a source of OH radicals [the H2O2/iron (II)–EDTA complex or the iron(III)/ascorbate system] yields a mixture of products which, after heating with thiobarbituric acid, forms a chromogen which is spectrophotometrically indistinguishable from that generated by reaction of thiobarbituric acid with MDA. In these conditions the degradation of deoxyribose is inhibited by any scavenger of OH to an extent that depends only on the scavenger to deoxyribose concentration ratio and on the scavenger’s second-order rate constant for reaction with OH [8]. 3. Scavenging capacity assays against stable, nonbiological radicals and redox-active species. In general, these methods involve a two-component reaction mixture which includes the antioxidant(s) and the oxidant. The oxidant is the probe itself, which on abstracting one electron from the antioxidant changes its color, and the decrease of the native absorbance of the radical, or the increase of the absorbance of the newly developed nonradical chromogen, is monitored spectrophotometrically usually at a fixed time (end point), but also by following the time course of the decrease/increase of the original/new species, respectively (kinetics). The degree of color change is proportional to the concentration and potency of the antioxidant(s). The most popular assays used for clinical applications are the 2,2diphenyl-1-picrylhydrazyl (DPPH) assay [9], the 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid assay (also known as Trolox equivalent antioxidant capacity assay) [10], and the ferric reducing/antioxidant power (FRAP) test [11]. The FRAP test directly measures the total antioxidant activity in biological fluids. It is based on a redox-linked colorimetric method which detects antioxidants and reductants employing the iron(III) tripyzidyltriazine (TPTZ) complex, which in ferrous form has an intense blue color that can be monitored spectrophometrically by measuring the change in absorbance at 593 nm. This last method therefore is directly related to the combined total reducing power of
Recent advances in the assessment of the antioxidant capacity of pharmaceutical drugs: from in vitro to in vivo evidence
the electron-donating antioxidants present in the reaction mixture [11]. 4. Assays based on oxidation probes. These assays are based on the use of (1) a thermal water-soluble radical generator [usually 2,2′-azobis(2-amidinopropane) dihydrochloride, AAPH] or of a biologically relevant oxidant (H2O2) to give a steady flux of peroxyl radicals in air-saturated solution; (2) a fluorescent oxidation probe for monitoring the reaction progress; (3) standard and test antioxidant(s) to obtain reaction kinetic parameters for the quantitation of antioxidant capacity. These methods have been adapted for high-throughput analysis using 96-well-plate fluorescence analyzers, and the most clinically used are the oxygen radical absorbance capacity (ORAC) assay, which applies the area under the oxidation kinetics curve approach (oxidation probe, fluorescein; lex = 485 nm, lem = 525 nm) [12], and the total radical absorbance power assay, TRAP based on the measurement of the luminolenhanced chemiluminescent response of human blood serum/plasma, where a lag phase type of measurement in used and the progress of the reaction is monitored for 60 min [13]. Total antioxidant assays of body fluids are useful to obtain a global picture of relative antioxidant activities in different fluids and how they change in clinical conditions. Anyway, the results should be interpreted in the light of the chemistry of the assay and can sometimes be misleading since rises in serum urate levels could obscure depletion of ascorbate and other antioxidants in certain diseases. 5. Molecular-marker-based assays for oxidative stress evaluation. F2-isoprostanes from the oxidation of arachidonic acid [14, 15], nitrotyrosine from peroxinitritemediated modification of proteins [16], oxidized DNA bases and glycoxidation and lipoxidation end products,
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i.e., carboxymethyl lysine, and methylglyoxal levels [17, 18] are considered as reliable indexes of oxidative stress in vivo, and different methods based on combined instrumental techniques such as gas chromatography (GC)–mass spectrometry (MS) and high-performance liquid chromatography (HPLC)–MS and ELISA commercial kits have been developed for their determination in plasma/serum, urine, cerebrospinal fluid, and sinovial fluid. Other molecular markers of current use in the diagnosis and therapy of specific diseases (i.e., rheumatoid arthritis) are plasma/serum total conjugated dienes monitored spectrophotometrically (lmax = 233 nm, second derivative [19]), IgG fluorescence and albumin fluorescence, and serum tocopherol, serum urate, ascorbic acid [20], and short-chain alkenals and carbonylated proteins such as 2,4-dinitrophenylhydrazone (DNPH) derivatives in plasma/serum and urine monitored by spectrophotometric or HPLC methods [21–23]. The technical details and experimental aspects of these techniques (summarized in Table 1) are described in the original works and are exhaustively reviewed elsewhere [24, 25]. Recently, fluorescent probes for the detection of ROS have been proposed as a promising tool for understanding their physiological roles [26]. Singlet oxygen, hydrogen peroxide (H2O2), ·OH radicals, and O2- have been targeted and evaluated with high selectivity and have been detected in several chemical and in vitro biological systems. In addition, electrochemistry, with the aid of electrodes of micrometer dimensions, has faced the problem of measuring markers of oxidative stress in single-cell systems [27]. The technique is well suited for the determination of redox ROS and RNS and exciting results have been obtained for electroactive species such as O2-, H2O2, NO,
Table 1 Principal general methodologies used in vitro and in vivo for the assessment of the antioxidant capacity of pharmaceutical drugs Assay /Marker
Principle
Free radicals [3, 4] Lipid peroxidation/ TBARS [8] DPPH [9] ABTS [10] FRAP [11] Total antioxidant capacity [12, 13]
Direct detection of unpaired electrons of free radicals by spin trapping ESR spectroscopy Inhibition of microsomal/serum oxidation Spectrophotometric, HPLC-UV Loss of absorbance of a colored, stable free radical or complexed Spectrophotometric heavy metal (Fe2+) in presence of the drug
Isoprostanes [14, 15] Nitro-tyrosine [16] Chloro-tyrosine [16] Conjugated dienes [19] AGE/ALE [18, 21]
Drug inhibition of fluorescence decay/formation of a chemical probe along time Determination of oxidation products from polyunsaturated lipids and amino acid protein residues
Inhibition of LDL oxidation time course Determination of end products from glycoxidation and lipoxidation in plasma and urines Protein carbonylation [22] (DNPH) Determination of total carbonylated proteins in plasma and urines
Method
Fluorometric, chemiluminometric ELISA, HPLC-MS
Spectrophotometric ELISA, HPLC-MS spectrofotometric
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peroxynitrite (ONOO-), and nitrite (NO2-). Unfortunately, until now the application of these promising techniques has been limited to the evaluation of single-cell secretion and drug permeation in tissue slices and in human skin [27]. This does not preclude the possibility that in the future the insertion of microdimensioned electrodes into a capillary vein/artery of a living organism could provide a fingerprint of all the electron-responsive species involved in cell signaling and oxidative stress with a sensitivity of detection at zeptomole to aptomole levels, thus allowing one to depict and follow in real time the basal level of the redox status before and after drug administration. The use of cyclic voltammetry for the evaluation of antioxidant capacity has been shown to be a convenient method through the quantitation of low molecular weight antioxidants (LMWA) in blood plasma and tissue homogenates [28]. However,the application of this interesting electroanalytical technique has been limited to the evaluation of the LMWA status of healthy and diseased subjects and has not been followed by further development related to drug intervention.
In vitro studies A literature search based on the use of keywords such as “drugs,” “antioxidant activity,” and “free radicals” indicated the existence of a significant number of studies on the antioxidant properties of drugs that can be classified into three main groups on the basis of their pharmacological activities: nonsteroidal anti-inflammatory drugs (NSAIDs), drugs for the treatment of cardiovascular diseases, and drugs for the treatment of central nervous system disorders.
Nonsteroidal anti-inflammatory drugs The antioxidant activity of a panel of 30 of the most commonly used anti-inflammatory drugs has been studied under different experimental conditions. Their chemical structures are reported in Fig. S1. Maffei Facino et al. [29] demonstrated the efficacy of tenoxicam in different homogeneous and heterogeneous in vitro models of oxidative stress: DPPH assay, scavenging of superoxide anion (DTNB test), quenching of peroxyl radicals (ORAC assay) and of hydroxyl radicals (ESR with DMPO as a spin trapper), and inhibition of (1) hyaluronic acid depolymerization induced by hydroxyl radicals produced by the xanthine/xanthine oxidase (XO)/iron(II) couple and (2) the hydroxyl-radical-driven peroxidation of phosphatidylcholine vesicles (markers, conjugated dienes monitored spectrophotometrically at 233 nm). The same authors studied the quenching activity of nimesulide and of its main metabolite (4-hydroxynimesulide) against
G. Beretta, R.M. Facino
superoxide anion and hydroxyl radicals by ESR, and against peroxyl radicals by monitoring in liposomes from synovial fluid the formation of conjugated dienes and of carbonyl groups (DNPH test) [30]. Asanuma et al. [7] demonstrated in cultured neuronal cells (marker, NO-donor-induced apoptotic cell death) the antioxidant activity of nimesulide, indomethacin, aspirin, mefenamic acid, ketoprofen, and naproxen against NO free radicals using an ESR-based method, compared with the antioxidant activity of hydroxycortisone and dexametasone. Conversely, Zheng et al. [31] used a chemiluminescence-based approach to investigate the antioxidant activity of nimesulide and of its metabolite 4-hydroxynimesulide in cell cultures using both horseradish peroxidase dependent and luminol-dependent chemiluminescence produced by human chondrocytes. Candelario-Jalil et al. [32] demonstrated the ability of nimesulide to inhibit kainate-induced oxidative damage in rat brain homogenates (TBARS assay). The ability of indomethacin to inhibit the luminol- and lucigenin-amplified chemiluminescence of human neutrophils was reported in the studies of Parij et al. [33], where the antioxidant activity of ketoprofene, naproxen, tolmetin, ibuprofen, acemetacine, flufenamic, acid, niflumic acid, tenoxicam, and piroxicam was investigated using this chemiluminescence approach. The antioxidant capacity of nimesulide and that of 4-hydroxynimesulide was unequivocally confirmed by Mouithys-Mickalad et al. [6] by different assays, based on measurement of peroxynitrite quenching by ESR spectroscopy and inhibition of luminol-enhanced chemiluminescence of activated human neutrophils, of erythrocyte membrane lipid peroxidation induced by the iron(II)/ascorbate couple, of NaOCl-induced chemiluminescence, and finally by the determination of their ability to directly scavenge peroxynitrite. In the same study, using the same models, the antioxidant potency was demonstrated for indomethacin, for aceclofenac, and for its metabolite 4-hydroxyaceclofenac. In 2005, Costa et al. [34] showed the H2O2 scavenging activity of indomethacin, acemetacin, etodolac, oxaprozin, ketorolac, sulindac sulfone, sulindac sulfide, sulindac, and tolmetin by measuring the H2O2-elicited lucigenin chemiluminescence with melatonin and GSH as positive controls. The results demonstrated that all the compounds were able to scavenge H2O2, with the following ranking order of potency: sulindac sulfone>sulindac sulfide>GSH> sulindac>indomethacin>acemetacin>etodolac>oxaprozin> ketorolac≈melatonin>tolmetin. In 2006, the same authors reported that the NSAIDs naproxen, ketoprofen, flurbiprofen, ibuprofen, fenoprofen, indoprofen, and fenbufen were able to inhibit the oxidative burst of activated human neutrophils monitored by a chemiluminescence-based approach [35].
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Drugs for cardiovascular diseases
Calcium blockers
β-blockers
Tong Mak et al. [40], studying the effects of selected calcium blockers—nicardipine, nifedipine, verapamil, and diltiazem—on free-radical injury in cultured endothelial cells, found that these drugs were able to inhibit the loss of cell viability and to spare intracellular GSH after the free-radical insult (O2-, ·OH) generated by the dihydroxyfumarate/iron(II)/ADP system, and that the antioxidant capacity of these drugs appears to be related to their partitioning ability within the biological membrane: dihydropyridines>verapamil>diltiazem. Kusmic et al. [41] demonstrated that dipyridamole, a compound endowed with antithrombotic and vasodilating actions, is able to spare in vitro vitamin E, GSH, and thiols in human red blood cells after exposure to oxidative stress induced by cumene hydroperoxide, by monitoring the loss of fluorescence of the oxidation probe parinaric acid.
Yue et al. [36] demonstrated the antioxidant properties of the β-blocker carvedilol (chemical structure in Fig. S2) using different approaches: (1) iron(II)/ascorbate-induced lipid peroxidation in rat brain homogenate; (2) oxygenradical-initiated α-tocopherol depletion in rat brain tissue in vitro; (3) ESR spin-trapping techniques using dihydroxyfumarate/iron(II)/ADP as an OH generator and DMPO as a trapping agent to determine whether carvedilol is a free-radical scavenger; (4) establishing structure–activity relationships of carvedilol for antioxidation by measuring the electrochemical potential of a series of carvedilol analogues. From these experiments, the authors concluded that carvedilol inhibits in vitro the oxygen-free-radical-initiated lipid peroxidation and α-tocopherol depletion in rat brain homogenate through a direct scavenging activity and with an efficiency at least 2 orders of magnitude greater than that of all the other β-blockers. More recently, Gomes et al. [37] through chemiluminescence-based techniques investigated in cell-free models the scavenging activity of a series of β-blockers: carvedilol, atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, and timolol against superoxide radical, hydrogen peroxide, hydroxyl radical, hypochlorous acid, peroxyl radical, nitric oxide, and peroxynitrate, demonstrating their antioxidant capacity using ascorbic acid, lipoic acid, melatonin, rutin, and ebselen as positive controls. The results of this study evidenced that some of these drugs are strong ROS and/or RNS direct scavengers. Angiotensin converting enzyme inhibitors Chopra et al reported the free radical scavenging activity of seven angiotensin converting enzyme inhibitors (captopril, zofenopril, enelaprilat, fentiapril, quinalaprilat, perindoprilat, and ramiprilat) using different experimental models: inhibition of riboflavin-mediated photooxidation of dianisidine, quenching of superoxide anion (DTNB test), hydrogen peroxide (thiol oxidation), hypochlorous acid (protection of α-antiprotease activity), and inhibition of microsomal lipid peroxidation (TBARS assay) [38]. In 1998, Benzie and Tomlinson [39] reported the antioxidant power of captopril, fosinopril, enalapril, perindopril, quinalapril, and ramipril using the FRAP assay, demonstrating that among the angiotensin converting enzyme inhibitors tested only captopril was able to reduce the complex Fe(III)(TPTZ)3 to Fe(II)(TPTZ)3, owing to the presence of the reducing thiol group in the structure of the compound.
Statins The in vitro antioxidant activity of fluvastatin and that of atorvastatin was demonstrated using the total oxyradical scavenging assay, a method for the evaluation of antioxidant capacity based on the inhibition of the formation of ethylene (measured by GC and a flame ionization detector) from the oxidation of the substrate α-keto-γ-methiolbutyric acid in the presence of different radical initiators [42]. Using this assay with uric acid and Trolox as positive controls, the authors found that the scavenging capacity for hydroxyl radicals was highest for simvastatin (3,375± 112 U/mg), a value 270.2% higher (Ppentoxiphilline.
In vivo studies A potential antioxidant effect of NSAIDs was evidenced by Situnayake et al. [20] in a limited number of rheumatoid arthritis patients (n=20) who were treated with full standard disease modifying antirheumatic drugs and full doses of NSAIDs. The authors noted a significant increase in the levels of conjugated dienes and TBARS after withdrawal of NSAIDs, an observation which may link 9,11-linoleic acid formation with increased prostaglandin formation and freeradical activity. More recently, Ozgocmen et al. [49] compared the in vivo effects on free-radical metabolism of two NSAIDs—
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celecoxib and tenoxicam—in patients with knee osteoarthritis. The serum levels of oxidative-stress-related enzymes [i.e., XO, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)], of MDA, and of nitrite were studied at the baseline and after a 4-week course of treatment with celecoxib (n = 11) and tenoxicam (n = 12). Celecoxibtreated patients had a significant decrease in nitrite levels (p =0.043), whereas SOD, XO, and GSH-Px enzyme activities and MDA levels did not change significantly compared with the baseline. Tenoxicam-treated patients had significant decrease in nitrite levels (p=0.036) and XO activity (p=0.01), but their SOD and GSH-Px enzyme activities and MDA levels were unchanged from the baseline. There was significant correlation between the patients’ (n=23) Western Ontario and McMaster universities (WOMAC) LK3.0 Osteoarthritis Index, WOMAC pain scores, and MDA levels (r = 0.50, p =0.014) and the patients’ WOMAC stiffness scores and XO enzyme activity (r=0.46, p=0.027) at the baseline. Significant improvement was found in Visual Analogue Scale pain, patients’ global assessment, and WOMAC pain, stiffness, and physical function scores in celecoxib- and tenoxicam-treated groups, confirming that tenoxicam may actually intervene through an antioxidant mechanism. In a prospective, open-label interventional trial, Shishehbor et al. [50] found that in hypercholesterolemic subjects with no known coronary artery disease treated with atorvastatin (10 mg/day, 12 weeks), in parallel with a significant reduction in total cholesterol, low-density lipoprotein(LDL) C, and apolipoprotein B-100 levels (25, 39, and 29%, respectively), there were comparable significant reductions in the levels of the oxidation markers ClTyr, diTyr, and NO2Tyr (30, 32, and 25%, respectively). More recently Tavridou et al. [51] demonstrated that simvastatin can significantly reduce circulating oxidized serum LDL levels (determined by the ELISA technique) both in subjects undergoing primary and in subjects undergoing secondary prevention of coronary heart disease. In a prospective, randomized clinical trial on patients with symptomatic stable heart failure, Kukin et al. [52] investigated the antioxidant effect of either carvedilol or metoprolol (β-blockers) administered in addition to standard therapy for congestive heart failure (CHF). Measured variables included symptoms, exercise, ejection fraction, and serum TBARS as an indirect marker of free-radical activity. Both patient groups showed beneficial effects from ß-blocker therapy. With metoprolol, serum TBARS values decreased from 4.7±0.9 nmol/mL at the baseline to 4.2± 1.5 nmol/mL at month 4 to 3.9±1.0 nmol/mL at month 6 (P
