Free Radical Biology and Medicine 70 (2014) 96–105

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Original Contribution

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides Tracey B. Kajer a,b, Kathryn E. Fairfull-Smith c, Toshihide Yamasaki d, Ken-ichi Yamada d, Shanlin Fu e, Steven E. Bottle c, Clare L. Hawkins a,b, Michael J. Davies a,b,n a

Heart Research Institute, Newtown, Sydney, NSW 2042, Australia Faculty of Medicine, University of Sydney, Sydney, NSW, Australia c School of Physical and Chemical Sciences, Queensland University of Technology, Brisbane, QLD, Australia d Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Kyushu, Japan e Centre for Forensic Science, University of Technology, Sydney, NSW, Australia b

art ic l e i nf o

a b s t r a c t

Article history: Received 4 November 2013 Received in revised form 23 January 2014 Accepted 12 February 2014 Available online 22 February 2014

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion,
OCl) generated by the myeloperoxidase (MPO)–H2O2–Cl
system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC50 values of  1 and  6 mM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC50 values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed. & 2014 Elsevier Inc. All rights reserved.

Keywords: Myeloperoxidase Neutrophil Nitroxide Hypochlorous acid Superoxide radicals Protein oxidation Free radicals

Activated neutrophils, monocytes, and some macrophages release the heme enzyme myeloperoxidase (MPO)1 both intraphagosomally and extracellularly. This enzyme uses H2O2 and halide/pseudohalide ions (Cl
, Br
, SCN
) to generate the potent oxidants hypochlorous acid (HOCl/
OCl), hypobromous acid (HOBr/
OBr), and hypothiocyanous acid (HOSCN/
OSCN), respectively [1–3]. The production of these species is important in immune defense against invading pathogens, as these oxidants, particularly HOCl and HOBr, show potent bactericidal activity [4,5]. The related enzyme eosinophil peroxidase (EPO), released extracellularly by activated eosinophils, uses H2O2 and Br
or SCN
to generate predominantly HOBr and

Abbreviations: DTNB, 5,50 -dithiobis(2-nitrobenzoic acid); EPO, eosinophil peroxidase; MPO, myeloperoxidase; TEEPO, 2,2,6,6-tetraethylpiperidine-1-oxyl radical; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl radical; TNB, 5-thio-2-nitrobenzoic acid n Corresponding author at: The Heart Research Institute, Free Radical Group, 7 Eliza Street, Newtown, Sydney, NSW 2042, Australia. Fax: þ 61 2 9565 5584. http://dx.doi.org/10.1016/j.freeradbiomed.2014.02.011 0891-5849 & 2014 Elsevier Inc. All rights reserved.

HOSCN to kill parasites [6,7]. A number of other heme peroxidases (e.g., lactoperoxidase and peroxidases present in the oral cavity and the stomach) also generate oxidants of this family [2,8–11]. Inappropriate formation of these oxidants has, however, been implicated in a number of major human inflammatory diseases, including atherosclerosis, asthma, rheumatoid arthritis, cystic fibrosis, kidney disease, and some cancers [1–3,12]. In light of these data, there is considerable interest in the development of therapeutically useful MPO inhibitors [12–14] and a number of novel materials have been examined (e.g., [13–18]). A number of recent studies have identified species that act as effective peroxidase substrates for MPO, thereby preventing rapid cycling of the enzyme via its halogenation cycle that generates HOCl/HOBr/HOSCN, and/or generate metabolites that induce irreversible heme modification [15,17]. Nitroxides are stable free radicals that have multiple biological effects, including acting as protective agents against oxidation in animal models of inflammation (e.g., [19–24]), though the

T.B. Kajer et al. / Free Radical Biology and Medicine 70 (2014) 96–105

mechanisms responsible are not fully understood. It has been reported that nitroxides can: (a) act as superoxide dismutase (SOD) mimetics [25–27], (b) reversibly or irreversibly scavenge radicals (e.g., carbon, oxygen, nitrogen, sulfur, and protein radicals [26,28–30]), and (c) reduce the oxyferryl (FeIV ¼O) center of heme proteins [19,31–34]. Recently we, and others, have also shown that a number of nitroxides can inhibit the chlorinating and nitrating activities of MPO [33–35]. In some cases this has been ascribed to radical scavenging (e.g., decreased protein nitration by MPO–H2O2– NO2
[33] and oxidative damage generated by MPO–H2O2–phenol [33,36]), but in other cases this inhibition seems to occur via the nitroxide interacting with compound I and compound II of the enzyme [33–35]. Whether inhibition also occurs with EPO is unknown. These data suggest that nitroxides may modulate MPO (and possibly EPO)-mediated damage in vivo, and it has been established that some of these species are well tolerated and show low toxicity in rodents [19,20,32]. Topical application of nitroxides has also been used to prevent radiation-induced alopecia in guinea pigs [37] and humans [38]. Nitroxides are, however, rapidly reduced in vivo to the corresponding hydroxylamines [39,40], which are much less effective inhibitors of MPO [34], and this may limit their activity and/or result in a requirement for high in vivo doses. Interestingly, it has been recently shown that novel members of this family with tetraethyl, instead of tetramethyl, substituents flanking the nitroxide function (i.e., TEEPO compared to TEMPO; for structure and nomenclature see Table 1) are reduced at a slower rate [41–43], with this ascribed to steric shielding and a reduced redox potential of the nitroxide function [44]. In light of these data we have investigated: (a) whether tetraethyl-substituted nitroxides (and related species) modulate oxidant formation by isolated MPO, isolated EPO, and activated human neutrophils and (b) the rates of reduction of these species by human plasma and unstimulated and stimulated human neutrophils to determine whether these species have a longer biological half-life and, hence, might be possible therapeutic agents in diseases that involve MPO-induced damage.

97

Materials and methods Materials All buffers and aqueous solutions were prepared using Nanopure water filtered through a four-stage Milli-Q system (Millipore Waters, Lane Cove, Australia). Unless otherwise stated, 0.1 M potassium phosphate buffer, pH 7.4, was used to prepare reaction mixtures. MPO (Planta Natural Products, Vienna, Austria) was resuspended in Nanopure water and stored at 4 1C. Catalase, dimethyl sulfoxide (DMSO), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), Hanks’ balanced salt solution without phenol red (HBSS), red blood cell lysing buffer Hybri-Max, sodium bromide, sodium chloride, sodium hydroxide, sodium thiocyanate, and phorbol myristate acetate (PMA) were obtained from Sigma–Aldrich (Sydney, Australia). 4-Oxo-TEMPO and 4-hydroxy-TEMPO were from Sigma–Aldrich, 4-amino-TEMPO was from Alexis Biochemicals (Lausen, Switzerland). 4-Hydroxy-TEEPO was prepared according to the method of Rajca (see supplementary material and [45]). 4-Oxo-TEEPO was synthesized and purified according to a previously reported method [42]. Initial batches of 4-aminoTEEPO were synthesized from 4-hydroxy-TEEPO as described under Method 1 in the supplementary material; subsequent samples were synthesized using Method 2 (see supplementary material) using a minor adaptation of a recently published method for 4-amino-TEMPO [46]. Dispirooxo-TEMPO was synthesized as described previously [47]. Polymorphprep density gradient solution was from Axis-Shield (Oslo, Norway). Stock solutions of hydrogen peroxide (Merck, Darmstadt, Germany) were prepared by dilution of a concentrated stock into Nanopure water and used immediately. Stock solutions of PMA (1 mg ml
1) were prepared in DMSO, stored at
80 1C, and diluted with HBSS before use. Quantification of oxidants using the TNB assay Oxidants generated by MPO, EPO, and stimulated neutrophils were quantified using TNB as described previously [48]. Briefly, HOCl or HOBr generated by isolated MPO, EPO, or activated

Table 1 Nitroxide structures.

Side chains

Name R2

R3

R4

X


CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3


CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3


CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH3


CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH3


OH
NH3þ ¼O
OH
NH3þ ¼O ¼O

4-Hydroxy-TEMPOa 4-Amino-TEMPOb 4-Oxo-TEMPOc 4-Hydroxy-TEEPOd 4-Amino-TEEPOe 4-Oxo-TEEPOf 4-Oxo-2,2-diethyl-6,6-dimethylpiperidin-1-yloxyl

¼O

Dispirooxo-TEMPOg

f f

R1


CH2CH2OCH2CH2a


CH2CH2OCH2CH2-

Also known as 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxyl. Also known as 4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl. Also known as 4-oxo-2,2,6,6-tetramethylpiperidin-1-yloxyl. d Also known as 4-hydroxy-2,2,6,6-tetraethylpiperidin-1-yloxyl. e Also known as 4-amino-2,2,6,6-tetraethylpiperidin-1-yloxyl. f Also known as 4-oxo-2,2,6,6-tetraethylpiperidin-1-yloxyl. g Also known as 7-aza-3,11-dioxa-15-oxodispiro[5.1.5.3]hexadec-7-yl-7-oxyl. b c

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neutrophils is trapped by added taurine to generate chloramines (RNHCl species), which react with the (yellow) TNB reagent added in the assay (ca. 45 mM, prepared by alkaline hydrolysis of the dimer DTNB followed by 40-fold dilution into 0.1 M potassium phosphate buffer, pH 7.4) to give colorless DTNB. HOSCN formed by these systems does not react with taurine [49], but reacts directly and rapidly with TNB [49]. The extent of TNB oxidation to DTNB arising from oxidant formation was quantified after 15 min incubation in triplicate wells on a 96-well microtiter plate using UV–visible spectrophotometry (Molecular Devices SpectraMax M2e microplate reader, Sunnyvale, CA, USA) at 412 nm with ε 14,150 M
1 cm
1 [50]. Isolated MPO reaction system Isolated MPO (50 nM) was incubated with taurine (10 mM), sodium chloride (NaCl; 100 mM), nitroxide (0–10 mM), and H2O2 (50 mM). Incubations were for 30 min at 37 1C, after which the reaction was quenched by placing tubes on ice before analysis using the TNB assay as described above. Isolated EPO reaction system Isolated EPO (20 nM) was incubated with taurine (10 mM), NaCl (100 mM), sodium bromide (NaBr; 100 mM), sodium thiocyanate (NaSCN; 100 mM), nitroxide (0–100 mM), and H2O2 (50 mM). Samples were incubated for 30 min at 37 1C, after which the reaction was quenched by placing the tubes on ice before analysis by the TNB assay as described above.

PMA-stimulated (100 ng ml
1) neutrophils (2  106 cells ml
1). The decay of nitroxide signal was monitored using the above spectrometer settings except with a fixed magnetic field corresponding to one of the nitroxide absorptions with a time constant of 2621 (plasma) or 1311 ms (neutrophils), over a period of 1342 s for plasma and 671 s for neutrophils. The initial linear rates of decay (100 s) of the kinetic scans were fitted to a linear function using OriginPro 8.6 (OriginLab Corp., Northampton, MA, USA). Statistics Statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software, San Diego, CA, USA). The inhibitory effects of nitroxides were determined using either one-way ANOVA with Dunnett’s post hoc test or two-way ANOVA with Bonferroni’s post hoc test, with p o 0.05 taken as significant. Unless otherwise stated, all results are reported as the mean 7 SEM for n Z 3 experiments with n Z 3 neutrophil donors. The concentration of nitroxide required for 50% inhibition of oxidant production (IC50) was determined by fitting a sigmoidal curve to dose–response data using nonlinear regression analysis where the bottom value was constrained to a value o1. Nitroxide concentrations were expressed as the base 10 logarithm. IC50 values are reported as the mean 7 SD for n Z 3 experiments. The stability of the TEEPO compounds, compared to TEMPO and derivatives, was evaluated using two-tailed paired t tests, with p o 0.05 taken as significant.

Results Human neutrophil incubations Neutrophils were isolated immediately after collection of blood into EDTA from healthy volunteers with informed consent and local ethical approval (Sydney South West Area Health Service, Protocols X09-0013 and X-12-0375) in accordance with the Declaration of Helsinki (2000) of the World Medical Association, as described previously [51]. Neutrophils were isolated using a Polymorphprep density-gradient method, followed by hypotonic lysis of residual erythrocytes. Once isolated, neutrophils were resuspended in HBSS at 4  106 cells ml
1 and used immediately. Cell viability was assessed by trypan blue dye exclusion. Purity of neutrophil suspensions was evaluated by microscopy of stained cytospins. Neutrophils (2  106 cells ml
1) were preincubated with taurine (10 mM) and nitroxide (0–20 mM) for 10 min at 37 1C, before stimulation by PMA (100 ng ml
1). Samples were incubated for 30 min at 25 1C, shaking every 5 min to ensure the cells remained suspended. Reactions were stopped with catalase (50 mg ml
1) and the tubes placed on melting ice. Cells were pelleted for 5 min at 4 1C at 14,000 g and supernatant was removed for the TNB assay as previously described. Electron paramagnetic resonance (EPR) spectroscopy EPR spectroscopy was performed at 21 1C using a Bruker EMX X-band spectrometer equipped with a cylindrical (ER4103TM) cavity and 100-MHz modulation. A flattened aqueous sample cell (WG-813-SQ; Wilmad, Buena, NJ, USA) contained solutions of nitroxides in plasma and neutrophil suspensions. Typical spectrometer settings for recording of nitroxide spectra were center field 348 mT, sweep width 10 mT, resolution 1024 points, microwave frequency 9.6 GHz, microwave power 25 mV, receiver gain 1.0  104, modulation amplitude 0.05 mT, time constant 163 ms, and sweep time 83 s. Scans were initiated within 2 min of mixing. Nitroxide (10 mM) decay kinetics were determined in the presence of freshly isolated human plasma or either unstimulated or

Effects of tetraethyl-substituted nitroxide radicals and related species on oxidant production by isolated MPO The production of chlorinating oxidants by isolated MPO (50 nM) in the presence of H2O2 (50 mM) and physiological Cl
concentrations (100 mM) was measured by trapping with taurine (10 mM) and assaying the resulting taurine chloramines using TNB (Fig. 1). Compared to reactions carried out in the absence of added nitroxide, all six nitroxides examined decreased the extent of chlorinating oxidant formation in a dose-dependent manner (Fig. 1). Significant inhibition occurred at r2.5 mM for all nitroxides. The concentrations of nitroxide required to produce a 50% decrease in oxidant formation (IC50 values) calculated from these data, for each of the species examined, are collected in Table 2. Comparison of the data for the TEMPO derivatives with TEEPO derivatives showed that tetraethyl substitution ortho to the nitroxide radical did not significantly decrease the extent of inhibition of oxidant production induced by tetramethyl substitution, and in some cases (4-hydroxy-TEEPO and 4-oxo-TEEPO) enhanced this. Experiments with the 4-oxo-2,2-diethyl-6,6-dimethyl derivative showed results comparable to both the TEMPO and the TEEPO series. In contrast, the dispiro-4-oxo-TEMPO (Fig. 1D) had no significant inhibitory effect, suggesting that larger and more rigid species are poor inhibitors. The parent amine of 4-hydroxy-TEEPO (4-hydroxy-2,2,6,6-tetraethylpiperidine; data not shown) also had no inhibitory effect, consistent with a requirement for the nitroxide function.

Effects of tetraethyl substitution of nitroxides on oxidant production by human neutrophils The production of oxidants by PMA-stimulated (100 ng ml
1) human neutrophils (2  106 cells ml
1) and the effects of nitroxides on this process were examined by trapping with taurine

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99

Fig. 1. Effects of tetraethyl versus tetramethyl substitution of nitroxide radicals on the formation of chlorinating oxidants by isolated MPO. The production of chlorinating oxidants was assayed with TNB and is reported as a percentage of the chloramines detected in the complete system in the absence of added nitroxide. The reaction mixtures contained purified MPO (50 nM), H2O2 (50 mM), NaCl (100 mM), taurine (10 mM), and nitroxide (0–10 mM). Black bars represent data for the TEMPO derivatives (4-hydroxyTEMPO, 4-amino-TEMPO, 4-oxo-TEMPO), white bars show data for the corresponding TEEPO derivatives. (A) 4-Hydroxy-substituted species, (B) 4-amino-substituted species, (C) 4-oxo-substituted species, where gray bars show data for the diethyl, dimethyl species and (D) dispirooxo-TEMPO. np o 0.05, statistically significant difference compared to system in absence of nitroxide, by one-way ANOVA using Dunnett’s post hoc test. #p o 0.05, statistically significant difference between tetramethyl- and tetraethylsubstituted nitroxides at a specified concentration, by two-way ANOVA using Bonferroni post hoc test. Data are the means 7 SEM for n Z 3 experiments.

Table 2 Half-maximal inhibitory concentrations of nitroxides for formation of halogenating oxidants generated by isolated MPO, activated neutrophils, and isolated EPO. Nitroxide

4-Hydroxy-TEMPO 4-Hydroxy-TEEPO 4-Amino-TEMPO 4-Amino-TEEPO 4-Oxo-TEMPO 4-Oxo-TEEPO 4-Oxo-2,2-diethyl-6,6-dimethylpiperidin-1-yloxyl Dispirooxo-TEMPO

IC50 value (mM) Isolated MPO

Neutrophils

Isolated EPO

2.6 7 0.5 1.4 7 0.6 1.0 7 0.02 1.9 7 0.8 1.9 7 0.4 1.0 7 0.03n 0.9 7 0.3n N/A

12.8 7 3.0 4.6 7 0.3n 2.2 7 0.6 3.6 7 1.0 22.8 7 9.8 11.5 7 1.8 12.5 7 2.4 N/A

43.6 724.5# 62.0 75.2# 46.0 79.0# 299.17 20.9n,# 125.7 743.0# 114.9 7 2.1# N/D N/D

The concentration of nitroxide required for 50% inhibition of oxidant production (IC50) was determined by fitting a sigmoidal curve to dose–response data using nonlinear regression analysis. IC50 values are reported as the mean 7 SD for n Z 3 experiments. The minimum concentration of nitroxide required for significant inhibition was determined by one-way ANOVA with Dunnett’s post hoc test where comparisons were made to control samples in the absence of nitroxide. N/D represents values that were not determined. N/A represents values that were not able to be quantified. n

#

p o 0.05, statistically significant difference compared with the corresponding TEMPO nitroxide as determined by t test. p o 0.05, statistically significant difference compared with the MPO system as determined by t test.

(10 mM) and assaying the resulting taurine chloramine using TNB. Inclusion of the nitroxides in the cell incubations resulted in a dose-dependent decrease in the concentration of oxidants detected in the presence of the TEMPO derivatives, the corresponding TEEPO derivatives, and 4-oxo-2,2-diethyl-6,6-dimethyl derivative (Fig. 2). No significant differences were detected between the TEMPO and the TEEPO series, with the exception of 4-hydroxy-TEEPO versus 4-hydroxy-TEMPO at a concentration of 5 mM. The IC50 values calculated from these data for each of the nitroxides examined are collected in Table 2.

Effects of tetraethyl- and tetramethyl-substituted nitroxides on oxidant production by isolated EPO The production of oxidants by isolated EPO (20 nM) in the presence of H2O2 (50 mM) and physiological concentrations of Cl
(100 mM), Br
(100 mM), and SCN
(100 mM) was assessed using the TNB assay, with oxidation induced either via the intermediacy of N-halogenated species or via direct HOSCN reaction (Fig. 3). Inclusion of both tetramethyl and tetraethyl nitroxides resulted in a decrease in the formation of oxidants in a dose-dependent

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Fig. 2. Effects of tetraethyl versus tetramethyl substitution of nitroxide radicals on the formation of chlorinating oxidants by human neutrophils. The production of chlorinating oxidants was assayed with TNB and is reported as a percentage of the chloramines detected in the complete system in the absence of added nitroxide. Incubations contained human neutrophils (2  106 cells ml
1) in HBSS stimulated with PMA (100 ng ml
1), taurine (10 mM), and nitroxide (0–20 mM). Black bars represent data for the TEMPO derivatives (4-hydroxy-TEMPO, 4-amino-TEMPO, 4-oxo-TEMPO), white bars represent data for the corresponding TEEPO derivatives. (A) 4-Hydroxysubstituted species, (B) 4-amino-substituted species, (C) 4-oxo-substituted species, where gray bars show data for the diethyl, dimethyl species. np o 0.05, statistically significant difference compared to system in absence of nitroxide, by one-way ANOVA using Dunnett’s post hoc test. #p o 0.05, statistically significant difference between tetramethyl- and tetraethyl-substituted nitroxides at the specified concentrations, by two-way ANOVA using Bonferroni’s post hoc test. Data are the means 7 SEM for n Z 3 experiments from three or more neutrophil donors.

Fig. 3. Effects of tetraethyl- and tetramethyl-substituted nitroxides on the formation of oxidants by isolated EPO. The production of oxidants was assayed with TNB and reported as percentage halamines remaining of the control system lacking nitroxide. Data represent incubations of isolated EPO (20 nM) with taurine (10 mM), NaCl (100 mM), NaBr (100 mM), NaSCN (100 mM), nitroxide (0–100 mM), and H2O2 (50 mM). Black bars show data for the TEMPO derivatives (4-hydroxy-TEMPO, 4-amino-TEMPO, 4-oxo-TEMPO), white bars show data for the corresponding TEEPO derivatives. (A) 4-Hydroxy-substituted species, (B) 4-amino-substituted species, (C) 4-oxo-substituted species. np o 0.05, statistically significant difference compared to system in absence of nitroxide, by one-way ANOVA using Dunnett’s post hoc test. #p o 0.05, statistically significant difference between tetramethyl- and tetraethyl-substituted nitroxides at the specified concentrations, by two-way ANOVA using Bonferroni’s post hoc test. Data are the means 7 SEM for n Z 3 experiments.

manner (Fig. 3). The concentration of nitroxides required to generate 50% inhibition was calculated as for the MPO system (see Table 2), but the levels required to achieve this level of inhibition were considerably higher than for the isolated MPO system. There was no significant difference between the TEMPO and the TEEPO derivatives except at the highest nitroxide concentrations studied. Effects of tetraethyl versus tetramethyl substitution on nitroxide radical stability in human plasma The rate of decay of nitroxides (10 mM) in the presence of fresh human plasma was monitored by EPR spectroscopy over 22 min at 21 1C. Spectral scans of each of the nitroxides gave the expected three-line signal with line intensities in the ratio 1:1:1 due to coupling to the nitroxide nitrogen. No additional couplings were detected with any of the compounds under the spectrometer conditions employed. Subsequent studies examined the decay of the radicals in the presence of fresh human plasma by using a fixed magnetic field set to the values for one of the absorption lines, with the decay of the peak examined in real time. For the TEMPO derivatives a rapid decay of the nitroxide signal was detected in the presence of human plasma, as expected on the basis of previous studies. The resulting reduction curves were analyzed over the first 100 s by curve fitting to give a decay rate in arbitrary units (Fig. 4). The resulting initial rate data are collected in Table 3. Compared to the TEMPO derivatives, each of the TEEPO species decayed at a much slower rate. These data suggest that

tetraethyl substitution markedly decreases the susceptibility of nitroxides to reduction by components of human plasma. These data are in accord with previous data for this family of radicals [42–44,52]. Effects of tetraethyl versus tetramethyl substitution on nitroxide radical stability in the presence of unstimulated and stimulated human neutrophils Although reduction by components of plasma has been suggested to be a major route to nitroxide decay in vivo [41–43,52,53], cells also contain many powerful enzymatic (reductase and thiolbased enzymes) and nonenzymatic reduction systems (e.g., thiols, ascorbate). Furthermore in the case of neutrophils, the oxidative burst generates high levels of superoxide radicals [54] that have been shown previously to react rapidly with some nitroxides (cf. the reported superoxide dismutase activity of these species [25,55]). As such it was important to determine the potential decay rates of nitroxide radicals in both the presence of unstimulated (i.e., decay induced by normal metabolic processes) and stimulated neutrophils (in which additional contributions from the oxidative burst and other released species might be expected to occur). Thus freshly isolated unstimulated or stimulated (100 ng PMA ml
1) human neutrophils (2  106 cells ml
1) were incubated with fixed (10 mM) concentrations of the nitroxides, and the decay of the radical was analyzed by EPR spectroscopy as indicated above (Fig. 5). All of the nitroxides studied decayed less rapidly in the presence of quiescent neutrophils compared to

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101

Fig. 4. Initial rates of decay of 4-hydroxy-, 4-amino-, and 4-oxo-TEMPO and -TEEPO species as detected by direct EPR spectroscopy in the presence of fresh human plasma. The rate of decay of the nitroxides (10 mM) in the presence of fresh human plasma was monitored by EPR spectroscopy over 22 min at 21 1C by setting the magnetic field of the EPR machine to one of the absorption lines of the nitroxide radical (determined in pilot experiments) and then scanning the intensity of this absorption over time. The initial rates of decay were subsequently calculated by fitting the kinetic data over the first 100 s. (A) Representative decay curves for (i) 4-hydroxy-TEMPO and (ii) 4-hydroxy-TEEPO and (iii) subsequent quantification. (B) Representative decay curves for (i) 4-amino-TEMPO and (ii) 4-amino-TEEPO and (iii) subsequent quantification. (C) Representative decay curves for (i) 4-oxo-TEMPO and (ii) 4-oxo-TEEPO and (iii) subsequent quantification. The tetraethyl derivatives showed significantly slower rates of decay compared to the corresponding tetramethyl-substituted species, when examined using paired Student’s t test, with np o 0.05. Data are the means 7 SEM for three independent experiments using three plasma donors. Table 3 Rates of decay of TEMPO and TEEPO nitroxides in plasma and in unstimulated and stimulated neutrophils. Nitroxide

Plasma Rate of decay (units s
1)

4-Hydroxy-TEMPO 4-Hydroxy-TEEPO 4-Amino-TEMPO 4-Amino-TEEPO 4-Oxo-TEMPO 4-Oxo-TEEPO

577 12 6.0 7 1.7n 1177 22 4.5 7 1.6n 2767 58 3.17 1.3n

Reduction in rate of decay

897 4.5% 96 7 1.9% 997 0.25%

Unstimulated neutrophils

Stimulated neutrophils

Rate of decay (units s
1)

Rate of decay (units s
1)

1.0 71.6 0.4 70.9 1.9 71.6 0.6 70.2
0.1 71.5
0.3 70.7

2.7 7 0.9# 1.0 7 0.6n 2.3 7 1.2 0.8 7 0.9n
0.5 7 0.3
0.2 7 1.5

Reduction in rate of decay

627 24% 757 29% N/S

The reduction in rate of decay is the percentage difference between the rate of decay of each TEMPO/TEEPO pair. Data are mean 7 SD from three or more experiments from three or more donors. N/S indicates no significant difference between the tetramethyl- and the tetraethyl-substituted species. n

#

p o 0.05, statistically significant difference compared with the corresponding TEMPO nitroxide as determined by t test. p o 0.05, statistically significant difference upon stimulation of neutrophils as determined by t test.

plasma. Unstimulated cells gave rise to less rapid reduction than the stimulated cells, and the presence of tetraethyl substituents gave rise to slower rates of decay than for the tetramethyl-substituted species.

Thus the rate of decay of 4-hydroxy-TEEPO was 62724% lower than that of 4-hydroxy-TEMPO, and for the corresponding 4-aminosubstituted pair, the decay rate of 4-amino-TEEPO was 75729%

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Fig. 5. Initial rates of decay of 4-hydroxy-TEMPO, 4-amino-TEMPO, and 4-oxo-TEMPO and the corresponding TEEPO derivatives in the presence of human neutrophils. Decay curves for the tetramethyl- and tetraethyl-nitroxides (10 mM) in the presence of freshly isolated human neutrophils (2  106 cells ml
1) stimulated with PMA (100 ng ml
1) monitored by EPR spectroscopy over 11 min at 21 1C as described in the legend to Fig. 4. Initial rates of decay were calculated by fitting the data over the first 100 s. Representative decay curves are shown for (A) (i) 4-hydroxy-TEMPO and (ii) 4-hydroxy-TEEPO and (iii) subsequent quantification, (B) (i) 4-amino-TEMPO and (ii) 4-aminoTEEPO and (iii) subsequent quantification, and (C) (i) 4-oxo-TEMPO and (ii) 4-oxo-TEEPO and (iii) subsequent quantification. The tetraethyl derivatives showed significantly slower rates of decay compared to the corresponding tetramethyl-substituted species, when examined using paired Student’s t test, with np o 0.05 and “ns” indicating not significant compared to the tetramethyl species. Data are the means 7 SEM for four independent experiments, using four neutrophil donors.

slower than for 4-amino-TEMPO (see Table 3). Both of the 4-oxosubstituted species were significantly more stable than the other nitroxides (Fig. 5C).

Discussion Nitroxides have been shown to inhibit the production of MPOderived oxidants [33–35]; however, these species are rapidly reduced in plasma and in experimental animals, to less or inactive hydroxylamines, by ascorbic acid, glutathione, and other reductants, thus limiting their effective lifetime [24,52,56–58]. As nitroxides can modulate a wide range of metabolic processes and have therapeutic potential [20,24,32], a number of studies have examined potential structural modifications that afford greater biological stability. A number of tetraethyl-substituted and related nitroxides have been reported to be less susceptible to reduction both in vitro and in vivo [41–44,52]. However, it is unclear whether such substitution perturbs the beneficial

functions of these compounds. One member of this family has recently been reported to protect against gastric damage induced by the nonsteroidal anti-inflammatory drug indomethacin [57]. This study therefore investigated whether tetraethyl substitution modulated the ability of the tetramethyl-substituted nitroxides to inhibit MPO in both isolated enzyme systems and human neutrophils. Their stability in the presence of both human plasma and unstimulated and stimulated neutrophils was also investigated by EPR spectroscopy. All of the piperidine nitroxides studied (4-hydroxy-, 4-amino-, and 4-oxo-TEMPO and their corresponding TEEPO derivatives) inhibited the production of chlorinating oxidants by isolated MPO and stimulated human neutrophils in a dose-dependent manner. Tetraethyl (and 2,2-diethyl-6,6-dimethyl) substitution did not have an impact on the ability of these nitroxides to inhibit MPO- and neutrophil-mediated oxidant formation, and in some cases inhibition was greater than that observed with the corresponding TEMPO species. Thus the calculated IC50 values for 4-hydroxy-TEEPO in the neutrophil system, and both 4-oxo-TEEPO

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and the 4-oxo-2,2-diethyl-6,6-dimethyl-substituted species for the isolated MPO system, were significantly lower (i.e., they were better inhibitors) than for the corresponding TEMPO species. The minimum concentrations required for significant inhibition of isolated MPO were lower for 4-hydroxy-TEEPO and 4-amino-TEEPO than for the corresponding tetramethyl species. Inhibition of MPO by nitroxides has been shown to be dependent on both the structure and the charge of the potential inhibitor, probably as a result of altered accessibility to the active site [34]. Access to the heme center is hindered by its location at the bottom of a deep crevice, and therefore only small substrates can reach the heme group [2,59]. Aromatic substrates have also been proposed to bind at the hydrophobic distal heme cavity [59]. Positively charged nitroxides have been reported to be more effective inhibitors than neutral and negatively charged nitroxides [34], with this attributed to the negatively charged Glu residue present near the heme group, which results in electrostatic interactions and hydrogen-bond formation with positively charged species and enhanced binding. Bulky nitroxides, such as isoindolin-2-yloxyl radicals, are less effective than 5- or 6-membered ring nitroxides probably because of steric interactions, and PEG-TEMPO has also been reported to be a poor MPO inhibitor because of the bulky polymer substituent [34]. Thus large groups close to the nitroxide hinder access and binding to the active site of MPO. The results obtained in the current study for the dispiro derivative of 4-oxo-TEMPO are consistent with these data [34], with this derivative proving to be a poor MPO inhibitor. In contrast substitution of the tetramethyl substituents ortho to the nitroxide function on the piperidine ring with tetraethyl groups does not appear to modulate activity, possibly as a result of their conformational flexibility. However, the presence of dispiro ring structures ortho to the nitroxide radical appear to prohibit this compound from interacting with the enzyme active site either as a result of the reduced flexibility of this structure or as a result of electronic interactions of the ether oxygen atoms with electron-rich sites on the protein. The inability of the parent amine, 2,2,6,6-tetraethylpiperidine, to inhibit MPO confirms a previous report that the nitroxide function is required for effective MPO inhibition [34]. The IC50 values determined here for MPO inhibition by 4-amino-TEMPO for the isolated enzyme system (1.070.02 mM) are in line with previously published data (1.270.1 mM) [34], whereas the values for the neutrophil system (2.2 70.6 mM) are somewhat lower (6.3 7 0.1 mM); this may reflect donor variation. For each compound, higher IC50 values were determined for the neutrophil system than for isolated MPO. This may arise from enhanced recycling of MPO intermediates (e.g., compound II) by neutrophil-derived reducing agents, including Od2
, that are not present in the isolated enzyme system [60]. This is in accord with a previous observation that SOD decreases the observed IC50 value for stimulated neutrophils [34]. Previous studies have provided considerable data to support a SOD-mimetic activity of nitroxides, particularly in isolated systems (e.g., [19,25,32]). Evidence has been presented for two mechanisms of reaction of Od2
with nitroxides. The first of these involves direct reduction to the hydroxylamine, and the second involves oxidation to the oxoammonium cation followed by subsequent reduction (i.e., processes analogous the oxidation–reduction processes that occur at the copper center in Cu/Zn SOD [61]). Considerable data support the latter process as the relevant mechanism [62]. Rate constants for reaction of Od2
with the nitroxide function of tetramethyl-substituted species have been reported as being structure dependent and in the range 104–108 M
1 s
1 [19,55]. On the basis of these values, and those for reaction of Od2
with SOD (k  109 M
1 s
1), it has been concluded that reaction of Od2
with nitroxides may be noncompetitive at typical cellular concentrations

103

of SOD [19,55]. It should, however, be noted that this conclusion does not take into consideration the potential accumulation of nitroxides in particular cellular compartments/locations. As the decay rates determined in this study for the tetraethyl-substituted nitroxides in the presence of both plasma and neutrophils are slower than for the tetramethyl species, we suggest that k for direct reaction of Od2
with the tetraethyl-substituted nitroxides is likely to be r104 M
1 s
1. Reaction with Od2
may therefore not be a kinetically viable removal pathway for these nitroxides in the presence of activated neutrophils where SOD is present. This proposed lower rate of SOD-mimetic activity may decrease other alternative beneficial effects of such nitroxides, and further examination of this point would seem to be warranted, but this is outside the scope of this study. Unlike the isolated MPO system, both the tetramethyl- and the tetraethyl-substituted nitroxides examined here were relatively poor inhibitors of isolated EPO, with much higher nitroxide concentrations required to produce significant inhibition of oxidant formation. This may be due to a number of factors, including differences in the structure of the protein compared to MPO and the significant variations in the kinetics of reaction and affinity of the heme center for the additional substrates (Br
and SCN
) used by this enzyme [59,63]. In light of the poor inhibition of the isolated enzyme by these nitroxides, experiments were not carried out with isolated eosinophils. Although the MPO inhibition results presented here are consistent with the study by Rees et al. [34], a recent study by Queiroz et al. [21] reported that 4-hydroxy-TEMPO reduces inflammation in a rat model by inhibiting the migration of neutrophils to sites of inflammation as opposed to inhibiting the production of MPOderived oxidants. However, these data may not be inconsistent as it has been reported that exposure of cells to HOCl results in an upregulation of intercellular adhesion molecules (ICAMs) and the formation of products that are signals for neutrophil migration [64,65]. It is therefore possible that nitroxide-mediated inhibition of MPO-derived oxidant formation may result in a decreased formation of HOCl-modified products and a downregulation of ICAM expression. The subsequent reduction in neutrophil migration (and MPO levels) would result in a dampening of the inflammatory cascade. All the TEEPO derivatives studied were more stable under the conditions employed than the corresponding TEMPO species in fresh human plasma, with the rates of decay being between 89 and 99% lower. A similar decrease in the rate of nitroxide loss was detected with stimulated human neutrophils, in which multiple competing or alternative removal pathways may play a role. The greater rates of nitroxide loss detected with the stimulated neutrophils compared to the unstimulated cells (cf. Table 3) are consistent with the major pathway for nitroxide loss being associated with neutrophil stimulation (and hence potential reaction with species arising from cell activation) rather than “resting” metabolic processes (e.g., cellular uptake and reduction by intracellular low-molecular-mass reductants or enzymes). Although the contribution of individual pathways has not been explored in detail, it seems that changes in redox properties of the nitroxide and/or steric shielding of the nitroxide against reducing agents may play a significant role [41–44,52]. Experimental redox potentials for the nitroxide/hydroxylamine couple have been reported (relative to a Ag/AgCl electrode) for 4-hydroxy-TEMPO (
0.158 V), 4-hydroxy-TEEPO (
0.240 V), 4-oxo-TEMPO (
0.095 V), and 4-oxoTEEPO (
0.245 V) [42]. Experimental data do not seem to have been reported for the corresponding 4-amino species, though some theoretical calculations have been reported [66,67]; these data are, however, difficult to compare to the current data as different solvents have been used. These limited data suggest that the tetraethyl-substituted species are more stable to reduction to the hydroxylamine than the

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tetramethyl-substituted species, which is in accord with the experimental data reported here and previous conclusions that the resistance of these types of compounds to ascorbic acid-mediated reduction is due to a positive value of the Gibbs energy (ΔG) for the tetraethyl species compared to a negative value for the tetramethylsubstituted nitroxides [42,44]. These data are, however, limited to these two pairs of compounds, so definitive conclusions as to the role of steric interactions versus differences in redox potential are premature; further work with a large range of compounds with known redox properties would clearly be beneficial in this respect. The prolonged lifetime afforded by the tetraethyl groups on the piperidine-1-oxyl structure, and particularly that for the 4-oxo species, may be of considerable utility for in vivo studies, especially when coupled with the observation of equal (or slightly enhanced) activity as inhibitors of chlorinating oxidant formation by MPO, as this would potentially allow lower levels of the nitroxide to be administered compared to the tetramethyl species to obtain a similar effect. This requires evaluation in future studies. Together, these data suggest that tetraethyl substitution may be an effective strategy for developing more effective nitroxide compounds that reduce MPO-derived oxidant damage in biological systems.

Acknowledgments We thank the Australian Research Council (through the ARC Centres of Excellence, CE0561607; to M.J.D. and S.E.B. and Discovery Programs, DP0988311; to M.J.D.) and the Japan Society for the Promotion of Science (KAKENHI Program, Nos. 24390011 and 24659020 to K.Y.) for financial support.

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