Free Radical Biology & Medicine 45 (2008) 943–949

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Free Radical Biology & Medicine

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Original Contribution

Pharmacokinetics of the potent redox-modulating manganese porphyrin,MnTE-2-PyP5+, in plasma and major organs of B6C3F1 mice

Ivan Spasojević a,⁎, Yumin Chen b, Teresa J. Noel b, Ping Fan a, Lichun Zhang a, Julio S. Rebouças c,Daret K. St. Clair b, Ines Batinić-Haberle c,⁎a Department of Medicine, Duke University Medical School, Durham, NC 27710, USAb Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USAc Department of Radiation Oncology, Duke University Medical School, Durham, NC 27710, USA

Abbreviations: SOD, superoxide dismutase; HIF-1, hκB, nuclear factor κB; AP-1, activator protein-1; TFMnTM-2(3,4)-PyP5+, Mn(III) meso-tetrakis{N-methylpy(2, 3, and 4 being the ortho, meta, and para isomer, res(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrinZn(II) meso-tetrakis(N-ethylpyridinium-2-yl)porphymeso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin; Ztetrakis(N-n-butylpyridinium-2-yl)porphyrin; meso, i5,10,15,20 positions on porphyrin core.⁎ Corresponding authors. I. Spasojević is to be conta

I. Batinić-Haberle, fax: +1 919 684 8885.E-mail addresses: spaso001@mc.duke.edu (I. Spasoje

(I. Batinić-Haberle).

0891-5849/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.freeradbiomed.2008.05.015

a b s t r a c t

a r t i c l e i n f o

Article history:

Mn(III) tetrakis(N-ethylpy Received 22 February 2008Revised 4 May 2008Accepted 19 May 2008Available online 28 May 2008

Keywords:MnTE-2-PyP5+

AEOL 10113SOD mimicPharmacokinetics in mouse plasmaPharmacokinetics in mouse organsPharmacokinetics in mouse tissuesFree radicals

ridinium-2-yl)porphyrin, MnTE-2-PyP5+, a potent catalytic superoxide andperoxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined.Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP5+

and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite highhydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels highenough (5.1 μM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the sameanalytical approach, which involves the reduction of MnTE-2-PyP5+ followed by the exchange of Mn2+ withZn2+ and HPLC/fluorescence detection of ZnTE-2-PyP4+, we measured levels of MnTE-2-PyP5+ in mouseplasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitonealinjection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile inplasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area underthe curve, cmax, tmax, and drug elimination half-time (t1/2). In terms of levels of MnTE-2-PyP5+ found, theorgans can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderatelevels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, andheart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed,with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continuedbeyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively chargedphospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t1/2=60 to135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosingregimens in future preclinical studies utilizing this and similar compounds.

© 2008 Elsevier Inc. All rights reserved.

For more than a decade we have been investigating metallopor-phyrin-based antioxidants, studying their in vitro efficacy in eliminat-ing reactive species and in vivo efficacy in oxidative stress-relatedmodels of diseases [1–8]. From our studies on structure–activity

ypoxia-inducible factor-1, NF-A, trifluoroacetic acid; MnP,ridinium-2(3,4)-yl}porphyrinpectively); MnTE-2-PyP5+, Mn(AEOL10113); ZnTE-2-PyP4+,rin; MnTnHex-2-PyP5+, MnnTnBu-2-PyP4+, Zn(III)meso-ndicates substitution in the

cted at fax: +1 919 684 9094.

vić), ibatinic@duke.edu

l rights reserved.

relationships between the redox potential of metalloporphyrins andtheir ability to dismute superoxide, O2

U−, MnTE-2-PyP5+1 emerged as astandard potent catalytic antioxidant now used in a wide range ofcellular and animal studies. The compound is highly effective atdismuting (disproportionating) O2

U− [1,2] (log kcat=7.76 compared tolog kcat=8.84–9.3 for SOD enzymes [9–14]). Moreover, its ability todismute O2

U− parallels its ability to reduce peroxynitrite, ONOO− (logkred=7.53) [15,16]. MnTE-2-PyP5+ is also very effective at scavengingcarbonate radical, CO3

U− [16]. The reduction of peroxynitrite or CO3U− is

catalytic in nature in vivo, as it is coupled to cellular reductants; theoverall reaction is more efficient with ascorbate and urate than withpotential targets such as thiols and amino acids [15–17]. The ability toeliminate O2

U−, ONOO−, and CO3U− exceeds that of other compounds

currently in preclinical and clinical studies, such as Mn salenderivatives [18,19], Mn cyclic polyamines [20], nitrones [21,22],nitroxides [23], MitoQ [24], and analogous compounds [25]. MnTE-

944 I. Spasojević et al. / Free Radical Biology & Medicine 45 (2008) 943–949

2-PyP5+ ameliorates oxidative stress-related diseases, presumablythrough elimination of reactive species, thus providing the protectionof most biologically relevant targets against primary oxidative stress.Moreover, the elimination of reactive species prevents activation ofthe transcription factors AP-1 [26,27], HIF-1α [28–31], and NF-κB[32,33], which in turn inhibits the expression of cytokines and/orenzymes that would otherwise perpetuate secondary oxidative stress.The catalytic power of MnTE-2-PyP5+ is enhanced by the presence ofelectron-withdrawing positively charged ortho alkylpyridyl groupsclose to the metal center. These proximal positive charges contributeto both thermodynamic (favorable redox potential) [1,6] and kinetic(electrostatic attraction of the negatively charged substrate) [6,34]facilitation of O2

U− dismutation and ONOO− and CO3U− reduction.

Several other metalloporphyrins [35–38] and corroles [39,71] havebeen developed that exploit the effects of cationic ortho quaternarynitrogens.

The beneficial effects of Mn alkylpyridylporphyrins have beenobserved in central nervous system injuries (stroke [40–42], spinalcord injury [43], ALS [44], Alzheimer disease [45]), radiation injury[46–49], cancer [26,29], diabetes [50,51], sickle cell disease [52],ischemia/reperfusion conditions [53–55], arthritis [56], and otherconditions. However, very little is known about the bioavailability, siteof action, and pharmacokinetics and toxicology of the porphyrin-based compounds. It has been assumed that excessive hydrophilicitywould limit the accumulation of MnTE-2-PyP5+ within the cell and itsorganelles, suggesting the extracellular localization of porphyrin as alikely site of action. However, the high efficacy observed in vivostrongly implicates not only cytosol, but also the mitochondrion[26,27] and the nucleus [32,33] as possible sites of action. To test thevalidity of such conclusions we have developed a sensitive HPLC/fluorescence method for the determination of the positively chargedMn(III) ortho N-alkylpyridylporphyrins in vivo and used it in a mousemitochondrial study [57]. Herein, utilizing a similar analyticalapproach, we undertook a pharmacokinetic study to detect levels ofporphyrin in mouse plasma and various organs over a period of 7 daysafter a single 10mg/kg intraperitoneal injection of MnTE-2-PyP5+. Thisis the first detailed pharmacokinetic study of such an important classof Mn porphyrin-based antioxidant therapeutics.

Experimental procedures

General

Sodium L-ascorbate, mannitol, albumin from bovine serum, andZnCl2 were from Sigma and Zn(CH3COO)2×2 H2O was from J. T. Baker.Acetonitrile and trifluoroacetic acid (TFA) were from Fisher Scientificand triethylamine was from Pierce. Methanol (anhydrous, absolute)was from Mallinckrodt. Glacial acetic acid was from EM Science.Phosphate-buffered saline (50 mM sodium phosphate, 0.9% NaCl, pH7.4) was from Gibco.

Porphyrins

MnTE-2-PyP5+ (λmax 454 nm, log ε=5.14) [1,2] and ZnTE-2-PyP4+

(λmax 425.5 nm, log ε=5.46) [57–59] were prepared as describedpreviously. ZnTnBu-2-PyP4+ was used as an internal standard and wasprepared and characterized as described [57].

UV/Vis spectroscopy

UV/Vis was done on a Shimadzu 2501 PC UV/Vis spectrophotometer.

Mice

The University of Kentucky Medical Center Research AnimalFacility has a continuously accredited program from AAALAC Interna-

tional. All experiments using animals were performed according tothe approved protocol for humane care and use of animals. TwoB6C3F1/J female mice were used for each time point. (This strain iscommonly used as a background strain for transgenic MnSOD miceused in studying oxidative stress injuries.) The mice weighed from 20to 25 g and the volumes of porphyrin saline solutions rangedaccordingly, from 200 to 250 μl. The mice were euthanized withhigh-dose pentobarbital. Before tissue was harvested, the animalswere perfused with saline to avoid artifacts related to the retention ofblood in the organs and tissue. The blood was drawn from the leftventricle of all mice perfused. The perfusion was by gravity flow ofsaline via a blunt needle placed into the aorta through the leftventricle, and the saline flow through the right atriumwas continueduntil the liver cleared out.

Protein levels

The protein levels were determined in plasma and tissuehomogenates by colorimetric Bradford assay as specified by themanufacturer (Bio-Rad, Richmond, CA, USA). Protein levels were usedto normalize tissue levels in homogenates.

MnTE-2-PyP5+ plasma/tissue extraction

Organs were cryo-pulverized in a Bessman tissue pulverizer(BioSpec Products, Bartlesville, OK, USA) under liquid nitrogen andthen homogenized in a rotary homogenizer (PTFE pestle and glasstube) with 2 volumes of deionized water. One hundred microliters ofeither plasma or tissue homogenate was transferred into a 2-mlpolypropylene screw-cap vial and 200 μl of deionizedwater and 300 μlof 1% acetic acid in methanol were added. The content was mixed in amultivortexer for 60 s and the homogenate was centrifuged 5 min at16,000 g. Four hundred microliters of the supernatant was pipettedinto a 5-ml polypropylene tube (10×50 mm) and the solvent wascompletely removed in a Savant Speed-Vac evaporator at 40°C within1 h. The dry residue was dissolved in 100 μl of deionized watercontaining 20 mM triethylamine, the pH of which was adjusted withdiluted TFA to pH 6.2, followed by 60 s vortexing and centrifugationfor 2 min at 2000 g. Eighty microliters of the supernatant wastransferred into a 2-ml polypropylene screw-cap tube, and 30 μl of 1.0M either Zn acetate or Zn chloride in water and 10 μl of freshlyprepared 0.11 M sodium ascorbate in water were added. The solutionwas left for 1 h at either 50 or 70°C. (If working at 70°C the more acidicsalt, Zn chloride, ought to be used owing to a lower degree ofhydrolysis compared to Zn acetate.) Twenty microliters of 2.7 M TFAand 20 μl of 800 nM ZnTnBu-2-PyP4+ were added, followed byvortexing for 60 s and centrifugation for 5 min at 16,000 g. Onehundred ten microliters of the supernatant was transferred to theHPLC autosampler polypropylene vial equipped with a silicone/PTFEseptum.

HPLC

HPLC equipment consisted of a Waters 2695 HPLC system (pump,autosampler, column oven) and Waters Model 2475 fluorescencedetector set on gain of 100, λexc 425 nm, and λabs 656 nm [60–62]. Thecolumnwas a YMC-Pack, ODS-AM, C18 column (3 μmparticle size, 120Å pore size, 150×4.6 mm) used at 45°C. Solvent A was 95% aqueous(deionized water, 20 mM triethylamine, pH 2.7 adjusted withconcentrated TFA) and 5% acetonitrile. Solvent B was acetonitrile.The elution gradient at 1.5 ml/min was 0–15–20 min, 100% A–80% A–100% A, followed by 5 min column conditioning. Autosamplertemperature was 4°C and injection volume was 80 μl.

The ortho isomers of MnTE-2-PyP5+, ZnTE-2-PyP4+, and theirparent metal-free ligand, H2TE-2-PyP4+, have atropoisomers, whichwe previously characterized by HPLC/UV/Vis/NMR and X-ray methods

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[59]. We observed that the abundance ratio of the four isomers ofZnTE-2-PyP4+ and their retention times on HPLC were the same inmouse heart mitochondrial homogenates [57], in processed mouseorgan homogenates, and in aqueous solution [58]. This indicated thatthe interactions of any of the four atropoisomers with cellularcomponents [57,58] are not strong enough to overcome the rotationalbarrier of the alkylpyridyl “arms,” which would cause a change in theabundance ratio of atropoisomers. The experiments also implied thatthe Mn porphyrin does not undergo oxidative degradation or anyother modification in vivo. Using any of the atropoisomers forcalculation of the levels of MnTE-2-PyP5+ in mitochondria gave thesame result. ZnTnBu-2-PyP4+ was used as an internal standard, and itsatropoisomers were also present in the chromatogram at the expectedabundance ratio [57,58]. The assignment of the individual atropoi-somers, αααα, αααβ, ααββ, and αβαβ, of both Zn porphyrins wasdone based on our previous HPLC/1H NMR/X-ray study [57,58]. In thisstudy, the most abundant atropoisomer, αααβ, was used for thequantification of both ZnTE-2-PyP5+ and ZnTnBu-2-PyP4+ (internalstandard).

Calibration curves

A set of serially diluted standard samples of MnTE-2-PyP5+ wasprepared for calibration. The range varied from 1.5 ng to 100 μg permilliliter of drug-free plasma or per gram of drug-free wet tissue, andwas dependent upon the type of organ studied. MnTE-2-PyP5+ wasadded into the tissue homogenate or plasma, followed by a 15-minincubation at room temperature. The rest of the assay procedure wasidentical to the procedure performedwith organs and plasma of drug-treated animals. Response was calculated as the ratio betweenstandard peak area/internal standard peak area.

Recovery of MnTE-2-PyP5+, i.e., the overall efficacy of extractionfrom plasma and organs plus Mn-to-Zn exchange, was determined intriplicate in the following way. A known amount of MnTE-2-PyP5+ wasadded to plasma or organ homogenate, and the procedure ofextraction/metal exchange was followed. The response from thisexperiment was compared to the response of the sample in which thecorresponding amount of ZnTE-2-PyP4+ (equal to 100% yield of MnTE-2-PyP5+) was diluted into the mobile phase. The obtained recoveryvalues are 83% in plasma, 89% in liver, 88% in kidney, 83% in spleen,70% in lung, 85% in heart, and 97% in brain.

Results and discussion

MnTE-2-PyP5+ is very effective in eliminating reactive species (RS),particularly O2

U− and ONOO−, with rate constants that are among thehighest compared to other synthetic antioxidants [1–8,15–17]. Thisallows the compound not only to decrease the primary insult of RS tobiological molecules, but also to inhibit activation of transcriptionfactors, which in turn leads to suppression of expression of thosecytokines and enzymes that perpetuate secondary oxidative stress[26–33]. Thus MnTE-2-PyP5+ has the potential to be an efficacious

Scheme 1. Representation of the method used to determine levels of MnTE-2-PyP5+ in plasmthe Zn salt was added along with sodium ascorbate into the supernatant to exchangeMn(II) wat 50 or 70°C was needed to yield a complete Zn for Mn exchange for the HPLC/fluorescenc

therapeutic in all diseases that have oxidative stress as a commonfactor [40–56]. Due to its high hydrophilicity and positive charge,crossing the blood–brain barrier and the mitochondrial envelope hasalways been a concern. Thus, we were surprised to find 5.1 μM (2.95ng/mg protein) MnTE-2-PyP5+ in mouse heart mitochondria 4 (or 7) hafter a single 10 mg/kg intraperitoneal injection; as a note, 10 mg/kg isthe most commonly used (as single or multiple) therapeutic dose ofthis porphyrin [57]. A previous study by Ferrer-Sueta et al. [17] onsubmitochondrial particles indicate that at ≥3 μM MnTE-2-PyP5+

nearly fully eliminates ONOO−-mediated damage to the componentsof the mitochondrial respiration machinery. The dose of 10 mg/kg waspicked up based upon the rodent studies on diabetes [50], cancer[28,29,63], and radioprotection [48,64] that have reported beneficialeffects (delayed onset of diabetes, tumor growth delay, radioprotec-tion) when MnTE-2-PyP5+ was administered at doses ranging from 6to 15 mg/kg. The TD50 was found to be 91.5 mg/kg when MnTE-2-PyP5+

was administered subcutaneously (M. Panni, unpublished data).We have reported that Mn porphyrins can mimic cytochrome

P450, yet to a lower extent than Fe analogous porphyrins [65]. It wasalso shown that Mn porphyrin can redox cycle with cytochrome P450reductase, which action was claimed to have possible beneficial and/or adverse effects [66]. Thus far our studies did not indicate anymetabolism of MnTE-2-PyP5+ or similar cationic porphyrins byenzymatic systems in vivo.

Herein, we studied the pharmacokinetics (PK) of MnTE-2-PyP5+ inmouse plasma and several important organs. Animal perfusion toremove blood from organs andmeasurement of porphyrin levels wereperformed as in themitochondrial study [57] but with some necessarymodifications, primarily in sample preparation procedures. The HPLC/fluorescence method is based on the reduction of MnTE-2-PyP5+ byascorbic acid, followed by in situ Mn2+ to Zn2+ exchange andfluorescence detection of ZnTE-2-PyP4+. Assay conditions are givenin Scheme 1. ZnTnBu-2-PyP4+ was used as an internal standard.Calibration curves, made with drug-free plasma and correspondingorgan homogenates, were linear in the ranges needed for quantifica-tion. Lower limits of quantification (LLQ) in ng/ml (plasma) or ng/gwet tissue (organs), with corresponding sample size in parentheses,were as follows: plasma 4.64 (100 μl), liver 11.9 (33 mg), spleen 72.5(33 mg), kidney 215 (33 mg), lung 35.8 (33 mg), heart 35.8 (33 mg),and brain 3.6 (100 mg). Accuracy (relative error) acceptance criteria ateach nominal concentration were b15% and b20% at LLQ [67]. Theconcentration in day 7 plasma samples was barely detectable andoutside the standard curve range (below LLQ). Nonetheless, during thepreparation of this article, a new LC/MS/MSmethod for MnTE-2-PyP5+

in plasmawas developed in our lab with LLQ of 0.5 ng/ml using a 250-μl sample. Thus, the pooled day 7 plasma sample (two animals) wasmeasured by LC/MS/MS; themethodwill be described in a subsequentpublication.

Pharmacokinetic profiles are shown in Figs. 1A–1G alongwith corresponding calibration curves. The levels of porphyrin aregiven in μg/g wet tissue or μg/ml plasma. For each time point thetwo concentrations measured, corresponding to two animals, are

a and organs. After the proteins were precipitated from plasma and tissue homogenates,ith Zn(II). Whereas the reduction of theMnP occurred instantaneously,1 h of incubatione measurement of ZnTE-2-PyP4+.

Fig. 1. Pharmacokinetics of MnTE-2-PyP5+ in (A) plasma, (B) liver, (C) spleen, (D) kidney, (E) lung, (F) heart, and (G) brain. Levels of porphyrin are given in micrograms per milliliter ofplasma or per gram of wet tissue. Drug was given at a single intraperitoneal dose of 10 mg/kg to female B6C3F1 mice. Animals were anesthetized and perfused and organs wereexcised. Twomice were used per data point. Calibration curves were done in plasma and related organs. The known amounts of MnTE-2-PyP5+ were added into the plasma and organhomogenates; the rest of the procedure was identical to that used for drug-treated mice.

946 I. Spasojević et al. / Free Radical Biology & Medicine 45 (2008) 943–949

Fig. 1 (continued).

947I. Spasojević et al. / Free Radical Biology & Medicine 45 (2008) 943–949

presented as open circles, whereas the average of these values isgiven by the solid bold line. Average concentrations at each timepoint were used in the pharmacokinetic calculations. Semilog plotswere chosen to illustrate the level of complexity in the PK behavior.Namely, a single-exponential rise/decay in concentration wouldindicate drug absorption/distribution from/into one or more“compartments” of very similar blood perfusion and/or drugaffinity properties. Such a single-exponential process would appearlinear in a semilog plot. Thus, as can be seen in Fig. 1A (far left),plasma pharmacokinetics over 7 days seems to be very complex—several linear regions (compartments) can be recognized. This is inagreement with the pharmacokinetics seen in different organs(Figs. 1B–1G). For this reason, and because of a limited number oftime points, no attempt was made to fit a multicompartmentalmodel into the data. Instead, the noncompartmental approach(WinNonlin, Pharsight, Inc.) was used: numerical integration of thePK profile was performed to calculate the area under the curve, and

Table 1Pharmacokinetic parameters of MnTE-2-PyP5+ in plasma, liver, kidney, spleen, lung,heart, and brain that indicate exposure of the organ to the drug

Organ tmax

(h)cmax

(μg/g)t1/2(h)

AUClast(μg d h/ml (g))

AUCinf(μg d h/ml (g))

Plasma 0.25 17.74 76.9a 18.7 18.8Liver 8 6.40 60.1 459.1 533.7Spleen 0.25 5.92 107.0 174.5 265.2Kidney 0.5 27.17 82.0 543.4 695.8Lung 0.75 0.12 55.5 24.4 26.5Heart 0.5 0.76 135.0 9.2 14.8Brain 0.75 0.11 (0.04)b —c 3.6 —c

tmax, time point at which cmax was achieved; cmax, maximal observed concentration;t1/2, half-life of MnTE-2-PyP5+ elimination obtained from terminal slope between days2 and 7; AUClast, area under the curve up to the last measurement, i.e., 7 days; AUCinf,projected total AUC at infinite time point. Clearance (=dose/AUC) of the drug frombody was calculated from plasma data to be 0.53 mg/kg/h.

a Half-time of elimination phase. Half-time of “drug in plasma” is t1/2(plasma) ~ 1 h.b Value at 7-day time point (cmax[last]).c No terminal phase achieved.

the terminal slope was used to calculate drug elimination half-time(t1/2) for each organ and plasma; cmax is the highest concentrationfound along the PK profile, and tmax is the corresponding time(Table 1). In terms of levels of MnTE-2-PyP5+ found, the organs can beclassified into three distinct groups: (1) high levels (kidney, liver, andspleen), (2) moderate levels (lung and heart), and (3) low levels(brain). The plots clearly reveal differences in drug penetration(diffusion or active transport) and drug tissue affinity (binding orretention) among organs. During the first 15 min, the drug is rapidlyabsorbed from the intraperitoneal cavity into the plasma. The levels inkidney, spleen, lung, and heart also increase rapidly, presumably dueto a passive diffusion—the drug levels in kidney reach especially highvalues, obviously owing to both high penetration and a high affinity.Consequently the concentration of the drug in plasma drops rapidly(t1/2(plasma) ~ 1 h). At the same time, in liver, the drug shows a slowpenetration (tmax=8 h) but reaches very high levels because of highaffinity. The drug also exhibits a very slow penetration into brain.Owing to different affinities (weak versus strong) for the differenttissues, an intensive distribution/redistribution of the drug in thebody goes on for 2 days, after which a slow drug elimination from allorgans (except brain) takes place at a similar rate. The MnTE-2-PyP5+

levels in brain, however, start increasing after 2 days and continue thetrend past 7 days, meaning that the drug binds very strongly to thebrain tissue; it is noteworthy that brain continues to accumulateMnTE-2-PyP5+ despite very lowand ever decreasing concentrations ofthe drug in circulating blood (plasma) (Fig. 1G). This accumulation ofthe drug in the brain at late times is maintained by the highconcentration of MnTE-2-PyP5+ in other tissues (liver, kidney, andspleen) from where it is supplied over days and weeks viaredistribution.

We have already found that positively charged isomeric Mn(III)methylpyridylporphyrins (MnTM-2(3,4)-PyP5+) associate with thenucleic acids of Escherichia coli [1]. Its cationic nature further drivesthe accumulation of MnTE-2-PyP5+ in mitochondria [57]. Finally, astudy in collaboration with Tse and Piganelli (I. Spasojević, I. Batinić-Haberle, H. Tse, J. Piganelli, unpublished) showed that MnTE-2-PyP5+

accumulates threefold more in the nucleus than in the cytosol ofmacrophages, presumably driven by the negatively charged phosphate

948 I. Spasojević et al. / Free Radical Biology & Medicine 45 (2008) 943–949

groups of the nucleic acids. Similarly, the accumulation of MnTE-2-PyP5+ in brain is probably driven by the abundance of negativelycharged phospholipids in this organ and may explain the beneficialeffects of MnTE-2-PyP5+ in ALS [44], stroke models [41,42], and painmanagement [68]. Further, it is well documented that overexpressionof MnSOD can suppress cancer phenotypes and the metastaticpotential of cancer cells, all suggesting a high therapeutic potentialof MnTE-2-PyP5+ for brain radioprotection and brain tumor treatment.

The ability of MnTE-2-PyP5+ to cross the blood–brain barrier hasbeen indicated in an independent study inwhich nearly full reversal ofmorphine tolerance was achieved by MnTE-2-PyP5+ therapy. More-over, identical effects were observed whether MnTE-2-PyP5+ wasadministered intraperitoneally or intrathecally [63,68]. Further, whenthe para methyl analogue MnTM-4-PyP5+ was used in a rabbit modelof cerebral palsy, its presence in fetal brain was observed by MRI (T.Sidhartha, unpublished). More thorough study is in progress to eva-luate whether brain levels achieved by the intraperitoneal or sub-cutaneous routes are close to the therapeutic levels in different animalmodels of central nervous system injuries.

The levels of MnTE-2-PyP5+ of ≥3 μM in submitochondrial particlesshown to be protective by Ferrer-Sueta et al. [17] coincide with levelsat which the aerobic growth of SOD-deficient E. coli achieves ≥60% ofthe growth of the wild type [1,69]. Based on data from Vinnakota andBassingthwaighte showing that the density of myocardial tissue is1.053 g/ml [70], and assuming that it is valid for other tissues as well,the 1 μg/g wet tissue of MnTE-2-PyP5+ corresponds to the concentra-tion of ~ 1 μM. Consequently, after a single intraperitoneal injection,therapeutic doses of MnTE-2-PyP5+ might have been achieved in liver,kidney, and spleen. This is in agreement with data from Saba et al. [54]showing that kidney ischemia/reperfusion injury was significantlydiminished by a single intravenous injection of the hexyl analogue,MnTnHex-2-PyP5+. More studies are needed to determine themagnitude and the frequency of administrations needed to achievetherapeutic doses in other organs.

Conclusions

We report here the first detailed pharmacokinetic study on animportant class of catalytic Mn-porphyrin-based antioxidant ther-apeutics. The findings presented are relevant for the proper dosing ofMnTE-2-PyP5+ in future in vivo studies. The PK analysis shows a veryefficient absorption of the drug from the intraperitoneal space into thebloodstream (within 15 min) and a fast disappearance of the drugfrom plasma due to the distribution into the tissues (t1/2(plasma) ~ 1 h).Due to the strong binding to the tissues, however, the elimination ofthe drug from the body is very slow (t1/2(elim) = 55–135 h). Unlike inother tissues, accumulation of the drug in the brain was observedbetween days 2 and 7. After a single dose, near therapeutic levels wereachieved in liver, kidney, and spleen. The observed high accumulationand slow release from most tissues suggest no need for frequentdosing. An initial higher loading dose may be followed by smallperiodic “maintenance” doses. However, in attempts to deliver ahigher dose to organs like lung, heart, and brain, one must bear inmind that very high and possibly toxic levels may be reached in liver,kidney, and spleen.

Acknowledgments

Ivan Spasojević acknowledges NIH/NCI Duke ComprehensiveCancer Center Core grant (5-P30-CA14236-29). Ines Batinić-Haberleappreciates financial support from NIH-IR21-ESO/3682 and NIH U19AI67798-01/pilot project. Yumin Chen, Teresa Noel, and Daret St.Clair are thankful for the support of NIH Grants CA 49797, CA 73599,and CA 94853. We are thankful to Huaxin Sheng for his helpwith animal perfusion and Irwin Fridovich for critically reading themanuscript.

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