Lipophilicity is a critical parameter that dominates the efficacy of metalloporphyrins in blocking the development of morphine antinociceptive tolerance through peroxynitrite-mediated pathways

Severe pain syndromes reduce the quality of life of patients with inflammatory and neoplastic diseases, partly because reduced analgesic effectiveness with chronic opiate therapy (i.e., tolerance) leads to escalating doses and distressing side effects. Peroxynitrite-mediated nitroxidative stress in the dorsal horn of the spinal cord plays a critical role in the induction and development of antinociceptive tolerance to morphine. This provides a valid pharmacological basis for developing peroxynitrite scavengers as potent adjuncts to opiates in the management of pain. The cationic Mn(III) ortho-N-alkylpyridylporphyrins MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+) are among the most potent peroxynitrite scavengers, with nearly identical scavenging rate constants (approximately 10(7) M(-1) s(-1)). Yet, MnTnHex-2-PyP(5+) is significantly more lipophilic and more bioavailable and, in turn, was 30-fold more effective in blocking the development of morphine antinociceptive tolerance than MnTE-2-PyP(5+) using the hot-plate test in a well-characterized murine model. The hydrophilic MnTE-2-PyP(5+) and the lipophilic MnTnHex-2-PyP(5+) were 10- and 300-fold, respectively, more effective in inhibiting morphine tolerance than the hydrophilic Fe(III) porphyrin FeTM-4-PyP(5+). Both Mn porphyrins decreased levels of TNF-alpha, IL-1 beta, and IL-6 to normal values. Neither of them affected acute morphine antinociceptive effects nor caused motor function impairment. Also neither was able to reverse already established morphine tolerance. We have recently shown that the anionic porphyrin Mn(III) tetrakis(4-carboxylatophenyl)porphyrin is selective in removing ONOO(-) over O(2)(-), but at approximately 2 orders of magnitude lower efficacy than MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), which in turn parallels up to 100-fold lower ability to reverse morphine tolerance. These data (1) support the role of peroxynitrite rather than superoxide as a major mechanism in blocking the development of morphine tolerance and (2) show that lipophilicity is a critical parameter in enhancing the potency of such novel peroxynitrite scavengers.     

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.     

Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP(5+) accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.     

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.     

Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection

Development of radiation therapy (RT)-induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP5+ is a catalytic Mn porphyrin mimic of SOD, already shown to protect lungs from RT-induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP5+ with a newly introduced analogue MnTnHex-2-PyP5+, which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured using breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP5+ (3 and 6 mg/kg) or MnTnHex-2-PyP5+ (0.3, 0.6, or 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post-RT, only MnTE-2-PyP5+ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1alpha and TGF-beta, and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP5+ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-beta and CA-IX expression. Significant loss of body weight was observed in animals receiving MnTnHex-2-PyP5+ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP5+ has the ability to dissolve lipid membranes, causing local irritation/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show an ability to ameliorate lung damage as measured using breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP5+ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury.     

Nitrosylation of manganese(II) tetrakis(N-ethylpyridinium-2-yl)porphyrin: a simple and sensitive spectrophotometric assay for nitric oxide.

Reaction between NO(*) and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) was investigated at 25 degrees C. At high excess of NO(*) (1.5 mM) the reaction with the oxidized, air-stable form Mn(III)TE-2-PyP(5+) (5 microM), proceeds very slowly (t(1/2) congruent with 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, Mn(II)TE-2-PyP(4+), which reacts with NO(*) stoichiometrically and in the time of mixing (k congruent with 1 x 10(6) M(-1) s(-1)). The high rate of formation and the stability of the product, Mn(II)TE-2-PyP(NO)(4+) (?Mn(NO)?(6)), make the reaction outcompete the reaction of NO(*) with O(2). Our in vitro measurements show a linear absorbance response upon addition of NO to a PBS, pH 7.4, solution containing an excess of ascorbate over Mn(III)TE-2-PyP(5+). Thus, the observed interactions can be the basis of a convenient and sensitive spectrophotometric assay for NO(*). Also, it may have important implications for the in vivo behavior of Mn(III)TE-2-PyP(5+) which is currently exploited as a possible therapeutic agent for various oxygen-radical related disorders.     

Manganese-porphyrin reactions with lipids and lipoproteins

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.     

Peroxynitrite flux-mediated LDL oxidation is inhibited by manganese porphyrins in the presence of uric acid

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.     

SOD-like activity of Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.     

Electrostatic contribution in the catalysis of O2*- dismutation by superoxide dismutase mimics. MnIIITE-2-PyP5+ versus MnIIIBr8T-2-PyP+

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) is a potent superoxide dismutase (SOD) mimic in vitro and was beneficial in rodent models of oxidative stress pathologies. Its high activity has been ascribed to both the favorable redox potential of its metal center and to the electrostatic facilitation assured by the four positive charges encircling the metal center. Its comparison with the non-alkylated, singly charged analogue Mn(III) beta-octabromo meso-tetrakis(2-pyridyl)porphyrin (Mn(III)Br(8)T-2-PyP(+)) enabled us to evaluate the electrostatic contribution to the catalysis of O(2)() dismutation. Both compounds exhibit nearly identical metal-centered redox potential for Mn(III)/Mn(II) redox couple: +228 mV for Mn(III)TE-2-PyP(5+) and +219 mV versus NHE for Mn(III)Br(8)T-2-PyP(+). The eight electron-withdrawing beta pyrrolic bromines contribute equally to the redox properties of the parent Mn(III)T-2-PyP(+) as do four quaternized cationic meso ortho pyridyl nitrogens. However, the SOD-like activity of the highly charged Mn(III)TE-2-PyP(5+) is >100-fold higher (log k(cat) = 7.76) than that of the singly charged Mn(III)Br(8)T-2-PyP(+) (log k(cat) = 5.63). The kinetic salt effect showed that the catalytic rate constants of the Mn(III)TE-2-PyP(5+) and of its methyl analogue, Mn(III)TM-2-PyP(5+), are exactly 5-fold more sensitive to ionic strength than is the k(cat) of Mn(III)Br(8)T-2-PyP(+), which parallels the charge ratio of these compounds. Interestingly, only a small effect of ionic strength on the rate constant was found in the case of penta-charged para (Mn(III)TM-4-PyP(5+)) and meta isomers (Mn(III)TM-3-PyP(5+)), indicating that the placement of the positive charges in the close proximity of the metal center (ortho position) is essential for the electrostatic facilitation of O(2)() dismutation.     

Impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: protection of SOD-deficient Escherichia coli via alternative mechanism where Mn porphyrin acts as a Mn carrier

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](3-) or MnTBAP(3-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](3-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-), respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-) being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the SOD activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](3-), E(1/2)=+213 mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](3-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](3-), [Mn(III)TSPP](3-), and [Mn(III)Br(8)TCPP](3-) did not rescue SOD-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](3-) to substitute for the SOD enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](3-) is comparable to or better than that of the potent SOD mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](3-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](3-) is not an SOD mimic per se but carries Mn into the E. coli cell.     

Manganese porphyrin, MnTE-2-PyP5+, Acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP(5+), augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H(2)O(2)](ss)) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP(5+) is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H(2)O(2)](ss) levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H(2)O(2) or depletion of glutathione by BSO prevents MnTE-2-PyP(5+) from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP(5+) glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF- κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP(5+). Taken together, these findings indicate that: 1) H(2)O(2) is important for MnTE-2-PyP(5+) activity; 2) Mn-TE-2-PyP(5+) cycles with GSH; and 3) MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP(5+) may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.     

MnTnBuOE-2-PyP treatment protects from radioactive iodine (I-131) treatment-related side effects in thyroid cancer

Treatment of differentiated thyroid cancer often involves administration of radioactive iodine (I-131) for remnant ablation or adjuvant therapy. However, there is morbidity associated with I-131 therapy, which can result in both acute and chronic complications. Currently, there are no approved radioprotectors that can be used in conjunction with I-131 to reduce complications in thyroid cancer therapy. It is well known that the damaging effects of ionizing radiation are mediated, in part, by the formation of reactive oxygen species (ROS). A potent scavenger of ROS, Mn(III)meso-tetrakis(N–n-butoxyethylpyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP), has radioprotective and anti-tumor effects in various cancer models including head and neck, prostate, and brain tumors exposed to external beam radiation therapy. Female C57BL/6 mice were administered I-131 orally at doses of 0.0085–0.01 mCi/g (3.145 × 105 to 3.7 × 105 Bq) of body weight with or without MnTnBuOE-2-PyP. We measured acute external inflammation, blood cell counts, and collected thyroid tissue and salivary glands for histological examination. We found oral administration of I-131 caused an acute decrease in platelets and white blood cells, caused facial swelling, and loss of thyroid and salivary tissues. However, when MnTnBuOE-2-PyP was given during and after I-131 administration, blood cell counts remained in the normal range, less facial inflammation was observed, and the salivary glands were protected from radiation-induced killing. These data indicate that MnTnBuOE-2-PyP may be a potent radioprotector of salivary glands in thyroid cancer patients receiving I-131 therapy.     

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics,MnTE-2-PyP5+ and MnTnHex-2-PyP5+

The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP⁵⁺ (AEOL10113, FBC-007) and MnTnHex-2-PyP⁵⁺ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O₂^•− and ONOO⁻ and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP⁵⁺ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP⁵⁺, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24 h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10 mg/kg MnTE-2-PyP⁵⁺ and 0.5 or 2 mg/kg MnTnHex-2-PyP⁵⁺. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (C_max, C_24 h, t_max, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUC_oral/AUC_iv) is 23% for MnTE-2-PyP⁵⁺ and 21% for MnTnHex-2-PyP⁵⁺. Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP⁵⁺ than for MnTE-2-PyP⁵⁺ (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP⁵⁺.     

Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP(5+), in plasma and major organs of B6C3F1 mice

Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite 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-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart 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 continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosing regimens in future preclinical studies utilizing this and similar compounds.     

Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnIIITE-2-PyP5+ (AEOL-10113) has proven effective in treating oxidative stress-induced conditions including cancer, radiation damage, diabetes, and central nervous system trauma. The ortho cationic pyridyl nitrogens of MnTE-2-PyP5+ are essential for its high antioxidant potency. The exceptional ability of MnIIITE-2-PyP5+ to dismute O2.- parallels its ability to reduce ONOO- and CO3-. Decreasing levels of these species are considered its predominant mode of action, which may also involve redox regulation of signaling pathways. Recently, Ferrer-Sueta at al. (Free Radic. Biol. Med. 41:503-512; 2006) showed, with submitochondrial particles, that>or=3 microM MnIIITE-2-PyP5+ was able to protect components of the mitochondrial electron transport chain from peroxynitrite-mediated damage. Our study complements their data in showing, for the first time that micromolar mitochondrial concentrations of MnIIITE-2-PyP5+ are obtainable in vivo. For this study we have developed a new and sensitive method for MnIIITE-2-PyP5+ determination in tissues. The method is based on the exchange of porphyrin Mn2+ with Zn2+, followed by the HPLC/fluorescence detection of ZnIITE-2-PyP4+. At 4 and 7 h after a single 10 mg/kg intraperitoneal administration of MnIIITE-2-PyP5+, the mice (8 in total) were anesthetized and perfused with saline. Mitochondria were then isolated by the method of Mela and Seitz (Methods Enzymol.55:39-46; 1979). We found MnIIITE-2-PyP5+ localized in heart mitochondria to 2.95 ng/mg protein. Given the average value of mitochondrial volume of 0.6 microL/mg protein, the calculated MnIIITE-2-PyP5+ concentration is 5.1 microM, which is sufficient to protect mitochondria from oxidative damage. This study establishes, for the first time, that MnIIITE-2-PyP5+, a highly charged metalloporphyrin, is capable of entering mitochondria in vivo at levels sufficient to exert there its antioxidant action; such a result encourages its development as a prospective therapeutic agent.     

Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site

Abstract

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP⁵⁺ accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.     

Effect of Molecular Characteristics on Cellular Uptake,Subcellular Localization,and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.