Zrinka Rajic

Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities

The most efficacious Mn(III) porphyrinic (MnPs) scavengers of reactive species have positive charges close to the Mn site, whereby they afford thermodynamic and electrostatic facilitation for the reaction with negatively charged species such as O2•− and ONOO−. Those are Mn(III) meso tetrakis(N-alkylpyridinium-2-yl)porphyrins, more specifically MnTE-2-PyP5+ (AEOL10113) and MnTnHex-2-PyP5+ (where alkyls are ethyl and n-hexyl, respectively), and their imidazolium analog, MnTDE-2-ImP5+ (AEOL10150, Mn(III) meso tetrakis(N,N′-diethylimidazolium-2-yl) porphyrin). The efficacy of MnPs in vivo is determined not only by the compound antioxidant potency, but also by its bioavailability. The former is greatly affected by the lipophilicity, size, structure, and overall shape of the compound. These porphyrins have the ability to both eliminate reactive oxygen species and impact the progression of oxidative stress-dependent signaling events. This will effectively lead to the regulation of redox-dependent transcription factors and the suppression of secondary inflammatory- and oxidative stress-mediated immune responses. We have reported on the inhibition of major transcription factors HIF-1α, AP-1, SP-1, and NF-κB by Mn porphyrins. While the prevailing mechanistic view of the suppression of transcription factors activation is via antioxidative action (presumably in cytosol), the pro-oxidative action of MnPs in suppressing NF-κB activation in nucleus has been substantiated. The magnitude of the effect is dependent upon the electrostatic (porphyrin charges) and thermodynamic factors (porphyrin redox ability). The pro-oxidative action of MnPs has been suggested to contribute at least in part to the in vitro anticancer action of MnTE-2-PyP5+ in the presence of ascorbate, and in vivo when combined with chemotherapy of lymphoma. Given the remarkable therapeutic potential of metalloporphyrins, future studies are warranted to further our understanding of in vivo action/s of Mn porphyrins, particularly with respect to their subcellular distribution.

A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity

The Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is preserved and even enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-) -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30 μM. MnTnHep-2-PyP(5+) was already toxic at 5 μM, and MnTnHex-2-PyP(5+) became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).

Differential Coordination Demands in Fe versus Mn Water-Soluble Cationic Metalloporphyrins Translate into Remarkably Different Aqueous Redox Chemistry and Biology

The different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose, the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli, and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ∼7.8. This is additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe relative to Mn center. The differences in the catalysis of O2(•-) dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 μM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 μM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 μM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 μM Fe salt, while the 20 μM Mn salt provides no protection to E. coli; and (2) 1 μM Fe porphyrin is fully degraded by 12 h in E. coli cytosol and growth medium, while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.

Radioprotection of the brain white matter by Mn(III) N-butoxyethylpyridylporphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP5+

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP5+(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP5+ before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP5+/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP5+/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP5+/RT. Our data demonstrate that MnTnBuOE-2-PyP5+ is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP5+ combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP5+ with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP5+ has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer. Mol Cancer Ther; 14(1); 70–79. ©2014 AACR.

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.

Challenges encountered during development of Mn porphyrin-based, potent redox-active drug and superoxide dismutase mimic, MnTnBuOE-2-PyP5 +, and its alkoxyalkyl analogues

We disclose here the studies that preceded and guided the preparation of the metal-based, redox-active therapeutic Mn(III) meso-tetrakis(N-n-butoxyethylpyridyl)porphyrin, MnTnBuOE-2-PyP5+ (BMX-001), which is currently in Phase I/II Clinical Trials at Duke University (USA) as a radioprotector of normal tissues in cancer patients. N-substituted pyridylporphyrins are ligands for Mn(III) complexes that are among the most potent superoxide dismutase mimics thus far synthesized. To advance their design, thereby improving their physical and chemical properties and bioavailability/toxicity profiles, we undertook a systematic study on placing oxygen atoms into N-alkylpyridyl chains via alkoxyalkylation reaction. For the first time we show here the unforeseen structural rearrangement that happens during the alkoxyalkylation reaction by the corresponding tosylates. Comprehensive experimental and computational approaches were employed to solve the rearrangement mechanism involved in quaternization of pyridyl nitrogens, which, instead of a single product, led to a variety of mixed N-alkoxyalkylated and N-alkylated pyridylporphyrins. The rearrangement mechanism involves the formation of an intermediate alkyl oxonium cation in a chain-length-dependent manner, which subsequently drives differential kinetics and thermodynamics of competing N-alkoxyalkylation versus in situ N-alkylation. The use of alkoxyalkyl tosylates, of different length of alkyl fragments adjacent to oxygen atom, allowed us to identify the set of alkyl fragments that would result in the synthesis of a single compound of high purity and excellent therapeutic potential.