Ursula Simonis

Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondrial damage in a rat model of ischemia/reperfusion injury.

The cardioprotective effectiveness of low-dose pyrroloquinoline quinone (PQQ, 3 mg/kg) was compared with metoprolol, a β1-selective adrenoceptor antagonist. Rats underwent 30 minutes of left anterior descending coronary artery occlusion and 2 hours of reperfusion. Metoprolol and/or PQQ were given at the onset of reperfusion to mimic clinical treatment. Metoprolol and/or PQQ reduced infarct size and protected against ischemia-induced left ventricular dysfunction after 2 hours of reper-fusion. Combined therapy augmented left ventricular developed pressure at the end of reperfusion. Metoprolol or PQQ alone enhanced mitochondrial respiratory ratios in ischemic and nonischemic myocardium. Although the PQQ/metoprolol combination therapy increased respiratory ratio values, the effects were small when compared with PQQ alone. Only PQQ decreased lipid peroxidation. Metoprolol and/or PQQ given at the onset of reperfusion reduce infarct size and improve cardiac function. Combination therapy further reduces infarct size. PQQ is superior to metoprolol in protecting mitochondria from ischemia/reperfusion oxidative damage

Proton NMR Spectroscopy of Model Hemes

Hemes and heme proteins are vital components of essentially every cell of every living organism. Their roles in cells include 1 the transport of dioxygen in the red blood cells of higher animals (hemoglobin); 2 the storage of dioxygen in the muscles of higher animals (myoglobin); 3 the transport of electrons in the respiratory chains of organisms as diverse as bacteria, yeasts, algae, plants, and animals, and in photosynthetic cells from those of the simplest photosynthetic bacteria to those of higher plants (cytochromes a, b, c, d, f); 4 synthesis, modification and degradation of fatty acids, steroid and adrenal hormones, anesthetics and xenobiotics (cytochromes P-450); 5 activation and metabolism of hydrogen peroxide (peroxidases, myeloperoxidase, haloperoxidases, catalases, etc.); and 6 metabolism of the oxides of nitrogen and sulfur (nitrite reductase, sulfite oxidase, etc.).

Rates of axial ligand rotation in diamagnetic d6 Co(III) and Fe(II) porphyrinates

Abstract In order to investigate the rates of rotation of pyridine and imidazole ligands in diamagnetic low-spin d 6 Co(III) and Fe(II) porphyrinate systems, we have synthesized tetramesitylporphyrinate (TMP) complexes of each of these metals with pyridine and imidazole ligands and investigated them as a function of temperature by 1 H NMR spectroscopy. We have already reported that for TMPFe(III) and -Co(III) complexes with hindered imidazoles the TMP o -CH 3 resonances can be used to measure the rates of rotation (N.V. Shokhirev, T.Kh. Shokhireva, J.R. Polam, C.T. Watson, K. Raffi, U. Simonis and F.A. Walker, J. Phys. Chem. A, 101 (1997) 2778). For the bis-1,2-dimethylimidazole complex, [TMPCo(1,2-Me 2 Im) 2 ]BF 4 , at ambient temperatures ligand rotation is slow but measureable on the NMP time scale, and four o -CH 3 resonances are observed, as we have already reported. In contrast, as shown in the present work, for the bis-4-dimethylaminopyridine complex, [TMPCo(4-NMe 2 Py) 2 ]BF 4 , ligand rotation is extremely rapid at ambient temperatures. At temperatures below −50°C at 300 MHz the o -CH 3 resonance broadens and the rates of rotation can be estimated using the modified Bloch equations simplified for the fast exchange regime. The activation parameters ΔH ≠ and ΔS ≠ have been determined, and the extrapolated rate constant at 25°C, k ex ≥ 1.1 × 10 6 s −1 . These results contradict previous reports (J. Huet and A. Gaudemer, Org. Magn. Reson., 15 (1981) 347; I. Cassidei, H. Bang, J.O. Edwards and R. G. Lawler, J. Phys. Chem., 95 (1991) 7186) that pyridine ligands bound to Co(III) porphyrinates do not rotate at room temperature in homogeneous solution. For unhindered imidazole complexes, such as [TMPCo(NMeIm) 2 ] + BF 4 − , no broadening of the o -CH 3 resonance is observed, even at −90°C, and thus the rate of axial ligand rotation is too fast to measure, even at that low temperature (or the difference in chemical shift of the two resonances expected if ligand rotation is slow is very small). For the corresponding Fe(II) porphyrinate complexes, the rates of pyridine and unhindered imidazole rotation are too fast to measure, even at −90°C. The 2-methylimidazole complex undergoes chemical reactions that prevent detailed study of this system by NMR spectroscopy, but the 1,2-dimethylimidazole complex is stable and of similar structure (ruffled porphyrinate ring, axial ligands in perpendicular planes) to the Co(III) and Fe(III) analogs, with the rate constant for ligand rotation, k ex ∼ 1 s −1 , at −90°C. Assuming a similar activation enthalpy to those of the Co(III) and Fe(III) systems, the rate of rotation of axial ligands in [TMPFe(1,2-Me 2 Im) 2 ] at 25°C is estimated to be about 2 × 10 4 s −1 .

Abstract 5111: Nitric oxide and its role in photodynamic therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Porphyrinic pigments are used as photosensitizers (PS) in photodynamic detection (PDD) and therapy (PDT) that is a minimally invasive modality in the fight against cancer. When the PS is activated by visible light at a given wavelength, reactive oxygen species (ROS) are generated, which cause cancer cells to undergo cell death. Despite significant advances, drawbacks of the PSs in clinical use include their non-selectivity in cellular-targeting causing cell death by necrosis leading to tissue inflammation. Nitric oxide (NO) has been shown to play a key role in modulating apoptotic cell death pathways and to react with reactive oxygen species to form additional lethal reactive nitrogen species (RNS). In our efforts to enhance the effectiveness of PDT, we set out to investigate the role of NO in PDT. We hypothesized that NO delivered to cancer cells at the time that the PS was administered would enhance the efficacy of PDT by promoting mitochondria-mediated apoptosis. To this end, we incubated androgen-sensitive human prostate adenocarcinoma (LNCaP) cells with both a PS and an NO releasing agent that was most effective in NO release as was spectrophotometrically determined by its oxidation of hemoglobin. Phototoxicity experiments were carried out at 37 OC with a noncoherent light source. Cell viability, damage and death were assessed in both illuminated and non-illuminated cells and were quantified by MTT staining as well as trypan blue and propidium iodide exclusion. To corroborate the cell viability results, we assayed clonogenic recovery in response to PDT in pigmented cells both in the presence and absence of NO. Our results indicate that the effectiveness of PDT in causing cell death depends on the NO concentration. PDT with NO alone was toxic to the cancer cells at high concentration of NO, whereas at low NO concentration no significant cell damage was observed after light illumination. PDT with the PS alone was not as effective in promoting cell death as PDT in the presence of both NO and the PS. Depending on the concentrations of the PS and NO, we observed that either necrosis or apoptosis were the prevailing modes of cell death after PDT. Our results indicate that the phototoxicity of the compounds is mainly determined by their intracellular concentration. Thus, understanding the combined effects of NO and the PSs in enhancing cell phototoxicity will aid in determining the roles that NO plays in improving the efficacy of PDT and may provide an alternate regimen to enhance PDT efficacy. Citation Format: Marco Monroy, Pooncharas Tipgunlakant, Ursula Simonis, Raymond Esquerra. Nitric oxide and its role in photodynamic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2014-5111

Synthesis and cellular localization of porphyrinic pigments

To determine factors that govern the uptake preference of photosensitizers in cellular organelles of human adenocarcinoma cells, diarginyl-dialkoxy- and diarginyl-dimethoxyphenylporphyrins (TPPs) and two of their corresponding indium(III) complexes were synthesized, characterized and incubated in androgen-sensitive human prostate adenocarcinoma cells LNCaP. The porphyrins revealed properties that are of importance for phototherapy. They are water-soluble, have their fourth Q-band absorbing at ≈ 650 nm, are taken up in relatively high concentrations in LNCaP cells, and are phototoxic. Colocalization and phototoxicity studies revealed that all porphyrins localized preferentially to the lysosomes and invoked cell death when excited with 650 nm light. Compared to the corresponding methoxy-substituted TPPs, the diargininyl-dialkoxy-substituted porphyrins localized to a small extent in the mitochondria. The corresponding In(III) chloride complexes that are slightly less water-soluble were also taken up in the lysosomes of LnCaP cells. When the TPPs were compared to a pheophorbide derivative recently synthesized in our laboratory, it was determined that the TPPs have a preference for lysosomal localization, whereas the pheophorbide derivative co-localized to the mitochondria. Phototoxicity studies revealed that the longer chain dialkoxyTPPs were more effective in cell killing and induced greater morphological changes typical of apoptotic cell death than the shorter chain methoxy substituted porphyrins. The In(III) complexes seemed to be the most phototoxic. These results highlight that the type, nature, and substitution pattern of the chromophore modulate the extent of apoptotic cell death and influence cellular targeting.

Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments

Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out. Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di-fluoro-substituted TPPs are trapped in the plasma membrane. Only a pheophorbide derivative recently synthesized in our laboratory localized to the mitochondria of LNCaP cells, which are at the center of cell death as is reflected in their key role during apoptosis, thus reassuring our attempts toward rational drug design.