Robert K. Ghazaryan

Rational Design of Superoxide Dismutase (SOD) Mimics: The Evaluation of the Therapeutic Potential of New Cationic Mn Porphyrins with Linear and Cyclic Substituents

Our goal herein has been to gain further insight into the parameters which control porphyrin therapeutic potential. Mn porphyrins (MnTnOct-2-PyP5+, MnTnHexOE-2-PyP5+, MnTE-2-PyPhP5+, and MnTPhE-2-PyP5+) that bear the same positive charge and same number of carbon atoms at meso positions of porphyrin core were explored. The carbon atoms of their meso substituents are organized to form either linear or cyclic structures of vastly different redox properties, bulkiness, and lipophilicities. These Mn porphyrins were compared to frequently studied compounds, MnTE-2-PyP5+, MnTE-3-PyP5+, and MnTBAP3–. All Mn(III) porphyrins (MnPs) have metal-centered reduction potential, E1/2 for MnIIIP/MnIIP redox couple, ranging from −194 to +340 mV versus NHE, log kcat(O2•–) from 3.16 to 7.92, and log kred(ONOO–) from 5.02 to 7.53. The lipophilicity, expressed as partition between n-octanol and water, log POW, was in the range −1.67 to −7.67. The therapeutic potential of MnPs was assessed via: (i) in vitro ability to prevent spontaneous lipid peroxidation in rat brain homogenate as assessed by malondialdehyde levels; (ii) in vivo O2•– specific assay to measure the efficacy in protecting the aerobic growth of SOD-deficient Saccharomyces cerevisiae; and (iii) aqueous solution chemistry to measure the reactivity toward major in vivo endogenous antioxidant, ascorbate. Under the conditions of lipid peroxidation assay, the transport across the cellular membranes, and in turn shape and size of molecule, played no significant role. Those MnPs of E1/2 ∼ +300 mV were the most efficacious, significantly inhibiting lipid peroxidation in 0.5–10 μM range. At up to 200 μM, MnTBAP3– (E1/2 = −194 mV vs NHE) failed to inhibit lipid peroxidation, while MnTE-2-PyPhP5+ with 129 mV more positive E1/2 (−65 mV vs NHE) was fully efficacious at 50 μM. The E1/2 of MnIIIP/MnIIP redox couple is proportional to the log kcat(O2•–), i.e., the SOD-like activity of MnPs. It is further proportional to kred(ONOO–) and the ability of MnPs to prevent lipid peroxidation. In turn, the inhibition of lipid peroxidation by MnPs is also proportional to their SOD-like activity. In an in vivo S. cerevisiae assay, however, while E1/2 predominates, lipophilicity significantly affects the efficacy of MnPs. MnPs of similar log POW and E1/2, that have linear alkyl or alkoxyalkyl pyridyl substituents, distribute more easily within a cell and in turn provide higher protection to S. cerevisiae in comparison to MnP with bulky cyclic substituents. The bell-shape curve, with MnTE-2-PyP5+ exhibiting the highest ability to catalyze ascorbate oxidation, has been established and discussed. Our data support the notion that the SOD-like activity of MnPs parallels their therapeutic potential, though species other than O2•–, such as peroxynitrite, H2O2, lipid reactive species, and cellular reductants, may be involved in their mode(s) of action(s).

Synthesis and in vitro anticancer activity of water-soluble cationic pyridylporphyrins and their metallocomplexes

Tetrapyrrolic compounds such as porphyrins are known to be prospective chemotherapeutics and photosensitizers for cancer treatment and diagnosis. In this work, water-soluble, meso-substituted cationic pyridyl-porphyrins and their metallocomplexes bearing various central metal atoms (Ag, Zn, Co, and Fe) in the porphine ring and various functional groups (allyl, oxyethyl, butyl, and methallyl) at the nitrogen atom in the pyridine ring were synthesized and characterized by 1H and 13C NMR and UV-visible spectroscopy. Cytotoxic and photodynamic activities of new porphyrins and their metal derivatives were investigated in vitro (KCL-22 cancer cell line of human chronic myeloid leukemia). The cytotoxicity of porphyrins was shown to be dependent on the presence and type of the central metal atom in the porphine ring. Ag-derivatives were more cytotoxic than Co−, Zn−, and Fe− metallocomplexes. The porphyrins bearing allyl-functional groups were evidenced to be more cytotoxic than those which included butyl-, oxyethyl-, and methallyl-groups. The change of nitrogen position in the pyridine ring of Ag-metalloporphyrins from 3(3-N-pyridylporphyrins) to 4(4-N-pyridylporphyrins) induced an increase in the cytotoxic activity of metallocomplexes. All synthesized Ag-metalloporphyrins, except, the oxyethyl-containing one were more cytotoxic than cisplatin. Allyl containing free porphyrin and its Zn-metallocomplex had higher phototoxicity than Ag−, Co−, and Fe-metalloporphyrins. The results obtained can be useful for further investigation of new porphyrins as potential chemotherapeutics and photosensitizers.

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods.

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800 nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.

Novel amphiphilic cationic porphyrin and its Ag(II) complex as potential anticancer agents

In the present study we have synthesized a novel amphiphilic porphyrin and its Ag(II) complex through modification of water-soluble porphyrinic structure in order to increase its lipophilicity and in turn pharmacological potency. New cationic non-symmetrical meso-substituted porphyrins were characterized by UV-visible, electrospray ionization mass spectrometry (ESI-MS), (1)H NMR techniques, lipophilicity (thin-layer chromatographic retention factor, Rf), and elemental analysis. The key toxicological profile (i.e. cytotoxicity and cell line- (cancer type-) specificity; genotoxicity; cell cycle effects) of amphiphilic Ag porphyrin was studied in human normal and cancer cell lines of various tissue origins and compared with its water-soluble analog. Structural modification of the molecule from water-soluble to amphiphilic resulted in a certain increase in the cytotoxicity and a decrease in cell line-specificity. Importantly, Ag(II) porphyrin showed less toxicity to normal cells and greater toxicity to their cancerous counterparts as compared to cisplatin. The amphiphilic complex was also not genotoxic and demonstrated a slight cytostatic effect via the cell cycle delay due to the prolongation of S-phase. As expected, the performed structural modification affected also the photocytotoxic activity of metal-free amphiphilic porphyrin. The ligand tested on cancer cell line revealed a dramatic (more than 70-fold) amplification of its phototoxic activity as compared to its water-soluble tetracationic metal-free analog. The compound combines low dark cytotoxicity with 5 fold stronger phototoxicity relative to Chlorin e6 and could be considered as a potential photosensitizer for further development in photodynamic therapy.

In Vitro Testing of Cyto- and Genotoxicity of New Porphyrin Water-Soluble Metal Derivatives

Porphyrins and porphyrin derivatives have an outstanding potential for discovery of novel pharmacological agents due to their ability for numerous chemical modifications and a variety of mechanisms of biological effects. New water-soluble Ag and Zn derivatives of tetrachloride meso-tetra (4-N-oxiethylpyridyl) porphyne were synthesized. Cyto- and genotoxicity of these substances were tested in vitro by the vital dye (trypan blue) exclusion and the micronucleus tests, respectively. Both metalloporphyrins were shown to be cytotoxic for Cos-7 (fibroblast-like African green monkey kidney cells transformed by simian virus 40 [SV40]), DU 145 (epithelial-like cells of human prostate carcinoma), and K-562 (human chronic myeloid leukemia cells) cell lines. At the same time they did not cause chromosome fragmentation in K-562 cell line at as high concentrations as IC50 (20 μmol/L for Ag and 70 μmol/L for Zn derivative). Thus, the metalloporphyrins tested meet at least two important demands to potential anticancer drugs as they combine the cytotoxicity with low genotoxicity. The three in vitro tumor models used are relevant to further in vitro and in vivo pre-clinical investigation of the studied metalloporphyrins as potential chemotherapeutics.

Complexes of bovine serum albumin with water-soluble Cu and Co containing cationic meso-tetra(4-N-oxyethylpyridyl)-porphyrins

Bovine serum albumin complexes with water-soluble cationic porphyrins, Cu- and Co-meso-tetra(4-N-hydroxyethylpyridyl)porphyrins (CuT4OEPyP, CoT4OEPyP), and their 3-N-analogs, meso-tetra(3-N-hydroxyethylpyridyl)porphyrins (CuT3OEPyP, CoT3OEPyP), have been investigated. The porphyrin-bovine serum albumin binding was monitored by the absorption in the visible region at 400-460 nm. The stoichiometry of binding and the binding constants of the porphyrins to bovine serum albumin were determined using binding isotherms and Scatchard plots. The Kb values obtained for these porphyrin- bovine serum albumin complexes are 1.7 × 105M−1, 3.2 × 105M−1, 1.4 × 105M−1 and 3 × 105M−1 respectively. Binding constants are sensitive to pH and ionic strength of the solution.

Investigation of photodynamic activity of water-soluble porphyrins in vitro and in vivo

Photodynamic therapy (PDT) is the method of photosensitized tumor treatment. It is based on the photosensitizer (PS) selective accumulation in tumors, its subsequent activation under the light influence and oxygen active form formation that results in tumor destruction. Photodynamic action of some new water-soluble porphyrins was investigated in our laboratory. Dose-dependent effect of these porphyrins was shown on PC-12 murine pheochromocytoma cell line. The results revealed that the efficiency of the investigated porphyrins decreased in the following way: TOEPyP (meso-tetra-(4-N-oxyethylpyridyl)porphyrin) > Zn-TOEPyP > Ag-TOEPyP. It was shown that TOEPyP possessed nearly the same photodynamic activity (LD50) as well-known photosensitizer chlorin e6. These porphyrins have also demonstrated quite high photodynamic activity in vivo. The results were obtained in the experiments on white mice with engrafted C-180 (Crokers sarcoma). Antitumor activity of these porphyrins in the dark was 30-40%, whereas photodynamic activity was 45-60%.

Complexation of porphyrins with nanoparticles of zeolite

The binding of nanoparticles zeolite with a number of cationic porphyrins are studied. Previously, it was established that the main mechanism of binding the zeolite nanoparticles with cationic porphyrins is an ionic bond. Since binding of porphyrins as ligands to nanoparticles at the initial stage of interaction is determined by the Brownian motion of porphyrins, it is obvious that the interaction of porphyrins with nanoparticles may depend on the temperature. In the present paper by methods of absorption and fluorescence spectroscopy was studied the complexation of porphyrins with zeolite nanoparticles at different temperature conditions. It was established that there is a clear temperature dependence of the complexation of cationic metalloporphyrins with zeolite naonoparticles, and for correct determination of the percentage of binding must be strict thermostating of the experimental conditions.

Photophysical properties and photodynamic efficiency of cationic porphyrins

Photodynamic inactivation of some microorganisms (St. aureus, E.coli) was investigated and their dependence on photo-physical properties of photosensitizers (PS) (cationic porphyrins and metalloporphyrins) was shown. One of the most important criteria for the effectiveness of the PS`s is the quantum yield of singlet oxygen (γΔ). Our investigations were shown that γΔ of metalloporphyrins, containing Zn, significantly higher than of metal-free porphyrins (85-97% and 77-79%, respectively). Previousl y experimentally we were found that under the action cationic porphyrins and metalloporphyrins on Gram (+) and Gram (-) microorganisms efficiency of metalloporphyrins Zn-TOEt4PyP and Zn-TBut4PyP in 3-5 times was higher than the metal-free porphyrins. In this study under the action of porphyrins and their Zn-derivatives on microorganism St. aureus such an effect was confirmed. Using the LED with a peak emission of 405 nm and a power density of 70 mW/cm2, and irradiation time of microorganisms from 5 to 30 minutes we have found, that at a concentration of 0.1 ug/ml the highest efficiency is observed of metalloporphyrin Zn-TBut3PyP. Upon irradiation of 10 and 15 min his efficiency is 3-5 times higher than the metal-free porphyrin TOEt4PyP, and irradiation for 30 min via Zn-TBut3PyP is practically completely kills microorganisms. These data correlate with the quantum yield of singlet oxygen for photosensitizers. The 30 mindirect sun exposure (power density of 70 mW/cm2) of photosensitizer solutions showed that a significant photobleaching of porphyrins and metalloporphyrins does not occur. Thus, Zn-containing cationic metalloporphyrins are highly efficient photosensitizers for photodynamic inactivation of microorganisms and PDT.

Stability of bilayer lipid membrane in the presence of Co-containing metalloporphyrins

Stability of bilayer lipid membrane (BLM) in an electric field in the presence of Co-porphyrin in the solution surrounding BLM is investigated. We have shown that the presence of porphyrin in a solution reduces line tension of the pore edge and surface tension of BLM and increases the number of pores on the BLM. It is also shown that a decrease in the stability of BLM in the electric field in the presence of the porphyrin is a result of the reduction of line tension of the pore edge and increase in the number of pores.