Kirpal S. Gulliya

Photochemistry of merocyanine 540 : the mechanism of chemotherapeutic activity with cyanine dyes

Abstract— The photophysical properties of merocyanine 540 have been determined in methanol solution over a modest temperature range. Triplet state population is inefficient (the limiting triplet quantum yield being 0.25) due to rapid isomerization of the central double bond from the first excited singlet state. Activation energies have been measured for isomerization from the excited singlet state (20 kJ mol‐1) and for conversion of the resultant cis‐isomer back to the original trans‐form (63 kJ mol‐1), both processes involving formation of a twisted species. The dye is easily oxidized to give an unstable adduct which decomposes on the sub‐ms timescale. Reversible redox chemistry occurs upon excitation in the presence of electron acceptors. These various observations are discussed in terms of the known chemotherapeutic activity of MC540 and it is concluded that the most probable mechanisms for cytotoxicity involve either local thermal disruption of cell membranes or in siru photogeneration of toxins derived from breakdown of the dye.

TUMOR CELL SPECIFIC DARK CYTOTOXICITY OF LIGHT‐EXPOSED MEROCYANINE 540: IMPLICATIONS FOR SYSTEMIC THERAPY WITHOUT LIGHT

Abstract— ‐Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light‐exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light‐exposed MC540 resulted in 70–90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte‐macrophage colony forming cells (CFU‐GM) survived the treatment. The observed cytotoxicity of light‐exposed MC540 to the tumor cells was significantly greater (P < 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light‐exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia‐bearing mice treated with light‐exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light‐exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light‐exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short‐lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.

In vitro PHOTODYNAMIC ACTIVITY OF KRYPTOCYANINE

Abstract— –The photophysical properties of 1,1′‐diethyl‐4,4′‐carbocyanine chloride (kryptocyanine) have been measured in methanol solution and for the dye bound to human serum albumin, incorporated in neutral micelles and after incubation with leukemia cells. In all cases, it is found that formation of the triplet state of the dye occurs with low efficiency and that illumination of the dye under aerobic conditions does not produce significant yields of O2(1δg). Instead, the only efficient photoprocess involves rapid internal conversion from the first excited singlet state to the ground state, probably via isomerization of the polymethine sequence. These findings are discussed with respect to the demonstrated ability of kryptocyanine to photodestroy leukemic cells.

Protein damage by photoproducts of merocyanine 540

Exposure of certain photoactive dyes to light prior to their use in biological systems (preactivation) has been shown to result in formation of long-lived cytotoxic photoproducts. The cytotoxic species responsible for the biological activity of preactivated merocyanine 540 (pMC540) appears to be a hydroperoxide generated by oxidation of ground-state dye by singlet molecular oxygen, formed via energy transfer from triplet excited-state dye to oxygen. A positive correlation (r = .93) exists between the levels of hydroperoxides and percent of tumor cells killed upon exposure to pMC540. Exposure of bovine serum albumin (BSA) (0.5 mg/mL) to pMC540 (0.2 mg/mL-1 mg/mL) results in loss of tryptophan fluorescence and 345 nm emission, suggesting a probable role of either hydroxyl (.OH) or .OH + superoxide (O2-). Polyacrylamide gel electrophoresis indicates fragmentation of treated BSA. Aggregation of pMC540-treated BSA is not detected. Bityrosine production is not observed. A dose-dependent decrease in BSA solubility is observed in treated samples, suggesting an increase in hydrophobicity. Amino acid analysis of BSA treated with pMC540 shows loss of some amino acids residues. The data presented here suggest that photoproducts of MC540 derived via the process of preactivation may mediate their effect (at least in part) by reactive oxygen species.

Elimination of leukemic cells by laser photodynamic therapy

SummaryWe studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 μg/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength. Normal bone marrow cells were treated under similar conditions. At this dose, 99.9999% of the leukemic cells were killed while 55% of the BM cell survived. Of the granulocyte-macrophage colony-forming cells (CFU-GM), 27% also survived this treatment. Photosensitization of a mixture of irradiated BM cells mixed with an equal number of nonirradiated HL-60 cell did not interfere with the killing of HL-60 cells. There was no significant reduction in the viability of cells when exposed to the laser light alone. In summary, laser light-induced photosensitization with MC540 has a selective cytotoxicity to leukemic cells; therefore, this procedure may be useful for purging neoplastic cells from autologous BM.

Preactivation—a novel antitumour and antiviral approach

Merocyanine 540 was activated by exposure to 514 nm laser light. This preactivated merocyanine 540 was then mixed (in the dark) with tumour cells, normal cells and envelope viruses to assess its antiproliferative activity. This treatment resulted in 70-90% killing of tumour cells from different cell lines while 85% of normal human peripheral blood mononuclear cells survived the treatment. However, not all types of tumour cells were affected. Preactivated merocyanine 540 was also effective in virtually completely inactivating cell-free herpes simplex and human immunodeficiency viruses. Preactivated photoactive compounds can exert their toxic effects in the dark without further dependence on light and may have potential systemic use.

Increased survival of normal cells during laser photodynamic therapy: Implications for ex vivo autologous bone marrow purging

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm2, 93.6 J/cm2 and 109.2 J/cm2, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.

Biodistribution and toxicity of photoproducts of merocyanine 540

SummaryLight-activated merocyanine 540 (pMC540) has been shown in our earlier studies to be effective against certain types of tumor cells and viruses, including human immunodeficiency virus (HIV-1). To test the potential extracorporeal and systemic use of pMC540, its toxicity was investigated in DBA/2 mice, pigs, and dogs. The lethal dose in DBA/2 mice after an i.p. injection was 370 mg/kg, and the 50% lethal dose (LD50) was 320 mg/kg; however, following i.v. administration, the lethal dose and the LD50 dose were 240 and 160 mg/kg, respectively. Tritium-labeled MC540 was used to study the biodistribution of pMC540 in DBA/2 mice. Almost 70% of the injected radioactivity was excreted within 6 h of injection. After 1 week, the pMC540 was almost completely cleared, with only 1.89% of the activity remaining, and had a plasma half life of 23 h. Pigs injected with an accumulated dose of 10 mg/kg and followed for a period of 30 days did not show adverse signs of toxicity as monitored by SMAC-28 analysis, CBC profile, and blood-coagulation studies. A dog injected with a single dose of 20 mg/kg showed induction of the hepatic enzymes glutamic oxaloacetic transminase (AST) and glutamic pyruvic transaminase (AST); however, serum levels of gamma-glutamyl transpeptidase (GGT) remained unchanged. The data presented herein may serve to identify certain drug-dose limitations in the systemic use of pMC540.

Effect Of Free Radical Quenchers On Dye-Mediated Laser Light Induced Photosensitization Of Leukemic Cells

The effect of free radical quenchers (ascorbate, catalase, and mannitol) on merocyanine 540 (MC540) mediated, laser light induced photolysis of human acute promyelocytic leukemia cell line (HL-60) was investigated. Results show that in the presence of human albumin (0.25%), dye-mediated (2014/m1), laser light induced photolysis of leukemic cells resulted in a 99.9999% cell kill. Seventy percent of the normal bone marrow cells survived the treatment. The addition of free radical quenchers prior to laser irradiation procedure increases the HL-60 cell survival. Increases of 5.5% and 4.4%, respectively, were observed in the presence of catalase and ascorbate or mannitol. In the presence of a mixture of catalase and mannitol or catalase and ascorbate, this increase in viability was not observed. However, the viability of normal bone marrow cells under these conditions also decreased from 70% to 63%. These findings may be useful in ex-vivo bone marrow purging.

Laser Light Induced Photosensitization Of Lymphomas Cells And Normal Bone Marrow Cells

Dye mediated, laser light induced photosensitization was tested in an in vitro model for its efficacy in eliminating the contaminating tumor cells for ex vivo autologous bone marrow purging. Daudi and U-937 cells (3 x 106/ml) in RPMI-1640 supplemented with 0.25% human albumin were mixed with 20 µg/ml and 25 µg/ml of MC-540, respectively. These cell-dye mixtures were then exposed to 514 nm argon laser light. Identical treatment was given to the normal bone marrow cells. Viability was determined by the trypan blue exclusion method. Results show that at 31.2 J/cm2 irradiation, 99.9999% Daudi cells were killed while 87% of the normal bone marrow cells survived. No regrowth of Daudi cells was observed for 30 days in culture. However, a light dose of 93.6 J/cm2 was required to obtain 99.999% U-937 cell kill with 80% normal bone marrow cell survival. Mixing of irradiated bone marrow cells with an equal number of lymphoma cells did not interfere with the photodynamic killing of lymphoma cells. Exposure of cells to low doses of recombinant interferon-alpha prior to photodynamic therapy increased the viability of lymphoma cells.