Réjean Langlois

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES-X. SYNTHESES AND ANALYSES OF SULFONATED PHTHALOCYANINES

Abstract— Synthetic methods to obtain selectively sulfonated metallo phthalocyanines are compared. Both condensation and direct sulfonation procedures lead to mixtures of mono‐ to tetrasulfonated products which are resolved by reverse phase liquid chromatography in buffered aqueous‐methanol. The proportion of sulfonated derivatives is examined as a function of the starting reagents in the case of the condensation method, and as a function of the temperature and reaction time in the case of the direct sulfonation procedure. The number of sulfonate groups per phthalocyanine molecule is determined by oxidative degradation of the phthalocyanine ring followed by quantitative chromatographic analysis of the sulfophthalamide and phthalamide fragments.

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VI. PHOTOOXIDATION OF L‐TRYPTOPHAN BY SELECTIVELY SULFONATED GALLIUM PHTHALOCYANINES: SINGLET OXYGEN YIELDS AND EFFECT OF AGGREGATION

Abstract— The photosensitized oxidation of L‐tryptophan by gallium phthalocyanines sulfonated to different degrees is studied as a function of both substrate and sensitizer concentrations in water and 50% MeOH‐H2O solutions. The maximum quantum yield of singlet oxygen was found to be nearly 0.5 for all sulfonated gallium complexes. The effect of adding sulfonate groups in the phthalocyanine backbone is to change the tendency of dye molecules to dimerize or aggregate in a particular solvent. A shift in the chemical equilibrium away from the monomeric state, which occurs at high dye concentrations and at lower degrees of dye sulfonation, results in a reduced photochemical yield. The variation of quantum yields in different solvent systems and at several wavelengths is similarly accounted for by the fraction of light absorbed by the productive monomer state.

Biological activities of phthalocyanines--VIII. Cellular distribution in V-79 Chinese hamster cells and phototoxicity of selectively sulfonated aluminum phthalocyanines.

Abstract— Water soluble chloro aluminum phthalocyanines sulfonated to different degrees are studied for phototoxicity and cellular distribution inV–79 Chinese hamster cells. The more hydrophobic disulfonated dyes, with sulfonate substituents on adjacent benzyl groups of the phthalocyanine ring structure, exhibited the best cell penetrating properties and the highest phototoxicity. Fluorescence microscopy revealed that the dye was uniformly distributed in the cytoplasm but absent in the nucleus. The greater cell membrane penetrating properties of the lower as compared to the higher sulfonated dyes are attributed to the amphiphilic nature of the former.

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES‐IX. PHOTOSENSITIZATION OFV–79 CHINESE HAMSTER CELLS ANDEMT–6 MOUSE MAMMARY TUMOR BY SELECTIVELY SULFONATED ZINC PHTHALOCYANINES

Abstract— Zinc phthalocyanines sulfonated to different degrees are tested for their ability to sensitizeV–79 Chinese hamster cells andEMT–6 mouse mammary tumors to red light. In vitro, the lower sulfonated derivatives were the most active with the exception of the poorly water‐soluble monosulfonated dye. An isomeric mixture of tetrasulfonated derivatives obtained via direct sulfonation was ten times more active than the homogeneous tetrasulfo derivative prepared via the condensation of sulfophthalic acid. In vivo, the latter dye was completely inactive, whereas the remainder of the sulfonated preparations exhibited a similar structure‐activity pattern as observed with theV–79 cells in vitro. The disulfonated zinc phthalocyanine showed the best tumoricidal activity in the series and also appeared to be a more efficient photosensitizer of cell inactivation and tumor cure than the aluminum or gallium complexes as well as hematoporphyrin derivative preparations. No significant differences in skin phototoxicity were observed among the various dyes.

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—V. PHOTODYNAMIC THERAPY OF EMT‐6 MAMMARY TUMORS IN MICE WITH SULFONATED PHTHALOCYANINES

Abstract— We tested water‐soluble sulfonated phthalocyanine and three metal chelate derivatives for their tumoricidal effect on the EMT‐6 mammary tumor in mice exposed to red light. The metal‐free sulfophthalocyanine had little effect, whereas the aluminum complex and the lower sulfonated fraction of the gallium complex exhibited tumoricidal activity similar to hematoporphyrin‐based photosensitizer (Photofrin II). The higher sulfonated fractions of the gallium complex were less active as compared to the lower sulfonated fraction. The cerium complex was the most active sensitizer in terms of dye and light doses required to induce tumor necrosis and cure but also showed the highest phototoxicity towards healthy skin. These results suggest that sulfonated phthalocyanines will offer a new alternative in photodynamic therapy of light‐accessible neoplasms.

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2′‐DEOXYGUANOSINE

Abstract— The photodynamic properties of the di‐and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2′‐deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2‐diamino [(2‐deoxy‐β‐d‐erythropentofuranosyl)‐4‐amino]‐5(2H)‐oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9‐(2‐deoxy‐β‐d‐erythropentofuranosyl)‐7,8‐dihydro‐4‐hydroxy‐8‐oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2′‐deoxyguanosine, confirming the major role of singlet oxygen in these processes.

Biological activities of phthalocyanines. III: Photoinactivation of V-79 chinese hamster cells by tetrasulfophthalocyanines

Abstract— Tetrasulfophthalocyanine and a series of its metal chelates were tested for their ability to photoinactivate V‐79 Chinese hamster cells. Incubation of cells for 1 h with tetrasulfophthalocyanine at 5 μM effectively sensitized cells towards red light. At the 1% survival level, the dye was 4 x more efficient than hematoporphyrin, efficiency being defined in terms of drug concentration in the medium and incident light fluence rather than on the basis of quanta absorbed. Chelation of the dye with metal ions resulted in most cases in a greatly diminished photosensitizing effect, except for cerium. The cerium complex was about 5 x more effective for cell killing than the metal free tetrasulfophthalocyanine and 20 x more efficient as compared to hematoporphyrin. Hypoxic conditions resulted in total loss of photoactivity indicating the involvement of oxygen in the action mechanism. The inactivation by near‐UV light by these drugs was also investigated. The potential of sulfonated phthalocyanines as novel photosensitizers for photodynamic cancer therapy is discussed.

Receptor‐Mediated Targeting of Phthalocyanines to Macrophages Via Covalent Coupling to Native or Maleylated Bovine Serum Albumin

Abstract— Targeted delivery of aluminum tetrasulfophthalocyanine (AlPcS4) to the scavenger receptor of macrophages, via coupling to maleylated bovine serum albumin (mal‐BSA), was explored as a means to improve photodynamic efficacy. The AlPcS4 was covalently coupled to BSA (9:1 molar ratio) via one or two sulfonamide‐hexanoic‐amide spacer chains, followed by treatment with maleic anhydride to yield the mal‐BSA‐phthalocyanine conjugates. The latter were tested for singlet oxygen production, receptor‐mediated cell uptake and phototoxicity toward J774 cells of macrophage origin and nonphagocytic EMT‐6 cells. Cell uptake of 125I‐mal‐BSA showed specific binding for J774 cells but not for EMT‐6 cells. Competition studies of the conjugates with 125I‐mal‐BSA showed that coupling of AlPcS4 to BSA resulted in recognition of the conjugate by the scavenger receptor, whereas coupling to mal‐BSA further enhanced its binding affinity. This suggests that affinity for the scavenger receptor is related to the overall negative charge of the protein. Phototoxicity of the conjugates toward J774 cells paralleled their relative affinity, with mal‐BSA‐AlPcS4 coupled via two spacer chains showing the highest activity. The conjugates were less phototoxic toward the EMT‐6 cell line. The activities in both cell lines of all conjugated AlPcS4 preparations were, however, lower than that of the free disulfonated AlPcS2. Possible implications for the in vivo use of protein‐photosensitizer conjugates to target selectively various macrophage‐associated disorders is discussed.

Biological activities of phthalocyanines: XIII: Synthesis tumor uptake and biodistribution of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine in C3H mice

The biodistribution and metabolism of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine (Ga-PcS) was studied in radiation-induced fibrosarcoma tumor-bearing C3H mice. The [14C]Ga-PcS compounds were prepared via the condensation of [14C]phthalic acid and sulfophthalic acid in the presence of gallium chloride and characterized by their spectroscopic and chromatographic properties. The tissue concentrations of the dyes was measured by scintillation counting of the 14C and by extraction and fluorescence measurements. Elevated dye levels were found in the liver, lungs, kidneys and spleen as well as in the tumor. Lower sulfonation of Ga-PcS favored liver and spleen uptake whereas higher dye sulfonation resulted in greater kidney uptake. Both dyes showed high tumor uptake with peak concentrations exceeding those of most tissues except for the liver in the case of Ga-PcS2. The highest tumor uptake was observed with Ga-PcS3. Both dyes were slowly excreted from the body. The liver-feces pathway was favored in the case of Ga-PcS2 with high activities persisting in the liver, even after 21 days. The Ga-PcS3 was preferentially excreted via the kidney-urine pathway. High performance liquid chromatography analysis of the liver and tumor extracts of [14C]Ga-PcS3-treated animals did not reveal desulfonation of the dye. However, urine analysis showed the presence of radioactive metabolites lacking the characteristic phthalocyanine absorption.

Biological activities of phthalocyanines--VII. Photoinactivation of V-79 Chinese hamster cells by selectively sulfonated gallium phthalocyanines.

Abstract Gallium chloride phthalocyanines sulfonated to different degrees were tested for their ability to inactivate V‐79 Chinese hamster cells in the presence of red light. The mono‐ and disulfonated compounds were the most active whereas the tri‐ and tetrasulfonated complexes were completely void of photoactivity. In addition, large variations in photoactivity were observed among the four isomeric disulfonated derivatives with the most hydrophobic isomer exhibiting the highest photoactivity. Prolonged exposure to the disulfonated complex resulted in increased photosensitization. Complexing the dye with Al instead of Ga resulted in a slightly increased photosensitizing effect.