Synthesis of phthalocyanine dimers,trimers, and oligomers bridged via phenyl groups

Several phthalocyanine dimers, trimers, and oligomers bridged via aryl (phenyl) groups were prepared using the Suzuki–Miyaura cross coupling reaction of phthalocyanine-boronate ester and various halide derivatives under palladium catalyst reaction conditions. Photophysical data reveal energy transfer between the Pc moieties resulting in the appearance of new red-shifted Q-bands. The shift and the nature of Q-band depend on the number of phenyl groups, the number of Pc, and the position of attachment on the phenyl ring.     

An easy route for the synthesis of pyrazine-2,3-dicarbonitrile 5,6-bis-substituted derivatives using a palladium catalyst

An easy synthetic method for the preparation of pyrazine-2,3-dicarbonitrile 5,6-bis-substituted derivatives using Pd-catalyzed cross coupling reaction (Sonogashira, Suzuki, Heck) is described. The reaction conditions allow preparing symmetrical and unsymmetrical bis-substituted products in moderate to high yields, as precursors for the preparation of highly substituted symmetrical and non-symmetrical aza-phthalocyanines with well-defined spectral properties.     

Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.     

PHOTODYNAMIC ACTIVITIES and SKIN PHOTOSENSITIVITY OF THE bis(DIMETHYLTHEXYLSILOXY)SILICON 2,3-NAPHTHALOCYANINE IN MICE

The photodynamic therapy (PDT) activity of the bis(dimethylthexylsiloxy)silicon 2,3-na-phthalocyanine (SiNc 8 ) was evaluated against the EMT-6 tumor implanted intradermally in BALB/c mice. The SiNc 8 was formulated in aqueous emulsions based on Cremophor EL or Solutol HS 15. The formulation was shown to affect plasma clearance and overall pharmacokinetics. Compared to Cremophor, Solutol promoted rapid plasma clearance and high liver retention of the dye, combined with a slight increase of dye tumor concentrations. The PDT action spectrum for tumor response of SiNc 8 in Cremophor (190 mW cm², 200 J cm², 24 h postinjection [p.i.] of 1 (jimol kg¹) showed a maximum at 780 nm, which corresponds to the absorption maximum of the monomelic dye as well as the in vivo maximum change in the “diffuse optical density” produced by the dye. The extent of tumor necrosis increased with augmented dye and light doses. Regardless of the formulation, at 1 h p.i. of 0.1 μmol kg^?! SiNc 8 , PDT efficiency (190 mW cm'², 400 J cm²) was high but accompanied by severe damage to normal tissues, at 24 h PDT resulted in complete tumor regression in 80% of the animals without adverse effects to adjacent tissues, while at 72 h p.i. PDT induced no tumor response with Cremophor and only a partial response with Solutol. At the latter time point, plasma dye clearance was nearly complete while tumor tissue levels remained high, suggesting that tumor response correlates with plasma rather than tumor dye levels. Skin sensitivity of SKhl mice to solar-simulated radiation was lower with SiNc 8 as compared to Photofrin®. Our data suggest the potential of SiNc 8 as a far-red absorbing photosensitizer in clinical PDT.     

Eradication of multiple myeloma and breast cancer cells by TH9402-mediated photodynamic therapy: implication for clinical ex vivo purging of autologous stem cell transplants

High-dose chemotherapy combined with autologous transplantation using bone marrow or peripheral blood-derived stem cells (PBSC) is now widely used in the treatment of hematologic malignancies as well as some solid tumors like breast cancer (BC). However, some controversial results were recently obtained in the latter case. The presence of malignant cells in the autograft has been associated with the recurrence of the disease, and purging procedures are needed to eliminate this risk. The aim of this study was to evaluate the potential of the photosensitizer 4,5-dibromorhodamine methyl ester (TH9402), a dibrominated rhodamine derivative, to eradicate multiple myeloma (MM) and BC cell lines, while sparing more than 50% of normal pluripotential blood stem cells from healthy volunteers. The human BC MCF-7 and T-47D and MM RPMI 8226 and NCI-H929 cell lines were used to optimize the photodynamic purging process. Cell concentration and the cell suspension thickness as well as the dye and light doses were varied in order to eventually treat 1-2 L of apheresis. The light source consisted of two fluorescent scanning tubes emitting green light centered about 515 nm. The cellular uptake of TH9402 was measured during the incubation and washout periods and after photodynamic treatment (PDT) using spectrofluorometric analysis. The limiting dilution assay showed that an eradication rate of more than 5 logs is obtained when using a 40 min incubation with 5-10 microM dye followed by a 90 min washout period and a light dose of 5-10 J/cm2 (2.8 mW/cm2) in all cell lines. Agitating the 2 cm thick cell suspension containing 20 x 10(6) cells/mL during PDT was essential for maximal photoinactivation. Experiments on mobilized PBSC obtained from healthy volunteers showed that even more drastic purging conditions than those found optimal for maximal eradication of the malignant cell lines were compatible with a good recovery of hematopoietic progenitors cells. The absence of significant toxicity towards normal hematopoietic stem cells, combined with the 5 logs eradication of cancer cell lines induced by this procedure suggests that TH9402 offers an excellent potential as an ex vivo photodynamic purging agent for autologous transplantation in MM and BC treatment.     

TYPES I AND II SENSITIZED PHOTOOXIDATION OF AMINOACID BY PHTHALOCYANINES: A FLASH PHOTOCHEMICAL STUDY

Abstract— In connection with the use of red light-photosensitizers for photodynamic therapy, the redox reactivity of excited metallophthalocyanines (M = Al, Ga) was investigated by flash photolysis in order to establish whether photooxidations proceed by Foote's mechanisms I or II. Aminoacids (tryptophan, tyrosine) were seen to function as electron transfer quenchers of the excited phthalocyanines with rate constants 10⁷ k 10⁴ M ^-1 s^-1. This was not the case of purines or ATP. The ability of the excited phthalocyanines to sensitize photooxidations by mechanisms I and II is discussed in terms of evaluated rate constants.     

Preparation, phototoxicity and biodistribution studies of anti-carcinoembryonic antigen monoclonal antibody-phthalocyanine conjugates

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of a conjugated dye. Aluminum tetrasulfophthalocyanine (AlPcS4) was covalently coupled to a 35A7 MAb directed against carcinoembryonic antigen (CEA) via a five-carbon spacer chain (A1) to yield conjugates with a molar ratio ranging from 5 to 16 mol of AlPcS4 per mol of 35A7 MAb. Conjugates were labeled with radioiodine for characterization. The immunoreactivity of the conjugates, determined in a direct binding assay on CEA coupled to sepharose, was not modified by the coupled AlPcS4A1 molecules. In vivo, these conjugates were evaluated in nude mice bearing human colon carcinoma xenografts (T380). 35A7 MAb-(AlPcS4A1)5, 35A7 MAb-(AlPcS4A1)12 and 35A7 MAb-(AlPcS4A1)16 conjugates displayed a tumor uptake of 35 +/- 5.0%, 40 +/- 5.7% and 32 +/- 3.3% of the injected dose per gram of tumor tissue, respectively, corresponding to an uptake of 97%, 104% and 91% as compared to that of the unconjugated 35A7 MAb. In each experimental group, the tumor-to-normal tissue ratios obtained with the conjugates were almost identical to those obtained with unconjugated 35A7 MAb. Average values of 1.8, 7 and about 30 were obtained for blood, liver and muscle, respectively. Phototoxic efficacy of the 35A7 MAb-(AlPcS4A1)12 conjugate was demonstrated in vitro on the LoVo cell line giving a 91% growth inhibition for a 2.50 micrograms/mL AlPcS4A1 concentration. We conclude that these conjugates demonstrate clear in vivo tumor-seeking capacity and in vitro photocytotoxic properties. Such conjugates could thus be promising candidate drugs for clinical photodynamic therapy of cancers expressing CEA.     

Receptor-mediated targeting of phthalocyanines to macrophages via covalent coupling to native or maleylated bovine serum albumin

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.