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.     

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.     

Semi-empirical molecular orbital studies of porphine and phthalocyanine derivatives,to simulate their intermolecular interactions

Gound-state electronic properties of porphine, phthalocyanine, and their derivatives, photosensitizers with potential use in the photodynamic therapy of tumors, were studied by semiempirical (MNDO ) molecular orbital calculations. Geometry, bond orders, electron populations, and net atomic charges were analyzed. The obtained models were applied to study the intermolecular interactions of these molecules utilizing a classical mechanics approach. The dimerization characteristics of different derivatives were obtained by a separate evaluation of the contribution of electrostatic and steric factors. © 1993 John Wiley & Sons, Inc.     

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.     

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.     

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.     

Carbon-14 labelling of biomolecules induced by14CO ionized gas

Ionized¹⁴CO gas provides a rapid method for producing¹⁴C-labelled biomolecules. The apparatus consists of a high vacuum system in which a small amount of¹⁴CO is ionized by electron impact. The resulting species drift towards a target where they interact with the molecule of interest to produce¹⁴C-labelled compounds. Since the reaction time is only 2 minutes, the method is particularly promising for producing tracer biomolecules with short-lived¹¹C at high specific activities. We have studied the applicability of the method to various classes of compounds of biological importance, including sterids, alkaloids, prostaglandins, nucleosides, amino acids and proteins. All compounds treated gave rise to¹⁴C addition and degradation products. Furthermore, for some compounds, chromatographic analysis in multiple systems followed by derivatization and crystallization to constant specific activity, indicated that carbon exchange may occur to produce the labelled, but otherwise unaltered substrate in yields of the order of 10–100 mCi/mol. More conclusive proof of radiochemical identity must await production of larger quantities of material and rigorous purification including at least two different chromatographic techniques. Supported by the Medical Research Council of Canada Grant MA-6137, and by the Banting Research Foundation.     

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.     

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.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES. XIII. THE EFFECTS OF HUMAN SERUM COMPONENTS ON THE in vitro UPTAKE AND PHOTODYNAMIC ACTIVITY OF ZINC PHTHALOCYANINE

Abstract— The effect of human serum components on the photodynamic activity of zinc phthalocyanine (ZnPc) toward Chinese hamster fibroblasts (lineV–79) was studied. Photodynamic activities were correlated with cellular uptake of radiolabeled [⁶⁵Zn]ZnPc, which allowed corrections to be made for the amount of sensitizer present in the cells at the time of irradiation and to express photodynamic efficiences on a cellular dye concentration basis. All serum components, with the exception of high-density lipoproteins, inhibit uptake of ZnPc byV–79 cells, when compared to incubation of ZnPc with the same cells in serum-free medium. High-density lipoproteins increased ZnPc uptake by 23%, but the photodynamic efficiency corrected for the cellular ZnPc concentration was unaffected. Very low-density lipoprotein and globulins decreased ZnPc cell uptake but likewise did not affect the cellular photodynamic efficiency of the dye. In contrast low-density lipoprotein and albumin, while inhibiting ZnPc cell uptake, increased the cellular photodynamic efficiency of ZnPc, suggesting that these proteins facilitate localization of the dye at cellular targers sensitive to photodynamic damage and vital to cell survival. We conclude from these results that association of ZnPc with serum components can have important, and widely differing, effects on both degree of uptake and cellular distribution of the photosensitizer.