Efficacy and mechanism of aluminium phthalocyanine and its sulphonated derivatives mediated photodynamic therapy on murine tumours.

The efficacy of photodynamic therapy (PDT) mediated by aluminium phthalocyanine (AlPc) and its mono- and disulphonated derivatives (AlPcS1 and AlPcS2, respectively) on murine EMT-6 tumour were compared in vivo. AlPc (0.25 mumol/kg) PDT resulted in no tumour recurrence in all treated mice. In contrast, PDT with AlPcS1 (2 mumol/kg) and AlPcS2 (1 mumol/kg) only produced tumour cure in 75% and 86% of mice, respectively. Immediately after AlPc-PDT, tumour cells were found to be viable as determined by in vitro clonogenicity, but progressive cell death occurred thereafter. In contrast, AlPcS1 and AlPcS2 PDT produced substantial cell death (approximately 35% and 70%, respectively, of entire tumour) immediately after phototherapy, and yet further loss of tumour cell viability continued after PDT. In all cases, few vascular effects were observed at 0 h post-PDT, as indicated by the retention of 99mTc-MIBI in the tumour. However, the reduction of blood flow in tumours progressed with time, such that blood flow in tumours fell to approximately 25% of the control level by 24 h after both AlPc and AlPcS1 PDT. With AlPcS2, there was only an approximate 50% fall in tumour blood flow by 24 h. These results demonstrate a greater PDT efficiency with AlPc on tumour destruction, which is an indirect mechanism involving damage of tumour vasculature, whereas AlPcS2 has a greater effect on direct tumour cytotoxicity and AlPcS1 exerts both direct and indirect modes of action against tumours.

Syntheses and photodynamic activities of novel trisulfonated zinc phthalocyanine derivatives.

The synthesis of water-soluble, unsymmetrical, trisulfonated zinc phthalocyanines (ZnPcS3) as single products of the ring expansion of boron tri(4-sulfo)subphthalocyanine (SubPc) is reported. The novel, water-soluble trisulfo-SubPcB(OH) was prepared via hydrolysis of the tris(4-chlorosulfonyl)SubPcB(Br) which in turn was obtained from the condensation of 4-(chlorosulfonyl)phthalonitrile with BBr3 in 1-chlorobenzene. A number of ZnPcS3 analogues were prepared via the reaction of S3SubPcB (OH) with different diiminoisoindoline derivatives of increasing hydrophobicity. The reaction proceeds at relative low temperature with acceptable yields. Metalation of free base Pc's with zinc acetate dihydrate afforded the corresponding zinc complexes. Photodynamic activities were measured against the EMT-6 mouse mammary tumor cell line and compared to those of the known ZnPcS3 and ZnPcS4. Added (t-Bu)benzo and (t-Bu)naphtho groups increased the in vitro cell photoinactivation efficacy of the ZnPcS3, whereas addition of a fourth sulfobenzo or bulky diphenylpyrazino group decreased the activity of the parent molecule. The (t-Bu)naphthotrisulfobenzoporphyrazine induced the best in vivo photodynamic tumor control which, combined with its good solubility and broad absorption spectrum, renders this compound an interesting dye for photodynamic applications in medicine.

High-resolution PET imaging for in vivo monitoring of tumor response after photodynamic therapy in mice.

UNLABELLED: The aim of this study was to investigate the use of [18F]fluoro-2-deoxy-D-glucose (FDG) and a small-animal PET scanner to assess early tumor response after photodynamic therapy (PDT) in mice. PDT consists of intravenous administration of a photosensitizer that accumulates preferentially in tumor tissue, followed by local illumination of the tumor with red light. Two different photosensitizers were used: Photofrin (PII), which has been approved for clinical use, and disulfonated aluminum phthalocyanine (AIPcS), which is a second-generation drug. These drugs have been shown to induce tumor necrosis through different action mechanisms, i.e., mainly initial vascular stasis (PII) or direct tumor cell kill (AIPcS). FDG PET was used to follow both perfusion and metabolic activity in the tumor tissue. METHODS: The study was performed using a mouse model implanted with two contralateral murine mammary tumors (5 mm diameter x 2.5 mm thickness) on the back. Only one tumor was subjected to PDT, whereas the other tumor served as a control. A total of 13 mice were studied, 1 without illumination, 3 at 30 min and 3 at 2 h after PDT with both PII-PDT and AIPcS-PDT. Dynamic PET imaging of the mice, which were placed in pairs in a prostate position parallel to the transaxial planes of the Sherbrooke animal PET scanner, was performed after a bolus injection of 11 MBq (300 microCi) FDG. Blood samples were collected concurrently from 1 mouse during each study using an automated microvolumetric blood sampler. RESULTS: Analysis of the tumor time-activity curves showed that (a) scans during the first 3 min provided an estimate of tumor perfusion, as confirmed by the blood samples; (b) the tumor FDG uptake after 15 min was a direct measurement of tumor metabolism clearly demonstrating the relative efficacy of the two PDT drugs; and (c) the tumor tracer concentration in the interval 3-15 min after FDG injection is an appropriate indicator of the different mechanisms of tumor necrosis through indirect vascular stasis (PII) or direct cell kill (AIPcS). CONCLUSION: This pilot study confirmed the feasibility of using dynamic in vivo PET imaging for assessing early tumor response to PDT in mice.

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.

Photodynamic therapy: tumor targeting with adenoviral proteins.

A brief summary of the mechanisms involved in photodynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocyanines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus penton base proteins contain the arginine-glycine-aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L-tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro photocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT-6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2-protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base-AlPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT-6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 mumol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adj. A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 mumol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.

Evidence for different mechanisms of EMT-6 tumor necrosis by photodynamic therapy with disulfonated aluminum phthalocyanine or photofrin: tumor cell survival and blood flow.

A comparison was made of photodynamic therapy (PDT) mediated by two photosensitizers, the disulfonated aluminum phthalocyanine (AlPcS2) and Photofrin* (PII) with regard to their mechanism of action on murine tumors. Balb/c mice bearing intradermally growing EMT-6 tumors were injected intravenously with either 1 mumol kg-1 body weight of AlPcS2 or 5 mg/kg of PII 24 h prior to red light irradiation from a Xenon lamp (650-700 nm, 200 mW cm-2, for AlPcS2 and 600-650 nm, 400 J cm-2 for PII. Tumor cell survival following in vivo PDT was determined by an in vitro clonogenicity assay on the dissociated tumors. Immediately after the completion of light irradiation, a reduction of approximately 72% in the number of clonogenic cells was seen with AlPcS2-treated tumor versus approximately 24% of that for PII-treated tumor. Further loss of clonogenic cell survival progressed as a function of time following PDT, and was considered to be the consequence of indirect PDT action, however, the decline in cell viability was steeper in the first 6 h with PII-PDT than with AlPcS2-PDT. 24 h after PDT, the clonogenic capacity of both AlPcS2-and PII-PDT treated tumor fell to approximately 3% of the control tumor. The PDT effect on tumor blood flow as a measure of the tumor vascular damage was monitored by the retention of 99mTc-MIBI in the tumor. Little effect on tumor blood flow was seen with AlPcS2-PDT at 0 h after the completion of light treatment. Thereafter the blood flow declined slowly and remained at approximately 50% the level of the control by 24 h post-PDT. In contrast, PII provoked a approximately 40% reduction of tumor blood flow immediately after the completion of photo irradiation, which then fell to approximately 20% within 2 h and approximately 7% by 24 h post-PDT. These results indicate the involvement of both direct and indirect mechanisms in the PDT induced tumor necrosis. However, AlPcS2-PDT exerted a larger direct tumor cell phototoxic effect, whereas PII-PDT induced tumor cell death to a greater extent via an indirect effect that parallels the extensive damage to the tumor vasculature.

Polyaspartamides as water-soluble drug carriers. Part 1: Antineoplastic activity of ferrocene-containing polyaspartamide conjugates.

BACKGROUND: Numerous detrimental side effects and/or lack of water-solubility of anticancer drugs often prove dose-limiting in chemotherapy. Water-soluble polymeric drug carriers may overcome/minimise many of these limitations. MATERIALS AND METHODS: Aspartic acid polymers to which ferrocene-containing antineoplastic agents are covalently bound, were tested for cytotoxicity against murine EMT-6 cancer cells. Cell survival was measured by means of the colorometric 3-(4,5-dimethylthiazol-2-yl)-diphenyltertrazolium bromide assay. RESULTS: The 90% lethal dosage of pure 3-ferrocenylbutanoic acid is 452 microg/mL LD90 for the polymeric derivative, expressed in terms of 3-ferrocenylbutanoic acid content, is only 65 microg/ml. A polymer structural effect in drug activity was evident: longer side chains linking drugs to polymer backbones enhanced drug activity. Drug activity is also enhanced if drug modifications (to enable drug anchoring) resulted in a lower ferrocenyl reduction potential. CONCLUSIONS: The effectivity of antineoplastic drugs may be enhanced by covalently anchoring them on suitable biodegradable water-soluble polymeric drug carriers.

Photodynamic activities and biodistribution of fluorinated zinc phthalocyanine derivatives in the murine EMT-6 tumour model.

The photodynamic properties and biodistribution pattern of zinc dodecafluoro-4-sulphophthalocyanine (ZnPcF12S1), zinc hexadecafluorophthalocyanine (ZnPcF16) and zinc phthalocyanine (ZnPc) were evaluated in the murine EMT-6 tumour model. All 3 dyes were formulated as a Cremophor oil-water emulsion after initial solubilization in methanol, acetone and pyridine, respectively. Comparison of their phototoxicity after in vitro incubation with EMT-6 cells and exposure to various fluences of red light showed that ZnPcF12S1 is about 50 times more active than ZnPcF16, reflecting better cell-penetrating properties. Solubilisation of ZnPc in 1-methyl-2-pyrrolidinone prior to formulation resulted in loss of photoactivity upon dilution in serum due to precipitation of the dye in the aqueous environment. In contrast, initial solubilisation in pyridine likely forms a ZnPc-pyridinium salt, and this preparation was 6 times more phototoxic than ZnPcF12S1. In vivo comparison of monosulphonated ZnPcF12S1 with perfluorinated ZnPcF16 showed improved pharmacokinetics in mice, including lower liver and spleen retentions and higher tumour-to-non-target tissue ratios. However, photodynamic therapy (PDT) of the EMT-6 tumour in BALB/c mice with red light, 24 or 48 hr post-injection of 1 micromol x kg(-1) of ZnPcF12S1 induced mortality. Lowering the drug and/or light dose or extending the time interval between drug administration and irradiation to 72 hr avoided adverse effects but also resulted in poor tumour response. The best tumour control (25% of animals) was obtained at 0.1 micromol x kg(-1) and a fluence of 400 J x cm(-2) at 24 hr post-injection. In contrast, ZnPcF16 required a 20-fold higher drug dose to induce a similar tumour response. The systemic shock following PDT with the amphiphilic ZnPcF12S1 likely results from extensive cellular effects.

Water-soluble aluminium phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice.

The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13,000-23,000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc-PEG and AlPc-PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75-100% of animals at a low drug dose (0.25 micromol kg(-1)) following PDT (400 J cm(-2), 650-700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 micromol kg(-1). In the non-cured animals, AlPc-PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc-PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this photosensitizer a valuable, water soluble candidate drug for clinical PDT of cancer.