Janusz M. Dąbrowski

New Halogenated Water-Soluble Chlorin and Bacteriochlorin as Photostable PDT Sensitizers: Synthesis, Spectroscopy, Photophysics, and in vitro Photosensitizing Efficacy

Chlorin and bacteriochlorin derivatives of 5,10,15,20‐tetrakis(2‐chloro‐5‐sulfophenyl)porphyrin have intense absorptions in the phototherapeutic window, high water solubility, high photostability, low fluorescence quantum yield, long triplet lifetimes, and high singlet oxygen quantum yields. Biological studies revealed their negligible dark cytotoxicity, yet significant photodynamic effect against A549 (human lung adenocarcinoma), MCF7 (human breast carcinoma) and SK‐MEL‐188 (human melanoma) cell lines upon red light irradiation (cutoff λ<600 nm) at low light doses. Time‐dependent cellular accumulation of the chlorinated sulfonated chlorin reached a plateau at 2 h, as previously observed for the related porphyrin. However, the optimal incubation time for the bacteriochlorin derivative was significantly longer (12 h). The spectroscopic, photophysical, and biological properties of the compounds are discussed in relevance to their PDT activity, leading to the conclusion that the bacteriochlorin derivative is a promising candidate for future in vivo experiments.

Synthesis, photophysical studies and anticancer activity of a new halogenated water-soluble porphyrin

A water‐soluble halogenated porphyrin, namely 5,10,15,20‐tetrakis(2‐chloro‐3‐sulfophenyl)porphyrin (TCPPSO3H), was prepared and evaluated as sensitizer for photodynamic therapy (PDT). Photophysical properties of TCPPSO3H, such as high photostability, long triplet lifetime and high singlet oxygen quantum yield suggest high effectiveness of this class of halogenated porphyrins in PDT. TCPPSO3H is non‐toxic in the dark and causes a significant photodynamic effect examined against MCF7 (human breast carcinoma), SKMEL 188 (human melanoma) and S91(mouse melanoma) cell lines upon red light irradiation (cutoff < 600 nm) at low light doses. Time‐dependent cellular uptake of TCPPSO3H reached plateau at 120 min and was the highest for S91, 20% lower for MCF7 and 70% lower for SKMEL 188. Our results show that this halogenated water‐soluble porphyrin is an efficient photosensitizer and reveal the potential of this class of compounds as PDT agents.

Combined effects of singlet oxygen and hydroxyl radical in photodynamic therapy with photostable bacteriochlorins: evidence from intracellular fluorescence and increased photodynamic efficacy in vitro.

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.

Biodistribution and Photodynamic Efficacy of a Water‐Soluble, Stable, Halogenated Bacteriochlorin against Melanoma

The in vitro phototoxicity of a photostable, synthetic, water‐soluble, halogenated bacteriochlorin, 5,10,15,20‐tetrakis(2‐chloro‐5‐sulfophenyl)bacteriochlorin (TCPBSO3H), toward mouse melanoma (S91) cells is ∼60‐fold higher than that of the analogous porphyrin, and is associated with very weak toxicity in the dark; 90 % of S91 cells were killed in response to a light dose of 0.26 J cm−2 in the presence of [TCPBSO3H]=5 μM. In vivo toxicity toward DBA mice is very low, even at doses of 20 mg kg−1. In vivo pharmacokinetics and biodistribution of TCPBSO3H were studied in DBA mice with S91 tumors; 24 h after intraperitoneal injection of 10 mg kg−1, TCPBSO3H demonstrated preferential accumulation in S91 mouse melanoma, with tumor‐to‐normal tissue ratios of 3 and 5 for muscle and skin, respectively. Photodynamic therapy (PDT) performed under these conditions, with 90 mW cm−2 diode laser irradiation at λ 750 nm for 20 min (total light dose of 108 J cm−2), resulted in tumor regression. Tumor recurrence was observed only approximately two months after treatment, confirming the efficacy of this PDT against melanoma.

Photodynamic Therapy Efficacy Enhanced by Dynamics: The Role of Charge Transfer and Photostability in the Selection of Photosensitizers

Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650-800 nm) and efficiently generate reactive oxygen species (ROS = singlet oxygen and oxygen-centered radicals). We show that the ratios between the triplet photosensitizer-O2 interaction rate constant (kD) and the photosensitizer decomposition rate constant (kd), kD/kd, determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer-oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high kD) must be tempered by photostability (low kd). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.

Tissue Uptake Study and Photodynamic Therapy of Melanoma‐Bearing Mice with a Nontoxic, Effective Chlorin

Chlorins have intense red absorptions and high tumor affinities that make them interesting candidates for photodynamic therapy (PDT) of cancer. This paper reports cytotoxicity, phototoxicity, in vitro cellular uptake, and in vivo biodistribution and PDT efficacy of a synthetic chlorin derivative (TCPCSO3H) towards Cloudman melanoma cells (S91). No cytotoxic effects were observed in vitro at concentrations up to 20 μm, and no toxicity was observed in vivo in DBA mice with doses up to 2 mg kg−1. Pharmacokinetics and biodistribution of TCPCSO3H were evaluated in vivo in DBA mice bearing S91 tumors. TCPCSO3H demonstrated preferential accumulation in S91 mouse melanoma, with tumor‐to‐normal tissue ratios of 5 and 11 for muscle and skin, respectively, 24 h after intravenous injection of 2 mg kg−1. Photodynamic therapy performed under these conditions with 70 mW cm−2 diode laser irradiation at 655 nm for 25 min (total light dose=105 J cm−2) resulted in scab formation, followed by temporary or permanent (>60 days) tumor remission. According to the Kaplan–Meier analysis, the median survival time of the control group was 9 days, whereas that of the treated group was 38 days.

The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy

Blood flow and pO2 changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F2BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F2BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO2 and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO2, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO2 compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.

Elimination of primary tumours and control of metastasis with rationally designed bacteriochlorin photodynamic therapy regimens.

Photodynamic therapy (PDT) with current photosensitisers focuses on local effects and these are limited by light penetration in tissues. We employ a stable near-infrared (NIR) absorbing bacteriochlorin with ca. 8h plasma half-life to increase the depth of the treatment and elicit strong systemic (immune) responses. Primary tumour growth delays and cures of BALB/c and nude mice bearing CT26 mouse colon carcinoma are related to the parameters that control PDT efficacy. The systemic anti-tumour protection elicited by the optimised PDT regimen is assessed by tumour rechallenges and by resistance to the establishment of metastasis after intravenous injection of CT26 cells. The optimised treatment regime offered 86% cure rate in BALB/c mice but no cures in BALB/c nude mice. Cured mice rechallenged over 3 months later with CT26 cells rejected the tumour cells in 67% of the cases. PDT of a subcutaneous CT26 tumour 5days after the additional intravenous injection of CT26 cells very significantly reduced lung metastasis. The PDT regimen optimised for the bacteriochlorin leads to remarkable long-term survival rates, effective immune memory and control of lung metastasis.

Improved biodistribution, pharmacokinetics and photodynamic efficacy using a new photostable sulfonamide bacteriochlorin

The bacteriochlorin-mediated PDT effects on melanoma tumors were investigated in correlation with its biodistribution. The pharmacokinetics of the photostable 5,10,15,20-tetrakis(2,6-dichloro-3-N-ethylsulfamoylphenyl)bacteriochlorin was determined in DBA mice bearing S91 melanoma tumors at different time intervals (2 h–72 h) after i.p. injection of a 10 mg kg−1 drug dose. PDT efficacy was maximal when irradiation was performed 24 h after i.p. administration, and led to the complete disappearance of tumors for nearly 2 months. Compared to the analogue sulfonated compound, the median tumor growth delay with respect to the control group increased from 27 to 44 days. This improvement is attributed to the higher stability, higher absorption in the NIR, amphiphilicity, and better selectivity of the sulfonamide bacteriochlorin.

Modulation of Biodistribution, Pharmacokinetics, and Photosensitivity with the Delivery Vehicle of a Bacteriochlorin Photosensitizer for Photodynamic Therapy

Intravenous (i.v.) formulations with various amounts of organic solvents [PEG400, propylene glycol (PG), cremophor EL (CrEL)] were used to deliver a fluorinated sulfonamide bacteriochlorin to mice, rats, and minipigs. Biodistribution studies in mice showed that a low‐content CrEL formulation combines high bioavailability with high tumor‐to‐muscle and tumor‐to‐skin ratios. This formulation was also the most successful in the photodynamic therapy of mice with subcutaneously implanted CT26 murine colon adenocarcinoma tumors. Pharmacokinetic studies in mice and minipigs revealed that with the same low CrEL formulation, the half‐life of the photosensitizer in the central compartment was longer in minipigs. Differences in biodistribution with the various formulations, and in pharmacokinetics between the two animal species with the same formulation, are attributed to the interaction of the formulations with low‐density lipoproteins (LDLs). Skin photosensitivity studies in rats showed that 30 min exposure of the skin to a solar simulator 7 days after i.v. administration of the fluorinated sulfonamide bacteriochlorin at 1 mg kg−1 did not elicit significant skin reactions.