Photodynamic Effect of Water Soluble Piperazinium and Imidazolium Salts of HPPH on A549 Cancer Cells (2008)

Photodynamic therapy (PDT) is a binary cancer therapy that relies on selective uptake of a photosensitizer in tumor tissues, followed by generation of singlet oxygen and other cytotoxic species upon irradiation with light of appropriate wavelength. Although a number of studies have documented preferential uptake of photosensitizers in tumor tissue, exact mechanism of PDT is not fully understood. There are several possible approaches including cellular specificity such as lower pH and more low-density lipoprotein (LDL) receptors in malignant tissue than normal one. In order to find optimal photosensitizers, different compounds have been synthesized and examined including porphyrins, chlorins, phthalocyanines and purpurins. Among them, chlorins represent the second generation of photosensitizers with promising physicochemical properties and high PDT efficiency. One of the challenging problems of chlorin based photosensitizers is their amphiphilic property due to a hydrophobic macrocycle of chlorins. If a photosenzitizer has too high lipophilicity, it has trouble to pass through blood vessel after intravenous injection. On the other hand, if a photosensitizer has too high hydrophilicity, it is difficult to penetrate cell membrane. Therefore it is important to compromise between hydrophilicity and lipophilicity. Introduction of hydrophilic groups imparts chlorin molecules with amphiphilic properties and, therefore, with good solubility in polar and non polar media. Such properties provide chlorin derivatives with good tumor/tissue ratio, high tumor efficacy and short clearance time. There have been several studies about protoporphyrin and chlorin e6 which are complexed with hydrophilic organic amine such as N-methyl-D-glucamine and arganine to improve their solubility in physiological solutions. As far as we know, no reports have been observed in the literature on 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), which is a promising photosensitizer under clinical trials, complexed with hydrophilic organic amine used as a photosensitizer for PDT. In this study, we have designed two new chlorin derivatives in which piperazine and imidazole groups chosen as a hydrophilic organic amine are non-covalently complexed to the propionic acid residue of HPPH to improve their amphiphilicity. A comparative study of their photodynamic activities on A549 cancer cells is carried out in order to reveal influences of piperazine and imidazole moieties on biological activities of HPPH. We have synthesized pyropheophorbide-a by several steps of chemical reactions including preparation of chlorophyll-a from Spirulina pacifica biomass, further conversion into HPPH, according to standard procedures. Salt-like complexes of HPPH, including complex of HPPH with piperazine (PIP) and complex of HPPH with imidazole (IMI), were obtained during HPPH treating with an organic free base-piperazine and imidazole, respectivily. Initial reagents were taken in 1:1 molar ratio in 1:3 methanol: dichloromethane. It is shown that formation of salt bond between carboxy group of parent chlorin and amino groups of piperazine and imidazole is a simple and effective method to obtain HPPH salt-like complexes (Fig. 1). We have determined octanol/water partition coefficient (logP) for PIP, IMI and HPPH to evaluate amphiphilic properties. The result demontstrated that hydrophilicity of PIP (logP ~ 2.8) and IMI (logP ~ 3.1) is more than that of HPPH (logP ~ 5.7). The biological activity of compound PIP and IMI complex of HPPH was evaluated in contrast to HPPH in A549 human