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Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy
Accepted Manuscript Title: Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy Author: Zhou Jiang Jingwei Shao Tingting Yang Jian Wang Lee JiaThe authors equally contribute to this work.
S0731-7085(13)00215-X http://dx.doi.org/doi:10.1016/j.jpba.2013.05.014 PBA 9075
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
2-4-2013 4-5-2013 11-5-2013
Please cite this article as: Z. Jiang, J. Shao, T. Yang, J. Wang, L. Jia, Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmaceutical development, composition and quantitative
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photodynamic therapy
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analysis of phthalocyanine as the photosensitizer for cancer
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Zhou Jiang†, Jingwei Shao†, Tingting Yang, Jian Wang, Lee Jia*
Cancer Metastasis Alert and Prevention Center, College of Chemistry
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and Chemical Engineering, Fuzhou University, 523 Industry Road, Fuzhou, Fujian 350002, China.
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* Corresponding author: Lee Jia, Email address: [email protected];
The authors equally contribute to this work
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†
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Tel. +86-15159630201; fax: +86 0591 83792563.
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Highlights Mechanisms of action of phthalocyanines (Pcs) used for photodynamic therapy
Quantitative methods for analyzing Pcs
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Current status of the Pcs under clinical investigation
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Pcs pharmaceutical development to improve their drugability & cellular localization
Abstract
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Phthalocyanine (Pc) and its related derivatives are a class of functional materials that are easily activated by the light at a special wavelength. As such
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photosensitizer, Pc has been applied to photodynamic therapy (PDT), in addition to its broad applications in many fields, for both malignant and benign diseases. One of our long-term research focuses is to develop Pc for cancer
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therapy. Herein we briefly review mechanisms of action of Pc used for
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photodynamic therapy, its pharmaceutical development and molecular modification to enhance its drugability and improve its intracellular localization.
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We also describe the current status of the Pc derivatives under clinical investigation, and analyze the methods used for quantitative analysis of those Pc derivatives.
Keywords:
Photodynamic therapy; Reactive oxygen species; Photosensitizer; Phthalocyanine
Abbreviations： Phthalocyanine
Pc
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phthalocyanine zinc
ZnPc
sulfonated phthalocyanine aluminum
AlPcS
PDT
reactive oxygen species
ROS
singlet molecular oxygen
1
single-chain Fv fragments
scFv
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O2
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photodynamic therapy
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di-(potassium sulfonate) -di-phthalimidomethyl ZnPcS2P2 phthalocyanine zinc
arginine-glycine-aspartic acid
RGD
EGFR
palmitoyl-oleoyl-phosphatidylcholine
OOPS
United States Pharmacopoeia
USP ER
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endoplasmic reticulum
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di-oleoyl phosphatidylserine
OPOC
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epidermal growth factor receptor
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1. Introduction
Phthalocyanine (Pc, Fig.1) and its derivatives are widely used as functional
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materials in many high technique fields, such as data storage, photoelectronic
generation, catalyst and so on. Using as photosensitizer in photodynamic
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therapy (PDT) is the most attractive application of Pc to various clinical trials
and has attracted intensive interests since 1990s. Nowadays, there are
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several phthalocyanines under clinical evaluation. However, there are only a few literature focused on pharmaceutical development and quantitative
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analysis of clinical phthalocyanines. Herein, we will comprehensively review the current status of Pc-based photosensitizer and our experience in analysis
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of sulfonic phthalocyanines.
development
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2. Mechanisms of photodynamic therapy and its pharmaceutical
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In the war on cancer, continues progresses have been achieved with the
great efforts to treat cancer mortality with drugs with better cure and less adverse effects. PDT is such a minimal invasive treatment which is currently used for malignant or benign diseases [1-4]. The therapy drug used for PDT is known as photosensitizer.
PDT involves two procedures. The first procedure is the topical or
systemical administration of a light-sensitive photosensitizer which should be selectively accumulated in the target tissue and/or cells. When the concentration of photosensitizer on the lesion is high enough and has a suitable ratio to adjacent normal tissue cells, the second procedure will be carried out, to deliver a specific wavelength of light to the lesion and activate 4
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the accumulated photosensitizer. The excited photosensitizer transfers the energy to surrounding oxygen to generate cytotoxic reactive oxygen species (ROS). The ROS, primarily singlet molecular oxygen (1O2), is responsible for the cascade of a series of cellular and molecular events that result in selective
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lesion destruction [5, 6]. The biological mechanisms of destroying the targeted lesion involve in direct cell killing of tumor cells (cellular PDT), indirect cell
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killing by vascular occlusion (vascular PDT), and the response of the immune
system [4, 5]. Selective accumulation of photosensitizer and controlled
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administration of light to lesion provide advantages of PDT over the three major treatments of malignant tumor (surgery, radiation therapy and
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chemotherapy), such as protection of functional structures, excellent cosmetic outcome, the safety of repeated uses, less side effects, fast convergence, and
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lower economic costs.
The development of photosensitizers plays a very important role in PDT.
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Therefore, photosensitizer has become an important field of research for both
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chemical and pharmaceutical sciences. The modern era of PDT started with the discovery of hematoporphyrin (Fig. 1) derivatives, the first generation of Photofrin® (a
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photosensitizers [7].
purified form
of hematoporphyrin
derivatives), the first commercial photosensitizer was approved for clinical use for bladder cancer in 1993 in Canada. From then on, PDT has been approved by regulatory authorities of other countries, such as the United States, Japan, the Netherlands and others in the world. PDT has been applied for more indications, such as esophagus, skin, head and neck, and lung cancers. At the same time, PDT is also applied to some non-malignant diseases, such as port wine stains, actinic keratoses, age-related macular degeneration, and localized infection [3, 4, 8-12]. Although Photofrin® has made considerable success and is still widely used on clinical, it has some shortages, such as moderate efficient and 5
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long-lasting skin photosensitization for several weeks, and composition with undefined mixture [7, 13]. This led to the development of new generation of photosensitizers. For the design of an ideal photosensitizer, the properties mentioned in the following should be fulfilled, such as: single and chemically
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pure compound; stability and good solubility in pharmaceutically acceptable
formulations and in biological media; high efficiency to yield ROS under
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illumination; fluorescence; no dark toxicity; fast clearance from the healthy
parts of the body and specific retention in diseased tissues; strong absorbance
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in Near-IR region and minimal absorbance between 400 and 600 nm [10, 13, 14]. Some photosensitizers of the second generation have been approved at
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clinical.
3. Pharmaceutical modification of Pc and its derivatives to increase their
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drugability
Various approaches have been made to improve the pharmaceutical
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properties of photosensitizers. One logical approach is through conjugation of
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photosensitizers to various linkers including antibodies, proteins and peptides. Phthalocyanine zinc was first tried to be conjugated to monoclonal antibodies
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that are specific for tumor-associated antigens [15]. However, the large size of antibodies hinders tissue penetration and lowers cellular uptake when used in vivo. The attachment of photosensitizers to monoclonal antibodies may reduce the antigenic specificity of monoclonal antibodies. As a result, smaller size of biomolecules has been searched for as the more effective alternative. Conjugation of photosensitizers with albumins and low-density lipoproteins has been explored, but only moderate target specificity has been achieved so far [16]. Smaller antibody fragments, such as single-chain Fv fragments (scFv) have received considerable attention as a good candidate [17]. Among various carriers for active drug targeting, synthetic peptides are of particular interest. Peptides with appropriate sequences can specifically bind to different surface 6
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biomarkers of cancer cells and circulating tumor cells [18, 19]. For instance, two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated
phthalocyanine
zinc
with
acetylenic
bombesin
or
arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid
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phase, have been explored to make a series of phthalocyanine zinc conjugates to target the gastrin-releasing peptide and integrin receptors [20].
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The peptide conjugation enhanced water solubility of phthalocyanine zinc and improved the latter’s photodynamic efficacy against cancer cell lines
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expressing gastrin-releasing peptide and integrin receptors. Ongarora et al [21] synthesized four Pc-peptide conjugates for targeting the epidermal growth
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factor receptor (EGFR) and evaluated the in vitro efficacy of the conjugates by using different cell lines. Two peptide ligands linked to 6 (EGFR-L1) and 13
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(EGFR-L2) amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical and experimental models. The Pc-EGFR-L1 conjugates efficiently targeted EGFR and were internalized,
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in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2
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conjugates targeted EGFR poorly probably because of their low aqueous
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solubility. All conjugates were nontoxic (IC50> 100 μM) to HT-29 colon cancer cells, both in the dark and upon light activation (1 J/cm2). Intravenous
administration of the conjugate into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a Near-IR fluorescence signal at 700 nm, 24 h after administration.
To increase the cell-targeting ability, two ZnPc-peptide conjugates, which
bore either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences, have been synthesized to evaluate the effect of the linker [22]. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The 7
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highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human
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HepG2 cells (IC50 77 μM) but were highly phototoxic (IC50< 2 μM at 1 J/ cm2). An alternative strategy to improve pharmaceutical properties of Pc
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involves direct formulations of Pc to increase its solubility (Table 1). ZnPc is lipophilic and slightly soluble in water or in a commonly-used pharmaceutical
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vehicle. This hydrophobic characteristics of ZnPc restricts its clinical studies. Cremophor EL is a polyethoxylated castor oil. Its major component is the
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material in which the hydroxyl groups of the castor oil triglyceride have ethoxylated with ethylene oxide to form polyethylene glycol ethers. This surfactant has been used to enhance solubility of those poorly-soluble
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materials including Pc by stabilizing emulsions of nonpolar materials in aqueous systems. We utilized Cremophor EL to increase solubility of
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Photocyanine [23] and ZnPc [24]. The formulation provided us the possibility to
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investigate in vitro activity and subcellular distribution of Pc analogues in cells.
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4. Intracellular localization of Pc and its derivatives Intracellular localization of Pc-like photosensitizers is usually determined by
using
confocal
fluorescence
microscopy.
Variations
in
intracellular
accumulation and the resulting phototoxicity between the conjugates correlate to
their
differences
in
hydrophobicity
as
well
as
cell
membrane
receptor-mediated uptake. It has been reported that the ZnPc conjugate bearing the long PEG-linker accumulated within cells 26 times more than the unconjugated ZnPc, and the short PEG-linker conjugate accumulated within cells 17 times more than the unconjugated ZnPc [22]. The result suggests the enhancement of PEG on transport of Pc across the cellular membrane.
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In the case of the phthalocyanine-bombesin conjugate, competition experiments confirmed the involvement of the membrane gastrin-releasing peptide receptor in both the photosensitizer activity as well as intracellular
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localization of Pc [20]. In our experiments, when the hepatocellular carcinoma HepG2 cells were
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incubated with ZnPc (10 μM) in dark, we could observe intracellular distribution of ZnPc under confocal laser microscopy with the aid of subcellular probes.
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Different emission wavelengths should be applied to corresponding probes of subcellular organelles. Usually, lysosome is stained with yellow probe and
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emitted at 577 nm; mitochondria is marked with green and emitted at 490 nm, and endoplasmic reticulum is stained blue and emitted at 587 nm. Using confocal laser microscopy with the aid of those probes, we demonstrated that
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ZnPc was predominately localized in polarized perinuclear regions such as
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mitochondria, lysosome and endoplasmic reticulum (Fig. 3).
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5. Pc-based photosensitizer and their quantitative analysis Phthalocyanine (Pc) and its related derivatives have been intensively
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investigated as the second generation photosensitizers since 1985. They are structurally related to porphyrins. With respect to the longest wavelength absorption of hematoporphyrin (near 630 nm), the absorption of Pc on Near-IR (670-780nm) is almost two orders of magnitude stronger [25]. With appropriate central atom, such as zinc, aluminum or silicon, phthalocyanine exhibits many other optimal properties that fulfill the requirements of an ideal photosensitizer mentioned above, such as high efficiency of ROS generation upon illumination, high stability, fluorescence, low intrinsic toxicity, high flexibility in structure modification and ease of synthesis [13, 14]. However, there are two principal shortages of Pc based photosensitizers: low water-solubility and tendency to aggregate. Most of the unsubstituted phthalocyanines are poorly soluble in 9
Page 9 of 27
water and normal organic solvents. Plate-like Pc molecules tend to form aggregation, which leads to the loss of PDT activity and fluorescence. The way to overcome these shortages lies on structure modification, suitable delivery system, or adds suitable solvent (such as pyridine) to reduce aggregation.
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Considerable and continuing study has been carried out during the past 3 decades. However, most of the researches are still in the preclinical stage. Up
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to date, there are only four Pc-based photosensitizers, i.e. Photosens, Pc 4, CGP55847 (ZnPc) and Photocyanine, available for clinical uses or trails in
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different countries (Table 1 and Fig. 2).
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A suitable quantitative analysis method is not only the need for quality control of photosensitizer drug with certified quality and composition, but also the requirements for clinical applications. Optical quantification methods,
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namely absorption and fluorescence spectroscopes are generally used. Fluorescence method has the potential of higher sensitivity over absorption,
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while they are both noninvasive and rapid. The phthalocyanines utilized as the
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photosensitizer all have fluorescence in Near-IR region, which gives Pc advantage to avoid interference from endogenous substances and makes
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quantitative analysis of Pc much easy without requirements for the labor-intensive separation. Fluorimetric assays were frequently used, especially for the early researches on biological samples [26-28]. A series of fluorescence diagnostic instruments have been developed and successfully used at clinical not only for measuring but also for controlling the PDT parameters [29]. High performance liquid chromatography (HPLC) is also a powerful tool in quantitative and qualitative analysis of pharmaceutical Pc. Various choices of both mobile and stationary phase enhance performance of HPLC in analyte separation, which will favor the research for drug metabolites and the drug with multi-component.
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6 Individual hydrophobic Pc-based photosensitizers 6.1. CGP55847 (ZnPc) CGP55847 is unsubstituted phthalocyanine zinc (ZnPc, Fig. 2) formulated in liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (OPOC) and
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di-oleoyl phosphatidylserine (OOPS) in a ratio of ZnPc: OPOC: OOPS at 1: 90: 10 (w/w/w). It was developed by QLT Phototherapeutics (Vancouver, Canada)
and sponsored by Ciba Geigy (Novartis, Basel, Switzerland). It is the first Pc
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reaching clinical trials (Phase I/II, Switzerland) for PDT of early cancer in the
upper aerodigestive tract. However, the clinical trials were discontinued and
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the reason is not clear [13, 30].
In the literature available to us, the quantitative analysis and in vivo
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pharmacokinetics of CGP55847 is based on the fluorescence method [31，32]. Some studies of other groups stated that ZnPc in biological matrices, which consists of a single isomer and can be prepared with high chemical purity, is used as internal standard [33,34]. 6.2. Pc 4
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also suitable for HPLC quantifications. Actually, in those reports, the ZnPc was
Fig. 2) and was developed for PDT application. Like ZnPc, It
has no isomer [35].
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-(CH2)3N(CH3)2,
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Pc 4 is a phthalocyanine silicon with axial substitutions (HOSiPcOSi(CH3)2
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Pc 4 had undertaken two phase I clinical trials approved by the FDA. Its
first clinical phase I trial aimed at systemic delivery of this photosensitizer for PDT of cutaneous malignancies initiated in 2001. The formulation for injection was prepared by dissolving 1 mg Pc 4 and 5 mg Povidone in 1 mL of Cremophor EL:ethanol=1:1 (NCI Diluent 12) following by being diluted with 9 volumes of normal saline.
The trial was terminated in 2006, because of
difficulty in accrual. However, three patients were treated with interesting results. The limit clinical trial showed Pc 4-PDT was well tolerated by subjects [36]. Another phase I clinical trial was conducted in 2004 for topical application of Pc 4. Pc 4 was dispensed after formulation in a vehicle of the United States Pharmacopoeia (USP) grade of propylene glycol and USP grade of ethanol(70/30%, v/v). Topical application is a convenient and safe method of 11
Page 11 of 27
specifically directing photosensitizers to accessible lesions, while avoiding the widespread distribution that occurs following intravenous administration. The trials were completed in 2010 and demonstrated the potential for topical delivery of Pc 4 to treat cutaneous neoplasms with good tolerability and no
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adverse effects [35, 37]. Pharmacology studies of Pc 4 in plasma or tissue were carried out with nile blue or a closely related molecular Pc 34 as the internal standard, by
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HPLC or HPLC/tandem mass spectrometry. The chromatographic separation
was accomplished on a reverse-phase column with an isocratic mobile phase
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consisting of methanol and ammonium formate buffer [36, 38]. 7． Sulfonic Pc-based hydrophilic photosensitizers
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The matrix of Pc is highly hydrophobic. Many hydrophilic groups, such as sulfonic, carboxyl, and phosphoric group, have been introduced to improve Pc’s solubility in physiological solution. Among the water soluble Pcs, sulfonic
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Pc has received the most intensive investigation as a photosensitizer. In the past tens of years, sulfonic Pc has been extensively studied [11, 14]. Unlike composition.
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the two phthalocyanines discussed above, sulfonic Pc is always complex in
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The synthesis of sulfonic Pc inevitably results in mixtures of its various isomers. Two main methods to produce sulfonic Pc are direct sulfonation
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method or condensation method. By direct sulfonation, all the four H atoms in each isoindole subunit of Pc have chance of substitution by sulfonic groups, thus resulting products are generally quite complex with compositions with different number of sulfonic groups on either α- or β-position (see Fig.1 for the
illustration of α- or β-position) [39-42]. The second method, which is also called the Weber-Busch method, involves the condensation of the mono-sulfonated precursors (such as phthalonitriles or phthalic acids) in the presence of urea, the selected metal salt and a catalyst. The mix condensation of sulfonic and unsubstituted precursors results in Pc components of 0 to 4 sulfonic groups. Expect for the unsubstituted and mono-sulfonated Pc, the other components are all mixture with different number of isomers [40, 43]. The much more complex substitution resulted from direct sulfonation leads to overlap of Pc with different degrees of sulfonation. But it has not been reported in the case of 12
Page 12 of 27
mixed condensation. In comparison of different methods for separating the sulfonic phthalocyanines, the van Lier’s group [40] found that HPLC with gradient elution was relatively satisfactory if phosphate buffer and methanol were used as the mobile phase with the C18 column. The adjacent forms of
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di-sulfonic Pc, i.e. the isomers with sulfonic groups on the same side of Pc ring are considered to be the most active photosensitizer for their amphiphilic
property [41, 44]. There are two sulfonic phthalocyanines are in clinical
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evaluation.
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7.1. Photosens® (Photosense)
Photosens® (Fig. 2) is a distilled-water solution of sodium salts of
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sulfonated phthalocyanine aluminum (AlPcS) that shows maximum absorption at 675 nm. This water soluble mixture is obtained by direct sulfonation and constituted of AlPcS with di- to tetra-sulfonic acid substituted. Photosense®
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was developed by the “NIOPIC” Moscow Research and Production Association. It is commercialized in Russia by NIOPIC [13, 14, 45, 46] and is highly effective in the treatment of various cancers, histological type and at
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various stages, such as squamous cell skin cancer, breast cancer,
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oropharyngeal cancer, lung cancer, eye and eyelid related tumors, bladder and cervical cancer, and so on. It is also used for the treatment of severe
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festering wounds, trophic ulcers, and some other nonmalignant maladies. The administration doses range from 0.5 to 2.0 mg per kg of the patient’s body weight. The introduction of sodium ascorbate in doses of 20–50 mg/kg body weight in addition to Photosens results in an improvement in therapeutic action and allows a twofold reduction of the Photosens dose. However, skin photosensitivity is a significant problem with this drug. The patient should remain heliophobic for 6 to 10 weeks after treatment [3, 10, 12, 47]. Fluorescence method had been developed to study pharmacokinetics of Photosens
[48,
49].
At
clinical,
the
computerized
fluorescent
spectrophotometry was used to monitor the photosensitizer in body before and after treatment for optimal therapy parameters [45, 47, 48, 50].
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7.2. Photocyanine (Suftalan Zinc) Suftalan Zinc (trade name: Photocyanine, Fig. 2) is an amphiphilic photosensitizer developed by our University, China. It is a mixture of four adjacent
form
isomers
(SSPP
type)
of
di-(potassium
sulfonate)
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-di-phthalimidomethyl phthalocyanine zinc (ZnPcS2P2, here, S and P represent sulfonate group and phthalimidomethyl group, respectively). It is injected in the 0.5 mg/mL solution formulated with 2% Cremophor EL, 20% 1,2-propylene
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glycol and 0.9% sodium chloride. The phase I clinical trials of Photocyanine began in 2008 with the approval of State Food and Drug Administration of
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China. Currently, Photocyanine is being applied for phase II clinical trials. The mix condensation method of 4-phthalimidomethyl phthalonitrile and
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4-sulfonic phthalonitrile has been developed instead of the previouly ‘in a plot reaction’ [51] to obtain less complex product. However, there are still 5 opposite (SPSP type) and 10 adjacent form (SSPP type) isomers existing in
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ZnPcS2P2 [52]. Several HPLC procedures have been carried out for preparation of Photocyanine. For quality control and quantitative analysis, great efforts have been taken to optimize HPLC method for separation of the
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four very close isomers. A complex mobile phase, i.e., phosphate-TEA: DMF:
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THF: CH3OH, 50: 37.5: 7.5: 5 (v/v/v/v) on a C18 column was developed. We found that addition of ion-pairing agents with positive charges to the mobile
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phase improved the resolution of di-, tri-, and tetra-sulfonic Pc isomers [43]. An ion-pair reversed-phase HPLC method using a mixture of acetonitrile and ion-pair buffer (0.01 M hexadecyl trimethyl ammonium bromide and 0.01 M potassium dihydrogen phosphate, adjusted the pH value to 6.8 with potassium hydroxide solution) was developed and gained similar efficiency [53].
8. Conclusions
Phthalocyanines are promising photosensitizers for PDT applications. Great emphasis has been placed on structural modification of the compounds or smart delivery system, while there are still only a few are on clinical application or trials. The experiences of those pioneers have shown the importance of quantitative methods for advancing the development of 14
Page 14 of 27
photosensitizers. The noninvasive optical method, and specific and sensitive HPLC method should both receive more attention than before. . Acknowledgements
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The authors would like to appreciate the financial support of the National Science Foundation of China (81201709, 81273548 and 21101028), the China
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Postdoctoral Science Foundation (No.2012M511441), the Science and
Technology Foundation of Fujian Province of China (2011J0104) and the
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Science Foundation of Education Department of Fujian Province of China
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an
(JK2010003).
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Legends for Figures: Fig.1.
Chemical
structures
of
representative
hematoporphyrin
and
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Fig. 2. Pc-based photosensitizers under clinical evaluation.
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phthalocyanines
Fig. 3. Intracellular distribution of ZnPc in HepG2 localized by fluorescence
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phase-contrast image without the irradiation. The upper, middle and lower panels showed images of cellular compartments lysosome, mitochondria and
an
endoplasmic reticulum (ER) respectively. B, E and H showed intracellular particles ZnPc in a granular pattern detected by red fluorescence. C, F and I showed the red granular particles ZnPc (10 μmol/L) merged on yellow
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lysosome (C), green mitochondria (F) and blue endoplasmic reticulum (I),
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respectively.
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References [1] Brown, S. B.; Brown, E. A.; Walker, I., The present and future role of photodynamic therapy in cancer treatment. The Lancet Oncology 2004, 5 (8), 497-508.
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[2] Yano, S.; Hirohara, S.; Obata, M.; Hagiya, Y.; Ogura, S.-i.; Ikeda, A.; Kataoka, H.; Tanaka, M.; Joh, T., Current states and future views in photodynamic therapy. Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2011, 12 (1), 46-67.
us
[3] Allison, R. R.; Sibata, C. H., Oncologic photodynamic therapy photosensitizers: A clinical review. Photodiagnosis and Photodynamic Therapy 2010, 7 (2), 61-75. [4] Dolmans, D. E.; Fukumura, D.; Jain, R. K., Photodynamic therapy for cancer. Nat Rev Cancer 2003, 3 (5), 380-387.
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[5] Macdonald, I. J.; Dougherty, T. J., Basic principles of photodynamic therapy. Journal of Porphyrins and Phthalocyanines 2001, 05 (02), 105-129.
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[6] Josefsen, L. B.; Boyle, R. W., Photodynamic therapy and the development of metal-based photosensitisers. Met Based Drugs 2008, 2008, 276109. [7] Allison, R. R.; Mota, H. C.; Sibata, C. H., Clinical PD/PDT in North America: An historical review. Photodiagnosis and Photodynamic Therapy 2004, 1 (4), 263-277.
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[8] Kharkwal, G. B.; Sharma, S. K.; Huang, Y. Y.; Dai, T.; Hamblin, M. R., Photodynamic therapy for infections: clinical applications. Lasers Surg Med 2011, 43 (7), 755-767.
Ac ce p
[9] Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T., Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chem Rev 2010, 110 (5), 2795-2838. [10] Allison, R. R.; Downie, G. H.; Cuenca, R.; Hu, X. H.; Childs, C. J. H.; Sibata, C. H., Photosensitizers in clinical PDT. Photodiagnosis and Photodynamic Therapy 2004, 1 (1), 27-42. [11] Pandey, R. K., Recent advances in photodynamic therapy. J. Porphyrins Phthalocyanines 2000, 4, 368-373. [12] Bonnett, R., Chemical Aspects of Photodynamic Therapy. Gordon & Breach Publishing Group: 2000. [13] Sekkat, N.; van den Bergh, H.; Nyokong, T.; Lange, N., Like a bolt from the blue: phthalocyanines in biomedical optics. Molecules 2012, 17 (1), 98-144. [14] Lukyanets, E. A., Phthalocyanines as Photosensitizers in the Photodynamic Therapy of Cancer. Journal of Porphyrins and Phthalocyanines 1999, 03 (06), 424-432. 17
Page 17 of 27
[15] Miller, G. G.; Lown, J. W., Immunophotodynamic therapy: Current developments and future prospects. Drug Development Research 1997, 42 (3-4), 182-197. [16] Sharman, W. M.; van Lier, J. E.; Allen, C. M., Targeted photodynamic therapy via receptor mediated delivery systems. Adv Drug Deliv Rev 2004, 56 (1), 53-76.
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[17] Bhatti, M.; Yahioglu, G.; Milgrom, L. R.; Garcia-Maya, M.; Chester, K. A.; Deonarain, M. P., Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments. Int J Cancer 2008, 122 (5), 1155-1163.
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[18] Jia, L.; Gorman, G. S.; Coward, L. U.; Noker, P. E.; McCormick, D.; Horn, T. L.; Harder, J. B.; Muzzio, M.; Prabhakar, B.; Ganesh, B.; Das Gupta, T. K.; Beattie, C. W., Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination. Cancer Chemother Pharmacol 2011, 68 (2), 513-524.
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[19] Jia, L.; Noker, P. E.; Piazza, G. A.; Leuschner, C.; Hansel, W.; Gorman, G. S.; Coward, L. U.; Tomaszewski, J., Pharmacokinetics and pharmacodynamics of Phor21-betaCG(ala), a lytic peptide conjugate. J Pharm Pharmacol 2008, 60 (11), 1441-1448.
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[20] Ranyuk, E.; Cauchon, N.; Klarskov, K.; Guerin, B.; van Lier, J. E., Phthalocyanine-Peptide Conjugates: Receptor-Targeting Bifunctional Agents for Imaging and Photodynamic Therapy. J Med Chem 2013, epub ahead of print.
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[21] Ongarora, B. G.; Fontenot, K. R.; Hu, X.; Sehgal, I.; Satyanarayana-Jois, S. D.; Vicente, M. G., Phthalocyanine-peptide conjugates for epidermal growth factor receptor targeting. J Med Chem 2012, 55 (8), 3725-3238.
Ac ce p
[22] Sibrian-Vazquez, M.; Ortiz, J.; Nesterova, I. V.; Fernandez-Lazaro, F.; Sastre-Santos, A.; Soper, S. A.; Vicente, M. G., Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates. Bioconjug Chem 2007, 18 (2), 410-420. [23] Shao, J.; Xue, J.; Dai, Y.; Liu, H.; Chen, N.; Jia, L.; Huang, J., Inhibition of human hepatocellular carcinoma HepG2 by phthalocyanine photosensitiser PHOTOCYANINE: ROS production, apoptosis, cell cycle arrest. Eur J Cancer 2012, 48 (13), 2086-2096. [24] Shao, J.; Dai, Y.; Zhao, W.; Xie, J.; Xue, J.; Ye, J.; Jia, L., Intracellular distribution and mechanisms of actions of photosensitizer Zinc(II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells. Cancer Lett 2013, 330 (1), 49-56. [25] Josefsen, L. B.; Boyle, R. W., Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics. Theranostics 2012, 2 (9), 916-966. [261] Lilge, L.; O'Carroll, C.; Wilson, B.C., A solubilization technique for photosensitizer quantification in ex vivo tissue samples. Journal of Photochemistry and Photobiology B: Biology 1997, 39 (3), 229-235. 18
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[27] Cubeddu, R.; Pifferi, A.; Taroni, P.; Torricelli, A.; Valentini, G.; Comelli, D.; D'Andrea, C.; Angelini, V.; Canti, G., Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine. Photochem Photobiol 2000, 72 (5), 690-695.
ip t
[28] Siqueira-Moura, M. P.; Primo, F. L.; Peti, A. P.; Tedesco, A. C., Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers. Pharmazie 2010, 65 (1), 9-14. [29] Loschenov, V.; Konov, V.; Prokhorov, A., Photodynamic therapy and fluorescence diagnostics. Laser Physics-Lawrence 2000, 10 (6), 1188-1207.
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[30] Hadjur, C.; Wagnières, G.; Ihringer, F.; Monnier, P.; van den Bergh, H., Production of the free radicals O.-2 and .OH by irradiation of the photosensitizer zinc(II) phthalocyanine. Journal of Photochemistry and Photobiology B: Biology 1997, 38 (2–3), 196-202.
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[31] Love, W. G.; Duk, S.; Biolo, R.; Jori, G.; Taylor, P. W., Liposome-mediated delivery of photosensitizers: localization of zinc (II)-phthalocyanine within implanted tumors after intravenous administration. Photochem Photobiol 1996, 63 (5), 656-661.
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[32] van Leengoed, H. L.; van der Veen, N.; Versteeg, A. A.; Ouellet, R.; van Lier, J. E.; Star, W. M., In vivo fluorescence kinetics of phthalocyanines in a skin-fold observation chamber model: role of central metal ion and degree of sulfonation. Photochem Photobiol 1993, 58 (2), 233-237.
te
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[33] Oliveira, L. T.; Garcia, G. M.; Kano, E. K.; Tedesco, A. C.; Mosqueira, V. C., HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies. J Pharm Biomed Anal 2011, 56 (1), 70-77.
Ac ce p
[34] Bohn, T.; Walczyk, T., Determination of chlorophyll in plant samples by liquid chromatography using zinc-phthalocyanine as an internal standard. J Chromatogr A 2004, 1024 (1-2), 123-128. [35] Miller, J. D.; Baron, E. D.; Scull, H.; Hsia, A.; Berlin, J. C.; McCormick, T.; Colussi, V.; Kenney, M. E.; Cooper, K. D.; Oleinick, N. L., Photodynamic therapy with the phthalocyanine photosensitizer Pc 4: the case experience with preclinical mechanistic and early clinical-translational studies. Toxicol Appl Pharmacol 2007, 224 (3), 290-299. [36] Kinsella, T. J.; Baron, E. D.; Colussi, V. C.; Cooper, K. D.; Hoppel, C. L.; Ingalls, S. T.; Kenney, M. E.; Li, X.; Oleinick, N. L.; Stevens, S. R.; Remick, S. C., Preliminary clinical and pharmacologic investigation of photodynamic therapy with the silicon phthalocyanine photosensitizer pc 4 for primary or metastatic cutaneous cancers. Front Oncol 2011, 1, 14. [37] Baron, E. D.; Malbasa, C. L.; Santo-Domingo, D.; Fu, P.; Miller, J. D.; Hanneman, K. K.; Hsia, A. H.; Oleinick, N. L.; Colussi, V. C.; Cooper, K. D., Silicon phthalocyanine (Pc 4) photodynamic therapy is a safe modality for cutaneous neoplasms: results of a phase 1 clinical trial. Lasers Surg Med 2010, 42 (10), 728-735. 19
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[38] Zuk, M. M.; Kenney, M. E.; Horowitz, B.; Ben-Hur, E., High-performance liquid chromatographic determination of the silicon phthalocyanine Pc 4 in human blood. J Chromatogr B Biomed Appl 1995, 673 (2), 320-324.
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[39] Brasseur, N.; Ali, H.; Langlois, R.; van Lier, J. E., Biological Activities of Phthalocyanines-IX. Photosensitization of V-79 Chinese Hamster Cells and EMT-6 Mouse Mammary Tumor by Selectively Sulfonated Zinc Phthalocyanines. Photochem Photobiol 1988, 47 (5), 705-711.
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[40] Ali, H.; Langlois, R.; Wagner, J. R.; Brasseur, N.; Paquette, B.; van Lier, J. E., Biological activities of phthalocyanines-X. Synthesis and Analyses of sulfonated phthalocyanines. Photochem Photobiol 1988, 47 (5), 713-717.
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[41] Bishop, S. M.; Khoo, B. J.; MacRobert, A. J.; Simpson, M. S.; Phillips, D.; Beeby, A., Characterisation of the photochemotherapeutic agent disulphonated aluminium phthalocyanine and its high-performance liquid chromatographic separated components. J Chromatogr 1993, 646(2), 345-350.
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[42] Schofield, J.; Asaf, M., Analysis of sulphonated phthalocyanine dyes by capillary electrophoresis. Journal of Chromatography A 1997, 770 (1–2), 345-348.
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[43] Jiang, Z.; He, W.; Yao, H.; Wang, J.; Chen, N.; Huang, J., Isomeric separation and identification of tetra-, tri-, and di-β-sulphonic phthalocyanine zinc complexes. Journal of Porphyrins and Phthalocyanines 2011, 15 (2), 140-148.
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[44] Brasseur, N.; Ali, H.; Langlois, R.; van Lier, J. E., Biological Activities of Phthalocyanines-VII. Photoinactivation of V-79 Chinese Hamster Cells by Selectively Sulfonated Gallium Phthalocyanines. Photochem Photobiol 1987, 46 (5), 739-744.
Ac ce p
te
[45] Sokolov, V. V.; Chissov, V. I.; Yakubovskaya, R. I.; Aristarkhova, E. I.; Filonenko, E. V.; Belous, T. A.; Vorozhtsov, G. N.; Zharkova, N. N.; Smirnov, V. V.; Zhitkova, M. B., Photodynamic therapy (PDT) of malignant tumors by photosensitzer photosens: results of 45 clinical cases. Proc. SPIE 2625, Photochemotherapy: Photodynamic Therapy and Other Modalities 1996, 281-287. [46] Filonenko, E. V.; Sokolov, V. V.; Chissov, V. I.; Lukyanets, E. A.; Vorozhtsov, G. N., Photodynamic therapy of early esophageal cancer. Photodiagnosis and Photodynamic Therapy 2008, 5 (3), 187-190. [47] Stranadko, E. P.; Skobelkin, O. K.; Markichev, N. A.; Riabov, M. V., Photodynamic therapy of recurrent cancer of oral cavity. Proc. SPIE 2887, Lasers in Medicine and Dentistry: Diagnostics and Treatment 1996, 233-235. [48] Zharkova, N. N.; Kozlov, D. N.; Smirnov, V. V.; Sokolov, V. V.; Chissov, V. I.; Filonenko, E. V.; Sukhin, G. M.; Galpern, M. G.; Vorozhtsov, G. N., Fluorescence observations of patients in the course of photodynamic therapy of cancer with the photosensitizer PHOTOSENS. Proc. SPIE 2325, Photodynamic Therapy of Cancer II 1995, 400-403. [49] Shirmanova, M.; Zagaynova, E.; Sirotkina, M.; Snopova, L.; Balalaeva, I.; Krutova, I.; Lekanova, N.; Turchin, I.; Orlova, A.; Kleshnin, M., In vivo study of 20
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photosensitizer pharmacokinetics by fluorescence transillumination imaging. Journal of Biomedical Optics 2010, 15 (4), 048004. [50] Sokolov, V. V.; Chissov, V. I.; Filonenko, E. V.; Yakubovskaya, R. I.; Sukhin, G. M.; Galpern, M. G.; Vorozhtsov, G. N.; Gulin, A. V.; Zhitkova, M. B.; Zharkova, N. N.; Kozlov, D. N.; Smirnov, V. V., First clinical results with a new drug for PDT. Proc. SPIE 2325, Photodynamic Therapy of Cancer II 1995, 364-366.
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[51] Jinling, H.; Naisheng, C.; Jiandong, H.; Ersheng, L.; Jinping, X.; Suling, Y.; Ziqiang, H.; Jiancheng, S., The preparation, characterization and anticancer activity of phthalocyanine metal complex for photodynamic therapy, amphiphilic phthlocyanine zinc complex ZnPcS2P2. Science in China (Series B) 2000, 30 (6), 481-488.
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[52] Wang, J.; Liu, H.; Xue, J. P.; Jiang, Z.; Chen, N. S.; Huang, J. L., The constitutes and isomer distribution of di-(potassium sulfonate)-di-phthatimidomethyl phthalocyanine zinc. Chinese Science Bulletin 2008, 53 (11), 1657-1664.
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te
d
M
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[53] Yang, B.-B.; Yao, H.-S.; Liu, H.; Jiang, Z.; Wang, J.; He, W.-Y.; Wang, Y.; Chen, N.-S.; Huang, J.-L., Structural identification and quality study on isomers of a novel anticancer photosensitiser Photocyanine. Acta Pharmaceutica Sinica 2010, 45 (12), 1545-1549.
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graphical abtract
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Photosensitizer
Trade name
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Wave
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Table 1. Pc-based photosensitizers used at clinical or clinical trials.
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Table(s)
length
M
(nm)
Photosens®
675
ep te
sulfonated AlPc
distilled-water solution
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Mixture of
Clinical trial
Formulation
Indications stage/ country Skin, breast, lung, oropharingeal, breast, larynx, head and neck cancers, commercially Sarcoma M1, epibulbal and choroidal available / tumors, eyes and eyelids tumors, Russia cervical cancer
Phase I (topical administration): Actinic
Topical administration: in
Keratosis, Bowen's Disease, skin cancer, or
HOSiPcOSi(CH3)2 -(CH2)3N(CH3)2
ZnPc
Ac c
propylene glycol and ethanol.
Pc 4
CGP55847
Intravenous administration: in
675
670
Stage I or Stage II Mycosis Fungoides clinical trials (September 2004-Agust 2010) (Phase I) / USA
solution of Cremophor EL and
Phase I (Intravenous administration): Skin
ethanol.
Cancer or Solid Tumors, met astatic to the Skin (August 2001- February 2006)
In liposomes composed of
clinical trials
Squamous cell carcinoma of upper
Page 23 of 27
di-sulfonic-di-phth
an
phosphatidylserine
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ip t holine and di- oleoyl
(Phase I/II) /
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palmitoyl-oleoyl-phosphatidylc
aerodigestive tract
Switzerland
0.5 mg/mL solution of 2% alimidomethyl phthalocyanine
Photocyanine
670
M
Cremophor
clinical trials
Skin cancer
(Phase I) /
esophageal cancer
China
(March 2009-)
EL, 20% 1,2-propylene glycol, zinc di-potassium
and 0.9% sodium chloride.
Ac c
ep te
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salt
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Figure(s)
Metallo phthalocyanine
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Metal free phthalocyanine
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Hematoporphyrin
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Fig.1. Chemical structures of representative hematoporphyrin and phthalocyanines
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Pc4
ZnPc(CGP55847)
Photocyanine
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Fig. 2. Pc-based photosensitizers under clinical evaluation.
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R=H or SO3H Photosense (Photosens)
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Fig. 3. Intracellular distribution of ZnPc in HepG2 localized by fluorescence phase-contrast image without the irradiation. The upper, middle and lower panels showed images of cellular compartments lysosome, mitochondria and endoplasmic reticulum (ER) respectively. B, E and H showed intracellular particles ZnPc in a granular pattern detected by red fluorescence. C, F and I showed the red granular particles ZnPc (10 μmol/L) merged on yellow lysosome (C), green mitochondria (F) and blue endoplasmic reticulum (I), respectively.
Page 27 of 27
S0731-7085(13)00215-X http://dx.doi.org/doi:10.1016/j.jpba.2013.05.014 PBA 9075
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
2-4-2013 4-5-2013 11-5-2013
Please cite this article as: Z. Jiang, J. Shao, T. Yang, J. Wang, L. Jia, Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmaceutical development, composition and quantitative
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photodynamic therapy
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analysis of phthalocyanine as the photosensitizer for cancer
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Zhou Jiang†, Jingwei Shao†, Tingting Yang, Jian Wang, Lee Jia*
Cancer Metastasis Alert and Prevention Center, College of Chemistry
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and Chemical Engineering, Fuzhou University, 523 Industry Road, Fuzhou, Fujian 350002, China.
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* Corresponding author: Lee Jia, Email address: [email protected];
The authors equally contribute to this work
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†
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Tel. +86-15159630201; fax: +86 0591 83792563.
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Page 1 of 27
Highlights Mechanisms of action of phthalocyanines (Pcs) used for photodynamic therapy
Quantitative methods for analyzing Pcs
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Current status of the Pcs under clinical investigation
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Pcs pharmaceutical development to improve their drugability & cellular localization
Abstract
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Phthalocyanine (Pc) and its related derivatives are a class of functional materials that are easily activated by the light at a special wavelength. As such
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photosensitizer, Pc has been applied to photodynamic therapy (PDT), in addition to its broad applications in many fields, for both malignant and benign diseases. One of our long-term research focuses is to develop Pc for cancer
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therapy. Herein we briefly review mechanisms of action of Pc used for
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photodynamic therapy, its pharmaceutical development and molecular modification to enhance its drugability and improve its intracellular localization.
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We also describe the current status of the Pc derivatives under clinical investigation, and analyze the methods used for quantitative analysis of those Pc derivatives.
Keywords:
Photodynamic therapy; Reactive oxygen species; Photosensitizer; Phthalocyanine
Abbreviations： Phthalocyanine
Pc
2
Page 2 of 27
phthalocyanine zinc
ZnPc
sulfonated phthalocyanine aluminum
AlPcS
PDT
reactive oxygen species
ROS
singlet molecular oxygen
1
single-chain Fv fragments
scFv
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O2
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photodynamic therapy
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di-(potassium sulfonate) -di-phthalimidomethyl ZnPcS2P2 phthalocyanine zinc
arginine-glycine-aspartic acid
RGD
EGFR
palmitoyl-oleoyl-phosphatidylcholine
OOPS
United States Pharmacopoeia
USP ER
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endoplasmic reticulum
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di-oleoyl phosphatidylserine
OPOC
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epidermal growth factor receptor
3
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1. Introduction
Phthalocyanine (Pc, Fig.1) and its derivatives are widely used as functional
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materials in many high technique fields, such as data storage, photoelectronic
generation, catalyst and so on. Using as photosensitizer in photodynamic
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therapy (PDT) is the most attractive application of Pc to various clinical trials
and has attracted intensive interests since 1990s. Nowadays, there are
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several phthalocyanines under clinical evaluation. However, there are only a few literature focused on pharmaceutical development and quantitative
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analysis of clinical phthalocyanines. Herein, we will comprehensively review the current status of Pc-based photosensitizer and our experience in analysis
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of sulfonic phthalocyanines.
development
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2. Mechanisms of photodynamic therapy and its pharmaceutical
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In the war on cancer, continues progresses have been achieved with the
great efforts to treat cancer mortality with drugs with better cure and less adverse effects. PDT is such a minimal invasive treatment which is currently used for malignant or benign diseases [1-4]. The therapy drug used for PDT is known as photosensitizer.
PDT involves two procedures. The first procedure is the topical or
systemical administration of a light-sensitive photosensitizer which should be selectively accumulated in the target tissue and/or cells. When the concentration of photosensitizer on the lesion is high enough and has a suitable ratio to adjacent normal tissue cells, the second procedure will be carried out, to deliver a specific wavelength of light to the lesion and activate 4
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the accumulated photosensitizer. The excited photosensitizer transfers the energy to surrounding oxygen to generate cytotoxic reactive oxygen species (ROS). The ROS, primarily singlet molecular oxygen (1O2), is responsible for the cascade of a series of cellular and molecular events that result in selective
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lesion destruction [5, 6]. The biological mechanisms of destroying the targeted lesion involve in direct cell killing of tumor cells (cellular PDT), indirect cell
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killing by vascular occlusion (vascular PDT), and the response of the immune
system [4, 5]. Selective accumulation of photosensitizer and controlled
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administration of light to lesion provide advantages of PDT over the three major treatments of malignant tumor (surgery, radiation therapy and
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chemotherapy), such as protection of functional structures, excellent cosmetic outcome, the safety of repeated uses, less side effects, fast convergence, and
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lower economic costs.
The development of photosensitizers plays a very important role in PDT.
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Therefore, photosensitizer has become an important field of research for both
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chemical and pharmaceutical sciences. The modern era of PDT started with the discovery of hematoporphyrin (Fig. 1) derivatives, the first generation of Photofrin® (a
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photosensitizers [7].
purified form
of hematoporphyrin
derivatives), the first commercial photosensitizer was approved for clinical use for bladder cancer in 1993 in Canada. From then on, PDT has been approved by regulatory authorities of other countries, such as the United States, Japan, the Netherlands and others in the world. PDT has been applied for more indications, such as esophagus, skin, head and neck, and lung cancers. At the same time, PDT is also applied to some non-malignant diseases, such as port wine stains, actinic keratoses, age-related macular degeneration, and localized infection [3, 4, 8-12]. Although Photofrin® has made considerable success and is still widely used on clinical, it has some shortages, such as moderate efficient and 5
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long-lasting skin photosensitization for several weeks, and composition with undefined mixture [7, 13]. This led to the development of new generation of photosensitizers. For the design of an ideal photosensitizer, the properties mentioned in the following should be fulfilled, such as: single and chemically
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pure compound; stability and good solubility in pharmaceutically acceptable
formulations and in biological media; high efficiency to yield ROS under
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illumination; fluorescence; no dark toxicity; fast clearance from the healthy
parts of the body and specific retention in diseased tissues; strong absorbance
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in Near-IR region and minimal absorbance between 400 and 600 nm [10, 13, 14]. Some photosensitizers of the second generation have been approved at
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clinical.
3. Pharmaceutical modification of Pc and its derivatives to increase their
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drugability
Various approaches have been made to improve the pharmaceutical
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properties of photosensitizers. One logical approach is through conjugation of
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photosensitizers to various linkers including antibodies, proteins and peptides. Phthalocyanine zinc was first tried to be conjugated to monoclonal antibodies
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that are specific for tumor-associated antigens [15]. However, the large size of antibodies hinders tissue penetration and lowers cellular uptake when used in vivo. The attachment of photosensitizers to monoclonal antibodies may reduce the antigenic specificity of monoclonal antibodies. As a result, smaller size of biomolecules has been searched for as the more effective alternative. Conjugation of photosensitizers with albumins and low-density lipoproteins has been explored, but only moderate target specificity has been achieved so far [16]. Smaller antibody fragments, such as single-chain Fv fragments (scFv) have received considerable attention as a good candidate [17]. Among various carriers for active drug targeting, synthetic peptides are of particular interest. Peptides with appropriate sequences can specifically bind to different surface 6
Page 6 of 27
biomarkers of cancer cells and circulating tumor cells [18, 19]. For instance, two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated
phthalocyanine
zinc
with
acetylenic
bombesin
or
arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid
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phase, have been explored to make a series of phthalocyanine zinc conjugates to target the gastrin-releasing peptide and integrin receptors [20].
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The peptide conjugation enhanced water solubility of phthalocyanine zinc and improved the latter’s photodynamic efficacy against cancer cell lines
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expressing gastrin-releasing peptide and integrin receptors. Ongarora et al [21] synthesized four Pc-peptide conjugates for targeting the epidermal growth
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factor receptor (EGFR) and evaluated the in vitro efficacy of the conjugates by using different cell lines. Two peptide ligands linked to 6 (EGFR-L1) and 13
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(EGFR-L2) amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical and experimental models. The Pc-EGFR-L1 conjugates efficiently targeted EGFR and were internalized,
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in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2
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conjugates targeted EGFR poorly probably because of their low aqueous
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solubility. All conjugates were nontoxic (IC50> 100 μM) to HT-29 colon cancer cells, both in the dark and upon light activation (1 J/cm2). Intravenous
administration of the conjugate into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a Near-IR fluorescence signal at 700 nm, 24 h after administration.
To increase the cell-targeting ability, two ZnPc-peptide conjugates, which
bore either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences, have been synthesized to evaluate the effect of the linker [22]. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The 7
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highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human
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HepG2 cells (IC50 77 μM) but were highly phototoxic (IC50< 2 μM at 1 J/ cm2). An alternative strategy to improve pharmaceutical properties of Pc
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involves direct formulations of Pc to increase its solubility (Table 1). ZnPc is lipophilic and slightly soluble in water or in a commonly-used pharmaceutical
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vehicle. This hydrophobic characteristics of ZnPc restricts its clinical studies. Cremophor EL is a polyethoxylated castor oil. Its major component is the
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material in which the hydroxyl groups of the castor oil triglyceride have ethoxylated with ethylene oxide to form polyethylene glycol ethers. This surfactant has been used to enhance solubility of those poorly-soluble
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materials including Pc by stabilizing emulsions of nonpolar materials in aqueous systems. We utilized Cremophor EL to increase solubility of
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Photocyanine [23] and ZnPc [24]. The formulation provided us the possibility to
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investigate in vitro activity and subcellular distribution of Pc analogues in cells.
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4. Intracellular localization of Pc and its derivatives Intracellular localization of Pc-like photosensitizers is usually determined by
using
confocal
fluorescence
microscopy.
Variations
in
intracellular
accumulation and the resulting phototoxicity between the conjugates correlate to
their
differences
in
hydrophobicity
as
well
as
cell
membrane
receptor-mediated uptake. It has been reported that the ZnPc conjugate bearing the long PEG-linker accumulated within cells 26 times more than the unconjugated ZnPc, and the short PEG-linker conjugate accumulated within cells 17 times more than the unconjugated ZnPc [22]. The result suggests the enhancement of PEG on transport of Pc across the cellular membrane.
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In the case of the phthalocyanine-bombesin conjugate, competition experiments confirmed the involvement of the membrane gastrin-releasing peptide receptor in both the photosensitizer activity as well as intracellular
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localization of Pc [20]. In our experiments, when the hepatocellular carcinoma HepG2 cells were
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incubated with ZnPc (10 μM) in dark, we could observe intracellular distribution of ZnPc under confocal laser microscopy with the aid of subcellular probes.
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Different emission wavelengths should be applied to corresponding probes of subcellular organelles. Usually, lysosome is stained with yellow probe and
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emitted at 577 nm; mitochondria is marked with green and emitted at 490 nm, and endoplasmic reticulum is stained blue and emitted at 587 nm. Using confocal laser microscopy with the aid of those probes, we demonstrated that
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ZnPc was predominately localized in polarized perinuclear regions such as
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mitochondria, lysosome and endoplasmic reticulum (Fig. 3).
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5. Pc-based photosensitizer and their quantitative analysis Phthalocyanine (Pc) and its related derivatives have been intensively
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investigated as the second generation photosensitizers since 1985. They are structurally related to porphyrins. With respect to the longest wavelength absorption of hematoporphyrin (near 630 nm), the absorption of Pc on Near-IR (670-780nm) is almost two orders of magnitude stronger [25]. With appropriate central atom, such as zinc, aluminum or silicon, phthalocyanine exhibits many other optimal properties that fulfill the requirements of an ideal photosensitizer mentioned above, such as high efficiency of ROS generation upon illumination, high stability, fluorescence, low intrinsic toxicity, high flexibility in structure modification and ease of synthesis [13, 14]. However, there are two principal shortages of Pc based photosensitizers: low water-solubility and tendency to aggregate. Most of the unsubstituted phthalocyanines are poorly soluble in 9
Page 9 of 27
water and normal organic solvents. Plate-like Pc molecules tend to form aggregation, which leads to the loss of PDT activity and fluorescence. The way to overcome these shortages lies on structure modification, suitable delivery system, or adds suitable solvent (such as pyridine) to reduce aggregation.
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Considerable and continuing study has been carried out during the past 3 decades. However, most of the researches are still in the preclinical stage. Up
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to date, there are only four Pc-based photosensitizers, i.e. Photosens, Pc 4, CGP55847 (ZnPc) and Photocyanine, available for clinical uses or trails in
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different countries (Table 1 and Fig. 2).
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A suitable quantitative analysis method is not only the need for quality control of photosensitizer drug with certified quality and composition, but also the requirements for clinical applications. Optical quantification methods,
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namely absorption and fluorescence spectroscopes are generally used. Fluorescence method has the potential of higher sensitivity over absorption,
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while they are both noninvasive and rapid. The phthalocyanines utilized as the
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photosensitizer all have fluorescence in Near-IR region, which gives Pc advantage to avoid interference from endogenous substances and makes
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quantitative analysis of Pc much easy without requirements for the labor-intensive separation. Fluorimetric assays were frequently used, especially for the early researches on biological samples [26-28]. A series of fluorescence diagnostic instruments have been developed and successfully used at clinical not only for measuring but also for controlling the PDT parameters [29]. High performance liquid chromatography (HPLC) is also a powerful tool in quantitative and qualitative analysis of pharmaceutical Pc. Various choices of both mobile and stationary phase enhance performance of HPLC in analyte separation, which will favor the research for drug metabolites and the drug with multi-component.
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6 Individual hydrophobic Pc-based photosensitizers 6.1. CGP55847 (ZnPc) CGP55847 is unsubstituted phthalocyanine zinc (ZnPc, Fig. 2) formulated in liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (OPOC) and
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di-oleoyl phosphatidylserine (OOPS) in a ratio of ZnPc: OPOC: OOPS at 1: 90: 10 (w/w/w). It was developed by QLT Phototherapeutics (Vancouver, Canada)
and sponsored by Ciba Geigy (Novartis, Basel, Switzerland). It is the first Pc
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reaching clinical trials (Phase I/II, Switzerland) for PDT of early cancer in the
upper aerodigestive tract. However, the clinical trials were discontinued and
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the reason is not clear [13, 30].
In the literature available to us, the quantitative analysis and in vivo
an
pharmacokinetics of CGP55847 is based on the fluorescence method [31，32]. Some studies of other groups stated that ZnPc in biological matrices, which consists of a single isomer and can be prepared with high chemical purity, is used as internal standard [33,34]. 6.2. Pc 4
M
also suitable for HPLC quantifications. Actually, in those reports, the ZnPc was
Fig. 2) and was developed for PDT application. Like ZnPc, It
has no isomer [35].
te
-(CH2)3N(CH3)2,
d
Pc 4 is a phthalocyanine silicon with axial substitutions (HOSiPcOSi(CH3)2
Ac ce p
Pc 4 had undertaken two phase I clinical trials approved by the FDA. Its
first clinical phase I trial aimed at systemic delivery of this photosensitizer for PDT of cutaneous malignancies initiated in 2001. The formulation for injection was prepared by dissolving 1 mg Pc 4 and 5 mg Povidone in 1 mL of Cremophor EL:ethanol=1:1 (NCI Diluent 12) following by being diluted with 9 volumes of normal saline.
The trial was terminated in 2006, because of
difficulty in accrual. However, three patients were treated with interesting results. The limit clinical trial showed Pc 4-PDT was well tolerated by subjects [36]. Another phase I clinical trial was conducted in 2004 for topical application of Pc 4. Pc 4 was dispensed after formulation in a vehicle of the United States Pharmacopoeia (USP) grade of propylene glycol and USP grade of ethanol(70/30%, v/v). Topical application is a convenient and safe method of 11
Page 11 of 27
specifically directing photosensitizers to accessible lesions, while avoiding the widespread distribution that occurs following intravenous administration. The trials were completed in 2010 and demonstrated the potential for topical delivery of Pc 4 to treat cutaneous neoplasms with good tolerability and no
ip t
adverse effects [35, 37]. Pharmacology studies of Pc 4 in plasma or tissue were carried out with nile blue or a closely related molecular Pc 34 as the internal standard, by
cr
HPLC or HPLC/tandem mass spectrometry. The chromatographic separation
was accomplished on a reverse-phase column with an isocratic mobile phase
us
consisting of methanol and ammonium formate buffer [36, 38]. 7． Sulfonic Pc-based hydrophilic photosensitizers
an
The matrix of Pc is highly hydrophobic. Many hydrophilic groups, such as sulfonic, carboxyl, and phosphoric group, have been introduced to improve Pc’s solubility in physiological solution. Among the water soluble Pcs, sulfonic
M
Pc has received the most intensive investigation as a photosensitizer. In the past tens of years, sulfonic Pc has been extensively studied [11, 14]. Unlike composition.
d
the two phthalocyanines discussed above, sulfonic Pc is always complex in
te
The synthesis of sulfonic Pc inevitably results in mixtures of its various isomers. Two main methods to produce sulfonic Pc are direct sulfonation
Ac ce p
method or condensation method. By direct sulfonation, all the four H atoms in each isoindole subunit of Pc have chance of substitution by sulfonic groups, thus resulting products are generally quite complex with compositions with different number of sulfonic groups on either α- or β-position (see Fig.1 for the
illustration of α- or β-position) [39-42]. The second method, which is also called the Weber-Busch method, involves the condensation of the mono-sulfonated precursors (such as phthalonitriles or phthalic acids) in the presence of urea, the selected metal salt and a catalyst. The mix condensation of sulfonic and unsubstituted precursors results in Pc components of 0 to 4 sulfonic groups. Expect for the unsubstituted and mono-sulfonated Pc, the other components are all mixture with different number of isomers [40, 43]. The much more complex substitution resulted from direct sulfonation leads to overlap of Pc with different degrees of sulfonation. But it has not been reported in the case of 12
Page 12 of 27
mixed condensation. In comparison of different methods for separating the sulfonic phthalocyanines, the van Lier’s group [40] found that HPLC with gradient elution was relatively satisfactory if phosphate buffer and methanol were used as the mobile phase with the C18 column. The adjacent forms of
ip t
di-sulfonic Pc, i.e. the isomers with sulfonic groups on the same side of Pc ring are considered to be the most active photosensitizer for their amphiphilic
property [41, 44]. There are two sulfonic phthalocyanines are in clinical
cr
evaluation.
us
7.1. Photosens® (Photosense)
Photosens® (Fig. 2) is a distilled-water solution of sodium salts of
an
sulfonated phthalocyanine aluminum (AlPcS) that shows maximum absorption at 675 nm. This water soluble mixture is obtained by direct sulfonation and constituted of AlPcS with di- to tetra-sulfonic acid substituted. Photosense®
M
was developed by the “NIOPIC” Moscow Research and Production Association. It is commercialized in Russia by NIOPIC [13, 14, 45, 46] and is highly effective in the treatment of various cancers, histological type and at
d
various stages, such as squamous cell skin cancer, breast cancer,
te
oropharyngeal cancer, lung cancer, eye and eyelid related tumors, bladder and cervical cancer, and so on. It is also used for the treatment of severe
Ac ce p
festering wounds, trophic ulcers, and some other nonmalignant maladies. The administration doses range from 0.5 to 2.0 mg per kg of the patient’s body weight. The introduction of sodium ascorbate in doses of 20–50 mg/kg body weight in addition to Photosens results in an improvement in therapeutic action and allows a twofold reduction of the Photosens dose. However, skin photosensitivity is a significant problem with this drug. The patient should remain heliophobic for 6 to 10 weeks after treatment [3, 10, 12, 47]. Fluorescence method had been developed to study pharmacokinetics of Photosens
[48,
49].
At
clinical,
the
computerized
fluorescent
spectrophotometry was used to monitor the photosensitizer in body before and after treatment for optimal therapy parameters [45, 47, 48, 50].
13
Page 13 of 27
7.2. Photocyanine (Suftalan Zinc) Suftalan Zinc (trade name: Photocyanine, Fig. 2) is an amphiphilic photosensitizer developed by our University, China. It is a mixture of four adjacent
form
isomers
(SSPP
type)
of
di-(potassium
sulfonate)
ip t
-di-phthalimidomethyl phthalocyanine zinc (ZnPcS2P2, here, S and P represent sulfonate group and phthalimidomethyl group, respectively). It is injected in the 0.5 mg/mL solution formulated with 2% Cremophor EL, 20% 1,2-propylene
cr
glycol and 0.9% sodium chloride. The phase I clinical trials of Photocyanine began in 2008 with the approval of State Food and Drug Administration of
us
China. Currently, Photocyanine is being applied for phase II clinical trials. The mix condensation method of 4-phthalimidomethyl phthalonitrile and
an
4-sulfonic phthalonitrile has been developed instead of the previouly ‘in a plot reaction’ [51] to obtain less complex product. However, there are still 5 opposite (SPSP type) and 10 adjacent form (SSPP type) isomers existing in
M
ZnPcS2P2 [52]. Several HPLC procedures have been carried out for preparation of Photocyanine. For quality control and quantitative analysis, great efforts have been taken to optimize HPLC method for separation of the
d
four very close isomers. A complex mobile phase, i.e., phosphate-TEA: DMF:
te
THF: CH3OH, 50: 37.5: 7.5: 5 (v/v/v/v) on a C18 column was developed. We found that addition of ion-pairing agents with positive charges to the mobile
Ac ce p
phase improved the resolution of di-, tri-, and tetra-sulfonic Pc isomers [43]. An ion-pair reversed-phase HPLC method using a mixture of acetonitrile and ion-pair buffer (0.01 M hexadecyl trimethyl ammonium bromide and 0.01 M potassium dihydrogen phosphate, adjusted the pH value to 6.8 with potassium hydroxide solution) was developed and gained similar efficiency [53].
8. Conclusions
Phthalocyanines are promising photosensitizers for PDT applications. Great emphasis has been placed on structural modification of the compounds or smart delivery system, while there are still only a few are on clinical application or trials. The experiences of those pioneers have shown the importance of quantitative methods for advancing the development of 14
Page 14 of 27
photosensitizers. The noninvasive optical method, and specific and sensitive HPLC method should both receive more attention than before. . Acknowledgements
ip t
The authors would like to appreciate the financial support of the National Science Foundation of China (81201709, 81273548 and 21101028), the China
cr
Postdoctoral Science Foundation (No.2012M511441), the Science and
Technology Foundation of Fujian Province of China (2011J0104) and the
us
Science Foundation of Education Department of Fujian Province of China
Ac ce p
te
d
M
an
(JK2010003).
15
Page 15 of 27
Legends for Figures: Fig.1.
Chemical
structures
of
representative
hematoporphyrin
and
cr
Fig. 2. Pc-based photosensitizers under clinical evaluation.
ip t
phthalocyanines
Fig. 3. Intracellular distribution of ZnPc in HepG2 localized by fluorescence
us
phase-contrast image without the irradiation. The upper, middle and lower panels showed images of cellular compartments lysosome, mitochondria and
an
endoplasmic reticulum (ER) respectively. B, E and H showed intracellular particles ZnPc in a granular pattern detected by red fluorescence. C, F and I showed the red granular particles ZnPc (10 μmol/L) merged on yellow
M
lysosome (C), green mitochondria (F) and blue endoplasmic reticulum (I),
Ac ce p
te
d
respectively.
16
Page 16 of 27
References [1] Brown, S. B.; Brown, E. A.; Walker, I., The present and future role of photodynamic therapy in cancer treatment. The Lancet Oncology 2004, 5 (8), 497-508.
cr
ip t
[2] Yano, S.; Hirohara, S.; Obata, M.; Hagiya, Y.; Ogura, S.-i.; Ikeda, A.; Kataoka, H.; Tanaka, M.; Joh, T., Current states and future views in photodynamic therapy. Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2011, 12 (1), 46-67.
us
[3] Allison, R. R.; Sibata, C. H., Oncologic photodynamic therapy photosensitizers: A clinical review. Photodiagnosis and Photodynamic Therapy 2010, 7 (2), 61-75. [4] Dolmans, D. E.; Fukumura, D.; Jain, R. K., Photodynamic therapy for cancer. Nat Rev Cancer 2003, 3 (5), 380-387.
an
[5] Macdonald, I. J.; Dougherty, T. J., Basic principles of photodynamic therapy. Journal of Porphyrins and Phthalocyanines 2001, 05 (02), 105-129.
M
[6] Josefsen, L. B.; Boyle, R. W., Photodynamic therapy and the development of metal-based photosensitisers. Met Based Drugs 2008, 2008, 276109. [7] Allison, R. R.; Mota, H. C.; Sibata, C. H., Clinical PD/PDT in North America: An historical review. Photodiagnosis and Photodynamic Therapy 2004, 1 (4), 263-277.
te
d
[8] Kharkwal, G. B.; Sharma, S. K.; Huang, Y. Y.; Dai, T.; Hamblin, M. R., Photodynamic therapy for infections: clinical applications. Lasers Surg Med 2011, 43 (7), 755-767.
Ac ce p
[9] Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T., Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chem Rev 2010, 110 (5), 2795-2838. [10] Allison, R. R.; Downie, G. H.; Cuenca, R.; Hu, X. H.; Childs, C. J. H.; Sibata, C. H., Photosensitizers in clinical PDT. Photodiagnosis and Photodynamic Therapy 2004, 1 (1), 27-42. [11] Pandey, R. K., Recent advances in photodynamic therapy. J. Porphyrins Phthalocyanines 2000, 4, 368-373. [12] Bonnett, R., Chemical Aspects of Photodynamic Therapy. Gordon & Breach Publishing Group: 2000. [13] Sekkat, N.; van den Bergh, H.; Nyokong, T.; Lange, N., Like a bolt from the blue: phthalocyanines in biomedical optics. Molecules 2012, 17 (1), 98-144. [14] Lukyanets, E. A., Phthalocyanines as Photosensitizers in the Photodynamic Therapy of Cancer. Journal of Porphyrins and Phthalocyanines 1999, 03 (06), 424-432. 17
Page 17 of 27
[15] Miller, G. G.; Lown, J. W., Immunophotodynamic therapy: Current developments and future prospects. Drug Development Research 1997, 42 (3-4), 182-197. [16] Sharman, W. M.; van Lier, J. E.; Allen, C. M., Targeted photodynamic therapy via receptor mediated delivery systems. Adv Drug Deliv Rev 2004, 56 (1), 53-76.
ip t
[17] Bhatti, M.; Yahioglu, G.; Milgrom, L. R.; Garcia-Maya, M.; Chester, K. A.; Deonarain, M. P., Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments. Int J Cancer 2008, 122 (5), 1155-1163.
us
cr
[18] Jia, L.; Gorman, G. S.; Coward, L. U.; Noker, P. E.; McCormick, D.; Horn, T. L.; Harder, J. B.; Muzzio, M.; Prabhakar, B.; Ganesh, B.; Das Gupta, T. K.; Beattie, C. W., Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination. Cancer Chemother Pharmacol 2011, 68 (2), 513-524.
an
[19] Jia, L.; Noker, P. E.; Piazza, G. A.; Leuschner, C.; Hansel, W.; Gorman, G. S.; Coward, L. U.; Tomaszewski, J., Pharmacokinetics and pharmacodynamics of Phor21-betaCG(ala), a lytic peptide conjugate. J Pharm Pharmacol 2008, 60 (11), 1441-1448.
M
[20] Ranyuk, E.; Cauchon, N.; Klarskov, K.; Guerin, B.; van Lier, J. E., Phthalocyanine-Peptide Conjugates: Receptor-Targeting Bifunctional Agents for Imaging and Photodynamic Therapy. J Med Chem 2013, epub ahead of print.
te
d
[21] Ongarora, B. G.; Fontenot, K. R.; Hu, X.; Sehgal, I.; Satyanarayana-Jois, S. D.; Vicente, M. G., Phthalocyanine-peptide conjugates for epidermal growth factor receptor targeting. J Med Chem 2012, 55 (8), 3725-3238.
Ac ce p
[22] Sibrian-Vazquez, M.; Ortiz, J.; Nesterova, I. V.; Fernandez-Lazaro, F.; Sastre-Santos, A.; Soper, S. A.; Vicente, M. G., Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates. Bioconjug Chem 2007, 18 (2), 410-420. [23] Shao, J.; Xue, J.; Dai, Y.; Liu, H.; Chen, N.; Jia, L.; Huang, J., Inhibition of human hepatocellular carcinoma HepG2 by phthalocyanine photosensitiser PHOTOCYANINE: ROS production, apoptosis, cell cycle arrest. Eur J Cancer 2012, 48 (13), 2086-2096. [24] Shao, J.; Dai, Y.; Zhao, W.; Xie, J.; Xue, J.; Ye, J.; Jia, L., Intracellular distribution and mechanisms of actions of photosensitizer Zinc(II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells. Cancer Lett 2013, 330 (1), 49-56. [25] Josefsen, L. B.; Boyle, R. W., Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics. Theranostics 2012, 2 (9), 916-966. [261] Lilge, L.; O'Carroll, C.; Wilson, B.C., A solubilization technique for photosensitizer quantification in ex vivo tissue samples. Journal of Photochemistry and Photobiology B: Biology 1997, 39 (3), 229-235. 18
Page 18 of 27
[27] Cubeddu, R.; Pifferi, A.; Taroni, P.; Torricelli, A.; Valentini, G.; Comelli, D.; D'Andrea, C.; Angelini, V.; Canti, G., Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine. Photochem Photobiol 2000, 72 (5), 690-695.
ip t
[28] Siqueira-Moura, M. P.; Primo, F. L.; Peti, A. P.; Tedesco, A. C., Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers. Pharmazie 2010, 65 (1), 9-14. [29] Loschenov, V.; Konov, V.; Prokhorov, A., Photodynamic therapy and fluorescence diagnostics. Laser Physics-Lawrence 2000, 10 (6), 1188-1207.
us
cr
[30] Hadjur, C.; Wagnières, G.; Ihringer, F.; Monnier, P.; van den Bergh, H., Production of the free radicals O.-2 and .OH by irradiation of the photosensitizer zinc(II) phthalocyanine. Journal of Photochemistry and Photobiology B: Biology 1997, 38 (2–3), 196-202.
an
[31] Love, W. G.; Duk, S.; Biolo, R.; Jori, G.; Taylor, P. W., Liposome-mediated delivery of photosensitizers: localization of zinc (II)-phthalocyanine within implanted tumors after intravenous administration. Photochem Photobiol 1996, 63 (5), 656-661.
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[32] van Leengoed, H. L.; van der Veen, N.; Versteeg, A. A.; Ouellet, R.; van Lier, J. E.; Star, W. M., In vivo fluorescence kinetics of phthalocyanines in a skin-fold observation chamber model: role of central metal ion and degree of sulfonation. Photochem Photobiol 1993, 58 (2), 233-237.
te
d
[33] Oliveira, L. T.; Garcia, G. M.; Kano, E. K.; Tedesco, A. C.; Mosqueira, V. C., HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies. J Pharm Biomed Anal 2011, 56 (1), 70-77.
Ac ce p
[34] Bohn, T.; Walczyk, T., Determination of chlorophyll in plant samples by liquid chromatography using zinc-phthalocyanine as an internal standard. J Chromatogr A 2004, 1024 (1-2), 123-128. [35] Miller, J. D.; Baron, E. D.; Scull, H.; Hsia, A.; Berlin, J. C.; McCormick, T.; Colussi, V.; Kenney, M. E.; Cooper, K. D.; Oleinick, N. L., Photodynamic therapy with the phthalocyanine photosensitizer Pc 4: the case experience with preclinical mechanistic and early clinical-translational studies. Toxicol Appl Pharmacol 2007, 224 (3), 290-299. [36] Kinsella, T. J.; Baron, E. D.; Colussi, V. C.; Cooper, K. D.; Hoppel, C. L.; Ingalls, S. T.; Kenney, M. E.; Li, X.; Oleinick, N. L.; Stevens, S. R.; Remick, S. C., Preliminary clinical and pharmacologic investigation of photodynamic therapy with the silicon phthalocyanine photosensitizer pc 4 for primary or metastatic cutaneous cancers. Front Oncol 2011, 1, 14. [37] Baron, E. D.; Malbasa, C. L.; Santo-Domingo, D.; Fu, P.; Miller, J. D.; Hanneman, K. K.; Hsia, A. H.; Oleinick, N. L.; Colussi, V. C.; Cooper, K. D., Silicon phthalocyanine (Pc 4) photodynamic therapy is a safe modality for cutaneous neoplasms: results of a phase 1 clinical trial. Lasers Surg Med 2010, 42 (10), 728-735. 19
Page 19 of 27
[38] Zuk, M. M.; Kenney, M. E.; Horowitz, B.; Ben-Hur, E., High-performance liquid chromatographic determination of the silicon phthalocyanine Pc 4 in human blood. J Chromatogr B Biomed Appl 1995, 673 (2), 320-324.
ip t
[39] Brasseur, N.; Ali, H.; Langlois, R.; van Lier, J. E., Biological Activities of Phthalocyanines-IX. Photosensitization of V-79 Chinese Hamster Cells and EMT-6 Mouse Mammary Tumor by Selectively Sulfonated Zinc Phthalocyanines. Photochem Photobiol 1988, 47 (5), 705-711.
cr
[40] Ali, H.; Langlois, R.; Wagner, J. R.; Brasseur, N.; Paquette, B.; van Lier, J. E., Biological activities of phthalocyanines-X. Synthesis and Analyses of sulfonated phthalocyanines. Photochem Photobiol 1988, 47 (5), 713-717.
us
[41] Bishop, S. M.; Khoo, B. J.; MacRobert, A. J.; Simpson, M. S.; Phillips, D.; Beeby, A., Characterisation of the photochemotherapeutic agent disulphonated aluminium phthalocyanine and its high-performance liquid chromatographic separated components. J Chromatogr 1993, 646(2), 345-350.
an
[42] Schofield, J.; Asaf, M., Analysis of sulphonated phthalocyanine dyes by capillary electrophoresis. Journal of Chromatography A 1997, 770 (1–2), 345-348.
M
[43] Jiang, Z.; He, W.; Yao, H.; Wang, J.; Chen, N.; Huang, J., Isomeric separation and identification of tetra-, tri-, and di-β-sulphonic phthalocyanine zinc complexes. Journal of Porphyrins and Phthalocyanines 2011, 15 (2), 140-148.
d
[44] Brasseur, N.; Ali, H.; Langlois, R.; van Lier, J. E., Biological Activities of Phthalocyanines-VII. Photoinactivation of V-79 Chinese Hamster Cells by Selectively Sulfonated Gallium Phthalocyanines. Photochem Photobiol 1987, 46 (5), 739-744.
Ac ce p
te
[45] Sokolov, V. V.; Chissov, V. I.; Yakubovskaya, R. I.; Aristarkhova, E. I.; Filonenko, E. V.; Belous, T. A.; Vorozhtsov, G. N.; Zharkova, N. N.; Smirnov, V. V.; Zhitkova, M. B., Photodynamic therapy (PDT) of malignant tumors by photosensitzer photosens: results of 45 clinical cases. Proc. SPIE 2625, Photochemotherapy: Photodynamic Therapy and Other Modalities 1996, 281-287. [46] Filonenko, E. V.; Sokolov, V. V.; Chissov, V. I.; Lukyanets, E. A.; Vorozhtsov, G. N., Photodynamic therapy of early esophageal cancer. Photodiagnosis and Photodynamic Therapy 2008, 5 (3), 187-190. [47] Stranadko, E. P.; Skobelkin, O. K.; Markichev, N. A.; Riabov, M. V., Photodynamic therapy of recurrent cancer of oral cavity. Proc. SPIE 2887, Lasers in Medicine and Dentistry: Diagnostics and Treatment 1996, 233-235. [48] Zharkova, N. N.; Kozlov, D. N.; Smirnov, V. V.; Sokolov, V. V.; Chissov, V. I.; Filonenko, E. V.; Sukhin, G. M.; Galpern, M. G.; Vorozhtsov, G. N., Fluorescence observations of patients in the course of photodynamic therapy of cancer with the photosensitizer PHOTOSENS. Proc. SPIE 2325, Photodynamic Therapy of Cancer II 1995, 400-403. [49] Shirmanova, M.; Zagaynova, E.; Sirotkina, M.; Snopova, L.; Balalaeva, I.; Krutova, I.; Lekanova, N.; Turchin, I.; Orlova, A.; Kleshnin, M., In vivo study of 20
Page 20 of 27
photosensitizer pharmacokinetics by fluorescence transillumination imaging. Journal of Biomedical Optics 2010, 15 (4), 048004. [50] Sokolov, V. V.; Chissov, V. I.; Filonenko, E. V.; Yakubovskaya, R. I.; Sukhin, G. M.; Galpern, M. G.; Vorozhtsov, G. N.; Gulin, A. V.; Zhitkova, M. B.; Zharkova, N. N.; Kozlov, D. N.; Smirnov, V. V., First clinical results with a new drug for PDT. Proc. SPIE 2325, Photodynamic Therapy of Cancer II 1995, 364-366.
cr
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[51] Jinling, H.; Naisheng, C.; Jiandong, H.; Ersheng, L.; Jinping, X.; Suling, Y.; Ziqiang, H.; Jiancheng, S., The preparation, characterization and anticancer activity of phthalocyanine metal complex for photodynamic therapy, amphiphilic phthlocyanine zinc complex ZnPcS2P2. Science in China (Series B) 2000, 30 (6), 481-488.
us
[52] Wang, J.; Liu, H.; Xue, J. P.; Jiang, Z.; Chen, N. S.; Huang, J. L., The constitutes and isomer distribution of di-(potassium sulfonate)-di-phthatimidomethyl phthalocyanine zinc. Chinese Science Bulletin 2008, 53 (11), 1657-1664.
Ac ce p
te
d
M
an
[53] Yang, B.-B.; Yao, H.-S.; Liu, H.; Jiang, Z.; Wang, J.; He, W.-Y.; Wang, Y.; Chen, N.-S.; Huang, J.-L., Structural identification and quality study on isomers of a novel anticancer photosensitiser Photocyanine. Acta Pharmaceutica Sinica 2010, 45 (12), 1545-1549.
21
Page 21 of 27
Ac
ce pt
ed
M
an
us
cr
ip t
graphical abtract
Page 22 of 27
Photosensitizer
Trade name
an
Wave
us
Table 1. Pc-based photosensitizers used at clinical or clinical trials.
cr
ip t
Table(s)
length
M
(nm)
Photosens®
675
ep te
sulfonated AlPc
distilled-water solution
d
Mixture of
Clinical trial
Formulation
Indications stage/ country Skin, breast, lung, oropharingeal, breast, larynx, head and neck cancers, commercially Sarcoma M1, epibulbal and choroidal available / tumors, eyes and eyelids tumors, Russia cervical cancer
Phase I (topical administration): Actinic
Topical administration: in
Keratosis, Bowen's Disease, skin cancer, or
HOSiPcOSi(CH3)2 -(CH2)3N(CH3)2
ZnPc
Ac c
propylene glycol and ethanol.
Pc 4
CGP55847
Intravenous administration: in
675
670
Stage I or Stage II Mycosis Fungoides clinical trials (September 2004-Agust 2010) (Phase I) / USA
solution of Cremophor EL and
Phase I (Intravenous administration): Skin
ethanol.
Cancer or Solid Tumors, met astatic to the Skin (August 2001- February 2006)
In liposomes composed of
clinical trials
Squamous cell carcinoma of upper
Page 23 of 27
di-sulfonic-di-phth
an
phosphatidylserine
cr
ip t holine and di- oleoyl
(Phase I/II) /
us
palmitoyl-oleoyl-phosphatidylc
aerodigestive tract
Switzerland
0.5 mg/mL solution of 2% alimidomethyl phthalocyanine
Photocyanine
670
M
Cremophor
clinical trials
Skin cancer
(Phase I) /
esophageal cancer
China
(March 2009-)
EL, 20% 1,2-propylene glycol, zinc di-potassium
and 0.9% sodium chloride.
Ac c
ep te
d
salt
Page 24 of 27
Figure(s)
Metallo phthalocyanine
ip t
Metal free phthalocyanine
cr
Hematoporphyrin
Ac
ce pt
ed
M
an
us
Fig.1. Chemical structures of representative hematoporphyrin and phthalocyanines
Page 25 of 27
Pc4
ZnPc(CGP55847)
Photocyanine
Ac
ce pt
ed
M
an
us
cr
Fig. 2. Pc-based photosensitizers under clinical evaluation.
ip t
R=H or SO3H Photosense (Photosens)
Page 26 of 27
ip t cr us an M ed ce pt
Ac
Fig. 3. Intracellular distribution of ZnPc in HepG2 localized by fluorescence phase-contrast image without the irradiation. The upper, middle and lower panels showed images of cellular compartments lysosome, mitochondria and endoplasmic reticulum (ER) respectively. B, E and H showed intracellular particles ZnPc in a granular pattern detected by red fluorescence. C, F and I showed the red granular particles ZnPc (10 μmol/L) merged on yellow lysosome (C), green mitochondria (F) and blue endoplasmic reticulum (I), respectively.
Page 27 of 27