Photosensitizer Radachlorin®: Skin cancer PDT phase II clinical trials

doi:10.1016/j.pdpdt.2010.07.006

Photodiagnosis and Photodynamic Therapy

Volume 7, Issue 4, December 2010, Pages 258-267

https://doi.org/10.1016/j.pdpdt.2010.07.006 Get rights and content

Summary

“Radachlorin”^®, also known in the EU as Bremachlorin, a composition of 3 chlorophyll a derivatives in an aqueous solution, was introduced into the Russian Pharmacopoeia. Its GMP (Good Manufacturing Practice) facility based manufacturing method was patented. Laboratory experiments and clinical phase I were performed.

Protocols were designed for PDT of basal cell carcinoma of the skin to result in GCP (Good Clinical Practice)-conformed randomized phase II clinical studies. “Radachlorin”^® solution for intravenous infusions 0.35% 10 mL in the doses of 0.5–0.6 and 1.0–1.2 mg/kg and a gel for topical application 0.1% 25 g in the dose of 0.1 g/cm² were photoactivated by 2.5W 662 nm semiconductor laser “LAKHTA-MILON^®” (St. Petersburg, Russia) in light doses of 200, 300 (solution), 400, 600, 800 (gel) J/cm².

Safety study showed no side effects and a good tolerability of “Radachlorin”^® by patients. There was no normal skin/subdermal tissue damage after both laser and sun light exposure. The main part (98%) of the drug was excreted or metabolized in the first 48 h. Drug administration at a dose of 1.0–1.2 mg/kg and irradiation at 3 h with 662 ± 3 nm light at a dose of 300 J/cm² (solution) and 4 PDT sessions at an interval of 1 week with 3 h gel exposure, followed by 400 J/cm² light exposure (gel) were found to be the optimal treatment regimes.

Having successfully passed clinical trials, “Radachlorin”^® achieved marketing authorization in Russia in 2009 and a conditional approval in South Korea in 2008. It is a candidate for phase III clinical trials in the EC and may be commercialized as a prospective second-generation photosensitizer.

Introduction

This paper represents the full report of a phase IIb clinical trials for the photosensitizer Radachlorin [1] in Russia (August 2003–August 2005), previously published partly [2], [3], [4], [5], set forth in 2 different clinical trial Protocols (3 hospitals for “Radachlorin”^®, i.v. administration (RCS); 2 hospitals for “Radachlorin”^®, topical administration, RCG), aimed at obtaining confirmation of previous data [6], [7], [8] on applicability, safety and tolerability of Radachlorin-LAKHTA-MILON photodynamic therapy, as well as optimizing PDT regimes for improving photodynamic tumor destruction examining various drug doses, and light exposures. This followed laboratory studies (1999–2001) and clinical phase 0 and I trials (conducted in Russia in 3 hospitals in July 2001–March 2003, involving 67 patients with i.v. and 35 patients with topical administration of photosensitizer), for cutaneous basal cell carcinoma (BCC) treatment protocols.

Section snippets

Photosensitizer

“Radachlorin”^® active pharmaceutical ingredient (API) as well as its finished formulation “Radachlorin”^® gel for topical application 0.1% 25 g (RCG) were produced in a GMP-certified facility of RADA-PHARMA Co. Ltd. (Moscow, Russia).

“Radachlorin”^® active pharmaceutical ingredient (API) is represented by the total sodium salts of chlorins (6.50/7.50 g), and purified water (up to 100.00 mL).

RCG is “Radachlorin”^® (1.43 g) (total sodium salts of chlorins—0.10 g), purified water (up to 100.00 mL), and

Results

Safety study showed no side effects and a good tolerability of RCS by patients, save for moderate pain, depending on individual sensitivity, tumor localization and irradiation field. There was no normal skin/subdermal tissue damage after both laser and sunlight exposure. The main part (98%) of the drug was excreted or metabolized in the first 48 h.

A low dark toxicity, 48 h clearance of RCS from the human's body and a low affinity to the skin helped to avoid the skin photosensitivity to daylight

Discussion

“Radachlorin”^® active substance is chemically stable in solutions for 1.5 years at 0 + 8 °C in the dark. When introduced to embryocarcinoma T36 bearing mice, it had maximal tumor uptake within 0.5–5 h post-injection with tumour-to-skin ratio around 14 by 3 h post-injection and clearance period about 24 h. In the animal PDT experiments “Radachlorin”^®, active substance showed an expressed specific PDT activity, causing an intensive but bearable by animals necrotic action to the tumors [10], [11], [12],

Acknowledgments

The authors are highly grateful to the supervisors of the clinical trials V.A. Privalov (Prof., M.D., D.Med.Sc.), V.V. Sokolov (Prof., M.D., D.Med.Sc.), A.V. Geinits (Prof., M.D., D.Med.Sc.), as well as to physicists making fluorescent diagnosis possible: A.V.Lappa (Prof., D.Med.Sc.), M.V.Evnevich (Ph.D.), N.N.Boulgakova (Ph.D.).

This work would certainly have been impossible without I.D. Zalevskiy (Ph.D.) and S.E.Goncharov, who provided the lasers.

We would also like to thank the staff member of

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Results of Phase II clinical trials of the photosensitizer Radachlorin in patients with cutaneous basal-cell carcinoma carried out at the Chelyabinsk municipal clinical hospital No. 1
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Photodynamic therapy and fluorescence diagnostics of head and neck cancer using second-generation photosensitizers
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Photodynamic therapy of cutaneous basal-cell carcinoma using photosensitizer from the chlorins family Radachlorin
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Five years’ experience of photodynamic therapy with new chlorin photosensitizer
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Clinical Trials of a New Chlorin Photosensitizer for Photodynamic Therapy of Malignant Tumors

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We present a thorough experimental investigation of fluorescence properties of Radachlorin photosensitizer in solutions of different acidity, viscosity and polarity. Experiments were performed using time-resolved fluorescence lifetime imaging and time-resolved analysis of polarized fluorescence. Variations of solution acidity resulted in considerable changes of Radachlorin fluorescence quantum yield and lifetime in the pH range from 4 to 7, but did not affect the rotational diffusion time, and almost did not change the quantum yield and characteristic times of singlet oxygen phosphorescence. Variations of solution polarity and viscosity were achieved by changing ethanol or methanol fraction in aqueous solution. The decrease of solution polarity resulted in nonlinear rise of Radachlorin fluorescence quantum yield and lifetime up to alcohol concentration of 50%–65%, as well as in considerable rise of singlet oxygen quantum yield and significant changes in characteristic times of its phosphorescence. Variations of solution viscosity resulted in changes of rotational diffusion time of Radachlorin molecules, which appeared to be in perfect correlation with methanol solution viscosity. Good correspondence with ethanol solution viscosity was observed only up to 50% alcohol fraction. Deviations of rotational diffusion time of Radachlorin molecules from direct proportionality with solution viscosity at higher ethanol concentrations were suggested to be due to different solvation conditions. The data obtained can give important reference points for analysis of microenvironment of Radachlorin molecules, their intracellular localization and performance in singlet oxygen generation.
Analysis of Radachlorin localization in living cells by fluorescence lifetime imaging microscopy
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Intracellular localization of photosensitizer molecules is influential on cell death pathway at photodynamic treatment and is thus an important aspect in achieving enhanced efficacy of photodynamic therapy. In this paper we performed thorough studies of the distribution of Radachlorin photosensitizer in three established cell lines: HeLa, A549, and 3T3 with fluorescence lifetime imaging microscopy through the analysis of lifetime distributions. Experiments carried out in Radachlorin solutions in phosphate buffered saline revealed the pronounced dependence of the fluorescence quantum yield and lifetime on solution pH. This finding was used for analysis of lifetime images of living cells and their phasor plot representations and allowed us to suggest that Radachlorin localized predominantly in lysosomes, known to have acidic pH values. Experiments on co-localization of Radachlorin fluorescence lifetimes and LysoTracker fluorescence intensity supported this suggestion. The results obtained show that the inhomogeneity of fluorescence quantum yield within a cell can be significant due to lower pH values in lysosomes than in other intracellular compartments. This finding suggests that the actual amount of accumulated Radachlorin can be underestimated if being evaluated solely by comparison of fluorescence intensities.
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Dendrimers play an integral part in advancement of drug delivery technology. Dendrimers are a type of polymeric nanoparticles that have wide variety of applications in the biomedical field such as drug or gene delivering and imaging. Their unique properties like controlled size, biocompatibility, low immunogenicity, high permeability, polyvalency, chemical stability, low toxicity, and structural perfection are responsible for their successful application in the abovementioned fields. Utilization of photosensitizers and laser irradiation for treatment of cancer is known as photodynamic therapy (PDT). Dendrimer-based PDT is a novel treatment method in the field of oncology that has been approved in various parts of the world as an alternative to chemotherapy. In this chapter, the dendrimer development and strategies to carry photosensitizers to the target tumor site are described. PDT possesses various drawbacks such as low solubility of photosensitizer, low specificity to tumor cells, and inefficient cellular uptake. These drawbacks of PDT were conquered by attaching photosensitizer drug molecules to dendrimer. Dendrimers improve the pharmacokinetic and pharmacodynamic properties of drug molecules. Dendrimer-based PDT most importantly reduces dark toxicity (i.e., toxicity of photosensitizers without irradiation of light). Moreover, application of dendrimers in PDT not only contributes to improve the delivery of photosensitizers to target sites, but also focuses on increasing the therapeutic value of those drugs. Various challenges related to the PDT were also discussed in this chapter by citing different literature reports.
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Efficient screening of photosensitizers (PS) as well as studying their photodynamic activity, especially PS excited in the near-infrared region, require informative in vitro models to adequately reflect the architecture, thickness, and intercellular interactions in tumors. In our study, we used spheroids formed from human colon cancer HCT-116 cells and liver cancer Huh7 cells to assess the phototoxicity of a new PS based on tetracationic derivative of synthetic bacteriochlorin (BC4). We optimized conditions for the irradiation regime based on the kinetics of BC4 accumulation in spheroids and kinetics of spheroid growth. Although PS accumulated more efficiently in HCT-116 cells, characterized by more aggressive growth and high proliferative potential, they were less susceptible to the photodynamic therapy (PDT) compared to the slower growing Huh7 cells. We also showed that 3D models of spheroids were less sensitive to BC4 than conventional 2D cultures with relatively identical kinetics of drug accumulation. Our findings suggest that BC4 is a perspective agent for photodynamic therapy against cancer cells.
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Citation Excerpt :
They exhibit a strong absorption band in the region between 650 and 690 nm with a higher molar absorption coefficient, allowing a greater penetration into the tumor tissue, low toxicity, and good photostability [9,14]. Several chlorin-type PSs are clinically used, such as Temoporfin or Foscan® [15,16], verteporfin [17,18], Bremachlorin® [19,20], Photodithazine® (chlorin e6) [21,22] and Laserphyrin® or LS11 [23,24]. The synthesis of new chlorins from protoporphyrin IX reacting with different maleimide substituents through the Diels-Alder reaction has significantly contributed to the chemistry of chlorin derivatives since these chlorins exhibited an “L-type” structure form with non-aggregation in solution and optimal chemical stabilities [25–27].
Photodynamic Therapy (PDT) is a promising treatment for various types of cancer and other non-oncological diseases. This technique uses a photosensitizer (PS) that generates reactive oxygen species (ROS), which lead cells to death in the presence of light and molecular oxygen. In this study, we evaluated the photoactivity of four chlorins that presented an L-type structure conformation (6a, 6b, 8a, and 8b) in two tumoral (HEp-2 and HeLa) and one non-tumoral (Vero) cell line under PDT conditions. Intracellular accumulation and subcellular localization were determined by confocal microscopy, showing that these chlorins exhibited an excellent (120 min) internalization through the plasmatic membrane with mitochondria and lysosome localization. The chlorins 6a-b were two and three-fold more selective than the chlorins 8a-b to HEp-2 and HeLa compared to Vero at 3 and 6 J cm⁻² doses, respectively. The fluorescence microscopy and flow cytometry analysis showed that the four chlorin derivatives lead tumor cells to death predominantly by apoptosis during the treatment with light. Considering that the chlorins 6a-b exhibited good water solubility and high phototoxicity to the tumor cells, we suggested that these tetrapyrrolic molecules have the potential for clinical treatment in cancer by PDT.
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Photodynamic therapy requires selection of effective photosensitizers that are selective with respect to the target and have a sufficient quantum yield of singlet oxygen under irradiation. In this work, we used Radachlorin, which is a second generation photosensitizer and is intended for fluorescence diagnostics and photodynamic therapy.63,64 The method of photodynamic therapy is based on the ability of Radachlorin to selectively accumulate in the tumor upon its administration and generate singlet oxygen under irradiation, which has a toxic effect on tumor cells.65
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^☆: This paper was supported by the Natural Health Foundation (Noordwijkerhout, The Netherlands, www.naturalhealthfoundation.com).

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Photosensitizer Radachlorin®: Skin cancer PDT phase II clinical trials☆

Summary

Introduction

Section snippets

Photosensitizer

Results

Discussion

Acknowledgments

Life Sci

Results of Phase II clinical trials of the photosensitizer Radachlorin in patients with cutaneous basal-cell carcinoma carried out at the Chelyabinsk municipal clinical hospital No. 1

Russian Biother J

Photodynamic therapy and fluorescence diagnostics of head and neck cancer using second-generation photosensitizers

Photodynamic therapy of cutaneous basal-cell carcinoma using photosensitizer from the chlorins family Radachlorin

Laser Med

Five years’ experience of photodynamic therapy with new chlorin photosensitizer

Clinical Trials of a New Chlorin Photosensitizer for Photodynamic Therapy of Malignant Tumors

Photosensitizer Radachlorin^®: Skin cancer PDT phase II clinical trials☆