Pharmacokinetics and biodistribution of Photolon® (Fotolon®) in intact and tumor-bearing rats

Photodiagnosis and Photodynamic Therapy (2009) 6, 97—104

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/pdpdt

Pharmacokinetics and biodistribution of Photolon® (Fotolon®) in intact and tumor-bearing rats S.V. Shliakhtsin MSc a,∗,2, T.V. Trukhachova a,1, H.A. Isakau a,2, Y.P. Istomin b,3 a

RUE ‘‘Belmedpreparaty’’, 220007 Minsk, Fabritsiusa str. 30, Belarus N.N. Alexandrov National Cancer Centre of Belarus, 223040 Minsk, p.o. Lesnoy-2, Belarus Available online 21 June 2009

b

KEYWORDS Photolon® ; Biodistribution; Blood—brain barrier; Pharmacokinetics

Summary Background: This paper provides the results of the non-clinical evaluation of biodistribution of the PS Photolon® in inner organs and tissues of intact and tumor-bearing rats with xenograft tumors of different morphologic types. Methods: The accumulation studies were performed in rats by means of intravital laser fluorimetry in situ and spectrophotometric determination ex vivo. Results: The biodistribution pattern of Photolon® does not depend on tumor carriage as well as on morphologic type of the xenograft tumor. We have also showed that Photolon® easily crosses an intact blood—brain barrier and accumulates in tissues of central nervous system. The relative bioavailability of brain tissues for Photolon® was estimated as 82%, Tmax —–30 min, mean residual time (MRT)—–1.6 h. Conclusions: In general, results of the experimental study of biodistribution of Photolon® in inner organs and tissues of rats, performed as in real time (by means of intravital laser fluorimetry in situ) as ex vivo (spectrophotometric assay) can be utilized while optimizing existing regimens of PDT with the purpose to increase safety and efficacy of treatment as well as for the development of new PDT protocols with Photolon® applied for new indications. Parameters of pharmacokinetics and biodistribution of Photolon® /Fotolon® as well as its’ ability to cross an intact blood—brain barrier, are optimal for the majority of modern clinical applications of PDT. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

Introduction

∗ Corresponding author at: Department of Pharmacology and Toxicology of RUE ‘‘Belmedpreparaty’’, 220007 Minsk, Fabritsiusa str. 30, Belarus. Tel.: +375 17 220 39 40; fax: +375 17 220 31 42. E-mail addresses: [email protected] (S.V. Shliakhtsin), [email protected] (T.V. Trukhachova), [email protected] (H.A. Isakau), [email protected] (Y.P. Istomin). 1 Tel.: +375 17 220 31 42; fax: +375 17 220 31 42. 2 Tel.: +375 17 220 39 40; fax: +375 17 220 39 40. 3 Tel: +375 17 220 39 40.

Photodynamic therapy (PDT) is one of the most promising modalities for the treatment of neoplastic and nonneoplastic diseases. PDT is based on the concept that certain chemicals, called photosensitizers (PS), can accumulate in target tissues with high selectivity and can be activated with the light of the appropriate wavelength to generate active molecular species, such as free radicals and singlet oxygen (1 O2 ) that are toxic to cells and tissues. A detailed description of cytotoxic action of PDT on target tissues was given by Dougherty and co-authors [1].

1572-1000/$ — see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2009.04.002

98 PDT makes possible selective therapeutic action versus pathologic (neoplastic) tissues. As it was shown in numerous experimental and clinical studies, after the introduction of the PS into the organism it preferably accumulates in tissues with high mitotic activity, particularly in the endothelium of neogenic blood vessels, in proliferative tissues with high vascularization, as well as in cells of tumor parenchyma [2—7]. The ability of the PS to accumulate preferably in tumor tissue and local irradiation of target tissue with laser light allow to pointedly destroy malignant and other atypical cells and mitigate damage of surrounding healthy tissues. The efficacy of PDT is substantially dependent on the concentration of the PS in target tissue. At the same time, the safety of the procedure is determined by levels of the PS in surrounding healthy tissues at the moment of irradiation of the treatment site [8,9]. Therefore, an evaluation of the selectivity of the PS accumulation in target tissue as well as evaluation of its’ biodistribution in normal tissues is of paramount importance during the development of novel photosensitizer and optimization of PDT protocols. Photolon® (also known as Fotolon® ) is a photosensitizer developed and manufactured by a Byelorussian pharmaceutical company RUE ‘‘Belmedpreparaty’’ (Republic of Belarus, Minsk). Photolon® represents a molecular complex of trisodium salt of chlorin e6 with low molecular polyvinylpirrolidone. In some papers Photolon® is also called as ‘‘Ce6 - PVP’’. Photolon® possesses a strong absorption maximum at 665 ± 2 nm and other physical and chemical properties with potential usefulness in PDT of neoplastic and non-neoplastic diseases [10—12]. Photolon® is officially approved for medical application as a remedy for photodynamic diagnostics and therapy of skin tumors and mucosal malignancies of hollow organs in the Republic of Belarus (since 2001, reg. no. 0106886) and in Russian Federation (since 2004, reg. no. 015948/01) [14]. This paper provides the results of the non-clinical evaluation of biodistribution of the PS Photolon® in inner organs and tissues of intact and tumor-bearing rats. The main objective of the present study was to estimate the influence of the xenograft tumor on the biodistribution pattern, as well as to evaluate the ability of the PS to cross through intact blood—brain barrier. The accumulation studies were performed by means of intravital laser fluorimetry in situ and spectrophotometric determination ex vivo.

Materials and methods Photosensitizer Photolon® was prepared for injection by dissolving of 12.5 mg of lyophilized powder in 10 ml of sterile sodium chloride 0.9%. Photolon® possesses absorption and fluorescence maximum in a red region (655—663 and 668—675 nm respectively) and high quantum yield of singlet oxygen (60%) [13]. Photolon® was administered intravenously (i.v.) by bolus injection into the tail vein at a dose of 5 mg/kg body weight.

S.V. Shliakhtsin et al.

Experimental animals All experiments were carried out in 72 random bred mature rats of both sex bred in the vivarium of National Cancer Center (Republic of Belarus, Minsk). Animals were housed at natural 12 h dark/light cycle according to standard specifications regarding animal keeping in vivariums. Humidity, illumination and temperature factors met requirements of the in-house sanitary code. Free access to food and drinking water was allowed throughout the experiments. All experiments were performed under neuroleptanalgesia (mixture of droperidol:phentanyl (2:1 vol/vol), 0.2 ml/100 g body weight i.m.). Animals were randomized according to sex and body weight into 3 equal experimental groups per 24 units in each.

Xenograft tumor models Experiments were performed using two models of xenograft tumors—–rat carcinosarcoma M-1 (Ca M-1) and alveolar liver carcinoma (PC-1). Tumors were obtained from the tumor strains collection of the N.N. Blokhin Cancer Research Center of the Russian Academy of Medical Sciences (Moscow, Russia). Tumors were implanted subcutaneously in a one flank of each animal by injection of 0.5 ml cell suspension, containing approximately 106 of tumor cells, in Hanks’ solution. Experimental animals were involved into experiments when xenograft tumors grew up to 1—1.5 cm in diameter (6—7 days after transplantation of Ca M-1 and 20—22 days after transplantation of PC-1, respectively). The lack of spontaneous tissue necrosis in the center of tumor nude was proven by histological investigations randomly performed in 2 animals from each group.

Intravital laser fluorimetry in situ One hour after i.v. injection of the PS animals were narcotized by i.m. injection of the mixture of 0.5 g droperidol and 0.005 g of phentanyl per each 100 g of body weight. After the narcotization tumor surface and an opposite to tumor femur muscle were denuded. Animals were also subjected to craniotomy and laparatomy incisions. In vivo fluorescence intensity measurements in inner organs, femur muscle, skin graft at the left flank and in xenograft tumors were performed superficially using a fiber laser spectrophotometer equipped with He—Ne diagnostic laser [15]. For the purpose to avoid the systematic bias due to fluorescent signals from endogenic porphyrins of biological tissues the levels of autofluorescence of target organs and tissues in intact rats were measured before the injection of Photolon® . In the sequel, the appropriate baseline autofluorescence was subtracted from the obtained PS fluorescence signal. After that operative fields were covered with sterile napkin wetted with isotonic saline solution to prevent the redundant graduation and fluid loss. Repeated measurements of fluorescence intensity in inner organs and tissues of experimental animals were performed 2—6 h and 24 h after injection of the PS. At each time point for each organ or tissue not less than 5 repetitive measurements of intravital fluorescence intensity were performed.

Pharmacokinetics and biodistribution of Photolon® (Fotolon® ) in rats

Quantification of Photolon® by means of spectrophotometric assay ex vivo At each time point after the measurement of intravital intensities of florescence signals in inner organs and tissues of intact and tumor-bearing rats 3 animals from each experimental group were mortified, blood samples and tissues of inner organs (liver, spleen, kidneys, bladder, femur muscle, encephalon, skin, xenograft tumors) were collected. Obtained samples of biological tissues were kept frozen at −35 ◦ C until a procedure of quantification of the PS. Immediately before PS assay samples were thawed at room temperature for 1 h and homogenized with tissue homogenizer (Polytron Ergonomic Homogenizer EW-0478000). Extraction of the PS from homogenized samples was performed according to the procedure described in details previously [15]. Briefly, 3 ml of a mixture of acetone and 30% ammonium hydroxide (7:1) was added to 1 g of tissue homogenate and intensively agitated for 10 min on a Vor-

99

tex mixer. The samples were centrifuged at 8000 × g for 10 min. The supernatant was filtered through 10 ␮m filter and an absorption spectra were recorded on the UV—vis spectrophotometer (Schimadzu UV-2401-PC) in the range of 250—750 nm. As control samples blank extracts from appropriate tissues of intact rats (which were not subjected to PS administration) were used. Content of the PS in a sample was calculated on the basis of characteristic absorption maximum at 665 ± 5 nm using calibration curve built with standard solutions.

Statistical analysis All data obtained was treated using methods of descriptive and non-parametric statistics. Description of data is given by mean and standard error of mean (mean ± SEM). Pharmacokinetic values were obtained by using pharmacokinetic functions for Microsoft Excel. Origin Stat Pro 8.0 software (OriginLab Corporation, Copyright©1991—2007) was used

Figure 1 Changes in time of intravital fluorescence intensity in inner organs and tumor tissues after single i.v. injection of Photolon® (Fotolon® ) at a dose of 5 mg/kg body weight: group 1 — intact healthy rats; group 2 — tumor-bearing rats with Sa M-1; group 3 — tumor-bearing rats with alveolar liver cancer PC-1.

100

S.V. Shliakhtsin et al.

Figure 2 Pharmacokinetics of Photolon® (Fotolon® ) in inner organs and tissues of intact and tumor-bearing rats after single i.v. injection of the PS at a dose of 5 mg/kg body weight: group 1 — intact healthy rats; group 2 — tumor-bearing rats with Sa M-1; group 3 — tumor-bearing rats with alveolar liver cancer PC-1.

for statistical analysis. To investigate differences between experimental groups of animals the Mann—Whitney U test was applied. P < 0.05 was considered significant.

Results It was established that after a single-dose i.v. injection at a dose of 5 mg/kg body weight Photolon® is rapidly distributed in inner organs and tissues of both intact and tumor-bearing animals. The biodistribution of the PS occurred nonuniformly. The maximum level of intravital fluorescence intensity in healthy tissues of inner organs was observed 1—2 h after the PS administration (liver (3035.37 ± 158.6 rel. units); spleen (1803.7 ± 98.2 rel. units); kidneys (2555.9 ± 247.3 rel. units); femur muscle (1627.2 ± 103.4 rel. units); brain (2883.9 ± 334.6 rel. units)). Accumulation of the PS in tumor xenografts occurred slower and the peak time was dependent of morphologic

type of the tumor. In particular, for the fast growing tumor Sa M-1 the maximum intensity of intravital fluorescent signal (3399.5 ± 29.4 rel. units) was observed 3 h after Photolon® administration. For the slow-growing tumor PC-1 the maximum intensity of intravital fluorescent signal (3739.8 ± 234.5 rel. units) was observed 4.0—4.5 h after the injection of the PS. The data obtained with intravital fluorimetry in situ was in a good agreement with the results of quantification of Photolon® in tissues by means of spectrophotometric assay performed after extraction of photosensitizer from tissues (Figs. 1 and 2). The maximum accumulation of Photolon® in brain tissues occurred in 30—45 min after injection (2883.9 ± 334.6 rel. units) and the maximum level of the PS concentration was comparable to that found in a highly perfused inner organs such as liver, kidney as well as in xenograft tumors of rats. The elimination of Photolon® from brain tissues also occurred rapidly (Fig. 3). In 6 h after injection only trace amounts of the PS were found in brain tissues of rats. The

Pharmacokinetics and biodistribution of Photolon® (Fotolon® ) in rats

Figure 3 Accumulation of Photolon® (Fotolon® ) in brain tissues of healthy rats after single i.v. injection of the PS at a dose of 5 mg/kg body weight.

relative bioavailability of brain tissues for Photolon® was estimated as 82%, Tmax —–30 min, mean residual time (MRT)— –1.6 h. The maximum level of the PS accumulation in skin (2034.29 ± 39.2 rel. units) was observed 24 h after injection (Fig. 4) however it was much lower than maximum level of the PS in xenograft tumors (3399.5 ± 29.4 rel. units and 3739.8 ± 234.5 rel. units for Sa M-1 and PC-1, respectively). Pharmacokinetic parameters of Photolon® calculated for each of the studied organs and tissues are shown in Tables 1—3. The ratios Cmax /AUC(0—24) and Cmax /AUC(0—inf) were calculated as estimators of the relative bioavailability of the PS in respect to each organ or tissue (Table 2) and the mean residual time for each organ or tissue was established as one of the estimators of PS selectivity to neoplastic tissue (Table 3).

Discussion Previously, on an animal model we have shown that the method of intravital laser fluorimetry in situ is valid for

Figure 4 Accumulation of Photolon® (Fotolon® ) in xenograft tumors and surrounding healthy skin of rats.

101

pharmacokinetic assays of different PS, particularly for investigation of pharmacokinetics of Photolon® [15]. In present study we applied this method to describe the biodistribution of Photolon® in inner organs and tissues of intact and tumor-bearing rats after its’ single-dose i.v. injection. Comparable dynamics of accumulation and elimination of Photolon® in inner organs and tissues, obtained by means of two different analytical methods proves that the method of intravital laser fluorimetry in situ is valid and attractive in application to pharmacokinetic studies of different photosensitizers. According to the data obtained the presence of xenograft tumor does not affect the pattern of Photolon® biodistribution in inner organs and normal tissues in comparison with control animals. The levels of the photosensitizer in normal tissues (brains, liver, spleen, kidneys, bladder, femur muscle) also do not depend on a morphological type of the xenograft tumor and are comparable in intact and tumorbearing rats in three groups. The data obtained prove that studied organs and tissues accumulate the PS in nearly the same amounts, as far as estimators of relative bioavailability of the PS for these tissues are of the same level. After the i.v. injection Photolon® allocates rapidly in the majority of inner organs and tissues. However, the time of maximum accumulation of the PS in target tissue as well as its’ halflife are quite different in respect to different organs and tissues. Thus, the selectivity of the PS to tumor tissue can be optimally described by such values as the mean residual time MRT, and the time of maximum concentration of the PS—Tmax (Table 3). The observed difference in time of maximum accumulation of the PS in two different morphologic types of tumors can be associated with different levels of tumor vascularization. More detailed study of this effect will be a subject of separate experiments. At the moment of maximum level of Photolon® accumulation in tumor tissues (Sa M-1 and PC-1, respectively) levels of the PS in normal (healthy) tissues were significantly lower. As far as the main problem of the most photosensitizers is skin photosensivity afterwards, we have evaluated levels of Photolon® uptake in mentioned tissue. At the time of maximum accumulation of PS in xenograft tumors (3 h post-injection for Sa M-1 and 4—4.5 h for PC-1, respectively) its level in skin is significantly lower. The mean tumor-to-skin ratio was estimated 2.5. The accumulation of the PS in skin occurred slower in comparison to other studied tissues and reached its maximum at 24 h after injection. However Photolon® can be characterized by relatively fast elimination from the skin: 72 h post-injection only trace amounts of the PS were found in this tissue (Fig. 4). The estimated half-life for Photolon® in skin was 14.9 h. Biodistribution profile of Photolon® brings some advantages in comparison with other clinically used photosensitizers, particularly in pharmacokinetics. Primary it refers to such characteristics as ‘‘time of maximum accumulation’’ (Tmax ) and ‘‘mean residual time’’ (MRT) and ‘‘half-life’’ for tumor and healthy tissues, respectively. The studies of pharmacokinetics and biodistribution of Foscan® (metatetra(hydroxyphenyl)chlorin) have shown that maximum accumulation of the PS in tumor tissue takes place within 48—72 h after its’ intravenous injection [16—18]. Moreover, Foscan® exhibits an extensive distribution pattern in the rat, particularly in the liver and also in adrenals, spleen, lungs

423.7 ± 45.2 184.5 ± 39.3 409.04 ± 11.7 — 398.8 ± 20.3 194.6 ± 44.4 367.5 ± 12.3 128.4 ± 22.2 391.08 ± 23.4 — 15.1 ± 0.4 10.5 ± 0.5 14.7 ± 0.8 11.6 ± 0.3 14.9 ± 1.1 —

PC-1 PC-1 PC-1

8.1 ± 0.5 15.3 ± 3.2 7.9 ± 0.6 13.9 ± 2.1 8.5 ± 0.2 —

± ± ± ± ± PC-1

160.0 62.0 128.0 182.2 42.0 16.5 4.4 11.1 37.8 12.3 ± ± ± ± ± 187.7 67.4 173.1 201.5 50.7

Sa M-1

42.9 11.3 24.0 34.1 15.6 ± ± ± ± ± Control

193.1 84.1 234.8 277.8 38.6 ± ± ± ± ± 135.8 58.8 121.9 118.6 34.0 ± ± ± ± ± 147.2 62.0 134.3 137.1 37.2 ± ± ± ± ± 14.1 7.4 12.4 16.8 2.2 ± ± ± ± ±

Control

11.1 10.9 17.7 22.2 4.8 ± ± ± ± ± 11.8 7.8 11.0 7.9 9.5

Liver Spleen Kidneys Bladder Femur muscle Skin Xenograft tumor

12.7 7.3 10.5 8.2 5.8

± ± ± ± ±

1.4 0.7 1.3 0.7 0.7

Sa M-1

1.5 0.4 1.5 1.1 1.2

12.2 7.7 8.2 6.7 9.7

± ± ± ± ±

1.3 0.6 0.9 0.9 2.1 Control

0.6 0.1 0.2 0.4 0.2

Sa M-1

0.6 0.2 0.3 0.2 0.1

10.6 8.3 10.6 17.3 1.3

± ± ± ± ±

0.8 0.7 0.1 0.3 0.1

control

32.4 11.2 15.2 19.2 12.2

Sa M-1

13.7 7.1 12.1 17.8 9.7

127.9 51.0 98.9 107.2 35.5

± ± ± ± ±

9.9 8.3 7.7 23.3 12.4

AUC (0-∞) (␮g × h/g) AUC (0—24) (␮g × h/g) T1/2 (h) Cmax (␮g/g)

Pharmacokinetic parameters of Photolon® (Fotolon® ) in intact and tumor-bearing rats after single i.v. injection at a dose of 5 mg/kg body weight. Table 1

487.4 ± 11.0 253.5 ± 58.5

S.V. Shliakhtsin et al. 19.2 7.1 13.3 26.4 10.5

102

and bone marrow. Release of the drug from the normal tissues is prolonged, with elimination half-lives in tissues of rat of 16, 137, 210, 227, 295, 369 and 489 h in plasma, lung, liver, adrenal, kidney, spleen and muscle, respectively [21]. A prolonged persistence of Foscan® in normal tissues significantly increases the risk of severe adverse reactions of phototoxicity and stipulates the necessity for the patient to comply a strictly limited light regimen for long period of time. In present study it was shown that maximum accumulation of Photolon® in Sa M-1 and PC-1 xenograft tumors takes place within 3—4 h after injection what is essentially faster than the accumulation of many commercially PSs. Elimination of Photolon® from studied tumor tissue of rats occurs with an average elimination half-life of 10.5—11.6 h (elimination rate constant is 0.1 ␮g/g h−1 ). At the same time, in majority of normal tissues studied maximum levels of PS accumulation were observed within 2 h after injection. In 24 h after injection in inner organs of rats only trace amounts of Photolon® were found (less than limit of quantification (LOQ) of the assay). Almost complete elimination of the PS from skin has occurred within 72 h after its administration. One of the major objectives in pharmacokinetic assays is the evaluation of the ability of the drug to cross-intact histohematogenous barriers, particularly a blood—brain barrier. In a number of published works [16,19,20] regarding experimental evaluation of different PSs it was shown, that not all of the clinically approved photosensitizers can cross the blood—brain barrier. In a paper [16] authors provide an experimental evidence that meta-tetra(hydroxyphenyl)chlorin, a second generation photosensitizer approved in EU and commonly known as Foscan, does not penetrate through hematoencephalic barrier and in this case is unsuitable for the systemic PDT of intracranial tumors. An ‘‘ideal photosensitizer’’ for PDT of intracranial tumors should correspond to a single ingredient preparation which accumulates with a high selectivity in tumor tissues, be able to cross the intact blood—brain barrier and to introduce into the infiltrated pathologic tissue but not into normal tissues of the central nervous system. It should exhibit a pronounced cytotoxic effect against tumor cell and to minimally affect surrounding healthy tissues of the brain. It also should possess a strong absorption maximum at the region of 650—800 nm providing a sufficient depth of tumor damage and do not have negative systemic effects on the whole organism. It should rapidly eliminate from the normal tissues and organs, especially from the skin. By means of intravital laser fluorimetry in situ and spectrophotometric assay performed ex vivo we studied the dynamics of Photolon® accumulation in brain tissues of intact and tumor-bearing rats after single i.v. injection of the PS at a dose of 5 mg/kg body weight (Fig. 3). The data obtained show that Photolon® easily crosses the intact blood—brain barrier and rapidly spreads within various parts of the brain of rat. In further studies, Gyneatullin et al. have successfully utilized Photolon® for PDT of intracranial tumors reproduced in rats [22]. The established ability of Photolon® to cross an intact blood—brain barrier and favorable pharmacokinetic properties were put into the basis for the design of a pilot clinical trial of the PS applied for intraoperative PDT in patients with high-grade gliomas. The

Pharmacokinetics and biodistribution of Photolon® (Fotolon® ) in rats

103

Table 2 Estimators of relative bioavailability of inner organs and xenograft tumors of rats for Photolon® (Fotolon® ) after its i.v. injection at a dose of 5 mg/kg body weight. Cmax /AUC (0—24) f

Liver Spleen Kidneys Bladder Femur muscle Skin Xenograft tumor

Cmax /AUC (0—inf) f

Control

Sa M-1

PC-1

Control

Sa M-1

PC-1

0.09 0.12 0.08 0.06 0.16 0.02

0.09 0.13 0.09 0.07 0.28 0.02 0.11

0.10 0.15 0.08 0.06 0.27 0.02 0.08

0.07 0.09 0.04 0.03 0.15 0.02

0.06 0.12 0.06 0.04 0.19 0.02 0.08

0.08 0.12 0.06 0.04 0.23 0.02 0.06

Table 3 Maximum accumulation time (Tmax ) and mean residual time (MRT) of Photolon® (Fotolon® ) in inner organs and xenograft tumors of rats. Tmax (h) Control Liver Spleen Kidneys Bladder Femur muscle Skin Xenograft tumor

1.0 2.0 2.0 5.0 4.0 24

MRT (h) Sa M-1 1.0 2.0 2.0 4.0 4.0 24 3

mentioned clinical trial was approved by the Ministry of Public Health of the Republic of Belarus and is currently ongoing. In general, results of our experimental study of biodistribution of Photolon® in inner organs and tissues of rats, performed as in real time (by means of intravital laser fluorimetry in situ) as ex vivo (spectrophotometric assay) can be utilized while optimizing existing regimens of PDT with the purpose to increase safety and efficacy of treatment as well as for the development of new PDT protocols with Photolon® .

Conclusion In present study we have investigated pharmacokinetics and biodistribution of Photolon® (Fotolon® ) in inner organs and tissues of intact and tumor-bearing rats by means of two independent analytical methods, namely by intravital laser fluorimetry in situ and spectrophotometric assay ex vivo. Estimated parameters of pharmacokinetics and biodistribution of Photolon® /Fotolon® are seemed to be favorable for the majority of modern clinical applications of PDT.

Acknowledgements Authors are sincerely grateful to express their gratitude towards all researchers and scientists who were involved into non-clinical and clinical investigations of Photolon® . We also thank all investigators who continue their researches directed to the widespread of applications of PDT with Photolon® . Furthermore, a special gratitude is

PC-1 1.0 2.0 2.0 5.0 4.0 24 4

Control 15.8 16.6 26.9 33.7 6.7 29.9

Sa M-1

PC-1

17.7 10.4 18.6 25.8 5.4 27.6 18.8

13.9 12.5 15.2 26.0 4.0 25.2 15.7

directed to research officers from Scientific Departments and to all other specialists and executive officers of RUE ‘‘Belmedpreparaty’’ (Minsk, Republic of Belarus) who are still working in the company or had already retired for their creative potential because of which the development of the unique medication Photolon® became possible.

References [1] Henderson BW, Dougherty TJ. How does photodynamic therapy work? J Photochem Photobiol B 1992;55:145—57. [2] Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part three—–photosensitizer pharmacokinetics, biodistribution, tumor localization and modes of tumor destruction. Photodiag Photodynam Ther 2005;2:91—106. [3] Freitas I. Lipid accumulation: the common feature to photosensitizer retaining in normal and malignant tissues. J Photochem Photobiol B 1990;7(November (2—4)):359—61. [4] Moan J, Peng Q, Sorensen R. The biophysical foundations of photodynamic therapy. Endoscopy 1998;4:387—91. [5] Andrejevic Blant S, Ballini JP, van den Bergh H, Fontolliet Ch, Wagnières G, Monnier Ph. Time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin and benzoporphyrin derivative monoacid ring A in the hamster model: comparative fluorescence microscopy study. J Photochem Photobiol B 2000;71(3):333—40. [6] Schastak S, Jean B, Handzel R, et al. Improved pharmacokinetics, biodistribution and necrosis in vivo using a new near infra-red photosensitizer: tetrahydroporphyrin tetratosylat. J Photochem Photobiol B 2005;78:203—13. [7] Rovers JP, Saarnak AE, de Jode M, Sterenborg HJCM, Terpstra OT, Grahn MF. Biodistribution and bioactivity of tetra-pegylated meta-tetra(hydroxyphenyl)chlorin compared

104

[8]

[9]

[10]

[11]

[12]

[13]

[14]

S.V. Shliakhtsin et al. to native meta-tetra(hydroxyphenyl)chlorin in a rat liver tumor model. J Photochem Photobiol B 2000;71(2):210—7. Cheung R, Solonenko M, Busch TM, et al. Correlation of in vivo photosensitizer fluorescence and photodynamic-therapyinduced depth of necrosis in a murine tumor model. J Biomed Optic 2005;8(2):248—52. Stewart F, Baas P, Star W. What does photodynamic therapy have to offer radiation oncologists (or their cancer patients)? Radiother Oncol 1998;48:233—48. Isakau HA, Parkhats MV, Knyukshto VN, Dzhagarov BM, Petrov EP, Petrov PT. Toward understanding the high PDT efficacy of chlorine e6—polyvinylpyrrolidone formulations: Photophysical and molecular aspects of photosensitizer—polymer interaction in vitro. J Photochem Photobiol B 2008;92: 165—74. Isakau HA, Trukhacheva TV, Petrov PT. Isolation and identification of impurities in chlorin e6. J Pharmaceut Biomed Anal 2007;45:20—9. Chin WW, Heng PW, Thong PS, et al. Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer. Eur J Pharm Biopharm 2008;69(3):1083—93. Epub 2008, Mar 10. Parkhats MV, Knyukshto VN, Isakov GA, et al. Spectralluminescent studies of the photosensitizer Photolon® in model systems and in blood of oncological patients. J Appl Spectroscop 2003;70(6):816—21. Trukhachova TV, Shliakhtsin SV, Isakau GA, et al. New aspects of clinical application of PDT with Photolon® . In: Book of abstracts of the 7-th international simposium on photodynamic therapy and photodiagnosis in clinical practice. Brixen; 2008. CT-03.

[15] Shliakhtsin SV, Trukhachova TV, Isakau GA, Istomin YP. Application of the method of vital in vivo fluorescence intensity measurement in biological tissues for pharmacokinetic studies of different chlorin-based photosensitizers. Biomed Chem 2009;(2). [16] Ronn AM, Batti J, Lee CJ, et al. Comparative biodistribution of meta-tetra(hydroxyphenyl)chlorin in multiple species: clinical implications for photodynamic therapy. Lasers Surg Med 1997;20(4):437—42. [17] Bossu E, A’amar O, Parache RM, et al. Determination of the maximal tumor/normal skin ratio after HPD or mTHPC administration in hairless mouse (SKH1) by fluorescence spectroscopy-non invasive method. Anti Cancer Drug 1997;8: 67—72. [18] Cramers P, Ruevekamp M, Oppelnar H, Dalesio O, Baas P, Stewart FA. Foscan uptake and tissue distribution in relation to photodynamic efficacy. Br J Cancer 2003;88:283—90. [19] Rosenthal M, Kavar B, Hill J, et al. Phase I and pharmacokinetic study of photodynamic therapy for high-grade gliomas using a novel boronated porphyrin. J Clin Oncol 2001;19(January (2)):519—24. [20] Stummer W, Stocker S, Novotny A, et al. In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid. J Photochem Photobiol 1998;45:160—9. [21] Foscan. European Public Assessment Report, Revision 10 - Published by EMEA 12/08/08. [22] Gyneatullin RU, Ismagilova ST, Eremeev DV, Astahova LN, Ignat’eva EN. Morphologic changes in brain tissues after photodynamic therapy of malignant tumors reproduced in rats (an experimental study). Bull Novel Med Technol 2008;3:64—85 (article in Russian).