Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

www.elsevier.com/locate/jphotobiol

Improved pharmacokinetics, biodistribution and necrosis in

vivo using a new near infra-red photosensitizer:
tetrahydroporphyrin tetratosylat q,qq
Stanislaw Schastak a,b,*, Benedikt Jean c, Romy Handzel a, Genady Kostenich d,
Ralf Hermann e, Ulrich Sack f, Arie Orenstein d, Yu-sheng Wang g, Peter Wiedemann a

a
Universitätsklinikum Leipzig AöR, Klinik und Poliklinik für Augenheilkunde, Liebig Straße 10-14, 04103 Leipzig, Germany
b
Laser Medizin Zentrum Leipzig, Liebig Straße 10-14, 04103 Leipzig, Germany
c
Universität Tübingen, Universitäts-Augenklinik I, Sektion Experimentelle Ophthalmochirurgie, Derendinger Straße 41, 72072 Tübingen, Germany
d
Advanced Technology Center, Sheba Medical Center, Tel-Hashomer 52621, Israel
e
Universität Leipzig, Fakultät für Chemie und Mineralogie, Permoserstraße 15, 04318 Leipzig, Germany
f
Universitätsklinikum Leipzig AöR, Institut für Klinische Immunologie und Transfusionsmedizin, Johannisallee 30, 04103 Leipzig, Germany
g
Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, 710032 XiÕan, PR China

Received 25 August 2004; received in revised form 9 November 2004; accepted 10 November 2004
Available online 7 January 2005

Abstract

The search for better photosensitizers for photodynamic therapy of malignancies has led to the investigation of a new water-
soluble, positively charged, and chemical stable tetrahydroporphyrin tetratosylat (THPTS) with a strong absorption at 760.5 nm,
belonging to the bacteriochlorophyll family.
THPTS undergoes a rapid uptake by human choroidal melanoma (CM) cells with a weak dark toxicity after a 24-h incubation
(LD10 = 150 lM, LD50 = 6.0 mM). In response to laser light at 760 ± 3 nm and doses of 10, 15 and 30 J/cm2, around 71%, 76%, and
92% of the CM cells were killed, respectively.
Studies of pharmacokinetics and biodistribution in vivo (living mice) and ex vivo (excised organs) were made in a Balb/c mice
bearing subcutaneously inoculated C26 colon carcinoma using fiber-optic spectrofluorimetry (FOS). Tumours were irradiated
3 h after intraperitoneal (i.p.) injection of 5.0 mg/kg THPTS with an incoherent light source at 750 ± 20 nm and an intensity
of 100 mW/cm2 and fluences of 60, 90 and 120 J/cm2.
THPTS demonstrated preferential accumulation in C26 colon carcinoma in comparison with most normal tissues except kidneys.
For the tissues of liver, colon, muscle, and spleen the tumour/normal tissue ratio (TNTR) ranged from 8.0 to 50. After irradiation
with 120 J/cm2 the depth of tumour necrosis reached 15 mm. Histological examination of the tumour samples 24 h after THPTS-
PDT, revealed severe stasis in the blood vessels and coagulative necrosis.
These results suggest that THPTS-PDT may be of particular importance in the treatment of accessible malignancies.

2004 Elsevier B.V. All rights reserved.

Keywords: BALB/c mice tumour model; Choroidal melanoma cell; Photodynamic therapy; Photosensitizer

q
Australian Patent No. AU 746710B, Canadian Patent No. CA 2323150A1, Europe Patent No. EP 1066293B1, US Patent No. US 6,410,568 B1,
WO Patent No. WO 1999050269A3.
II
Data presented in part at the 100th Annual Meeting of the Deutsche Ophthalmologische Gesellschaft, Berlin, September 26, 2002
(Ophthalmologe 2002; 99; Suppl.1: DO.08.17).
*
Corresponding author. Tel.: +49 341 972 1582; fax: +49 341 972 1659.
E-mail address: stas@medizin.uni-leipzig.de (S. Schastak).

1011-1344/$ - see front matter
2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2004.11.006
204 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

1. Introduction (UV–Vis) spectroscopy. The measurements were per-

formed using a Bruker Avance DPX 250 MHz spec-
Photodynamic Therapy (PDT) is a well-known pro- trometer in D2O (2.5 mg/ml of THPTS) at room
cedure in the field of clinical medicine for the treat- temperature. Chemical shifts were referenced to the sol-
ment of various diseases. At present, it is mostly vent resonance, which was set to dH = 4.72 ppm. The
used for the treatment of cancers [1–6], although there water signal was suppressed by presaturation.
has also been research into its application for non- UV–Vis spectra of THPTS in distilled water and in
malignant conditions such as age related macular methanol were recorded with a UV–Vis spectrophoto-
degeneration [7,8]. meter CARY 3 (UV-2101 PC, Shimadzu, Tokyo,
PDT is based on the discovery that cells are destroyed Japan). Fluorescence emission spectra of THPTS were
when treated with certain photosensitising drugs and ex- measured in distilled water and methanol using a Lumi-
posed to light at a particular wavelength (photoactiva- nescence Spectrometer LS 50B (Perkin–Elmer).
tion). It uses a fixed wavelength light source that is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
able to activate photosensitising drugs, which have accu- razoliumbromide), dissolved to 5 mg/ml in distilled
mulated in the body tissues. Photodynamic therapy of water, SDS (sodium dodecyl sulphate), dissolved to 40
cancers using Photofrin and laser light of 630 nm wave- mg/100 ml in distilled water, isopropanol, dimethyl
length is already clinically useful but confined to a formamide (DMF), and paraformaldehyde were ob-
tumouricidal tissue penetration of 4 mm, which might tained in highest available purity from Serva, Heidel-
be increased threefold with laser light between 700 and berg, Germany. Trypsin/EDTA, cell culture media and
800 nm. supplements, and animal serum were purchased from
On the basis of the promising clinical results ob- Biochrom, Berlin, Germany. The disposable cell culture
tained with Photofrin [2], and because of various dis- equipment was from Technoplastics Products Ltd., Tra-
advantages of this substance (chemical heterogeneity, sadingen, Switzerland.
high and enduring phototoxicity in daylight), second
generation photosensitizers [9–18] are currently being 2.2. In vitro studies
tested. This group comprises numerous substance clas-
ses such as anthraquinones, anthrapyrazoles, perylene 2.2.1. Cell cultures
quinones, xanthenes, cyanines, acridines, phenoxa- An undifferentiated human choroidal melanoma cell
zines, and phenothiazines. These photosensitizers are line (CM) [6,7] and the C26 colon carcinoma cell line
selected according to the following criteria [1]: high (Harlan Laboratories Ltd., Jerusalem, Israel) were inoc-
absorption (at minimal drug dose) in the range of ulated into plastic tissue culture flasks at a concentration
700–800 nm, to achieve maximal tissue penetration, of 5 · 105 cells/ml and maintained in RPMI 1640 med-
minimal dark toxicity but high photocytotoxicity, ium supplemented with 10% heat-inactivated fetal calf
good water solubility for easier drug administration serum (FCS), penicillin (100 U/ml) and streptomycin
and rapid clearance from the body to avoid cutaneous (100 U/ml) and placed in an incubator at 37 C in a
phototoxicity, predominant localization in pathologi- 8% CO2 atmosphere.
cal tissue (e.g., tumour) with rapid clearance from
normal tissue. 2.2.2. Cellular uptake studies
In this work, we present the novel cationic dye Analyses were performed on a Fluorescence Acti-
5,10,15,20-tetrakis-(1-methyl-3-pyridyl)-21H,23H-7,8, vated Cell Sorter (FACS) Calibur flow cytometer (Bec-
17,18-tetrahydroporphyrin tetratosylat (THPTS), which ton-Dickinson, San Jose, CA). To prepare a cell
strongly absorbs in the near infra-red (NIR) spectral mixture for flow cytometric analysis, CM cells were
region around 760.5 nm [9–11]. We report also results incubated in a suspension of 2 · 105 cells/ml with 5
of PDT treatment with this water soluble and chemically lM THPTS dissolved in phosphate buffered saline
stable agent. (PBS, pH 7.4) for 1, 3, 6, 12, 24, 30, 36 or 48 h in a
humidified incubator enriched with 8% CO2 at 37 C.
CM cells were removed by trypsinization, washed twice
2. Materials and methods in PBS, stabilized for 20 min at 4 C in 1% paraformal-
dehyde. The fluorescence intensity of THPTS in the CM
2.1. General cells was measured at an excitation wavelength of
630 nm. Experiments were performed in quadruplicate.
The photosensitizer tetrahydroporphyrin tetratosylat
(C72H70N8O12S4; molecular weight 1367.66 Da) was ob- 2.2.3. Dark toxicity study
tained from Astrid Schastak Inc., D-04519 Rackwitz/ To determine dark toxicity of THPTS, CM cells were
Germany. THPTS was characterized by nuclear mag- incubated in the dark with increasing concentrations of
netic resonance (1H-NMR) and ultra violet–visible the agents in RPMI up to 6 mM for 24 h in a humidified
 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213 205

incubator enriched with 8% CO2 at 37 C. After 24 h passed via the second branch of the fiber bundle and
incubation in the dark, the cells were washed three times was set to 5 mW at the fiber tip, as measured with a laser
with PBS and incubated in 100 ll RPMI 1640 with 10 ll power meter (Laserstar, Ophir, Israel). An average of
MTT solution for 4 h. After extraction in 100 ll SDS– three recordings was used for each data point.
DMF the absorbance of the formazan product was mea- The fluorescence intensity data for each tissue sample
sured at 570 nm in a microplate spectrophotometer per time point was calculated as mean ± standard error
(Spectramax 250, Molecular Devices Inc., Sunnyvale, (SE). In comparison of the fluorescence intensity
CA) [12]. between the tumour and the appropriate normal tissue
All experiments were done in quadruplicate. Values (tumour/normal tissue ratio, TNTR), statistical signifi-
are expressed as percentages of non-incubated controls. cance (p < 0.05) was determined using the StudentÕs
For each incubation concentration, four independent t-test.
experiments were also performed in quadruplicate.
2.3.3. Effect assessment of THPS-PDT
2.2.4. Study of cellular phototoxicity THPTS was injected intraperitoneally (i.p.) at a dose
Cells resuspended in 50 ll DMEM for 4 h at a con- of 5.0 mg/kg, 3 h before light treatment. The mice were
centration of 50,000 cells per well were incubated in qua- anesthetized with Nembutal (60 mg/kg; i.p.) and
druplicates in the dark with photosensitizer at a final the dorsa were depilated by hair removal cream (Vito,
concentration of 150 lM THPTS (LD10-value) for 24 Dexon). The mice were covered with aluminium foil,
h in flat-bottom 96 well microtiter plates. After incuba- exposing only the tumour area.
tion, the cells were washed three times with PBS, cul- Irradiation of each group (n = 3) was performed
tured in 100 ll RPMI medium supplemented with 10% using a fiber-optic non-coherent light delivery system
FCS, penicillin, and streptomycin, and were irradiated (SeNet Ltd., Israel) in combination with a broadband
at 760 ± 3 nm by a Nd:YAG pumped Ti:Sapphire oscil- interference filter at 750 ± 20 nm, at an intensity of
lator laser in cw-mode (MIRA 900 F, Coherent Inc., 100 mW/cm2, measured with a Model IL 1400A radio-
Santa Clara, CA) with 40 mW/cm2 and fluences of 10, meter/photometer (International Light Inc., Newbury-
15, and 30 J/cm2. Cell activity was determined 19 h post port, MA).
exposure using the MTT test. Tumour necrosis depth was examined 24 h after
PDT. Vital staining was performed by i.p. injection
2.3. Animal experiments of 0.4 ml 1% Evans Blue (EB) solution. Six hours later,
animals were sacrificed, tumours were excised, and 2–3
2.3.1. Animals and tumour model mm thick cross-section slices were cut. A section from
Female BALB/c nude mice, at 8 weeks of age, the central area of each tumour was examined macro-
were obtained from Harlan Ltd. (Jerusalem, Israel) scopically using a morphometrical system (video cam-
and maintained under pathogen-limited conditions. era OLYMPUS DP 50, in conjunction with a PC
Animals were given Purina food and water ad libitum. Image-Pro program). The unstained area was attrib-
Tumours were induced by subcutaneous injection of uted to tissue necrosis, whereas the stained area
0.5 ml suspension containing 106 cells of colon C26 showed tissue with preserved blood supply. Five mea-
carcinoma in saline into the flank area. Experiments surements of necrosis depth were made on each
were started when the tumour diameter was 9–15 section.
mm. All experiments were conducted in compliance
with regulations of the Animal Welfare Committee of 2.3.4. Histological examination
the Sheba Medical Center. For histological examination, the samples were fixed
in 4% buffered formaldehyde, embedded in paraffin, and
2.3.2. In vivo assessment of pharmacokinetics and sectioned into 5 lm thick sections. The sections were
biodistribution stained with hematoxylin–eosin (H&E) and examined
Measurements of in vivo (living mice) and ex vivo under a light microscope.
(excised organs) fluorescence were made using fiber-
optic spectrofluorimetry (FOS). An argon ion laser
(Melles Griot, 43 series) was coupled to a spectrofluoro- 3. Results
photometer (Shimadzu, model RF-5301PC) using a
bifurcated fiber bundle (Oriel, Stratford, CT, model 3.1. Spectroscopic characterization
77533). The common-end tip of the bundle was fixed
at a distance of 1 mm from a mouse (in vivo) or from The structure of THPTS was investigated by proton
a tissue sample (ex vivo), while the distal tip of one NMR experiments. The 1H-NMR (250 MHz) spectrum
branch was mounted in front of the entrance slit of (Fig. 1) showed following chemical shifts: d 1.7 (s,
the spectrofluorophotometer. Laser light (514 nm) was 3H · 4, (a)); d 6.5 (d, 2H · 4, (b)); d 7.2 (d, 2H · 4,
206 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

10 9 8 7 6 5 4 3 2 1
ppm
1
Fig. 1. H-NMR-spectrum at 250 MHz of 5,10,15,20-tetrakis-(1-alkyl-3-pyridyl)-21H, 23H-7,8,17,18-tetrahydroporphyrin tetratosylat (THPTS)
photosensitizer in D2O with presaturation.

(c)); d 4.1 (s, 8H, (d)); d 8.1 (s, 4H, (e)); d 8.5 (m, 4H, >90%. The toxicity increases slightly reaching the
(f)); d 9.5 (s, 4H, (g)); d 9.2 (d, 4H, (h)), d 9.1 (d, 4H, LD50 value (cell survival 650%) at a concentration of
(i)). The shifts were assigned to the functional groups about 6 mM.
CH, CH2, CH3 [13] and the structure of the compound During incubation of CM cells with THPTS rapid
was determined as shown in Fig. 2. Considering purity accumulation of the drug occurred within 3 h after the
it can be concluded that the 1H-NMR spectrum is free beginning of incubation. Accumulation increased slowly
of impurities and the purity of the drug is better reaching a plateau at about 24 h (Fig. 6).
than 90%, related to hydrogen-containing organic To assess phototoxicity, CM cells were preloaded for
compounds. 24 h with THPTS at concentrations of 50, 100 and 150
Fig. 3 demonstrates that wavelengths and full widths lM and irradiated with 760 ± 3 nm laser light. Photo-
at half maximum of the absorption bands were not af- toxicity was dependent on both drug and light doses.
fected by the polarity of the solvent. Absorption of At 150 lM of THPTS, irradiation with 10, 15 and 30
THPTS in the UV spectral region was somewhat more J/cm2 caused cell death of 70.8 ± 3.9%, 75.9 ± 0.9%
pronounced in water as compared with methanol. and 91.6 ± 2.2%, respectively (Fig. 7).
Steady-state fluorescence emission spectra of THPTS
(Fig. 4) were characterized by two maxima at 665 nm 3.3. In vivo spectroscopy, pharmacokinetics and
and at 770 nm. The measurements showed that biodistribution
THPTS is much weaker fluorescing than the corre-
sponding chlorin by-product. Fig. 8 depicts in vivo fluorescence kinetics in mice
skin over 72 h. Fluorescence reached a maximum 0.5–
3.2. In vitro studies 1 h after administration, followed by a rapid decrease
due to elimination of the compound.
Fig. 5 depicts dark toxicity as a function of THPTS Fig. 9 shows comparative biodistribution kinetics of
concentration on CM cells at 24 h incubation time. In THPTS fluorescence in various tissues ex vivo over 72
the dark, the sensitizer exhibits low cytotoxicity. At h. Highest overall fluorescence of the agent was
THPTS concentration of 150 lM, cell survival was observed in the kidneys. Tumour and skin were very
 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213 207

+
N R
(i)
HH (d)

H
R H
N
+
NH N O
-
(h) CH3 S O
+ (a)
N NH N O
(g) (b) (c) 4
H R
H (e)
H
H
+
R N (f)

Fig. 2. Structure formula of the 5,10,15,20-tetrakis-(1-alkyl-3-pyridyl)-21H,23H-7,8,17,18-tetrahydroporphyrin tetratosylat (THPTS)

photosensitizer.

0,8

Methanol
Water
0,6
Absorption (a.u.)

0,4

0,2

0,0

200 300 400 500 600 700 800

Wavelength(nm)

Fig. 3. Absorption spectrum of THPTS in distilled water and in methanol. Note the main absorption maximum at 760.5 nm wavelength and absence
of solvatochromic shift of the Soret band. The spectrum was measured at THPTS concentration of 5 lM using a UV–Vis scanning
spectrophotometer (UV-2101 PC, Shimadzu, Tokyo, Japan).

15000
Chlorin
Fluorescence intensity (a.u.)

THPTS

10000

5000

400 500 600 700 800 900

Wavelength (nm)

Fig. 4. Steady-state fluorescence emission spectrum of THPTS in distilled water. The spectrum has two maxima around 665 and 770 nm. The
maximum at 665 nm belongs to a chlorin by-product and is selectively excited at 420 nm. THPTS fluorescence emission peak at 770 nm is selectively
excited at 516.5 nm.
208 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

110
100
90
80

Cell survival (%)

70
60
50
40
30
20
10
0
0,1 1 10 100 1000
Concentration of THPTS (µM)

Fig. 5. Dark toxicity of THPTS for choroidal melanoma cells, incubated in RPMI medium in the dark for 24 h in presence of the indicated
concentrations of photosensitizer. After incubation, the percentage of cells killed was determined by MTT assay [22]. Each point represents the
mean ± SD of 4 experiments.

200
Fluorescence intensity (a.u.)

150

100

50

0 10 20 30 40 50
Incubation time (h)

Fig. 6. Uptake kinetics of THPTS in choroidal melanoma cells. The cells were incubated with THPTS (5 lM) in RPMI culture medium for 1, 3, 6,
12, 24, 30, 36 or 48 h. After incubation the cells were washed in PBS, stabilized and measured at 630 nm excitation wavelength by FACS.

similar in both kinetics and level of accumulation, which are shown in Fig. 10. Morphometric measurements
significantly exceeded liver, spleen, lung, muscle, and co- showed an increase in the extent of damage as a function
lon tissues. Table 1 presents relative tumour to normal of light dose. The results of these experiments are pre-
tissue ratios (TNTR) at different time points. sented in Table 2. Maximal tumour necrosis depth
13.2 ± 1.2 mm was noted after irradiation with
3.4. Necrosis depth assessment 120 J/cm2.

Twenty-four hours after treatment, viable and dam- 3.5. Histology

aged tumour tissues were identified using vital staining
with EB. Strong blue staining was observed in untreated Fig. 11 demonstrates histological sections of C26
controls, whereas in PDT-treated tumours damage was colon carcinoma in untreated control (A) and 24 h after
clearly distinguishable as an unstained area in the upper THPTS-PDT at 90 J/cm2 (B). THPTS-PDT caused se-
part of the tumour. Typical images of C26 colon carci- vere stasis in the blood vessels and coagulation necrosis
noma samples after THPTS-PDT and EB vital staining in the tumour.
 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213 209

100
0 µM
50 µM
80 100 µM

Cell survival (%)

150 µM

60

40

20

0
0 10 20 30
Light dose (J/cm²)
Fig. 7. Phototoxicity of THPTS on cultured human CM cells. Cells were incubated with photosensitizer at various concentrations for 24 h, washed,
then exposed to laser light at 760 ± 3 nm with an irradiation dose of 0–30 J/cm2. The percentage of cells killed was determined by MTT assay at 19 h
after laser light exposure. Each point represents the mean ± SE of 4 experiments.

200
Fluorescence intensity (a.u.)

150

100

50

0 20 40 60 80
Time (h)

Fig. 8. In vivo kinetics of THPTS fluorescence at 770 nm in mice skin (mean ± SE, 3 mice) after i.p. administration of the dye (5 mg/kg). Excitation
at 514 nm.

120
Kidney
Tumour
Fluorescence intensity (a.u.)

100 Skin
Liver
80 Colon
Muscle
60 Lung
Spleen
40

20

0 20 40 60 80
Time (h)

Fig. 9. Biodistribution of THPTS (i.p. administration, 5 mg/kg) in C26 colon carcinoma and in normal tissues of mice at different time points
(mean ± SE, four mice per point). Ex vivo fluorescence registered by FOS at 770 nm; excitation at 514 nm.
210 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

Table 1
Tumour/normal tissue ratio (TNTR) data for THPTS after i.p. administration of 5 mg/kg
Time (h) Tumour/normal tissue ratio
Skin Liver Kidney Spleen Lung Muscle Colon
3 1.4 9.0 1.7 25.0 10.0 16.7 3.3
8 1.2* 3.8 0.6 11.5 9.2 11.5 6.6
16 1.1* 3.3 0.6 20.0 13.3 16.0 8.0
24 1.3 2.5 0.7 19.3 16.5 19.0 3.8
48 1.2* 3.0 0.6 12.0 6.0 12.0 3.6
72 2.0 5.0 1.3 50.0 8.3 33.3 3.3
*
The difference in fluorescence intensity between tumour and appropriate normal tissue is statistically insignificant (P > 0.05).

Fig. 10. Vitally stained C26 colon carcinoma tumour samples in untreated control (a) and after THPTS-PDT at 120 J/cm2 (b). Unstained area –
tumour necrosis, stained area – viable tissue.

Table 2
Tumour necrosis depth after THPTS-PDT
Treatment group Light dose [J/cm2] Necrosis
Frequency* (%) Depth (mm)
1. THPTS + light 60 80 5.2 ± 0.6
2. THPTS + light 90 100 9.5 ± 0.8
3. THPTS + light 120 100 13.2 ± 1.2
4. THPTS alone 0 0 0
5. Light alone 120 0 0
5 mg/kg THPTS was injected 3 h before photo irradiation using 750 ± 20 nm light at a fluence rate of 100 mW/cm2.
Three animals in each group were examined.
Data are shown as mean ± standard deviation.
*
Percent of samples with necrosis in the treatment group.

4. Discussion agents are poor water solubility, reduced chemical sta-

bility or low tumour selectivity [26,27].
There is a great interest in synthesis of second- We have studied general characteristics of a novel
generation photosensitizers with improved PDT char- water soluble, positively charged and chemically stable
acteristics. Such photoactive compounds are under NIR-absorbing photosensitising agent belonging to the
development in many laboratories in various phases of bacteriochlorophyll family.
preclinical and clinical trials [3,5,14,15,17–24]. Bacterio- Analyzing 1H-NMR, absorption and fluorescence
chlorins and bacteriochlorophylls particularly are under emission spectra of THPTS we have concluded that
strong investigations because of their unique spectral the purity degree of the agent must have been better
properties [25–28]. Some of the problems with these than 95%, related to hydrogen-containing organic
 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213 211

Fig. 11. C26 colon carcinoma in control (a) and 24 h after THPTS-PDT (b) (THPTS 5.0 mg/kg, 90 J/cm2). H&E staining, · 400. Light irradiation
(750 ± 20 nm, 90 J/cm2) was performed 3 h after intraperitoneal injection of THPTS at a dose of 5 mg/kg body weight.

compounds. The major absorption bands, both at 373 The results of the present study showed that THPTS
nm and at 760.5 nm, are not red shifted as a result of is a potent and effective photosensitizer warranting fur-
a solvatochromic effect (Fig. 3). Absorption of THPTS ther investigations.
in water and methanol grew linearly as a function of
drug concentration up to 6 mM, and obeyed the Beer–
Lambert law. Wavelengths and full widths at half 5. Conclusion
maximum of the absorption bands were not affected
by solvent polarity. These results showed that THPTS Evaluations in human CM cells in vitro and in a co-
in aqueous solution was chemically stable, and that no lon adenocarcinoma model in Balb/c mice showed that
aggregation of the photosensitizer occurred. Extinction THPTS is a chemically stable photoactive compound
coefficient (e) of THPTS in water at 760.5 nm is with low cellular dark toxicity and an excellent
e = 105,000 M 1 cm 1. phototoxicity.
In vitro, THPTS demonstrated fast intracellular pen- THPTS was relatively rapidly accumulated in C26
etration into CM cells with low dark toxicity and strong colon carcinoma and had low systemic toxicity and
phototoxic effect (Figs. 5–7). Previously, it has been high uptake in most of the normal tissues except
shown by confocal laser microscopy in a model of bili- kidneys.
ary tract cancer cells that THPTS localized in distinct THPTS-PDT of C26 colon carcinoma with near-
subcellular compartments within the cytosol and within infrared photoirradiation at 750 ± 20 nm caused sig-
the cell core [9]. nificant damage to tumour tissue. The preliminary
In vivo, rapid uptake of THPTS by C26 colon carci- results demonstrate that THPTS is a potent agent that
noma was observed already at 3 h post i.p. injection. can be used for fluorescence diagnosis as well as for
This may be explained by highly developed functional efficient photodynamic therapy of thick tumours. Fur-
neovascular network in this tumour model [29]. The bio- ther studies will be necessary to define the clinical po-
distribution assay revealed preferential tumour accumu- tency of this dye as a tumour-targeting agent for PDT
lation of THPTS in comparison with all normal tissues of cancer.
with TNTR ranging from 1.4 to 25 (Fig. 9, Table 1).
For the time interval between 8 and 48 h, kidneys
showed the highest fluorescence, which is not surprising 6. Abbreviations
as they are the main route for agent elimination from the
body. Importantly, liver and normal colon tissue dem- AMD age related macular degeneration
onstrated relatively low uptake of THPTS. Therefore, CM choroidal melanoma
TNTRs of THPTS observed for liver, lung, spleen and CW continuous wave
muscles were higher than those published for some other DMEM DulbeccoÕs Modified Eagle Medium
photosensitizers [28,30–33]. DMF dimethyl formamide
THPTS-PDT caused severe damage to the tumour EDTA ethylenediaminetetraacetic acid
vasculature resulting in coagulative necrosis. Depth of MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
necrosis increased with fluence and reached 13.2 ± 1.2 tetrazolium bromide
mm at 120 J/cm2 (Table 2, Fig. 10). At this light dose, PDT photodynamic therapy
adjacent normal tissues demonstrated only moderate THPTS 5,10,15,20-tetrakis-(1-methyl-3-pyridyl)-
temporary damage (oedema), thus tumours could be 21H,23H-7,8,17,18-tetrahydroporphyrin tetra-
eradicated selectively. tosylat
212 S. Schastak et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 203–213

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