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Pharmacokinetic and tumour-photosensitizing properties of the cationic porphyrin meso-tetra(4N-methylpyridyl)porphine
CANCER LETTERS Cancer
Letters 73 (1993) 59-64
Pharmacokinetic and tumour-photosensitizing properties of the cationic porphyrin meso-tetra(4Wmethylpyridyl)porphine Angeles
Villanueva* a, Giulio Jori b
“Departamento de Biologia, Universidad Autdnoma de Madrid, 28049 Mudrid, Spain ‘Dipartimento di Biologia. Universitir di Padova, Via Triesfe 7.5, 35121 Padova, Ital)
(Received 19 April 1993; revision received 21 June 1993; accepted
23 June
1993)
Abstract The pharmacokinetic behaviour and the photodynamic properties of the cationic porphyrin meso-tetra (4N-methylpyridyl) porphine (T,MP,P 2.1 mgikg) were examined in Balb/c mice bearing an MS-2 fibrosarcoma. The porphyrin shows good tumour localizing properties; 24 h after drug administration the tumour concentration of T,MP,P was approximately 1.2 ng/mg, while the concentrations in normal tissues were substantially lower, except for liver and spleen. In the serum, T,MP,P is preferentially transported by albumin and globulins (80.5X), while minor amounts are associated to lipoproteins (19.5%). The phototherapeutic efficiency of T,MP,P was tested by following the growth curves of fibrosarcoma irradiated by 600-680 nm (450 J/cm’) 24 h after the i.v. injection of T,MP,P (2.1 mgikg). PDT-treated tumours showed a temporary delay in their growth compared with control tumours. The excellent selectivity of T,MP,P and its antitumour activity on photoexcitation encourage further studies for assessing the usefulness of this porphyrin in photodynamic therapy. Key words: Meso-tetra(4N-niethyIpyridyl)porphine; sitizer
Pharmacokinetic
1. Introduction Photodynamic therapy (PDT) of tumours is based on the systemic administration of a photosensitizer that is retained in sufficiently large amounts by malignant tissues so that illumination of the tumour area with wavelengths specifically absorbed by the photosensitizer leads to tumour necrosis. Essentially all clinical applications of
* Corresponding
author.
0304-3835193/$06.00 0 1993 Elsevier Scientific SSDI 0304-3835(93)03117-N
Publishers
Ireland
properties;
Photodynamic
therapy;
Photosen-
PDT use one derivative of hematoporphyrin (HpD) or its partially purified version, known as Photofrin II, as phototherapeutic agents [l]. The intrinsic limitations of Photofrin, such as the reduced selectivity of tumour targeting and the heterogeneous chemical composition, prompted several investigations aimed at identifying secondgeneration photosensitizers with improved phototherapeutic properties. In general, such photosensitizers, including chlorins, phthalocyanines and naphthalocyanines, are typically associated with cell membranes [2]. Ltd. All rights reserved.
60
A. Villanueva. G. Jori/Cancer
On the other hand, no thorough investigation has been performed on the phototherapeutic potential of DNA-interacting photosensitizers. A number of investigators have reported the capability of the cationic porphyrin meso-tetra(4N-methylpyridyl)porphine (T,MP,P) (Fig. 1) to bind to DNA by intercalation between the base pairs [3] and photoinduce DNA damage in vitro 1451. T4MP,P appears to have several interesting photochemical properties, such as a high quantum yield of production of the cytotoxic species IO2 [6] and a monomeric molecular behaviour over a wide concentration range [7]. Most porphyrintype photosensitizers undergo aggregation in aqueous solution at concentrations used for in vivo experiments, which depresses the photosensitizing efficiency [8]. This situation may change on systemic injection of the porphyrins, which are largely converted to monomeric species on interaction and binding with plasma proteins. One notable exception is Photofrin II, which remains aggregated, at least in part, in the bloodstream [9]. Recent studies have shown that T4MP,P provokes the photodynamic inactivation of HeLa cells [5]. However, to our knowledge, in vivo studies with experimental tumours have not been carried out with this porphyrin.
‘CH,
Fig. 1, Chemical structure of meso-tetra (4N-methyl-pyridyl) porphine tetraiodide (T,MP,P).
73 (1993) 59-64
2. Materials and methods 2.1. Drug Meso-tetra (4N-methylpyridyl)porphine tetraiodide (T,MP,P) was obtained from Ventron-Alfa Produkte (Karlsruhe) and stored at -4°C. Solutions were prepared in 0.9% NaCl, maintained in the dark and used within 24 h. 2.2. Animals and tumour model Female Balb/c mice (18-22 g body weight) obtained from Charles River (Como) were kept in a temperature-controlled and light-controlled room with free access to water and dietary chow. When necessary, animals were anaesthetized with ketalar (150 mg/kg i.p.). The experimental tumour MS-2 librosarcoma was intramuscularly implanted in the right hind leg by injection of a cell suspension containing at least lo6 cells/ml. Experiments were performed within 6-8 days, when the tumour diameter was 5-7 mm. Spontaneous tumour necrosis was minimal or absent for these tumour sizes. New Zealand rabbits were used for studies of T4MP,P distribution in serum. In all cases, animals were treated according to the guidelines for animal welfare established by the Italian Committee for Experiments on Animals. 2.3. Pharmacokinetic
H3C
Lett.
studies
T4MP,P (2.1 mg/kg) was administered by iv. injection. Animals were sacrificed at different times (six mice at 24 h and three mice for other times) between 1 h and 1 week after administration. About 200 mg of tissue was homogenized in 2% sodium dodecyl sulfate (SDS) (4 ml) and incubated for 1 h. The homogenates were centrifuged at 3000 rev/min for 15 min. and the fluorescence of the supernatants was measured setting the excitation wavelength at 420 nm and recording the emission spectrum from 655 to 720 nm. Serum samples, isolated from blood by centrifugation, were diluted with suitable volumes of 2% SDS so that the absorbance of T,MP,P at 423 nm was lower than 0.1 and analysed at the spectrophoto-
A. Villanueva, G. Jori / Cancer Lett.
61
73 (19931 59-64
fluorimeter. T4MP,P levels were determined by interpolation on a standard curve plotted with known amounts of T4MP,P in 2% SDS, and reported in terms of ng/mg tissue or @ml of serum. The standard curve was periodically repeated to compensate for any time-dependent variability in the instrumental response. In all cases the measurements were performed under conditions yielding a linear relationship between fluorescence intensity and porphyrin concentration. Serum samples were also obtained from New Zealand rabbits 2 h after injection of 1 mg/kg of T,MP,P. Blood samples (14 ml) were taken from the ear vein and centrifuged to separate the plasma. The total lipoprotein fraction was isolated by ultracentrifugation at 39 000 revimin (Ultra Centrikon T-2060, Kontron Instruments). The density gradient was obtained by controlled addition of aqueous KBr to the serum [ 111. After centrifugation the top, containing at least 95% of the lipoproteins, was separated from the bottom, which contained heavier serum proteins. The amount of T4MP,P bound with each protein fraction was evaluated by spectrofluorimetric analysis after dilution of the samples with 2% SDS [l 11. 2.4. Tumour response
When tumours were of the appropriate size (as indicated above), T4MP,P was injected i.v. at a dose of 2.1 mgikg. Twenty-four hours later, animals were anaesthetized and the depilated tumour area was exposed to 450 J/cm’ of 600-680 nm light, which was selected by optical filters from the emission of a 250 W halogen lamp (Teclas, Sorengo). The light was delivered at a dose rate of 230 mW/cm’ by means of an optical fibre whose tip was positioned at a distance of 10 mm from the irradiated tumour area. Tumour sizes were measured daily with a calliper in three perpendicular directions. Tumour response was evaluated as the percentage of tumour growth relative to day 0 (day of light treatment) taken as 100%. In this experiment, the control and irradiated groups comprised five animals each.
3. Results 3.1. Clearance
of serum and plasma studies
The concentration of T4MP,P in the serum (Table 1) rapidly decreases over 1 to 3 h and falls below detection limits beyond 24 h. Table 2 shows the relative percentages of T4MP,P in rabbit plasma fractions 2 h after i.v. injection (1 mg/kg). The drug is largely associated with albumin and globulin (bottom fraction), and only approximately 20% of T4MP,P is associated with lipoproteins. The observed recovery from the lipoprotein fraction is likely to represent on upper limit, since the possibility exists that a fraction of T4MP,P weakly bound to albumin migrates to the lipoprotein domain during centrifugation. 3.2. Tissue distribution
of T&P,.P
As shown in Table 1, liver and spleen yield the highest recoveries of T,MP,P. The maximum levels are found at 48 h, while an appreciable amount remains 1 week after injection. The T4MP,P concentration in the kidney reaches a maximum at 24-48 h, then rapidly decreases, and no T,MP,P is found in this organ after 1 week. T,MP,P accumulates quite rapidly in the tumour. Significant levels of this porphyrin are observed 1 h after administration, with maximum concentration at 24 h. Lastly, only at 1 h are low T,MP,P concentrations detected in skin and muscle. No detectable levels of T,MP,P are found in the brain at any times examined by us. This porphyrin does not appears to cross the blood-brain barrier, so toxic effects at the level of the central nervous system would seem unlikely. 3.3.
Tumour response
to PDT
The response of MS-2 tibrosarcoma to T4MP,P PDT treatment was assessed by measuring the percentage growth of irradiated versus untreated tumours. The data reported in Fig. 2 (average of five animals for both control and phototreated groups) shows that the control tumours grow at a relatively uniform rate. However, the PDT-treated
A. Villanueva. G. Jori/Cancer
62
Table 1 Recoveries of T,MP,P standard deviation
from tumour-bearing
Balbic mice injected
with 2. I mgkg
Lett. 73 (1993) 59-64
of drug. Values within parentheses
represent
the
Time lapse alter injection lh
3h
24 h
48 h
96 h
1 week
Serum
686.00 (77.38)
0.00
0.00
0.00
0.00
Tumour
0.60 (0.11) 0.09 (0.03) 1.1 I (0.13) 0.79 (0.14) 0.53 (0.01) 0.60 (0.09) 0.00
179.17 (7.22) 0.71 (0.12) 0.00
1.19 (0.21) 0.00
0.60 (0.05) 0.00
0.62 (0.23) 0.00
0.37 (0.09i 0.00
1.38 (0.43) 0.81 (0.18) 0.69 (0.16) 0.00
2.64 (0.45) 1.29 (0.30) 0.76 (0.21) 0.00
3.27 (0.44) 1.44 (0.17) 0.77 (0.08) 0.00
1.92 (0.39) 1.11 (0.21) 0.13 (0.07) 0.00
0.99 (0.60) 1.04 (0.29) 0.00
0.00
0.00
0.00
0.00
Muscle Liver Spleen Kidney Skin Brain Data expressed
as ng of T,MP,P
0.00
per mg of tissue or per ml of serum (average
tumours show a temporarily delayed growth during the first five days after irradiation (Fig. 2B). Thereafter an exponential growth is resumed, and at the eleventh day no significant differences in growth rate are observed between control and treated tumours. Tumours exposed to T4MP,P alone or to light alone grow at the same rate as untreated controls, thus ruling out any significant contribution of thermal processes to the observed tumour response.
Table 2 Distribution of T,MP,P among injected with 1 mg/kg of drug Protein
fraction
Plasma Lipoprotein Bottom (albumin
the serum proteins
in a rabbit
ng T,MP,P
‘X
716.5 140.0 576.5
100.0 19.5 80.5
+ globulins)
The serum was taken 2 h after injection. Data expressed as total recovery of T,MP,P (rig). The percentages are relative to the plasma levels.
0.00
of at least three mice)
4. Discussion Our results indicate that T4MP,P is a good tumour localizer, since significant concentrations are accumulated and slowly eliminated in our tumour model. Twenty-four hours after administration, the photosensitizer concentration in the tumour tissue is higher than in several healthy tissues (Table 1). The liver and, to a minor degree, the spleen are remarkable exceptions, retaining detectable amounts of T4MP,P for prolonged periods. Several reports indicate that many photosensitizers are accumulated in high concentrations by the components of the reticuloendothelial system [ 12,131. However, T4MP,P appears to have a clear advantage over HpD, since its elimination from liver occurs at a faster rate (see the high HpD recoveries from liver after 1 week in refs. 14, 15). The small amounts of TaMP,P accumulated by kidneys suggest that this porphyrin is mostly eliminated from the organism via the bile-gut pathway. Likewise, only negligible amounts of T4MP,P accumulate in the muscle, indicating a selective tumour uptake, since the muscle represents the peritumoural tissue in our
A. Villanueva. G. Jori / Cancer Lert. 73 (1993) 59-64
63
experimental model. This should minimize the risk of damage to tumour-adjacent tissues during PDT. In the same way, the undetectable values of T4MP,P in the skin (minimal values only after 1 h) make skin photosensitivity less likely. At present, one undesired side effect of PDT is the persistent sensitivity to sunlight induced by Photofrin. There appears to be a relationship between the mechanism of drug transport in the blood and the type of damage produced in the tumour [16,17]. Sensitizers transported by serum lipoproteins (in particular by low-density lipoproteins) cause a direct necrosis of malignant cells, while drugs associated to albumin induce destruction of the tumour vasculature [ 181. Our findings show that in the serum, T,MP,P is mainly associated (about 80%) with heavy proteins. The remaining 20% of T,MP,P is bound to lipoproteins. Similarly, the tumour uptake of tetraporphine sulphonates was found to be independent of the pattern of their
distribution among plasma proteins [19]. Thus it appears that photosensitizer transport via lipoproteins is not an essential requisite for selectivity of tumour targeting. The results reported in Fig. 2 show that the growth rate of the MS-2 tibrosarcoma after PDT with T,MP,P is slower than that observed in control mice. This decrease in the growth rate possibly reflects the presence of a large non-growing mass, which could be the result of the combined effect of destruction of tumour microvasculature and a direct killing of tumour cells, as suggested by the heterogeneous T4MP,P transport by serum proteins. However, 7 days after treatment the tumours start to grow again, possibly because some tumour cells were not killed by treatment. Similar regrowth of experimental tumours after PDT treatment has been described by Evensen Moan [20] and Peng et al. [21], using HpD and other photosensitizers. In summary, the present results show that
1000
B 600
-
CONTROL
.-----
T, MPyP
2
4
400
6 Days
Fig. 2. A, Growth curves of fibrosarcoma exposure: 450 J/cm’ (dose rate 230 mW/cm’)
6
10
12
-
0
1
2
3
4
6
6
Daya MS-2 after PDT. T,MP,P was administered i.v. at a dose of 2.1 mg/kg. Total light 24 h after injection. B, Expansion of the five first days. Means of five animals f S.E.M.
A. Villanueva, G. Jori/ Cancer Lett.
64
T,MP,P is a potential tumour photosensitizer. It possesses a high selectivity to the tumour tissue and exhibits antitumour activity when excited by red light. Further studies are in progress to explore the usefulness of this porphyrin as a PDT agent. 5. Acknowledgements This work was supported in part by the Associazione Italiana Ricerca sul Cancro, in part by CNR, under the special project ‘Tecnologie Elettroottiche’, grant 9 1.O1089, and in part by the Direction General de Investigation Cientifica y Tecnica. Spain (PM 91-0021).
9
IO
II
12
I3
6. References Henderson. B.W. and Dougherty, T.J. (1992) How does photodynamics work? Photochem. Photobiol., 55. 145-157. Jori, G. and Reddi. E. (1991) Second generation photosensitizers for PDT of tumors. In: Light in Biology and Medicine, pp. 253-266. Editors: R.H. Douglas, J. Moan and G. Ronto. Plenum. London. Fiel, R.J., Howard, J.C.. Mark, E.H. and Datta-Gupta, N. (1979) Interaction of DNA with a porphyrin Iigand: evidence for intercalation. Nucleic Acids Res.. 6, 3093-3118. Fiel. R.J.. Datta-Gupta. N.. Mark, E.H. and Howard, J.C. (1981) Induction of DNA damage by porphyrin photosensitizers. Cancer Res., 14, 3543-3545. Villanueva. A., Juarranz, A.. Diaz, V.. Gomez. J. and Canete. M. (1992) Photodynamic effects of a cationic mesosubstituted porphyrin in cell cultures. Anti-Cancer Drug Design. 7. 297-303. Verlhac. J.B.. Gaudemer, A. and Kraljic. I. (1984) Watersoluble porphyrins and metalloporphyrins as photosensitizers in aerated aqueous solutions. I: Detection and determination of quantum yield of formation of singlet oxygen. Nouv. J. Chim.. 8. 401-406. Pasternack, R.F.. Gibbs, E.J.. Gaudemer. A.. Antebi. A.. Bassner, S.. De Poy. L., Turner, D.H.. Williams. A., Laplace. F., Lansard. M.H.. Merienne. C. and PerreeFauvet. M. (1985) Molecular complexes of nucleosides and nucleotides with a monomeric cationic porphyrin and some of its metal derivatives. J. Am. Chem. Sot.. 107, 8 179-8 186. Jori, G. (1990) Photosensitized processes ‘in viva’: proPhotochem. posed phototherapeutic applications. Phorobiol., 52. 439-443.
14
15
16
17
18
19
20
21
73 (19931 59-64
Kongshaug. M. (1992) Distribution of tetrapyrrole photosensitizers among human plasma proteins. Int. J. Biochem., 24. 1239-1265. Jori, G. (1989) ‘In viva’ transport and pharmacokinetic behaviour of tumour photosensitizers. In: Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use (Ciba Foundation Symposium. 146). pp. 78-94. Wiley, Chichester. Ginevra, F., Biffanti, S.. Pagnan. A.. Biolo. R.. Reddi, E. and Jori. G. (1990) Delivery of the tumour photosensitizer zinc(II)-phthalocyanine to serum proteins by different liposomes: studies ‘in vitro’ and ‘in viva’. Cancer Lett.. 49. 59-65. Bugelski, P.J.. Porter. C.W. and Dougherty, T.J. (1981) Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res.. 41, 4606-4612. Chan. W.S.. Marshall. J.F.. Lam, G.Y.F. and Hart. I.R. (1988) Tissue uptake, distribution. and potency of the photoactivable dye chloroaluminium sulfonated phthalocyanine in mice bearing transplantable tumours. Cancer Res.. 48, 3040-3044. Bellnier. D.A.. Ho, Y., Pandey, R.K.. Missert. J.R. and Dougherty, T.J. (1989) Distribution and elimination of Photofrin II in mice. Photochem. Photobiol.. 50, 221-228. Kessel, D. (1984) Hematoporphyrin and HPD: photophysics, photochemistry and phototherapy. Photochem. Photobiol., 39. 851-859. Jori. G., Tomio. L.. Reddi. E.. Rossi. E. and Corti. L. (1983) Preferential delivery of liposome-incorporated porphyrins to neoplastic cells m tumour-bearing rats. Br. J. Cancer. 48. 307-309. Kessel, D. (1986) Porphyrin-lipoprotein association as a factor in porphyrin localization. Cancer Lett., 33. 183-188. Kessel. D.. Thompson, P., Saatio, K. and Nantwi, K.D. (1987) Tumor localization and photosensitization by tetraphenylporphine. derivatives of sulfonated Photochem. Photobiol.. 45. 787-790. Winkelman, J. (1985) Quantitative studies of tetrdphenylporphine sulfonate and hematoporphyrin derivative distribution in animal tumor systems. In: Methods in Porphyrin Photosensitization. pp 91-96. Editor: D. Kessel. Plenum. New York. Evensen. J.F. and Moan, J. (1987) A test of different photosensitizers for photodynamic treatment of cancer in a murine tumor model. Photochem. Photobiol.. 46. 859-865. Peng. Q.. Moan, J., Warloe. T.. Nesland. J.M. and Rimington. C. (1992) Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma. Int. J. Cancer, 52. 433-443.
Letters 73 (1993) 59-64
Pharmacokinetic and tumour-photosensitizing properties of the cationic porphyrin meso-tetra(4Wmethylpyridyl)porphine Angeles
Villanueva* a, Giulio Jori b
“Departamento de Biologia, Universidad Autdnoma de Madrid, 28049 Mudrid, Spain ‘Dipartimento di Biologia. Universitir di Padova, Via Triesfe 7.5, 35121 Padova, Ital)
(Received 19 April 1993; revision received 21 June 1993; accepted
23 June
1993)
Abstract The pharmacokinetic behaviour and the photodynamic properties of the cationic porphyrin meso-tetra (4N-methylpyridyl) porphine (T,MP,P 2.1 mgikg) were examined in Balb/c mice bearing an MS-2 fibrosarcoma. The porphyrin shows good tumour localizing properties; 24 h after drug administration the tumour concentration of T,MP,P was approximately 1.2 ng/mg, while the concentrations in normal tissues were substantially lower, except for liver and spleen. In the serum, T,MP,P is preferentially transported by albumin and globulins (80.5X), while minor amounts are associated to lipoproteins (19.5%). The phototherapeutic efficiency of T,MP,P was tested by following the growth curves of fibrosarcoma irradiated by 600-680 nm (450 J/cm’) 24 h after the i.v. injection of T,MP,P (2.1 mgikg). PDT-treated tumours showed a temporary delay in their growth compared with control tumours. The excellent selectivity of T,MP,P and its antitumour activity on photoexcitation encourage further studies for assessing the usefulness of this porphyrin in photodynamic therapy. Key words: Meso-tetra(4N-niethyIpyridyl)porphine; sitizer
Pharmacokinetic
1. Introduction Photodynamic therapy (PDT) of tumours is based on the systemic administration of a photosensitizer that is retained in sufficiently large amounts by malignant tissues so that illumination of the tumour area with wavelengths specifically absorbed by the photosensitizer leads to tumour necrosis. Essentially all clinical applications of
* Corresponding
author.
0304-3835193/$06.00 0 1993 Elsevier Scientific SSDI 0304-3835(93)03117-N
Publishers
Ireland
properties;
Photodynamic
therapy;
Photosen-
PDT use one derivative of hematoporphyrin (HpD) or its partially purified version, known as Photofrin II, as phototherapeutic agents [l]. The intrinsic limitations of Photofrin, such as the reduced selectivity of tumour targeting and the heterogeneous chemical composition, prompted several investigations aimed at identifying secondgeneration photosensitizers with improved phototherapeutic properties. In general, such photosensitizers, including chlorins, phthalocyanines and naphthalocyanines, are typically associated with cell membranes [2]. Ltd. All rights reserved.
60
A. Villanueva. G. Jori/Cancer
On the other hand, no thorough investigation has been performed on the phototherapeutic potential of DNA-interacting photosensitizers. A number of investigators have reported the capability of the cationic porphyrin meso-tetra(4N-methylpyridyl)porphine (T,MP,P) (Fig. 1) to bind to DNA by intercalation between the base pairs [3] and photoinduce DNA damage in vitro 1451. T4MP,P appears to have several interesting photochemical properties, such as a high quantum yield of production of the cytotoxic species IO2 [6] and a monomeric molecular behaviour over a wide concentration range [7]. Most porphyrintype photosensitizers undergo aggregation in aqueous solution at concentrations used for in vivo experiments, which depresses the photosensitizing efficiency [8]. This situation may change on systemic injection of the porphyrins, which are largely converted to monomeric species on interaction and binding with plasma proteins. One notable exception is Photofrin II, which remains aggregated, at least in part, in the bloodstream [9]. Recent studies have shown that T4MP,P provokes the photodynamic inactivation of HeLa cells [5]. However, to our knowledge, in vivo studies with experimental tumours have not been carried out with this porphyrin.
‘CH,
Fig. 1, Chemical structure of meso-tetra (4N-methyl-pyridyl) porphine tetraiodide (T,MP,P).
73 (1993) 59-64
2. Materials and methods 2.1. Drug Meso-tetra (4N-methylpyridyl)porphine tetraiodide (T,MP,P) was obtained from Ventron-Alfa Produkte (Karlsruhe) and stored at -4°C. Solutions were prepared in 0.9% NaCl, maintained in the dark and used within 24 h. 2.2. Animals and tumour model Female Balb/c mice (18-22 g body weight) obtained from Charles River (Como) were kept in a temperature-controlled and light-controlled room with free access to water and dietary chow. When necessary, animals were anaesthetized with ketalar (150 mg/kg i.p.). The experimental tumour MS-2 librosarcoma was intramuscularly implanted in the right hind leg by injection of a cell suspension containing at least lo6 cells/ml. Experiments were performed within 6-8 days, when the tumour diameter was 5-7 mm. Spontaneous tumour necrosis was minimal or absent for these tumour sizes. New Zealand rabbits were used for studies of T4MP,P distribution in serum. In all cases, animals were treated according to the guidelines for animal welfare established by the Italian Committee for Experiments on Animals. 2.3. Pharmacokinetic
H3C
Lett.
studies
T4MP,P (2.1 mg/kg) was administered by iv. injection. Animals were sacrificed at different times (six mice at 24 h and three mice for other times) between 1 h and 1 week after administration. About 200 mg of tissue was homogenized in 2% sodium dodecyl sulfate (SDS) (4 ml) and incubated for 1 h. The homogenates were centrifuged at 3000 rev/min for 15 min. and the fluorescence of the supernatants was measured setting the excitation wavelength at 420 nm and recording the emission spectrum from 655 to 720 nm. Serum samples, isolated from blood by centrifugation, were diluted with suitable volumes of 2% SDS so that the absorbance of T,MP,P at 423 nm was lower than 0.1 and analysed at the spectrophoto-
A. Villanueva, G. Jori / Cancer Lett.
61
73 (19931 59-64
fluorimeter. T4MP,P levels were determined by interpolation on a standard curve plotted with known amounts of T4MP,P in 2% SDS, and reported in terms of ng/mg tissue or @ml of serum. The standard curve was periodically repeated to compensate for any time-dependent variability in the instrumental response. In all cases the measurements were performed under conditions yielding a linear relationship between fluorescence intensity and porphyrin concentration. Serum samples were also obtained from New Zealand rabbits 2 h after injection of 1 mg/kg of T,MP,P. Blood samples (14 ml) were taken from the ear vein and centrifuged to separate the plasma. The total lipoprotein fraction was isolated by ultracentrifugation at 39 000 revimin (Ultra Centrikon T-2060, Kontron Instruments). The density gradient was obtained by controlled addition of aqueous KBr to the serum [ 111. After centrifugation the top, containing at least 95% of the lipoproteins, was separated from the bottom, which contained heavier serum proteins. The amount of T4MP,P bound with each protein fraction was evaluated by spectrofluorimetric analysis after dilution of the samples with 2% SDS [l 11. 2.4. Tumour response
When tumours were of the appropriate size (as indicated above), T4MP,P was injected i.v. at a dose of 2.1 mgikg. Twenty-four hours later, animals were anaesthetized and the depilated tumour area was exposed to 450 J/cm’ of 600-680 nm light, which was selected by optical filters from the emission of a 250 W halogen lamp (Teclas, Sorengo). The light was delivered at a dose rate of 230 mW/cm’ by means of an optical fibre whose tip was positioned at a distance of 10 mm from the irradiated tumour area. Tumour sizes were measured daily with a calliper in three perpendicular directions. Tumour response was evaluated as the percentage of tumour growth relative to day 0 (day of light treatment) taken as 100%. In this experiment, the control and irradiated groups comprised five animals each.
3. Results 3.1. Clearance
of serum and plasma studies
The concentration of T4MP,P in the serum (Table 1) rapidly decreases over 1 to 3 h and falls below detection limits beyond 24 h. Table 2 shows the relative percentages of T4MP,P in rabbit plasma fractions 2 h after i.v. injection (1 mg/kg). The drug is largely associated with albumin and globulin (bottom fraction), and only approximately 20% of T4MP,P is associated with lipoproteins. The observed recovery from the lipoprotein fraction is likely to represent on upper limit, since the possibility exists that a fraction of T4MP,P weakly bound to albumin migrates to the lipoprotein domain during centrifugation. 3.2. Tissue distribution
of T&P,.P
As shown in Table 1, liver and spleen yield the highest recoveries of T,MP,P. The maximum levels are found at 48 h, while an appreciable amount remains 1 week after injection. The T4MP,P concentration in the kidney reaches a maximum at 24-48 h, then rapidly decreases, and no T,MP,P is found in this organ after 1 week. T,MP,P accumulates quite rapidly in the tumour. Significant levels of this porphyrin are observed 1 h after administration, with maximum concentration at 24 h. Lastly, only at 1 h are low T,MP,P concentrations detected in skin and muscle. No detectable levels of T,MP,P are found in the brain at any times examined by us. This porphyrin does not appears to cross the blood-brain barrier, so toxic effects at the level of the central nervous system would seem unlikely. 3.3.
Tumour response
to PDT
The response of MS-2 tibrosarcoma to T4MP,P PDT treatment was assessed by measuring the percentage growth of irradiated versus untreated tumours. The data reported in Fig. 2 (average of five animals for both control and phototreated groups) shows that the control tumours grow at a relatively uniform rate. However, the PDT-treated
A. Villanueva. G. Jori/Cancer
62
Table 1 Recoveries of T,MP,P standard deviation
from tumour-bearing
Balbic mice injected
with 2. I mgkg
Lett. 73 (1993) 59-64
of drug. Values within parentheses
represent
the
Time lapse alter injection lh
3h
24 h
48 h
96 h
1 week
Serum
686.00 (77.38)
0.00
0.00
0.00
0.00
Tumour
0.60 (0.11) 0.09 (0.03) 1.1 I (0.13) 0.79 (0.14) 0.53 (0.01) 0.60 (0.09) 0.00
179.17 (7.22) 0.71 (0.12) 0.00
1.19 (0.21) 0.00
0.60 (0.05) 0.00
0.62 (0.23) 0.00
0.37 (0.09i 0.00
1.38 (0.43) 0.81 (0.18) 0.69 (0.16) 0.00
2.64 (0.45) 1.29 (0.30) 0.76 (0.21) 0.00
3.27 (0.44) 1.44 (0.17) 0.77 (0.08) 0.00
1.92 (0.39) 1.11 (0.21) 0.13 (0.07) 0.00
0.99 (0.60) 1.04 (0.29) 0.00
0.00
0.00
0.00
0.00
Muscle Liver Spleen Kidney Skin Brain Data expressed
as ng of T,MP,P
0.00
per mg of tissue or per ml of serum (average
tumours show a temporarily delayed growth during the first five days after irradiation (Fig. 2B). Thereafter an exponential growth is resumed, and at the eleventh day no significant differences in growth rate are observed between control and treated tumours. Tumours exposed to T4MP,P alone or to light alone grow at the same rate as untreated controls, thus ruling out any significant contribution of thermal processes to the observed tumour response.
Table 2 Distribution of T,MP,P among injected with 1 mg/kg of drug Protein
fraction
Plasma Lipoprotein Bottom (albumin
the serum proteins
in a rabbit
ng T,MP,P
‘X
716.5 140.0 576.5
100.0 19.5 80.5
+ globulins)
The serum was taken 2 h after injection. Data expressed as total recovery of T,MP,P (rig). The percentages are relative to the plasma levels.
0.00
of at least three mice)
4. Discussion Our results indicate that T4MP,P is a good tumour localizer, since significant concentrations are accumulated and slowly eliminated in our tumour model. Twenty-four hours after administration, the photosensitizer concentration in the tumour tissue is higher than in several healthy tissues (Table 1). The liver and, to a minor degree, the spleen are remarkable exceptions, retaining detectable amounts of T4MP,P for prolonged periods. Several reports indicate that many photosensitizers are accumulated in high concentrations by the components of the reticuloendothelial system [ 12,131. However, T4MP,P appears to have a clear advantage over HpD, since its elimination from liver occurs at a faster rate (see the high HpD recoveries from liver after 1 week in refs. 14, 15). The small amounts of TaMP,P accumulated by kidneys suggest that this porphyrin is mostly eliminated from the organism via the bile-gut pathway. Likewise, only negligible amounts of T4MP,P accumulate in the muscle, indicating a selective tumour uptake, since the muscle represents the peritumoural tissue in our
A. Villanueva. G. Jori / Cancer Lert. 73 (1993) 59-64
63
experimental model. This should minimize the risk of damage to tumour-adjacent tissues during PDT. In the same way, the undetectable values of T4MP,P in the skin (minimal values only after 1 h) make skin photosensitivity less likely. At present, one undesired side effect of PDT is the persistent sensitivity to sunlight induced by Photofrin. There appears to be a relationship between the mechanism of drug transport in the blood and the type of damage produced in the tumour [16,17]. Sensitizers transported by serum lipoproteins (in particular by low-density lipoproteins) cause a direct necrosis of malignant cells, while drugs associated to albumin induce destruction of the tumour vasculature [ 181. Our findings show that in the serum, T,MP,P is mainly associated (about 80%) with heavy proteins. The remaining 20% of T,MP,P is bound to lipoproteins. Similarly, the tumour uptake of tetraporphine sulphonates was found to be independent of the pattern of their
distribution among plasma proteins [19]. Thus it appears that photosensitizer transport via lipoproteins is not an essential requisite for selectivity of tumour targeting. The results reported in Fig. 2 show that the growth rate of the MS-2 tibrosarcoma after PDT with T,MP,P is slower than that observed in control mice. This decrease in the growth rate possibly reflects the presence of a large non-growing mass, which could be the result of the combined effect of destruction of tumour microvasculature and a direct killing of tumour cells, as suggested by the heterogeneous T4MP,P transport by serum proteins. However, 7 days after treatment the tumours start to grow again, possibly because some tumour cells were not killed by treatment. Similar regrowth of experimental tumours after PDT treatment has been described by Evensen Moan [20] and Peng et al. [21], using HpD and other photosensitizers. In summary, the present results show that
1000
B 600
-
CONTROL
.-----
T, MPyP
2
4
400
6 Days
Fig. 2. A, Growth curves of fibrosarcoma exposure: 450 J/cm’ (dose rate 230 mW/cm’)
6
10
12
-
0
1
2
3
4
6
6
Daya MS-2 after PDT. T,MP,P was administered i.v. at a dose of 2.1 mg/kg. Total light 24 h after injection. B, Expansion of the five first days. Means of five animals f S.E.M.
A. Villanueva, G. Jori/ Cancer Lett.
64
T,MP,P is a potential tumour photosensitizer. It possesses a high selectivity to the tumour tissue and exhibits antitumour activity when excited by red light. Further studies are in progress to explore the usefulness of this porphyrin as a PDT agent. 5. Acknowledgements This work was supported in part by the Associazione Italiana Ricerca sul Cancro, in part by CNR, under the special project ‘Tecnologie Elettroottiche’, grant 9 1.O1089, and in part by the Direction General de Investigation Cientifica y Tecnica. Spain (PM 91-0021).
9
IO
II
12
I3
6. References Henderson. B.W. and Dougherty, T.J. (1992) How does photodynamics work? Photochem. Photobiol., 55. 145-157. Jori, G. and Reddi. E. (1991) Second generation photosensitizers for PDT of tumors. In: Light in Biology and Medicine, pp. 253-266. Editors: R.H. Douglas, J. Moan and G. Ronto. Plenum. London. Fiel, R.J., Howard, J.C.. Mark, E.H. and Datta-Gupta, N. (1979) Interaction of DNA with a porphyrin Iigand: evidence for intercalation. Nucleic Acids Res.. 6, 3093-3118. Fiel. R.J.. Datta-Gupta. N.. Mark, E.H. and Howard, J.C. (1981) Induction of DNA damage by porphyrin photosensitizers. Cancer Res., 14, 3543-3545. Villanueva. A., Juarranz, A.. Diaz, V.. Gomez. J. and Canete. M. (1992) Photodynamic effects of a cationic mesosubstituted porphyrin in cell cultures. Anti-Cancer Drug Design. 7. 297-303. Verlhac. J.B.. Gaudemer, A. and Kraljic. I. (1984) Watersoluble porphyrins and metalloporphyrins as photosensitizers in aerated aqueous solutions. I: Detection and determination of quantum yield of formation of singlet oxygen. Nouv. J. Chim.. 8. 401-406. Pasternack, R.F.. Gibbs, E.J.. Gaudemer. A.. Antebi. A.. Bassner, S.. De Poy. L., Turner, D.H.. Williams. A., Laplace. F., Lansard. M.H.. Merienne. C. and PerreeFauvet. M. (1985) Molecular complexes of nucleosides and nucleotides with a monomeric cationic porphyrin and some of its metal derivatives. J. Am. Chem. Sot.. 107, 8 179-8 186. Jori, G. (1990) Photosensitized processes ‘in viva’: proPhotochem. posed phototherapeutic applications. Phorobiol., 52. 439-443.
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Kongshaug. M. (1992) Distribution of tetrapyrrole photosensitizers among human plasma proteins. Int. J. Biochem., 24. 1239-1265. Jori, G. (1989) ‘In viva’ transport and pharmacokinetic behaviour of tumour photosensitizers. In: Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use (Ciba Foundation Symposium. 146). pp. 78-94. Wiley, Chichester. Ginevra, F., Biffanti, S.. Pagnan. A.. Biolo. R.. Reddi, E. and Jori. G. (1990) Delivery of the tumour photosensitizer zinc(II)-phthalocyanine to serum proteins by different liposomes: studies ‘in vitro’ and ‘in viva’. Cancer Lett.. 49. 59-65. Bugelski, P.J.. Porter. C.W. and Dougherty, T.J. (1981) Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res.. 41, 4606-4612. Chan. W.S.. Marshall. J.F.. Lam, G.Y.F. and Hart. I.R. (1988) Tissue uptake, distribution. and potency of the photoactivable dye chloroaluminium sulfonated phthalocyanine in mice bearing transplantable tumours. Cancer Res.. 48, 3040-3044. Bellnier. D.A.. Ho, Y., Pandey, R.K.. Missert. J.R. and Dougherty, T.J. (1989) Distribution and elimination of Photofrin II in mice. Photochem. Photobiol.. 50, 221-228. Kessel, D. (1984) Hematoporphyrin and HPD: photophysics, photochemistry and phototherapy. Photochem. Photobiol., 39. 851-859. Jori. G., Tomio. L.. Reddi. E.. Rossi. E. and Corti. L. (1983) Preferential delivery of liposome-incorporated porphyrins to neoplastic cells m tumour-bearing rats. Br. J. Cancer. 48. 307-309. Kessel, D. (1986) Porphyrin-lipoprotein association as a factor in porphyrin localization. Cancer Lett., 33. 183-188. Kessel. D.. Thompson, P., Saatio, K. and Nantwi, K.D. (1987) Tumor localization and photosensitization by tetraphenylporphine. derivatives of sulfonated Photochem. Photobiol.. 45. 787-790. Winkelman, J. (1985) Quantitative studies of tetrdphenylporphine sulfonate and hematoporphyrin derivative distribution in animal tumor systems. In: Methods in Porphyrin Photosensitization. pp 91-96. Editor: D. Kessel. Plenum. New York. Evensen. J.F. and Moan, J. (1987) A test of different photosensitizers for photodynamic treatment of cancer in a murine tumor model. Photochem. Photobiol.. 46. 859-865. Peng. Q.. Moan, J., Warloe. T.. Nesland. J.M. and Rimington. C. (1992) Distribution and photosensitizing efficiency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma. Int. J. Cancer, 52. 433-443.