Meso-TETRAPHENYLPORPHYRIN DIMER DERIVATIVES AS POTENTIAL PHOTOSENSITIZERS IN PHOTODYNAMIC THERAPY -PART 2

PHOTOTHERAPEUTIC PROPERTIES OF meso-TETRAPHENYLPORPHYRIN DIMER DERIVATIVES: A COMPARATIVE STUDY.

Maria A. F. Faustino¹, Maria G. P. M. S. Neves¹, José A. S. Cavaleiro¹, Marcus Neumann², Hans-D. Brauer², Giulio Jori³

²Institut für Physikalische und Theoretische Chemie der Universität Frankfurt,

Preliminary in vivo biological activity, of three different dimeric porphyrins with an amide linkage (D2 - D4) are described and compared with the results recently reported for a dimeric porphyrin (D1). The pharmacokinetic behaviour and photodynamic properties of all dimers were examined in Balb/c mice bearing MS-2 fibrosarcomas. The maximal efficiency and selectivity of photosensitizer accumulation in each tumour tissue takes place at 24 h after drug administration of 1.0 mg kg^-1 into DL-a-dipalmitoylphosphatidylcholine liposomes by i.v. injection. The phototherapeutic efficiency of all dimers was evaluated by following the growth curves of fibrosarcoma irradiated with red light (600-700 nm) with a total dose of 400 J cm^-2, at 24 h after intravenous injection. Photodynamic therapy –treated tumours showed a significant delay in growth as compared to untreated control mice. The results obtained suggest that the evaluated dimeric structures may be promising candidates for further use in PDT experiments. The results also allow the possibility to establish a correlation between the chemical structure of the dyes and the efficiency/selectivity of the PDT of tumours and can be used for building up optimal photosensitizing agents for tumours.

Photodynamic Therapy (PDT) is a tumour phototherapy currently using a sensitizing drug and it was recognized as a promising medicinal technique [1-2]. The development of new compounds as potential photosensitizers in PDT is of a great scientific interest. A sensitizer in PDT has to fulfil several chemical, physical and biological requirements and because of that, an ideal photosensitizer is not yet known[3].

Based on the promising photophysical, pharmacokinetic and PDT properties showed by a dimeric meso-tetraphenylporphyrin derivative D1 [4], we extended our work to the synthesis of others dimeric derivatives D2, D3 and D4 [5] and to the evaluation of their PDT properties by following the same experimental procedures as previously described for dimer D1. All dimers are structurally related; all of them contain an amide function linking the p-positions of the meso-phenyl groups involved in the coupling of both monomer moieties. The differences between these four dimers are in the position of the methoxy substituents in the phenyl groups of one porphyrin moiety (dimer D1 and D3) and porphyrin-chlorin (m-methoxy substituents on the phenyl groups of the chlorin moiety, dimer D4). The phenyl groups of dimer D2 are unsubstituted (Fig. 1).

In the present work we examined the PDT properties exhibited by these new synthetic dimers (D2-D4) and to compare their behaviour with the corresponding data previously found for dimer D1 [4].

MATERIALS AND METHODS

The porphyrin-porphyrin dimers D2, D3 and porphyrin-chlorin dimer D4 were prepared by methods described previously [4,5]. As an additional check on purity, analytical thin-layer chromatography of all samples was carried out on Merck 60 silica gel (pre-coated sheets 0.2 mm thick).

DL-a-dipalmitoylphosphatidylcholine (DPPC Sigma Chemicals Co) was used as received. All other chemicals and solvents were of analytical grade.

The two porphyrin-porphyrin (D2,D4) and the porphyrin-chlorin dimers (D4) were incorporated into DPPC liposomes using a sonication procedure described elsewhere [6]. The photosensitizer:lipid molar ratio in the liposomes was 1:150 in order to obtain a predominantly monomeric state of the porphyrin in the phospholipid bilayer and to inject a photosensitizer dose of 1.0 mg/kg body weight. The dimers concentrations in the liposome suspensions were calculated by diluting a known volume of each suspension into a known excess of tetrahydrofuran and measuring the corresponding absorbance at 420 nm for dimer D2 (e = 5.18 x 10⁵ M^-1 cm^-1), 421 nm for dimer D3 (e = 7.14 x 10⁵ M^-1 cm^-1) and 418 nm for dimer D4 (e = 5.6 x 10⁵ M^-1 cm^-1).

Female Balb/c mice (20-22 g body weight) were supplied by Charles River (Como, Italy) and kept in cages with free access to tap water and standard dietary chow. The MS-2 fibrosarcoma was originally supplied by Istituto Nazionale dei Tumori, Milan, Italy. For tumour implantation 2 x 10⁵ cells in 0.2 mL of sterile physiological solution were intramuscularly injected into the right hind leg of the mouse; the tumour growth took place at a rather aggressive rate, reaching an external diameter of ca. 0.7 cm on the seventh day. All phototherapeutic studies were performed within 7 days after tumour implantation when no detectable spontaneous tumour necrosis had generally occurred. When necessary, the mice were anaesthetised by intraperitoneal injection of Ketalar (150 mg kg^-1). Animal care was performed according to the guidelines established by the Italian Committee for Experimental Animals.

For experimental PDT studies the tumour-bearing mice were injected with the dimers in DPPC liposomes at a dose of 1.0 mg/kg b.w.. Phototreatments were performed at 24 h after administration of the drug. The animals were anesthetized and the depilated tumour area were irradiated with light from a 250 W quartz/halogen lamp (Teclas, Lugano, Switzerland). This light was focused into a bundle of optical fibres having an external diameter of 0.6 cm. In order to establish a correlation between the chemical structure of dyes and the efficiency/selectivity of PDT of tumours we performed our experiments with the same irradiation protocol used in our previous studies with the dimer D1 [4]. The lamp was equipped with a set of optical cut-on and cut-off filters which eliminated all light wavelengths outside the 600-700 nm interval. The fibre tip was placed at 1 cm from the surface of the tumour and the light was delivered at a fluence rate of 230 mW cm^-2 (measured at the end of the fibre bundle) and a total light dose of 400 J cm^-2. The tumour volume was measured daily, by means of a calliper, in three mutually perpendicular directions (a, b, c) i.e. assuming a hemiellipsoidal structure for the tumour. The rate of tumour growth was compared with that typical of the tumour in control mice that had been transplanted simultaneously with the phototreated mice but had not been injected with photosensitizer or exposed to light. The volume was calculated using the formula V = 2/3 p (a/2 x b/2 x c), as previously described [4]. In this experiment, the control and irradiated groups comprised five animals each.

As the pharmacokinetic studies of these dimers were shown [5], the maximal recoveries from the malignant lesion were measured at 24 h after the drug administration (Fig. 2-4). The ratios of dye concentration in the tumour to that in the muscle and in the tumour to that in the skin for the three dimers were maximum at this time.

Figure 2. Recovery of porphyrin dimer D2 from selected tissues of Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma at various times after i.v. injection of 1 mg.kg^-1 photosensitizer incorporated into DPPC liposomes [the recoveries are expressed as ng of drug per mg of tissue. The data referred to three independently analyzed mice: (average ± standard deviation)]

Figure 3. Recovery of porphyrin dimer D3 from selected tissues of Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma at various times after i.v. injection of 1 mg.kg^-1 photosensitizer incorporated into DPPC liposomes [the recoveries are expressed as ng of drug per mg of tissue. The data referred to three independently analyzed mice: (average ± standard deviation)]

Figure 4. Recovery of porphyrin-chlorin dimer D4 from selected tissues of Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma at various times after i.v. injection of 1 mg.kg^-1 photosensitizer incorporated into DPPC liposomes [the recoveries are expressed as ng of drug per mg of tissue. The data referred to three independently analyzed mice: (average ± standard deviation)]

On the basis of that data, the tumour was irradiated with red light at 24 h after porphyrin administration, which corresponds with the largest endotumoral concentration and the highest tumour:muscle ratio of sensitizer concentration.

The response of MS-2 fibrosarcoma to PDT treatment was assessed by measuring the tumour volume of irradiated and untreated tumours. All experiments showed a similar behaviour. The data reported for D3 (Fig. 5, average of five animals for both control and phototreated groups) point out that the control tumours grew at a relatively uniform rate. The PDT-treated tumours showed some delay of tumour growth as compared with untreated control mice. Quite similar results were obtained by irradiation in the presence of D2 and D4. Table 1 shows the delay of tumour growth found for the different photosensitizers studied. The dimer D3 caused a maximum growth delay as compared to D2 and D4 (2.4 days).

Figure 5. Rate of tumour growth for mice (five per group) injected with 1.0 mg kg^-1 of D3 incorporated in DPPC liposomes and irradiated with 230 mW cm^-2 and 400 J cm^-1 of red light (600-700 nm) (·^......·). Comparison with the rate of tumour growth in untreated and unirradiated mice (■^_____■). Each point represents the average of five mice ± SE.

Table 1. Tumour growth delay for the tumour-bearing mice irradiated with 230 mW/cm² and 400 J/cm² at 24 h after i.v.-injection of different porphyrin dimers (1.0 mg/kg b.w.) in DPPC liposomes

Drug

Growth Delay

(days)

1.3

2.4

1.2

Under our irradiation conditions, the tumour temperature, as monitored by means of a thermocouple contained within a hypodermic needle connected to a digital thermometer (Omega Engineering Stamford, CT), never increased beyond 37.5ºC, namely about 5ºC above the basal temperature (32 ± 1ºC) of anesthetized Balb/c mice. This temperature rise is not supposed to generate any significant thermal effect [7]. This conclusion was further supported by the observation that the rate of tumour growth in mice that received no porphyrin and were irradiated by the same protocol as used for sensitized mice was identical with that of control unirradiated animals.

When the porphyrin-porphyrin dimers D2, D3 and porphyrin-chlorin dimer D4 were irradiated in THF solution by using the same irradiation protocol as used for PDT tests in vivo, no change in the optical spectrum was observed over a 30 min irradiation period, thus any red light induced photobleaching of the dimers appears to be negligible.

The phototherapeutic properties of the dimers D2, D3 and D4 show several similarities with those found for the meso-tetra-phenylporphyrin dimer (D1) previously studied [4]. In all cases, treated tumours showed a significant delay in growth as compared to untreated control mice. Actually, the highest tumour response to our PDT treatment was observed in the case of the dimer D3 in spite of the fact that this photosensitizer reaches a largest tumour concentration of 1.4 ng/mg as compared with 2.2 ng/mg measured for dimer D2 [5]. The differences in the phototherapeutic efficiency cannot be due to differences in the photophysical properties or in the photosensitization mechanism, since both dimers D2 and D3 exhibit quite similar triplet lifetimes and quantum yields for the generation of the singlet oxygen [5]. Moreover, all dimers appear to have very low yields of the photobleaching under our irradiation conditions. Finally, the efficiency of absorption of the incident light should be largest for dimer D4 (had a chlorin moiety), which displays the greatest molar extinction coefficient in the therapeutically useful red spectral region, yet induces a smaller delay of post-irradiation tumour regrowth as compared with dimer D3.

As a consequence, we propose that the observed differences in the photoactivity are determined by a different distribution of the examined porphyrin dimers among the various compartments of the malignant cells and/or tissues. It is well known that tumours are highly heterogeneous systems and the distribution of porphyrin-type photosensitizers in the districts constituting the tumour is strongly influenced by the chemical structure of the photosensitizing agents [8] as well as by the association of the photosensitizer with select delivery vehicles [9]. From this point of view, it may be relevant to underline the circumstance that dimer D3 shows a phototherapeutic activity towards the MS-2 fibrosarcoma which is essentially identical with that exhibited by dimer D1 in the same tumour model [4]. Both dimers are characterized by the presence of the methoxy functional groups in the phenyl rings which occupy the meso positions of the tetrapyrrolic macrocycles (see the chemical structure in Fig. 1). A high PDT efficiency has been previously reported for alkoxy-substituted metallo-phthalocyanines in our animal model [10].

A more precise interpretation of our results cannot be provided at the present stage of our investigations, since only limited information is available as regards the structural factors which control the biodistribution of the photosensitizer in a neoplastic tissue [11], as well as the role of the photodamage to specific tumour compartments in determining the extent of tumour response to the PDT treatment [12]. While both the direct photodamage of malignant cells and the impairment of blood flow in the tumour capillaries have been invoked as causative factors of tumour necrosis after PDT [13], an overall consideration of the functional and structural features of PDT-treated tumours strongly suggests that other compartments, such as the neoformed collagen and non-vascular stroma, may be heavily involved [12]. Thus, the present findings clearly support the favourable tumour-localizing and – photosensitizing properties of dimeric porphyrin derivatives, although further investigations appear to be required in order to obtain more detailed indications on the physico-chemical parameters which enhance their phototherapeutic properties and modulate their subtissular and subcellular localization.

Financial support from the Human Capital and Mobility Programme (PDT Euronet, contract ERBCHRXCT930178), from Praxis Project 2/2.1/Qui/145 and from the JNICT/DAAD and JNICT/CNR Protocols are gratefully acknowledged. One of us (M. A. F. F.) thanks PRAXIS XXI for the award of a Ph. D. grant.

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