Effect of photosensitizer delivery system and irradiation parameters on the efficiency of photodynamic therapy of B16 pigmented melanoma in mice.

Previous studies (Biolo et al., Photochem. Photobiol. 59, 362-365, 1994) showed that liposome-delivered Si(IV)-naphthalocyanine (SiNc) photosensitizes B16 pigmented melanoma subcutaneously transplanted in C57 mice to the action of 776 nm light. However, the efficacy of the phototreatment was limited by a lack of selectivity of tumor targeting by SiNc as well as by incomplete necrosis of the neoplastic mass. The present investigations show that the use of a different delivery system (Cremophor emulsion vs liposomes of dipalmitoylphosphatidylcholine) causes no significant increase in the selectivity of tumor targeting for three injected doses of SiNc (0.5, 1, 2 mg/kg). However, upon 776 nm light irradiation (300 mW/cm2; 520 J/cm2), the delay in the rate of tumor growth was maximal (7-8 days) for the highest naphthalocyanine dose. On the other hand, a remarkable improvement in the tumor response was obtained by inducing an intratumoral temperature increase to 44 degrees C immediately after PDT. The thermal effect appeared to be due to photoexcitation of melanin by 776 nm light (550 mW/cm2; 520 J/cm2) and subsequent partial conversion of absorbed energy into heat.

Effect of delivery system on the pharmacokinetics and tissue distribution of bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC), a photosensitizer for tumor therapy.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in tumor-bearing rats as a function of delivery system and time following administration. The tumor model was an N-(4-[5-nitro-2-furyl]-2-thiazolyl) formamide (FANFT)-induced urothelial cell carcinoma transplanted into one hind leg of male Fischer 344 rats; isoBOSINC was delivered to the rats by intravenous injection of 0.50 mg/kg of body weight as a suspension either in 10% Tween 80 in saline (Tween) or 10% (Cremophor EL + propylene glycol) in saline (Cremophor). The isoBOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin. Quantitation was by a high-performance liquid chromatographic technique with detection that utilizes the native fluorescence of the naphthalocyanine derivative. Independent of the delivery system, the dye was retained in tumors at higher concentrations than in normal tissues, except for spleen and liver. The isoBOSINC retention in tumors was high and was vehicle dependent. For Tween, the maximal ratio of dye in tumor versus peritumoral muscle occurred 12 h after injection; for Cremophor, the maximal ratio occurred later, 336 h postinjection. When the drug was delivered in Tween, isoBOSINC in serum showed two compartment kinetics: half-lives of about 2 and 11 h were found for the distribution and the elimination phases, respectively. When Cremophor was the vehicle, the elimination half-life was about 20 h, and one compartment kinetics was observed. The latter findings may explain the generally higher levels of the dye attained by the tissues at later times with Cremophor as the vehicle. An interesting exception was that after 7 and 14 days postinjection in Tween, the levels of dye found in testes were six- to seven-fold higher than those found after Cremophor delivery. Levels of dye were very low or not detectable in the brain. Optimal parameters for PDT of tumors with this novel photosensitizer are clearly time- and vehicle-dependent, and future PDT studies will need to incorporate these modulators.

Unusually high affinity of Zn(II)-tetradibenzobarrelenooctabutoxy-phthalocyanine for low-density lipoproteins in a tumor-bearing mouse.

An important factor in determining the efficacy of photosensitizing compounds in photodynamic therapy of tumors is the level to which tumors take up the photosensitizers after systemic injection. This parameter seems to be related to the transport modalities of the photosensitizer in the bloodstream. In this work the photosensitizer Zn(II)-tetradibenzobarrelenooctabutoxyphthalocyanine was shown to have an unprecedentedly high association with low-density lipoproteins (71% of the phthalocyanine in the plasma) when delivered in Cremophor micelles to tumor-bearing mice. This was accompanied by a particularly high tumor uptake at 24 h post-injection.

Photodynamic therapy of B16 pigmented melanoma with liposome-delivered Si(IV)-naphthalocyanine.

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (epsilon = 5 x 10(5) M-1 cm-1), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg-1 of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 microgram g-1, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm-2; at higher dose rates, thermal effects are likely to become important.

Pharmacokinetic and tissue distribution studies of the photosensitizer bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) in normal and tumor-bearing rats.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in normal and in tumor-bearing rats as a function of time following intravenous injection of isoBOSINC as a suspension in 10% Tween 80 in saline. The dose studied was 0.25 mg/kg of body weight. The compound isoBOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin, and quantitated by a high-performance liquid chromatographic technique. The tumor model, an N-(4-[5-nitro-2-furyl]-2-thiazolyl)formamide (FANFT)-induced urothelial cell carcinoma, was transplanted into the hind legs of Fischer 344 rats. The dye was retained in tumors at higher concentrations than in all tissues and organs examined, except for spleen and liver. The highest concentration ratio of dye in tumor versus peritumoral muscle (24.5) occurred 9 h after injection. Serum clearance of isoBOSINC showed similar kinetic behavior for both groups of rats, with a t 1/2 of elimination of approximately 10 h. At 7 and 14 days postinjection, the levels of dye found in testes were generally higher than in most other tissues, except spleen and liver. Concentrations of isoBOSINC were either very low or not detectable in rat brain. Trace amounts of the dye were excreted in the urine, and by day 14 approximately 17% of the dose was accounted for in the feces. The significant levels of the drug in tumors, as well as the excellent ratios of tumor-to-muscle concentration observed, have promising implications for PDT of tumors.

Photodynamic effects of new silicon phthalocyanines: in vitro studies utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources.

Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with approximately 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH3]2[CH2]3N[CH3]2) and SiPc V (HOSiPc-OSi[CH3]2[CH2]3N[CH3]3+I-) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in microsomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.

New phthalocyanine photosensitizers for photodynamic therapy.

Six new aluminum and silicon phthalocyanines have been synthesized and their photocytotoxicity toward V79 cells has been studied. The compounds that have been prepared are: A1PcOSi(CH3)2(CH2)3N(CH3)2, I; A1Pc-OSi(CH3)2(CH2)3N(CH3)3+I-, II; CH3SiPcOSi(CH3)2(CH2)3N(CH3)2, III; HOSiPcOSi(CH3)2(CH2)3N(CH3)2, IV; HOSiPcOSi(CH3)2(CH2)3N(CH3)3+I-, V; and SiPc[OSi(CH3)2(CH2)3N(CH3)3+I-]2, VI. Relative growth delay values for compounds I-VI and relative cytotoxicity values for compounds I, II, IV, V and VI have been determined. Compounds I and II have been shown to be comparable in photocytotoxicity to what is presumed to be A1PcOH.xH2O, and compound IV has been shown to have greater activity. The classes of compounds to which these six compounds belong appear to have potential for photodynamic therapy.

Si(IV)-naphthalocyanine: modulation of its pharmacokinetic properties through the use of hydrophilic axial ligands.

A water-soluble derivative of the highly hydrophobic molecule Si(IV)-naphthalocyanine was synthesized by the addition of two polyethyleneglycol ligands in axial positions to the centrally coordinated Si(IV) ion. The compound can be intravenously injected in homogeneous aqueous solution, where it is largely aggregated and remains mostly in an unbound state. While the naphthalocyanine is accumulated in significant amounts by an intramuscularly transplanted MS-2 fibrosarcoma in Balb/c mice, the dye shows little tumour selectivity and essentially no phototherapeutic activity. The situation is not improved by association of the naphthalocyanine with dipalmitoyl-phosphatidylcholine liposomes, probably owing to a lack of incorporation of the bulky dye molecule into the phospholipid bilayer.

Two new sterically hindered phthalocyanines: synthetic and photodynamic aspects.

The synthesis and the properties of 1,4-dibutoxy-2,3-dicyanotriptycene, of a metal-free tetradibenzobarrelenooctabutoxyphthalocyanine, and of the corresponding zinc phthalocyanine are described. The two phthalocyanines do not aggregate when dissolved in benzene at concentrations up to 450 microM. For the metal-free and the zinc compounds, the red band maxima are at 736 and 757 nm, the triplet maxima are at 590 and 605 nm, the triplet state lifetimes are 58 and 177 microseconds, and the protoporphyrin-IX dimethyl ester-to-compound bimolecular rate constants for triplet energy transfer are 2.61 x 10(8) and 1.47 x 10(8) M-1 s-1. Triplet energy transfer from the metal-free compound to O2 is endoergonic by 1.0 kcal mol-1. The potential of the zinc compound for photodynamic therapy is touched upon.

Quenching of singlet molecular oxygen by phthalocyanines and naphthalocyanines.

Using the direct measurement of the photosensitized luminescence of singlet molecular oxygen (1O2) the rate constants (kq) have been determined for 1O2 quenching by the monomeric molecules of the following phthalocyanines and naphthalocyanines in chloroform: tetra-(4-tert-butyl) phthalocyanine (I); octa-(3,6-butoxy) phthalocyanine (II), tetra-(6-tert-butyl)-2,3 naphthalocyanine (III), aluminium tetra-(1-tert-phenyl)-2,3 naphthalocyanine (IV), tri-(n-hexyl-siloxy) derivatives of silicon- (V), tin- (VI), aluminium- (VII) and gallium- (VIII) 2,3 naphthalocyanine. The following kq values were obtained (kq x 10(-8) M-1 s-1): 2.9 (I), 59 (II), 100 (III), 20 (IV), 3.9 (V), 53 (VI), 33 (VII), 110 (VIII). As most of the quenchers have the low-lying triplet levels, a contribution of the quenching mechanism based on the energy transfer from 1O2 to these levels has been analysed. A formula is proposed describing the relation between kq values caused by this mechanism, and photophysical constants of the quencher triplet state. This formula was applied to phthalocyanines, naphthalocyanines, beta-carotene and bacterochlorophyll a. The data suggest that the energy transfer can fully explain the activity of V and strongly contributes into the activities of II, III and VI-VIII. A charge transfer interaction might be an additional mechanism involved in 1O2 quenching by compounds studied. As some phthalocyanines and naphthalocyanines are strong physical quenchers of singlet oxygen they can be used as efficient inhibitors for photodestructive processes in photochemical systems.

Protection by the fluoride ion against phthalocyanine-induced photodynamic killing of Chinese hamster cells.

When a dilute F- solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCl, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F-. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA-protein crosslinks caused by light and this sensitizer were also inhibited by F-. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA-protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3-N(CH3)2 were not inhibited by F-. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F-, and that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA-protein crosslink formation indicates that the effects of F- can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F-.

Photosensitizers for tumor therapy: determination of bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) in rat tissue and serum by high-performance liquid chromatography.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a synthetic potential photosensitizer for tumor therapy. A new method, which combines solvent extraction and several purification steps, has been developed to determine its presence in tissues. Separation and quantitation of isoBOSINC is done by high-performance liquid chromatography on a silica column with toluene as a mobile phase and using fluorescence detection (lambda ex = 365 nm, lambda em = 750 nm). For recovery studies, isoBOSINC was added to muscles at levels of 0.067 and 0.67 micrograms/g; the mean recoveries were 100%, with coefficients of variation of 6.1 and 6.4%, respectively. For liver samples, the amounts added were 0.67 and 6.7 micrograms/g and for serum 0.67 and 6.70 micrograms/ml. The mean recoveries for liver were 86 and 93%, with coefficients of variation of 7.7 and 4.4%, respectively. For serum, the mean recoveries were 99 and 96%, with coefficients of variation of 2.6 and 6.9%, respectively. Due to its low detection limit and selectivity, the method is appropriate for pharmacokinetic as well as tumor uptake studies following in vivo exposure to isoBOSINC. Preliminary data on tissue distribution of the photosensitizer in normal rats are also presented.

Liposome-delivered Si(IV)-naphthalocyanine as a photodynamic sensitiser for experimental tumours: pharmacokinetic and phototherapeutic studies.

The pharmacokinetic behaviour and phototherapeutic effectiveness of bis(di-isobutyloctadecylsil-oxy)-2,3-naphthalocyanatosilicon (iso-BOSiNc) incorporated into dipalmitoyl-phosphatidylcholine (DPPC) liposomes have been studied in Balb/c mice bearing an MS-2 fibrosarcoma. We found that iso-BOSiNc i.v.-injected at a dose of 0.5 mg kg-1 b.w. is preferentially transported by serum lipoproteins; in particular, the photosensitiser is associated with LDL (57.8% of total recovery in the serum) and HDL (35.7%) while minor amounts are associated to VLDL (2.63%) and other serum proteins (3.89%), Iso-BOSiNc concentrations greater than 1 microgram g-1 of tissue are recovered from the tumour at 12-48 h after administration while the ratio of iso-BOSiNc concentration in tumour and peritumoral tissue is greater than 10. Upon increasing the injected dose, the additional iso-BOSiNc is almost exclusively bound by HDL, which leads to large uptake of the photosensitiser by liver and spleen. The efficiency of iso-BOSiNc as a photodynamic agent was measured upon irradiation with a different dose-rate for a total light dose of 450 J cm-2. The extent of tumour necrotic area increases as a function of the time after the end of PDT treatment and reaches a maximum level after about 24 h. Moreover, the necrotic area is linearly dependent on the irradiation dose-rate up to 100 mW cm-2. In all there is substantial evidence that iso-BOSiNc delivered in a liposomal dispersion is a highly effective photosensitizer for PDT of tumours.

Photoproperties of alkoxy-substituted phthalocyanines with deep-red optical absorbance.

Triplet-state properties of 1,4,8,11,15,18,22,25-octa-n-butoxyphthalocyanine and its zinc derivative were determined for the first time. The T1 state of the metal-free phthalocyanine was characterized by a short lifetime (tau T = 17 microseconds) and low quantum yield (phi T = 0.095), and quenching of the triplet by O2 occurred with a bimolecular rate constant (kT sigma = 1.3 x 10(8) M-1 s-1) that is indicative of an endogonic reaction. The zinc complex (ZnPc(OBu)8) was markedly better as a triplet photosensitizer with respect to both tau T (60 microseconds) and phi T (0.5). Quenching by O2 produced singlet oxygen with nearly 100% efficiency, and kT sigma (1.7 x 10(9) M-1s-1) was close to the spin-statistical diffusion-controlled limit. Phosphorescence measurements showed the energy of the T1 state of ZnPc(OBu)8 to be 100 kJ/mol, which is 6 kJ/mol above the 1 delta g state of O2. These photoproperties, together with Q-band absorption maxima in the mid-700 nm range indicate that metal-centered 1,4,8,11,15,18,22,25-octaalkoxyphthalocyanines have excellent potential as sensitizers in photodynamic therapy.