Identification of porphyrin modified photosensitizer porfimer sodium and its precursors by high performance liquid chromatography and mass spectrometry.

Photodit (porfimer sodium) is a complex mixture of porphyrin-modified oligomers that has demonstrated high photoactivity during photodynamic therapy of malignant tumors. Hematoporphyrin derivative monomer precursors of Photodit and its oligomers were analyzed using liquid chromatography and mass spectrometry to determine their chemical composition and structures. Hematoporphyrin (M-3), O,O'-diacetylhematoporphyrin (M-6), and isomers of 8(-3)-hydroxyethyl-3(-8)-vinyldeuteroporphyrin (M-2), 8(-3)-(1-acetoxyethyl)-3(-8)-vinyldeuteroporphyrin (M-4), and O-acetylhematoporphyrin (M-5), in ratio 0.59:0.94:0.21:0.34:1.00, were identified as hematoporphyrin derivative monomer precursors. The main compounds of Photodit as a porfimer sodium salt were dimers: D-1 [di-[8(-3)-hydroxyethyl-3(-8)-vinyldeuteroporphyrin] ether], D-2 [[Hematoporphyrin-8(-3)-hydroxyethyl-3(-8)-vinyldeutroporphyrin] ether], and D-3 [Dihematoporphyrin ether] in ratio 0.51:1.00:0.50, and trimers: T-1 [(M-2)-(D-2) ether], T-2 [(D-2)-(M-3) ether], and T-3 [(D-3)-(M-3) ether] in ratio 0.98:1.00:0.44, respectively. An average oligomer length of 2.23 porphyrin units was determined.

Effect of delivery system on the pharmacokinetic and phototherapeutic properties of bis(methyloxyethyleneoxy) silicon-phthalocyanine in tumor-bearing mice.

A Si(IV)-phthalocyanine bearing two methoxyethyleneglycol axial ligands bound to the central metal ion (SiPc) has been prepared by chemical synthesis and analyzed for its phototherapeutic activity after administration in a Cremophor or liposome formulation to C57B1/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma (LLC). The maximum drug accumulation in the tumor is found at 24 h after intraperitoneal injection, independent of the delivery system. However, the tumor concentration of SiPc in the Cremophor formulation is about two-fold higher, while the drug concentration in liver and skin shows similar trends with the two delivery systems. The drug accumulation and retention in the brain is much larger when using Cremophor emulsion. Photodynamic therapy (672 nm, 370 mW m-2, 360 J cm-2) at 24 h after the injection of Cremophor emulsion- or DPPC liposome-formulated SiPc causes a very efficient and similar response for the LLC (approximately 8 versus 22 mm mean tumor diameter for the control groups at 21 days after phototreatment). These very promising effects, obtained both at higher and lower tumor drug concentrations, clearly demonstrate the potential phototherapeutical activity of the newly synthesized SiPc.

Naphthalocyanine complexes as potential photosensitizers for photodynamic therapy of tumors.

In the present paper information about the synthesis and results on the pharmacokinetic and experimental photodynamic therapy (PDT) of naphthalocyanines are given. The photodynamic activity of differently substituted zinc(II)- and silicon(IV)-naphthalocyanines using liposomes or Cremophor EL as drug-delivery systems is shown on different tumor models. For the evaluation of the phototherapeutic effect different assessment criteria were used, including light and electron microscopy observations. The main conclusions which can be arrived at on the basis of our findings are the following: silicon(IV)-naphthalocyanine seems to be not a very effective tumor sensitizer, especially in the treatment of pigmented melanoma, while zinc(II)-naphthalocyanines appear to be very promising for PDT of tumors. Their selective targeting and slow clearance from tumor tissue, fast clearance from skin and pronounced phototherapeutic effect on different tumor models and especially at melanotic tumors, even after application of low drug doses, make this group of photosensitizers very attractive for successful PDT of cancer. © 1999 Society of Photo-Optical Instrumentation Engineers.

Fluence rate effects on photodynamic therapy of B16 pigmented melanoma.

In vivo experiments were performed to evaluate the effect of fluence rate on the efficiency of Zn(II)-2,3 naphthalocyanine (ZnNc) photosensitization of B16 pigmented melanoma subcutaneously transplanted in C57B1/6 mice. The tumour was irradiated with 774 nm light at 24 h after an injection of liposome--which incorporated ZnNc (0.5 mg kg-1 b.w.). A total light dose of 360 J cm-2 was delivered at fluence rates of 260, 320, 380, 440 and 500 mW cm-2. Separate groups of mice utilized to monitor tumour temperature changes during irradiation without or after anaesthesia. Tumour response was determined by measuring the mean tumour diameter of the treated towards the untreated animals for a period of 21 days following PDT, as well as the percentage of cured animals. The most promising result (40% complete tumour response) was obtained with anaesthetized mice following 380 mW cm-2. Higher dose rates (440 and 500 mW cm-2) led to less promising results for both anaesthetized and non anaesthetized mice. These results outline the potential of PDT with longer wavelengths for the treatment of highly pigmented tumour tissues. The optimal fluence rate for tumour treatment should be chosen in order to avoid inflammatory effects (tissue swelling) and oxygen suppression with sublethal injury to the tumour cells.

Si(IV)-methoxyethylene-glycol-naphthalocyanine: synthesis and pharmacokinetic and photosensitizing properties in different tumour models.

A Si(IV)-naphthalocyanine bearing two methoxyethylenglycol axial ligands to the centrally coordinated metal ion (SiNc) was prepared by chemical synthesis and assayed for the phototherapeutic activity after administration in a Cremophor formulation to C57BI/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma or B16 pigmented melanoma. Pharmacokinetic studies indicate that the maximal accumulation in the tumour occurs at 24 h after intraperitoneal injection of 0.5 mg kg-1 of SiNc, although the naphthalocyanine concentration in the Lewis lung carcinoma (0.70 microgram g-1) is significantly larger than that in the B16 pigmented melanoma (0.15 microgram g-1). This results in a higher selectivity of tumour targeting in the case of the lung carcinoma. Photodynamic therapy (782 nm, 370 mW cm-2, 360 J cm-2) at 24 h after SiNc injection causes an efficient tumour response for Lewis lung carcinoma (50% lower tumour diameter on day 19 post-treatment as compared to untreated controls) while the pigmented melanoma shows only a minor response regarding the rate of tumour growth.

Tetraamido-substituted 2,3-naphthalocyanine zinc(II) complexes as phototherapeutic agents: synthesis, comparative photochemical and photobiological studies.

The aim of this study is to report the synthesis and photochemical and phototherapeutic activities of tetraamido-substituted 2,3-naphthalocyanine zinc(II) complexes (ZnNcs 5-8). Four naphthalocyanine complexes, tetrabenzamido- (ZnNc 5), tetramethoxybenzamido- (ZnNc 6), tetrahexylamido- (ZnNc 7) and tetradodecylamido- (ZnNc 8) naphthalocyanine zinc complexes absorbing at around 770 nm were synthesized. The dye-sensitized photo-oxidation of 1,3-diphenylisobenzofuran via 1O2 was studied in more detail in order to compare the quantum yields of these different sensitizers. Pharmacokinetic and photodynamic therapy studies of Lewis lung carcinoma in mice were carried out after administration of liposome incorporated ZnNcs 5-8. The phototherapeutic efficiency was evaluated by changes in the mean tumour diameter with time, regrowth delay (days), average survival time (days) and electron microscopy observations. According to all assessment criteria used, the most promising photosensitizer seems to be tetrabenzamido-substituted ZnNc 5. We suggest that mainly structural properties are the reason for the better results observed. The morphological analysis confirms the mechanism of photonecrosis, which was observed in our previous work with unsubstituted and substituted ZnNcs 1-4. Direct photodamage to the membrane, mitochondria and rough endoplasmic reticulum in the neoplastic cells and delayed photodestruction of the endothelial cells surrounding the tumour tissue were detected. Also, changes in lysosomes were observed. The data presented through different parameters are compared with such obtained for other photodynamic therapy sensitizers.

Inhibition of UDP-glucuronyltransferase activity in rat liver microsomes by pyrimidine derivatives.

Thirty-one differently substituted pyrimidine bases were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol and phenolphthalein by rat liver microsomes. 5-Nitrouracil (compound 1) and its isomer 4,6-dihydroxy-5-nitropyrimidine (compound 2) were the most potent and selective inhibitors of 4-nitrophenol glucuronidation, without any effect on the phenolphthalein conjugating activity of UDP-glucuronyltransferase (UGT). Kinetic studies with compound 1 revealed a mixed type of inhibition toward the acceptor substrate 4-nitrophenol and an atypical competitive type of inhibition toward UDP-glucuronic acid, with apparent Ki values of 0.11 and 0.2 mM, respectively. Two benzylamino-substituted pyrimidines (compounds 10 and 12) and an orotic acid derivative (compound 25) inhibited both 4-nitrophenol and phenolphthalein UGT activities.

Hydrophobic Zn(II)-naphthalocyanines as photodynamic therapy agents for Lewis lung carcinoma.

Four Zn(II) 2,3-naphthalocyanines (unsubstituted ZnNc1, tetracetylamido substituted ZnNc2, tetramino substituted ZnNc3 and tetramethoxy substituted ZnNc4) incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine have been injected intra-peritoneally (i.p.) (0.25-0.3 mg kg-1) to male C57/Black mice bearing a transplanted Lewis lung carcinoma. The pharmacokinetic investigations show that three of the four studied ZnNcs, 1, 2 and 4, are good tumor-localizers in Lewis lung carcinoma. The highest concentration is detected after ZnNc1 administration. The lowest tumor concentration as well as the lowest phototherapeutic effect were established with ZnNc3. In previous work it was shown that this ZnNc did not differ from the other three studied ZnNcs regarding the quantum yield of 1O2-formation and the photoinduced electron transfer. Obviously not only the good photochemical properties but also the tumor drug uptake can be an important factor of effective PDT. The biodistribution investigations also show that 72 h after drug injection, the skin concentration of the studied ZnNcs returns to the original base line. Indeed, we can expect that the skin photosensitivity will last for no longer than three days after PDT. The established higher drug concentration in the tumor rather than in the liver tissue (20 h after injection) shows again the tumor targeting selectivity of the applied liposome-sensitiser delivered procedure. Evaluating the PDT effect as reflected in the dynamics of the mean tumor diameter, we obtained unambiguous data on the potential capacity of ZnNcs 1,2,4 as PDT-photosensitisers. The data obtained from the assessment of the cytotoxic effect of PDT on the basis of the degree of induced necrosis, gave an adequate characterization of the tumor tissue destruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Liposome-delivered Zn(II)-2,3-naphthalocyanines as potential sensitizers for PDT: synthesis, photochemical, pharmacokinetic and phototherapeutic studies.

The aim of this investigation is to report the synthesis and fundamental photochemical properties of naphthalocyanines with potential interest for photodynamic therapy (PDT), as well as their pharmacokinetics and phototherapeutic effects in a tumor model. Four zinc naphthalocyanines (ZnNc), unsubstituted ZnNc 1, tetraacetylamido-substituted ZnNc 2, tetraamino-substituted ZnNc 3 and tetramethoxy-substituted ZnNc 4 absorbing around 760-770 nm, were synthesized. The dye-sensitized photo-oxidation of 1,3-diphenylisobenzofuran via 1O2 was studied in dimethylsulfoxide (DMSO). Quantum yields for this photoreaction are 0.135-0.164 and are relatively independent of the kind of substituent. In addition, the photoinduced electron transfer studied in N,N-dimethylformamide-water in the presence of methylviologen and mercaptoethanol is only slightly influenced by the kind of substituent. The pharmacokinetic properties of ZnNc 1 in hamsters bearing a transplanted rhabdomyosarcoma were studied using dipalmitoylphosphatidylcholine liposomes. Experimental PDT of rhabdomyosarcoma was carried out using liposome-delivered ZnNc 1-4. The phototherapeutic effect was evaluated by tumor photonecrosis, the mean tumor diameter during the observation period and the percentage of cured animals. The best effect was found after PDT with ZnNc 2 (50% of the treated animals were cured). A slightly lower effect was observed after application of ZnNc 4 (40% cured animals). No effect at all was noted after PDT with ZnNc 3 and a very low efficiency was found after treatment with ZnNc 1 as photosensitizer. Obviously, the photodynamic effect depends on the biological characteristics as well as on the nature of the substituents.

Comparative pharmacokinetic and photodynamic studies with zinc(II) phthalocyanine in hamsters bearing an induced or transplanted rhabdomyosarcoma.

Comparative pharmacokinetic studies in hamsters bearing an induced or first-generation transplanted rhabdomyosarcoma that were injected with liposome-incorporated zinc(II) phthalocyanine (ZnPc) show a higher drug concentration in the induced tumour. The selectivity of tumour targeting is underlined by the fact that, 24 h after injection, larger amounts of ZnPc are found in the tumour than in the liver. Photodynamic therapy investigations were carried out using 673 nm light from an argon-dye laser. On the basis of different assessment criteria (changes in mean tumour diameter with time, tumour mass regression, survival time of the treated groups of animals, and histological determination of the necrotic tissue) the photosensitizing effect of ZnPc appears to be comparable for both kinds of tumour in spite of the higher uptake of photosensitizer by the induced tumour.

Hydrated electron formation in nanosecond and picosecond laser flash photolysis of hematoporphyrin in aqueous solution.

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.

Photodynamic therapy in gastrointestinal cancer.

Six patients with an early stage of gastrointestinal (GI) cancer (T1N0M0, stage I) were successfully treated by photodynamic therapy (PDT) as follows: esophagus-1, stomach-2, rectum-3. The patients were photosensitized 72 hrs prior to treatment with pure hematoporphyrin at a dose of 5.10(-6) kg/kg b.w. in a slow intravenous infusion. Argon-pumped dye laser light at 0.630 microns wavelength was used in single and multiple treatment sessions with the power density ranging from 0.015 to 0.192 W.m-2 and a dose varying from 0.320 to 1.600 kJ.m-2. Tumor eradication (complete response) was obtained in each of the patients. No early or late treatment related complications were recorded. The patients were followed-up in the course of 7-16 months after treatment and no local recurrence or general development of disease (metastases) were reported. PDT in the early stage of GI carcinoma was recognized as a radical therapeutic method in clinical oncology.

Studies on the photodynamic effect of haematoporphyrin derivative.

A case-control photodynamic therapy (PDT) was studied on adenocarcinoma (AC755) in BDF1 mice. Haematoporphyrin derivative (HPD; Porphyrin Products, U.S.A.) and a Bulgarian HPD were used as photosensitizers at doses of 10 mg kg-1. An argon dye laser system with lambda em=630 nm (400 mW cm-2) was used for PDT with a total light dose of 400 J cm-2. The therapeutic effect was assessed by the changes in tumour dimensions, the size of photonecrosis and the mean survival time of the animals. Histologic and ultrastructural studies were carried out. No significant difference was recorded between the antitumour effects of the two photosensitizers. Best results were obtained in small tumours (less than 10 mm) with incision of covering skin. Results are discussed in an attempt to advocate an optimal regimen for PDT in experimental tumours.

Comparison of the photosensitizing efficiencies of haematoporphyrin (HP) and its derivative (HPD) with that of free-base tetrasulphophthalocyanine (TSPC-H2) in homogeneous and microheterogeneous media.

Using solutions of different polarity and various reducing (oxidizing) substrates, the type I (free radical) and type II (singlet oxygen) photosensitizing efficiencies of haematoporphyrin (HP), haematoporphyrin derivative (HPD, Photofrin II) and free-base tetrasulphophthalocyanine (TSPC-H2) were investigated. The quantum yields of 1O2-mediated oxidation of 2,2,6,6-tetramethyl-4-oxopiperidine (TEMP) followed the order QHP greater than QHPD greater than QTSPC-H2 in all the reaction media investigated. Monomeric TSPC-H2 effectively generates O2-. as shown by spin-trapping measurements. It is probable that both HPD and TSPC-H2 can oxidize L-tryptophan (Trp) via a mixed type I and type II mechanism, depending on the polarity of the medium. Both HP and TSPC-H2 in the monomeric form can be readily photoreduced by mild electron donors (ethylenediaminetetraacetic acid (EDTA), cysteine, sodium ascorbate, etc.). However, the reaction efficiencies differ because of the higher net negative electrical charge of TSPC-H2.

Photodynamic therapy in lung and gastrointestinal cancers.

Twelve central bronchial carcinoma patients and two gastrointestinal (GI) tract (oesophageal and colonic) early-stage cancer patients were treated with photodynamic therapy (PDT). Haematoporphyrin (HP/5, Jacopo Monico, Italy) at a dose of 5 mg kg-1 body weight was used as photosensitizer. Laser light at 628.2-630 nm generated by two different laser systems (gold vapour laser (I.P. Optics, Sofia, Bulgaria) in lung cancer cases and an argon dye laser system (Spectra Physics, Mountain View, U.S.A.) in GI tract cancers) was used. Lung cancers were irradiated 48 h after drug administration and GI tract cancers were irradiated 72 h after infusion of the photosensitizer. Both tumour sites were treated with a total energy dose in the range 350-600 J cm-2. Efficiency of PDT in lung cancer was evaluated by X-rays and endoscopic and functional respiratory tests for bronchial de-obstruction. Complete remission after PDT of GI tract cancers was considered to be tumour eradication (histologically and cytologically proved) and a tumour-free interval of at least 12 months.

Characterization of spin-labelled fatty acids and hematoporphyrin binding sites interactions in serum albumin.

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).

Influence of medium polarity on the photosensitizing properties of hematoporphyrin and hematoporphyrin derivative (Photofrin II).

The influence of the polarity of the medium on the efficiency of Type I and Type II photosensitization as carried out by hematoporphyrin and its derivative Photofrin II was studied by EPR techniques. Our results suggest that porphyrin aggregates do not participate as such in the photosensitized processes. Thus, the solvent-induced breakdown of the aggregated components of Photofrin II appears to determine the photosensitizing efficiency of this porphyrin.