ACUTE SKIN RESPONSE IN ALBINO MICE FOLLOWING PORPHYRIN PHOTOSENSITIZATION UNDER OXIC AND ANOXIC CONDITIONS

Abstract Acute normal skin toxicity induced by porphyrin photosensitization has been examined using albino mice. Oxic and anoxic (clamped) skin was exposed to red light (630 nm) 24 h following administration of hematoporphyrin derivative (HpD) or Photofrin II (the active component of HpD). Experiments were also performed to determine the effect of sodium pentobarbital anesthesia on HpD and Photofrin II photosensitization of normal skin. Results from this study demonstrated that comparable levels of acute skin damage were induced by HpD and Photofrin II under oxic conditions but neither porphyrin produced any apparent phototoxicity under anoxic conditions. In addition, the level of skin damage induced by porphyrin photosensitization was not affected by sodium pentobarbital anesthesia.     

CHLORIN AND PORPHYRIN DERIVATIVES AS POTENTIAL PHOTOSENSITIZERS IN PHOTODYNAMIC THERAPY

In order to find a photosensitizer with better optical properties and pharmacokinetics than Photofrin II, a series of new photosensitizers related to methyl pheophorbide-a and chlorin-e6 were synthesized. These compounds absorb at substantially longer wavelengths (lambda max 660 nm) than does Photofrin II (630 nm) and show promise for use in photodynamic therapy. Among the porphyrins, we observed that long carbon chain ether derivatives are better photosensitizers than their ester analogs. These sensitizers were tested for in vivo photosensitizing activity vis-a-vis Photofrin II, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. Most of these photosensitizers were found to have better tumoricidal photosensitizing activity than Photofrin II and demonstrated more rapid attenuation of normal tissue photosensitivity with time after administration vis-a-vis Photofrin II.     

Fluorescence imaging and phototoxicity effects of new formulation of chlorin e6-polyvinylpyrrolidone

Evaluations of the efficiency of a new formulation of chlorin consisting of a complex of trisodium salt chlorin e6 (Ce6) and polyvinylpyrrolidone (PVP) in photodynamic therapy (PDT) and fluorescence diagnosis was performed on poorly differentiated human bladder carcinoma murine model with the following specific aims: (i) to qualitatively evaluate the fluorescence accumulation in human bladder tumor, (ii) to determine fluorescence distribution of Ce6-PVP using the tissue extraction technique and fluorescence imaging technique, (iii) to compare the fluorescence distribution of Ce6, Ce6-PVP and Photofrin in skin of nude mice, and (iv) to investigate phototoxicity caused by different parameters (drug-light interval, drug dose, irradiation fluence rate and total light fluence) in PDT. The fluorescence of the Ce6-PVP formulation was determined either by fluorescence imaging measurements or by chemical extraction from the tissues displaying similar trends of distribution. Our results demonstrated that the Ce6-PVP formulation possesses less in vivo phototoxic effect compared to Ce6 alone. The phototoxicity revealed a strong dependence on the drug and light dosimetry as well as on the drug-light interval. In PDT, the Ce6-PVP compound was most toxic at the 1h drug-light interval at 200J/cm(2), while Ce6 alone was most toxic at a light dose of more that 50J/cm(2) at the 1 and 3h drug-light interval. We also confirmed that Ce6-PVP has a faster clearance compared to Ce6 alone or Photofrin. This eliminates the need for long-term photosensitivity precautions. In conclusion, the Ce6-PVP formulation seems to be a promising photosensitizer for fluorescence imaging as well as for photodynamic treatment.     

Photobleaching of photofrin II as a means of eliminating skin photosensitivity

One of the effects of using Photofrin II as a sensitizer in clinical photodynamic therapy is skin photosensitivity. Avoiding the harmful effect of this photosensitivity typically requires the patient to remain indoors during daylight hours for 4 weeks post injection. Photosensitivity can last up to 8 weeks. A possible method of quickly eliminating skin photosensitivity is the use of photobleaching to eliminate the DHE (dihematoporphyrin ether, the active agent in Photofrin II) from the skin. The Photofrin II was used as supplied (Photomedica, Raritan, NJ) and will be referred to as DHE. We report a series of experiments using Swiss mice which demonstrate the ability to reduce the period of photosensitivity to 1 week. The Swiss mice received 5 mg kg^-1 DHE intraperitoneal. Beginning at 24 h post injection they were given daily increasing light doses starting with 18 J cm^-2 (at 630 nm). Only the left hind feet were treated with light. At 7 days post injection both hind feet were treated with 135 J cm^-2. The pretreated left foot showed no reaction to this treatment whereas the right foot reacted as strongly as control mice which had received no pretreatment with light. The control mice were also injected 7 days prior to treatment. As a check for tolerance animals received a second injection of 5 mg kg^-1 DHE intraperitoneally 96 h after the conclusion of the bleaching protocol. Twenty-four h later the left hind foot (previously desensitized by light) reacted to 135 J cm^-2 as strongly as controls which had received the 5 mg kg^-1 of DHE but no pretreatment.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

Photoproducts formed from photofrin II in cells

Fluorescence and absorption spectra of light-exposed cells containing the tumour-localizing porphyrin preparation Photofrin II (PII) have been studied. Light exposure results in spectral changes that may be due to a photoinduced modification of the porphyrins without breakage of the porphyrin macrocycle and/or to a photoinduced displacement of the porphyrins in the cells. Photochemical reaction involving breakage of the porphyrin macrocycle also occur as can be seen from the loss of absorbance within the Soret band region during light exposure. Singlet oxygen may be involved in the photodegradation of PII in cells since the process is slowed down on bubbling N2 through the samples and is slightly faster in suspensions in Dulbecco's phosphate buffered saline (PBS) made of D2O compared with suspensions in PBS made of H2O. During light exposure a fluorescent product is formed in the cells with fluorescence excitation and emission characteristics similar to those of the "age pigment" lipofuscin (lambda exc = 350 nm, lambda em = 440 nm).     

THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.     

MOUSE SKIN PHOTOSENSITIVITY WITH DIHAEMATOPORPHYRIN ETHER (DHE) AND ALUMINIUM SULPHONATED PHTHALOCYANINE (AlSPc)

Skin photosensitivity of sun exposed sites is the major side effect of dihaematoporphyrin ether (DHE) photodynamic therapy (PDT). Reports of severe oedema and erythema have generally been anecdotal. We have studied aluminium sulphonated phthalocyanine (AlSPc) as a potential photosensitiser for PDT. In this paper we report our work comparing the skin photosensitivity reactions of DHE and AlSPc. We have studied: (i) the time course of the skin reactions, (ii) the effect of increasing time from administration of photosensitiser to irradiation, (iii) drug-skin reaction dose response. Groups of Skh I female hairless albino mice were given an intravenous bolus dose of either 0.9% saline solution, AlSPc or DHE (Photofrin II). Drug doses ranged from 0.5 to 50 mg/kg. At times ranging from 1 h to 1 month animals were irradiated with a range of doses of solar simulated radiation (SSR). The skin reaction was observed over a 2 week period. DHE reactions were always more severe than those with AlSPc. Peak skin reaction was seen at 3 h for DHE and 6 h for AlSPc. DHE reactions were still visible 2 weeks after irradiation whereas the AlSPc reaction disappeared by 48 h. Irradiation evoked a reaction up to 2 months after administration of DHE but only up to 2 weeks with AlSPc. The mean SSR dose at which a skin reaction was seen decreased with increasing dose of both agents. The rate of decrease was slower with AlSPc than DHE. This study suggests that in PDT, AlSPc will cause much less skin photosensitivity than DHE.     

Carrier thiols are targets of Photofrin II photosensitization of isolated rat liver mitochondria

To gain further insight into the ability of Photofrin II to photosensitize mitochondrial translocators, and to ascertain whether mitochondrial thiols are specific targets of Photofrin II, the activity of phosphate carrier was measured in isolated rat liver mitochondria irradiated with 365 nm light in the presence of Photofrin II. Photodynamic treatment decreased the maximum rate of phosphate uptake, without changing the phosphate affinity for its own carrier. The ability of the thiol reagent mersalyl (an inhibitor of phosphate, dicarboxylate and oxodicarboxylate carriers) to protect these carriers against Photofrin II photosensitization was also tested. Protection was observed, indicating the involvement of carrier thiols in mitochondrial photosensitization.     

Tissue-localizing properties of some photosensitizers studied by in vivo fluorescence imaging

Using fluorescence imaging, the tissue-localizing properties of five photosensitizers were studied in vivo in tumours in 'sandwich' observation chambers and in tumours growing on thigh muscle. The preliminary results indicate that of the three photodynamically active dyes tested (haematoporphyrin derivative, Photofrin II and aluminium phthalocyanine tetrasulphonate), the phthalocyanine possesses the best tumour-localizing properties. This makes it possible to combine tumour fluorescence detection and photodynamic therapy with reduced skin photosensitivity. The two photodynamically inactive dyes tested (uroporphyrin I and acridine red) may be useful for application in fluorescence imaging to localize superficial tumours without inducing skin photosensitivity. In particular, acridine red has remarkable tumour-localizing properties, but is rather toxic.     

THE TIME COURSE OF CUTANEOUS PORPHYRIN PHOTOSENSITIZATION IN THE MURINE EAR

This study was designed to investigate the time course of acute cutaneous photosensitivity following administration of Photofrin II using the murine ear swelling response (ESR) as an in vivo end-point. Ros:(ICR) mice were injected with 5 mg/kg Photofrin II and illuminated 7.5 h to 31 days later with 630-nm laser light; ESR was measured 24 h after illumination. There was a direct correlation between ESR and the concentration of [14C]Photofrin II in blood, while no relationship between ESR and the level of [14C]Photofrin II in the ear tissue of exsanguinated mice was evident. Photosensitivity in the mouse foot can be suppressed by preexposure to low doses of light via a photochemical destruction of tissue-bound sensitizer (Boyle and Potter, 1987, Photochem. Photobiol. 46, 997-1001). However, mouse ears pretreated with 84 J/cm2 of 630-nm light (28 J/cm2/day, given 2, 4 and 6 d after injection), a dose sufficient to reduce porphyrin fluorescence in ear tissue by about 75%, prior to the usual light dose (88.6 J/cm2, 630 nm, day 9 after injection) showed a mean ESR not significantly different (P less than 0.5) from that for ears which received only a single dose of 88.6 J/cm2 on day 9. It is concluded, for this animal model, that circulating porphyrin is the source of photoinduced ear-tissue edema and that photobleaching of tissue-bound sensitizer does not attenuate ear-tissue photosensitivity.     

Topical use of liposomal copper palmitate formulation blocks porphyrin-induced photosensitivity in rats

Photodynamic therapy (PDT) is a new treatment modality that uses porphyrin derivatives and visible light, especially for the treatment of cancer. However, PDT with certain photosensitisers can cause prolonged skin photosensitization. This is particularly true for Photofrin II (Photofrin)-mediated PDT where patients are required to avoid direct exposure to sunlight for a period of 4-6 weeks. This is the only long-term adverse reaction to the drug. Recent studies have shown that topical copper treatment avoids this type of inflammatory reaction. In this study, we have tested the efficiency of the liposomal formulation of copper palmitate on porphyrin-photosensitized rats. Initially, adult male Sprague-Dawley rats were rendered photosensitive either by administration of Photofrin or aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX). Prior to this, their dorsal skin was shaved and treated topically with a cream consisting of either empty or copper palmitate-encapsulated liposomal formulation. After being kept in a dimmed light environment, the rats were exposed to visible light, and inflammatory responses were inspected. Histological studies revealed that no inflammatory cells were present at the skin sites treated with liposomal cream containing copper palmitate in the Photofrin-sensitized group while no reduction in the number of inflammatory cells was observed at the skin samples treated with the empty liposomes. In conclusion, the data demonstrate the significant protective effect of topically-applied liposome-encapsulated copper palmitate against both Photofrin and ALA-induced PpIX photosensitivity.     

THE PREPARATION OF RADIOLABELED PORPHYRINS and THEIR USE IN STUDIES OF PHOTODYNAMIC THERAPY

Abstract— We tested water-soluble sulfonated phthalocyanine and three metal chelate derivatives for their tumoricidal effect on the EMT-6 mammary tumor in mice exposed to red light. The metal-free sulfophthalocyanine had little effect, whereas the aluminum complex and the lower sulfonated fraction of the gallium complex exhibited tumoricidal activity similar to hematoporphyrin-based photosensitizer (Photofrin II). The higher sulfonated fractions of the gallium complex were less active as compared to the lower sulfonated fraction. The cerium complex was the most active sensitizer in terms of dye and light doses required to induce tumor necrosis and cure but also showed the highest phototoxicity towards healthy skin. These results suggest that sulfonated phthalocyanines will offer a new alternative in photodynamic therapy of light-accessible neoplasms.     

Preclinical Assessment of Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: Progress Update

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-( N,N -dimethylami-no)-propylamine-HB]. Extinction coefficients at 630 nm (φ₆₃₀) are 6230, 6190 and 4800, respectively; and ¹O₂ quantum yields, φ, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMToVEd tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhi-murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.     

MOUSE SKIN PHOTOSENSITIZATION WITH BENZOPORPHYRIN DERIVATIVES AND PHOTOFRIN®: MACROSCOPIC AND MICROSCOPIC EVALUATION

A comparative study, at both the macroscopic and microscopic level, of skin photosensitivity caused by four isomeric forms of benzoporphyrin derivative (BPD) has been carried out, and compared to effects of Photofrin. Animals injected intravenously with BPD analogues and exposed to light 3 h later showed extensive photosensitivity. Animals receiving the monoacid derivatives of BPD (BPD-MA and BPD-MB) showed markedly more photosensitivity than those receiving the diacid derivatives (BPD-DA and BPD-DB). Animals receiving BPD analogues which were exposed to light 24 h or more later showed only minimal reactivity. Histological examination of biopsies taken after photosensitizer injection and light exposure showed extensive changes in epidermis and dermis, including epidermal erosion, degranulation of the stratum granulosum, spongiosis, depletion in cellularity and mast cell degranulation. These changes were noted to be similar to changes caused by Photofrin.     

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

PHARMACOKINETICS OF FLUORESCENT POLYPORPHYRIN PHOTOFRIN II IN NORMAL RAT TISSUE AND RAT BLADDER TUMOR

The transient behavior of the molecular components responsible for fluorescence emission of the photosensitizing polyporphyrin Photofrin II has been studied quantitatively in the liver, small intestines, bladder and muscles of rats. Relative concentrations of the substance were determined fluorometrically in vivo using a Kr(+)-laser (wavelength = 406.7 nm) and a mercury arc lamp (wavelength = 405 or 550 nm) for fluorescence excitation of Photofrin II. Fluorescence was detected at the maxima of the emission bands, at 630 or 690 nm. The results of the experiments show that Photofrin II can be clearly detected by its fluorescence in all the organs investigated from 3 h up to at least 28 days after systemic application of the substance. Within this investigational period the fluorescing components of Photofrin II are released continuously from the organs. In all the tissues examined, an initial decrease with time constants between 2 and 42 h followed by a slow decay with time constants between about 300 and 600 h can be observed. In addition the pharmacokinetics of the fluorescent components of Photofrin II in chemically induced rat bladder tumors with different stages of malignancy were compared to healthy rat bladder tissue. In a time range of 2-10 days after intravenous injection Photofrin II shows a fluorescence 2-5 times brighter in rat bladder tumors than in healthy bladder tissue.     

In vitro phototoxicity of nifedipine: sequential induction of toxic and non-toxic photoproducts with UVA radiation.

Anecdotal reports suggest that the dihydropyridine calcium antagonist, nifedipine (NIF), may be phototoxic in human skin. We have studied NIF phototoxicity in vitro using UVA fluorescent tubes (Sylvania PUVA). NIF was phototoxic to Candida albicans and induced photohaemolysis both with NIF present during irradiation and with pre-irradiated drug. In V79 hamster fibroblasts, NIF (10 micrograms ml-1) was phototoxic MTT assay) 24 h after irradiation (0-112 kJ m-2); at 7.5 kJ m-2, about 70% of cells were damaged whilst at 37.5 kJ m-2, only about 45% of cells were damaged. A similar pattern was seen with pre-irradiated NIF. Absorption spectroscopy showed that the NIF absorption maximum (Amax approximately 340 nm) blue-shifted to 314 nm at low UVA doses (7.5 kJ m-2 or less) and red-shifted to 345 nm at higher doses (isosbestic point, 325 nm). Thin layer chromatography of irradiated NIF showed a single photoproduct (PP1; Amax approximately 314 nm) formed at 7.5 kJ m-2 or less which disappeared at higher UVA doses to give further photoproducts. PP1 was highly dark toxic to V79 cells (50% damage at about 5 micrograms ml-1) but PP1 pre-irradiated with UVA was non-toxic. Preliminary gas chromatography-mass spectroscopy studies suggest that PP1 is the nitroso derivative of NIF. These results indicate that NIF phototoxicity in vitro is partially mediated by initial formation of a toxic photoproduct (PP1) but, paradoxically, subsequent UVA irradiation may reduce phototoxicity. The NIF concentrations required to induce in vitro phototoxicity are much greater than therapeutic plasma levels. Unless there is skin accumulation of NIF or PP1, our in vitro results suggest that NIF may not be an important skin-photosensitizing agent in vivo.     

ACTIVATION OF BENZOPORPHYRIN DERIVATIVE IN THE CIRCULATION OF MICE WITHOUT SKIN PHOTOSENSITIVITY

In vitro experiments with benzoporphyrin derivative monoacid ring A (BPD) confirmed earlier studies that it was taken up rapidly (within 30 min) to maximum concentrations by all cells tested. It was also confirmed that rapidly dividing tumor cell lines and mitogen-activated murine T lymphocytes took up significantly more (5-10-fold) BPD than did normal splenic lymphocytes. Further experiments were undertaken to determine whether BPD could be activated by whole-body irradiation with red light in the blood of animals, shortly after intravenous (i.v.) administration, in the absence of skin photosensitivity. It was found that shaved and depilated mice injected i.v. 60 min earlier with BPD at between 0.5 and 1.0 mg/kg could tolerate 160 J/cm² of broad-band red light (560-900 nm) delivered, at a relatively low rate, over a 90 min time interval without developing skin photosensitivity or general phototoxicity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 μg/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When LI 210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total-body light treatment (160 J/cm² of 590-900 nm light delivered over 90 min), significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This indicated that sufficient light was being delivered to BPD in the blood flowing in the peripheral vasculature to effect cytotoxicity to cells containing the photosensitizer without causing either vascular or skin photosensitivity. Thus, activation of this photosensitizer in the circulation can be achieved by transdermal light exposure without causing skin photosensitivity provided that light exposure is performed at a time when the first phase of plasma clearance is complete and when the drug has not yet accumulated in skin.