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.     

A COMPARISON OF SERUM KINETICS AND TISSUE DISTRIBUTION OF PHOTOFRIN II FOLLOWING INTRAVENOUS AND INTRAPERITONEAL INJECTION IN THE MOUSE

Abstract Although hematoporphyrin derivative (HPD) and its 'purers' variety Photofrin II are the most widely used tissue sensitizers in both clinical and experimental photodynamic therapy (PDT), quantitative studies of tissue distribution have been few. We have extracted and measured Photofrin II in several organs of the normal mouse including those of relevance to urological practice. In view of the reported heterogeneities in the distribution within tissues of various cytotoxics when administered intraperitoneally. we have compared results for Photofrin II given by this route with those for intravenous injection. Although both routes of administration gave equally consistent results, differences in absolute tissue concentration as a function of time after injection were found for several but not all tissues. Furthermore, the porphyrin accumulated following intravenous administration seemed to contain more of the non-polar photodynamically active component than that accumulated following the intraperitoneal route. We attempt to explain these differences by reference to published data on porphyrin binding to serum proteins.     

The composition of Photofrin II

The compositions of Photofrin II and haematoporphyrin derivative (HPD) were examined. Dihaematoporphyrin ester was unambiguously synthesized and shown by high performance liquid chromatography (HPLC) not to be a significant component of Photofrin II. Hydrolysis studies showed the presence of an acid-stable oligomer in Photofrin II which comprised about 40% of of an acid-stable oligomer in Photofrin II which comprised about 40% of the total sample. A dimer derivative was isolated from this oligomeric material by fully dehydrating the methyl-esterified acidic hydrolysis product of Photofrin II. The structure of this dimer, in which two porphyrin units are linked by a three-carbon bridge containing a double bond and a methyl group, was determined by nuclear magnetic resonance (NMR) and fast atom bombardment (FAB) mass spectrometry. An analogous trimer was also isolated and identified. Structures for the hydroxyl-containing precursors to the dimer and trimer, and for the acid-stable oligomeric material in Photofrin II, are suggested. The acid-stable oligomeric material is produced in the sodium hydroxide polymerization step of the preparation of Photofrin II.     

Hematoporphyrin derivatives: an oligomeric composition study

The oligomeric composition of HpD, Photofrin II and other hematoporphyrin derivatives useful for the diagnosis and therapy of tumors has been studied. Gel chromatographic procedures were used that excluded porphyrin aggregation. Photofrin and hematoporphyrin derivatives were shown to contain different quantities of monomer, dimer and other oligomeric porphyrins.     

QUANTITATIVE ANALYSIS OF INTRACELLULAR BEHAVIOUR OF PORPHYRINS

Abstract Fluorometric analysis performed on L 1210 cells after treatment with Photofrin indicated that the interactions with cellular structures induce a significant modification of the equilibria among the different porphyrin species. This modification turned out to be dependent on the uptake and release processes. Thus, a comparative analysis of the dynamic aspects of the drug accumulation process was performed on cells treated with hematoporphyrin, Photofrin and Photofrin II. The results obtained were interpreted taking into account the different chemical composition of the drugs employed. The porphyrin species mainly released seem to be the monomeric ones and 'unfolded oligomers'. The release process results in further modifications of the aggregation and/or configu-rational state of intracellular porphyrins due to altered internal equilibrium.     

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.     

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)     

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.     

A COMPARISON OF THREE PHOTOSENSITIZERS WITH RESPECT TO EFFICIENCY OF CELL IN ACTIVATION, FLUORESCENCE QUANTUM YIELD AND DNA STRAND BREAKS

Intracellular properties of three photosensitizers relevant to photodynamic cancer therapy were compared using cultured human NHIK 3025 cells. When taken up in the cells, the hydrophilic photosensitizer aluminum phthalocyanine tetra sulfonate required about 10 times more quanta of light absorbed per cell to kill 90% of the cells than did the hydrophobic dyes Photofrin II and tetra(3-hydroxyphenyl)porphyrin. In spite of this, the phthalocyanine molecule was the most efficient dye per quantum of excitation light, since the extinction coefficient of the phthalocyanine is more than 10 times higher than that of the two hydrophobic photosensitizers at therapeutic wavelengths. The two hydrophobic dyes had significantly higher fluorescence quantum yields when taken up by cells than when bound to human plasma or human serum albumin, whereas the opposite was true for the hydrophilic phthalocyanine dye--suggesting intracellular aggregation. Finally, the potential genetic toxicities of the drugs in the form of DNA strand breaks were compared. The aluminum phthalocyanine tetra sulfonate photosensitized more DNA strand breaks than did Photofrin II and tetra(3-hydroxyphenyl)porphyrin when compared at the same level of cell survival.     

UPTAKE and PHOTODYNAMIC EFFICIENCY OF HEM ATOPORPH YRIN, HYDROXYETHYLVINYLDEUTEROPORPHYRIN and HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II®): A STUDY WITH ISOLATED MITOCHONDRIA

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.     

PHOTOSENSITIZATION BY PHOTOFRIN II DELIVERED TO WI26VA4 SV40-TRANSFORMED HUMAN FIBROBLASTS BY LOW DENSITY LIPOPROTEINS: INHIBITION OF LIPID SYNTHESIS AND FATTY ACID UPTAKE

Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.     

Photofrin as a specific radiosensitizing agent for tumors: studies in comparison to other porphyrins, in an experimental in vivo model

The use of ionizing radiation for tumor treatment represents a well established therapeutic modality. The efficiency and selectivity of radiotherapeutic protocols can be often enhanced by the addition of specific chemical compounds that optimise the response of the tumor to the incident radiation as compared with peritumoral tissue districts. The results of this study showed that Photofrin, a porphyrin derivative which is presently used as a tumor-photosensitizing agent in photodynamic therapy (PDT), can also act as an efficient tumor radiosensitizer. To test this possibility, we used nude mice subcutaneously implanted with human bladder cancer RT4. The mice were injected with different porphyrin-type photosensitizing agents, including Photofrin, 5-aminolevulinic acid, chlorin e(6), haematoporphyrin, protoporphyrin, Zn-tetrasulphophtalocyanine, and irradiated with 5 and 15 Gy using a Siemens X-ray device. Even though all the porphyrins accumulated in significant amounts in the neoplastic lesion, only Photofrin significantly improved the response of the tumor to irradiation by increasing the doubling time of the tumor volume from 6.2 days in the untreated control group to 10.9 days in the 5 and 15 Gy-irradiated groups. The tumor response was maximal with injected Photofrin doses of 7.5 mg/kg, and was not further enhanced by injection of higher doses. Our hypothesis is, that the radiosensitizing effect of Photofrin seems to be due to some oligomeric constituents which could specifically react with radiogenerated-radicals thereby amplifying the effect of the X-ray radiation.     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

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).     

INTRACELLULAR FLUORESCENCE OF PHOTOSENSITIZING PORPHYRINS AT DIFFERENT CONCENTRATIONS OF MITOCHONDRIA

Abstract— Photofrin II, which contains the most efficient components of hematoporphyrin derivative with regard to tumor phototherapy, was measured fluorometrically in single cells of three squamous cell lines with different mitochondrial concentrations. Using the method of time-resolved fluorescence microscopy, two components—probably monomeric and dimeric species—were differentiated according to the lifetimes of their excited states. The intensity ratio between these components was found to depend on the concentration of mitochondria. This may be due to a monomerization of those porphyrin molecules which are retained in mitochondrial sites.     

PHOTOINDUCED DEGRADATION AND MODIFICATION OF PHOTOFRIN II IN CELLS in vitro

Abstract— Human cells of the line NHIK 3025 were incubated with Photofrin II (PII) and exposed to light. Fluorescence- and absorption spectra of PII in the cells were measured. Light exposure resulted in a degradation of PII in the cells and changes in the shape of the fluorescence spectra. These changes are probably partly due to a photochemical modification of PII and to a relocalization of PII in the cells. Notably, a destruction of binding sites for PII on or close to proteins was caused by the light exposure. The rate of the light-induced decay of the porphyrin fluorescence intensity was only slightly increasing with the PII concentration, indicating that each porphyrin molecule is mainly degraded by photoproducts originating from itself. On the other hand, the rate of the degradation of porphyrin binding sites on the proteins increased with increasing PII concentrations.
The excitation spectrum of PII in cells has a peak at285–290 nm attributed to energy transfer from proteins to porphyrins located close to the proteins. The intensity of this peak relative to the intensity of the Soret band increases with decreasing porphyrin concentrations. This might indicate that some of the binding sites close to proteins have a higher affinity for the porphyrin than binding sites at longer distances from the proteins.     

Modulation of Porphyrin Derivatives Accumulation in C6 Glioma Cells by Drugs Acting on β-Adrenergic Receptors. A Spectrofluorometric Study

The pharmacological modulation of the uptake of porphyrin derivatives in cultured C6 glioma cells was investigated by means of spectrofluorometric analysis both in single cells and in cell homogenates. The influence of drugs acting as β-receptor agonists or antagonists was studied in cells grown to semiconfluency. Isoproterenol (ISO), a β-receptor agonist, enhanced the intracellular fluorescence intensity of both Photofrin and protoporphyrin IX (PpIX). A treatment with a β-receptor antagonist I-propranolol (PRO), simultaneous with ISO, resulted in an intracellular Photofrin fluorescence signal comparable to that of the control cells, indicating the specificity of the pharmacological action. The pharmacological treatment seemed particularly effective with the aggregated species. This is suggested by the relative increase of the band at 670 nm, being greater than that in the 630 nm band in the emission spectra of Photofrin and PpIX, and by the comparison of the fluorescence intensity on cell homogenates measured both in the absence and in the presence of cetyltrimethyl-ammonium bromide as a detergent.     

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.     

CHEMISTRY OF HEMATOPORPHYRIN-DERIVED PHOTOSENSITIZERS

Abstract The hematoporphyrin-derived tumor-localizing preparation HPD consists of porphyrin monomers, which are'inactive'(not tumor-localizing), and a dimer/oligomer fraction which is responsible for the localizing phenomenon. In an organic solvent system, gel-exclusion chromatography can separate HPD into fractions containing porphyrin monomers, dimers or oligomers. The relative amount of the dimer/oligomer fraction of HPD was a function of the pH of the mixture used to transform HP mono/di acetate to HPD. HPD prepared by the'Upson'procedure contained dimer/oligomer linkages which are labile to 1 M NaOH (in 50% tetrahydrofuran), and are reduced by LiAlH₄ to alcohols. These properties are characteristic of esters. But a commercial product, Photofrin II, contained approx. 50% of material refractory to both reagents described above. This behavior is characteristic of an ether linkage. These observations show that the nature of the linkage joining the porphyrin units is sensitive to conditions employed in HPD preparation. Tumor localization derives, in part, from affinity of these oligomers for plasma lipoprotein, and is associated with conformational alterations characteristic of these porphyrin-porphyrin linkages.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: II. THE EFFECTS OF HUMAN versus MOUSE AND BOVINE SERUM

The effects of human serum (HS), mouse serum (MS) and fetal bovine serum (FBS) on cellular delivery and retention of Photofrin were examined using human lung tumor cells (A549) cultured in vitro. The results show that these three kinds of sera exhibit substantial differences in: (i) degree of inhibition of Photofrin cellular uptake, (ii) retention capacity of Photofrin delivered to the cells in their presence and (iii) efficacy of promoting the clearance of Photofrin from the cells. It is suggested that these differences originate from unequal interaction of each of the sera with Photofrin material, which in turn is the consequence of variability in composition and in the levels of serum proteins in HS, MS and FBS. The highest degree of Photofrin disaggregation and and competitive binding of its constituents was attributed to HS. The lowest degree of Photofrin disaggregation, and the competitive binding limited mostly to monomeric porphyrin forms was implicated for FBS. For MS, the spectroscopic and cellular data indicated a lesser degree of Photofrin disaggregation than with HS, with little if any consequence in Photofrin retention characteristics. The implication of this comparative analysis is that in vitro studies using FBS may underestimate the extent of interaction of Photofrin with serum proteins in humans, and overestimate the retention capacity of the photosensitizer in human tissues. Studies in vivo using a mouse model may also underestimate the degree of disaggregation of Photofrin in human circulation, and give different photosensitizer tissue retention levels than in humans.