PORPHYRIN ACCUMULATION BY ATHEROMATOUS PLAQUES OF THE AORTA

Abstract A complex mixture of porphyrins termed hematoporphyrin derivative (HPD) has been clinically useful for tumor localization. When sections of human aorta containing atheromatous plaques were incubated with HPD, accumulation of fluorescent porphyrin was observed within the plaques. Analytic studies showed that the plaques had accumulated hematoporphyrin (HP), which is substantially more hydrophilic than that HPD fraction generally associated with tumor localization. Fluorescence spectra suggest that the plaque binding sites of HP resemble the relatively aqueous micelles formed by the detergent sodium dodecylsulfate. This result has implications for tumor-localization procedures, since accumulation of hydrophilic porphyrins by tumors has been reported.     

PROBING THE STRUCTURE OF HPD BY FLUORESCENCE SPECTROSCOPY

Fluorescence emission spectra indicate that oligomers containing both hematoporphyrin and its dehydration products (vinyl porphyrins) comprise the tumor-localizing fraction of HPD. In the relatively polar solvent methanol, the vinyl porphyrins exhibit reduced fluorescence yields while the hematoporphyrin residues are relatively resistant to fluorescence quenching by Fe+3. In the less polar solvent tetrahydrofuran, fluorescence from oligomeric vinyl porphyrins was enhanced, and Fe+3-induced quenching of oligomeric hematoporphyrin promoted. These, together with other studies in biological systems, suggest a substantial degree of interaction among the porphyrin units contained in these oligomers, as a function of the polarity of the environment.     

On the preparation and properties of dihematoporphyrin ether, the tumor-localizing component of HPD

Abstract— A porphyrin mixture derived from hematoporphyrin and termed HPD (hematoporphyrin derivative) is used for localization and therapy of tumors. The major localizing component of HPD has been provisionally identified as a dihematoporphyrin ether (DHE). In this study, we describe HPLC procedures for analytical and preparative separation of HPD components. Optimal conditions for DHE formation were determined. A comparison of absorbance and fluorescence spectra in different solvents indicate that DHE aggregates are substantially more stable than are the analogous hematoporphyrin structures. Studies on the interaction between DHE and tumor cells indicate an initial binding of the drug at lipophilic membrane sites, followed by gradual relocation to intracellular loci.     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

Abstract
The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5–6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero-and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N -methylated porphyrins show clearance times in the vicinity of 15–22 h. Monoaspartyl chlorin e₆ shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

PHOTOSENSITIZATION BY DIPORPHYRINS JOINED VIA METHYLENE BRIDGES

Abstract— Photodynamic and biophysical properties of three porphyrin dimers joined by methylene bridges were examined. Fluorescence emission spectra and fluorescence lifetimes of the methylene-linked dimers were similar to values obtained with porphyrin monomers. Singlet oxygen quantum yields were not significantly different when the three diporphyrins were compared. The diporphyrins were short-acting tumor photosensitizers in vivo, and were rapidly cleared from plasma. Of the 3 diporphyrins examined, one was essentially ineffective as a sensitizer in vivo. This could not have been predicted from in vitro studies which indicated photodamage to membrane and mitochondrial loci. The methyiene-linked diporphyrins were hydrophilic dyes (water/octanol distribution ratio =120–200) and bound mainly to plasma high-density lipoprotein. In contrast, the more hydrophobic diporphyrin ester/ether fraction from HPD was a long-persisting photosensitizer in vivo. Compared with hematoporphyrin, this hematoporphyrin derivative (HPD) fraction demonstrated a red-shift in fluorescence emission and a shortened fluorescence lifetime. These comparisons suggest that ring-ring interactions occur in the ester/ether-linked diporphyrins from HPD, but not in the methyiene-linked diporphyrins.     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5-6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero- and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N-methylated porphyrins show clearance times in the vicinity of 15-22 h. Monoaspartyl chlorin e6 shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

QUANTITATIVE in vivo MEASUREMENT OF THE FLUORESCENT COMPONENTS OF PHOTOFRIN II

Abstract Photofrin II which contains the most efficient components of hematoporphyrin derivative with regard to photodynamic therapy of cancer, was measured fluorometrically in tumor and tumor-free tissues in vivo over a period up to 8 days. Using time-resolving (nanosecond and picosecond) microscopic techniques, the fluorescence of different components was quantitated and attributed to monomelic, dimeric, and possibly aggregated porphyrin species. The long-lasting retention of the porphyrins in live tissues was in contrast to the rapid removal from cultured cells. This might be due to monomerization or dimerization of non-fluorescent aggregates. Tumor-selective accumulation was found to be similar for two different (probably monomeric and dimeric) components. This indicates that the integral fluorescence of these components may also correlate with the distribution of the main photosensitizing species.     

DISTRIBUTION OF PORPHYRINS IN THE VARIOUS COMPARTMENTS OF UNILAMELLAR LIPOSOMES OF DIPALMITOYL-PHOSPHATIDYLCHOLINE AS PROBED BY FLUORESCENCE SPECTROSCOPY

Abstract— The spectroscopic properties of hematoporphyrin, hematoporphyrin-dimethyl ester, uroporphyrin and uroporphyrinoctamethyl ester, incorporated into unilamellar liposomes of dipalmitoylphos-phatidylcholine, have been studied with the aim to assess the distribution of porphyrins within the various liposomal compartments.
The results obtained indicate that the highly hydrosoluble uroporphyrin is partitioned in the endoliposomal aqueous pool while its octamethylester is homogeneously distributed in the inner lipid monolayer. Hematoporphyrin and its dimethylester show an heterogeneous distribution within the phospholipid bilayer. At T = 25°C these porphyrins are preferentially located in the outer phospholipid monolayer.
Detailed studies on hematoporphyrin indicate that the distribution between the inner and outer phospholipid monolayer is a function of temperature and liposome dimensions. In particular, the increase of temperature above the critical temperature for the liquid-gel phase transition of the liposomes causes a partial shift of the porphyrin molecules toward the inner phospholipid monolayer. Moreover, the increase of liposome dimensions leads to a greater accessibility of porphyrin to the external medium.     

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.     

PHOTOPHYSICAL AND PHOTOBIOLOGICAL PROPERTIES OF DIPORPHYRIN ETHERS

Spectral properties of several diporphyrin ethers were assessed in different solvents and after accumulation by leukemia L1210 cells in vitro. To facilitate studies in a variety of solvents, both tetramethylesters of the diporphyrin ethers and free acids were employed. For comparison, studies on the corresponding porphyrin monomers were also carried out. The joining of two porphyrins by an ether linkage had several consequences. We observed a blue shift in the Soret band of the ethers, but not of the corresponding simple porphyrins, in protic solvents. This phenomenon is likely related to ether aggregation under conditions which promote H-bonding. The presence of an ether linkage was associated with enhanced fluorescence at 630-640 nm and decreased fluorescence lifetimes and yields, especially in protic solvents. The ether linkage was unaffected by intracellular enzymes, but porphyrin esters were readily hydrolyzed upon accumulation by L1210 cells. The joining of two hematoporphyrin molecules by an ether linkage promoted dye accumulation by L1210 cells. In contrast, accumulation of mesoporphyrin and protoporphyrin was thereby retarded.     

SITES OF PHOTOSENSITIZATION BY DERIVATIVES OF HEMATOPORPHYRIN

Leukemia L1210 cells were incubated in vitro with the tumor-localizing product HPD (hem-atoporphyrin derivative) for 0.5. 4 and 18 h. Effects of subsequent irradiation on viability, membrane transport and integrity, DNA synthesis and intracellular ATP concentration were assessed. Intracellular porphyrin pools were analyzed by HPLC. A 30 min incubation led to concentration of a readily-exchangeable pool of monomeric HPD components at plasma membrane loci; irradiation resulted in photodamage to membrane transport and a loss in capacity for dye exclusion. In contrast, increasing the incubation time led to a corresponding increase in the size of a non-exchangeable intracellular pool of other HPD components. Subsequent irradiation led to depletion of intracellular ATP and loss of capacity for biosynthesis of DNA, but little plasma membrane damage.     

PHOTODYNAMIC INACTIVATION OF R3230AC MAMMARY CARCINOMA IN VITRO WITH HEMATOPORPHYRIN DERIVATIVE: EFFECTS OF DOSE, TIME, and SERUM ON UPTAKE and PHOTOTOXICITY

Abstract Primary cultures of the R3230AC mammary adenocarcinoma were used for pharmacokinetic studies of hematoporphyrin derivative (HPD), a preparation containing several porphyrin species and useful as a photoactivatable anti-tumor agent. Uptake of HPD in vitro was shown to be time-, dose- and temperature-dependent with an apparent plateau reached at 2 - 4 h. An increase in the amount of serum in the medium progressively reduced the amount of HPD taken up by the cells; at a level of 10% serum, uptake of HPD was reduced by >95%. The time-course of efflux of HPD from these cells demonstrated a complex pattern, with an initial rapid component followed by a more gradual rate of efflux up to 4 h. Assessment of photoradiation-induced cytotoxicity was performed by a method developed to quantitatively measure trypan blue exclusion. Relative cytotoxicity was determined by use of heat-killed cells as a standard. At two different concentrations of HPD, cytotoxicity was dependent on light exposure time. The presence of serum, which reduced uptake of HPD was correctable to reduced cytotoxicity. Based on the amount of light exposure to produce 50% cell kill, an order of potency was obtained for HPD > hematoporphyrin > hydroxyethylvinyldeuteroporphyrin in vitro. This order of potency correlated with the relative proportion of hydrophobic components as estimated by HPLC analysis. The results indicate that HPD is an effective cytotoxic agent in vitro in a well-differentiated mammary adenocarcinoma model.     

FLOW CYTOMETRIC ANALYSIS OF INTRACELLULAR HEMATOPORPHYRIN DERIVATIVE IN HUMAN TUMOR CELLS AND MULTICELLULAR SPHEROIDS

Flow cytometry (FCM) has been used to investigate the intracellular fluorescence of hematoporphyrin derivative (HPD) in monolayer and spheroid cultures of WiDr cells. For exponentially-growing monolayer cultures mean cellular fluorescence was directly proportion to the external HPD levels in the range 5-100 micrograms ml-1 (r = 0.99). Heterogeneity of cellular fluorescence was quantified by determining the ratio of the fluorescence value below which were observed values for 98% of the cell population compared to the fluorescence value for 2%. In exponentially-growing cultures, decreasing levels of HPD in the medium led to an increase in the 98:2% ratio, i.e. an increase in heterogeneity of intracellular drug levels. The growth of cells as multicellular spheroids confers a spheroid-size-dependent resistance to photodynamic treatment. With increasing spheroid size (100, 250, 500, 750 and 1000 microns diam.) there was a decrease in mean intracellular HPD levels and a large linear increase in the 98:2% ratio (r = 0.94).     

PICOSECOND FLUORESCENCE OF R3230AC MAMMARY CARCINOMA MITOCHONDRIA AFTER TREATMENT WITH HEMATOPORPHYRIN DERIVATIVE and PHOTOFRIN® II in vivo

Hematoporphyrin derivative (HPD) and other porphyrin samples were excited by 20-ps 532-nm laser pulses. Fluorescence was detected using a low-jitter streak camera. Data were fitted to a sum of exponential decay times on the order of picoseconds. Fluorescence of porphyrins in aqueous solution show various behaviors depending on the hydrophobicity of the porphyrins. The most hydrophilic porphyrins show long decays only (greater than 500 ps). Porphyrins intermediate in hydrophobicity have intensity-dependent fast decays. The most hydrophobic have fast decays (less than 20 ps). Picosecond fluorescences of mitochondria prepared from rat tumors treated in vivo with HPD or Photofrin II show an increase in the ratio of fast to slow decays when compared to the injected porphyrins. These results are consistent with the concentration of the more hydrophobic porphyrins in mitochondria in photosensitization treatment. Thus picosecond fluorescence studies of porphyrins may provide a means to obtain photoproperties which differentiate between effective and ineffective in vivo photosensitizers.     

PHOTOCHEMICAL PROPERTIES OF PORPHYRIN c: AN AGENT FOR USE IN TUMOR PHOTOTHERAPY

Abstract— The absorption and fluorescence properties of porphyrin c (P c ), the porphyrin chromophore present in cytochrome c , have been determined in several solvents and micellar environments. In aqueous buffer solutions at pH 7.5 Pc may exist in both a fluorescent monomeric form with quantum yield of fluorescence, (Φ_f,) ∼ 0.03, and fluorescence lifetime, (τ_f) ∼ 8 ns, and as a non-fluorescent aggregate. The proportion of monomeric form is higher in organic solvents and micelles but is reduced with increasing porphyrin concentrations in aqueous solutions. Porphyrin c readily complexes with Zn²⁺ to produce a fluorescent chelate (Zn-P c ) with Φ_f, ∼ 0.02 and τ_f, ∼ 2 ns at pH 7.5. The yields of singlet excited oxygen formation from Pc and the Zn-P c complex are higher than observed for hematoporphyrin derivative (HpD). Both P c and Zn-P c are effective agents in tumor phototherapy and do not induce the prolonged cutaneous photosensitivity observed with the use of HpD.     

ENHANCEMENT OF PORPHYRIN-PHOTOSENSITIZATION OF YEAST CELLS BY ETHANOL

Abstract In porphyrin photosensitization, the localization of porphyrin in the cell and the sensitizing activity have been of recent concern. Hydrophobic porphyrins are usually in a highly aggregated state in aqueous systems. This study was designed to see whether the change in the polarity of the environment by adding ethanol could modify the sensitizing effects of porphyrins using a fermentable (alcohol tolerant) yeast ( Saccharomyces cerevisiae ) cells. The results showed that (1) the addition of ethanol (˜15%) to the aqueous suspension remarkably increased inactivation and cell membrane damage both in the hematoporphyrin (HP) and protoporphyrin (PP) photosensitizations, and (2) a sharp induction of genetic changes occurred concomitantly both in HP and PP sensitized cells in the presence of ethanol. In view of the fact that the addition of ethanol modified the absorption spectra and fluorescence intensity of porphyrins in favor of deaggregation, these results may be interpreted to mean that deaggregation of porphyrins promoted by ethanol enhanced their solubility in the lipophilic environment of the cell membrane and even further inside, thereby increasing the sensitizing activities.     

THERMODYNAMICS OF THE BINDING OF HEMATOPORPHYRIN ESTER, A HEMATOPORPHYRIN DERIVATIVE-LIKE PHOTOSENSITIZER, AND ITS COMPONENTS TO HUMAN SERUM ALBUMIN, HUMAN HIGH-DENSITY LIPOPROTEIN AND HUMAN LOW-DENSITY LIPOPROTEIN

The phenomena of the high affinity of porphyrins to the human serum proteins, albumin, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) is well established. Yet, evaluation of the activities of these proteins as endogenous porphyrin carriers, especially with respect to receptor-mediated porphyrin uptake into tumor cells, the merits of which are still in dispute, requires more quantitative protein-porphyrin binding data. As a continuation of previous studies on this issue, the binding of several porphyrin systems to each of the three proteins, employing previously developed spectral methodologies, was studied. The specific systems reported here are hematoporphyrin ester (HPE), which is a novel hematoporphyrin derivative (HPD)-like system, two porphyrin trimers (denoted O1 and O2) and a porphyrin dimer (denoted O3) isolated from HPE. Human serum albumin (HSA) was found to have a single high-affinity site for the monomeric components of HPE, with an equilibrium binding constant of 3.6 × 10⁶. The equilibrium parameters determined for the binding of the three HPE-isolated oligomers to each of the serum proteins are: (1) Binding constants (K_b') of 2.3 × 10⁶, 6.9 × 10⁴ and 1.5 × 10⁴ and number of sites per protein molecule (n) of 3, 1 and 5, for the binding of 01, 02 and 03, respectively, to HSA. (2) K_b’values of 15.5 × 10³, 15.3 × 10³ and 6.6 × 10³ and n values of 1, 2 and 2, for the binding of O1, O2 and O3, respectively, to HDL. (3) K_b’values of 3.3 × 10³, 2.28 × 10⁴ and 8.0 × 10³ and n values of 50, 20 and 16 for the binding of O1, O2 and O3, respectively, to LDL. These data are direct and clear support not only for the high affinity of porphyrins to serum proteins but specifically of stable oligomers that have been assigned critical roles in the photodynamic treatment of tumors. Of the three proteins, LDL is clearly the best camer, providing the highest drug payload with a moderate affinity (enough to bind and not too much to prevent release). These data are suggested to be promising for the postulated role of LDL in porphyrin uptake into tumor cells and to be useful in the future as benchmarks for novel porphyrin systems.     

PROPOSED STRUCTURE OF THE TUMOR-LOCALIZING FRACTION OF HPD (HEMATOPORPHYRIN DERIVATIVE)

Abstract— Synthesis of the tumor-localizing preparation HPD (hematoporphyrin derivative) results in the formation of dimers and oligomers of hematoporphyrin joined by labile linkages. Studies with HPD and an HPD analog containing the chlorin analog of mesoporphyrin suggest the presence of two different linkages, either of which yields a tumor-localizing product. One such linkage is apparently derived from a hematoporphyrin-based oligomer present in commercial preparations of hematoporphyrin as an impurity. The other linkage is formed during the chemical steps leading to the conversion of hematoporphyrin to "HPD".     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.     

EQUILIBRIUM AMONG HEMATOPORPHYRIN-DERIVATIVE COMPONENTS: INFLUENCE OF THE INTERACTION WITH CELLULAR STRUCTURES

Abstract— Hematoporphyrin-derivative (HpD) is a complex mixture of porphyrins in different aggregation states. The spectral analysis of HpD in aqueous solution shows the presence of monomeric species through the fluorescence emission spectrum and of both monomeric and aggregated species through the absorption spectrum.
The interaction with biopolymers and cellular components results in the appearance of new emission bands at 630 nm and in the640–670 nm spectral region which can be evidenced under suitable excitation conditions. Correspondingly, two new decay times (∼ 0.6 ns, and ∼ 3 ns), are observable. The new fluorescent species detected can be considered as the result of the hydrophobic effect induced by cellular structures on porphyrin aggregates.