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.     

UPTAKE AND RETENTION OF PHOTOFRIN® BY CULTIVATED HUMAN LYMPHOBLASTIC CELLS (Reh6): PREFERENTIAL AFFINITY OF THE CELLS FOR A MINOR COMPONENT DEMONSTRATED BY NORMAL PHASE CHROMATOGRAPHY

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.     

PHOTOFRIN ACCUMULATION IN MALIGNANT AND HOST CELL POPULATIONS OF A MURINE FIBROSARCOMA

Abstract— Photofrin (25 mg/kg) was administered to the FsaR fibrosarcoma-bearing mice (either syngeneic or severe combined immunodeficient [SOD]) and the tumors were excised 24 h later. The photosensitizer content in the cells dissociated from tumor tissue was analyzed using flow cytometry. Staining the cell suspensions with the monoclonal antibodies against specific membrane markers served to identify the malignant cells and various types of host immune cells infiltrating the tumor. Photofrin content was also examined in the cells from normal tissues of the tumor-bearing mice (spleen, heart muscle, peritoneal macrophages). The results show a marked heterogeneity in the Photofrin cellular content of FsaR tumor, particularly within the population of tumor-associated macrophages (TAM). The Photofrin levels in some TAM were lower or similar to those in the malignant cells. In contrast, a subpopulation of TAM accumulated very high levels of the photosensitizer, which exceeded by far the levels found in the other tumor cell populations. This TAM fraction was characterized by particularly high expression of interleukin-2 receptors and increased cell size and granularity when compared to the other TAM, which suggests that these macrophages are in the activated state. Their average Photofrin content was almost 13 times higher than in the malignant cells. The lowest photosensitizer levels in the tumor were found in tumor-infiltrating leukocytes other than TAM. In FsaR tumors growing in SCID mice, the pattern of Photofrin distribution in TAM and other cellular populations was similar to that found in tumors growing in syngeneic mice. Due to a presumably better perfusion, these tumors accumulated higher levels of Photofrin in all cellular populations. The findings of this study suggest that the tumor-localizing effect of Photofrin can be attributed to the accumulation of extremely high levels of the photosensitizer in a subpopulation of TAM.     

ACTIVITY AND PHYSICOCHEMICAL PROPERTIES OF PHOTOFRIN®

Upon certain conditions of storage, Photofrin samples changed their photodynamic therapy activity in mice. These samples showed similar absorption and high performance liquid chromatography (HPLC) profiles. The Photofrin samples that were active, however, had higher relative fluorescence yields than those which failed. Active and inactive samples also had different characteristics when analyzed by gel filtration chromatography using Sephacryl S-300; the failed samples had 60% or more high-molecular-weight fraction. The change of the amount of this high-molecular-weight fraction with the time of storage at room temperature could be clearly demonstrated. Results from fluorescence, HPLC and mass spectrometry studies suggest that the high-molecular-weight fraction may form by dehydration from or condensation between various components of Photofrin.     

BINDING OF HEMATOPORPHYRIN DERIVATIVE TO HUMAN SERUM ALBUMIN

Abstract Dialysis of hematoporphyrin derivative fraction A (HpD-A) off human serum albumin at 38°C followed the Hill equation for cooperative binding with saturation at 5 to 8. 600 dalton porphyrin units. Approximately 15% of the HpD-A was free for concentrations typical of human serum in photoradiation therapy. Possible structures of the tumor-localizing and -photosensitizing component in HpD-A are considered. Of these, a folded-over, covalent dimer appears to be more consistent with the photophvsical properties.     

Chlorins as photosensitizers in biology and medicine

The photodynamic therapy (PDT) of tumors involves illumination of the tumorous area following the administration of a tumor-localizing photodynamic sensitizer. Hematoporphyrin derivative (HPD) and Photofrin II (a purified form of HPD), the main sensitizers used clinically for PDT to date, are complex mixtures of porphyrins; furthermore, these preparations absorb light very poorly in the red region of the spectrum (wavelengths greater than 600 nm) where light penetration into mammalian tissues is greatest. Thus there is considerable interest in identifying new sensitizers that localize more effectively in tumors, absorb more strongly at longer wavelengths and can be prepared in high purity. Much of this interest has been directed towards chlorins (reduced porphyrins), which typically absorb strongly in the red. This review summarizes research that has been carried out on selected types of chlorins, some of which may have important applications as sensitizers for PDT.     

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.     

In vitro UPTAKE OF DICARBOXYLIC PORPHYRINS BY HUMAN ATHEROMA. KINETIC and ANALYTICAL STUDIES

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.     

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".     

Apoptosis or Necrosis Following Photofrin® Photosensitization: Influence of the Incubation Protocol

Photosensitization using the tumor-localizing porphyrin Photofrin® induces cell death both in vitro and in vivo, but the mechanism of cell death is not well understood. Cell lysis (necrosis) and apoptosis have both been observed. The latter seems restricted mainly to lymphoma and epithelial cell lines. To check the influence of the incubation protocol on the cell death mechanism, CV-1 cells were loaded with Photofrin using two different protocols. In both protocols, photosensitized CV-1 cells underwent severe morphological changes before cell death. Many cells treated with protocol 1 (24 h with 1 μ g/mL of Photofrin in culture medium) underwent apoptosis, as demonstrated by plasma membrane blebbing and fragmentation into vesicles, condensation of the chromatin and fragmentation of the nucleus with oligonucleosomic degradation of the DNA. In contrast, cells treated with protocol 2 (1 h with 10 μg/mL of Photofrin in phosphatebuffered saline) lysed instead of fragmented, without oligonucleosomic degradation of the DNA. This type of cell death looks much like necrosis. However, early morphological changes suggest that it is, in fact, apoptosis stopped by plasma membrane leakage. It is concluded that apoptosis is primarily induced in CV-1 cells but may be arrested by membrane lysis, depending on the incubation protocol.     

Purification and analysis of hematoporphyrin and hematoporphyrin derivative by gel exclusion and reverse-phase chromatography

A ternary-solvent HPLC method for analysis of the components of the tumor-localizing product HPD is described. This HPLC system was used to assess the utility of a new method, based on gel exclusion chromatography, for purification of hematoporphyrin.     

IN VIVO FLUORESCENCE OF TUMORS AFTER TREATMENT WITH DERIVATIVES OF HEMATOPORPHYRIN

Abstract— Administration of a mixture of porphyrins termed HPD (hematoporphyrin derivative) to mice bearing the Lewis lung tumor leads to preferential accumulation of fluorescence at tumor loci in vivo after 48 h. HPLC analysis shows that the fluorescent species consist of hematoporphyrin and its dehydration products. But injection of these porphyrins does not lead to fluorescence localization. The intracellular fluorescence which is observed apparently arises from intracellular degradation of the tumor-localizing component of HPD. These fluorescent species represent only a small fraction of the total accumulated porphyrin pool; a larger weakly-fluorescent porphyrin pool is also present, and may be the major factor in tumor photosensitization.     

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.     

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.     

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.     

A rapid method for isolation and purification of an anticoagulant from Whitmania pigra

Whitmania pigra is common in China and has been used as a traditional Chinese anticoagulant medicine for years, but its effective components are unknown to scientists. In this article we report a rapid method for isolation and purification of an anticoagulant from W. pigra for the first time. An acetone-water extract of W. pigra was subjected to anion-exchange chromatography on a Sephadex DEAE A-50 column, and gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns successively, which afforded a fraction with potent anticoagulant activity. An anticoagulant was isolated and purified from this fraction by reversed-phase high-performance liquid chromatography (RP-HPLC). It was identified as a single pure substance by RP-HPLC and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This component was named whitmanin and its molecular weight was determined as 8608 Da by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS).     

EQUILIBRIUM AMONG HEMATOPORPHYRIN DERIVATIVE COMPONENTSs—II. EFFECT OF ESTERASE ACTIVITY

Photofrin II is the hematoporphyrin-derivative fraction enriched in covalently-linked oligomers, characterized by a high degree of folding. Interaction with hydrophobic structures, such as biomolecules and cell structures, results in a modification of the equilibria among the different species, as a consequence of an unfolding effect exerted towards the electrostatic aggregates. The effect of esterase activity was evaluated, taking into account the nature suggested for the covalent linkage of the oligomers (ether and/or ester). The study was performed in Photofrin II aqueous solution by means of absorption and fluorescence spectral analysis. The results showed that the esterase is active only towards the unfold oligomers: that is, in Photofrin II solution supplemented with albumin. In these conditions, spectral analysis revealed the presence of a monomerization process, which is clearly evident during the first four hours of incubation. The monomerization effect induced by the enzyme was also proven by both equilibrium-dialysis measurements and zinc ion complexation. Zinc ion complexes with high affinity for monomeric species, giving rise to a very distinct emission band at 580 nm. The amount of ester linkage shown in the oligomers through enzyme hydrolysis appeared to be less than might have been expected, owing to the inhibiting effect of the monomer produced on the enzyme. The results are a step toward clarifying the intracellular and intratissue turnover of the drug observed after administration.     

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.     

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 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.