John D. Spikes

Phthalocyanines as photosensitizers in biological systems and for the photodynamic therapy of tumors.

Several photosensitizing porphyrins are selectively retained in solid tumors and other rapidly growing tissues in humans and other mammals. On subsequent illumination with light of wavelengths absorbed by the porphyrin, tumors can be destroyed with relatively little damage to the surrounding normal tissue; it is generally assumed that tumor damage is mediated by singlet oxygen generated by the light excited sensitizer. In clinical practice the patient is injected intravenously with a few mg/kg of the porphyrin. Then, 48-72 h later (an interval necessary to attain the maximum differential concentration of porphyrin in the tumor) the tumor area is illuminated with an appropriate dose of light (25-50 J/cm); typically a dye laser tuned to an absorption peak of the porphyrin is used. The only side effect observed in patients is a skin photosensitivity that persists for a month or so. This photochemotherapeutic procedure, termed photodynamic therapy (PDT)*, has been used on several thousand patients, with generally encouraging results (Dougherty ,1984, 1985). In spite of the utility of the technique, PDT as presently used suffers from some disadvantages. The photosensitizer preparation most generally used is a complex mixture of porphyrins termed hematoporphyrin derivative (HPD). This is prepared by subjecting hematoporphyrin acetates to alkaline hydrolysis. The composition of the mixture varies in different preparations and with time of storage. The most active component has been described as a dihematoporphyrin ether (Dougherty, 1985) or ester (Kessel, 1985); however these are not available as pure chemicals. Red light, usually from a dye laser tuned to 630 nm (corresponding to the longest wavelength absorption peak of HPD), is most commonly used in PDT to permit the maximum depth of

Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins

Cationic porphyrins have been shown to photoinduce the direct inactivation of Gram-positive (G+) and Gram-negative (G-) bacteria, thereby differing from anionic or neutral porphyrins which can photosensitize the G- bacteria only after permeabilization of their outer membrane. The present data show that the differences between these positively and negatively charged porphyrins are not related by a difference in the intrinsic photosensitizing efficiency, as determined by the photo-oxidation of model substrates or the yield of 1O2 generation; moreover, there are only minor differences in the quantum yield of porphyrin photobleaching. Rather, it appears that the positive charge promotes an electrostatic binding of the porphyrin to the outer cell surface inducing an initial limited damage which favours the penetration of the photosensitizer. Actually, the overall photoprocess is inhibited by the preincorporation of the porphyrin into liposomes, while it is enhanced by using amphiphilic dicationic porphyrins which bind to endocellular sites in larger amounts and in a more stable form.

New trends in photobiology: 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.

QUANTUM YIELDS AND KINETICS OF THE PHOTOBLEACHING OF HEMATOPORPHYRIN, PHOTOFRIN II, TETRA(4-SULFONATOPHENYL)-PORPHINE AND UROPORPHYRIN

Abstract

Photodynamic therapy of tumours and other diseases using porphyrins

Photodynamic therapy (PDT) with porphyrins and red light (620–630 nm) is finding increasing clinical application for both the eradication of relatively small tumours and the palliation of inoperable or obstructive tumours. PDT also shows some promise for the sterilization of the tumour bed after surgical removal of neoplastic masses. Several porphyrins have been found to be accumulated and retained by tumour tissues; however, a chemically prepared derivative of haematoporphyrin, termed HpD, and a purified form of HpD, termed DHE (dihaematoporphyrin ether or ester), are most frequently used in clinical practice owing to their optimal tumour-localizing properties and low systemic toxicity in the dark. The efficiency of HpD/DHE photoactivation by red light is very low, since their extinction coefficient at wavelengths above 600 nm is below 103m−1 cm−1. Therefore, a large number of investigations are being performed in order to improve the efficacy of PDT. One approach involves the use of porphyrin analogs (e.g., chlorins, phthalocyanines) which retain a high affinity for tumours and possess intense absorption bands in the red spectral region. Moreover, the selectivity of tumour targeting can be enhanced by transport of the photosensitizing drug with some types of lipoproteins or monoclonal antibodies. These developments are of interest also in view of the proposed extension of PDT to the treatment of other diseases, including viral and microbial infections, atheroma and psoriasis.

Photophysical, photochemical and antibacterial photosensitizing properties of a novel octacationic Zn(II)-phthalocyanine

A novel Zn(II)-phthalocyanine (1). peripherally substituted with four bis(N,N,N-trimethyl)amino-2-propyloxy groups prepared by chemical synthesis is shown to be an efficient photodynamic sensitizer with a quantum yield of 0.6 for singlet oxygen generation in neat water, which is reduced to about 0.3 in phosphate-buffered saline. The physicochemical properties of 1 in both the ground and the electronically excited states strongly depend on the nature of the medium; in particular, aggregation of 1 was favoured by polar media of high ionic strength. Compound 1 exhibited an appreciable affinity for a typical Gram-positive bacterium (Staphylococcus aureus) and a typical Gram-negative bacterium (Escherichia coli). Both bacterial strains were extensively inactivated upon 5 min-irradiation with 675 nm light in the presence of 1 microM photosensitizer, even though the binding of 1 to the two bacterial cells appears to occur according to different pathways. In particular, E. coli cells underwent initial photodamage at the level of specific proteins in the outer wall, thus promoting the penetration of the photosensitizer to the cytoplasmic membrane where some enzymes critical for cell survival were inactivated.

A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and/or light

Three water soluble copolymers based on N-(2-hydroxypropyl)methacrylamide were prepared. Copolymer I contains adriamycin, a chemotherapeutic agent, attached via enzymatically degradable oligopeptide (glycylphenylalanylleucylglycine; G-F-L-G) side chains. The other two copolymers contained the photosensitizer, meso-chlorin e6 monoethylene diamine disodium salt (Mce6). In Copolymer II, the chlorin is attached via the degradable G-F-L-G sequence, and it was bound by the nondegradable glycyl spacer in Copolymer III. Initially, the copolymers were characterized separately in vitro and in vivo. Combinations of the copolymer bound chemotherapeutic agent and each of the copolymer bound photosensitizers were then assessed for antitumor effect in vivo. Localization/retention studies (A/J mice; Neuro 2A neuroblastoma solid tumor) were performed with the two copolymers containing Mce6 as well as the free drug. Results of these experiments demonstrated a very different tumor uptake profile for the two copolymers. While the free drug was rapidly cleared from tumor tissue, the copolymer containing Mce6 attached via the non-degradable bond was retained for an extended period; drug concentrations in the tumor were high even after 5 days. On the other hand, a high concentration of the copolymer containing Mce6 bound via the degradable sequence was taken up by the tumor, yet its concentration in the tumor was substantially diminished at 48 h after administration. This shows indirect evidence of in vivo cleavage of Mce6 from the copolymer in the lysosomal compartment which is supported by direct evidence of cleavage by cathepsin B (a lysosomal enzyme) in vitro. Antitumor effects were assessed on Neuro 2A neuroblastoma induced in A/J mice for all three copolymers. Photodynamic therapy (PDT) proved the copolymer with Mce6 bound via the degradable oligopeptide sequence to be a more effective photosensitizer in vivo than the other chlorin containing copolymer. The difference in activity was consistent with the results obtained by photophysical analyses in which the free drug had a higher quantum yield of singlet oxygen generation than the polymer bound drug in buffer. The quantum yield of singlet oxygen generation increased with the enzymatic cleavage of the chlorin from the copolymer. Conditions were subsequently determined for which chemotherapy or PDT would show some antitumor effect, yet be incapable of curing tumors. Finally, combination therapy experiments were performed in which the copolymer bound adriamycin was mixed with either of the copolymer bound chlorin compounds and injected intravenously (i.v.) into the tail veins of mice.(ABSTRACT TRUNCATED AT 400 WORDS)

FLASH PHOTOLYSIS STUDIES OF HEMATO-AND COPRO-PORPHYRINS IN HOMOGENEOUS AND MICROHETEROGENEOUS AQUEOUS DISPERSIONS

This paper describes an experimental study of the photo properties of the triplet (T,) states of hematoporphyrin (HP) and coproporphyrin (CP), particularly in relation to their medium dependence and reactivity towards oxygen. Triplet‐triplet absorption spectra of HP and CP have been determined in aqueous buffer at pH = 7.4 and in water‐methanol and water‐formamide mixtures. The spectra corrected for ground state contributions show major absorption peaks near 400 nm and lesser peaks near 500 nm. Extinction coefficient measurements have been made and their dependences on solvent composition investigated. Natural lifetimes of the T1 states of HP and CP and the bimolecular quenching constants with oxygen have been determined. The quantum yields of T1 formation are ca. 0.6 in buffer rising to 0.8 and higher in predominantly organic media. Incorporation of the porphyrins into micellar phases similarly causes φT, to increase. Quantum efficiencies of O−2 and O2(lΔg) formation have been determined for HP in buffer, some binary mixtures and micellar dispersions. Superoxide yields are low and may result from photo‐ionization processes. O2(lΔg) yields are large but appear to have an unexpected dependence on the medium.

Photosensitizing properties of mono-l-aspartyl chlorin e6 (NPe6): A candidate sensitizer for the photodynamic therapy of tumors

There is a large amount of interest in chlorins as photosensitizers for the photodynamic therapy of tumors because of their strong absorption in the red, where light penetration into mammalian tissues is efficient. Mono-L-aspartyl chlorin e6 (NPe6), in phosphate buffer of pH 7.4, had absorption peaks at 400 and 654 nm with molar absorption coefficients of 180,000 and 40,000 M-1 cm-1 respectively. In buffer, the NPe6 triplet had a peak at 440 nm and a lifetime under argon of approximately 300 microseconds. The triplet was efficiently quenched by ground state oxygen (kQ = 1.9 x 10(9) M-1 s-1) with the formation of singlet oxygen, as identified by its near infrared luminescence. The quantum yield of singlet oxygen production was 0.77. A number of substrates were efficiently photo-oxidized by NPe6, including furfuryl alcohol, cysteine, histidine, tryptophan and human serum albumin. These reactions were efficiently inhibited by azide (which did not quench NPe6 triplets), indicating that they are probably mediated by singlet oxygen. Thus, NPe6 has a desirable array of photoproperties for a sensitizer to be used in the clinical photodynamic therapy of tumors.

Photodynamic crosslinking of proteins. III. Kinetics of the FMN- and rose bengal-sensitized photooxidation and intermolecular crosslinking of model tyrosine-containing N-(2-hydroxypropyl)methacrylamide copolymers.

Abstract. As part of a study on the role of Tyr residues in the photosensitized intermolecular crosslinking of proteins, we have surveyed the kinetics of the rose bengal‐ and flavin mononucleotide (FMN)‐sensitized photooxidation and crosslinking of a water‐soluble N‐(2‐hydroxypro‐pyl)methacrylamide copolymer with attached 6‐carbon side chains terminating in tyrosinamide groups (thus the ‐OH group of the Tyr is free, but both the amino and carboxyl groups are blocked, simulating the situation of a nonterminal Tyr in a protein). The intermolecular photodynamic crosslinking of the Tyr copolymer can result only from the formation of Tyr‐Tyr (dityrosine) bonds, because the copolymer itself is not photooxidizable. Rose bengal, primarily a Type II (singlet oxygen) sensitizer, sensitized the rapid photooxidation of the Tyr residue in the Tyr copolymer only at high pH, where the Tyr phenolic group is ionized; crosslinking did not occur with rose bengal under any of the reaction conditions used. In contrast, FMN, which can sensitize by both Type I (free radical) and Type II processes, sensitized the photooxidation of the Tyr copolymer over the pH range 4–9.5. Also, significant photocrosslinking occurred, but only from pH 4 to 8, with a maximum rate at pH 6. Crosslinking required the presence of oxygen. Studies with inhibitors, D2O as solvent, cataiase and superoxide dismutase indicated that the photooxidation and photocrosslinking of the Tyr copolymer with FMN at pH 6 were not mediated by singlet oxygen, superoxide or hydrogen peroxide. It appears that crosslinking involves the abstraction of an H atom from the Tyr phenolic group to give Tyr and FMN radicals. The Tyr radical in one Tyr copolymer can then react with a Tyr radical in another Tyr copolymer to give an intermolecular dityrosine crosslink.