Leonard I. Grossweiner

SINGLET OXYGEN GENERATION BY PHOTODYNAMIC AGENTS

The singlet oxygen quantum yield of photodynamic agents was measured at 546nm, 630 nm and on the far-red absorption peak. The technique employed is available in most laboratories, in which the photosensitization of lysozyme is used as an internal actinometer. Measurements in a pH 7.4 phosphate buffer plus 1% Triton X-100 (PB/X100) are scaled to 0.52 for methylene blue in the phosphate buffer. The average quantum yields are: hematoporphyrin IX (0.73), protoporphyrin IX (0.56), zinc protoporphyrin IX (0.91), mesotetra-(4-sulfonato-phenyl)porphine (0.61), PhotofrinR (0.89), benzoporphyrin derivative monoacid ring-A (0.84), chlorin e6 in PB (0.64), pheophorbide a (0.69), and aluminum phthalocyanine tetrasulfonate (0.38). Protection factors were measured for added azide ion, 1,4-diazabicyclo[2,2,2]-octane, and superoxide dismutase. Spectral evidence is presented for chlorin e6 interactions with PB/TX100 and for binding to lysozyme.

SINGLET OXYGEN GENERATION BY HEMATOPORPHYRIN IX, UROPORPHYRIN I and HEMATOPORPHYRIN DERIVATIVE AT 546 nm IN PHOSPHATE BUFFER and IN THE PRESENCE OF EGG PHOSPHATIDYLCHOLINE LIPOSOMES

Abstract— The quantum yield of singlet oxygen generation (φ) was measured at 546 nm with the p‐nitrosodimethylaniline (RNO) method. The results obtained in pH 7.4 phosphate buffer (PB) were: φ(HP) = 0.44 ± 0.05: φ= 0.71 ± 0.07: φ(HpD‐A) = 0.06 ± 0.02. The value of φ was constant from 3 to 67 μM, attributed to the dominance of HP dimers; φ (HP) increased to 0.77 ± 0.13 in the presence of small egg phosphatidylcholine liposomes (SUV), attributed to solubilization and monomerization: φ (HpD‐A) increased to 0.87 ± 0.17 in the presence of SUV. attributed to monomerization of the impurity porphyrins and unfolding of the covalent dimer constituents. The quantum efficiency of the RNO system (100 μM RNO plus 10 mM histidine) was approximately 0.015 at pH 7.4 and increased significantly at lower pH.

Photochemistry of proteins: a review.

Proteins are an important target of photochemical damage to the eye. The wavelength of irradiation is a major determinant of the initial mechanisms. The effects of ultraviolet radiation are initiated by absorption in the aromatic amino acid residues and cystine. Photoionization of tyrosine and tryptophan residues leads to aromatic free radicals. The ejected electrons are stabilized in the aqueous medium as hydrated electrons and may be temporarily trapped by cystyl bridges. N-formylkynurenine is an important tryptophan photoproduct, which can act as an endogenous photosensitizer of near-ultraviolet radiation by generating singlet oxygen and superoxide. Singlet oxygen is produced also by exogenous sensitizers absorbing in the visible and near ultraviolet regions. The mechanisms are illustrated by porphyrins and furocoumarins, in which dark binding interactions influence the photosensitization pathways.

Photochemical Generation of the Hydrated Electron

A transient species with properties similar to the hydrated electron produced previously by electron irradiation has been observed by flash photolysis of aqueous solutions of inorganic salts and aromatic compounds including amino acids.

Applications of the 1-D diffusion approximation to the optics of tissues and tissue phantoms.

Optical constants of animal and plant tissues were measured over a wide spectral range using an integrating sphere spectrophotometer. The data were analyzed with two formulations of the 1-D diffusion approximation differing in the phase function. The accuracy of the similarity transformation was examined with tissue phantoms incorporating known chromophores in light scattering media.

Photosensitization of aqueous model systems by hypericin

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.

PHOTODYNAMIC THERAPY OF HEAD and NECK SQUAMOUS CELL CARCINOMA: OPTICAL DOSIMETRY and CLINICAL TRIAL

Abstract This paper reports the retrospective comparison of a PDT dosimetry model with the current results of an ongoing clinical trial on photodynamic therapy (PDT) for head and neck squamous cell carcinoma (HNSCC). The model is based on the assumption that tumor eradication requires a minimum absorption of radiant energy by the tumor‐localized porphyrins. The diffusion approximation was employed to calculate the incident light dose required to attain the minimum absorbed energy density at tumor boundaries most distant from the light source. Dosimetry tables for HNSCC were calculated with estimated tissue parameters, giving the PDT light dose for front surface exposure (FS) and illumination by interstitial cylindrical diffuser fibers (CI) in terms of the tumor dimensions. The model includes a correction for the photobleaching of the localized photosensitizer by the therapeutic light. The PDT trial was carried out on nine patients with previously untreated or recurrent early stage tumors and one patient with a recurrent advanced stage tumor. A complete response was obtained in 83% (10/12) of the sites treated. The calculated doses for FS and CI exposures vary from comparable with to three‐fold lower than the actual doses for each complete response tumor site.

PHOTOSENSITIZED LYSIS OF LIPOSOMES BY HEMATOPORPHYRIN DERIVATIVE

The spectral properties and efficiency for photosensitizing the lysis of phosphatidylcholine liposomes have been measured for the components of hematoporphyrin derivative (Hpd) after alkaline hydrolysis and fractionation by polyacrylamidc gel chromatography. Two major and two minor Hpd fractions have been identified whose spectral properties correlate with the anoxic sensitizing efficiency and the oxygen enhancement ratio (OER). The fastest moving fraction, which is the putative biologically active component, comprised one‐third of the starting material and had OER = 2.7. Liposome lysis by this fraction was inhibited in the presence of human serum albumin at concentration ratios comparable to those employed for photoradiation therapy. The present results show that Hpd can act as an oxic and anoxic photosensitizer of a model biomembrane and suggest that separation from serum proteins is required for in vivo photosensitization.

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.

PDT light dosimetry revisited

A versatile PDT light dosimetry model is described incorporating the effects of drug photobleaching, drug elimination, and normal tissue damage. The dependence of the necrosis depth (dn) on the incident light dose for the four major modes of PDT light delivery has the form: dn = delta loge(DG), where delta is the optical penetration depth of the tumor tissue, D is the ratio of the incident light dose to the energy fluence at the necrosis threshold, and G is a function of the tissue optical constants. Light dosimetry graphs were calculated for Photofrin at standard conditions.