Hydroxyethylvinyldeuteroporphyrin Research Articles

Chlorin-Type Photosensitizers Photochemically Derived from Vinyl Porphyrins¶

The reaction of singlet oxygen with four vinyl-substituted dicarboxylic porphyrins, vinyldeuteroporphyrin (VD), ethylvinyldeuteroporphyrin (EVD), hydroxyethylvinyldeuteroporphyrin (HVD) and protoporphyrin (PP) in organic solutions is investigated. The main products, the “hydroxyaldehyde” chlorin-type derivatives, are formed with a concentration-dependent photochemical quantum yield that reaches a maximum of 7.4 ± 1.6 × 10−3. However, owing to the high turnover of singlet-oxygen production, these chlorin-type compounds are easily prepared photochemically with a chemical yield of 70% and little side product formation. In chemical ionization mass spectrometry, these compounds display an unusual fragmentation with a loss of 16 mass units. This is attributed to the loss of the oxygen bound to the saturated carbon of the modified pyrrole unit. All these compounds sensitize the formation of singlet oxygen with a yield around 0.8. They interact with singlet oxygen with rate constants of 5 × 106–9 × 106M−1 s−1, lower than those measured for vinyl porphyrins. These data are likely to help in the characterization of photoproducts of vinyl porphyrins relevant to photodynamic therapy (PP, HVD). As exemplified with VD and EVD, they also point out the reaction of singlet oxygen as an efficient route to chlorin-type photosensitizers.

Preparation of Photodynamic Hydrolyzates from Bovine Hemoglobin

Porphyric peptides were prepared from an agricultural byproduct, bovine hemoglobin. Hemoglobin was hydrolyzed with alcalase. After removal of heme iron, the total hydrolysate was separated on gel filtration. It consisted of three fractions; the major one was separated by reversed-phase performance chromatography. FAB analysis indicated that protoporphyrin, hematoporphyrin, and hydroxyethylvinyl deuteroporphyrin were the major porphyrin constituents. These porphyrins were maintained in solution by peptidic hydrolysates. The hydrophilic and photosensitizing properties of porphyrin-peptide complexes were demonstrated. The water-solubilizing ability of hydrolysates was significant and allowed amphiphilic porphyrins to be obtained

Capillary electrophoresis analysis of polyhaematoporphyrin, a photosensitizer used in photodynamic therapy

Free solution capillary electrophoresis and micellar electrokinetic capillary chromatography are used to separate components in polyhaematoporphyrin (PHP), a preparation similar to Photofrin which is used as a photosensitizing agent in photodynamic therapy. PHP is known to contain haematoporphyrin (HP), protoporphyrin (PP), hydroxyethylvinyldeuteroporphyrin (HVD) and a mixture of ester, ether and carbon-carbon linked oligomers. The use of reference samples of HP and PP, together with partially dehydrated HP, has allowed assignment of HP and HVD peaks in the electropherograms of PHP in 20 m M phosphate pH 6.9 and 20 m M disodium tetraborate pH 9.2 buffers using sodium dodecyl sulphate in the range 0–100 m M. Changes in the pattern and areas of monomer peaks and oligomer distribution following base hydrolysis (which cleaves ester linkages) and acid hydrolysis (which cleaves ester and ether linkages) yields information consistent with that from HPLC studies.

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.

Binding of porphyrin to human serum albumin. Structure–activity relationships

The equilibrium binding of hydroxyethyl vinyl deuteroporphyrin (HVD) and of irreversible porphyrin aggregates to human serum albumin was studied at the molecular level. This protein may function as an endogenous drug carrier for porphyrins in photodynamic therapy of tumours. HVD-protein binding studies revealed two types of binding sites, which are attributed to the two HVD isomers. The binding constant for the high-affinity isomer, 2.1 (+/- 0.3) x 10(8) M-1, is similar to that previously determined for protoporphyrin. At the same time the binding constant for the lower-affinity HVD isomer, 1.8(+/- 0.3) x 10(6) M-1, is similar to that previously determined for haematoporphyrin. Irreversible porphyrin aggregates were purified from the haematoporphyrin derivative and from Photofrin and are defined by spectral and chromatographic data. Gel-exclusion studies indicate that the dominant size of these aggregates is ten porphyrin monomeric units. The protein-binding constant of these aggregates is 1.7(+/- 0.2) x 10(5) M-1, with four binding sites per protein molecule. The distinction between the HVD isomers along the porphyrin-protein affinity sequence gives insight into the relationship of porphyrin structure to porphyrin-albumin binding. On the basis of this study an evaluation of human serum albumin as an endogenous carrier for porphyrins (at various aggregation states) in photodynamic therapy of tumours is presented.

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.

Protection of NIH 3T3 Cells from Infection by Trypomastigotes and Sphaeromastigotes of Trypanosoma cruzi, Telahuen Strain, by Porphyrins in the Presence and Absence of Light (630 and 690 NM)

The light and dark toxicities of naturally occurring and synthesized porphyrins were investigated for their potential to protect NIH 3T3 cells from infection with mammalian tissue culture-derived trypomastigotes of Trypanosoma cruzi. The fluorescent nucleic acid dye Hoechst 33342 was employed to trace T. cruzi intracellular development following porphyrin exposure with and without irradiation. The synthetic porphyrins investigated included isomers A and B of mono- and diacid benzoporphyrin (BPD-MA, BPD-DA, BPD-MB, and BPD-DB). derivatives of naturally occurring hematoporphyrin dihematoporphyrin ether (DHE), and hydroxyethylvinyl deuteroporphyrin (HEVD). These porphyrins provided positive protection following irradiation with light Protection also was obtained due to dark toxicity phenomena. HEVD at a concentration of 1.0 micrograms/ml followed by irradiation of 20 J/cm2 of light at 630 nm provided 90% protection, whereas BPD-MA and BPD-MB provided greater than 85% protection using irradiation at 690 nm. Dark protection was greatest at 1.0 micrograms/ml with the BPD (55-70%), but HEVD provided the greatest toxicity (89%) at a concentration of 10 micrograms/ml. Further investigations are in progress to find methods to increase the protection obtained and to determine the mechanism(s) involved in protection.

Cellular uptake of hydroxyethylvinyldeuteroporphyrin (HVD) and photoinactivation of cultivated human leukemia (REH6) cells.

Abstract— The in vitro incorporation of purified hydroxyethylvinyldeuteroporphyrin (HVD) into cells (Reh6) derived from an acute lymphocytic human leukemia is investigated using quantitative extraction techniques and fluorescence spectroscopy. A fast incorporation step (< 2 min) is characterized by its dependence on the porphyrin concentration in the incubation medium which suggests a saturation process. It is followed by a slower uptake, the rate of which linearly depends on the porphyrin concentration. No preferential uptake of aggregated form of HVD, which is shown to dimerize with an equilibrium constant of 9.7 × 105M−1, can be evidenced. As inferred from fluorescence spectra of cell suspensions and those of HVD dissolved in aqueous and micellar solutions as references, the porphyrin is mainly located in membrane structures and to a lower extent in cytoplasm. Cell photoinactivation does not depend on the incubation time but is only related to the intracellular porphyrin concentration.