Phototherapeutic Agent Research Articles

Photodynamic action of a water-soluble hypocrellin derivative with enhanced absorptivity in the phototherapeutic window II. Photosensitized generation of active oxygen species

Cysteine-substituted hypocrellin B (Cys-HB) is a water-soluble perylenequinonoid derivative with significantly enhanced absorptivity in the range of wavelength longer than 600 nm. Electron paramagnetic resonance (EPR) measurements, quenching experiments and 9,10-diphenyl-anthracene bleaching studies were used to investigate the photodynamic action of Cys-HB in the presence of oxygen. Illumination of Cys-HB solution, in the presence of oxygen, generated singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. The accumulation of active oxygen species was transformed into that of the semiquinone anion radical with the depletion of oxygen, detected by the spin counteraction of TEMPO radical formed via the reaction of TEMP with singlet oxygen produced by Cys-HB photosensitization. Oxygen content, Cys-HB concentration and reaction environment affected the transformation and the competition between the Type I and Type II reactions. Compared with hypocrellin B (HB), Cys-HB primarily remained similar and slightly lower capability of active oxygen-generation, confirmed to be a favorable phototherapeutic agent.

Synthesis of a new water-soluble phototherapeutic sensitizer from hypocrellin B with enhanced red absorption

Hypocrellins are new photodynamic agents. To improve the red absorption and water-solubility of these new type photosensitizers, we have synthesized a new hypocrellin derivative, i.e. di-cysteine substituted hypocrellin B (DCHB), using the photoreaction of hypocrellin B with cysteine in an aerated ethanol-buffer (1:3 by volume, pH>10) solution. Thiylation, followed by amination, afforded this new compound. The yield of DCHB is significantly influenced by the oxygen content, the pH value and the solvent used in the reaction. Compound DCHB is completely water-soluble, and its longer absorption band shifts to 684 nm, failing in the domain of the phototherapeutic window (600–900 nm). Moreover, DCHB is photodynamically active in terms of both the type I and type II mechanisms. These observations confirm that DCHB is a promising and potential phototherapeutic agent. ©

ESR and UV-Vis studies of semiquinone radical anion and hydroquinone generated by irradiation of 15-Deacetyl-13-glycine substituted hypocrellin B

15-Deacetyl-13-glycine substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with enhanced red absorption longer than 600 nm and water solubility. When an anaerobic DMSO or DMSO-buffer (pH 7.4) solution of GDHB was illuminated with > 470 nm light, a strong electron spin resonance (ESR) signal was formed. The ESR signal was assigned to the semiquinone anion radical of GDHB (GDHB·-) based on a series of experiments. GDHB·- was predominantly photoproducted via the self-electron transfer between the excited- and ground-state species. Decay of this species, both in the presence and absence of electron donor, was consistent with second-order kinetics. In aqueous solution, the TEMPO counterspin experiment indicated the formation of GDHB·- that could not be detected by ESR method directly. The formation of GDHB·- and hydroquinone of GDHB (GDHBH·-) was also confirmed by spectrometric method. These findings suggested that GDHB was at least a favorable type I phototherapeutic agent.

Photodynamic action of a water-soluble hypocrellin derivative with enhanced absorptivity in the phototherapeutic window I: ESR and UV-Vis studies on the photogeneration of the semiquinone anion radical and hydroquinone

Cysteine-substituted hypocrellin B (Cys-HB) is a hypocrellin derivative with enhanced water solubility and absorption in the domain of phototherapeutic window (600–900 nm). Illumination of Cys-HB in DMSO or DMSO-buffer (pH 8.0) generated a strong Electron Spin Resonance (ESR) signal. The ESR signal was assigned to the semiquinone anion radical of Cys-HB (Cys-HB •−) based on a series of experimental results. Decay of Cys-HB •− in the presence and absence of electron donors, were both consistent with second-order kinetics. Spin counteraction of TEMPO by Cys-HB photosensitization indicated the formation of Cys-HB •− which couldn't be detected by ESR method directly in aqueous solution or acidic media. Spectrophotometric measurements showed that the absorption bands at 623 nm, and 504 nm (pH 8.0) or 520 nm (pH 11.0) were those of Cys-HB •− and hydroquinone of Cys-HB (Cys-HBH 2), respectively. The kinetical results of Cys-HB •− decay were in agreement with those by ESR measurements. Compared with hypocrellin B (HB), Cys-HB •− was more stable and less susceptible in strong alkaline media. Strong intramolecular hydrogen bonding was considered to be present in the chromophore of CysHB •−. The results in this study showed that Cys-HB was at least a favorable Type I phototherapeutic agent.

Interaction between hypocrellin A and some biological substrates with emphasis on an electron transfer mechanism

Hypocrellin A (HA) is an efficient phototherapeutic agent. Laser flash photolysis was used to produce and investigate the properties of the lowest excited triplet state (T 1) and the semiquinone radical anion of hypocrellin A (HA •−) at room temperature. In the presence of biological substrates, such as ascorbic acid and cysteine, the formation and decay of HA •−, attributed to electron transfer between HA and the substrates, were observed. Production of the superoxide radical anion (O 2 •−) by photoactivated HA in the presence of biological substrates was examined by using the Nitro Blue Tetrazolium (NBT) trapping method in order to elucidate the mechanism of formation of superoxide and to quantify this formation. Specifically, production of O 2 •− was demonstrated unequivocally by reaction with superoxide dismutase. In addition, in weak acidic solution, HA •− could also be produced and observed by laser flash photolysis, but it decays much faster than that in neutral or basic solution. The values of the free energy change for electron transfer between HA* and some biological substrates are less than zero, indicating that this process is permissible thermodynamically. Based on the experimental results, an electron transfer (Type I) mechanism may play a hitherto unrecognized role in the photodynamic interaction between HA and some biological substrates.

Electron transfer interaction between hypocrellin A and biological substrates and quantitative analysis of superoxide anion radicals

Hypocrellin A (HA), a hydroxyperylenequinone derivative, is an efficient phototherapeutic agent. Laser flash photolysis is used to produce and investigate the properties of the lowest excited triplet state (T1) and semiquinone radical anion of hypocrellin A (HA˙–) at room temperature. In the presence of some biological substrates (such as ascorbic acid and cysteine), the formation and decay of HA radical anion at different pH, attributed to the electron transfer between triplet HA and substrates, are observed. Meanwhile, the superoxide radical anion (O2;˙–) production by photoactivated HA in the presence of biological substrates is examined by using the nitro blue tetrazolium (NBT) trapping method in order to elucidate the mechanism of formation of superoxide and to quantify this formation. Specifically, production of O2;˙– is demonstrated unequivocally to be by reaction with the superoxide dismutase. The rate of reduction of NBT is dependent not only on the concentration of NBT but also on the pH of the system. The relative quantum yield of superoxide anion radicals increases considerably in alkaline solution: ϕO2;˙– = 6.97 × 10–3 at pH 9.0 as compared with ϕO2;˙– = 2.49 × 10–4 at pH 5.8 in the presence of ascorbic acid. Based on the experimental results, electron transfer (Type I) mechanism may play a hitherto unrecognized role in the photodynamic interaction between HA and some biological substrates.

Photophysics and intracellular distribution of a boronated porphyrin phototherapeutic agent.

The absorption and emission spectra, fluorescence quantum yields and lifetimes and triplet state properties of a boronated porphyrin, the tetrakiscarborane carboxylate ester of 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX (BOPP), have been determined. This compound is an alternative photodynamic therapy (PDT) agent that exhibits highly selective tumor localization, with the potential to be used in conjunction with boron neutron capture therapy. The photophysical characteristics of BOPP are similar to other porphyrins and it exhibits marked aggregation and acid-base speciation under typical physiological conditions. In particular, protonation of the porphyrin imino (-N=) nitrogens occurs in the pH 5-7 region and influences the photophysical properties. Time-resolved confocal fluorescence imaging of the intracellular distribution of BOPP in C6 glioma cells indicates distinct subcellular localization and heterogeneity of emission. The results are interpreted and discussed in terms of the possible mechanisms for cellular uptake and localization.

Pertussis toxin reverses the inhibition of the adenylyl cyclase system by khellin in HeLa cells

Khellin, a furochromone structurally related to furocoumarins, is a phototherapeutic agent used against psoriasis and vitiligo. This paper reports that pretreatment of HeLa cells with pertussis toxin reverses both the inhibition of NaF-stimulated adenylyl cyclase activity and the stimulation of GTPase by khellin alone and plus UVA light, as previously reported. Our results demonstrate, for the first time, the khellin is able to elicit an intracellular response by Giα protein.

Characteristics of the reaction between semiquinone radical anion of hypocrellin A and oxygen in aprotic media

Hypocrellin A (HA), a hydroxylperylenequinone derivative, is an efficient phototherapeutic agent. The reactivity of the semiquinone radical anion of HA (HA −) with O 2 in aprotic medium has been studied. Although the standard redox potential of the HA −HA couple (−360 mV vs. a normal hydrogen electrode (NHE)) is less negative than that of the O 2 −O 2 couple (−500 mV vs. NHE), it appears that HA − can still be oxidized with oxygen. The electron transfer is accompanied by a proton transfer between one of the phenolic hydroxyl groups of HA and O 2 − yielding the conjugated base of HA (HA b −) and hydrogen peroxide. On the basis of the results obtained from spectrophotometric, electron spin resonance spin-trapping and CCl 4-inhibiting experiments, a reasonable reaction pathway was proposed for the oxidation of HA − with O 2 in aprotic media.

Spectroscopic study on the photoreduction of hypocrellin A: generation of semiquinone radical anion and hydroquinone

Hypocrellin A (HA) is an efficient phototherapeutic agent. Illumination of HA in dimethylsulphoxide (DMSO) or DMSO-buffer (1:1 by volume, pH > 6.7) generated a strong electron paramagnetic resonance (EPR) signal. This EPR signal was intensified in the presence of reductants. The EPR spectrum obtained was assigned to the semiquinone radical anion of HA (HA .−) based on a series of experimental results. Decay of HA .−, attributed to a radical-radical reaction, follows second-order kinetics. In acidic DMSO-buffer (1:1 by volume) solution, no EPR signal of HA .− was detected in the absence or presence of reductant. This was explained by a fast disproportionation of radicals, facilitated by protonation of radicals. The spectrophotometric measurements indicated that on illumination HA was directly reduced to its two-electron reduction product, i.e. hydroquinone, in acidic solution. The absorption maximum of the hydroquinone of HA which is at 496 nm at pH 5.8 shifts bathochromically with increase in pH of the medium. In DMSO or DMSO-buffer (1:1 by volume, pH > 6.7) solutions the semiquinone radical anion of HA was also observed spectrophotometrically. The absorption maximum of HA .− is at around 628 nm. Strong intramolecular hydrogen bonding was considered to exist in the chromophore of HA .−.

Studies on the chelation of hypocrellin A with aluminium ion and the photodynamic action of the resulting complex

Hypocrellin A (HA), a perylenoquinone derivative, is an efficient phototherapeutic agent. The chelation of HA with aluminium ion (Al 3+) results in the formation of a 1:1 complex (Al 3+-HA). The probable structure for this complex has been proposed. Compared with HA, this complex has a much higher solubility in water. In addition, Al 3+-HA preserves the 1O 2-generating function of HA as demonstrated by diphenylanthracene photobleaching experiments. In irradiated oxygen-saturated solutions of Al 3+-HA superoxide radical anion and hydroxyl radical are formed as verified using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the electron spin resonance (ESR) spin trapping agent. The production of superoxide radical anion is enhanced significantly in the presence of electron donors such as 1-benzyl-1,4-dihydronicotinamide (BNAH). These results suggest that the photodynamic action of Al 3+-HA proceeds via both type 1 and type 2 reactions.

Studies of the triplet state properties of hypocrellin A by nanosecond flash photolysis

Abstract Hypocrellin A (HA) is an efficient phototherapeutic agent. Its triplet properties have been investigated in cyclohexane using the nanosecond flash photolysis technique. The triplet—triplet (T—T) absorption spectrum of HA gives three bands with absorption maxima at around 520, 555 and 600 nm. The triplet energy level for HA has been estimated to be about 42.5 kcal mol −1 by the energy transfer method. The extinction coefficients for the T—T absorption of HA have been determined by the ground state depletion method. The triplet quantum yield of HA is 0.78±0.04 (by reference to benzophenone).

Studies of the sulfonation of hypocrellin A and the photodynamic actions of the product

Hypocrellin A (HA), a perylenoquinone derivative, is an efficient phototherapeutic agent. The reaction of HA with Na 2SO 3 in alkaline solution under reflux led to the formation of the sodium salt of 14-dehydroxy-15-deacetyl-hypocrellin A-13-sulfonate (13-SO 3Na-DDHA). ESR studies in combination with chemical methods showed that 13-SO 3Na-DDHA was an active photosensitizer. An ESR spectrum, assigned to the semiquinone radical anion of 13-SO 3Na-DDHA was generated by illumination of a DMF buffer (1:1 by volume) solution of 13-SO 3Na-DDHA in the presence of a series of reductive biomaterials. Using 13-SO 3Na-DDHA as photosensitizer and 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) as a spin trap, the ESR spectra of DMPO/S̊O 3 − radical adduct, ascorbyl radical and DMPO/O 2 ·− radical adduct were obtained. Singlet oxygen formed from 13-SO 3Na-DDHA photosensitization was detected by means of the 13-SO 3Na-DDHA photosensitized oxidation of anthracene and ESR spectroscopy. It was therefore concluded that the photodynamic actions of 13-SO 3Na-DDHA proceeded via both Type 1 and Type 2 reactions.

Steady state and time-resolved spectroscopic studies on zinc(II) phthalocyanine in liposomes

Zinc(II) phthalocyanine (ZnPc), a potential second-generation phototherapeutic agent for tumours, has been incorporated into small unilamellar vesicles (SUVs) (diameter, 52 nm) and large unilamellar vesicles (LUVs) (diameter, 84 nm) of dipalmitoyl-phosphatidylcholine (DPPC). Absorption spectroscopy, as well as steady state and time-resolved fluorescence emission studies, indicate that ZnPc is monomeric in SUVs at a stoichiometric concentration below 0.25 μM (corresponding to an actual endoliposomal concentration of about 0.5 mM), while in LUVs it is monomeric below 2 μM. The fluorescence lifetime of the monomer is 3–3.5 ns. Upon increasing the ZnPc concentration, aggregated derivatives are formed, which are characterized by shorter fluorescence lifetimes (1.2–1.5 ns; 0.4–0.6 ns). The possible implications of these observations for the phototherapeutic efficiency of ZnPc are briefly discussed.

A New Porphyrin Photosensitizer (PH1008) in Model Membranes, Normal Cells, and Bladder Cancer Cells

Newer photosensitizers continue to be sought for photodynamic therapy of bladder cancer particularly since local rather than systemic application is desired. Recent studies have indicated that a cationic dye, PH1008, a 13,17-N,N,N-dimethylethylethanolamine ester of protoporphyrin, sensitizes the photolysis of red blood cells. The study described in this report was designed to investigate the plasma membrane partitioning of PH1008 model lipid system and to compare partitioning of PH1008 in normal transitional cells and bladder cancer cells in vitro.Partition coefficient (Kp) values characterizing the distribution of PH1008 between aqueous buffer and normal and malignant transitional cells were 3.4 ± 0.7 × 104 (CRL-7881) and 9.5 ± 1.4 × 104 (HTB-9), resulting in a 20% difference in membrane photosensitizer concentration at a particular photosensitizer concentration. Significantly higher (2-5 fold) differences are observed between tumor and surrounding normal tissue for systemically delivered photofrin II. Cell-bound drug was 30-fold (CRL-7881) and 80-fold (HTB-9) more fluorescent when compared to aqueous buffer. The combined effects of partitioning and bound fluorescence suggest that a 3.2-fold increase in fluorescence of transformed vs. normal bladder urothelium exists. This difference in fluorescence suggests that PH1008 might be more useful as a diagnostic tool than as a phototherapeutic agent.

Factors controlling the selectivity and efficiency of tumour damage in photodynamic therapy

The scope and potential of the photodynamic therapy of tumors can be enhanced through an adequate control of the factors which improve the selectivity of tumour targeting by the systemically injected photosensitizer and increase the efficiency of photosensitized tumour damage. Promising results are obtained by using hydrophobic photosensitizers which can be specifically transported and released to the tumour by serum lipoproteins, especially low-density lipoproteins. The photosensitizer molecule should possess those structural features which induce a high probability of photoactivation by 700–800 nm light, as well as a high yield of long-lived triplet state. The use of liposome-delivered Zn-phthalocyanine as a second generation phototherapeutic agent for tumours is proposed.

PHOTOCHEMISTRY OF HYPOCRELLIN A (V)——PHOTOREACTION OF HYPOCRELLIN A AND MERCAPTOETHANOL

I.INTRODUCTION Hypocrellin A(abbreviated HA) from Hypocrellia bambusae (B. ct Br.)sacc. growing in Yunnan Province, China, is an efficient phototherapeutic agent. Recently HA irradiation with visible light has been successfully used for the treatment of white lesions of vulva and vitiligo. In molecular level, studies on phototherapeutic mechanism have been focused on the structure, photophysical and photochemical properties of HA and its derivatives. The photoreactions, which are closely related to phototherapeutic activities, have not been re-

Accumulation of zinc-phthalocyanine (Zn-Pc) in the aorta of atherosclerotic rabbits

Phthalocyanines are structural analogs of porphyrins and are characterized by a closely similar mechanism of transport in the bloodstream. We evaluated the accumulation of liposomebound Zn(II)-phthalocyanine (Zn-Pc) by atherosclerotic aortas in rabbits. The biodistribution of intravenously injected Zn-Pc (0.023 mg/kg body weight) in the serum and some tissues resembled that previously observed for other experimental animals. Moreover, significant amounts of Zn-Pc were recovered from the aorta of the atherosclerotic and not of the normal rabbits. The maximum accumulation (circa 0.04μg of Zn-Pc g−1 tissue) was observed at 3 h after injection. We conclude that Zn-Pc might act as a fluorescence marker for atheromas and it has a potential as a phototherapeutic agent in atherosclerosis.

Tetra- n-propylporphycene as a tumour localizer: pharmacokinetic and phototherapeutic studies in mice

The porphin isomer tetra-n-propyl-porphycene (TPP) was incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine and intravenously injected at a dose of 2 mg/kg to BALB c mice bearing a MS-2 fibrosarcoma. Pharmacokinetic studies show that TPP is selectively transported by serum lipoproteins and delivered to the tumour tissue with good efficiency (approx. 1 μg of the TPP per g of tissue at 24 h after injection) and selectivity (ratio of TPP concentration in the tumour to the peritumoural tissue 16.7 at 24 h). Large doses of TPP are also accumulated by the liver, in agreement with the elimination of the drug via the biliary route, while no TPP is recovered from the brain. Red light-irradiation (300 J/cm 2) of the tumour area caused extensive necrosis, while only little cutaneous photosensitivity was observed. Since TPP has a large absorbance in the 630–640 nm region, can be synthesized with a high degree of purity and is an efficient generator of singlet oxygen, this drug represents a potential candidate as a phototherapeutic agent for tumours.