Photoactivation of corticosteroids in UVB-exposed skin.

The photodegradation of flumethasone (FM) and fluocinolone acetonide (FC) was studied in solution and in the pig skin. Both glucocorticosteroids applied to the pig skin were unstable under UVB light. The photoproducts formed in the skin were the lumi-, photolumi- and andro-derivatives for FM, the same found in vitro. Instead, FC hydroperoxide formed in solution was not found in the skin: the reactivity and oxidative ability of this photoproduct towards biological substrates (lipids, proteins) seems the reason of the lack of its detection in the ex vivo model. In fact, it demonstrated to quickly oxidize amino acids and peptides, and to react with BSA both in the dark and under irradiation. Moreover, the presence in the irradiated pig skin of the FC andro-derivative, which usually forms in H-donating environment, seems consistent with the mechanism of Norrish I fragmentation followed by H-abstraction, likely from the surrounding biological substrates. These findings indicate that photoreactivity of these compounds may take place in the skin of patients exposing themselves to sunlight and is a warning about possible skin damage as a result of that. Furthermore, photolability of these drugs in the skin might cause loss of their therapeutic activity.

In vitro phototoxicity of phenothiazines: involvement of stable UVA photolysis products formed in aqueous medium.

This paper reports the results of an in vitro evaluation of the phototoxic potential of stable photoproducts formed by UVA photolysis of three phenothiazines, perphenazine, fluphenazine, and thioridazine, in a water environment. Perphenazine gave a single product due to dechlorination. From thioridazine, the two major products formed; the endocyclic sulfoxide and the endocyclic N-oxide in which the 2-SCH3 substituent was replaced by a hydroxy group were tested. From fluphenazine, two products have been examined as follows: an exocyclic N-piperazine oxide and a carboxylic acid arising from hydrolysis of the 2-CF3 group. The phototoxicity of the isolated photoproducts has been studied in order to determine their possible involvement in the photosensitizing effects exhibited by the parent drugs, using hemolysis and 3T3 fibroblasts viability as in vitro assays. As fluphenazine, perphenazine, and thioridazine did, some photoproducts proved phototoxic. In particular, the perphenazine dechlorinated photoproduct and the thioridazine N-oxide were found to exert phototoxic properties similar to the parent compounds. Therefore, our data suggest that some phenothiazine photoproducts may play a role in the mechanism of photosensitivity of these drugs. Because some of these photoproducts correspond to metabolic products of phenothiazines found in humans, it cannot be ruled out that metabolites of phenothiazines can be phototoxic in vivo.

Reversed-phase high-performance liquid chromatography (RP-HPLC) determination of lipophilicity of furocoumarins: relationship with DNA interaction.

The partition coefficient (P) of 82 furocoumarins and analogs (psoralens, angelicins, allopsoralens, 8-azapsoralens, angular furoquinolin-2-ones, sulfur and selenium isosters of psoralen and angelicin) has been determined by reversed-phase HPLC. The chromatographic behavior correlates well with the in vitro affinity of the compounds toward DNA.

Photoaddition of 4,6-dimethyltetrahydrobenzoangelicin to thymine in DNA: X-ray studies and experiments with model oligonucleotides.

The crystal structures of 4,6-dimethyltetrahydrobenzoangelicin (THBA), a furocoumarin analog, and of its furan-side cis-syn cycloadduct with thymine formed in the photoreaction with DNA, have been determined. The crystal structure of the latter compound contained only one enantiomeric form corresponding to the addition to a 5'-XpT site. Contrary to most psoralen derivatives studied, THBA showed higher photoreactivity toward synthetic oligonucleotides containing that sequence than toward those with the 5'-TpX sequence.

PUVA (psoralen + UVA) photochemotherapy: processes triggered in the cells.

Photochemotherapy using psoralens and UVA is a treatment used widely in some skin diseases, in cutaneous lymphomas and in autoimmune diseases. This review has selected recent publications dealing with the photochemical processes triggered in the cells by UVA radiation and psoralen treatment. The photochemical changes initiated in the cell membranes were described.

New benzoquinolizin-5-one derivatives as furocoumarin analogs: DNA-interactions and molecular modeling studies.

Three derivatives of 1H,5H and 3H,5H-benzo[ij]quinolizin-5-one (BQZ1), previously prepared by chemical synthesis with the aim of obtaining furocoumarin analogs, have been studied. These are able to intercalate inside DNA and by subsequent irradiation with UVA light, to photoreact with DNA. Compound I (10-methoxy-7-methyl-1H,5H-benzo[ij]quinolizin-5-one) has a potentially photoreactive 2,3 double bond because of its conjugation with the pyridine ring of quinolinone, while compounds II (10-acetoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) and III (10-methoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) have a potentially photoreactive 1,2 double bond conjugated with the benzene ring of quinolinone. Compounds I and III, having a tricyclic planar structure, intercalate inside the DNA, while compound II cannot intercalate efficiently because of the steric hindrance of the acetoxy group in 10, lying outside the plane of the molecule and rotated by an angle of 77.6 degrees with respect to the tricyclic plane. The photoreaction of BQZ with DNA structure, as already known for psoralen and angelicin derivatives, consists of a [2 + 2] photocycloaddition reaction with the pyrimidine bases. The main photoadduct between the 2,3 double bond of I and the 5,6 double bond of thymine has been isolated and characterized by NMR, showing a cis-anti structure. Theoretical calculations, using AM1 Hamiltonian, have been carried out to describe the photocycloaddition reaction mechanism better. From a theoretical point of view, in the case of BQZ both the 1,2 or 2,3 double bonds and the 6,7 double bond may be involved in the [2 + 2] photocycloaddition. Spin densities and molecular orbital symmetries of compound I, in its triplet state, suggest that the 2,3 double bond interacts favorably with the 5,6 double bond of thymine moiety. On the contrary, the acetoxy substituent in position 10 of II seems to play a negative role in the DNA intercalation process.

Photochemical and photobiological studies on methylthioangelicins.

4,4',5'-Trimethyl-1'-thioangelicin (1) and 4,6,4',5'-tetramethyl-1'-thioangelicin (2), two newly synthesised isosters of furocoumarins having a sulfur atom in their five-membered ring, were studied in terms of interactions with DNA, both in the ground state and after UVA light absorption. The compounds were able to intercalate the macromolecule and to photobind efficiently, forming C4-cycloadducts with thymine. The antiproliferative effect of this binding was shown in Ehrlich and HeLa cells and by T2 phage inactivation. Tests on Salmonella typhimurium indicated low mutagenic activity. In particular, compound 1 has photobiological activity comparable with that of 4,6,4'-trimethylangelicin, but is less mutagenic.

Photoreactions of psoralens with lecithins.

The formation of cyclobutane (cb) photoadducts of psoralen with a model lecithin has been shown. The adducts are formed both in ethanol solution and in micellar suspension in water. In spite of their sensitivity to various factors such as light, temperature, air, etc., they are isolated by thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) and characterized by mass spectrometry, NMR and UV absorption spectroscopy. The NMR analysis indicates the similarity of isomeric forms of cb adducts in lecithin to those formed with free oleic acid.

Characterization of psoralen-oleic acid cycloadducts and their possible involvement in membrane photodamage.

By UVA irradiation of an ethanol solution of psoralen and oleic acid, four main photoproducts have been isolated and characterized: two have cis,cis structure; the other two are trans,cis. The same adducts have been isolated from the photoreaction of psoralen with beta-oleoyl-gamma-stearoyl-1-alpha-phosphatidylcholine followed by enzymatic hydrolysis with phospholipase A2. The four isomers stimulate protein kinase C to almost the same extent.

Dark and photochemical interactions of dimethyltetrahydrobenzoangelicin with DNA.

A study of dark interaction and photoreaction between 4,6-dimethyltetrahydrobenzoangelicin (THBA) and DNA is described. 4,6-Dimethyltetrahydrobenzoangelicin is a furocoumarin derivative in which 4' and 5' carbons are linked by a four-methylene bridge. In spite of the bulky aliphatic ring, THBA forms a complex with DNA in the dark and, on UVA irradiation, reacts with pyrimidine bases of DNA yielding monoadducts only involving its furan side double bond. Two main photoproducts form: they derive from a C4-cycloaddition to thymine and cytosine, respectively, and account for 56% and 39% of the total photoreaction yield. Both show cis-syn configuration. Two other isomers, one with thymine and one with cytosine, formed with so much lower yield (ca 3 and 1%, respectively) that their structure could not be assigned. Furthermore, in spite of its angular structure, THBA induces a small number of crosslinks in DNA.

Estimation of the effect of increasing UVB exposure on the human immune system and related resistance to infectious diseases and tumours.

Exposure to UV light has, besides some beneficial effects (vitamin D production), many harmful effects on human health. UVB irradiation has been shown to suppress both systemic and local immune responses to a variety of antigens, including some microorganisms. However, it is still not known whether such immunomodulating effects may lead to an increase in the number and severity of certain tumours and/or infections in humans. We report herein the data provided by a project that was funded by the European Union (Programme Environment), and that was aimed at the estimation of the risk associated with increased UVB exposure due to ozone depletion regarding the deleterious effects on the immune system and related resistance to tumours and infections in humans. The data, obtained by the different research groups involved, were assembled and used to calculate for the first time a risk assessment for increased environmental exposure to UVB in human subjects.

Activity of 3-carbethoxyangelicin photolysis products.

3-Carbethoxyangelicin (3-CA), carrying an electron-withdrawing group at the pyrone side, has been prepared to have a fully monofunctional angelicin derivative. 3-CA does not photoreact with DNA and induces a moderate antiproliferative activity. 3-CA proved to be extremely sensitive to ultraviolet A (UVA) light, undergoing rapid photolysis. Only one photolysis product has been isolated and identified. By means of alkaline elution, we observed that 3-CA and its photolysis products are able to induce a large amount of single-strand breaks in DNA in vivo. The results obtained from studying the capacity to produce singlet oxygen suggest that the photodynamic mechanism of action of 3-CA very likely results from its capacity--as well as that of its photolysis products--to produce singlet oxygen.

1-Thiopsoralen, a new photobiologically active heteropsoralen. Photophysical, photochemical and computer aided studies.

1-Thiopsoralen (7H-thieno[3,2-g]benzofuran-7-one) 1, a lead compound of a series of heteropsoralens, was investigated. The electronic transitions involved were studied. Fluorescence quantum yield is very low, while laser flash photolysis showed that the triplet state is practically the sole transient of 1. Fluorescence quantum yield (phi F) and triplet lifetime (tau F) as well as triplet quantum yield (phi T) and lifetime (tau T) were determined. The production of singlet oxygen was also evaluated by photophysical measurements. Photophysical data suggest that DNA photobinding of 1, owing to short fluorescence lifetime value and high triplet quantum yield, occurs likely through triplet mechanism. Interactions between 1 and DNA were studied both in the ground and the excited state. In the ground state 1 undergoes intercalation inside duplex DNA. This fact is also supported by molecular modeling studies. By UVA-light activation 1 photobinds covalently to DNA forming mono and diadducts. The furan side 1-thymine monoadduct, isolated from DNA photomodified by thiopsoralen, shows a cis-syn stereochemistry, in agreement with quantum mechanics studies. Compound 1 photobinds also with linolenic acid, component of lecithins, giving a C4-cycloaddition, and supporting that this compound also induces photolesions at the level of cell membrane, like psoralen. Compound 1 exhibits strong skin-phototoxicity.

4'-Methyl derivatives of 5-MOP and 5-MOA: synthesis, photoreactivity, and photobiological activity.

The synthesis and photobiological activity of four new 4'-methyl derivatives of 5-MOP (5-methoxypsoralen) and 5-MOA (5-methoxyangelicin), i.e., 4,4'-dimethyl-5-methoxypsoralen, 3,4'-dimethyl-5-methoxypsoralen, 4,4'-dimethyl-5-methoxyangelicin, and 3,4'-dimethyl-5-methoxyangelicin, are described. All these compounds photobind efficiently to DNA. The DNA-photobinding process was investigated using various nucleic acid structures such as double-helix DNA, bacterial DNA, and synthetic polydeoxyribonucleotides. Photoreaction experiments showed that, unlike 8-MOP (8-methoxypsoralen) and 5-MOP, both angular derivatives bind thymine and cytosine with the same efficiency. The principal nucleoside-psoralen monoadducts were isolated and characterized after enzymatic digestion or acid hydrolysis. Biological activity studies revealed a good correlation with the extent of covalent photoaddition. Moreover, the two angular derivatives and the 4,4'-dimethyl-5-methoxypsoralen were unable to induce skin erythema, in striking contrast with the reference drugs, 8-MOP and 5-MOP; only the 3,4'-dimethyl-5-methoxypsoralen caused erythema, although to a substantially lower extent than that induced by the two parent compounds.

Photobiological properties of a new tetramethylfuroquinolinone.

1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) is a new isoster of angelicin characterised by an extremely strong photosensitizing activity, which is several times higher than that of 8-MOP and 4,6,4'-trimethylangelicin (TMA). Following treatment with 1.2 microM FQ and a dose as low as 0.05 kJ m(-2) of UVA irradiation, survival (colony forming ability) of HeLa cells was abolished, while TMA and 8-MOP (even at five times the concentration for the latter) were practically ineffective. Upon UVA irradiation FQ induces various types of lesions in mammalian cells in DNA: single-strand breaks (SSBs), many monoadducts and covalent DNA-protein cross-links (DPC), but not interstrand cross-links (ISC). Using the two step irradiation procedure, DPC induced by FQ appeared to be severe lesions, having a high antiproliferative activity; their formation requires the successive absorption of two photons, thus, in this respect, resembling ISC formation. In spite of its higher capacity for damaging DNA, FQ showed a skin-phototoxicity potency very similar to 8-MOP. As some benzopsoralens, FQ induced a certain antiproliferative activity also in the dark, which was accompanied by the formation of double-strand breaks into DNA associated with DPC. This lesion is generally induced by topoisomerase inhibitors. On the basis of these features, FQ can be expected to show useful activities in photochemotherapy and photopheresis. However, before medical use careful studies on its genotoxicity are required.

The lack of efficacy of 4,6,6'-trimethylangelicin to induce immune suppression in an animal model for photopheresis: a comparison with 8-MOP.

Photopheresis is an extracorporeal form of photochemotherapy with 8-methoxypsoralen (8-MOP) and UVA (PUVA). Patients ingest 8-MOP and then a psoralen-rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell-mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8-MOP. Upon UVA exposure 8-MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross-links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross-links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4'-trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8-MOP under conditions relevant to the in vivo situation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppression in vivo, even after extensive irradiation. In addition a dose dependency of 8-MOP/UVA versus the induced immune suppression was carried out. It was shown that there is a log dose/response correlation of r=0.9205.

Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases. Synthesis, biological activity, mechanism of action, and computer-aided studies.

With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones. Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one (10). These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism. The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration. In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8. The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9-olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA. In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored. These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark. They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy. Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP. On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases.

Psoralen photosensitization: damages to nucleic acid and membrane lipid components.

For decades the therapeutic properties of psoralens were related to their photochemical reactions with DNA and RNA. Such approach, although fruitful in treatments, did not explain satisfactorily the way of healing of the multitude of diseases manifested through skin disorders. The new research field presented in our review is directed to another target: the lipid components of the cell. The studies on the photobiology of phospholipids may help to elucidate the phototoxic and pigment inducing activity of psoralens.

Sulphur and selenium analogues of psoralen as novel potential photochemotherapeutic agents.

Some heteropsoralens, obtained by replacing one or both the intracyclic oxygen atoms with sulphur and/or selenium, were studied. In preliminary tests, these compounds showed strong photobiological activity, in some cases more than two orders of magnitude higher than that of psoralen. Heteropsoralens containing sulphur undergo intercalation inside duplex DNA, showing evident affinity for the macromolecule; when selenium replaces furan oxygen, the psoralen isoster also undergoes intercalation but with lower efficiency, while psoralen isosters in which pyrone oxygen is replaced by selenium practically do not intercalate. Parallel behaviour was also observed for DNA photobinding and crosslink formation. The cycloadduct between furan selenium and pyrone sulphur isoster and thymine was isolated and characterized. The capacity of the various psoralen isosters to generate singlet oxygen and superoxide radical anion was studied. For the former the yield varies markedly for the various compounds, while for the latter the yield is similar for all compounds.

Pyrroloquinolinone methylderivatives, furocoumarin analogues: interaction with biomolecules and computer-aided studies.

Pyrroloquinolinones, furocoumarin analogues, contain a divinilbenzene moiety, suggesting possible photoreactivity. Quantum mechanics calculations indicate that the pyrrole-side double bond exhibits strong photoreactivity, while the pyridone-side double bond is only poorly photoreactive. Intercalation models obtained by molecular mechanics calculations suggest that, in the cis-syn intercalation arrangement, the pyridone-side double bond is well aligned with the nearby thymine, supporting possible C4-cycloaddition with the 5,6 double bond of thymine, while the pyrrole-side double bond assumes an unfavourable position for photobinding. These data suggest that photoreaction between the pyridone-side and thymine double bonds may takes place, although with very low yield. Experimental evidence concerning DNA-photobinding exhibited by 2,6-dimethyl-9-methoxy-4H-pyrroloquinolinone (Compound I) confirms theoretical predictions. The formation of C4-cycloadducts between the pyridone side double bond and thymine also takes place with very low yield. Compound I shows marked BSA photobinding, suggesting that pyrroloquinolinones may photoreact with proteins. The three pyrroloquinolinones examined show high yields of singlet oxygen generation, suggesting that photobiological effects may be obtained through this photodynamic pathway, rather than through DNA photobinding.