pH-Dependent Photoinduced Interconversion of Furocoumaric and Furocoumarinic Acids.

Photo-controlled or photo-regulated molecules, especially biologically active and operating in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the alkaline solvolysis of psoralen led to furocoumaric acid, which was further Z → E photoisomerized (365 nm) to furocoumarinic acid. The kinetics of Z → E photoisomerization was monitored by HPLC and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids, as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic acid was found to be visibly fluorescent at pH 2.0-12.0, with the maxima of fluorescence emission spectra being pH-dependent. The reverse E → Z photoisomerization predicted by quantum chemistry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is particularly valuable in view of a wide range of pharmacological effects previously predicted for this compound.

The effect of pathogen inactivation on cryoprecipitate: a functional and quantitative evaluation.

BACKGROUND: As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. MATERIALS AND METHODS: Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. RESULTS: Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. DISCUSSION: Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.

Photoionization of psoralen derivatives in micelles: Imperatorin and alloimperatorin.

The fluorescence properties of psoralen derivatives, 8-methoxypsoralen (8-MOP), imperatorin (IMP) and alloimperatorin (ALLOI), were investigated in various solvent and micellar solutions. The variation in intensity and maxima of the fluorescence in micellar solutions suggest that psoralens are located in the micelle-water interface region. Radical cations and hydrated electrons were generated by photoionization in micellar solution upon excitation at 266 nm. A nonlinear relationship between transient yield and photon fluency was obtained for each compound, indicating that a two-photon mechanism is predominant in the photoionization of the sensitizers. The photoionization efficiencies are significantly higher in anionic sodium dodecyl sulfate (SDS) than in cationic cetyltrimethylammonium bromide (CTAB) micelles, reflecting the influence of micelle charge on the efficiency of the separation of the photoproduced charge carriers. The photoionization efficiencies of IMP and ALLOI are similar.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

Quantitative and qualitative analysis of proteins in fresh frozen plasma obtained from whole blood donations and prepared with two photochemical treatments.

Thirty-six fresh frozen plasma (FFP) units obtained from whole blood donations were used for 12 replicate experiments. For each replicate experiment, three ABO-matched FFP units were pooled and divided into three units containing different volumes of identical plasma. One unit was used as control FFP, one unit was treated with methylene blue plus visible light and one unit was treated with amotosalen and UVA light. The overall coagulation factor levels were better maintained in untreated FFP than in photochemically treated plasma. However, treated-plasma by both photochemical methods maintained coagulation factor levels that met or exceeded the European Pharmacopeia requirements for therapeutic plasma.

Furocoumarins photolysis products induce differentiation of human erythroid cells.

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.

Photoinduced crosslinking of double-helical DNA by psoralen covalently linked to a triple helix-forming oligonucleotide under near-physiological conditions.

Stable triplexes have been generated under near-physiological conditions by the introduction of the C and T base analogues 3-methyl-2-aminopyridine-2'-deoxyriboside and 5-(3-aminoprop-2-ynyl)-'-deoxyuridine into psoralen-conjugated triplex-forming oligonucleotides. After irradiation with UV light at 365 nm, photo-induced cross-linking of the TFO to double-helical DNA was observed by UV-melting analysis and fluorescence measurements.

Selective regulation of mutant K-ras mRNA expression by photo-cross-linking antisense oligonucleotide.

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop new types of anticancer drugs. In this report, we report new types of antisense molecules that photo-cross-link to target mRNA having a point mutation site in a sequence specific manner. We designed and synthesized photo-reactive antisense oligonucleotides containing a 2'-O-psoralen-conjugated adenosine (2'-Ps-oligo). They contain a psoralen via an ethoxymethylene linker (2'-Ps-eom), a propoxymethylene linker (2'-Ps-pom) and a buthoxymethylene linker (2'-Ps-bom), respectively. We evaluated the photo-cross-linking efficiency and the sequence specificity toward complementary RNA. 2'-Ps-eom showed the highest photo-cross-linking efficiency among 2'-Ps-oligos.

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells.

Psoralens are widely used for the treatment of hyperproliferative skin disease. In this work, we prepared nanoparticles (NP) containing a benzopsoralen (3-ethoxy carbonyl-2H-benzofuro[3,2-f]-1-benzopiran-2-one) by the solvent evaporation technique. We evaluated important NP parameters such as particle size, drug encapsulation efficiency, effect of the encapsulation process over the drug's photochemistry, zeta potential, external morphology, and in vitro release behavior. We also investigated the nanoparticle as a drug delivery system (DDS), as well as its target delivery to the action site, which is a very important parameter to increase the therapeutic use of psoralens and to reduce their side effects. The uptake of benzopsoralen-loaded PLGA nanoparticles by different kinds of cells found in rat peritoneal exudates was also studied. The photodamage promoted by irradiation with UV light revealed morphological characteristics of cell damage such as cytoplasmic vesiculation, mitochondrial damage, and swelling of both the granular endoplasmatic reticulum and nuclear membrane. This encapsulation method maintained the drug's properties and improved drug delivery to the target cell.

Properties of novel antisense oligonucleotides containing 2'-O-modified adenosine with a photo-reactive group.

In order to enhance the efficiency of antisense molecules for target RNA regulation, a novel photo-reactive antisense oligonucleotide was developed. We designed and synthesized a photo-reactive antisense oligonucleotide containing an adenosine in which 2'-OH was modified with 4,5',8-trimethylpsoralen (Ps) via an ethoxymethylene linkage (2'-Ps-eom). We evaluated the photo-cross-linking efficiency and sequence specificity toward complementary RNA (match-RNA). 2'-Ps-eom was selectively photo-cross-linked to the match-RNA. The photo-cross-linking efficiency was about 75% upon UVA-irradiation (365 nm) for 10 min. Previously, we reported oligonucleotides that had an adenosine anchoring Ps at 2'-O-position via a methylene linkage (2'-Ps-met). The photo-cross-linking efficiency of 2'-Ps-met and match-RNA was about 35% upon UVA-irradiation for 120 min. The photo-cross-linking efficiency of 2'-Ps-eom was dramatically enhanced in comparison with the one of 2'-Ps-met.

UV-irradiated grapefruit juice loses pharmacokinetic interaction with nifedipine in rats.

In the present study, UV-irradiated grapefruit juice was used to investigate the effects of UV light on nifedipine pharmacokinetics. Grapefruit juice in quartz vessels was UV irradiated (302 nm) with a transilluminator for 0 to 6 h at 4 degrees C, and furanocoumarins, potent contributors to the pharmacokinetic interaction, in each juice sample were measured using HPLC. The concentrations of all three types of furanocoumarins, bergamottin, 6',7'-dihydroxybergamottin, and bergaptol, decreased in a time-dependent manner. Concentrations of bergamottin, 6',7'-dihydroxybergamottin, and bergaptol were decreased to 1.66, 1.98, and 5.58%, respectively, after UV irradiation for 6 h. Two milliliters of untreated and UV-irradiated grapefruit juice were preadministered into the duodenum in rats to assess the effects of UV irradiation on nifedipine pharmacokinetics in vivo. After 30 min, nifedipine was intraduodenally administered at a dose of 3 mg/kg body weight. The nifedipine concentrations in the plasma samples were determined using HPLC. A significant increase in the area under the concentration-time curve of nifedipine was observed in untreated grapefruit juice to 1.6-fold that in the control group, but not in the UV-irradiated grapefruit juice. These findings suggest that UV irradiation is useful to eliminate pharmacokinetic interactions with grapefruit juice.

Stem cell transplantation with S-59 photochemically treated T-cell add-backs to establish allochimerism in murine thalassemia.

Hematopoietic cell transplantation (HCT) from HLA-identical sibling donors has curative potential for beta-thalassemia. The probability of surviving free of thalassemia under these conditions is approximately 85%. The application of this therapy is limited because many patients lack an HLA-identical sibling donor. HLA-mismatched stem cell transplantation for thalassemia is severely restricted by graft rejection and the risks for graft-versus-host disease (GVHD). Thus, the development of a novel method that facilitates immunological tolerance and improves the safety of HLA-mismatched HCT would greatly expand the opportunity of HCT for thalassemia patients. We hypothesized that removal of T cells from the donor hematopoietic stem cell preparation and subsequent add-back after photochemical treatment with S-59, a psoralen, would promote and stabilize the engraftment and significantly reduce the risk of GVHD. This was tested in a MHC-mismatched HCT model of murine thalassemia. S-59-treated T cells were infused simultaneously with bone marrow-derived stem cells into mice with a heterozygous deletion of one beta-globin alleles that had been conditioned with a sublethal dose of total body irradiation. Mice that received treated T cells showed increased engraftment compared to those that did not receive T cells. T-cell treatment improved survival without GVHD compared to recipients that received untreated T cells. We conclude that photochemical treatment of T cells facilitates engraftment and minimizes GVHD in allo-HCT for murine thalassemia, and sets the stage for further development of such protocols for the treatment of patients with thalassemia.

Photodynamic and photo-cross-linking potential of bergamottin.

Bergamottin (5-geranoxypsoralen) is a main component of bergamot and grapefruit oil. In order to investigate the photophysical and photochemical behaviour of bergamottin, absorption and fluorescence properties, production of singlet oxygen and superoxide radical anions and further cross-linking of DNA were studied. Strong photochemical reactions were not observed.

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets.

BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function. MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days. RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting. CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

Epoxidation of some natural furocoumarins and furochromones using gamma-ray.

Epoxidation of imperatorin (1a) with m-chloroperbenzoic acid (mcpba) under the irradiation of gamma-ray gave a mixture of epoxide 3a and dioxofuran 4a. Whereas, alloimperatorin (1b) under the same condition obtained the epoxide 3b as a sole product. On the other hand, it was successfully epoxidized xanthotoxin (1c) with mcpba under the same condition as above. In addition, visnagin (2a) was epoxidized to give a mixture of epoxides 5a, 6a. While, khellin (2b) gave the epoxide 5b, no other products were observed.

A bioassay using the human hepatoblastoma cell line HepG2 for detecting phototoxicity of furocoumarins.

We successfully evaluated the human hepatoblastoma cell line HepG2 as a model to assess phototoxicity of coumarins. Five natural furocoumarins were tested and their phototoxic activities, obtained by measuring cell viability in the presence of UV using the MTT test, were as follows: xanthotoxin (8-MOP) >> heraclenol = trichoclin = imperatorin >> peucedanin, both in growing and confluent cell cultures. This easy-to-perform, miniaturised, quantitative and sensitive method could therefore be used as a primary screening test for phototoxicity of a large number of compounds and plant extracts.

Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen.

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.

Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides.

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.