8-MOP Concentration Research Articles

8‐METHOXYPSORALEN INCREASES DAYTIME PLASMA MELATONIN LEVELS IN HUMANS THROUGH INHIBITION OF METABOLISM

It is well established that in healthy humans oral intake of 5- or 8-methoxypsoralen (5- and 8-MOP) is followed by a significant increase in plasma melatonin concentrations. The effect of psoralen on rat melatonin has been studied in vitro and in vivo and a stimulation of release or secretion from the pineal gland has been suggested. In this study we examined the time-related changes in plasma concentrations of 8-MOP, melatonin and 6-sulfatoxymelatonin in 15 patients admitted for routine psoralen plus UVA therapy. On the first day of treatment blood samples were collected before, and 30, 60, 66 and 90 min after intake of 8-MOP (0.6 mg/kg). Although the rate of 8-MOP absorption varied greatly, a significant increase (P = 0.0002) in melatonin levels was found 60 min after 8-MOP intake. During UVA exposure a strongly correlated decrease in mean melatonin and mean 8-MOP concentrations was found, indicating an effect of UVA radiation, either direct or 8-MOP mediated, on circulating melatonin levels. Plasma 6-sulfatoxymelatonin concentrations decreased significantly between all time points, suggesting inhibition of melatonin metabolism.

Photochemical inactivation of pathogenic bacteria in human platelet concentrates

Platelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 10(5) to 10(7) colony- forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 micrograms/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 10(5) CFU/mL of all 5 gram-positive (Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes, Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 10(1) to 10(3.7) CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 micrograms/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 10(5) CFU/mL of 4 resistant gram-negative organisms (Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 10(4.1) CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 micrograms/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.

Photochemical Inactivation of Pathogenic Bacteria in Human Platelet Concentrates

AbstractPlatelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 105 to 107 colony-forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 μg/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 105 CFU/mL of all 5 gram-positive (Staphylococcus aureus. Streptococcus epid-ermidis, Streptococcus pyogenes. Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 101 to 103.7 CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 μg/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 105 CFU/mL of 4 resistant gram-negative organisms [Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 104.1 CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 μg/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.

PUVA erythemal sensitivity depends on plasma psoralen concentration and UVA sensitivity

The variation in erythemal sensitivity of the skin during PUVA therapy with oral 8-methoxypsoralen (8-MOP) was examined by measuring both UVA and PUVA erythemal responses, together with plasma 8-MOP concentration, in 27 patients about to start PUVA therapy for psoriasis. The erythema responses were judged visually, and also measured using a reflectance instrument in order to construct dose-response curves. No significant association was found between the UVA and PUVA minimal erythema responses. The plasma psoralen concentration showed significant association with the slope of the PUVA erythema dose-response curve. The slopes of the UVA and PUVA erythema dose-response curves were significantly associated, and this association became much stronger when allowance was made for plasma psoralen concentration. These results show that erythemal sensitivity during PUVA therapy is related to both plasma psoralen concentration and inherent UVA sensitivity, but that this relationship is not apparent when sensitivity is judged visually as the minimal erythema response. The association between PUVA and UVA erythemal sensitivity suggests a common pathway in the vascular response induced by UVA radiation, with or without psoralen.

Skin concentration of 8-methoxypsoralen, 5-methoxypsoralen and 4,5,8-trimethylpsoralen in guinea pigs

The skin concentrations of 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and 4,5,8-trimethylpsoralen (TMP) were studied in the albino guinea pig following oral administration and intraperitoneal injection. The skin concentration of phototoxic drugs after oral administration peaked at 1.5 h, and the concentration of 8-MOP was 3.5 times greater than that of 5-MOP. The skin concentration of TMP was not detected in our study (limit of sensitivity 5 ng/ml). The highest skin concentrations of 8-MOP and 5-MOP after intraperitoneal injection were achieved for both drugs at 0.5 h, with the level of 8-MOP approximately 1.3 times higher than that of 5-MOP. The level of TMP could not be measured even in the case of intraperitoneal injection.

Photoinduced cutaneous inflammatory response by psoralens

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA—visible irradiation (320–700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24–48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 μg 8-MOP cream followed by irradiation for 2 h (9.4 J cm −2) produced 3–7 times iVP, 2–4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.

Bioavailability of two 8-methoxypsoralen formulations

The aim of the study was to compare the pharmacokinetics of two formulations of 8-methoxypsoralen: a commercial tablet formulation (B) and capsules containing 8-MOP dissolved in a mixture of polyethylene glycol and glycerol (A). The pharmacokinetic parameters studied were the time and value of the maximum peak concentration of 8-MOP (Tmax and Cmax) and the area under the curve (AUC) of concentration versus time. The main results were: an earlier peak plasma concentration for A (30 min versus 126 min); less variability in the absorption of 8-MOP and in Cmax for A; a faster rate of elimination with A; and no significant difference between ratios of ingested dose and plasma levels reached. Thus, the more stable bioavailability of the new formulation was clearly demonstrated.

Selective Cytotoxicity of Low-Density Lipoprotein to Helper T Cells of Cutaneous T-Cell Lymphoma After Photoperoxidation With 8-Methoxypsoralen

Lymphocyte-containing plasma subjected to photolysis in the presence of 8-methoxypsoralen (methoxsalen, 8-MOP) has previously been shown to be effective against cutaneous T-cell lymphoma and the AIDS-related complex. The mechanism of this effect was thought to involve photoreaction of 8-MOP with DNA, based on certain in vitro experiments. The results of this study suggest a different mechanism. Low-density lipoprotein (LDL) from fresh human plasma was photosensitized by addition of 8-MOP and exposure to UV light (mp-LDL), and the reactions of the LDL lipids and the chemical actions induced by these reactions were monitored. In a separate procedure, LDL was peroxidized with hydrogen peroxide and peroxidase (p-LDL). mp-LDL and p-LDL were then tested in cytotoxicity assays on HuT-78 helper T cells of cutaneous T-cell lymphoma. These results indicate that (a) LDL in plasma in the presence of very low concentrations of 8-MOP (200 ng/mL) can be peroxidized by UV light; (b) this photoperoxidized LDL is cytotoxic to helper T cells of cutaneous T-cell lymphoma in a dose-dependent manner; but (c) it does not kill normal lymphocytes under similar conditions. The findings also suggest alternative therapeutic strategies for treatment of cutaneous T-cell lymphoma, such as direct utilization of peroxidized LDL.

The relationship between plasma psoralen concentration and psoralen-UVA erythema

The plasma 8-methoxypsoralen (8-MOP) concentration was measured in 60 patients commencing psoralen photochemotherapy (PUVA). At the time of blood sampling each patient was phototested using a series of 10 exposures to UVA. The resulting erythema was measured objectively 72 h after irradiation and dose-response curves for psoralen-UVA erythema were constructed. Although the dose of 8-MOP was calculated according to body weight, patients receiving 30 mg of 8-MOP had a significantly lower mean plasma concentration than those receiving higher doses. There was no significant correlation between plasma 8-MOP concentration and minimal phototoxic dose, either estimated visually or calculated from the dose-response curves. However the slope of the dose-response curve showed significant correlation with plasma 8-MOP concentration. The variation between patients in the rate of increase of the erythemal response, but not the variation in threshold sensitivity, can be explained by difference in plasma psoralen concentration.

Study of the skin concentrations after administration of the various phototoxic drugs

The skin concentrations of 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and 4, 5', 8-trimethylpsoralen (TMP) were studied in the guinea pig following oral administration and bathing. The skin concentration of phototoxic drugs after oral administration peaked at 1.5 hours, and the concentration of 8-MOP was 3.5 times greater than that of 5-MOP. The skin concentration of TMP was not detected in our study (limit of sensitivity 5ng/ml). The skin concentrations of phototoxic drug after bathing decreased in the order of 5-MOP, TMP, and 8-MOP.

Serum 8-methoxypsoralen (8-MOP) concentrations after bath water delivery of 8-MOP plus UVA

The percutaneous absorption of 8-methoxypsoralen in patients with psoriasis during bath water delivery of 8-MOP plus UVA was determined by a reverse high-performance liquid chromatographic method.

Ultrastructural Modification of the Plasma Membrane in HUT 102 Lymphoblasts by Long-Wave Ultraviolet Light, Psoralen, and PUVA

Ultrastructural alterations of the plasma membrane in HUT 102 lymphoblasts were assessed after a 2-h interaction with a suprapharmacologic (15 micrograms/ml) concentration of 8-MOP, 2-h irradiation with UVA (2.1 mW/cm2), and the exposure of the HUT 102 cells to PUVA under the same conditions. The dark reaction of HUT cells with 8-MOP resulted in the disappearance of microvilli, the emergence of plasma-membrane-associated spherical bodies, formation of lamellar fungiform membrane evaginations, and, in approximately 1% of the cells, formation of uropods and cell capping. Except for uropod formation and cell capping, UVA has induced the same plasma-membrane alterations, and was more deleterious to structural cytoplasmic integrity than 8-MOP. Morphologic changes of the plasma membrane in PUVA-exposed cells tended to replicate structural alterations elicited independently during the dark reaction by suprapharmacologic 8-MOP concentrations. Partial retention of microvilli by cells after PUVA was the sole exception. In light of all available evidence we conclude that psoralen during the dark reactions interacts with plasma membrane lipids by as yet undisclosed mechanisms and that in addition to lipids, membrane proteins are also the primary target of the initial interaction of HUT 102 cells with psoralen during PUVA treatment.

Pharmacokinetics of 8‐methoxypsoralen in the dog

The pharmacokinetics of 8-MOP were studied in six dogs following intravenous administration of 2 mg kg-1. In most animals a bi-exponential decline of the plasma level profile was observed. The calculated pharmacokinetic parameters varied considerably between the different dogs. The mean half-lives of distribution and of elimination were 0.20 and 2.17 h, respectively. The average total plasma clearance was 0.51 l kg-1 h-. Following both oral and intravenous administration of doses of 1, 3, and 10 mg kg-1, non-linear kinetics were found. Non-linearity was confirmed following administration of the substance at increasing infusion rates. Finally in four dogs, extraction by the liver was calculated by measuring 8-MOP concentration in the femoral artery and in a hepatic vein after intravenous administration of 1 mg kg-1. The extraction varied from animal to animal (18 per cent to nearly 100 per cent). Administration of an additional dose of 3 mg kg-1 in two of the dogs led to a decreased extraction.

Binding of 8-methoxypsoralen to human serum proteins and red blood cells.

Serum binding of 8-methoxypsoralen (8-MOP) was studied by equilibrium dialysis. In therapeutic concentrations, 8-MOP binding in serum was high, 91.4%, and constant, indicating concentration-independent kinetics. This binding involved the two main proteins, human serum albumin and alpha 1-acid glycoprotein, in a saturable process with one class of binding sites (n) and affinity constants (Ka) of 1.295 X 10(4) mol/l and 2.115 X 10(4) mol/l, respectively. Binding to lipoproteins and gamma globulins was negligible and non-saturable in therapeutic concentrations, with nKa values of 0.35, 0.024, 0.013 and 0.0004 mumol/l for VLDL, LDL, HDL and gamma globulins, respectively. Inhibition of 8-MOP serum binding was observed with salicylic acid and indomethacin, but not with diazepam, warfarin or erythromycin. Over a range of therapeutic concentrations, the ration of 8-MOP concentration in red blood cells (RBCs) and in serum was constant at 20.3% and three times higher than would be expected if a simple diffusion of the 8-MOP plasma free fraction (fu) occurred. According to the measured and calculated parameters, simulations of 8-MOP blood binding in pathological states (hypoalbuminaemia with or without inflammation) showed variations of fu which were partially 'buffered' by RBCs. Simulation of 8-MOP protein binding at cutaneous interstitial fluid level showed that fu is approximately 30% and permitted prediction of a decrease of fu available to the epidermis in case of local or systemic inflammation. This may imply an increase in the minimum phototoxic dose relevant for PUVA and explain some cases of 'poor' responsiveness of psoriatic patients to PUVA therapy.

PUVA therapy damages psoriatic and normal lymphoid cells within milliseconds.

Results of previous investigations have indicated that photochemotherapy (PUVA) attacks membranes of target cells. Using the combination of a stopped-flow technique and laser irradiation we were able to prove that the fast PUVA effect is explainable solely by the membrane damage. Lymphoid cells of healthy persons or psoriatics were taken, within 1 ms mixed with 8-methoxy-psoralen (8-MOP) at concentrations of 1.0, 0.1, and 0.5 microgram/ml, and then irradiated by a 337-nm laser pulse (0.5 mJ/cm2) lasting some picoseconds. Approximately 1 ms after administration of 8-MOP to the cell surface at least 10% of the cells were damaged, as could be judged using the standard trypan blue exclusion test. This happened at 8-MOP concentrations of 1.0 or 0.1 microgram/ml plus laser irradiation, but a concentration of 0.05 microgram/ml 8-MOP plus laser exposure did not cause any effect within 8 ms after mixing. There was no difference between using lymphoid cells from healthy persons or from psoriatics. The fact that only a very short time is necessary before cell damage occurs means that, as far as the fast PUVA effect is concerned, a photochemical reaction involving nuclear DNA can be discounted.

Neoplastic Transformation of C3H Mouse Embryo 10T1/2 Cells by 8-Methoxypsoralen Plus UVA Radiation

The effect of 8-methoxypsoralen plus UVA radiation (PUVA) on cell killing and induction of transformation was studied in the C3H mouse embryo 10T1/2 cell line. Dose-response data for both survival and transformation were obtained as a function of 8-methoxypsoralen (8-MOP) concentration and UVA dose. PUVA treatment caused cell death and induced transformation in a dose-dependent manner. Treatment of cells with 8-MOP alone (10 micrograms/ml) or UVA alone (90 J/m2) had no effect on either cell killing or transformation. The product of 8-MOP concentration and UVA dose calculated at 10% survival and 10(-3) transformation frequency levels were quite similar regardless of 8-MOP concentration or UVA dose. This suggests that there exists a simple reciprocal relationship between 8-MOP concentration and UVA dose. Both type II and type III foci induced by PUVA treatment were tumorigenic in vivo. These data provide further evidence for the carcinogenicity of PUVA treatment. In addition, the system described here could serve as a valuable model for studying the relationships between transformation and the specific cellular and molecular lesions induced by PUVA treatment.

Phototoxicity and mutagenicity of 4,5′-dimethylangelicin and long-wave ultraviolet irradiation in Chinese hamster cells and human skin fibroblasts

The phototoxicity and mutagenicity of 4,5′-dimethylangelicin (Ang) were assessed in cultures of hamster cells and human skin fibroblasts after long-wave ultraviolet irradiation. To obtain an impression of the clinical usefulness of Ang, we compared the phototoxicity and mutagenicity of this compound with those of 8-methoxypsoralen (8-MOP). To reach the same cell-killing rate in hamster cells and human skin fibroblasts, the concentration of Ang had to be 15 times higher than that of 8-MOP at comparable doses of radiation. Whether we used the same concentrations and an adjusted radiation dose or adjusted concentrations of Ang and 8-MOP at a constant radiation dose, the mutation induction in hamster cells and human skin fibroblasts was much higher after incubation with Ang than with 8-MOP

The Relationship Between 8-Methoxypsoralen Skin and Blood Levels

A method is described to determine the 8-methoxypsoralen (8-MOP) concentration in vivo in the skin by means of high-performance liquid chromatography (HPLC). Skin and blood samples were taken from 80 rats at specific intervals after oral administration of [3H]8-MOP. The pharmacokinetic results obtained for the skin levels were compared to the blood levels. In addition, liquid scintillation counting (LSC) was done on all the samples to compare the concentrations of 8-MOP plus metabolites to the concentrations of 8-MOP alone. There was a good correlation between the 8-MOP skin and blood levels. The values obtained with LSC were higher in function of time than the corresponding values obtained by HPLC, which indicates the presence of metabolites in both the skin and the blood. No statistically significant difference in the time of peaking was noted for the skin and blood levels. The blood levels seem to be a good parameter for the 8-MOP skin concentration.

Photobiological activity of suction blister fluid from patients treated with 8-methoxypsoralen.

Suction blister fluid was collected from normal human volunteers before (SBF) and 2 h after (SBF 8-MOP) oral 8-methoxypsoralen (0.6 mg/kg) ingestion without irradiation. In SBF 8-MOP the concentration of 8-MOP was 150 ng/ml, and fluorescent metabolites were also present. The toxicity of SBF 8-MOP was then determined with and without UV-A irradiation in the diploid strain D7 of the yeast Saccharomyces cerevisiae which is suitable for the detection of lethal, mutagenic and recombinogenic events. It was compared with that of SBF, SBF with 8-MOP added in vitro (150 ng/ml) and 8-MOP (150 ng/ml) in water. SBF showed no effect with UV-A doses up to 360 kJ m-2; SBF 8-MOP showed a slight decrease in survival and a dose-dependent increase in nuclear mutations, mitotic gene conversion and crossing over; SBF with 8-MOP added in vitro showed increased photobiological activity compared with SBF 8-MOP. This photobiological activity was increased by a factor of six when using 8-MOP in water. These results indicate a different bioavailability of 8-MOP in water and in interstitial fluid containing proteins. The metabolites of 8-MOP in human skin did not increase the photobiological activity of the drug. We conclude that the 8-MOP present in human skin during PUVA therapy is mutagenic and recombinogenic for eukaryotic cells.

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5′,8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA −(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5′,8-trimethylpsoralen (4,5′,8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors). The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5′,8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted. Both 8-MOP and 4,5′,8-TMP were mutagenic in WP2uvrA −(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5′,8-TMP to psoriatic patients may present reduced risk of malignant disease.