Pharmacokinetic behaviour of sublingually administered 8-methoxypsoralen for PUVA therapy.

BACKGROUND: Conventional oral PUVA therapy is hampered by large inter- and intraindividual variations in the bioavailability of 8-methoxypsoralen (8-MOP), caused by its low solubility in the gastrointestinal juices and large interindividual differences in hepatic metabolism rate (hepatic first pass). AIMS: New galenic formulations of 8-MOP based on solid dispersions, suspensions, and saturated solutions containing penetration enhancers were developed for sublingual administration, a drug delivery route which avoids the hepatic first pass metabolism. METHODS: Solubility properties of 8-MOP were tested in 22 potential penetration enhancers and solubilizers. Following preliminary in vivo tests of 13 sublingual 8-MOP formulations, five were administered to groups of volunteers at a nominal dose of 0.6 mg/kg body weight: two solid dispersions based on PEG 1540 (with and without Xylitol); a solution in Labrasol (glyceryl and PEG-8 caprylate/caprate), PEG 400, Transcutol (ethoxydiglycol) (1:1:1); a micronized suspension in sorbitol, water, ethanol, propylene glycol (ca. 3:1:1:5 w/v); and Oxsoralen capsules. Pharmacokinetic behaviour of 8-MOP was examined in serum; samples were analysed by HPLC. Photosensitivity was measured in seven subjects. RESULTS: The peak of maximum 8-MOP concentration in blood was sharp, rapid and reproducible: tmax of 8-MOP in blood averaged 23+/-3 min, independent of the particular formulation. Cmax was higher when 8-MOP was presented in dissolved form (solution and capsule formulations, 85+/-29 and 85+/-35 ng/ml, respectively) and lowest with the suspension (42+/-15 ng/ml). Photosensitivity peaked reproducibly at 45 min. post dosing. CONCLUSIONS: Sublingual PUVA therapy is suitable for patients with skin types I and II, in particular patients who are less suitable candidates for standard PUVA therapy (due to hepatic, renal, or cardiac insufficiency) or who have experienced side effects with standard PUVA.

Carcinogenic risk of bath PUVA in comparison to oral PUVA therapy.

The potential carcinogenic risk of bath PUVA therapy was compared to that of systemic (oral) PUVA. An analysis of the epidemiological data on cancer risk following bath PUVA with trimethylpsoralen does not support the conclusion that bath PUVA per se is less carcinogenic than systemic PUVA with 8-methoxypsoralen (8-MOP). Pharmacokinetic studies indicate that both the concentration of 8-MOP in the target organ for PUVA carcinogenicity (skin) at the relevant time point (time point of UVA irradiation) and the extents of biological effects in the skin are comparable following bathwater or systemic 8-MOP administration. Furthermore, the therapeutic effects of PUVA arise from the same photochemical reaction mechanisms as do the carcinogenic effects. Theoretically, the ratio of (desired) cytotoxic versus (undesired) mutagenic effects could increase with increasing efficiency of the PUVA therapy itself. On the basis of the available evidence, it is concluded that all forms of PUVA therapy, independently of the route of 8-MOP administration, contribute to a small but dose-dependent increase in nonmelanoma skin cancer risk.

Measurement of 5-methoxypsoralen and 8-methoxypsoralen in saliva of PUVA patients as a noninvasive, clinically relevant alternative to monitoring in blood.

BACKGROUND: Monitoring of psoralen concentration and time-course in PUVA patients is vital for efficient PUVA therapy. Blood sampling is invasive and labour-intensive and thus unsuited for routine use and repeat measurements over the course of therapy. OBJECTIVE: Psoralen pharmacokinetics in saliva were investigated and validated as a noninvasive, simple and biologically relevant alternative to measurements in blood. METHODS: The time-course of psoralen concentration was measured in saliva and serum of volunteers and patients receiving PUVA or extracorporeal photopheresis therapy. The samples were analysed by high-performance liquid chromatography. Three commonly used oral psoralen preparations were tested: Psoraderm5 (5-methoxypsoralen; 5-MOP), Meladinine and Oxsoralen (both 8-methoxypsoralen; 8-MOP). RESULTS: The pharmacokinetic parameter Cmax in saliva averaged 10% (range 6-20%) of the serum values for 8-MOP, and < or = 4% for 5-MOP. These concentrations correspond to the therapeutically relevant, non-albumin-bound fraction of psoralen in serum that is available to diffuse into the tissues. The parameter tmax in saliva and serum coincided, indicating that psoralens diffuse rapidly between the two compartments. CONCLUSION: Monitoring of psoralens in saliva is a valuable, noninvasive alternative to measurements in serum, suitable for routine use. A series of five or six saliva samples is sufficient to determine tmax in a patient beginning photochemotherapy. To determine Cmax, three independent saliva measurements at t = tmax are recommended.

Pharmacokinetics of 8-methoxypsoralen during extracorporeal photopheresis.

BACKGROUND/PURPOSE: Extracorporeal photopheresis (ECP) is a widely used therapy for the treatment of diverse diseases such as cutaneous lymphomas and graft-vs-host disease. Knowledge of the effective concentration of 8-methoxypsoralen (8-MOP) in the photopheresis apparatus and the photodegradation time-course of 8-MOP during ECP is a prerequisite for a successful therapy. METHODS: The time course of 8-MOP concentration was measured in patients' serum and in the photoactivation chamber (so-called buffy coat fraction) during ECP. Samples were analyzed by high-performance liquid chromatography. Half-lives of 8-MOP in both fractions were calculated assuming first-order kinetics (exponential decay). Losses due to adsorption and photodegradation were investigated and the recovery of bioavailable 8-MOP calculated. RESULTS: In female patients (average age 61+/-9 years) given 0.4-0.6 mg 8-MOP/kg body weight in the form of Oxsoralen capsules, peak serum concentrations averaged 420+/-80 ng/ml (n=8). In contrast, peak concentrations in the photoactivation chamber averaged only 134 ng/ml, or 32% of serum values. In serum, peak 8-MOP concentrations were reached < or =40 min following ingestion; the half-life of 8-MOP in the serum was 50+/-14 min (n=7). The effective half-life of 8-MOP in the photoactivation chamber was considerably longer (about 4 h). The recovery of free, bioavailable 8-MOP in the photoactivation chamber at the end of ECP averaged 42% of the applied dose; losses stemmed mainly from photodegradation of 8-MOP and from adsorption of 8-MOP to the surfaces of the apparatus. CONCLUSION: We conclude that interpretation of investigations on clinical success and dose-response aspects of ECP must take into account the complex pharmacokinetic behaviour of 8-MOP during the ECP procedure.

Covalent binding of agaritine to DNA in vivo.

14C-Ring-labelled agaritine was administered orally to eight C57BL/6 mice at a chemical dose of 7.5 mg and radioactive dose of 1.2 x 10(9) dpm/kg body weight. After 24 hr, the animals were killed and DNA from stomach, liver and kidneys was purified by a phenol-free method involving proteinase K digestion of chromatin and coprecipitated proteins, followed by hydroxylapatite chromatography, dialysis and precipitation with ethanol. An increase in radioactivity was found in DNA of all three organs examined. Stomach DNA had the highest levels: 160 and 30 dpm/mg DNA in males and females, respectively. Liver and kidney DNA both showed levels of approximately 1 dpm/mg, with no measurable gender differences. Expressed in the units of the covalent binding index (CBI), agaritine has a potency of 42 in mouse stomach in males and 8 in females. The CBI of agaritine in liver and kidney was 0.2-0.3 in both sexes. The genotoxic activity of agaritine is thus very weak. The cumulative lifetime cancer risk of agaritine consumption in mushrooms is estimated to lie at approximately 10(-5).

Comparison of 8-MOP aqueous bath and 8-MOP ethanolic lotion (Meladinine) in local PUVA therapy.

BACKGROUND AND OBJECTIVE: Local PUVA (psoralen plus UVA light) is an effective outpatient treatment for patients with palmoplantar eczema or psoriasis. In this study, the efficacy, applicability and patient acceptance of two local forms of 8-methoxypsoralen (8-MOP) PUVA therapy were compared. METHODS: The study design was a left-right comparison (n = 37): the left hand or foot was treated with (aqueous) 8-MOP bath PUVA whereas the right received (ethanolic) 8-MOP lotion PUVA. After 1 month, the more successful treatment was continued on both sides until lesions cleared. RESULTS: Both therapies were effective and both useful for particular clinical applications: patients with erosive lesions and rhagades appreciated the gentleness of bath PUVA. Those with pustules or hyperkeratotic lesions appreciated the greater effectiveness of 8-MOP lotion PUVA. The total UVA dose and number of sessions to clearance were smaller with 8-MOP lotion. There was no difference in the length of the relapse-free period. Therapy nonresponders usually became apparent within the first 12 sessions. CONCLUSIONS: The difference between bath PUVA and lotion PUVA can be described as 'gentle' versus 'strong' therapy. The better therapy depends on clinical indication.

Genotoxicity of agaritine in the lacI transgenic mouse mutation assay: evaluation of the health risk of mushroom consumption.

The mutagenic potency of the common mushroom Agaricus bisporus and crude agaritine extracted from mushrooms was determined in vivo using a new mutagenesis assay with lacI transgenic mice (Big Blue mice). Pairs of female lacI mice were fed one of three diets for 15 wk: (1) fresh mushrooms 3 days/wk followed by normal lab chow for 4 days/wk; (2) freeze-dried mushrooms mixed at 25% (w/w) into powdered chow; or (3) a mushroom extract containing 30% agaritine (w/w) mixed into powdered chow. The corresponding daily doses of agaritine were 30 (averaged over the whole week), 80 and 120 mg/kg body weight, respectively. Positive control animals received N-nitrosodimethylamine, N-nitrosomethylurea or urethane, mixed into powdered chow at concentrations corresponding to daily doses of 0.3, 3 and 130 mg/kg body weight, respectively. DNA of the forestomach, kidney, liver, lung and glandular stomach of the lacI mice was examined for increases in mutant frequency (MF). Control MFs ranged from 5 x 10(-5) to 10 x 10(-5). Positive control substances induced a two- to seven-fold increase in MF in their respective target organs. Of the mushroom diets, significant effects were seen only with the crude agaritine extract: it induced an increase in MF of 100% in the kidney and 50% in the forestomach. The other two A. bisporus diets, with lower agaritine doses, showed slightly but not significantly, raised MF values in the kidney alone. Thus, agaritine was weakly genotoxic in vivo; no genotoxic activity other than that attributable to agaritine was detected in A. bisporus. Substances or processes that might influence carcinogenicity by means of non-genotoxic mechanisms (e.g. increase in fibre, or decrease in calorie intake) are not detected in the lacI assay. Using a previously derived quantitative correlation between mutagenicity in the lacI test and carcinogenic potency, the carcinogenicity of agaritine in mushrooms was estimated: the average Swiss mushroom consumption of 4 g/day would be expected to contribute a lifetime cumulative cancer risk of about two cases per 100,000 lives.

The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo.

The detection limit of the lacI transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lacI transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacI mutant frequency the mice had to be treated with a total dose equal to 50 times the TD50 daily dose level. This total dose could be administered either at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacI experiment using a 250-day exposure period would give a detection limit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute studies with the presently available lacI system offered no increase in sensitivity. However, subchronic lacI studies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). It is concluded that a positive result in the lacI test can be highly predictive of carcinogenicity but that a negative result does not provide a large margin of safety.

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene.

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques). At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.

Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation.

Naturally occurring dipeptides, cholecystokinine (CCK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10-30 min with 40 mM-nitrite (pH 3.5, 37 degrees C), and the resultant products examined for mutagenicity in Salmonella typhimurium TA100. Specific mutagenicities (net revertants per mumol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/mumol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/mumol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/mumol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/mumol, respectively). The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/mumol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

Alkylating potency of nitrosated amino acids and peptides.

The alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, Tyr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, Tyr-Tyr, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present during the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artificial sweetener aspartame); only Met under these conditions had a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Met produces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the first-order reaction rate for nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat.

In a colorimetric assay using 4-(p-nitrobenzyl)pyridine (NBP) as a nucleophilic scavenger of alkylating agents, the nitrosation and alkylation reactions were investigated for a number of amino acids and derivatives. The alkylating activity increased with the square of the nitrite concentration. The nitrosation rate constants for aspartic acid, aspartame, and glycine ethylester (= precursors C) were 0.08, 1.4 and less than or equal to 0.2, respectively, expressed in terms of the pH-dependent k2 rate constant of the equation dNOC/dt = k2.[C].[nitrite]2. The rates correlated inversely with the basicity of the amino group. The stability of the alkylating activity was astonishingly high, both in acid and at neutral pH. Half-lives of 500, 200, and 30 min were determined for aspartic acid (pH 3.5), aspartame (pH 2.5), and glycine ethylester (pH 2.5). Values of 60, 15, and 2 min, respectively, were found at pH 7. It is concluded that rearrangement of the primary N-nitroso product to the ultimate alkylating agent could be rate-limiting. The potential of nitrosated alpha-amino acids to bind to DNA in vivo was investigated by oral gavage of radiolabelled glycine ethylester to rats, followed immediately by sodium nitrite. DNA was isolated from stomach and liver and analysed for radioactivity and modified nucleotides. No indication of DNA adduct formation was obtained. Based on an estimation of the dose fraction converted from glycine ethylester to the nitroso product under the given experimental conditions, the maximum possible DNA-binding potency of nitroso glycine ethylester is about one order of magnitude below the methylating potency of N-nitrosomethylurea in rat stomach. The apparent discrepancy to the in vitro data could be due to efficient detoxification processes in mammalian cells.

Nitrosation of dietary precursors.

The diet contains a large number of constituents which can be nitrosated in the gastrointestinal tract (especially in the stomach) to potentially carcinogenic nitroso compounds (NOC). The nitrosation of food mixtures has been investigated with a number of assays, such as chemical analysis or detection of alkylating potential, mutagenicity and carcinogenicity. Relatively good information is available on the formation of stable nitrosamines using high nitrite concentrations. Little is known, however, about the formation of chemically unstable NOC at low nitrite concentration and their genotoxicity in target cells. A comparison of the precursor classes, alkylamines, aromatic amines, amino acids, amides and peptides, ureas and guanidines, reveals a vast range, both with respect to daily intake (10(5)-fold) and nitrosation rate (10(4)-fold both for 1st and 2nd order nitrite dependence). A total span of 10(8) results for the relative yield of NOC in the stomach. The endogenous NOC burden from dietary ureas and aromatic amines may represent as large a hazard as the intake of preformed NOC. Recent evidence also indicates that heterocyclic amines and phenols must be considered and that the half-life of nitrosated alpha-amino acids can be much longer than that of nitrosated primary alkylamines. In these classes, more information should be collected on dietary concentrations, on the nitrosation under realistic conditions and on the genotoxicity in stomach lining cells. Within a chemical precursor class, a wide range is seen with respect to alkylating potency. It cannot, therefore, be excluded that individual precursors within the top ranking classes might become more important than single preformed NOC. Not considered in the above analysis but probably just as important for a risk evaluation in a population is the knowledge of the nitrosation conditions and target cell susceptibility in individuals.

In-vitro assays to detect alkylating and mutagenic activities of dietary components nitrosated in situ.

Nitrosation of dietary components has been combined with the 4-(para-nitrobenzyl)pyridine (NBP) colorimetric test for screening alkylating agents and with the Ames test for the detection of mutagenic activity. This allowed the investigation of short-lived nitrosation products of dietary components which generate electrophilic degradation products requiring no metabolic activation (natural amino acids and some derivatives, ureas, guanidines, primary alkyl and aryl amines). In a first system, precursor, nitrous acid and NBP were present simultaneously. All amino acids tested, except glutamic acid and glutamine, gave positive results. The reactivities spanned more than three orders of magnitude, with the aromatic amino acids and methionine the most active; two primary amines, tryptamine and histamine, were also strongly reactive. All guanidines tested, except the amino acid arginine, gave negative results. A second system consisted of two phases: NBP was added only after destruction of residual nitrite and adjustment of the pH to neutrality. This system was useful for the study of ureas, which are stable in acid but not in neutral media. The range of responses covered more than two orders of magnitude. Most amino acids and primary amines also gave positive results, but could be assessed only after analysing the kinetics of the competing reactions and choosing appropriate reaction times. In a third system, Salmonella typhimurium strain TA100 replaced NBP. Representatives of the class of amino acids, ureas, the primary amine tryptamine, and aniline became highly mutagenic upon nitrosation. Methylguanidine was only weakly mutagenic under the present assay conditions. The results indicate that further studies with unstable nitrosation products of dietary components are required to understand more thoroughly the role of endogenous nitrosation in gastric cancer.

Model risk analysis of nitrosatable compounds in the diet as precursors of potential endogenous carcinogens.

The potential health risk posed by the endogenous formation of N-nitroso compounds (NOC) from nitrosation of dietary ureas, guanidines, amides, amino acids and amines (primary, secondary and aromatic) was estimated according to the model: Risk = [daily intake of precursor] X [gastric concentration of nitrite]n X [nitrosatability rate constant] X [carcinogenicity of derivative]. The daily intakes of these compound classes span five orders of magnitude (100 g/day amides, top; 1-10 mg/day secondary amines, ureas, bottom); the nitrosation rate constants span seven orders of magnitude (aryl amines, ureas, top; amides, secondary amines, bottom); and the carcinogenicity estimates span a 10,000-fold range from 'very strong' to 'virtually noncarcinogenic'. The resulting risk estimates likewise span an enormous range (nine orders of magnitude): dietary ureas and aromatic amines combined with high nitrite concentration could pose as great a risk as the intake of preformed N-nitrosodimethylamine in the diet. In contrast, the risk posed by the in-vivo nitrosation of primary and secondary amines is probably negligible. The risk contributed by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes.

Assessment of the risk of formation of carcinogenic N-nitroso compounds from dietary precursors in the stomach.

A literature review has shown that the daily intakes of various N-nitroso-precursor classes in a typical European diet span five orders of magnitude. Amides in the form of protein, and guanidines in the form of creatine and creatinine, are the nitrosatable groups found most abundantly in the diet, approaching levels of 100 g/day and 1 g/day, respectively. Approximately 100 mg of primary amines and amino acids are consumed daily, whereas aryl amines, secondary amines and ureas appear to lie in the 1-10 mg range. The ease of nitrosation of each precursor was estimated, the reactivities being found to span seven orders of magnitude, with ureas at the top and amines at the bottom of the scale. From this information and an assessment of the carcinogenicity of the resulting N-nitroso derivatives, the potential health risk due to gastric in vivo nitrosation was calculated. The combined effects of these risk variables were analysed using a simple mathematical model: Risk = [daily intake of precursor] X [gastric concentration of nitrite]n X [nitrosatability rate constant] X [carcinogenicity of derivative]. The risk estimates for the various dietary components spanned nine orders of magnitude. Dietary ureas and aromatic amines combined with a high nitrite burden could pose as great a risk as the intake of preformed dimethylnitrosamine in the diet. In contrast, the risk posed by the in vivo nitrosation of primary and secondary amines is probably negligibly small. The risk contribution by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. Thus three priorities for future work are a comprehensive study of the sources and levels of arylamines and ureas in the diet, determination of the carcinogenic potencies of key nitrosated products to replace the necessarily vague categories used so far, and the development of short-term in situ tests for studying the alkylating power of genotoxicity of N-nitroso compounds too unstable for inclusion in long-term studies.