PHOTOCHEMICAL SENSITIZATION BY AZATHIOPRINE AND ITS METABOLITES—II. AZATHIOPRINE AND NITROIMIDAZOLE METABOLITES

Abstract— The photochemical reactivity of the immunosuppressant drug, azathioprine (AZT) and two representative metabolites of the nitroimidazole moiety, 1-methyl-4-nitroimidazole (MNI) and lmethyl-4-nitro-5-thioimidazole (MNTI) has been examined. No transient species of AZT were observed by laser flash photolysis, inferring a low triplet yield or its rapid deactivation. Consequently AZT, MNI and MNTI displayed little or no photodynamic sensitization via photooxidation reactions. However, AZT and MNI are highly reactive radical scavengers when photoexcited, as determined by reaction with polyacrylamide radicals. The photoreactivity of the metabolites of AZT is greater than that of the parent drug. an observation which may be relevant to the photocarcinogenicity of AZT.     

An Improved Method for Micro-Fluorimetric Determination of 6-Mercaptopurine

Abstract

A sensitive and specific fluorimetric assay for 6-mercaptopurine (6-MP) is described. The method consists of enzymatic (xanthine oxidase) conversion of 6- MP to the oxypurine derivatives, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The good recovery and reproducibility were obtained. The sensitivity (the smallest amount detectables) was about 22 pg/ml for 6- MP in water. Different spiked serum samples containing 6-MP and azathioprine (AZA) were also analyzed and the sensitivity for 6-MP was about 2.2 ng/ml.     

Determination of the orientation of azathioprine adsorbed on a silver electrode by SERS and ab initio calculations

We report the SERS spectrum of azthioprine (AZA) on a silver electrode surface and the results of normal mode calculations using empirical and ab initio calculations of the 6-mercaptopurione (6-MP) component of AZA. The empirical calculations were done with a Urey-Bradley force field (UBFF) and the ab initio calculations with the STO-3G basis set using the UHF, MP2 and BLYP methods. From the difference between the SERS and solid spectra, we determined that AZA attaches edge-on to the surface through the N3 site on the 6-MP component of the molecule. The UBFF calculation on an Ag adatom-molecule model reproduced most of the main observed frequency shifts in the SERS spectrum. With a similar model, the ab initio calculations yielded frequency shifts in the same direction as the one observed for the in-plane normal modes, but they yielded opposite shifts for the out-of-plane normal modes. This phenomenon may be attributed to a face-on interaction of the 6-MP component with a neighboring adatom made possible by an inclination of the molecule on the surface.     

DNA ASSOCIATED WITH THE CELL MEMBRANE IS INVOLVED IN THE INHIBITION OF THE SKIN REJECTION RESPONSE INDUCED BY INFUSIONS OF PHOTODAMAGED ALLOREACTIVE CELLS THAT MEDIATE REJECTION OF SKIN ALLOGRAFT

Abstract Cell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T-cells. It has recently been characterized as a target for photomodification by 8-methoxypsoralen (8-MOP) and long-wave ultraviolet light (UV-A). Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDN A photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8-MOP/UV-A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed-type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8-MOP and UV-A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8-MOP and UV-A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.     

DNA ASSOCIATED WITH THE CELL MEMBRANE IS INVOLVED IN THE INHIBITION OF THE SKIN REJECTION RESPONSE INDUCED BY INFUSIONS OF PHOTODAMAGED ALLOREACTIVE CELLS THAT MEDIATE REJECTION OF SKIN ALLOGRAFT

Cell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T-cells. It has recently been characterized as a target for photomodification by 8-methoxypsoralen (8-MOP) and long-wave ultraviolet light (UV-A). Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDNA photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8-MOP/UV-A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed-type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8-MOP and UV-A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8-MOP and UV-A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.     

Quantification of Thiopurine/UVA-Induced Singlet Oxygen Production

Thiopurines were examined for their ability to produce singlet oxygen ((1)O(2)) with UVA light. The target compounds were three thiopurine prodrugs, azathioprine (Aza), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), and their S-methylated derivatives of 6-methylmercaptopurine (me6-MP) and 6-methylthioguanine (me6-TG). Our results showed that these thiopurines were efficient (1)O(2) sensitizers under UVA irradiation but rapidly lost their photoactivities for (1)O(2) production over time by a self-sensitized photooxidation of sulfur atoms in the presence of oxygen and UVA light. The initial quantum yields of (1)O(2) production were determined to be in the range of 0.30-0.6 in aqueous solutions. Substitution of a hydrogen atom with a nitroimidazole or methyl group at S decreased the efficacy of photosensitized (1)O(2) production as found for Aza, me6-MP and me6-TG. (1)O(2)-induced formation of 8-oxo-7,8-dihydro-2'-dexyguanosine (8-oxodGuo) was assessed by incubation of 6-methylthiopurine/UVA-treated calf thymus DNA with human repair enzyme 8-oxodGuo DNA glycosylase (hOGG1), followed by apurinic (AP) site determination. Because more 8-oxodGuo was formed in Tris D(2)O than in Tris H(2)O, (1)O(2) is implicated as a key species in the reaction. These findings provided quantitative information on the photosensitization efficacy of thiopurines and to some extent revealed the correlations between photoactivity and phototoxicity.     

RELATION BETWEEN SOME PHOTOBIOLOGICAL PROPERTIES OF FUROCOUMARINS AND THEIR EXTENT OF SINGLET OXYGEN PRODUCTION

Abstract— The production of singlet oxygen (¹O₂) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8-methoxypsoralen (8-MOP) to generate ¹O₂.
The following compounds: 5-methoxypsoralen (5-MOP), psoralen, 4,5',8-trimethylpsoralen (TMP) and 5,8–dimethoxypsoralen (5,8–DMOP), are able to generate ¹O₂ when irradiated with long–wave ultraviolet light. With the photobiologically inactive angelicin no ¹O₂ production has been found. The relative extent of ¹O₂ formation has been determined for the various furocoumarins and has been compared with literature data for the skin photosensitizing effect. The observed relation between experimental data on the one side and the literature data on the other side is discussed.     

FAILURE OF UVR DOSE RECIPROCITY FOR SKIN TUMORIGENESIS IN HAIRLESS MICE TREATED WITH 8-METHOXYPSORALEN

Abstract— 8-Methoxypsoralen (8-MOP) phototumorigenesis was studied in hairless albino mice irradiated with solar simulated radiation (SSR). Animals were exposed to three different daily doses of SSR after application of an oily vehicle with or without 8-MOP. Tumor production was dependent on daily SSR dose irrespective of topical treatment. The phototumorigenic potency of SSR was increased by the vehicle and to a much greater extent by inclusion of 8-MOP. Irrespective of topical treatment, a failure in dose reciprocity for tumorigenesis was demonstrated; in terms of cumulative SSR dose, the lower daily doses were more tumorigenic than the higher doses. This reciprocity failure in 8-MOP photosensitized mice is similar to that previously observed for UV-B phototumorigenesis. The relevance of these findings to therapeutic use of psoralens is discussed.     

SYNTHESIS AND PHOTOBIOLOGICAL PROPERTIES OF 4-HYDROXYMETHYL-4'-METHYLPSORALEN DERIVATIVES

The synthesis and the photobiological activity of two new hydroxymethyl derivatives of psoralen namely 4-hydroxymethyl-4'-methyl- and 4-hydroxymethyl-4'-methyl-8-rnethoxypsoralen are described. Both compounds exhibited efficient photobinding to DNA and RNA. The DNA-photobinding process was investigated using different nucleic acid structures such as double-helical DNA, ribosomal RNA, bacterial DNA and DNA organized in the nucleosomal arrangement. The test derivatives were able to induce cross-links to a similar extent as 8-methoxypsoralen (8-MOP), used as a reference photochemotherapeutic drug. In contrast to 8-MOP, they produced relatively high levels of ^lO₂. Most photobiological effects (DNA synthesis inhibition, T₂ phage sensitization, inhibition of tumor transmitting capacity) showed a good correlation with the extent of covalent photoaddition. On the other hand, the new 4-hydroxymethylpsoralens were unable to induce skin erythema, in striking contrast with 8-MOP. Thus, neither cross-linking of the nucleic acid nor ¹O₂ production were coupled with skin phototoxicity in this class of compounds. The new derivatives appear to represent an important beginning to development of new active photochemotherapeutic agents devoid of undesired phototoxic side effects.     

FLUORESCENCE DETECTION OF AN 8-METHOXYPSORALEN METABOLITE IN HUMAN INTERSTITIAL FLUID

Abstract— A fluorescent method has been used to study the suction blister fluid of human volunteers collected after 8-methoxypsoralen (8-MOP) oral intake. A fluorescent chromophore with spectral characteristics (Λ_max= 390 nm, Λ_max=470nm) distinct from 8-MOP has been detected. Our results suggest the existence of a metabolite form of 8-MOP within the patients's skin prior to any UV irradiation. This form might result in the opening of the4–5' double bond of the 8-MOP molecule.     

4, 4'', 5''-TRIMETHYL-8-AZAPSORALEN, A NEW-PHOTOREACTIVE AND NON-SKIN-PHOTOTOXIC BIFUNCTIONAL BIOISOSTER OF PSORALEN

Photochemical and photobiological properties of a new isoster of psoralen, 4,4',5'-trimethyl-8-azapsoralen (4,4',5'-TMAP), have been studied. This compound shows a high DNA-photobinding rate, higher than that of 8-methoxypsoralen (8-MOP), forming both monoadducts and inter-strand cross-links. The yield of cross-links, however, is markedly lower than that of 8-MOP. Antiproliferative activity of 4,4',5'-TMAP, in terms of DNA synthesis inhibition in Ehrlich ascites tumor cells, is higher than that of 8-MOP. Mutagenic activity on E. coli WP2 R46+ cells appeared similar to or even lower than that of 8-MOP. This new compound applied on depilated guinea pig skin and irradiated with UVA did not show any skin-phototoxicity. On the basis of these properties 4,4',5'-TMAP appears to be a potential photochemotherapeutic agent.     

PHOTOBIOLOGICAL ACTIVITY OF 3,4''-DIMETHYL-8-METHOXYPSORALEN, A LINEAR FUROCOUMARIN WITH UNUSUAL DNA-BINDING PROPERTIES

The furocoumarin derivative 3,4'-dimethyl-8-methoxypsoralen (DMe-8-MOP) exhibits remarkable antiproliferative activity, but is devoid of skin phototoxicity. To gain insight into this peculiar behaviour we investigated non-covalent and covalent binding of DMe-8-MOP to calf thymus DNA, along with DNA-synthesis inhibition and mutagenic activity. The non-covalent interaction of DMe-8-MOP with the nucleic acid is quite poor as shown by equilibrium dialysis, spectroscopic, chiroptical and hydrodynamic techniques. However, it exhibits relevant photobinding ability to DNA using both isolated nucleic acid samples and cellular systems. Unlike the large majority of congeners, DMe-8-MOP undergoes predominantly photochemical monoaddition to the double helical polynucleotide. Upon examination of the products obtained by enzymatic hydrolysis of DMe-8-MOP photomodified DNA, the formation of an unusual furan side adduct is proposed, which could account for the peculiar photochemical and photobiological properties of the 3,4'-dimethyl furocoumarin derivative.     

ANTIGENICITY OF DNA INDUCED BY PHOTOADDITION OF 8-METHOXYPSORALEN

Abstract— Rabbits immunized with sonicated DNA UV-irradiated (365 nm) in the presence of 8-methoxypsoralen (8-MOP) produced a specific antiserum directed against DNA-8-MOP-photoadduct. None of the long chain DNA preparations modified photochemically in the same way elicited an immunological response, although all of them gave a positive immunodiffusion test on agarose with the antiserum directed against DNA-8-MOP-photoadduct. A positive reaction has also been demonstrated in the indirect immunofluorescence test, using as a source of cellular native DNA Crithidia luciliae cells treated with 8-MOP and irradiated at 365 nm (UVA). A possible use of the specific antiserum for detecting the formation of DNA-8-MOP-photoadduct, in the skin and/or lymphocytes of patients with psoriasis treated by combination of 8-MOP and UVA irradiation, is suggested.     

Determination of 6-mercaptopurine and azathioprine in plasma by high-performance liquid chromatography.

Using 1-ml plasma samples, levels of 6-mercaptopurine (6MP) as low as 5 ng/ml and azathioprine (AZA) as low as 40 ng/ml can be detected using a high-performance liquid chromatography reversed-phase column procedure following extraction. Both compounds were stable in frozen plasma for seven weeks. AZA stability in blood was temperature dependent; the half-lives of AZA breakdown to 6MP at 37 degrees were 28 and 46 min in blood drawn from two rhesus monkeys. Plasma levels of 6MP were measured in a rhesus monkey following 6MP (1.47 mg/kg) and AZA (3 mg/kg) intravenous administration. 6MP levels were also measured in three renal transplant patients on daily 50- and 100-mg AZA doses. Peak levels (45-75 ng/ml) were reached within an hour and 6MP levels were detected for up to 7 h.     

THE TREATMENT OF MASTOCYTOMA CELLS WITH 8-METHOXYPSORALEN AND LONG-WAVELENGTH ULTRAVIOLET RADIATION ENHANCES CELLULAR IMMUNOGENICITY: PRELIMINARY RESULTS

Evidence for the increased immunogenicity of mastocytoma cells (P815) treated with 8-methoxypsoralen (8-MOP) and long-wavelength ultraviolet radiation (UVA) is presented. A highly tumorigenic clone (P1) became much less tumorigenic (tum^-) after repetitive phototreatments with 8-MOP (16 ng/mL) and UVA (1 J/cm²). The yield of tum^- clones was proportional to the number of phototreatments. In a pilot study in which P1 cells were treated with three successive rounds of 8-MOP/UVA, one clone out of 73 was tum^-. In a second series of experiments, the P1 cells were treated 10 times and 4 out of 100 clones were much less tumorigenic. When some of the tum^-clones were administered intraperitoneally to DBA/2 mice, significant protection against challenge with the original P1 clone was observed. In addition, the transfer of immune cells from tum^--treated mice allowed the transfer of resistance to other tum^- clones to immunosuppressed mice (650 rad). These results are consistent with earlier literature showing the potent mutagen, N -methyl- N' -nitrosoguanidine, led to mutations in P1 that altered the expression of new surface antigens, which stimulated the murine immune system such that there was also cross recognition of shared antigens on untreated P1 cells used to challenge the immunized mice. The increased immunogenicity that resulted from the less mutagenic 8-MOP/UVA treatment may arise by a similar mechanism and may be responsible in part for the efficacy of 8-MOP/UVA photochemotherapy for the treatment of cutaneous T cell lymphoma.     

4,4'',6-TRIMETHYLANGELICIN, A NEW VERY PHOTOREACTIVE AND NON SKIN-PHOTOTOXIC MONOFUNCTIONAL FUROCOUMARIN

Abstract— 4,4',6-Trimethylangelicin is a new monofunctional furocoumarin which appears to be a very promising potential agent for the photochemotherapy of psoriasis. Actually, it is capable of photoreacting with DNA to a large extent (four times more than 8-MOP), forming only monoadducts; it produces singlet oxygen to an insignificant extent. Its antiproliferative effect (tested in Ehrlich ascites tunior cells) appears to be several times higher than that induced by the most active angelicins now known and by 8-MOP itself. In spite of this high photosensitized effect, 4,4',6-trimethylangelicin seems to be non-phototoxic on guinea-pig skin and much less mutagenic than 8-MOP in E. coli WP2 uvr -A, a strain in which cross-links show lethality rather than mutations.     

Successful spectrofluorometric and chemiluminescence methods for the estimation of azathioprine as an immunosuppressive drug in pharmaceutical preparation

The determination of azathioprine (AZA), a mercaptopurine derivative, has attracted a particular attention in most researches. Herein, spectrofluorometric (I) and chemiluminescence (II) methods were successfully confirmed to estimate azathioprine (AZA) in bulk powder form and pharmaceutical preparation. The optimum conditions to improve the fluorescence and chemiluminescence intensity of AZA were investigated. The method (I) measured the native fluorescence intensity of AZA in methanol as solvent, without any addition. The micelle-enhanced fluorescence of AZA was affected by the surfactant addition, type of solvent, pH value, and the volume of sodium dodecyl sulfate (SDS). The method (II) consisted in the improvement of the weak signal of calcein–KMnO₄ chemiluminescence system by introducing synthesized silver nanoparticles (AgNPs) in the presence of AZA. The analytical curves were linear in the concentrations range 5.0 × 10⁻⁸ − 1.0 × 10⁻⁴ M and 5.0 × 10⁻⁹ − 2.0 × 10⁻³ M with a detection limit equal to 1.5 × 10⁻⁹ M and 2.6 × 10 ⁻¹⁰ M for techniques (I) and (II), respectively. The statistical analysis indicated no significant difference between the proposed and reported methods. The suggested techniques were successfully used to determine azathioprine in its tablet form and were validated according to ICH guidelines.     

3-CARBETHOXYPYRANOCOUMARIN, A PHOTOREACTIVE DERIVATIVE OF XANTHYLETIN WITH INTERESTING PHOTOBIOLOGICAL PROPERTIES

Abstract— The photobiological activity of the newly synthesized pyranocoumarin derivative 3-carbethoxypyranocoumarin, so-called 3-carbethoxyhomopsoralen (3-CHPs) was studied in comparison to the known bifunctional furocoumarin 8-methoxypsoralen {8-MOP) and to the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) in the presence of 365 nm irradiation using two eukaryotic cell systems, the yeast Saccharomyces cerevisiae and cultured normal human skin fibroblasts. 3-Carbethoxyhomopsoralen is shown to be a photobiologically active compound capable of effectively photoinducing cytoplasmic "petite" mutants (mitochondrial damage), nuclear reversions and mitotic gene conversion in the diploid yeast strain D₇. Per unit dose it is more effective than 8-MOP and 3-CPs for the induction of cytoplasmic "petite" mutants but less effective than 8-MOP for the induction of nuclear reversions and mitotic gene conversion. A very moderate effect on cell survival is accompanied by a relatively strong genetic activity per viable cell. In human fibroblasts 3-CHPs produces a stronger inhibition of DNA synthesis than 8-MOP and 3-CPs at low doses of 365 nm radiation. During post-treatment incubation human fibroblasts recovered more easily from DNA synthesis and growth inhibitions photoinduced by 3-CHPs than from those photoinduced by 8-MOP. The results are in accord with the notion that 3-CHPs is a highly photoreactive monofunctional compound inducing easily repairable lesions with a low lethal but significant mutagenic potential.     

THE EFFECT OF DARK COMPLEXING ON THE PHOTOSENSITIZED FORMATION OF 8-METHOXYPSORALEN CROSS-LINKS WITH DNA*

The near-UV dose at 365 nm required for the formation of 8-methoxypsoralen (8-MOP) cross-links with calf thymus DNA was found to be insensitive to the concentration ratio of nucleotides to 8-MOP over the range 100:1 to 5:1. The proposed explanation is that only the dark-complexed 8-MOP contributes to the photosensitized formation of monoadducts and cross-links. A kinetics analysis indicates that the fluence required for cross-linking should be inversely proportional to the square root of the fraction of dark-complexed 8-MOP per nucleotide (the binding ratio), a quantity that is insensitive to the ratio of nucleotides to 8-MOP. These findings predict a small dependence of photosensitivity on the total 8-MOP concentration in cellular systems in which cross-links are the major lethal lesion.     

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

Abstract 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 rnin after intake of 8-MOP (0.6 mg/kg). Although the rate of 8-MOP absorption vaned 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.