The benefits of photoacoustics for the monitoring of drug stability and penetration through tissue-mimicking membranes.

The paper demonstrates the potential of photoacoustics for the identification of the mechanisms underlying drug transport through tissue-mimicking systems. Photoacoustic experiments were performed for a model transdermal delivery system, consisting of drug dithranol (in pharmaceutical form) and dodecanol-collodion (DDC) membrane. The spectroscopic data revealed a single-path photodegradation of dithranol in Vaseline (dithranol â†’ danthrone, characterized by the 1st order decay rate of (7.85 ± 0.31)·10-4 s-1), and a multipath degradation in the DDC system, involving danthrone and the unknown compound (characterized by the absorption band at ~400 nm) as the final products. The desorption experiments performed enabled the identification of the unknown compound as the photodegradation product of dithranol molecules bound to the membrane matrix. The result led to the incorporation of the adsorption effects and heterogeneous structure of the membrane into the hydrodynamical model of mass transport. The model was tested against the photoacoustic depth-profiling data for dithranol permeation through DDC. The analysis allowed the dispersion and advection coefficients to be determined (D' = (2.05 ± 0.03)⋅10-9 cm2 s-1 and va' = (-5.55 ± 0.05)⋅10-7 cm s-1, respectively). Moreover, it was found, that the dithranol photodegradation rate in the non-steady state system is slower compared to the steady-state case; the effect was attributed to different permeation rates of dithranol and mobile Vaseline particles through the membrane.

Endophytic association of bioactive and halotolerant Humicola fuscoatra with halophytic plants, and its capability of producing anthraquinone and anthranol derivatives.

Halophytic plants growing in harsh desert environments are rich reservoirs of unique endophytic microorganisms. Here, healthy fresh plants of the families Tamaricaceae and Amarantaceae at three saline locations in Iran were investigated for their bioactive endophytic fungi. Among a vast number of isolates, eight isolates were identified as Humicola fuscoatra (Sordariomycetes, Pezizomycotina, Ascomycota) by microscopy and representative DNA sequences of the 5.8S rDNA (ITS) and partial ß-tubulin (TUB2). Those isolates were halotolerant, and highly bioactive, so that their intra- and extra-cellular metabolites possessed in vitro antifungal, antibacterial and antiproliferative activities, against a number of fungal and bacterial plant pathogens including the fungi Arthrobotrys conoides, Pyrenophora graminea, Pyricularia grisea and the bacteria Agrobacterium tumefaciens, Pseudomonas syringae and Xanthomonas oryzae. Chemical analyses of metabolites from the endophytes using HNMR, CNMR, NOESY, COSY, HMBC, HSQC, DEPT, TOCSY and EI MASS techniques identified 3,8-dihydroxy-1-methyl-9,10-anthracenedione (aloesaponarin II; an anthraquinone derivative), 1,8,9-anthracenetriol structure (chrysarobin; an anthranol derivative) and 2,4-di-tert-butylthiophenol in fungal extracts. To the best of our knowledge, this is the first report of endophytic association of halotolerant H. fuscoatra isolates with Tamaricaceae and Amarantaceae, and their bioactivity against plant pathogens. Also, the capability of chrysarobin and aloesaponarin II production is new to the fungal kingdom. These findings may find application in agriculture, pharmacology, and biotechnology.

Natural anthraquinone derivatives from a marine mangrove plant-derived endophytic fungus Eurotium rubrum: structural elucidation and DPPH radical scavenging activity.

There is considerable interest in the isolation of potent radical scavenging compounds from natural resources to treat diseases involving oxidative stress. In this report, four new fungal metabolites including one new bisdihydroanthracenone derivative (1, eurorubrin), two new seco-anthraquinone derivatives [3, 2-O-methyl-9-dehydroxyeurotinone and 4, 2-Omethyl- 4-O-(alpha-D-ribofuranosyl)-9-dehydroxyeurotinone], and one new anthraquinone glycoside [6, 3-O-(alpha-D-ribofuranosyl)- questin], were isolated and identified from Eurotium rubrum, an endophytic fungal strain that was isolated from the inner tissue of the stem of the marine mangrove plant Hibiscus tiliaceus. In addition, three known compounds including asperflavin (2), 2-O-methyleurotinone (5), and questin (7) were also isolated and identified. Their structures were elucidated on the basis of spectroscopic analysis. All of the isolated compounds were evaluated for 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity.

Defensive components in insect eggs: are anthraquinones produced during egg development?

Eggs of several insect species are protected against natural enemies by noxious components. However, almost nothing is known about the fate of these defensive substances during egg development nor their site of biosynthesis. The eggs of several leaf beetle species of the taxon Galerucini contain components that are unusual in insects: 1,8-dihydroxylated anthraquinones and anthrones that deter predators such as ants and birds. These components, i.e., the anthrones dithranol and chrysarobin, and the anthraquinones chrysazin and chrysophanol, are not sequestered from host plants. We asked whether the amounts of these components in the overwintering eggs of Galeruca tanaceti change from deposition to larval hatching. Gas chromatography-mass spectroscopy (GC-MS) analyses of eggs revealed a significant decrease in total amounts of dithranol and chrysophanol from egg deposition in autumn to the next spring 5 months later. Thus, these results do not provide any hint of active anthraquinone biosynthesis within eggs. Instead, the anthrones and anthraquinones that must be incorporated by the female into the eggs seem to be degraded to some extent either by the embryo or endosymbionts. GC-MS analyses showed that parasitization of eggs had some effects on the quantities of anthrones and anthraquinones.

Location of anthralin radical generation in mouse skin by UV-A irradiation: an estimation using microscopic EPR spectral-spatial imaging.

In vivo location of the anthralin radical generated in mouse skin by ultraviolet A (UV-A) irradiation was estimated by microscopic electron paramagnetic resonance (EPR) spectral-spatial imaging. An X-band EPR spectrometer equipped with specially designed high-power imaging coils and a TE-mode cavity was employed. The maximum field gradient used in this study was 6.77 mT/mm. Anthralin was applied to the dorsal skin of live mice, which were then exposed to UV-A irradiation. A broad singlet EPR spectrum (peak-to-peak line width = 0.6 mT and g = 2.004) was obtained. Microscopic EPR spectral-spatial imaging of the skin tissue showed that the anthralin radical was located mainly in the epidermis (27 microm from the skin surface). This result was consistent with the finding that the proportions of the radical in the dermis and epidermis were about 15% and 85%, respectively.

The anti-psoriatic drug anthralin accumulates in keratinocyte mitochondria, dissipates mitochondrial membrane potential, and induces apoptosis through a pathway dependent on respiratory competent mitochondria.

Anthralin is a potent topical drug, inducing clearance of psoriatic plaques. Anthralin disrupts mitochondrial function and structure, but its mechanism of action remains undefined. This study aimed to determine whether anthralin induced keratinocyte apoptosis as well as to investigate molecular mechanisms and the role of mitochondria. We studied human keratinocytes and human 143B rho(0) cells, which lack mitochondrial DNA and a functional respiratory chain. We show that anthralin disrupts mitochondrial membrane potential (DeltaPsim) and causes endogenous cytochrome c release, resulting in the activation of caspase-3 and characteristic morphological changes of apoptosis. Disruption of DeltaPsim and cytochrome c release were independent of mitochondrial permeability transition or caspase activation. Human 143B rho(0) cells were resistant to anthralin-induced cell death, disruption of DeltaPsim, and cytochrome c release compared with the isogenic 143B rho+ cell line. Using the intrinsic fluorescence of anthralin, rapid accumulation within mitochondria was observed independent of DeltaPsim. Using assays that measure individual respiratory chain complexes, we show that anthralin specifically interacts with ubiquinone pool. These data indicate that anthralin induces apoptosis through a novel mitochondrial pathway dependent on oxidative respiration and involving electron transfer with the ubiquinone pool. These studies identify keratinocyte apoptosis as a potentially important mechanism involved in the clearance of psoriasis.

Anthranoid free radicals found in pseudomelanosis coli.

Pseudomelanosis coli occurs after prolonged intake a anthranoids. After discontinuation of intake the pigmentation disappears apparently without noxious effects, including carcinogenicity and genotoxicity. We are presenting ESR spectra of pseudomelanosis coli specimen, compared to ESR spectra of pigmented skin scales taken from psoriatic patients treated topically with anthralin, and with ESR spectra of anthralin brown material formed in vitro. The ESR spectra show comparable g values within the accuracy of measurements. The examined specimens reveal remarkable stability: the intensity of the ESR signal remained practically constant over the period of four years. The chemical and physicochemical properties of the brown pigments formed from anthranoids explain the observed bio-inertness of these materials including that of melanosis coli pigment derived from anthranoids.

Antipsoriatic anthrones: aspects of oxygen radical formation, challenges and prospects.

1. Antipsoriatic anthrones are among the most commonly used topical agents for the treatment of psoriasis. 2. These drugs generate reactive oxygen species during their auto-oxidation under physiological conditions. 3. Recent studies have indicated that activation of molecular oxygen by anthrones may play a critical role in their mechanism of action at the molecular level. 4. This article summarizes the evidence pointing toward the significance of oxygen activation and radical formation in the antipsoriatic action and induction of skin inflammation of anthrones. 5. Also, the role of oxygen radical formation in the development of novel anthrones devoid of inflammatory and staining properties is discussed.

Anti-psoriatic drug anthralin activates transcription factor NF-kappa B in murine keratinocytes.

Anthralin is one of the most effective and safest therapeutic agents for the treatment of psoriasis, a skin disease characterized by epidermal hyperproliferation and hyperkeratosis. The drug induces and inflammatory response in the skin involving the expression of cytokine and cell adhesion molecule genes that is thought to be essential for its therapeutic efficacy. Reactive oxygen intermediates (ROIs) generated in vivo during the auto-oxidation of anthralin were discussed as mediators of the inflammatory response, but it is not yet understood how this is translated into novel inflammatory gene expression. In this study, we show that at little as 10 microM anthralin can activate a prototypic form of transcription factor NF-(kappa)B, a central transcriptional regulator of inflammatory and immune responses. Two different lines of evidence show that ROIs, in particular H2O2, are second messengers for the anthralin-induced NF-(kappa)B activation. Firstly, the activation could be inhibited by the structurally unrelated antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate. Secondly, keratinocytes stably overexpressing catalase showed a significant reduction of NF-(kappa)B activation, while stable overexpression of Cu/Zn-superoxide dismutase augmented the anthralin effect. Our data suggest that ROI-induced NF-(kappa)B plays a role in the anti-psoriatic activity of the drug anthralin.

In vivo detection of anthralin-derived free radicals in the skin of hairless mice by low-frequency electron paramagnetic resonance spectroscopy.

Free radicals were directly detected in vivo in the skin of hairless mice by low-frequency electron paramagnetic resonance spectroscopy after topical application of anthralin under pertinent therapeutic conditions. The electron paramagnetic resonance signal intensity increased steadily, reaching a maximum after about 1 d and decreased slowly in the following days, probably because of desquamation of the skin. We conclude from the spectroscopic features (single line with a line width of 6 gauss; g = 2.0036) and from the pharmacokinetic pattern that the observed signal arises from the final products of anthralin metabolism (ether-insoluble polymeric structures--"anthralin brown"). Two potential antioxidants, vitamin E and the spin trap tert-butylphenylnitrone, decreased the amount of the anthralin-derived radical that was formed. Neither vitamin E radicals nor tert-butylphenylnitrone spin adducts were observed. We suggest that electron paramagnetic resonance is a valuable tool for the noninvasive and direct in vivo monitoring of drug-induced radical formation in the skin under therapeutic conditions.

Reduction of nitroxides by anthralin and some of its derivatives.

In DMSO solution, anthralin and its C-10 monosubstituted derivatives reduce nitroxides to the corresponding hydroxylamine derivatives, which are not further transformed. The reaction rate depends on the solvent used, the nitroxide, and the structure of the reducer. It is faster in DMSO than in DMF, piperidine type of nitroxides are reduced faster than the pyrrolidine type, and the substitution on C-10 of anthralin has a significant influence on the reaction rate. Anthralin derivatives without protons at C-10 are not able to reduce nitroxides.

Treatment of psoriasis with anthrones-chemical principles, biochemical aspects, and approaches to the design of novel derivatives.

Antipsoriatic anthrones are probably the most commonly used topical agents in the treatment of psoriasis. There is growing evidence that the biochemical basis for their mechanism of action at the molecular level is related to their redox activity leading to the production of active oxygen species, which include singlet oxygen, superoxide anion radical, and hydroxyl radical. These species are involved in a variety of oxidative effects affecting cellular targets that have been implicated both in the mode of action and the skin-irritating properties of antipsoriatic anthrones: interaction with DNA, inhibition of various enzyme systems associated with cell proliferation and inflammation, such as glucose-6-phosphate dehydrogenase and inflammation, such as glucose-6-phosphate dehydrogenase and 5-lipoxygenase, and destruction of membrane lipids. Furthermore, the application of this information to the design of novel derivatives is discussed. In particular, compounds with diminished oxygen radical-generating properties have been developed, which may permit a separation of antipsoriatic and inflammatory effects. Some of the novel anthrone analogs which produced significantly less amounts of oxygen radicals than dithranol compared favorably in biological tests with this known antipsoriatic drug as an alternative method of treating psoriasis.

Modelling of drug penetration into human skin using a multilayer membrane system.

In this article, a new in vitro model system is presented with a multilayer membrane system serving as acceptor. Matrix-stabilized membranes are applied with dodecanol as lipid and collodion as matrix. Using the drug dithranol it was shown that the acceptor system can be varied over a wide range by changing the dodecanol content of the membranes and by addition of the hydrophilic substance propylene glycol to the membranes. It was demonstrated that the variability of the acceptor system can be used to adapt the penetration profiles of dithranol in a six-layer membrane system to those in excised human skin. It is shown that it is possible to study the penetration of dithranol from different semisolid formulations using the 'adapted' model system.

Structure-activity relationships for epidermal ornithine decarboxylase induction and skin tumor promotion by anthrones.

The present study was designed to compare the skin tumor promoting and epidermal ornithine decarboxylase (ODC) inducing activities of various structural analogs of anthralin (1,8-dihydroxy-9-anthrone) and chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone). Groups of 30 SENCAR mice each were initiated with 7,12-dimethylbenz[a]anthracene and 2 weeks later promoted with once- or twice-weekly applications of various doses of these anthrone derivatives. Carbon-10 (C10)-acyl derivatives of anthralin were active skin tumor promoters in the range of 25-440 nmol per mouse. 10-Acetylanthralin was significantly more active than 10-myristoyl-anthralin at low doses (e.g. 25 and 50 nmol per mouse) and nearly as potent as the unsubstituted compound. Higher doses (greater than or equal to 100 nmol per mouse) of this derivative were toxic, hence, reducing the final papilloma response. On a relative activity scale where anthralin is 1.0, these derivatives had activities that were approximately 0.7 and 0.2, respectively. 10,10-Dipropylanthralin was totally inactive at the doses tested. C6-Substituted derivatives of chrysarobin demonstrated diverse tumor promoting activities when tested in the range of 25-440 nmol per mouse. On a relative activity scale where chrysarobin is 1.0, 6-methoxychrysarobin (physcion anthrone) was approximately 0.9, whereas 6-hydroxychrysarobin (emodin anthrone) had no activity. Chrysophanic acid (1,8-dihydroxy-3-methyl-9,10-anthraquinone) was also inactive as a tumor promoter at the doses tested. In general, the tumor promoting activities of these anthrone derivatives correlated very well with their ability to induce epidermal ODC after a single topical application indicating an important role for this enzyme in skin tumor promotion by anthones. The ability of C10-substituted derivatives of anthralin to undergo base catalyzed oxidation in vitro correlated with both ODC inducing and tumor promoting activities. In addition, copper(II)bis(diisopropylsalicylate) was found to inhibit both ODC induction and skin tumor promotion by chrysarobin. These latter data, when taken together, suggest a role for oxidation at C10 in skin tumor promotion by anthrone derivatives.

The release and percutaneous permeation of anthralin products, using clinically involved and uninvolved psoriatic skin.

The release and permeation of 1% Westragel and 1% Westrastick and three commercial 1% anthralin products were investigated in vitro with the use of a Franz diffusion cell unit. An inert Teflon membrane with a mesh opening of 74 mu was used for measuring the rate of release. Involved psoriatic and uninvolved human skin collected from the same subjects were used for the permeation study. The permeation of danthron and dianthrone, the major degradation products of anthralin, was also studied with the use of microemulsion gels of 1% danthron and 1% dianthrone, which were prepared in the same way as 1% Westragel. The penetrating anthralin, danthron, and dianthrone were stabilized by a modified receptor fluid, and sample solutions were analyzed by a high-power liquid chromatography method. Involved psoriatic skin was found to be much more permeable to anthralin than was uninvolved psoriatic skin. The slow permeation rate of anthralin, danthron, and dianthrone through normal skin and uninvolved skin indicates that the stratum corneum is the rate-limiting barrier. In involved psoriatic skin, the release rate of anthralin from the topical product becomes the rate-determining step. Large individual variations were found in the permeation of anthralin through involved psoriatic skin, suggesting that the permeation rate of anthralin also may depend on the disease state of psoriatic patients. The anthralin molecule, possessing both hydrophilic and lipophilic centers, diffused significantly faster than did danthron and dianthrone. Westragel, 1%, showed the highest diffusion rate as well as the highest driving force when compared to other commercial 1% anthralin products. This suggests that 1% Westragel may be an optimal design, especially for short-contact anthralin therapy.

Antipsoriatic drug action of anthralin: oxidation reactions with peroxidizing lipids.

Reactions of anthralin with peroxidizing lipids were investigated. Using ESR spectroscopy and quantitative HPLC analysis radical species and decomposition products respectively were analysed in the reaction mixture as a function of time. Directly after mixing anthralin with peroxidized lipids, the 1,8-dihydroxy-9-anthron-10-yl radicals (primary radical) and small amounts of 1,8-dihydroxy-anthraquinone (AQ) were formed. After a few days of reaction, two secondary radicals were observed in addition to the primary radical. At the same time, 1,8,1'8'-tetrahydroxy-10,10'-bis-9(10H)-anthrone (DI) and an increasing amount of AQ and other nonidentified decomposition products were found. As the reaction proceeded further on the amount of AQ and the nonidentified decomposition products increased, the primary radical disappeared (within about 40 d) and the concentration of DI decreased to zero (within 1 yr). Nonidentified decomposition products are tentatively assigned to polymeric degradation products (anthralin brown) formed from DI via the observed secondary radical species. These radical reactions of anthralin with peroxidized lipids help to elucidate speculations on radical type reactions of anthralin in psoriasis indications, e.g., the role of peroxidized skin lipids as radical reaction initiators.