Dithranol-loaded lipid-core nanocapsules improve the photostability and reduce the in vitro irritation potential of this drug.

Dithranol is a very effective drug for the topical treatment of psoriasis. However, it has some adverse effects such as irritation and stain in the skin that make its application and patient adherence to treatment difficult. The aims of this work were to prepare and characterize dithranol-loaded nanocapsules as well as to evaluate the photostability and the irritation potential of these nanocarriers. Lipid-core nanocapsules containing dithranol (0.5 mg/mL) were prepared by interfacial deposition of preformed polymer. EDTA (0.05%) or ascorbic acid (0.02%) was used as antioxidants. After preparation, dithranol-loaded lipid-core nanocapsules showed satisfactory characteristics: drug content close to the theoretical concentration, encapsulation efficiency of about 100%, nanometric mean size (230-250 nm), polydispersity index below 0.25, negative zeta potential, and pH values from 4.3 to 5.6. In the photodegradation study against UVA light, we observed a higher stability of the dithranol-loaded lipid-core nanocapsules comparing to the solution containing the free drug (half-life times around 4 and 1h for the dithranol-loaded lipid-core nanocapsules and free drug solution containing EDTA, respectively; half-life times around 17 and 7h for the dithranol-loaded lipid-core nanocapsules and free drug solution containing ascorbic acid, respectively). Irritation test by HET-CAM method was conducted to evaluate the safety of the formulations. From the results it was found that the nanoencapsulation of the drug decreased its toxicity compared to the effects observed for the free drug.

An EPR method for estimating activity of antioxidants in mouse skin using an anthralin-derived radical model.

Inhibitory effects of intravenously or orally administered antioxidants on the anthralin-derived radical generated in skin (mainly in the epidermis) of living mice by ultraviolet-A (UVA) irradiation were estimated. Anthralin was applied to the dorsal skin of living mice and the mice were then exposed to UVA. The EPR signal intensity in skin tissue strips obtained from mice after anthralin-UVA treatment was measured by an X-band EPR spectrometer. Several common antioxidants such as ascorbate, glutathione and Trolox (a vitamin E analogue) intravenously administered to mice reduced anthralin-derived radical generation. Trolox showed the most prolonged and powerful effect. Intravenous injection of a clinically used cerebral neuroprotective drug, Edarabone (Radicut), also showed depletion for the anthralin-derived radical. Oral administration of a commercialized nutritional supplement (a cocktail of 17 herbals and vitamins) also attenuated the anthralin-derived radical. The anthralin-UVA treatment model for antioxidant activity in the epidermis is a potentially feasible method to estimate activity of antioxidants in the body.

Inhibition of the nuclear factor-kappaB signaling pathway by leflunomide or triptolide also inhibits the anthralin-induced inflammatory response but does not affect keratinocyte growth inhibition.

We performed this study to determine the relationship between activation of nuclear factor (NF)-kappaB and inhibition of keratinocyte growth by anthralin, which not only might be useful for a better understanding of the role of NF-kappaB in the pathogenesis of psoriasis, but also indicate whether the inflammatory reaction induced by anthralin is inseparable from its antipsoriatic activity. The involvement of NF-kappaB was assessed using the antipsoriatic drugs leflunomide and triptolide (T0) as effectors, since they can inhibit NF-kappaB activation induced by anthralin. The results showed that the inhibition of keratinocyte growth by anthralin was not related to the activation of NF-kappaB. Using sodium salicylate, a known NF-kappaB inhibitor, further confirmed this conclusion. Thus it might be possible to inhibit the inflammatory response induced by anthralin via repression of NF-kappaB activation. We found that leflunomide or T0 could significantly inhibit the mRNA overexpression of interleukin-8 and intercellular adhesion molecule-1 in keratinocytes induced by anthralin. Taken together, our data indicate that the growth inhibition of anthralin is related to the NF-kappaB-independent signaling pathway, and that leflunomide or T0 could control proinflammatory cytokine expression induced by anthralin via inhibiting the activation of NF-kappaB.

Low-dose dithranol treatment and tape stripping induce tolerance to dithranol in a mouse ear oedema model.

BACKGROUND: It is well known from clinical practice that repeated treatment with dithranol leads to the development of tolerance. OBJECTIVES: To investigate the characteristics and mechanism of such dithranol tolerance. METHODS: The mouse ear was pretreated with a low dose of dithranol or croton oil or, in previously sensitized animals, with dinitrofluorobenzene (DNFB). Twenty-four hours later irritant dermatitis was elicited by painting the mouse ear with a high dose of dithranol, croton oil or DNFB, and the dermatitis was characterized by measurement of ear thickness. RESULTS: Low-dose dithranol significantly suppressed dithranol-induced oedema, whereas it had no effect on croton oil- or DNFB-induced dermatitis, suggesting that dithranol-induced tolerance is specific. Tolerance to dithranol could not be induced by pretreatment of the mouse ear with a low dose of croton oil or DNFB. Mild tape stripping of the mouse ear also inhibited the inflammatory effect of dithranol applied 24 h later. Superoxide dismutase treatment abolished the tolerance-inducing effect of low-dose dithranol or stripping. CONCLUSIONS: These results suggest that superoxide anion radicals are involved not only in the inflammatory effect of dithranol, but also in the induction of tolerance.

Heterocyclic substituted anthralin derivatives as inhibitors of keratinocyte growth and inducers of differentiation.

Heterocyclic substituted derivatives of the antipsoriatic anthralin were synthesized and evaluated in vitro for their antiproliferative action against keratinocytes and their ability to induce keratinocyte differentiation. The indole-2-carboxylic acid analogue 2e exhibited the same excellent antiproliferative activity as anthralin and also induced terminal differentiation of keratinocytes. As a benefit of its strongly diminished potential to generate oxygen radicals, 2e did not induce damage of keratinocyte membranes.

Tumorigenicity of a combination of psoriasis therapies.

Coal tar, a tumour initiator, and dithranol, a tumour promoter, are used in the treatment of psoriasis. Topical treatment of mice with pharmaceutical formulations of these two agents, at therapeutic doses, induced skin papillomas in a classical two-stage carcinogenesis protocol, while treatment with either agent alone did not. This finding has implications for the use of both agents in combination in the treatment of psoriasis.

Hydroxyl radical damage to DNA sugar and model membranes induced by anthralin (dithranol).

The antipsoriatic anthrones anthralin and butantrone caused degradation of the DNA sugar deoxyribose in the presence of ferric salt. The degradation was substantially inhibited by iron-binding hydroxyl radical scavengers, iron chelators, superoxide dismutase (SOD) and catalase, suggesting a mechanism in which antipsoriatic anthrones generate hydroxyl radicals via the Fenton reaction or an iron-catalysed Haber-Weiss reaction. Butantrone was markedly less efficient at generating hydroxyl radicals than anthralin. Using bovine brain phospholipid liposomes as model membranes to study the effects of antipsoriatic anthrones on lipid peroxidation, the peroxidation of liposomal membranes in the presence of ferric salt was maximally enhanced by anthralin and butantrone at 12.5 and 5 microM, respectively. Higher concentrations of the drugs resulted in less peroxidation. Chain-breaking antioxidants and iron chelators strongly decreased anthralin-enhanced lipid peroxidation, suggesting the involvement of hydroxyl, peroxyl or alkoxyl radicals. In contrast to their stimulatory effects on liposomal membrane peroxidation, both anthralin and butantrone diminished Fe3+/ascorbate-induced lipid peroxidation in liposomes. Butantrone was more effective as an inhibitor of lipid peroxidation than was anthralin. The antioxidant properties of antipsoriatic anthrones were determined in terms of their reactivities with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Antioxidant activity of antipsoriatic anthrones requires the presence of free hydroxyl groups at C-1 and C-8 and at least one hydrogen atom at C-10 of the anthrone nucleus. The role of active oxygen species produced by antipsoriatic anthrones and the biological effects on cellular targets are discussed with respect to the mode of action and manifestation of side effects of these drugs.

Dithranol (anthralin)-induced skin irritation in C57BL/6, NMRI and SENCAR mice.

Dithranol-induced skin irritation was compared in C57BL/6, NMRI and SENCAR mice, the strains representing different sensitivity to tumour promotion. Skin irritation was assessed using ear thickness and skin weight measurements, visual estimation of back skin irritation and histopathology. Both single and repeated applications of dithranol caused a delayed skin irritation resulting in the maximal response between 7-11 days after the beginning of the treatment. Contrary to the findings with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), C57BL/6 mice were the most sensitive and SENCAR mice the most resistant to the dithranol-induced skin irritation up to 30 days from the beginning of the treatment. NMRI mice were intermediate. Differences were found in the ear swelling, epidermal hyperplasia, amount of inflammatory cell infiltrate and skin ulceration. During repeated treatment of about 40 days, however, the responsiveness of SENCAR mice increased over that of C57BL/6 and NMRI mice. SENCAR mice had also more epidermal hyperplasia than the other strains at the end of the 74 day period of 3 times weekly applications. The magnitude of epidermal hyperplasia after long term treatment seems to correlate with the sensitivity to tumor promotion in the different mouse strains.

Effect of tumor-promoting agents on density and morphometric parameters of mouse epidermal Langerhans and Thy-1+ cells.

Topical application of tumor-promoting agents to the dorsal skin of female SENCAR mice on a twice-weekly basis resulted in a reduction in density per unit area of bone marrow-derived Thy-1+ dendritic cells. Activity was observed for well-established tumor-promoting doses of promoting agents of several different chemical types, including 12-O-tetradecanoylphorbol-13-acetate (TPA, diterpene diester), anthralin (dihydroxyanthrone), and n-dodecane (n-alkane). A reduction in density of the same cells was also observed on the basis of the asialoGM1 lipid as a surface marker after TPA treatment. No parallel effect was observed for epidermal Langerhans (Ia+) cells, the second major epidermal immunofunctional cell type, except in the case of anthralin, a finding which is consistent with the reported toxicity of this agent. The stage 2 promoting agent mezerein was unique in inducing a consistent increase in Langerhans cell densities, but did not affect the density of Thy-1+ cells when applied for a prolonged period unless applied following four doses of TPA. In contrast to the SENCAR strain, the promotion-resistant Balb/c and C56BL/6 strains showed no response with respect to TPA-induced reduction of Thy-1+ cell density. In addition to effects on density, the above tumor-promoting agents induced morphological changes in both Thy-1+ and Langerhans cells. When these changes were placed on a quantitative basis by the calculation of shape and area fraction parameters, marked and significant effects were observed for the above agents, but not for the partial promoting agent mezerein nor the non-promoting phorbol diester 4-O-methyl-TPA. The effects of TPA were largely blocked by the potent anti-promoting agent fluocinolone acetonide, moreover. These findings further support an important role for quantitative and qualitative alterations in dendritic epidermal cells in tumor promotion.

Pro-oxidative interactions of dithranol with human phagocytes promote oxidative damage to DNA of bystander leucocytes.

Dithranol at therapeutic concentrations (5-40 micrograms ml-1) induced strand breaks in human leucocyte DNA in vitro in a dose-related manner. Leucocytes from individuals with chronic granulomatous disease (CGD) incurred substantially less DNA strand breaks than did normal leucocytes during exposure to dithranol indicating that activated phagocytes are involved. H-7, 4-beta-bromophenacyl bromide (BPB) and staurosporine, all inhibitors of protein kinase C, decreased both dithranol-mediated activation of the phagocyte respiratory burst and induction of DNA strand breaks. Similar effects were observed with the hydrogen peroxide scavenger catalase. These results suggest that dithranol induces DNA strand breaks, mainly as a result of pro-oxidative interactions with phagocytes.

Cutaneous NAD(P)H: quinone reductase: a xenobiotica-metabolizing enzyme with potential cancer and oxidation stress-protecting properties.

The toxicity of quinones--including certain chemotherapeutic agents such as doxorubicin--have been related to the enzymatic or nonenzymatic formation of the corresponding semiquinones and their subsequent reaction with molecular oxygen yielding superoxide anion radicals by spontaneous regenerating of the quinones. This semiquinone redox cycling is prevented by the NAD(P)H:quinone reductase (NQR; EC 1.6.99.2) because it mediates a 2-electron reduction which results in the formation of hydroquinones instead of semiquinones. Interestingly, inducers of this enzyme such as butylated hydroxytoluene protect against the severe ulceration of accidental infiltration of doxorubicin into the area around the intravenous infusion. Recently, it has been shown that this highly protective enzyme has a very high basal activity in the epidermis which is in the same range as in the liver. The human gene of the NQR is localized on chromosome 16 and has been cloned recently as well as the gene of the murine liver NQR. We determined NQR in the cytoplasma of murine skin, liver, and human keratinocytes using 2,6-dichlorophenol-indophenol as substrate. In order to characterize this enzyme, induction by polycyclic hydrocarbones and inhibition with several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were determined. There was a similar pattern of inhibition of the basal and induced activity in all tissues so far investigated. Pyrazole, progesterone and phenobarbital did not inhibit; however, rutin and indomethacin inhibited dose-dependently. The most potent inhibitor was dicoumarol. These findings suggest that the same enzymatic form is present in liver and skin, and in murine skin and human keratinocytes.

Studies on the contact sensitizing activity of dithranol (anthralin) and 10-butyryl dithranol (butantrone).

The contact sensitizing activity of dithranol and butantrone (10-butyryl dithranol) was studied in 3 animal models: the guinea pig maximization test (GPMT), the closed patch test (CPT), and the mouse ear swelling test (MEST) in 2 different mouse strains. In the GPMT, both dithranol and, to a greater extent, butantrone showed sensitizing potential. Because butantrone was less irritant, the concentrations used were 10x higher than those of dithranol. In the CPT, only butantrone was slightly positive. In the MEST, with both CF-1 and Balb/c mice, dithranol caused less swelling of the test ear after challenge than butantrone. According to the evaluation criteria of the MEST, only butantrone caused sensitization in 50% of the CF-1 mice and in 40% of the Balb/c mice. Thus, the GPMT was the only test which indicated the minor contact sensitizing potential of dithranol. On the other hand, the 10-butyryl analogue of dithranol showed undoubtedly stronger contact sensitizing potential than the parent compound in all tests. Therefore, as compared to dithranol, an increased risk of sensitization should be considered.

Identification of tumor promoters by their inhibitory effect on intercellular transfer of lucifer yellow.

The effect of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein, teleocidin, anthralin, the Ca2+-ionophore A23187, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT) and phenobarbital (PB) on lucifer yellow transfer in cultures of SV-40-transformed Djungarian hamster fibroblasts was studied. TPA, mezerein, teleocidin, A23187, DDT and BHT exerted a strong inhibitory effect on cell-to-cell dye transfer. Anthralin uncoupled cells in 3 experiments out of 6. PB appeared to enhance lucifer yellow transfer. Sodium nitrite, a substance with unknown promoting activity, effectively uncoupled cells. All the promoters investigated had a reversible effect on the dye transfer. The value of the dye transfer method for promoter screening is discussed.

Irritancy of dithranol in normally pigmented and depigmented skin of patients with vitiligo.

In order to ascertain the extent to which the pigmentary system plays a protective role in dithranol-induced irritancy, a within-subject comparison was carried out between normally pigmented and depigmented skin of patients with vitiligo. In open patch tests, various concentrations of dithranol in a cream base were applied to the normally pigmented and depigmented skin of 6 patients with vitiligo. The responses were assessed 48 h after application. A mild to moderate inflammation occurred in the pigmented and depigmented skin and no statistically significant difference was shown between the two test areas. The present study does not support the hypothesis that the pigmentary system might be involved in dithranol-induced irritancy.

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.

Cocarcinogenic and tumor-promoting capabilities of anthralin.

Numerous chemicals to which humans are exposed either therapeutically or as a result of living in an industrial environment constitute a potential threat as carcinogens, mutagens, and/or tumor promoters and cocarcinogens. Anthralin, and antipsoriatic agent, acts as a tumor promoter for Balb/c-3T3 mouse embryo cell cultures that were previously exposed to a low dose of either benzo-a-pyrene (BaP), an indirect-acting carcinogen needing metabolic conversion for its carcinogenic action, or beta-propiolactone (BPL), a direct-acting carcinogen which needs no metabolic conversion. As a cocarcinogen, i.e., when exposure of cells to anthralin was simultaneous with exposure to the carcinogen, anthralin enhanced neoplastic transformation only when the carcinogen was BaP. Several explanations are explored. The possibility that cocarcinogens and tumor promotion occur by separate mechanisms is suggested.

Effect of arachidonic acid on anthralin inflammation.

1 The effect of topical arachidonic acid on anthralin inflammation was studied using sequential measurements of erythema (reflectance photometry) and oedema (calipers). 2 Topical arachidonic acid in concentrations which produced a small short-lived inflammatory response greatly augmented the initial phase and depressed the later phase of the inflammatory response to anthralin. 3 The initial augmentation was inhibited by concomitant administration of alpha-tocopherol. 4 It is suggested that free radical formation by anthralin has a direct action on membrane substrates such as arachidonic acid forming inflammatory products by a non-enzymic process.

Keratinocyte expression of MHC class II antigens in allergic sensitization and challenge reactions and in irritant contact dermatitis.

Keratinocytes expressed major histocompatibility complex class II antigens during the development of irritant contact dermatitis, and during the induction of contact hypersensitivity, as well as in established allergic contact dermatitis. A battery of anti-class II monoclonal antibodies, some of which are specific for class II subregion products (DP, DQ, DR), was used in an immunohistochemical study of the sequential changes in the allergic challenge reactions to dinitrochlorobenzene (DNCB) and nickel, the irritant response to anthralin, and the induction of sensitization to DNCB. The induction of keratinocyte class II expression paralleled the influx of Leu-3a+ T cells into the skin and had occurred by 24 or 48 h in each type of reaction. Differential expression of class II subregion products on keratinocytes was noted: DR was the most frequently expressed molecule, followed by DP and DQ, although in the irritant response, DP expression was not observed. The importance of these observations can be decided only by functional studies.