RESUMEN
Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lympho-epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast-populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin-like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin-like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.
Asunto(s)
Calicreínas/metabolismo , Síndrome de Netherton/genética , Síndrome de Netherton/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Piel/metabolismo , Quimotripsina/química , Desmocolinas/metabolismo , Desmogleína 1/metabolismo , Epidermis/metabolismo , Fibroblastos/metabolismo , Proteínas Filagrina , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Calicreínas/genética , Queratinocitos/citología , Fenotipo , Proteínas Inhibidoras de Proteinasas Secretoras/genética , ARN Interferente Pequeño/metabolismo , Inhibidor de Serinpeptidasas Tipo Kazal-5 , Inhibidores de Serina Proteinasa/metabolismo , Piel/patología , Técnicas de Cultivo de Tejidos/métodosAsunto(s)
Acné Vulgar/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Piperazinas/farmacología , Piridazinas/farmacología , Glándulas Sebáceas/efectos de los fármacos , Piel/efectos de los fármacos , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Acné Vulgar/enzimología , Acné Vulgar/patología , Animales , Atrofia , Inhibidores Enzimáticos/química , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos , Piperazinas/química , Piridazinas/química , Glándulas Sebáceas/enzimología , Glándulas Sebáceas/patología , Piel/patología , Estearoil-CoA Desaturasa/metabolismoRESUMEN
The potent antiproliferative agent SDZ LAP 977, which has shown efficacy in a clinical proof of concept study in actinic keratosis patients, has been previously demonstrated to block the cell cycle in mitosis. In the present study, we further explored the mode of action: SDZ LAP 977 binds to the "colchicine binding site" on tubulin and, thus, inhibits tubulin polymerization in vitro. Moreover, we established structure-activity relationships for the effect of modifications in the 2,5-dimethoxyphenyl moiety ("ring A") of the molecule on in vitro antiproliferative activity.
Asunto(s)
Fenoles/química , Fenoles/farmacología , Sitios de Unión , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular , Colchicina/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Fenoles/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismoRESUMEN
Pimecrolimus is an ascomycin macrolactam derivative that has been recently approved for the topical treatment of atopic dermatitis. In this study we report for the first time on a direct comparison of the inhibitory activity of pimecrolimus and the glucocorticosteroids betamethasone 17-valerate, dexamethasone and hydrocortisone at the level of T-cell proliferation and cytokine production. Stimulated human peripheral blood mononuclear cell (PBMC) systems were used that are either sensitive or resistant to calcineurin inhibitors or glucocorticosteroids. Pimecrolimus and the glucocorticosteroids inhibited dose-dependently T-cell proliferation and cytokine production in a sensitive system (anti-CD3 mAb-stimulated PBMC) with the following rank order of potency: pimecrolimus approximately betamethasone 17-valerate approximately dexamethasone > hydrocortisone. In resistant systems (anti-CD3 plus anti-CD28- or Staphylococcal enterotoxin B-stimulated PBMC), pimecrolimus or the glucocorticosteroids alone exerted either no effect, or only a partial inhibitory effect. However, combinations of pimecrolimus with a glucocorticosteroid synergistically and strongly inhibited T-cell proliferation. Taken together, the data indicate that medium potency glucocorticosteroids, such as betamethasone 17-valerate and dexamethasone, are as potent T-cell inhibitors as pimecrolimus. Furthermore, the experimental evidence suggests that combinations of glucocorticosteroids and pimecrolimus could be used clinically to achieve superior therapeutic efficacy, when monotherapy with the individual agents is unsatisfactory.
Asunto(s)
Glucocorticoides/farmacología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Tacrolimus/análogos & derivados , Anticuerpos Monoclonales/farmacología , Antígenos Bacterianos/farmacología , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , División Celular/inmunología , Células Cultivadas , Citocinas/metabolismo , Sinergismo Farmacológico , Enterotoxinas/farmacología , Humanos , Técnicas In Vitro , Linfocitos T/citología , Linfocitos T/metabolismo , Tacrolimus/farmacologíaRESUMEN
Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A(2alpha). Much of the present knowledge has relied on the use of C1P of various acyl chain lengths, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here, we show that addition of C1P in ethanol-dodecane to culture medium leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation, and cell death. Not only the toxicity of C1P, but also its ability to trigger prostaglandin E2 release, is fully dependent upon addition of a premade C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and, in turn, help in unraveling C1P signaling, which is still poorly understood.
Asunto(s)
Alcanos/farmacología , Ceramidas/farmacología , Dilatación Mitocondrial/efectos de los fármacos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Cartilla de ADN , Etanol/farmacología , Microscopía Fluorescente , Plásmidos , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Recent studies in murine models of allergic contact dermatitis have shown that systemic treatment with pimecrolimus in contrast to tacrolimus did not inhibit the sensitization phase, whereas both compounds equivalently suppressed the inflammatory response in sensitized animals. This finding indicated a differential sensitivity of antigen-naïve and primed T cells towards pimecrolimus and tacrolimus. METHODS: T cells obtained from healthy and allergic donors were subjected to primary and secondary stimulation by allogeneic or staphylococcal superantigen-presenting dendritic cells (DC). Human skin-derived, allergen-specific T cell clones from an atopic dermatitis patient were activated by anti-CD3 antibodies or by specific allergen-presenting DC. The inhibition of T cell proliferation and cytokine release by graded doses of calcineurin inhibitors was evaluated. RESULTS: Primary stimulation of T cells was inhibited by pimecrolimus with an approximately 8-fold lower potency as compared with tacrolimus. In contrast, the secondary response of ex vivo expanded T cells activated by allogeneic or staphylococcal superantigen-presenting DC was inhibited by both compounds with equivalent potency. Likewise, both drugs showed very similar potency to inhibit the proliferation and cytokine synthesis from antigen- stimulated T cell clones and the induction of cytokines in Jurkat T cells. CONCLUSION: These data indicate that pimecrolimus has a selectivity for antigen-primed memory T cells not seen with tacrolimus.
Asunto(s)
Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Células Dendríticas/inmunología , Dermatitis Atópica/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Células Jurkat , Prueba de Cultivo Mixto de Linfocitos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Steroid sulfatase (STS) offers a new target for the treatment of steroid hormone-dependent diseases, such as breast and prostate cancer and androgen-dependent skin diseases. We here characterize a novel non-estrogenic inhibitor of the enzyme, namely 6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate (AHBS), with special attention to its potential use in the treatment of acne. The compound blocks STS activity in homogenates of human skin with IC(50)=16 nM. Following a single oral dose (5 mg/kg) in rats, the compound blocks STS in the skin by 95% at 8 h, followed by recovery of activity over 5 days. Following topical application to the skin, both in vitro and in vivo, AHBS passes through the stratum corneum leading to inhibition of STS activity in the dermal compartment with rapid onset and long duration. Topical application of AHBS to Göttingen minipigs for a period of 2 weeks does not induce symptoms of ichthyosis as seen in STS-deficient human subjects, but leads to a reduction of sebum secretion to the skin surface. Based on these data, clinical studies with AHBS in acne patients are warranted, in order to verify the hypothesis on the importance of the sulfatase pathway in androgen-dependent skin diseases.
Asunto(s)
Adamantano/análogos & derivados , Adamantano/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Esteril-Sulfatasa/antagonistas & inhibidores , Absorción , Acné Vulgar/tratamiento farmacológico , Adamantano/administración & dosificación , Adamantano/uso terapéutico , Administración Cutánea , Administración Oral , Anciano , Andrógenos/metabolismo , Animales , Línea Celular , Estabilidad de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/uso terapéutico , Estrógenos/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Ratas , Ratas Wistar , Piel/efectos de los fármacos , Piel/enzimología , Piel/patología , Esteril-Sulfatasa/metabolismo , PorcinosRESUMEN
We report the synthesis and results from the in vitro evaluation of 6-(adamantan-2-ylidene-hydroxybenzoxazole)-O-sulfamate 1 as an irreversible inhibitor of human steroid sulfatase (STS). Highly straightforward, condensation of 2-methyl-6-hydroxybenzoxazole with 2-adamantanone, subsequent elimination of water and sulfamoylation provide the title compound in 45% overall yield from the inexpensive 2,4-dihydroxyacetophenone. 1 was found to be a potent irreversible inhibitor of purified human steroid sulfatase (STS) and specific for this enzyme relative to human arylsulfatases A and B. In cellular assays with human keratinocytes, sebocytes and fibroblasts, 1 blocked STS activity with IC(50) values in the range of 0.15-0.8 nM, and in MCF-7 breast cancer cells with IC(50)=2.3 nM, while it did not bind to estrogen receptors alpha and beta. Thus, 1 is a candidate for further investigation of its potential as a drug to be used in androgen- and estrogen-dependent diseases.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Guanina/análogos & derivados , Guanina/síntesis química , Guanina/farmacología , Esteril-Sulfatasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Guanina/química , Humanos , Cinética , Modelos Moleculares , Conformación Molecular , Relación Estructura-ActividadRESUMEN
The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.
Asunto(s)
Antineoplásicos/uso terapéutico , Queratosis/tratamiento farmacológico , Fenoles/uso terapéutico , Animales , Antineoplásicos/efectos adversos , Apoptosis , Ciclo Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Tolerancia a Medicamentos , Humanos , Queratinocitos/efectos de los fármacos , Fenoles/efectos adversos , Enfermedades de la Piel/inducido químicamente , PorcinosRESUMEN
2-Alkylchromen-4-one 6-O-sulfamates, a new class of potent steroid sulfatase (STS) inhibitors, were evaluated for their estrogenic potential. Structure-activity relationships for estrogenic activity were identified; however, no correlation with STS inhibition was found. Estrogenicity is favored by bulky side chains and can be effectively abrogated by an (additional) linear substituent. Compound 2g, which lacks estrogenicity while potently inhibiting STS, has an ideal in vitro profile for the treatment of breast cancer.