Anthony Winiski

Effects of ceramide-1-phosphate on cultured cells: dependence on dodecane in the vehicle.

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 A2α. 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.

SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7

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.

Differential Inhibition of Primary versus Preactivated T Cells by Pimecrolimus but Not by Tacrolimus in vitro

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.

Pharmacological Inhibition of Stearoyl CoA Desaturase in the Skin Induces Atrophy of the Sebaceous Glands

Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the formation of delta9-monounsaturated fatty acids from saturated precursors. Deficiency of SCD-1 in mice causes atrophy of sebaceous glands. We asked whether local pharmacological inhibition of this enzyme in the skin would lead to a similar effect. To this end, we characterized the low-molecular-weight compound XEN103 as a potent and selective inhibitor of SCD-1 activity. The compound blocks microsomal and cellular SCD-1 activity across species with IC50 values in the range of 10-15 nM. Upon topical application to the skin of mice as a 1% solution, XEN103 induces pronounced sebaceous gland atrophy with a rapid onset after a few days of dosing, both sebaceous gland numbers and size being reduced by 50 to 75%, and without any signs of skin irritation. We show that the effect is due to the local action of the compound on SCD-1 in the skin and occurs in the presence of only minimal plasma exposure. Based on these observations, SCD-1 inhibitors such as XEN103 may provide an opportunity to develop a novel topical therapy for acne, as a disease characterized by overproduction of sebum from hypertrophic sebaceous glands.

6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate, a steroid sulfatase inhibitor for the treatment of androgen- and estrogen-dependent diseases.

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.

Inhibition of T-cell activation in vitro in human peripheral blood mononuclear cells by pimecrolimus and glucocorticosteroids and combinations thereof.

Abstract:  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 ∼ betamethasone 17‐valerate ∼ 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.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis

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.

SDZ 281‐977: A modified partial structure of lavendustin A that exerts potent and selective antiproliferative activities in vitro and in vivo

The chemical derivatization of biologically active microbial metabolites continues to be a promising approach to the identification of new drugs. We recently synthesized the novel antiproliferative compound SDZ 281‐977, 5‐[2‐(2,5‐dimethyoxyphenyl)ethyl]‐2‐hydroxy‐benzoic acid methylester, a derivative of the EGF receptor tyrosine kinase inhibitor lavendustin A. Here we report on our studies of the anticancer efficacy and the mode of action of SDZ 281‐977. The growth of both the human pancreatic tumor cells MIA PaCa‐2 and the human vulvar carcinoma cells A431 was inhibited in the low micromolar range. Tumors from these cells were induced in nude mice and were shown to respond to orally or intravenously administered SDZ 281‐977. In contrast, no antitumor effect was detected in rats bearing dimethylbenzanthracene‐induced mammary tumors. Studies in mice indicated that SDZ 281‐977 was neither immunosuppressive nor hematosuppressive at doses effectively inhibiting tumor growth. Surprisingly, the mode of action of SDZ 281‐977 apparently does not involve inhibition of EGF receptor tyrosine kinase, because, in contrast to lavendustin A, SDZ 281‐977 failed to inhibit this enzyme in a cell‐free assay. The mechanism of the antiproliferative effect can be explained on a cellular level by the ability of the compound to arrest cells in mitosis. SDZ 281‐977 is thus the first example of an antimitotic agent derived from the potent tyrosine kinase inhibitor lavendustin A. The therapeutic potential of SDZ 281‐977 is enhanced by the fact that it is not subject to multidrug resistance, because tumor cells expressing the multidrug resistance phenotype were as sensitive to SDZ 281‐977 as their nonresistant counterparts. In conclusion, SDZ 281‐977 represents a novel lavendustin A derivative with potent antiproliferative properties in vitro and in vivo that may be explained on the basis of its antimitotic effects. SDZ 281‐977 may be a candidate drug for the treatment of selected cancers, including those expressing the multidrug resistance phenotype.