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. © 1996 Wiley-Liss, Inc.     

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.     

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.     

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-na?ve 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.     

Estrogenic potential of 2-alkyl-4-(thio)chromenone 6-O-sulfamates: potent inhibitors of human steroid sulfatase

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.     

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

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.