Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure-activity relationship, in vitro and in vivo evaluation as antitumor agents.

The design, synthesis, and biological evaluation of a series novel N1­methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11-13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 µM, respectively. Compounds 14-16 inhibited the polymerization of tubulin with IC50s near ∼1 µM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented ßIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9-17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (ßIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.

The 3-D conformational shape of N-naphthyl-cyclopenta[d]pyrimidines affects their potency as microtubule targeting agents and their antitumor activity.

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6'-methoxynaphthyl-1'-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5'-methoxynaphthyl-2'-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.

Evaluation of skin irritation following weathered crude oil exposure in two mouse strains.

Petroleum crude oil spills are common and vary in size and scope. Spill response workers throughout the course of remediation are exposed to so-called weathered oil and are known to report diverse health effects, including contact dermatitis. A murine model of repeated exposure to weathered marine crude oil was employed utilizing two strains of mice, C57BL/6 and BALB/c, to investigate the pathology of this irritant and identify the principal hydrocarbon components deposited in skin. Histopathology demonstrated clear signs of irritation in oil-exposed skin from both mouse strains, characterized by prominent epidermal hyperplasia (acanthosis). BALB/c mice exposed to oil demonstrated more pronounced irritation compared with C57BL/6 mice, which was characterized by increased acanthosis as well as increased inflammatory cytokine/chemokine protein expression of IL-1ß, IL-6, CXCL10, CCL2, CCL3, CCL4, and CCL11. A gas chromatography/mass spectrometry method was developed for the identification and quantification of 42 aliphatic and EPA priority aromatic hydrocarbons from full thickness skin samples of C57BL/6 and BALB/c mice exposed to oil samples. Aromatic hydrocarbons were not detected in skin; however, aliphatic hydrocarbons in skin tended to accumulate with carbon numbers greater than C16. These preliminary data and observations suggest that weathered crude oil is a skin irritant and this may be related to specific hydrocarbon components, although immune phenotype appears to impact skin response as well.

IL-6 Negatively Regulates IL-22Rα Expression on Epidermal Keratinocytes: Implications for Irritant Contact Dermatitis.

Irritant Contact Dermatitis (ICD) is characterized by epidermal hyperplasia and inflammatory cytokine release. IL-6 has been shown to be involved in the pathogenesis of ICD; however, the involvement of the IL-22/IL-22Rα axis and its relation to IL-6 in the inflammatory response following irritant exposure are unknown. Using a chemical model of ICD, it was observed that mice with a keratinocyte-specific knockout of IL-6Rα (IL-6Rα Δker) presented with increased inflammation and IL-22Rα and IL-22 protein expression relative to WT following irritant exposure, indicating that IL-6Rα deficiency in epidermal keratinocytes leads to the upregulation of IL-22Rα and its ligand during ICD. Furthermore, it was shown that IL-6 negatively regulates the expression of IL-22Rα on epidermal keratinocytes. This effect is functional as the effects of IL-22 on keratinocyte proliferation and differentiation were markedly reduced when keratinocytes were pretreated with IL-6 prior to IL-22 treatment. These results show that IL-6 modulates the IL-22/IL-22Rα axis in the skin and suggest that this occurrence may be associated with the increased epidermal hyperplasia and exacerbated inflammatory response observed in IL-6Rα Δker mice during ICD.

Interleukin 6 Function in the Skin and Isolated Keratinocytes Is Modulated by Hyperglycemia.

Diabetes currently affects over twenty-five million Americans. Annual health care cost of diabetes exceeds $254 billion and is associated with a distinct set of diabetic complications that include delayed wound healing and diabetic ulcers. Interleukin 6 (IL-6) plays an important role in wound healing and is known to be elevated in the serum of both type I and type II diabetes patients. This study assesses the expression and function of IL-6 in the hyperglycemic epidermis and keratinocyte culture. Streptozotocin-treated mice were wounded six weeks after induction of hyperglycemia. Wound closure, protein, and mRNA expression were assessed up to 13 days of postwounding. Wound closure was delayed 4-5 days in hyperglycemic animals. Hyperglycemic wounds displayed greater IL-6 and IL-6Rα protein expression at 1, 7, and 10 days of postwounding compared to euglycemic control. However, IL-6Rα mRNA expression was reduced at all time points beyond day 1, while IL-6 mRNA expression did not significantly differ at any time point. SOCS3 mRNA expression was higher in the hyperglycemic skin at every time point. Imaging of fluorescent immunohistology also revealed significantly lower expression of SOCS3, but higher nuclear pSTAT3 in the epidermis of the hyperglycemic skin. Primary mouse keratinocytes cultured in high glucose for 7 days displayed 2-fold higher IL-6Rα mRNA and higher rmIL-6-induced nuclear pSTAT3, but lower SOCS3 basal levels compared to normal glucose-cultured cells. Thus, it appears that delayed diabetic skin wound healing is associated with increased induction and expression of IL-6 and its receptor, but its function in epidermal keratinocytes may be impaired.

Transcriptional profiling of irritant contact dermatitis (ICD) in a mouse model identifies specific patterns of gene expression and immune-regulation.

BACKGROUND: Irritant contact dermatitis (ICD) is a cutaneous inflammatory response to a variety of triggers that requires no sensitization and accounts for up to 80% of occupational dermatitis cases. IL-6 has been alternately associated with both allergic and irritant dermatitis and is closely linked to skin wound healing, therefore making it an ideal candidate to investigate in the mechanism of ICD. RESULTS: Despite being a well-known pro-inflammatory cytokine, IL-6 deficient (IL-6KO) mice show much more severe ICD than controls. Transcriptome analysis was employed to examine irritant-exposed and control skin samples from C57BL/6 and IL-6KO mice. Over 1900 transcripts were found differentially modulated between C57 (1184 total) and IL-6KO (802 total) mice with the magnitude of expression significantly disparate. Overall gene ontology revealed metabolic and cellular enriched functional processes but numerous pro-inflammatory and immune associated genes (Cxcl2, Cxcl3, Cxcl5, Acod, Hamp, c-Lectins, for example), keratin associated genes (Krt6b and various Krtaps), and members of the Sprr and Lce family, which promote skin barrier integrity and keratinocyte functions, were also differentially modulated. CONCLUSIONS: The altered expression of these genes may provide a potential mechanism to explain the increased ICD severity in IL-6-deficient mice. Overall, this study offers new insight into the pathogenesis of ICD, indicates new mediators/biomarkers that may influence the variability of responses to irritants and provides potential targets for therapeutic development.

Interleukin (IL)-6 modulates transforming growth factor-ß receptor I and II (TGF-ßRI and II) function in epidermal keratinocytes.

It been shown that IL-6 modulates TGF-ß1 expression in fibroblasts, however, what role IL-6 plays concerning TGF-ßR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL-6 might modulates TGF-ß receptors in skin. Skin from WT, IL-6 over-expressing mice and IL-6 treated keratinocyte cultures was analysed for TGF-ßRI and TGF-ßRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co-immunoprecipitation. IL-6 overexpression and treatment increased TGF-ßRII expression in the epidermis. IL-6 treatment of keratinocytes induced TGF-ßRI and II expression and augmented TGF-ß1-induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL-6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL-6 regulates keratinocyte function by modulating TGF-ßRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.

SOCS3 modulates interleukin-6R signaling preference in dermal fibroblasts.

AIMS: This study aims to investigate the mechanisms in the apparent preference for mitogen-activated protein kinase /ERK signaling through interleukin (IL)-6R in dermal fibroblasts. METHODS: Dermal fibroblasts isolated from IL-6KO mice were pretreated with specific ERK or STAT3 chemical inhibitors or SOCS3 specific siRNA and treated with rmIL-6. Phosphorylation was monitored via enzyme-linked immunosorbent assay or immunohistology. SOCS3 interaction with p120Ras-Gap was examined by co-immunoprecipitation and Western blot. Expression of MMP2 mRNA was assessed via real-time quantitative polymerase chain reaction. RESULTS: A dose response phosphorylation of ERK1/2 occurred while no STAT3 activation (p-Tyr705) was induced after IL-6 treatment, despite an increase in Ser727 phosphorylation. Inhibition of STAT3 in fibroblasts potentiated IL-6R induced ERK phosphorylation and vice versa. Phosphorylated SOCS3 and p120 RasGAP co-immunoprecipitated in response to IL-6 treatment. SOCS3 siRNA knockdown allowed STAT3 phosphorylation after rmIL-6 treatment. Chemical inhibition of IL-6R signaling altered the IL-6 modulated mRNA expression of MMP-2. CONCLUSIONS: SOCS3 interaction with p120 Ras-Gap plays a role in determining the preference for IL-6R signaling through ERK in dermal fibroblasts. This study provides insight into the pleiotropic nature of IL-6 and the selective signaling mechanism elicited by the IL-6R system in dermal fibroblasts. It may further indicate a method for manipulation of IL-6R function.