PLAG (1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol) Modulates Eosinophil Chemotaxis by Regulating CCL26 Expression from Epithelial Cells

<div><p>Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (<i>Cervus nippon</i> Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.</p></div

IL-4 induced CCL26 expression is inhibited by PLAG.

<p>(A) A549 cells were treated with various doses of PLAG (1, 5, 10, 50 μg/mL) for 1 h and stimulated with IL-4 (10 ng/mL) for 24 h. The mRNA levels of following genes, VCAM-1, ICAM-1, MIF, CCL5, CCL7, CCL11, CCL13, CCL17, CCL24, and CCL26 were analyzed by conventional RT-PCR. Each reaction was repeated three times and representative images were displayed. GAPDH was used as an internal control. (B) The culture supernatant was harvested at 48 h, and the protein level of CCL26 was evaluated by ELISA. PLAG decreased CCL26 secretion from A549 cells in a dose-dependent manner. *<i>p</i><0.001, ***<i>p</i><0.05. (C) A549 cells were pretreated with PLAG (10 μg/mL) for 1 h and stimulated with IL-4 (10 ng/mL) for various lengths of time. PLAG decreased CCL26 secretion from A549 cells from the early time point. NC; negative control. *<i>p</i><0.001, **<i>p</i><0.01, *** <i>p</i><0.05. (D) HaCaT cells were treated with various doses of PLAG and stimulated with IL-4 (20 ng/mL) for 24 h. The mRNA levels of CCL 26 were analyzed by RT-PCR. (E) PLAG also decreased CCL26 secretion from HaCaT cells in a dose-dependent manner. *<i>p</i><0.001, ***<i>p</i><0.05.</p

PLAG inhibits eosinophil migration in vitro.

<p>(A) Murine eosinophils were differentiated from BM cells by treating SCF, FLT3 ligand and IL-5 for 2w. The differentiated cells were analyzed by FACS staining with SiglecF and CD11b. SiglecF+/CD11b+ cells were increased in the differentiated cells to ~25%. (B) CCR3 expression was increased in the differentiated murine eosinophils. (C-D) The supernatant of IL-4 and/or PLAG-treated A549 cells was prepared and used for the migration assay as described in Materials and Methods. The transmigration of eosinophil was decreased by PLAG treatment in both murine BM-derived eosinophils (C) and human eosinophil cell line, EoL-1 (D). *<i>p</i><0.001, **<i>p</i><0.01. (E) PLAG did not exhibit any cytotoxic effects on A549 cells at various concentrations.</p

PLAG decreases eosinophil infiltration into the airways of asthmatic mice.

<p>(A) Cells were harvested from the BALFs of OVA-challenged mice and analyzed by CBC. The number of infiltrated immune cells were increased in OVA-challenged mice. PLAG or DEX treatment inhibited immune cell infiltration into airways to the level of normal control. (B) After CBC, cells were collected from the BALF, and analyzed by flow cytometry. PLAG and DEX treatment induced the recovery of PMN cell populations which were increased in the allergic mice. (C) SiglecF+/CD11b+ eosinophils were increased in the OVA-challenged mice, which were decreased in PLAG- or DEX-treated mice.</p

PLAG alleviates inflammatory phenotypes in allergic asthma model.

<p>(A) Mice were sensitized and challenged by OVA, and the efficacy of PLAG and DEX was tested in the generated asthma model as depicted. (B-C) The lung tissues from the OVA-sensitized and–challenged mice were stained with H&E, and the representative images were displayed (X200 magnification, B). Infiltrated immune cells were counted, calculated and displayed as inflammation index. Eosinophils from each of the three mice per treatment group were analyzed morphometrically and mean eosinophil counts per 50 × 50 μm area ± SD were displayed. Four such areas were measured from each tissue section. PLAG was the most effective with a dose of 50 mg/Kg as showed in (B) and calculated in (C). *<i>p</i><0.001. (D) The expression level of CCL26 in BALFs of OVA-challenged mice was analyzed by ELISA. Each sample was analyzed in triplicate and displayed as mean ± SD. *<i>p</i><0.001.</p

Discovery and Early Clinical Evaluation of BMS-605339, a Potent and Orally Efficacious Tripeptidic Acylsulfonamide NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

The discovery of BMS-605339 (<b>35</b>), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropyl­acylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1′ site of the protease. The identification of <b>35</b> was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound <b>35</b> which demonstrated antiviral activity in HCV-infected patients

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (<b>2</b>) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to <b>2</b>, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like <b>2</b>, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (<b>1</b>) from clinical trials. Structure–activity relationships (SARs) at each of the structural subsites in <b>2</b> were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (<b>29</b>)