Oleylamine-carbonyl-valinol inhibits auto-phosphorylation activity of native and T315I mutated Bcr-Abl, and exhibits selectivity towards oncogenic Bcr-Abl in SupB15 ALL cell lines

Chronic myeloid leukemia (CML) is characterized by the presence of p210Bcr-Abl which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of CML. Despite high rates of clinical response, CML patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein kinase domain. Previously, we have identified oleic acid as the active component in the mushroom Daedalea gibbosa that inhibited the kinase activity of Bcr-Abl. Here, we report that the oleyl amine derivatives, S-1-(1-Hydroxymethyl-2-methyl-propyl)- 3-octadec-9-enyl-urea [oleylaminocarbonyl-L-Nvalinol, oroleylaminocarbonyl-S-2-isopropyl-N-ethanolamine, oleylamine-carbonyl-L-valinol] (cpd 6) and R-1-(1-Hydroxymethyl- 2-methyl-propyl)-3-octadec-9-enyl-urea [oleylamineocarbonyl- D-N-valinol, oleylaminocarbonyl-R-2-isopropyl- N-ethanolamine, or oleylamine-carbonyl-D-valinol] (cpd 7), inhibited the activity of the native and T315I mutated Bcr-Abl. Furthermore, cpd 6 and 7 exhibited higher activity towards the oncogenic Bcr-Abl in comparison to native c-Abl in SupB15 Ph-positive ALL cell line.This work was supported, in part, by DFG-RU 728/3-2 to MR, YN and JM

US Patent App. 14/415,863

The invention provides fatty acid derivatives for use in a method of treatment of at least one disease, disorder or condition selected from anxiety, depression, conditions associated menopause, stress, bipolar disorder, neuropathic pain and fibromyalgia

The effect of Relacin on Rel-ribosomes interaction.

<p>(<b>A</b>) Relacin inhibits dissociation of Rel/Spo from the ribosome. The relative amount of Rel/Spo (<i>D. radiodurans</i>) bound to purified ribosomes was quantified following the addition of increasing levels of Relacin. Rel/Spo molecules associated with 70S complexes were detected by Western blot analysis (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>). Histogram indicates the average of two independent biological repeats. Error bars represent the range. (<b>B</b>) Ribosome independent inhibition of (p)ppGpp synthesis. The constitutively active, ribosome-independent RelAC638F (<i>E. coli</i>) protein was treated with increasing concentrations of Relacin, as indicated (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>) in the presence or absence of isolated ribosomes. Shown is the average of duplicates of a representative experiment. Error bars represent the range.</p

Relacin affects bacterial growth and survival.

<p>(<b>A</b>) Relacin influences entry into stationary phase. Shown are growth curves of wild type <i>B. subtilis</i> (PY79) cells grown in CH medium at 37°C in the absence or presence of increasing concentrations of Relacin added at OD₆₀₀ 0.2. (<b>B</b>) Relacin exerts a toxic effect. The viability of <i>B. subtilis</i> (PY79) cells was evaluated by counting colony forming units (CFU) after 24 hours of incubation in CH medium at 37°C in the absence or presence of increasing concentrations of Relacin added at OD₆₀₀ 0.2. Shown is a representative experiment, in which SD was calculated from at least three repeats for each concentration. (<b>C</b>) Long term effect of Relacin treatment. The effect of Relacin (2 mM) on the viability of wild type <i>B. subtilis</i> (PY79) cells or <i>ΔrelA</i> (ME215) cells was measured. Cells were incubated in CH medium at 37°C, and viability was determined by counting colony forming units (CFU). Relacin was added at OD₆₀₀ 0.2. Shown is a representative experiment, in which SD was calculated from at least three repeats for each point. (<b>D</b>) The toxic effect of Relacin on GAS. The effect of Relacin (2 mM) on the viability of wild type GAS (JRS4) cells, incubated in THY medium at 37°C, was evaluated as in (C).</p

Relacin influences the sporulation process in <i>Bacilli</i>.

<p>(<b>A</b>) Relacin inhibits sporulation. Microscopy images of sporulating wild type <i>B. subtilis</i> (PY79) cells in the absence or presence of Relacin, added at time 0 of sporulation at the indicated concentrations. Upper panels: cells at t = 2 hr of sporulation stained with the fluorescent membrane dye FM1–43. Arrows indicate position of polar septa. Lower panels: phase contrast images of cells at t = 24 hr of sporulation. Scale bars correspond to 1 µm. (<b>B</b>) Relacin inhibits expression of the mid-sporulation protein SpoIIQ. Fluorescence microscopy images of <i>B. subtilis</i> (PE128) cells harboring <i>spoIIQ-gfp</i> at t = 4 hr of sporulation, in the absence (upper panels) or presence (lower panels) of Relacin (1 mM), added at time 0 of sporulation. Shown are phase contrast (red), GFP fluorescence (green) and overlay images. Scale bar corresponds to 1 µm. (<b>C–D</b>) Relacin inhibits <i>Bacilli</i> spore formation. The formation of heat resistant <i>B. subtilis</i> (PY79) (C) and <i>B. anthracis</i> (Sterne) (D) spores was monitored in the absence or presence of Relacin, added at the indicated concentrations at time 0 of sporulation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). Shown are representative experiments, in which SD was calculated from at least three repeats for each concentration. (<b>E</b>) Relacin added at different time points during sporulation inhibits spore formation. Inhibition of spore formation by wild type <i>B. subtilis</i> (PY79) cells was evaluated after addition of Relacin (1 mM) at the indicated time points of sporulation. Inhibition was determined using a heat resistance assay (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>) and is expressed relative to untreated cultures. Shown is a representative experiment, in which SD was calculated from at least three repeats for each time point.</p

Relacin inhibits the activity of Rel proteins.

<p>(<b>A</b>) Chemical structure of Relacin. (<b>B–C</b>) Relacin inhibits (p)ppGpp synthesis <i>in vitro</i>. Representative autoradiograms of PEI thin-layer chromatography showing a decrease in labeled (p)ppGpp synthesized from α-³²P-GTP precursor by purified RelA (<i>E. coli</i>) (B) or Rel/Spo (<i>D. radiodurans</i>) (C) with increasing concentrations of Relacin, as indicated (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>). Shown is the average of duplicates of a representative experiment. Error bars represent the range. (<b>D</b>) Relacin inhibits (p)ppGpp synthesis in living <i>B. subtilis</i> (PY79) cells. The accumulation of (p)ppGpp in response to amino acid starvation, induced by the addition of SHX, was monitored in the absence or presence of increasing concentrations of Relacin, as indicated. The (p)ppGpp level was determined using PEI thin-layer chromatography as in (B–C) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>). Shown is the average of duplicates of a representative experiment. Error bars represent the range.</p

Relacin affects biofilm formation in <i>Bacillus subtilis.</i>

<p>(<b>A</b>) Relacin inhibits pellicle biofilm formation. Wild type <i>B. subtilis</i> (3610) cells were induced to form biofilms in liquid standing cultures in the absence or presence of Relacin at the indicated concentrations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). Cultures were photographed after 3 days. Scale bar corresponds to 5 mm. (<b>B</b>) Relacin inhibits biofilm colony formation. Wild type <i>B. subtilis</i> (3610) cells were induced to form biofilms on solid medium, in the absence or presence of Relacin (1 mM) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). Colonies were photographed after 24 hours. Scale bar corresponds to 3 mm. (<b>C</b>) Relacin causes biofilm disintegration. <i>B. subtilis</i> (YA224) cells harboring P<i>_rrnE-gfp</i> fusion were induced to form biofilm in liquid standing cultures in the absence or presence of Relacin (1 mM) as indicated (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). Biofilms were visualized after 3 days using confocal microscopy (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>). Green signal corresponds to GFP produced from P<i>_rrnE</i>. Scale bar corresponds to 50 µm. (<b>D</b>) Relacin reduces biofilm biomass. <i>B. subtilis</i> (YA224) cells harboring P<i>_rrnE-gfp</i> fusion were induced to form biofilm in liquid standing cultures in the absence or presence of Relacin (1 mM) as indicated. Biofilm pellicles were disintegrated and cell biomass was evaluated by GFP fluorescence measurements and is displayed in arbitrary units [AU] (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). Shown is the average of two independent biological repeats. Error bars represent the range. (<b>E</b>) Relacin leads to cell death within the biofilm. <i>B. subtilis</i> (YA224) cells harboring P<i>_rrnE-gfp</i> fusion were induced to form biofilm in liquid standing cultures in the absence or presence of Relacin (1 mM) as indicated (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#ppat.1002925.s007" target="_blank">Text <i>S1</i></a>). After 3 days, biofilms were stained with PI to indicate cell death and observed by confocal microscopy (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002925#s4" target="_blank">Materials and Methods</a>). Shown are GFP fluorescence produced from P<i>_rrnE</i> (green), PI staining (red), and overlay images. Scale bar corresponds to 50 µm.</p

The effect of pH and ionic strength of dissolution media on in-vitro release of two model drugs of different solubilities from HPMC matrices

The evaluation of the effects of different media ionic strengths and pH on the release of hydrochloroth-iazide, a poorly soluble drug, and diltiazem hydrochloride, a cationic and soluble drug, from a gel forming hydrophilic polymeric matrix was the objective of this study. The drug to polymer ratio of formulated tablets was 4:1. Hydrochlorothiazide or diltiazem HCl extended release (ER) matrices containing hypromellose (hydroxypropyl methylcellulose (HPMC)) were evaluated in media with a pH range of1.2–7.5, using an automated USP type III, Bio-Dis dissolution apparatus. The ionic strength of the media was varied over a range of 0–0.4 M to simulate the gastrointestinal fed and fasted states and various physiological pH conditions. Sodium chloride was used for ionic regulation due to its ability to salt out polymers in the mid range of the lyotropic series. The results showed that the ionic strength had a pro-found effect on the drug release from the diltiazem HCl K100LV matrices. The K4M, K15M and K100M tablets however withstood the effects of media ionic strength and showed a decrease in drug release to occur with an increase in ionic strength. For example, drug release after the 1 h mark for the K100M matrices in water was 36%. Drug release in pH 1.2 after 1 h was 30%. An increase of the pH 1.2 ionicstrength to 0.4 M saw a reduction of drug release to 26%. This was the general trend for the K4M and K15M matrices as well. The similarity factor f2 was calculated using drug release in water as a reference.Despite similarity occurring for all the diltiazem HCl matrices in the pH 1.2 media (f2= 64–72), increases of ionic strength at 0.2 M and 0.4 M brought about dissimilarity. The hydrochlorothiazide tablet matrices showed similarity at all the ionic strength tested for all polymers (f2= 56–81). The values of f2 however reduced with increasing ionic strengths. DSC hydration results explained the hydrochlorothiazide release from their HPMC matrices. There was an increase in bound water as ionic strengths increased. Texture analysis was employed to determine the gel strength and also to explain the drug release for the diltiazem hydrochloride. This methodology can be used as a valuable tool for predicting potential ionic effects related to in vivo fed and fasted states on drug release from hydrophilic ER matrices