<i>In Vivo</i> Activity and Pharmacokinetics of Nemorosone on Pancreatic Cancer Xenografts

<div><p>Pancreatic cancer is one of the leading cancer-related causes of death in the western world with an urgent need for new treatment strategies. Recently, hyperforin and nemorosone have been described as promising anti-cancer lead compounds. While hyperforin has been thoroughly investigated <i>in vitro</i> and <i>in vivo</i>, <i>in vivo</i> data for nemorosone are still missing. Thus, we investigated the growth-inhibitory potential of nemorosone on pancreatic cancer xenografts in NMRI nu/nu mice and determined basic pharmacokinetic parameters. Xenograft tumors were treated with nemorosone and gemcitabine, the current standard of care. Tumor sections were subjected to H&E as well as caspase 3 and Ki-67 staining. Nemorosone plasma kinetics were determined by HPLC and mass spectrometry. Induction of CYP3A4 and other metabolizing enzymes by nemorosone and hyperforin was tested on primary hepatocytes using qRT-PCR. At a dose of 50 mg/kg nemorosone per day, a significant growth-inhibitory effect was observed in pancreatic cancer xenografts. The compound was well tolerated and rapidly absorbed into the bloodstream with a half-life of approximately 30 min. Different metabolites were detected, possibly resembling CYP3A4-independent oxidation products. It is concluded that nemorosone is a potential anti-cancer lead compound with good bioavailability, little side-effects and promising growth-inhibitory effects, thus representing a valuable compound for a combination therapy approach.</p> </div

Hyperforin and nemorosone.

<p>Closely related chemical structures of hyperforin (left) and nemorosone (right), two polycyclic polyprenylated acylphloroglucinols.</p

Tumor volume and body weight development in MIA-PaCa-2 xenograft mice.

<div><p>Mice were treated with daily i.p. injections of 50 mg/kg nemorosone, vehicle only or 120 mg/kg gemcitabine at the indicated time points (green dots). Tumor volume was measured 2-3 times per week using a digital caliper. Values represent the mean ± SD of 8 animals per group.</p> <p>* p < 0.05, ** p < 0.01 (compared to the vehicle control).</p></div

Plasma concentration of nemorosone and its metabolites.

<p>Plasma samples were taken at pre-defined time points after i.p. application of 50 mg/kg nemorosone. Nemorosone and its metabolites were quantified by RP-HPLC with the help of a calibration curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074555#pone.0074555.s003" target="_blank">Figure S3</a>). (<b>A</b>) Plasma concentration of nemorosone (upper green line) and its potential metabolites (M01 to M09). (<b>B</b>) Pharmacokinetics of nemorosone were best described by a two-compartment model with a half-life of 30 min.</p

Induction of selected genes involved in metabolism.

<p>Primary human hepatocytes were treated with the indicated concentrations of rifampicin, hyperforin, nemorosone or vehicle only. RNA was extracted after 48 h and subjected to qRT-PCR analysis to detect expression changes of selected genes involved in drug metabolism. Expression values are relative to the vehicle control and represent means ± SD of triplicate measurements.</p

Hematoxylin-eosine and immunohistochemical staining of tumor sections.

<p>Sections of 5 µm of nemorosone-treated and control tumors were stained with hematoxylin-eosine (H&E; <b>A</b> and <b>B</b>) or immunohistochemically analyzed with antibodies directed against active caspase 3 (<b>C</b> and <b>D</b>) or Ki-67 (<b>E</b> and <b>F</b>). Treated tumors demonstrated reduced tumor mass due to apoptosis/necrosis (black arrows) and a lower number of proliferating cells as compared to the control (dark-brown cells).</p

Primary cilia (PC) in intraductal papillary-mucinous neoplasia (IPMN).

<p><b>A/B)</b> Two benign and <b>(C)</b> one malignant IPMN without PC. <b>(D)</b> In stromal tissue surrounding IPMN lesions, an increased fraction of cilia carrying cells (<b>E</b>, Kruskal-Wallis test: <i>p</i> < 0. 01) and an increased length of PC (<b>F</b>, Kruskal-Wallis test: <i>p</i> < 0. 001) were detected compared to normal pancreas (donor). Post-hoc Dunns test: *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0. 001, acetylated α-tubuline: red, γ-tubuline: green, DAPI: blue. Mean ± SEM.</p

Schematical structure and visualization of primary cilia (PC).

<p><b>(A)</b> Cross section of primary cilia, consisting of 9 microtubule duplets containing α and β tubline. <b>(B)</b> Structure of PC. Cilia are fixed by basal bodies on the cell membrane. <b>(C)</b> Immunofluorescent visualization of PC in Panc1 cells. The axonem is stained in red (acetylated α-tubline), the basal body is stained green (γ-tubline). <b>(D)</b> Immunofluorescent visualization of PC and the spindle apparatus in the BxPx3 cancer cell line.</p

Re-Distribution of primary cilia (PC) from epithelial to stromal cells in pancreatic carcinogenesis.

<p>The fraction of primary cilia (PC) carrying cells decreases in epithelia, while there is a simultaneous increase of ciliated cells in stromal tissue during pancreatic carcinogenesis (from PanIN 1A to pancreatic G1/G2 ductal adenocarcinomas (PDAC). Mean ± SEM.</p

Primary cilia (PC) in pancreatic intra-epithelial neoplasia (PanIN) and pancreatic cancer cells (PDAC).

<p><b>(A)</b> Comparison between PanIN 1a (*) and PanIN 1B (#), the latter showing papillary epithelium and reduced number of cilia. <b>(B)</b> PanIN 3 lesion. Epithelial cells do not carry cilia while PC are present in stromal cells (*). <b>(C)</b> Loss of epithelial PC in PDAC (indicated by arrows), while in the stromal cells there is a noticeable increase of both the length of PC and the number of PC carrying cells (*). <b>(D)</b> Length of epithelial PC is decreased in PanIN lesions in comparison to normal pancreas (donor). <b>(E)</b> Gradual loss of epithelial PC in PanIN lesions. In PDAC, almost no epithelial PC were detected. <b>(F)</b> In stromal tissue around PanIN lesions and PDAC (G1/G2), an increased length of PC and <b>(G)</b> increased fraction of cilia carrying cells was evident compared to normal pancreas (donor). Kruskal-Wallis test: <i>p</i> < 0.0001, post-hoc Dunns test: ***<i>p</i> < 0.001 vs. donor, **<i>p</i> < 0.01 vs donor, *<i>p</i> < 0.01 vs donor, ###<i>p</i> < 0. 001 vs PanIN 1A, ##<i>p</i> < 0. 01 vs PanIN 1A, +++<i>p</i> < 0. 001 vs PanIN 1B, +<i>p</i> < 0. 05 vs PanIN 1B, acetylated α-tubuline: red, γ-tubuline: green, DAPI: blue. Mean ± SEM.</p