Data_Sheet_1_Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.docx

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3–4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.</p

Data_Sheet_2_Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.docx

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3–4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.</p

Important Role of CYP2J2 in Protein Kinase Inhibitor Degradation: A Possible Role in Intratumor Drug Disposition and Resistance

<div><p>We have investigated <i>in vitro</i> the metabolic capability of 3 extrahepatic cytochromes P-450, CYP1A1, 1B1 and 2J2, known to be over-expressed in various tumors, to biotransform 5 tyrosine kinase inhibitors (TKI): dasatinib, imatinib, nilotinib, sorafenib and sunitinib. Moreover, mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in 6 hepatocellular and 14 renal cell carcinoma tumor tissues and their surrounding healthy tissues, was determined.</p><p>Our results show that CYP1A1, 1B1 and especially 2J2 can rapidly biotransform the studied TKIs with a metabolic efficiency similar to that of CYP3A4. The mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in tumor biopsies has shown i) the strong variability of CYP expression and ii) distinct outliers showing high expression levels (esp. CYP2J2) that are compatible with high intratumoral CYP activity and tumor-specific TKI degradation.</p><p>CYP2J2 inhibition could be a novel clinical strategy to specifically increase the intratumoral rather than plasma TKI levels, improving TKI efficacy and extending the duration before relapse. Such an approach would be akin to beta-lactamase inhibition, a classical strategy to avoid antibiotic degradation and resistance.</p></div

Michaelis-Menten kinetic parameters.

<p>Michaelis-Menten kinetic parameters determined in microsome incubations with cDNA expressed CYP1A1, 1B1, 2J2 and 3A4 isozymes. A) Affinity constants, Km in µM, for different TKI metabolic pathways. B) Relative intrinsic clearance (Vmax/Km; in arbitrary unit [arb]) for the same TKI metabolic pathways, expressed as fold differences of the intrinsic clearance of CYP3A4, the major hepatic enzymes involved in TKI degradation. Abbreviations: OH-: hydroxyl-metabolites, NO-: N-oxide metabolite. Imatinib metabolites are numbered in accordance to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095532#pone.0095532-Rochat4" target="_blank">[18]</a>.</p>(*)<p>Results obtained in our previous published study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095532#pone.0095532-Rochat4" target="_blank">[18]</a>.</p

Disappearance of TKI in in culture media.

<p>Disappearance of TKI (2.5 µM added in the culture media at T = 0) in culture media of HepG2 cell infected with lacZ (control), CYP1A1, 1B1, 2J2 and 3A4. Disappearance of TKI was determined by LC-MS analysis and is given in % of the levels at T = 0. A) Remaining TKI in the culture media after 72 hours (representative experiment from 3 experiments in percent of initial TKI levels). Time course of B) dasatinib, C) nilotinib and D) sorafenib disappearance in the incubations are depicted for cytochrome P-450 (CYP) 1A1, 1B1 and 2J2 isozymes.</p

mRNA relative expression of CYP1A1, 1B1, 2J2 and 3A4.

<p>Mean +/− SD of the total mRNA relative expression of CYP1A1, 1B1, 2J2 and 3A4 in A) hepatocellular carcinoma (HCC; N = 6 and B) renal cell carcinoma (RCC; N = 14) and the healthy tissues counterpart surrounding the tumors. Correlation between total mRNA expression found in healthy tissues for C) CYP2J2 for HCC and for D) CYP1B1 for RCC and E) CYP2J2 for RCC. Outliers with very high mRNA expression can be observed and are highlighted with arrows.</p

Disappearance rates of TKI.

<p>Disappearance rates of TKI (2.5 µM at T = 0) in microsomal incubations in the first 10 minutes. Microsomal incubations were performed with 0.25 nM of TKI, 20 picomoles of CYP1A1, 1B1, 2J2 or 3A4. The disappearance rate is expressed in nmole of TKI degraded per min and nmole of CYP.</p

Disappearance of TKI in <i>in vitro</i> incubations.

<p>Disappearance of dasatinib, imatinib, nilotinib, sorafenib and <i>Z</i>-sunitinib (each TKI = 2.5 µM at T = 0) from <i>in vitro</i> incubations performed with cDNA expressed CYP1A1, 1B1, 2J2 and 3A4 isozymes. A) Remaining TKI in the microsomal incubations after 30 min., are given in percent of initial TKI levels (T = 0; SD corresponds to the maximum analytical deviation found in the LC-MS assays). Time course of B) dasatinib, C) nilotinib and D) sorafenib degradations in the incubations are depicted for cytochrome P-450 (CYP) 1A1, 1B1, 2J2 and 3A4 isozymes.</p

Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian <i>Eudistoma</i> sp. as active. Novel heterocyclic alkaloids eudistidines A (<b>1</b>) and B (<b>2</b>) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (<b>1</b>) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)­pyrimidin-2-amine (<b>3</b>) and 4-methoxy-phenylglyoxal (<b>4</b>), while eudistidine B (<b>2</b>) was synthesized in a similar fashion with glyoxylic acid (<b>5</b>) in place of <b>4</b>. Naturally occurring eudistidine A (<b>1</b>) effectively inhibited CH1/C-TAD binding with an IC₅₀ of 75 μM, and synthetic <b>1</b> had similar activity. The eudistidine A (<b>1</b>) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein–protein binding events

World of Children as Media Users

This dissertation consists of two parts - a theoretical and an analytical, empirical part. In the theoretical segment, there is a summary of theoretical knowledge about celebrities and their relationship to society and the public, a comparison between celebrities and real authorities, an overview of mass-communication and its influence on the socialisation of children and a series of advantages and disadvantages of the impact of mass-communication on children. The final stage of the theoretical segment characterises, from the point of view of developmental psychology, the two age groups that will be compared in the second segment. The empirical segment answers such questions as how much do children use media in their leisure time, what types of media are most popular amongst them, which celebrities do the children admire and why are these celebrities important for them in particular. The empirical segment is based on analysis of data taken from questionnaires created purposely for qualitative-quantitative research. These questionnaires were given to students in the 4th and 6th grades of an elementary school in Polepy in the Czech Republic (ages 10/11 and 12/13 respectively). The goal of this dissertation is to show the trending habits of children as mass- media users