Histolopathology.

<p>Histologic features of liver PHEO lesions stained with H&E 24 hours after <i>i.p</i>. vehicle alone (left) or with <i>i.p.</i> LB1 at 1.5 mg/kg plus TMZ by gavage at 80 mg/kg (right). Exposure to a single <i>i.p.</i> injection of vehicle showed a homogeneous field of healthy appearing tumor cells, whereas combination treatment resulted in extensive necrosis of tumor cells.</p

Western blot on MPC cells.

<p>Changes in pAKT, p53, pMDM2, and pPlk-1 after 24 hours treatment on MPC cells with 5 µM of LB1, 50 µM of TMZ and combination of both drugs. (A) Western blots show that LB1 exposure increases pAKT compared to control MPC cells (untreated, only vehicle). TMZ does not noticeably change the expression of pAKT and combination of LB1 and TMZ highly increases pAKT expression. (B) A demonstration of markedly increased expression of p53 after TMZ treatment but inhibition of expression by exposure to LB1. Noticeable increase in the expression of pMDM2 in MPC cells treated with LB1 alone or in combination with TMZ. (C) Noticeable increases in expression of pPlk1 in MPC cells after exposure to the combination of drugs.</p

Effect of LB1 and TMZ on tumor cell cycle and apoptosis.

<p>(A) Cell cycle analysis of MPC cells in exponential growth exposed for 48 hours to vehicle alone; LB1 alone at 5 µM; TMZ at 50 µM and; LB1 at 5 µM and TMZ at 50 µM. (B) PARP expression changes in 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses.</p

Western blot on PHEO tumors.

<p>Changes in the state of phosphorylation and abundance of small pAKT, p53, pMDM2, and pPlk-1 24 hours after treatment of mice bearing hepatic tumors with vehicle, LB1 alone at 1.5 mg/kg by gavage, TMZ alone by gavage at 80 mg/kg, of both drugs at the same doses. (A) Western blots show that LB1 exposure increases pAKT in control-untreated tumors and treated tumors. TMZ does not change the expression of pAKT and combination of LB1 plus TMZ highly increases pAKT expression. (B) Western blots demonstrate marked increased expression of p53 after TMZ but complete inhibition of this induction by exposure to LB1 accompanied by an increase in the expression of pMDM2 in tumor cells exposed to LB1 alone or in combination with TMZ (C) Expression of pPlk1 shows a marked increase in tumors after exposure to the combination of drugs.</p

<i>In vivo</i> anti-tumor activity of LB1 and TMZ and histological examination.

<p>Effects of treatment upon growth and molecular changes in hepatic tumors: (A) MRI images of untreated mice at weeks 5, 6, and 7 following intravenous injection of MPC cells and a photomicrograph showing the growth hepatic lesions and Alzet minipump. Barbed arrow indicates the gall bladder. Plain arrows indicate the same tumor nodules over time. (B) Inhibition of total hepatic tumor volume by LB1 alone at 1.5 mg/kg daily for 14 days by <i>c.i.</i> administered at 5^th day after MPC cells injection; TMZ alone at 80 mg/kg for 3 doses administered at 15^th day after MPC cells injection; and, combination of both drugs. (C) Survival curve combining the data from the study depicted in (B) a total of 10 control animals, 10 animals with combination treatment LB1 and TMZ, 5 animals with TMZ alone, and 5 animals with LB1 alone. Kaplan-Meier analysis revealed that survival following LB1 plus TMZ was significantly greater than with LB1 alone and TMZ alone (log rank, <i>P</i><0.0001). (D) MRI images of mice, treated with the combination of LB1 by <i>c.i</i>. for 14 days and 3 doses of TMZ, at week 7, 9, and 12. Partial response of treatment is presented with delayed appearance of hepatic tumors compared to untreated group. Complete response presents absence of hepatic tumors after treatment. A photomicrograph of the liver of one treated animal at week 12, showing the absence of gross tumor, and the presence of fibrous scar tissue (arrows). (E) Inhibition of estimated total hepatic tumor volume by combination treatment of LB1 and TMZ. LB1 at 1.5 mg/kg daily for 14 days by <i>c.i</i>. administered at 5^th day after MPC cells injection and TMZ at 80 mg/kg every 3 days for 14 doses beginning on 15^th day after MPC cells injection (with combination or alone). (F) Survival curve combining the data described in E. Total of 12 control animals, 5 animals for TMZ and 7 animals for combination treatment LB1 and TMZ. Survival of animals with combined treatment were significantly greater compared to controls (log rank, <i>P</i><0.0001). (G) Serial MRI images of mice, treated with the combination of LB1 and 14 doses TMZ with partial and complete responses. (H) Histologic features of liver PHEO at week 12 stained with H&E receiving no treatment or LB1 by <i>c.i.</i> and three doses of TMZ as described in D. Untreated animals showed intrahepatic deposits of cancer cells whereas the liver of an animal receiving both drugs that had no gross evidence of tumor revealed normal parenchyma and fibrous tissue, believed to be scarring at former sites of tumor masses. (I) Survival curves of animals treated with LB1 and 14 doses of TMZ when administration started at the same time, on day 5 after MPC cells injection. (n = 5 treated animals with LB1 plus TMZ, n = 5 for controls; log rank, <i>P</i> = 0.0035).</p

<i>In vitro</i> anti- proliferative activity of LB1 and TMZ and their combination.

<p>Inhibition of growth of MPC cells in culture: (A and B) Exposure for 3 days to increasing concentrations of LB1 or TMZ. (C–E) Exposure to increasing concentrations of LB1 plus TMZ. (F) Synergy analysis was done based on data from (A–E) using CalsuSyn software. CI values: C = 1 as additivity; C<1 as synergy; C>1 as antagonism. Combo 1 presents combination of 5 µM of LB1 and 100, 200, 300 µM of TMZ; Combo 2 presents combination of 7.5 µM of LB1 and 100, 200, 300 µM of TMZ; and Combo 3 10 µM of LB1 and 100, 200, 300 µM of TMZ.</p

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure