Synthesis, Biological Evaluation, and <i>in Vivo</i> Imaging of the first Camptothecin–Fluorescein Conjugate

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. <i>In vivo</i> and <i>ex vivo</i> imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution <i>in vivo</i>

Evaluation of the Impact of the Cancer Therapy Everolimus on the Central Nervous System in Mice

<div><p>Cancer and treatments may induce cognitive impairments in cancer patients, and the causal link between chemotherapy and cognitive dysfunctions was recently validated in animal models. New cancer targeted therapies have become widely used, and their impact on brain functions and quality of life needs to be explored. We evaluated the impact of everolimus, an anticancer agent targeting the mTOR pathway, on cognitive functions, cerebral metabolism, and hippocampal cell proliferation/vascular density in mice. Adult mice received everolimus daily for 2 weeks, and behavioral tests were performed from 1 week after the last treatment. Everolimus-treated mice displayed a marked reduction in weight gain from the last day of the treatment period. <i>Ex vivo</i> analysis showed altered cytochrome oxidase activity in selective cerebral regions involved in energy balance, food intake, reward, learning and memory modulation, sleep/wake cycle regulation, and arousal. Like chemotherapy, everolimus did not alter emotional reactivity, learning and memory performances, but in contrast to chemotherapy, did not affect behavioral flexibility or reactivity to novelty. <i>In vivo</i> hippocampal neural cell proliferation and vascular density were also unchanged after everolimus treatments. In conclusion, two weeks daily everolimus treatment at the clinical dose did not evoke alteration of cognitive performances evaluated in hippocampal- and prefrontal cortex-dependent tasks that would persist at one to four weeks after the end of the treatment completion. However, acute everolimus treatment caused selective CO modifications without altering the mTOR effector P70S6 kinase in cerebral regions involved in feeding behavior and/or the sleep/wake cycle, at least in part under control of the solitary nucleus and the parasubthalamic region of the hypothalamus. Thus, this area may represent a key target for everolimus-mediating peripheral modifications, which has been previously associated with symptoms such as weight loss and fatigue.</p></div

Impact of everolimus treatment on the mean body weight of mice.

<p>Body weight of vehicle- and everolimus-treated mice was evaluated at long term after the end of the treatment period (gray bar). Mice received vehicle or everolimus once a day during 14 continuous days. Bars represent standard error of the mean. ANOVA, Treatment x Day interaction <i>p</i><.001 followed by LSD post hoc: **<i>p</i><.01, ***<i>p</i><.001.</p

Cytochrome oxidase activity in cerebral areas of vehicle and everolimus-treated mice.

<p>Percentage of optical densities of cytochrome oxidase activity staining after everolimus treatment in the telencephalon, the diencephalon, the mesencephalon, the metencephalon, the myelecephalon and the cerebellum.</p><p>Data are means +SEM. (Student <i>t</i> test, *<i>p</i><.05). (a, area; ant, anterior; Diag, diagonal; horiz, horizontal; genic, geniculate; mesenc, mesencephalic; n, nucleus; ret, reticular; vert, vertical).</p><p>Cytochrome oxidase activity in cerebral areas of vehicle and everolimus-treated mice.</p

Effect of everolimus on precursor cell proliferation <i>in vivo</i> and <i>in vitro.</i>

<p>(A) Effect of <i>in vivo</i> administration of everolimus on neural precursor cell proliferation in the hippocampus. Left, BrdU-positive cells (intense green cells, white arrows) were counted in the subgranular zone (SGZ) and the remaining part of the dentate gyrus (outside SGZ). Bregma −2.16 mm; Scale bar, 100 µm. Right, Number of BrdU⁺ cells in the SGZ or outside SGZ (Mann whitney U tests, <i>p</i>>.05). (B and C) Effect of increasing concentrations of vehicle and everolimus on neural stem cell growth in culture. (B) <i>Upper</i>, Phase-contrast image of floating neurospheres at 24 hours after treatment with vehicle and everolimus (10⁻⁸ and 10⁻⁵ M). <i>Below</i>, Neurosphere volume in the absence (control: 0) and presence of vehicle and everolimus after 24 hours and 48 hours of treatment (Kruskal-Wallis ANOVA followed by Dunn's tests ***<i>p<.001 vs</i> 0; Mann Whitney U-tests, ^#<i>p<.05,</i>^##<i>p<.01</i> everolimus <i>vs</i> vehicle. Data represent means +SEM. (C) Proportion of neurospheres according to different categories of volumes. The increase of neurosphere mean volume might be explained by an increased proportion of bigger neurospheres and a decreased proportion of smaller neurospheres associated with everolimus treatment.</p

Spatial learning and memory, and behavioral flexibility of mice treated with vehicle or everolimus in the Morris water maze test.

<p>(A) Motivation and visuo-motor abilities after vehicle or everolimus treatment (Student <i>t</i> test, <i>p</i>>.05). (B and C) During the training and retrieval (R) phases, latency (B) and distance crossed (C) after vehicle or everolimus treatment (ANOVA, Day effect ***<i>p</i><.001). (D) During the probe test, time spent by animals of both groups in the quadrant where the platform was located during the training phase (χ², ***<i>p</i><.001 <i>vs</i> chance). During the transfer phase, escape latency (E), distance crossed (F), and swimming speed (G) in vehicle- or everolimus-treated mice (ANOVA, Treatment effect *<i>p</i><.05, Trial effect *** <i>p</i><.001 followed by LSD post hoc ^##<i>p</i><.01). (H) During the 1st day of the transfer phase, time spent in the previously correct northwest quadrant after vehicle or everolimus treatment (Student <i>t</i> test, <i>p</i>>.05). Data are means +SEM.</p

Schematic representation of brain areas presenting cytochrome oxidase activity modifications in mice treated with everolimus.

<p>Everolimus does not induce alteration of the phosphorylated P70S6K in the studied brain areas. It is here hypothesized that everolimus direct action on visceral organs would impact some cerebral areas through the vagus nerve. Among the brain areas with CO activity modifications are the solitary nucleus (Sol) as the principal recipient of visceral information conveyed by the vagus afferents and connected with the autonomic nervous system, motor nuclei of cranial nerves, nuclei in the brainstem and hypothalamus; the parasubthalamic nucleus (PSTh) within the hypothalamus, and motor trigeminal nucleus (MTN) involved in ingestion; the accumbens (Acc) shell important for reward and motivation processes; the preoptic area (LPO) regulating sleep/wake state; and the thalamic reuniens (Re) and rhomboid (Rh) nuclei and cortical areas that integrate arousal, somatosensory and visceral information, and participate to the modulation of learning and memory, and maintenance of arousal and wakefulness. Thus brain areas with modified CO activity are involved in the integration of autonomic and neuroendocrine information important for regulation of food intake, weight, metabolic, motivational processes, and arousal, that could be associated with symptoms suggestive of fatigue and energy homeostasis alterations. Arrows in purple show connections between brain areas metabolically modified. AH, anterior hypothalamic area; BBB, brain blood barrier; DM, dorsomedial hypothalamic nucleus; Ect, ectorhinal cortex; LH, lateral hypothalamic area; PH, posterior hypothalamic nucleus; PrL, prelimbic cortex; PVN, paraventricular hypothalamic nucleus; VM, ventromedial hypothalamic nucleus.</p

Vascular component density in hippocampus and endothelial cell proliferation <i>in vitro.</i>

<p>(A) Vascular density in the dentate gyrus of the hippocampus. <i>Upper</i>, IQGAP1-immunolabeled vascular niches in the dentate gyrus were illustrated from brain slices of vehicle- and everolimus-treated mice (Bregma −1.68 mm). Scale bar, 100 µm. <i>Below</i>, Mean IQGAP1-positive surface labeling and number of branches/nm² (+SEM) in the dentate gyrus (Mann-Whitney U-tests, <i>p</i>>.05). (B) Effects of increasing concentrations of vehicle and everolimus on the bEND.3 endothelial cell growth, as evaluated by endothelial cell number after 24 hours and 48 hours of treatment with vehicle or everolimus. Values are normalized to the mean number of cells in the control condition (white bar). Effect of everolimus, Kruskal-Wallis ANOVA followed by Dunn's tests *<i>p</i><.05, **<i>p</i><.01, ***<i>p</i><.001 <i>vs</i> control; Effect of everolimus compared with vehicle (Mann-Whitney tests, #<i>p</i><.05, ##<i>p</i><.01, ###<i>p</i><.001).</p

Longitudinal assessment of K103N mutants frequency by allele-specific PCR during the intermittent off-therapy periods.

<p>w, week; Y, yes; N, no; *, K103N mutants also detected by direct sequencing; dashes, samples not available.</p

Higher efavirenz half-life in patients in whom K103N emerged than in whom it did not.

<p>The Wilcoxon rank sum test was used for the comparison.</p