Finally, the binding of GOXG 1 to the GnRH-R and the associated elicitation of testosterone release in mice were also determined. NMR spectroscopy and in vitro radioligand binding assays were exploited for GOXG 1 to validate that upon conjugating the drug to the peptide, the peptide microenvironment responsible for its GnRH-R binding is not perturbed and to confirm its high binding potency to the GnRH-R. The major metabolite at low pH emanated from the cleavage of the pH-labile oxime bond, validating our design approach. The metabolism of GOXG 1 was examined in liver microsomes, as well as in buffers mimicking the pH of intracellular organelles, resulting in the identification of two metabolites. The most potent analog, designated GOXG 1, was used for pharmacokinetic studies in mice. We then assessed the ability of the developed PDCs to internalize in prostate cancer cells and to release gemcitabine. Their stability and the release of gemcitabine over time were monitored in vitro in cell culture and in human plasma using LC-MS/MS. Subsequently, the three PDCs were evaluated for their in vitro antiproliferative effect in prostate cancer cells. The internalization of the tumor-homing peptide unit in cancer cells, overexpressing the GnRH-R, was first validated through confocal laser microscopy and flow cytometry analysis. We developed a rapid (1 hour) and cost-effective “click” oxime bond ligation platform to assemble in one-pot the 3 desired PDCs that does not require purification, surpassing traditional time-ineffective and low yield methods. The challenging metabolic properties of gemcitabine were addressed during design of the PDCs. We exploited the oxime bond responsiveness against the acidic pH of the tumor microenvironment and the GnRH endocytosis via the GnRH-R GPCR which is overexpressed on cancer cells. The pro-drugs were designed to enable the selective targeting of malignant tumor cells with linker guided differential drug release rates.
![rapidclick targeting rapidclick targeting](https://vonkohnsearchgroup.com/wp-content/uploads/2017/03/target-blue-01.png)
These units were tethered via acid-labile programmable linkers to guide a differential drug release rate from the PDC through a combination of ester or amide and “click” type oxime ligations. Herein, we have developed and preclinically evaluated three PDCs with gemcitabine as the anticancer cytotoxic unit and D-Lys 6-GnRH (gonadotropin-releasing hormone GnRH) as the cancer-targeting unit. However, their development is often laborious and time-consuming. High-resolution archival tags provide new insights into the underwater foraging and echolocation behavior of resident killer whales capturing Pacific salmonPeptide-drug conjugates (PDCs) are gaining considerable attention as anti-neoplastic agents. Investigating differences in foraging efficiency between these two populations, including the impact of vessel noise exposure, will help to clarify factors impeding the recovery of southern residents, and identify strategies to best mitigate these threats.
![rapidclick targeting rapidclick targeting](https://www.tintoyarcade.com/image/cache/data/product/main/tta1641_mini_dart_board_game__93907-1000x1000.jpg)
DFO and NOAA are now conducting a transboundary study using Dtag data to compare the foraging behavior of endangered southern residents with that of the growing northern resident population. Distinctive ‘crunches’ were audible at shallow depths (m) following captures, indicating that killer whales brought most fish to the surface for handling and consumption. Once salmon were caught, both the mean proportion of time that whales spent echolocating and the mean clicking rate decreased significantly, confirming that echolocation plays an important role in prey detection and tracking. Most buzzes occurred at depths >100 m and preceded prey captures, which is consistent with their presumed function of close-range prey targeting. For each capture, whales produced an average of 14.3 regular click trains with mean repetition rates of 10 clicks s-1, and 2.5 ‘buzzes’ with rapid click rates exceeding 50 clicks s-1. Echolocation by resident killer whales during 17 successful salmon captures was also examined in detail. Foraging dive structure also reflected the deeper vertical distribution of Chinook salmon and revealed several predator evasion strategies used by salmonids. Foraging whales dove deeper, remained submerged longer, swam faster, increased their dive path tortuosity, and rolled their bodies more than during other activities. We deployed Dtags on 32 northern residents off Vancouver Island, BC, and compared reconstructed whale dive paths to distributions of different salmonid prey. They provide a valuable tool for examining the foraging behavior of resident killer whales, a specialist predator that relies on echolocation to find and pursue Pacific salmon, its primary prey. Dtags contain sensors for recording both high resolution animal movement and underwater sound.