Lipid droplet-mediated scavenging as novel intrinsic and adaptive resistance factor against the multikinase inhibitor ponatinib.

Ponatinib is a small molecule multi-tyrosine kinase inhibitor clinically approved for anticancer therapy. Molecular mechanisms by which cancer cells develop resistance against ponatinib are currently poorly understood. Likewise, intracellular drug dynamics, as well as potential microenvironmental factors affecting the activity of this compound are unknown. Cell/molecular biological and analytical chemistry methods were applied to investigate uptake kinetics/subcellular distribution, the role of lipid droplets (LDs) and lipoid microenvironment compartments in responsiveness of FGFR1-driven lung cancer cells toward ponatinib. Selection of lung cancer cells for acquired ponatinib resistance resulted in elevated intracellular lipid levels. Uncovering intrinsic ponatinib fluorescence enabled dissection of drug uptake/retention kinetics in vitro as well as in mouse tissue cryosections, and revealed selective drug accumulation in LDs of cancer cells. Pharmacological LD upmodulation or downmodulation indicated that the extent of LD formation and consequent ponatinib incorporation negatively correlated with anticancer drug efficacy. Co-culturing with adipocytes decreased ponatinib levels and fostered survival of cancer cells. Ponatinib-selected cancer cells exhibited increased LD levels and enhanced ponatinib deposition into this organelle. Our findings demonstrate intracellular deposition of the clinically approved anticancer compound ponatinib into LDs. Furthermore, increased LD biogenesis was identified as adaptive cancer cell-defense mechanism via direct drug scavenging. Together, this suggests that LDs represent an underestimated organelle influencing intracellular pharmacokinetics and activity of anticancer tyrosine kinase inhibitors. Targeting LD integrity might constitute a strategy to enhance the activity not only of ponatinib, but also other clinically approved, lipophilic anticancer therapeutics.

Synthesis and biological evaluation of biotin-conjugated anticancer thiosemicarbazones and their iron(III) and copper(II) complexes.

Triapine, the most prominent anticancer drug candidate from the substance class of thiosemicarbazones, was investigated in >30 clinical phase I and II studies. However, the results were rather disappointing against solid tumors, which can be explained (at least partially) due to inefficient delivery to the tumor site. Hence, we synthesized the first biotin-functionalized thiosemicarbazone derivatives in order to increase tumor specificity and accumulation. Additionally, for Triapine and one biotin conjugate the iron(III) and copper(II) complexes were prepared. Subsequently, the novel compounds were biologically evaluated on a cell line panel with different biotin uptake. The metal-free biotin-conjugated ligands showed comparable activity to the reference compound Triapine. However, astonishingly, the metal complexes of the biotinylated derivative showed strikingly decreased anticancer activity. To further analyze possible differences between the metal complexes, detailed physico- and electrochemical experiments were performed. However, neither lipophilicity or complex solution stability, nor the reduction potential or behavior in the presence of biologically relevant reducing agents showed strong variations between the biotinylated and non-biotinylated derivatives (only some differences in the reduction kinetics were observed). Nonetheless, the metal-free biotin-conjugate of Triapine revealed distinct activity in a colon cancer mouse model upon oral application comparable to Triapine. Therefore, this type of biotin-conjugated thiosemicarbazone is of interest for further synthetic strategies and biological studies.

Lysosomal Sequestration Impairs the Activity of the Preclinical FGFR Inhibitor PD173074.

Knowledge of intracellular pharmacokinetics of anticancer agents is imperative for understanding drug efficacy as well as intrinsic and acquired cellular resistance mechanisms. However, the factors driving subcellular drug distribution are complex and poorly understood. Here, we describe for the first time the intrinsic fluorescence properties of the fibroblast growth factor receptor inhibitor PD1703074 as well as utilization of this physicochemical feature to investigate intracellular accumulation and compartmentalization of this compound in human lung cancer cells. Cell-free PD173074 fluorescence, intracellular accumulation and distribution were investigated using analytical chemistry and molecular biology approaches. Analyses on a subcellular scale revealed selective drug accumulation in lysosomes. Coincubation with inhibitors of lysosomal acidification strongly enhanced PD173074-mediated fibroblast growth factor receptor (FGFR) inhibition and cytotoxicity. In conclusion, intrinsic fluorescence enables analysis of molecular factors influencing intracellular pharmacokinetics of PD173074. Lysosome-alkalinizing agents might represent candidates for rational combination treatment, preventing cancer cell-intrinsic PD173074 resistance based on lysosomal trapping.

Nanoformulations of anticancer FGFR inhibitors with improved therapeutic index.

Fibroblast growth factor receptor (FGFR) inhibitors like ponatinib and nintedanib are clinically approved for defined cancer patient cohorts but often exert dose-limiting adverse effects. Hence, we encapsulated the FGFR inhibitors ponatinib, PD173074, and nintedanib into polylactic acid nanoparticles and liposomes to enable increased tumor accumulation/specificity and reduce side effects. Different methods of drug loading were tested and the resulting formulations compared regarding average size distribution as well as encapsulation efficiency. Appropriate encapsulation levels were achieved for liposomal preparations only. Nanoencapsulation resulted in significantly decelerated uptake kinetics in vitro with clearly decreased short-term (up to 72 h) cytotoxicity at higher concentrations. However, in long-term clonogenic assays liposomal formations were equally or even more active as compared to the free drugs. Accordingly, in an FGFR inhibitor-sensitive murine osteosarcoma transplantation model (K7M2), only liposomal but not free ponatinib resulted in significant tumor growth inhibition (by 60.4%) at markedly reduced side effects.

Electromagnetic navigated condylar positioning after high oblique sagittal split osteotomy of the mandible: a guided method to attain pristine temporomandibular joint conditions.

OBJECTIVES: Reproduction of the exact preoperative proximal-mandible position after osteotomy in orthognathic surgery is difficult to achieve. This clinical pilot study evaluated an electromagnetic (EM) navigation system for condylar positioning after high-oblique sagittal split osteotomy (HSSO). STUDY DESIGN: After HSSO as part of 2-jaw surgery, the position of 10 condyles was intraoperatively guided by an EM navigation system. As controls, 10 proximal segments were positioned by standard manual replacement. Accuracy was measured by pre- and postoperative cone beam computed tomography imaging. RESULTS: Overall, EM condyle repositioning was equally accurate compared with manual repositioning (P > .05). Subdivided into 3 axes, significant differences could be identified (P < .05). Nevertheless, no significantly and clinically relevant dislocations of the proximal segment of either the EM or the manual repositioning method could be shown (P > .05). CONCLUSIONS: This pilot study introduces a guided method for proximal segment positioning after HSSO by applying the intraoperative EM system. The data demonstrate the high accuracy of EM navigation, although manual replacement of the condyles could not be surpassed. However, EM navigation can avoid clinically hidden, severe malpositioning of the condyles.

Can electromagnetic-navigated maxillary positioning replace occlusional splints in orthognathic surgery? A clinical pilot study.

INTRODUCTION: Because of the inaccuracy of intermaxillary splints in orthognathic surgery, intraoperative guidance via a real time navigation system might represent a suitable method for enhancing the precision of maxillary positioning. Therefore, in this clinical trial, maxillary repositioning after Le Fort I osteotomy was guided splintless by an electromagnetic navigation system. MATERIALS AND METHODS: Conservatively planned maxillary reposition in each of 5 patients was transferred to a novel software module of the electromagnetic navigation system. Intraoperatively, after Le Fort I osteotomy, the software guided the maxilla to the targeted position. Accuracy was evaluated by pre- and postoperative cone beam computer tomography imaging (the vectorial distance of the incisal marker points was measured in three dimensions) and compared with that of a splint transposed control group. RESULTS: The repositioning of the maxilla guided by the electromagnetic navigation system was intuitive and simple to accomplish. The achieved maxillary position with a deviation of 0.7 mm on average to the planned position was equally accurate compared with that of the splint transposed control group of 0.5 mm (p > 0.05). DISCUSSION: The data of this clinical study display good accuracy for splintless electromagnetic-navigated maxillary positioning. Nevertheless, this method does not surpass the splint-encoded gold standard with regard to accuracy. Future investigations will be necessary to show the full potential of electromagnetic navigation in orthognathic surgery.

Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer.

BACKGROUND: Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. METHODS: 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. RESULTS: Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification (bafilomycin A1, chloroquine) exerted distinctly synergistic growth inhibitory effects. CONCLUSION: Our findings provide a powerful tool to dissect molecular factors impacting organismal and intracellular pharmacokinetics of nintedanib. Regarding clinical application, prevention of lysosomal trapping via lysosome-alkalization might represent a promising strategy to circumvent cancer cell-intrinsic nintedanib resistance.

Computer assisted positioning of the proximal segment after sagittal split osteotomy of the mandible: Preclinical investigation of a novel electromagnetic navigation system.

INTRODUCTION: Modifications of the temporomandibular joint position after mandible osteotomy are reluctantly accepted in orthognathic surgery. To tackle this problem, we developed a new navigation system using miniaturized electromagnetic sensors. Our imageless navigation approach is therefore optimized to avoid complications of previously proposed optical approaches such as the interference with established surgical procedures and the line of sight problem. MATERIAL AND METHODS: High oblique sagittal split osteotomies were performed on 6 plastic skull mandibles in a laboratory under conditions comparable to the operating theatre. The subsequent condyle reposition was guided by an intuitive user interface and performed by electromagnetic navigation. To prove the suitability and accuracy of this novel approach for condyle navigation, the positions of 3 titanium marker screws placed on each of the proximal segments were compared using pre- and postoperative Cone Beam Computed Tomography (CBCT) imaging. RESULTS: Guided by the electromagnetic navigation system, positioning of the condyles was highly accurate in all dimensions. Translational discrepancies up to 0,65 mm and rotations up to 0,38° in mean could be measured postoperatively. There were no statistically significant differences between navigation results and CBCT measurements. CONCLUSION: The intuitive user interface provides a simple way to precisely restore the initial position and orientation of the proximal mandibular segments. Our electromagnetic navigation system therefore yields a promising approach for orthognathic surgery applications.

Electromagnetic navigated positioning of the maxilla after Le Fort I osteotomy in preclinical orthognathic surgery cases.

OBJECTIVES: Inaccuracies in orthognathic surgery can be caused during face-bow registration, model surgery on plaster models, and intermaxillary splint manufacturing. Electromagnetic (EM) navigation is a promising method for splintless digitized maxillary positioning. STUDY DESIGN: After performing Le Fort I osteotomy on 10 plastic skulls, the target position of the maxilla was guided by an EM navigation system. Specially implemented software illustrated the target position by real-time multistage colored three-dimensional imaging. Accuracy was determined by using pre- and postoperative cone beam computed tomography. RESULTS: The high accuracy of the EM system was underlined by the fact that it had a navigated maxilla position discrepancy of only 0.4 mm, which was verified by postoperative cone beam computed tomography. CONCLUSIONS: This preclinical study demonstrates a precise digitized approach for splintless maxillary repositioning after Le Fort I osteotomy. The accuracy and intuitive illustration of the introduced EM navigation system is promising for potential daily use in orthognathic surgery.

Impact of Stepwise NH2-Methylation of Triapine on the Physicochemical Properties, Anticancer Activity, and Resistance Circumvention.

One of the most promising classes of iron chelators are α-N-heterocyclic thiosemicarbazones with Triapine as the most prominent representative. In several clinical trials Triapine showed anticancer activity against hematological diseases, however, studies on solid tumors failed due to widely unknown reasons. Some years ago, it was recognized that "terminal dimethylation" of thiosemicarbazones can lead to a more than 100-fold increased activity, probably due to interactions with cellular copper depots. To better understand the structural requirements for the switch to nanomolar cytotoxicity, we systematically synthesized all eight possible N-methylated derivatives of Triapine and investigated their potential against Triapine-sensitive as well as -resistant cell lines. While only the "completely" methylated compound exerted nanomolar activity, the data revealed that all compounds with at least one N-dimethylation were not affected by acquired Triapine resistance. In addition, these compounds were highly synergistic with copper treatment accompanied by induction of reactive oxygen species and massive necrotic cell death.

Approach to intraoperative electromagnetic navigation in orthognathic surgery: A phantom skull based trial.

INTRODUCTION: Intraoperative guidance using electromagnetic navigation is an upcoming method in maxillofacial surgery. However, due to their unwieldy structures, especially the line-of-sight problem, optical navigation devices are not used for daily orthognathic surgery. Therefore, orthognathic surgery was simulated on study phantom skulls, evaluating the accuracy and handling of a new electromagnetic tracking system. MATERIAL AND METHODS: Le-Fort I osteotomies were performed on 10 plastic skulls. Orthognathic surgical planning was done in the conventional way using plaster models. Accuracy of the gold standard, splint-based model surgery versus an electromagnetic tracking system was evaluated by measuring the actual maxillary deviation using bimaxillary splints and preoperative and postoperative cone beam computer tomography imaging. The distance of five anatomical marker points were compared pre- and postoperatively. RESULTS: The electromagnetic tracking system was significantly more accurate in all measured parameters compared with the gold standard using bimaxillary splints (p < 0.01). The data shows a discrepancy between the model surgical plans and the actual correction of the upper jaw of 0.8 mm. Using the electromagnetic tracking, we could reduce the discrepancy of the maxillary transposition between the planned and actual orthognathic surgery to 0.3 mm on average. DISCUSSION: The data of this preliminary study shows a high level of accuracy in surgical orthognathic performance using electromagnetic navigation, and may offer greater precision than the conventional plaster model surgery with bimaxillary splints. CONCLUSION: This preliminary work shows great potential for the establishment of an intraoperative electromagnetic navigation system for maxillofacial surgery.