Search

Supplementary Video 5 shows a confocal time-lapse series, showing MDCK cells treated with 40 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 2 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 1000 ug K16ApoE intravenously.

Supplementary Video 4 shows a confocal time-lapse series, showing MDCK cells treated with 20 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 1 shows sonographic visualization of the axial position of the heart of a NOD/SCID mouse injected with 9 mg/mL NaCl intravenously.

Supplementary Video 3 shows a confocal time-lapse series, showing a control sample of MDCK cells labeled with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Supplementary Video 6 shows a confocal time-lapse series, showing MDCK cells treated with 80 ug of K16ApoE, immediately after labeling with Calcein-AM (green), which indicates live cells, and Ethidium homodimer-1 (red), indicating dead cells.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood–brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Supplementary Materials shows supplementary methods, Supplementary Figures S1-S9 with legends and Supplementary Table 1. Supplementary Table 1: Mean Ve, Kep and AUC after DCE-MRI; Supplementary Figure S1: In vivo study design; Supplementary Figure S2: Blood smear from mice subjected to 1,000 µg K16ApoE; Supplementary Figure S3: In vitro cell viability: Supplementary Figure S4: Mechanistic studies of the in vitro effects of K16ApoE; Supplementary Figure S5: In vitro BBB model; Supplementary Figure S6: Biodistribution of 125I-K16ApoE; Supplementary Figure S7: Histopathological evaluation of tissue samples after long- and short-term treatment of NOD/SCID mice with the peptide; Supplementary Figure S8: In vivo treatment control experiment; Supplementary Figure S9: Mass spectrometry of treated and untreated brain tissue

Antibodies are attractive as radioligands due to their outstanding specificity and high affinity, but their inability to cross the blood–brain barrier (BBB) limits their use for CNS targets. To enhance brain distribution, amyloid-β (Aβ) antibodies were fused to a transferrin receptor (TfR) antibody fragment, enabling receptor mediated transport across the BBB. The aim of this study was to label these bispecific antibodies with fluorine-18 and use them for Aβ PET imaging. Bispecific antibody ligands RmAb158-scFv8D3 and Tribody A2, both targeting Aβ and TfR, were functionalized with trans-cyclooctene (TCO) groups and conjugated with 18F-labeled tetrazines through an inverse electron demand Diels–Alder reaction performed at ambient temperature. 18F-labeling did not affect antibody binding in vitro, and initial brain uptake was high. Conjugates with the first tetrazine variant ([18F]T1) displayed high uptake in bone, indicating extensive defluorination, a problem that was resolved with the second and third tetrazine variants ([18F]T2 and [18F]T3). Although the antibody ligands’ half-life in blood was too long to optimally match the physical half-life of fluorine-18 (t1/2 = 110 min), [18F]T3-Tribody A2 PET seemed to discriminate transgenic mice (tg-ArcSwe) with Aβ deposits from wild-type mice 12 h after injection. This study demonstrates that 18F-labeling of bispecific, brain penetrating antibodies is feasible and, with further optimization, could be used for CNS PET imaging.

Background: Noninvasive molecular imaging using peptides and biomolecules labelled with positron emitters has become important for detection of cancer and other diseases with PET (positron emission tomography). The positron emitting radionuclide fluorine-18 is widely available in high yield from cyclotrons and has favorable decay (t1/2 109.7 min) and imaging properties. 18F-Labelling of biomolecules and peptides for use as radiotracers is customarily achieved in a two-step approach, which can be challenging to automate. 6-[18F]Fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([18F]F-Py-TFP) is a versatile 18F-prosthetic group for this purpose, which can be rapidly be produced in an one-step approach on solid support. This work details an automated procedure on the cassette-based GE FASTlabTM platform for the labeling of a peptidomimetic, exemplified by the case of using the Glu-CO-Lys motif to produce [18F]DCFPyL, a ligand targeting the prostate specific membrane antigen (PSMA). Results: From fluorine-18 delivery a fully automated two-step radiosynthesis of [18F]DCFPyL was completed in 56 min with an overall end of synthesis yield as high as 37% using SPE purification on the GE FASTlabTM platform. Conclusions: Putatively, this radiolabeling methodology is inherently amenable to automation with a diverse set of synthesis modules, and it should generalize for production of a broad spectrum of biomolecule-based radiotracers for use in PET imaging.

Noninvasive molecular imaging using peptides and biomolecules labelled with positron emitters has become important for detection of cancer and other diseases with PET (positron emission tomography). The positron emitting radionuclide fluorine-18 is widely available in high yield from cyclotrons and has favorable decay (tFrom fluorine-18 delivery a fully automated two-step radiosynthesis of [Putatively, this radiolabeling methodology is inherently amenable to automation with a diverse set of synthesis modules, and it should generalize for production of a broad spectrum of biomolecule-based radiotracers for use in PET imaging.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood–brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was conducted to find the most suitable ring systems for further optimization, whereas a second screen explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT signaling activity at 10 μM concentration was achieved, however, only low affinities towards MET were observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards the development of a dual inhibitor of MET and WNT signaling.

Introduction: Cabozantinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of medullary thyroid cancer, renal cell carcinoma and hepatocellular carcinoma, and is currently in clinical trials for the treatment of prostate cancer and others. It exerts its therapeutic effect mainly through inhibition of the tyrosine kinases MET (hepatocyte growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2), in addition to several other kinases involved in cancer. PET imaging with TKIs such as [18F]cabozantinib could potentially aid in cancer diagnosis and guide treatment. This study aims to evaluate the utility of [18F]cabozantinib as a PET imaging probe in PC3 tumor xenografted mice. Methods: [18F]cabozantinib was evaluated in non-tumor and tumor bearing (PC3 xenografted) male mice by ex vivo biodistribution studies and in vivo μPET imaging. Pretreatment studies were performed in the tumor bearing mice with the MET inhibitor PF04217903. Mouse plasma was analyzed with HPLC to quantify radiometabolites. To further evaluate the binding specificity of [18F]cabozantinib, in vitro autoradiography studies on heart and PC3 tumor sections were performed in the presence of authentic cabozantinib or specific MET and VEGFR2 inhibitors. Results: Tissue distribution studies in non-tumor bearing mice revealed slow blood clearance, absence of brain uptake and a high myocardial uptake. In the tumor bearing mice, tumor uptake was low (0.58 ± 0.20% ID/g at 30 min post tracer injection), which was confirmed by μPET imaging. No differences in tissue distribution and kinetics were observed in both biodistributions and μPET studies after pretreatment with the MET inhibitor PF04217903. At 30 min post tracer injection, 60 ± 3% of the recovered radioactivity in plasma in non-tumor bearing mice was present as intact tracer. [18F]cabozantinib binding in vitro to heart and tumor tissues was partly blocked in the presence of selective MET and VEGFR2 inhibitors (up to 40% block). The fraction of non-specific binding was relatively high for both tissues (66% for heart and 39% for tumor). Conclusion: [18F]cabozantinib exhibits non-favorable properties as a PET imaging probe, demonstrated by slow excretion kinetics along with low tumor uptake and high non-specific binding in tumor and heart tissue. The results reflect cabozantinibs multi-kinase activity, making PET imaging of tumor specific kinase expression with [18F]cabozantinib challenging., SCOPUS: ar.j, info:eu-repo/semantics/published

The tyrosine kinase MET (hepatocyte growth factor receptor) is abnormally activated in a wide range of cancers and is often correlated with a poor prognosis. Precision medicine with positron emission tomography (PET) can potentially aid in the assessment of tumor biochemistry and heterogeneity, which can prompt the selection of the most effective therapeutic regimes. The selective MET inhibitor PF04217903 (1) formed the basis for a bioisosteric replacement to the deoxyfluorinated analogue [18F]2, intended as a PET tracer for MET. [18F]2 could be synthesized with a “hydrous fluoroethylation” protocol in 6.3 ± 2.6% radiochemical yield and a molar activity of >50 GBq/µmol. In vitro autoradiography indicated that [18F]2 specifically binds to MET in PC3 tumor tissue, and in vivo biodistribution in mice showed predominantly a hepatobiliary excretion along with a low retention of radiotracer in other organs.

The single-crystal X-ray diffraction investigation of a substituted quinoline derivative, which may serve as a basis for the development of a family of cytotoxic agents, confirms the anti­cipated covalent structure with an unusual twisted conformation and reveals a densely packed mol­ecular lattice., The protein tubulin is central for maintaining normal cellular processes, and mol­ecules inter­fering with the tubulin dynamics have potential in the treatment of cancerous diseases. The title compound, C17H14N2O5, was prepared as a lead compound in a project dedicated to the development of therapeutic agents binding to the colchicine binding site on tubulin, thereby inter­fering with the cell division in cancer cells. It holds many of the main structural characteristics for colchicine binding and has the potential for further modification and functionalization. In the title mol­ecule, the benzene ring is inclined to the quinoline ring by 76.10 (8)°. In the crystal, mol­ecules are linked by two pairs of C—H⋯O hydrogen bonds, forming tubular-like arrangements, propagating along the direction of the diagonals of the ab plane, and enclosing R 2 2(26) and R 2 2(16) ring motifs.

Based on the cabozantinib scaffold, novel c‐Met inhibitors were rationalized from the limited knowledge of structure‐activity relationships for the quinoline 6‐position. Emphasis was given to modifications capable of engaging in additional polar interactions with the c‐Met active site. In addition, ortho‐fluorinations of the terminal benzene ring were explored. Fifteen new molecules were synthesized and evaluated in a c‐Met enzymatic binding assay. A wide range of substituents were tolerated in the quinoline 6‐position, while the ortho‐fluorinations performed were shown to give considerable reductions in the c‐Met binding affinity. The antiproliferative effects of the compounds were evaluated in the NCI60 cancer cell line panel. Most notably, compounds 15b and 18b were able to inhibit cell proliferation more efficiently than cabozantinib in leukemia, CNS, and breast cancer cell lines. The in vitro data agreed well with the in silico docking results, where additional hydrogen bonding was identified in the enzymatic pocket for the para‐amino substituted 15b and 18b.

Introduction The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [ 18 F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18 F-labeled compounds in brain of living rats. Methods We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18 F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo . Results A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18 F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18 F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo . Conclusions The novel small molecule 18 F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro , and may prove useful for quantitative autoradiographic studies in vitro . The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.

Correction for ‘Towards dual inhibitors of the MET kinase and WNT signaling pathway; design, synthesis and biological evaluation’ by Vegard Torp Lien et al., RSC Adv., 2019, 9, 37092–37100.

Individuals with brain metastasis usually await a poor prognosis. Even with the ongoing development of novel targeted therapies, a major impediment in the treatment of brain metastasis is delivering of drugs across the blood-brain/tumor barrier. Different strategies to accomplish temporary opening of the heterogenous blood-brain barrier have been described. Here, we report on a synthetic peptide, K16ApoE, which is able to open the blood-brain barrier for improved drug-delivery in a non-permanent manner. We carried out extensive dynamic contrast enhanced magnetic resonance imaging experiments to study the ability of the peptide to open the blood-brain barrier in healthy mice. We further performed dynamic positron emission/computer tomography, with F18-labeled albumin and IgG to validate an open blood-brain barrier after co-injecting with K16ApoE. Further, we studied the effects of the peptide on cells and tissue in vitro and in vivo. The biodistribution of the peptide was studied in healthy mice by labeling I-125 to the imidazole ring of histidine in K16ApoE and tracking the peptide in plasma and tissue. We also conducted a treatment study on BRAF(V600E)mutant brain metastases, where the treatment groups received K16ApoE before a BRAF inhibitor was administered, BRAF inhibitor only, K16ApoE only or vehicle. We found a peptide-induced therapeutic window at approximately 30 minutes using dynamic contrast enhanced magnetic resonance imaging. The dynamic positron emission/computer tomography showed cerebral uptake of albumin (∼67 kDa) and IgG (∼150 kDa) in healthy mice with a presumed intact blood-brain barrier. Further, we identified lysis of cells treated with K16ApoE in addition to in part endocytosis-mediated peptide uptake. In vitro blood-brain barrier modeling demonstrated endothelial and astrocyte cell layer regrowth within approximately three and 15 hours, respectively, after treatment with the peptide. We observed a significant treatment effect of co-injecting the peptide with a BRAF inhibitor on BRAF mutated brain metastases.

Protectin D1 is a specialized pro-resolving mediator with potent pro-resolving and anti-inflammatory effects in vivo in several human disease models. Herein the preparation of the first synthetic analog of protectin D1, named 22-F-PD1, is presented together with data from in vivo investigations. This analog showed potent pro-resolving and anti-inflammatory properties. These results inspired the preparation of the radiotracer 22-[(18)F]F-PD1-ME that was used in a positron emission tomography proof of concept study. Altogether, the findings presented contribute to new knowledge on the biomolecular properties of protectin D1 analogs. In addition, an improved formal synthesis of the metabolite 22-OH-PD1 is reported.

The decapeptide gonadotropin-releasing hormone, also referred to as luteinizing hormone-releasing hormone with the sequence (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) plays an important role in regulating the reproductive system. It stimulates differential release of the gonadotropins FSH and LH from pituitary tissue. To date, treatment of hormone-dependent diseases targeting the GnRH receptor, including peptide GnRH agonist and antagonists are now available on the market. The inherited issues associate with peptide agonists and antagonists have however, led to significant interest in developing orally active, small molecule, non-peptide antagonists. In this review, we will summarize all developed small molecule GnRH antagonists along with the most recent clinical data and therapeutic applications.

© 2017 Bentham Science Publishers Background: Histone deacetylases (HDACs) emerged as important epigenetic regulators of gene expression. Method: In order to identify potential positron emission tomography (PET) tracers for imaging HDACs, we evaluated in vitro and in cellulo activities of some compounds that were reported as potent HDAC2-selective inhibitors. We observed marked differences between reported activity values and the values obtained in our assays for some of the compounds. To understand the structural basis of the activity of some of these inhibitors, we also performed molecular docking studies to understand their interaction patterns and binding modes with HDAC2. Results and Conclusion: We observed the low affinity compounds 4, 6 and 7 did not showed equal number of key ?-? interactions and hydrogen bonding when compared to high affinity compounds, and could be the possible reason for poor inhibition as reflected in in vitro assays. These preliminary experimental and computational results will help to interpret the HDAC affinity values of these key compounds with caution. ispartof: Letters in Drug Design & Discovery vol:14 issue:7 pages:787-797 status: published

To date the majority of 18F-peptide radiolabeling approaches are multi-step, low yielding and time-consuming processes. Given the short half-life of 18F (109.8 min), it is critical that methods are developed to increase the efficiency of this process with simpler, higher yielding and faster reactions that can be rapidly translated into clinical use. Here, we demonstrate the first microfluidic synthesis of the [18F]F-Py-TFP prosthetic group with radiochemical yields of up to 97% and a synthesis time of 3 min. In addition, we utilized a single microfluidics device to prepare the [18F]F-Py-YGGFL peptide using [18F]F-Py-TFP, from [18F]fluoride in a two-step, fully automated approach. The model peptide NH2-YGGFL was radiolabeled with [18F]F-Py-TFP in up to 28% overall radiochemical yield within 8 minutes starting from anhydrous [18F]fluoride.

BACKGROUND: Melanoma patients have a high risk of developing brain metastases, which is associated with a poor prognosis. The blood-brain barrier (BBB) inhibits sufficient drug delivery into metastatic lesions. We investigated the ability of a synthetic peptide (K16ApoE) to permeabilize the BBB for more effective drug treatment. METHODS: DCE-MRI was performed to study the therapeutic window of BBB opening facilitated by K16ApoE. In vivoand in vitroassays were used to determine K16ApoE toxicity and also to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on melanoma metastases was determined together with dabrafenib, which is otherwise known not to cross an intact BBB. RESULTS: DCE-MRI exhibited an effective K16ApoE-mediated BBB opening for up to 1h. Mechanistic studies displayed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At higher concentrations, the peptide also showed unspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice compared to dabrafenib. We also showed by PET/CT that the peptide facilitated the delivery of compounds up to 150 kDa into the brain. CONCLUSIONS: We demonstrate a transient opening of the BBB, caused by K16ApoE, that facilitates improved drug-delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Patients with brain metastases usually have a poor prognosis. Regardless of the constant progress in novel drug establishments, a crucial obstacle is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. Different study designs to achieve temporary BBB opening have previously been studied. Here, we describe a synthetic peptide termed K16ApoE, which is able to transiently open the BBB for drug-delivery into experimental brain metastases.We conducted a systemic study of the ability of the peptide to open the BBB by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, we studied the mechanisms of the peptide using flow cytometry, electron microscopy and dynamic positron emission tomography (PET). The biodistribution of the peptide was studied in blood plasma and organs using I-125 labeled K16ApoE. Finally, a treatment study using the peptide in combination with the B-RAF inhibitor dabrafenib was conducted.An opening of the BBB for up to 60 mins was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The treatment study demonstrated that the group of animals receiving K16ApoE followed by dabrafenib had smaller tumor volumes than the other two animal groups.In conclusion, the synthetic K16ApoE peptide in combination with dabrafenib decreased the number of experimental brain metastases. Thus, the current strategy could have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Christopher Florian Holte, Olivier Keunen, Tine Veronica Karlsen, Olav Tenstad, Habib Baghirov, Dag Erlend Olberg, Rolf Bjerkvig, Gobinda Sarkar, Frits Thorsen. The good drug, the bad barrier and the handy peptide: Improved treatment of experimental melanoma brain metastases using a synthetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-314.

Introduction: Patients with brain metastases await a dismal prognosis. Regardless of the continuous progress in drug development, a major problem is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all compounds, in particular highly charged, hydrophilic or large compounds, and most of the current chemotherapeutic agents are thus unable to penetrate the BBB. Varying strategies to transiently open the BBB have been studied previously. Here, we describe a peptide transporter comprising 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that the peptide (K16ApoE) is able to transiently open the BBB for drug-delivery into experimental brain metastases. Experimental procedures: A systemic study of the ability of the peptide to open the BBB was conducted by dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. The BBB permeability was studied after administering 200 μg of the peptide intravenously. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using 125I labeled K16ApoE. Finally, a treatment study was initiated, treating the animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle. Summary: After injecting the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers, reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and a dose-dependent cell death pattern was observed at higher concentrations of K16ApoE.The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. The treatment study displayed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two animal groups. Conclusions: We have shown that the peptide opens the BBB and facilitates a therapeutic window of 4 hours. The peptide did in combination with Dabrafenib decrease the number of experimental brain metastases in our studies. Thus, the current strategy could also have the potential to improve the treatment of patients with brain metastatic disease. Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Olivier Keunen, Christopher Florian Holte, Habib Baghirov, Rolf Bjerkvig, Tine Veronica Karlsen, Olav Tenstad, Dag Erlend Olberg, Gobinda Sarkar, Robert B Jenkins, Frits Thorsen. The peptide transporter K16ApoE increases drug delivery across the blood brain barrier in an experimental animal model of melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2865. doi:10.1158/1538-7445.AM2017-2865

Individuals with metastatic disease within the brain usually await a poor prognosis. Regardless of the ongoing development of novel drugs, a significant challenge is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. The BBB excludes almost all therapeutic compounds, as highly charged, hydrophilic or large compounds are unable to penetrate the BBB. Different strategies to temporarily open the BBB have been studied previously. Here, we describe a peptide transporter encompassing 16 lysine residues and 20 amino acid residues corresponding to the low density lipoprotein receptor (LDLR) binding domain of apolipoprotein E (ApoE). We show that this peptide, K16ApoE, is able to transiently open the BBB for drug-delivery into experimental brain metastases. The ability of the peptide to open the BBB was studied in vivo using dynamic contrast enhanced magnetic resonance imaging (DCE MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, cellular effects after treatment with the peptide was investigated in vitro using confocal microscopy, flow cytometry and impedance experiments. The biodistribution of the peptide was studied in blood plasma and several organs using isotope labeled K16ApoE. Finally, we treated animals with the peptide in combination with the B-RAF inhibitor Dabrafenib, only Dabrafenib or vehicle. After intravenously administering the K16ApoE peptide into the mice, a transient opening of the BBB for up to 4 hours was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The impedance experiments displayed that the permeability through endothelial cell barriers was increased after treatment with K16ApoE, and dose-dependent cell death was seen. The peptide did not affect endothelial cell tight junctions. The biodistribution study showed that the peptide was eliminated from blood plasma in less than five minutes through the kidneys. In the treatment study it was observed that the group of animals receiving K16ApoE followed by Dabrafenib had smaller tumor volumes than the other two experimental groups. K16ApoE did in combination with Dabrafenib decrease the number of experimental brain metastases, as a therapeutic window up to 4 hours was seen when using the peptide. The described strategy could thus have the potential to improve the treatment of patients with brain metastatic disease.