Decreased eukaryotic initiation factors expression upon temozolomide treatment-potential novel implications for eIFs in glioma therapy.

PURPOSE: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment. METHODS: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX. RESULTS: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis. CONCLUSION: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.

Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures.

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

M01 as a novel drug enhancer for specifically targeting the blood-brain barrier.

Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.

Optimized creation of glioblastoma patient derived xenografts for use in preclinical studies.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults, highlighting the need for novel treatment strategies. Patient derived xenografts (PDX) represent a valuable tool to accomplish this task. METHODS: PDX were established by implanting GBM tissue subcutaneously. Engraftment success was compared between NMRI Foxn1nu and NOD/SCID as well as between fresh and cryopreserved tissue. Established PDX were analyzed histologically and molecularly. Five PDX were experimentally treated with different drugs to assess their potential for preclinical drug testing. RESULTS: Establishment of PDX was attempted for 36 consecutive GBM cases with an overall success rate of 22.2% in NMRI Foxn1nu mice. No difference was observed between fresh or cryopreserved (20-1057 days) tissue in direct comparison (n = 10 cases). Additionally, engraftment was better in NOD/SCID mice (38.8%) directly compared to NMRI Foxn1nu mice (27.7%) (n = 18 cases). Molecular data and histology of the PDX compare well to the primary GBM. The experimental treatment revealed individual differences in the sensitivity towards several clinically relevant drugs. CONCLUSIONS: The use of vitally frozen GBM tissue allows a more convenient workflow without efficiency loss. NOD/SCID mice appear to be better suited for initial engraftment of tumor tissue compared to NMRI Foxn1nu mice.

Improved Treatment of MT-3 Breast Cancer and Brain Metastases in a Mouse Xenograft by LRP-Targeted Oxaliplatin Liposomes.

The anti-cancer drug oxaliplatin (OxP) has rarely been used to treat breast carcinoma, as it cannot cross the BBB to treat the frequently subsequent brain metastases. Here, we encapsulated OxP in liposomes prepared to reduce side effects and to simultaneously treat primary tumor and brain metastasis. The angiopep LRP-receptor ligand was bound to the vesicular surface for targeting. Targeted and non-targeted OxP liposomes were tested in vitro (binding, uptake, and transcytosis) and in vivo. Liposomes contained 0.65 mg OxP/mL, their mean diameter was 165 nm, and they released 50% of OxP within 8 days at 4 degrees C and within 22 h at 36 degrees C. MDCK cells were used for uptake and transcytosis quantification. Compared to non-targeted liposomes, targeted liposomes showed 12-fold greater uptake, and 2.25-fold higher transcytosis. In vivo efficacy was tested using human MT-3 breast cancer cells transplanted subcutaneously and intracerebrally into female nude mice, and tumor growth inhibition was measured. OxP was injected (6 mg OxP/kg) four times. The best results were obtained with targeted liposomes (T/C: 21% for subcutaneous and 50% for intracerebral). OxP liposomes with a fluid membrane all inhibited MT-3 tumors significantly better than free OxP, with no significant difference between targeted and non-targeted liposomes. The therapeutic effect was accompanied with strong leukopenia and mild thrombocytopenia with all formulations. The newly developed OxP liposomes significantly improved the treatment of subcutaneously and intracerebrally growing breast cancer, but the targeted angiopep-equipped liposomes showed no superior effect in vivo.

How perifosine affects liposome-encapsulated drug delivery across a cell barrier.

BACKGROUND: The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells. METHODS: To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured. RESULTS & CONCLUSION: Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general.

Treatment of experimental brain metastasis with MTO-liposomes: impact of fluidity and LRP-targeting on the therapeutic result.

PURPOSE: To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain. METHODS: Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting. RESULTS: Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t(1/2) was extended for liposomes. CONCLUSION: Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes.

Improving the transport of chemotherapeutic drugs across the blood-brain barrier.

The successful treatment of brain tumors or metastases in the brain is still hampered by the very efficient blood-brain barrier, which prevents the cerebral accumulation of a pharmacologically sufficient amount of a drug. Beside the possibility of disintegrating the functionality of this effective working barrier, a nanocarrier-mediated transport is presently an interesting and promising method to increase the drug concentration in the brain. Nanocarriers are small vesicles (<200 nm) and can be prepared by polymerization, resulting in nanoparticles, or by producing superficial lipid structures to incorporate the drug. In this context, liposomes are of importance owing to their ability to adapt their properties to the pharmacological requirements. In this article, we will give an overview of current possibilities of enhancing anticancer drug transport across the blood-brain barrier, based on its structure and functionality. Special consideration will be given to recent liposomal approaches that use active targeting for receptor-mediated transport across this physiological barrier.

Impact of membrane properties on uptake and transcytosis of colloidal nanocarriers across an epithelial cell barrier model.

The aim of this study was to investigate the effect of liposomal membrane properties on cellular uptake and transcytosis across a tight Madin-Darby canine kidney (MDCK) cell barrier in vitro. More than 25 small vesicles were prepared by lipid film hydration/extrusion to generate small unilamellar vesicles. The fluorescence marker calcein was encapsulated to mimic hydrophilic drug transport. Marker uptake by MDCK cells seems to be mediated by different mechanisms for the liposomes used. It was mainly depending on membrane fluidity and vesicle charge. Liposomes L2 with a positive charge (325 +/- 3 pmol/well) and vesicles L3 containing the helper lipid dioleylphosphatidylethanolamine (DOPE) in their membrane (216 +/- 42 pmol/well) were taken up to the most. Selected liposomes were tested for their transcytotic transport across a MDCK monolayer. Liposomes L4 containing equimolar DOPE and octadecyl-1,1-dimethylpiperidin-1-ium-4-yl phosphate (OPP) were the most efficient vesicles for transcellular transport resulting in 808 +/- 30 pmol calcein/cm(2) in the basal medium (28.1% of total liposomal marker added). Transcytosis was positively correlated with membrane fluidity in the outer part of the bilayer, as electron paramagnetic resonance measurements revealed. We expect that an increase in membrane fluidity of vesicles should also improve the restricted transport of hydrophilic drugs across the blood-brain barrier.