Blood-tumor barrier opening by MRI-guided transcranial focused ultrasound in a preclinical breast cancer brain metastasis model improves efficacy of combinatorial chemotherapy.

Patients with metastatic breast cancer have high and continually increasing rates of brain metastases. During the course of the disease, brain metastases can occur in up to 30% of these patients. In most cases, brain metastases are diagnosed after significant disease progression. The blood-tumor barrier increases the difficulty of treating brain metastasis by preventing accumulation of chemotherapy within metastases at therapeutically effective concentrations. Traditional therapies, such as surgical resection, radiotherapy, and chemotherapy, have poor efficacy, as reflected by a low median survival rate of 5-8% after post-diagnosis. Low-intensity focused ultrasound (LiFUS) is a new treatment for enhancing drug accumulation within the brain and brain malignancies. In this study, we elucidate the effect of clinical LiFUS combined with chemotherapy on tumor survival and progression in a preclinical model of triple-negative breast cancer metastasis to the brain. LiFUS significantly increased the tumor accumulation of 14C-AIB and Texas Red compared to controls (p< 0.01). LiFUS-mediated opening of the BTB is size-dependent, which is consistent with our previous studies. Mice receiving LiFUS with combinatorial Doxil and paclitaxel showed a significant increase in median survival (60 days) compared to other groups. LiFUS plus combinatorial chemotherapy of paclitaxel and Doxil also showed the slowest progression of tumor burden compared to chemotherapy alone or individual chemotherapy and LiFUS combinations. This study shows that combining LiFUS with timed combinatorial chemotherapeutic treatment is a potential strategy for improving drug delivery to brain metastases.

Characterization of passive permeability after low intensity focused ultrasound mediated blood-brain barrier disruption in a preclinical model.

BACKGROUND: Systemic drug delivery to the central nervous system is limited by presence of the blood-brain barrier (BBB). Low intensity focused ultrasound (LiFUS) is a non-invasive technique to disrupt the BBB, though there is a lack of understanding of the relationship between LiFUS parameters, such as cavitation dose, time of sonication, microbubble dose, and the time course and magnitude of BBB disruption. Discrepancies in these data arise from experimentation with modified, clinically untranslatable transducers and inconsistent parameters for sonication. In this report, we characterize microbubble and cavitation doses as LiFUS variables as they pertain to the time course and size of BBB opening with a clinical Insightec FUS system. METHODS: Female Nu/Nu athymic mice were exposed to LiFUS using the ExAblate Neuro system (v7.4, Insightec, Haifa, Israel) following target verification with magnetic resonance imaging (MRI). Microbubble and cavitation doses ranged from 4-400 µL/kg, and 0.1-1.5 cavitation dose, respectively. The time course and magnitude of BBB opening was evaluated using fluorescent tracers, ranging in size from 105-10,000 Da, administered intravenously at different times pre- or post-LiFUS. Quantitative autoradiography and fluorescence microscopy were used to quantify tracer accumulation in brain. RESULTS: We observed a microbubble and cavitation dose dependent increase in tracer uptake within brain after LiFUS. Tracer accumulation was size dependent, with 14C-AIB (100 Da) accumulating to a greater degree than larger markers (~ 625 Da-10 kDa). Our data suggest opening of the BBB via LiFUS is time dependent and biphasic. Accumulation of solutes was highest when administered prior to LiFUS mediated disruption (2-fivefold increases), but was also significantly elevated at 6 h post treatment for both 14C-AIB and Texas Red. CONCLUSION: The magnitude of LiFUS mediated BBB opening correlates with concentration of microbubbles, cavitation dose as well as time of tracer administration post-sonication. These data help define the window of maximal BBB opening and applicable sonication parameters on a clinically translatable and commercially available FUS system that can be used to improve passive permeability and accumulation of therapeutics targeting the brain.

Circadian Influences on Chemotherapy Efficacy in a Mouse Model of Brain Metastases of Breast Cancer.

Chemotherapy is more effective in the treatment of peripheral tumors than brain metastases, likely reflecting the reduced ability of chemotherapy to cross the blood-brain barrier (BBB) and blood-tumor barrier at efficacious concentrations. Recent studies demonstrate circadian regulation of the BBB. Thus, we predicted that optimally timed chemotherapy would increase anti-tumor efficacy in a model of brain metastases of breast cancer (BMBC). First, we characterized novel daily alterations in BBB permeability to a commonly used chemotherapeutic, 14C-paclitaxel, within BMBC following injections given at four time points across the day. Peak and trough 14C-paclitaxel concentrations within BMBC occurred during the mid-dark phase and at the beginning of the light phase, respectively. Notably, chemotherapy injections during the dark phase increased cell death within BMBC and delayed onset of neurological symptoms relative to injections during the light phase. These data provide strong evidence for the beneficial effects of chrono-chemotherapy for the treatment of BMBC.

Modulation of the blood-tumor barrier to enhance drug delivery and efficacy for brain metastases.

The blood-brain barrier is the selectively permeable vasculature of the brain vital for maintaining homeostasis and neurological function. Low permeability is beneficial in the presence of toxins and pathogens in the blood. However, in the presence of metastatic brain tumors, it is a challenge for drug delivery. Although the blood-tumor barrier is slightly leaky, it still is not permissive enough to allow the accumulation of therapeutic drug concentrations in brain metastases. Herein, we discuss the differences between primary brain tumors and metastatic brain tumors vasculature, effects of therapeutics on the blood-tumor barrier, and characteristics to be manipulated for more effective drug delivery.

Overcoming the acquired resistance to gefitinib in lung cancer brain metastasis in vitro and in vivo.

In our previous work, PC-9-Br, a PC-9 brain seeking line established via a preclinical animal model of lung cancer brain metastasis (LCBM), exhibited not only resistance to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib in vitro, but also chemotherapy regimens of cisplatin plus etoposide in vivo. Using this cell line, we investigated novel potential targeted therapeutics for treating LCBM in vitro and in vivo to combat drug resistance. Significant increases in mRNA and protein expression levels of Bcl-2 were found in PC-9-Br compared with parental PC-9 (PC-9-P), but no significant changes of Bcl-XL were observed. A remarkable synergistic effect between EGFR-TKI gefitinib and Bcl-2 inhibitors ABT-263 (0.17 ± 0.010 µM at 48 h and 0.02 ± 0.004 µM at 72 h), or ABT-199 (0.22 ± 0.008 µM at 48 h and 0.02 ± 0.001 µM at 72 h) to overcome acquired resistance to gefitinib (> 0.5 µM at 48 h and 0.10 ± 0.007 µM at 72 h) in PC-9-Br was observed in MTT assays. AZD9291 was also shown to overcome acquired resistance to gefitinib in PC-9-Br in MTT assays (0.23 ± 0.031 µM at 48 h and 0.03 ± 0.008 µM at 72 h). Western blot showed significantly decreased phospho-Erk1/2 and increased cleaved-caspase-3 expressions were potential synergistic mechanisms for gefitinib + ABT263/ABT199 in PC-9-Br. Significantly decreased protein expressions of phospho-EGFR, phospho-Akt, p21, and survivin were specific synergistic mechanism for gefitinib + ABT199 in PC-9-Br. In vivo studies demonstrated afatinib (30 mg/kg) and AZD9291 (25 mg/kg) could significantly reduce the LCBM in vivo and increase survival percentages of treated mice compared with mice treated with vehicle and gefitinib (6.25 mg/kg). In conclusion, our study demonstrated gefitinib + ABT263/ABT199, afatinib, and AZD9291 have clinical potential to treat LCBM.

Nanoparticles as a Tool in Neuro-Oncology Theranostics.

The rapid growth of nanotechnology and the development of novel nanomaterials with unique physicochemical characteristics provides potential for the utility of nanomaterials in theranostics, including neuroimaging, for identifying neurodegenerative changes or central nervous system malignancy. Here we present a systematic and thorough review of the current evidence pertaining to the imaging characteristics of various nanomaterials, their associated toxicity profiles, and mechanisms for enhancing tropism in an effort to demonstrate the utility of nanoparticles as an imaging tool in neuro-oncology. Particular attention is given to carbon-based and metal oxide nanoparticles and their theranostic utility in MRI, CT, photoacoustic imaging, PET imaging, fluorescent and NIR fluorescent imaging, and SPECT imaging.

A Review of Mathematics Determining Solute Uptake at the Blood-Brain Barrier in Normal and Pathological Conditions.

The blood-brain barrier (BBB) limits movement of solutes from the lumen of the brain microvascular capillary system into the parenchyma. The unidirectional transfer constant, Kin, is the rate at which transport across the BBB occurs for individual molecules. Single and multiple uptake experiments are available for the determination of Kin for new drug candidates using both intravenous and in situ protocols. Additionally, the single uptake method can be used to determine Kin in heterogeneous pathophysiological conditions such as stroke, brain cancers, and Alzheimer's disease. In this review, we briefly cover the anatomy and physiology of the BBB, discuss the impact of efflux transporters on solute uptake, and provide an overview of the single-timepoint method for determination of Kin values. Lastly, we compare preclinical Kin experimental results with human parallels.

Irradiator Commissioning and Dosimetry for Assessment of LQ α and ß Parameters, Radiation Dosing Schema, and in vivo Dose Deposition.

Radiation dosimetry is critical in the accurate delivery and reproducibility of radiation schemes in preclinical models for high translational relevance. Prior to performing any in vitro or in vivo experiments, the specific dose output for the irradiator and individual experimental designs must be assessed. Using an ionization chamber, electrometer, and solid water setup, the dose output of wide fields at isocenter can be determined. Using a similar setup with radiochromic films in the place of the ionization chamber, dose rates for smaller fields at different depths can also be determined. In vitro clonogenic survival assays of cancer cells in response to radiation treatment are inexpensive experiments that provide a measure of inherent radio-sensitivity of cell lines by fitting these data with the traditional linear-quadratic model. Model parameters estimated from these assays, combined with the principles of biologic effective doses, allows one to develop varying fractionation schedules for radiation treatment that provide equivalent effective doses in tumor-bearing animal experiments. This is an important factor to consider and correct for in comparing in vivo radiation therapy schedules to eliminate potential confounding of results due to variance in the delivered effective doses. Taken together, this article provides a general method for dose output verification preclinical animal and cabinet irradiators, in vitro assessment of radio-sensitivity, and verification of radiation delivery in small living organisms.

Drug resistance occurred in a newly characterized preclinical model of lung cancer brain metastasis.

BACKGROUND: Cancer metastasis and drug resistance have traditionally been studied separately, though these two lethal pathological phenomena almost always occur concurrently. Brain metastasis occurs in a large proportion of lung cancer patients (~ 30%). Once diagnosed, patients have a poor prognosis surviving typically less than 1 year due to lack of treatment efficacy. METHODS: Human metastatic lung cancer cells (PC-9-Br) were injected into the left cardiac ventricle of female athymic nude mice. Brain lesions were allowed to grow for 21 days, animals were then randomized into treatment groups and treated until presentation of neurological symptoms or when moribund. Prior to tissue collection mice were injected with Oregon Green and 14C-Aminoisobutyric acid followed by an indocyanine green vascular washout. Tracer accumulation was determined by quantitative fluorescent microscopy and quantitative autoradiography. Survival was tracked and tumor burden was monitored via bioluminescent imaging. Extent of mutation differences and acquired resistance was measured in-vitro through half-maximal inhibitory assays and qRT-PCR analysis. RESULTS: A PC-9 brain seeking line (PC-9-Br) was established. Mice inoculated with PC-9-Br resulted in a decreased survival time compared with mice inoculated with parental PC-9. Non-targeted chemotherapy with cisplatin and etoposide (51.5 days) significantly prolonged survival of PC-9-Br brain metastases in mice compared to vehicle control (42 days) or cisplatin and pemetrexed (45 days). Further in-vivo imaging showed greater tumor vasculature in mice treated with cisplatin and etoposide compared to non-tumor regions, which was not observed in mice treated with vehicle or cisplatin and pemetrexed. More importantly, PC-9-Br showed significant resistance to gefitinib by in-vitro MTT assays (IC50 > 2.5 µM at 48 h and 0.1 µM at 72 h) compared with parental PC-9 (IC50: 0.75 µM at 48 h and 0.027 µM at 72 h). Further studies on the molecular mechanisms of gefitinib resistance revealed that EGFR and phospho-EGFR were significantly decreased in PC-9-Br compared with PC-9. Expression of E-cadherin and vimentin did not show EMT in PC-9-Br compared with parental PC-9, and PC-9-Br had neither a T790M mutation nor amplifications of MET and HER2 compared with parental PC-9. CONCLUSION: Our study demonstrated that brain metastases of lung cancer cells may independently prompt drug resistance without drug treatment.

MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes.

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a-/- mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.

Improving CNS Delivery to Brain Metastases by Blood-Tumor Barrier Disruption.

Brain metastases encompass nearly 80% of all intracranial tumors. A late stage diagnosis confers a poor prognosis, with patients typically surviving less than 2 years. Poor survival can be equated to limited effective treatment modalities. One reason for the failure rates is the presence of the blood-brain barrier (BBB) and blood-tumor barrier (BTB) that limit the access of potentially effective chemotherapeutics to metastatic lesions. Strategies to overcome these barriers include new small molecule entities capable of crossing into the brain parenchyma, novel formulations of existing chemotherapies, and disruptive techniques. Here, we review BBB physiology and BTB pathophysiology. Additionally, we review the limitations of routinely practiced therapies and three current methods being explored for BBB/BTB disruption for improved delivery of chemotherapy to brain tumors.

Targeting the Blood-Brain Barrier to Prevent Sepsis-Associated Cognitive Impairment.

Sepsis is a systemic inflammatory disease resulting from an infection. This disorder affects 750 000 people annually in the United States and has a 62% rehospitalization rate. Septic symptoms range from typical flu-like symptoms (eg, headache, fever) to a multifactorial syndrome known as sepsis-associated encephalopathy (SAE). Patients with SAE exhibit an acute altered mental status and often have higher mortality and morbidity. In addition, many sepsis survivors are also burdened with long-term cognitive impairment. The mechanisms through which sepsis initiates SAE and promotes long-term cognitive impairment in septic survivors are poorly understood. Due to its unique role as an interface between the brain and the periphery, numerous studies support a regulatory role for the blood-brain barrier (BBB) in the progression of acute and chronic brain dysfunction. In this review, we discuss the current body of literature which supports the BBB as a nexus which integrates signals from the brain and the periphery in sepsis. We highlight key insights on the mechanisms that contribute to the BBB's role in sepsis which include neuroinflammation, increased barrier permeability, immune cell infiltration, mitochondrial dysfunction, and a potential barrier role for tissue non-specific alkaline phosphatase (TNAP). Finally, we address current drug treatments (eg, antimicrobials and intravenous immunoglobulins) for sepsis and their potential outcomes on brain function. A comprehensive understanding of these mechanisms may enable clinicians to target specific aspects of BBB function as a therapeutic tool to limit long-term cognitive impairment in sepsis survivors.

Development of a Cx46 Targeting Strategy for Cancer Stem Cells.

Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

Long non-coding RNA FAM83H-AS1 is regulated by human papillomavirus 16 E6 independently of p53 in cervical cancer cells.

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.

Conditional knockout of SHP2 in ErbB2 transgenic mice or inhibition in HER2-amplified breast cancer cell lines blocks oncogene expression and tumorigenesis.

Overexpression of the human epidermal growth factor receptor 2 (HER2) is the cause of HER2-positive breast cancer (BC). Although HER2-inactivating therapies have benefited BC patients, development of resistance and disease recurrence have been the major clinical problems, pointing to a need for alternative therapeutic strategies. For that to happen, proteins that play critical roles in the biology of HER2-induced tumorigenesis have to be identified and characterized. Here, we show that the Src homology phosphotyrosyl phosphatase 2 (Shp2) encoded by the Ptpn11 gene is a requisite for ErbB2-induced tumorigenesis. We report that conditional knockout of Shp2 alleles in the ErbB2 BC model mice abrogates mammary tumorigenesis by blocking the expression of the ErbB2 transgene. We also show that inhibition of SHP2 encoded by the PTPN11 gene in the HER2-amplified BC cells induces a normal-like cellular phenotype and suppresses tumorigenesis and metastasis by blocking HER2 overexpression. These findings demonstrate that ErbB2-induced tumors in mice or xenograft tumors induced by transplantation of HER2-amplified BC cells are vulnerable to SHP2 inhibition since it abrogates the expression of the very oncogene that causes of the disease. This report paves the way for developing SHP2-targeting therapies for BC treatment in the future.

Permeability changes and effect of chemotherapy in brain adjacent to tumor in an experimental model of metastatic brain tumor from breast cancer.

BACKGROUND: Brain tumor vasculature can be significantly compromised and leakier than that of normal brain blood vessels. Little is known if there are vascular permeability alterations in the brain adjacent to tumor (BAT). Changes in BAT permeability may also lead to increased drug permeation in the BAT, which may exert toxicity on cells of the central nervous system. Herein, we studied permeation changes in BAT using quantitative fluorescent microscopy and autoradiography, while the effect of chemotherapy within the BAT region was determined by staining for activated astrocytes. METHODS: Human metastatic breast cancer cells (MDA-MB-231Br) were injected into left ventricle of female NuNu mice. Metastases were allowed to grow for 28 days, after which animals were injected fluorescent tracers Texas Red (625 Da) or Texas Red dextran (3 kDa) or a chemotherapeutic agent 14C-paclitaxel. The accumulation of tracers and 14C-paclitaxel in BAT were determined by using quantitative fluorescent microscopy and autoradiography respectively. The effect of chemotherapy in BAT was determined by staining for activated astrocytes. RESULTS: The mean permeability of texas Red (625 Da) within BAT region increased 1.0 to 2.5-fold when compared to normal brain, whereas, Texas Red dextran (3 kDa) demonstrated mean permeability increase ranging from 1.0 to 1.8-fold compared to normal brain. The Kin values in the BAT for both Texas Red (625 Da) and Texas Red dextran (3 kDa) were found to be 4.32 ± 0.2 × 105 mL/s/g and 1.6 ± 1.4 × 105 mL/s/g respectively and found to be significantly higher than the normal brain. We also found that there is significant increase in accumulation of 14C-Paclitaxel in BAT compared to the normal brain. We also observed animals treated with chemotherapy (paclitaxel (10 mg/kg), erubilin (1.5 mg/kg) and docetaxel (10 mg/kg)) showed activated astrocytes in BAT. CONCLUSIONS: Our data showed increased permeation of fluorescent tracers and 14C-paclitaxel in the BAT. This increased permeation lead to elevated levels of activated astrocytes in BAT region in the animals treated with chemotherapy.

An Effective Approach to Teaching Pharmacogenomics in the First Year of Pharmacy Curriculum.

Objective. To develop an effective method in teaching pharmacogenomics as a part of a new course, Biopharmaceutics and Pharmacogenomics. Methods. Teaching effectiveness was measured by quizzes, retrospective pre- and post-surveys, team activities, and journal reflections. Four team activities were included in the course: genomic disease, patient case, genetic counselor and a debate about personalized medicine. Outcomes and course impact were evaluated at the end of the course. The evaluation methods included the assessment of knowledge, students' perceptions regarding the utility of team activities, the impact of the course on students' confidence to discuss pharmacogenomics with health care providers or patients, and long-term knowledge retention, measured in the following P2 semester. Results. Seventy-six students were enrolled in the course. Multiple assessments during the course demonstrated that students' knowledge of pharmacogenomics improved. The team activities had a positive impact on student learning, and the course improved their confidence level to discuss pharmacogenomics with another health care provider or a patient. While 86% of the students considered themselves "unconfident," "somewhat unconfident" or "neither confident nor unconfident" at the beginning of the course, 91% reported being "confident" or "somewhat confident" by the end of the course. This increase in confidence was statistically significant. Furthermore, students showed knowledge retention six months after taking the course. Conclusion. Implementation of a new course in pharmacogenomics was effective and well received by the students. It also prepared students for system-based therapeutics courses later in the curriculum.

Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases.

In women, breast cancer is the most common cancer diagnosis and second most common cause of cancer death. More than half of breast cancer patients will develop metastases to the bone, liver, lung, or brain. Breast cancer brain metastases (BCBM) confers a poor prognosis, as current therapeutic options of surgery, radiation, and chemotherapy rarely significantly extend life and are considered palliative. Within the realm of chemotherapy, the last decade has seen an explosion of novel chemotherapeutics involving targeting agents and unique dosage forms. We provide a historical overview of BCBM chemotherapy, review the mechanisms of new agents such as poly-ADP ribose polymerase inhibitors, cyclin-dependent kinase 4/6 inhibitors, phosphatidyl inositol 3-kinaseinhibitors, estrogen pathway antagonists for hormone-receptor positive BCBM; tyrosine kinase inhibitors, antibodies, and conjugates for HER2+ BCBM; repurposed cytotoxic chemotherapy for triple negative BCBM; and the utilization of these new agents and formulations in ongoing clinical trials. The mechanisms of novel dosage formulations such as nanoparticles, liposomes, pegylation, the concepts of enhanced permeation and retention, and drugs utilizing these concepts involved in clinical trials are also discussed. These new treatments provide a promising outlook in the treatment of BCBM.

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer.

PURPOSE: The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect. METHODS: Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated. RESULTS: Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle. CONCLUSIONS: Liposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.

Trastuzumab distribution in an in-vivo and in-vitro model of brain metastases of breast cancer.

BACKGROUND: Drug and antibody delivery to brain metastases has been highly debated in the literature. The blood-tumor barrier (BTB) is more permeable than the blood-brain barrier (BBB), and has shown to have highly functioning efflux transporters and barrier properties, which limits delivery of targeted therapies. METHODS: We characterized the permeability of 125I-trastuzumab in an in-vivo, and fluorescent trastuzumab-Rhodamine123 (t-Rho123) in a novel microfluidic in-vitro, BBB and BTB brain metastases of breast cancer model. In-vivo: Human MDA-MB-231-HER2+ metastatic breast cancer cells were grown and maintained under static conditions. Cells were harvested at 80% confluency and prepped for intra-cardiac injection into 20 homozygous female Nu/Nu mice. In-vitro: In a microfluidic device (SynVivo), human umbilical vein endothelial cells were grown and maintained under shear stress conditions in the outer compartment and co-cultured with CTX-TNA2 rat brain astrocytes (BBB) or Met-1 metastatic HER2+ murine breast cancer cells (BTB), which were maintained in the central compartment under static conditions. RESULTS: Tissue distribution of 125I-trastuzumab revealed only ~3% of injected dose reached normal brain, with ~5% of injected dose reaching brain tumors. No clear correlation was observed between size of metastases and the amount of 125I-trastuzumab localized in-vivo. This heterogeneity was paralleled in-vitro, where the distribution of t-Rho123 from the outer chamber to the central chamber of the microfluidic device was qualitatively and quantitatively analyzed over time. The rate of t-Rho123 linear uptake in the BBB (0.27 ± 0.33 × 104) and BTB (1.29 ± 0.93 × 104) showed to be significantly greater than 0 (p < 0.05). The BTB devices showed significant heterogenetic tendencies, as seen in in-vivo. CONCLUSIONS: This study is one of the first studies to measure antibody movement across the blood-brain and blood-tumor barriers, and demonstrates that, though in small and most likely not efficacious quantities, trastuzumab does cross the blood-brain and blood-tumor barriers.