Aging and CNS Myeloid Cell Depletion Attenuate Breast Cancer Brain Metastasis.

PURPOSE: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. EXPERIMENTAL DESIGN: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. RESULTS: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. CONCLUSIONS: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.

Vinorelbine Delivery and Efficacy in the MDA-MB-231BR Preclinical Model of Brain Metastases of Breast Cancer.

PURPOSE: To evaluate vinorelbine drug exposure and activity in brain metastases of the human MDA-MB-231BR breast cancer model using integrated imaging and analysis. METHODS: Brain and systemic metastases were created by administration of cancer cells in female NuNu mice. After metastases developed, animals were administered vinorelbine at the maximal tolerated dose (12 mg/kg), and were evaluated thereafter for total and unbound drug pharmacokinetics, biomarker TUNEL staining, and barrier permeability to Texas red. RESULTS: Median brain metastasis drug exposure was 4-fold greater than normal brain, yet only ~8% of non-barrier systemic metastases, which suggests restricted brain exposure. Unbound vinorelbine tissue/plasma partition coefficient, Kp,uu, equaled ~1.0 in systemic metastases, but 0.03-0.22 in brain metastases, documenting restricted equilibration. In select sub-regions of highest drug-uptake brain metastases, Kp,uu approached 1.0, indicating complete focal barrier breakdown. Most vinorelbine-treated brain metastases exhibited little or no positive early apoptosis TUNEL staining in vivo. The in vivo unbound vinorelbine IC50 for TUNEL-positive staining (56 nM) was 4-fold higher than that measured in vitro (14 nM). Consistent with this finding, P-glycoprotein expression was observed to be substantially upregulated in brain metastasis cells in vivo. CONCLUSIONS: Vinorelbine exposure at maximum tolerated dose was less than one-tenth that in systemic metastases in >70% of brain metastases, and was associated with negligible biomarker effect. In small subregions of the highest uptake brain metastases, compromise of blood-tumor barrier appeared complete. The results suggest that restricted delivery accounts for 80% of the compromise in drug efficacy for vinorelbine against this model.

Highly sensitive LC-MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study.

A selective, sensitive and rapid LC-MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200µL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1→105.1 and m/z 375.3→201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5min and the elution of PZ and HZ occurred at 2.53, and 1.92min, respectively; this was achieved with a mobile phase consisting of 10mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51ng/mL. A linear response function was established for the range of concentrations 0.51-25.2ng/mL (r>0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

Design and synthesis of a C7-aryl piperlongumine derivative with potent antimicrotubule and mutant p53-reactivating properties.

Small molecules that can restore biological function to the p53 mutants found in human cancers have been highly sought to increase the anticancer efficacy. In efforts to generate hybrid anticancer drugs that can impact two or more targets simultaneously, we designed and developed piperlongumine (PL) derivatives with an aryl group inserted at the C-7 position. This insertion bestowed a combretastatin A4 (CA4, an established microtubule disruptor) like structure while retaining the piperlongumine configuration. The new compounds exhibited potent antiproliferative activities against eight cancer cell lines, in particular, were more cytotoxic against the SKBR-3 breast cancer cells which harbor a R175H mutation in p53 suppressor. KSS-9, a representative aryl PL chosen for further studies induced abundant ROS generation and protein glutathionylation. KSS-9 strongly disrupted the tubulin polymerization in vitro, destabilized the microtubules in cells and induced a potent G2/M cell cycle block. More interestingly, KSS-9 showed the ability to reactivate the p53 mutation and restore biological activity to the R175H mutant protein present in SKBR3 cells. Several procedures, including immunocytochemistry using conformation-specific antibodies for p53, immunoprecipitation combined with western blotting, electrophoretic shift mobility shift assays showed a reciprocal loss of mutant protein and generation of wild-type like protein. p53 reactivation was accompanied by the induction of the target genes, MDM2, p21cip1 and PUMA. Mechanistically, the redox-perturbation in cancer cells by the hybrid drug appears to underlie the p53 reactivation process. This anticancer drug approach merits further development.

CNS Anticancer Drug Discovery and Development Conference White Paper.

Following the first CNS Anticancer Drug Discovery and Development Conference, the speakers from the first 4 sessions and organizers of the conference created this White Paper hoping to stimulate more and better CNS anticancer drug discovery and development. The first part of the White Paper reviews, comments, and, in some cases, expands on the 4 session areas critical to new drug development: pharmacological challenges, recent drug approaches, drug targets and discovery, and clinical paths. Following this concise review of the science and clinical aspects of new CNS anticancer drug discovery and development, we discuss, under the rubric "Accelerating Drug Discovery and Development for Brain Tumors," further reasons why the pharmaceutical industry and academia have failed to develop new anticancer drugs for CNS malignancies and what it will take to change the current status quo and develop the drugs so desperately needed by our patients with malignant CNS tumors. While this White Paper is not a formal roadmap to that end, it should be an educational guide to clinicians and scientists to help move a stagnant field forward.

Capecitabine and lapatinib uptake in surgically resected brain metastases from metastatic breast cancer patients: a prospective study.

BACKGROUND: Breast cancer brain metastases (BCBM) are challenging complications that respond poorly to systemic therapy. The role of the blood-tumor barrier in limiting BCBM drug delivery and efficacy has been debated. Herein, we determined tissue and serum levels of capecitabine, its prodrug metabolites, and lapatinib in women with BCBM resected via medically indicated craniotomy. METHODS: Study patients with BCBM requiring surgical resection received either single-dose capecitabine (1250 mg/m(2)) 2-3 h before surgery or 2-5 doses of lapatinib (1250 mg) daily, the last dose 2-3 h before surgery. Serum samples were collected serially on the day of surgery. Drug concentrations were determined in serum and BCBM using liquid chromatography tandem mass spectrometry. RESULTS: Twelve patients were enrolled: 8 for capecitabine and 4 for lapatinib. Measurable drug levels of capecitabine and metabolites, 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine, and 5-fluorouracil, were detected in all BCBM. The ratio of BCBM to serum was higher for 5-fluorouracil than for capecitabine. As for lapatinib, the median BCBM concentrations ranged from 1.0 to 6.5 µM. A high variability (0.19-9.8) was noted for lapatinib BCBM-to-serum ratio. CONCLUSIONS: This is the first study to demonstrate that capecitabine and lapatinib penetrate to a significant though variable degree in human BCBM. Drug delivery to BCBM is variable and in many cases appears partially limiting. Elucidating mechanisms that limit drug concentration and innovative approaches to overcome limited drug uptake will be important to improve clinical efficacy of these agents in the central nervous system. Trial registration ID: NCT00795678.

ß-Hydroxybutyric acid grafted solid lipid nanoparticles: a novel strategy to improve drug delivery to brain.

Delivery of drugs to brain is an elusive task in the therapy of many serious neurological diseases. With the aim to create a novel formulation to enhance the drug uptake to brain, betreliesoxybutyric acid (HBA) grafted docetaxel loaded solid lipid nanoparticles (HD-SLNs) were explored. Transportation of HD-SLNs relies on the transport of novel ligand, HBA, by monocarboxylic acid transporter (MCT1). Expression of MCT1 transporter on brain endothelial cells (bEnd cells) was studied using immunocytochemistry. Stearylamine-HBA conjugate was used to modify the surface of SLNs and it was confirmed using XPS (X-Ray Photon Spectroscopy) analysis. In vitro release studies revealed the controlled release of drug from HD-SLNs. Cytotoxicity and cell uptake studies revealed the increased uptake of docetaxel with HD-SLNs. Mechanism involved in the uptake of HD-SLNs was studied in bEnd cells by saturating MCT1 with excess HBA. Pharmacokinetic and brain distribution demonstrated increased docetaxel concentrations in brain compared with Taxotere®. FROM THE CLINICAL EDITOR: The authors of this study demonstrate enhanced drug delivery to the brain using a novel formulation of beta-hydroxybutyric acid grafted docetaxel loaded solid lipid nanoparticles. The results show increased uptake of docetaxel compared with Taxotere.

Lapatinib distribution in HER2 overexpressing experimental brain metastases of breast cancer.

PURPOSE: Lapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following (14)C-lapatinib administration (100 mg/kg p.o. or 10 mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer. METHODS: Drug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography. RESULTS: (14)C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7-9-fold greater than surrounding brain tissue at 2 and 12 h after oral administration. However, average lapatinib concentration in brain metastases was still only 10-20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains. CONCLUSIONS: Results show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

The ketogenic diet changes metabolite levels in hippocampal extracellular fluid.

Despite successful use of the ketogenic diet (KD) for the treatment of drug-resistant epilepsy, its mechanism of action is unclear. After KD-feeding, increased plasma D-beta-hydroxybutyrate (BHB) levels appear to be important for protection against seizures. We hypothesized that the KD leads to metabolic changes in the brain, which are reflected in the hippocampal extracellular fluid (hECF). CD1 mice were fed control or KD for 2-3 weeks since weaning. In vivo microdialysis of hECF was used to measure the levels of glucose, lactate, as well as BHB under basal conditions and during 30 min stimulation with 60 mM K(+), which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4±10.1 µM using the zero-flow method and 50.7±5.5 µM based on in vitro recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180 nmol/g. The intracellular BHB concentration is therefore estimated to be about 3-fold higher than the extracellular level, which suggests that BHB in adolescent mouse brains may not be quickly metabolized. The basal hECF glucose concentration was 30% lower in KD-fed mice, indicating that glucose may be less important as an energy source. Lactate levels were similar in control and KD-fed mice. High potassium stimulation elevated lactate by 3-3.5-fold and decreased glucose by 40-50% in both diet groups, consistent with similar anaerobic and aerobic metabolism in both diet groups during high hippocampal activity. Overall, these data (1) defined the BHB concentration in the hippocampal extracellular fluid in KD-fed mice and (2) showed lower glucose metabolism compared to control diet-fed mice. This work will now enable other researchers to mimic the hippocampal extracellular environment in experiments aimed at deciphering the mechanisms of the KD.

Eicosapentaenoic and docosahexaenoic acids are not anticonvulsant or neuroprotective in acute mouse seizure models.

Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or vegetable oil (control) were added to standard rodent chow (6 g/kg) and fed to mice ad lib for 4 weeks to determine if polyunsaturated fatty acids (PUFA) are anticonvulsant or neuroprotective in mice. The seizure susceptibility of these mice was compared using the fluorothyl, pentylenetetrazole (PTZ), 6 Hz, and kainate models. We found that PUFA feeding significantly altered the fatty acid profile in both plasma and brain, but did not change seizure thresholds in the fluorothyl, PTZ, or 6 Hz models nor did it significantly alter seizure behavior or hippocampal damage following kainate injection. In conclusion, DHA or EPA feeding did not show anticonvulsant or neuroprotective activity in four acute seizure models. Chronic seizure models remain to be examined.

Anticonvulsant profile of a balanced ketogenic diet in acute mouse seizure models.

Anticonvulsant effects of the ketogenic diet (KD) have been reported in the mouse, although previous studies did not control for intake of vitamins, minerals and antioxidants. The aim of this study was to examine the effects of balanced ketogenic and control diets in acute mouse seizure models. The behavior in four mouse seizure models, plasma d-beta-hydroxybutyrate (d-BHB) and glucose levels were determined after feeding control diet, 4:1 and 6:1 KDs with matched vitamins, minerals and antioxidants. Feeding 4:1 and 6:1 KDs ad lib to 3-week-old (adolescent) mice resulted in 1.2-2.2mM d-BHB in plasma, but did not consistently change glucose levels. The 6:1 KD reproducibly elevated the CC50 (current that initiates seizures in 50% mice tested) in the 6-Hz model after 14 days of feeding to adolescent CD1 mice. Higher plasma d-BHB levels correlated with anticonvulsant effects. Despite ketosis, no consistent anticonvulsant effects of KDs were found in the fluorothyl or pentylenetetrazole CD1 mouse models. The 4:1 KD was neither anticonvulsant nor neuroprotective in hippocampus in the C3H mouse kainate model. Taken together, the KD's anticonvulsant effect was limited to the 6-Hz model, required chronic feeding with 6:1 fat content, and was independent from lowering plasma glucose.