An important property in any central nervous system (CNS) drug is the ability to cross into the brain and reach therapeutic concentrations at safe and acceptable systemic doses. Multiple parameters influence drug availability to the brain. One of the most important of these is the blood-brain barrier (BBB). The vasculature of the brain differs from that of other organs of the body in that it greatly restricts the exchange of most solutes into the brain from the systemic circulation. Equilibration, which only requires seconds to minutes for low molecular weight drugs in the interstitial fluid of most tissues of the body, can require days to weeks for many agents in the brain. The restricted neurovascular exchange is based upon the unique properties of the endothelial cell membranes lining the brain blood vessels which limit the passive diffusion of many polar solutes into the brain and avidly pump out a broad array of polar and nonpolar agents through a series of active efflux transporters. This chapter presents a conceptual overview of the primary methods to assess brain drug distribution in vivo, providing an insider’s guide to many of the critical steps to use the methods appropriately. Then, two case examples are provided in detail illustrating application and interpretation of specific methods. The entire chapter is written with a perspective of providing an “insider’s view” of the level of drug necessary to reach therapeutic action in the brain. Several parameters are broadly used to explain CNS drug passage and equilibration. One of these is the cerebrovascular permeability-surface area product (PS), which reflects how rapidly a solute can cross in or out of the brain. Another is the brain distribution volume or partition coefficient (Kp,brain), which characterizes the extent (either high or low) that a drug equilibrates in the brain. Because most drugs bind or associate reversibly to proteins, lipids, and other biologic macromolecules, a third parameter is the fraction to which a solute travels freely in the tissue or blood (fu, the free or unbound fraction). This parameter can be used to calculate the free and bound drug concentrations from the total concentration that is measured by many analytical methods. Together with the time course of drug in the circulation, the above parameters can be used to predict drug total, free, and bound concentrations in brain tissue at all time points after administration. This information can then be used to calculate biologic activity if the binding constant (KD) of the receptor or the inhibitory constant (Ki) of the signaling process is known. Specific methods, such as in situ brain perfusion, brain efflux index, and in situ brain microdialysis, are valuable to dissect the specific mechanisms operational at the barrier that mediate or regulate drug transport across the brain endothelial cell membranes. In the end, the investigator has a broad array of approaches to assess drug availability to the brain and to make recommendations that would improve outcomes. In some cases, such as for drugs that act in other tissues, the desire may be to limit brain exposure to avoid adverse drug reactions. A specific focus of the chapter is to promote accurate measurements and avoid nonspecific approaches that are error bound and have led to a lot of confusion in the field.

Abstract 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.

Abstract Women diagnosed with breast cancer at a younger age (typically defined as < 40 years old) often have a poorer prognosis and an increased risk of brain metastasis compared to their older counterparts. Multivariate analyses accounting for differences in tumor characteristics have shown that age is an independent predictor of worse outcome. We therefore hypothesized that rather than intrinsic tumor properties, extrinsic microenvironmental factors contribute to age-related differences in aggressiveness. The effect of age was examined by injecting brain-selected breast cancer cells into young (2 – 6 months) and older (>12 months) mice. In four brain metastasis models examined, young mice developed 2- to 16-fold (p < 0.05) more brain metastases compared to older mice. The effect of age was not observed in mouse breast cancer models that metastasize to liver and lungs, suggesting that this is an organ-specific phenomenon. Flow cytometry-based immune-profiling of mouse brains showed that T-cells (CD4+, CD8+, and FOXP3+CD25+ regulatory T-cells), monocytes and neutrophils were elevated in brains with metastases, but the abundance of these populations did not vary dramatically with age. Furthermore, antibody-based depletion of T-cells, monocytes and neutrophils did not significantly alter brain metastasis development. Microglia, which are resident CNS myeloid cells, were 1.5-fold more abundant in young brains compared to older brains. Depletion of CNS myeloid cells using the colony stimulating factor-1-receptor inhibitor PLX3397 reduced brain metastatic tumor burden in young mice by 2.1-fold (p < 0.001). Importantly, loss of CNS myeloid cells/microglia, which are normally more activated in aged mice and thus may protect the older brain against metastasis, did not augment brain metastasis formation in older mice. These results suggest that the younger brain is more permissive for breast cancer metastasis and that targeting resident CNS myeloid cells may be an effective strategy to prevent brain metastasis development in younger patients.

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.

In this paper a simple and efficient method for the unsymmetrical terphenyls via sequential one-pot Suzuki coupling reactions using Pd(OAc)(2) without isolation of the intermediate is described. The prepared terphenyls were found to possess potent anticancer properties against a panel of cancer cells which includes A549, HeLa, MCF7, DU145, HT29 and BxPC-3. Structural similarity with combretastatin A4, these terphenyls disrupted the tubulin polymerization in vitro and destabilized the microtubules in cells. Flow cytometry studies indicated growth arrest of cells in the G2/M phase of the cell cycle corresponding to antimitotic action. Furthermore, compound 4c showed potent anti-mitotic activity even in zebrafish model and could likely be a potential therapeutic compound as it is active both in in vitro and in vivo.

To evaluate vinorelbine drug exposure and activity in brain metastases of the human MDA-MB-231BR breast cancer model using integrated imaging and analysis.

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.

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.

BACKGROUNDBreast 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.METHODSStudy 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.RESULTSTwelve 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.CONCLUSIONSThis 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.

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 (R).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. (C) 2013 Elsevier Inc. All rights reserved.

An important property in any central nervous system drug is the ability to cross the blood–brain barrier (BBB) and to reach therapeutic concentrations in brain at safe and acceptable doses. Multiple parameters influence brain drug bioavailability, including solubility, membrane permeation, and affinity for influx and efflux transporters. This chapter overviews the primary in vivo methods to assess brain drug distribution in preclinical and clinical reports. In most studies, two parameters, the BBB permeability-surface area product (PS) and brain distribution volume or partition coefficient (K p,brain), are used to characterize the ability of a drug compound to gain access to and distribute in brain. Together with the time course of systemic drug exposure, these two parameters can be used to predict total drug concentration within brain. Further, because unbound drug concentration often correlates better with drug activity, a number of studies also determine the drug free fraction (f u) so that the free concentrations can be calculated. Specific methods, such as in situ brain perfusion, brain efflux index, and brain microdialysis, are valuable to dissect specific elements of BBB drug permeation or transport as well as equilibration in brain interstitial fluid and cellular elements. Overall, these approaches complement in vivo drug distribution studies and in vitro BBB permeation methods.

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 14C-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.

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 30min stimulation with 60mM K+, which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4±10.1μM using the zero-flow method and 50.7±5.5μM based on in vitro recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180nmol/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.

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 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.