Quentin R. Smith

Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer

Purpose: Brain metastases of breast cancer appear to be increasing in incidence, confer significant morbidity, and threaten to compromise gains made in systemic chemotherapy. The blood–tumor barrier (BTB) is compromised in many brain metastases; however, the extent to which this influences chemotherapeutic delivery and efficacy is unknown. Herein, we answer this question by measuring BTB passive integrity, chemotherapeutic drug uptake, and anticancer efficacy in vivo in two breast cancer models that metastasize preferentially to brain. Experimental Design:Experimental brain metastasis drug uptake and BTB permeability were simultaneously measured using novel fluorescent and phosphorescent imaging techniques in immune-compromised mice. Drug-induced apoptosis and vascular characteristics were assessed using immunofluorescent microscopy. Results: Analysis of over 2,000 brain metastases from two models (human 231-BR-Her2 and murine 4T1-BR5) showed partial BTB permeability compromise in greater than 89% of lesions, varying in magnitude within and between metastases. Brain metastasis uptake of 14C-paclitaxel and 14C-doxorubicin was generally greater than normal brain but less than 15% of that of other tissues or peripheral metastases, and only reached cytotoxic concentrations in a small subset (∼10%) of the most permeable metastases. Neither drug significantly decreased the experimental brain metastatic ability of 231-BR-Her2 tumor cells. BTB permeability was associated with vascular remodeling and correlated with overexpression of the pericyte protein desmin. Conclusions: This work shows that the BTB remains a significant impediment to standard chemotherapeutic delivery and efficacy in experimental brain metastases of breast cancer. New brain permeable drugs will be needed. Evidence is presented for vascular remodeling in BTB permeability alterations. Clin Cancer Res; 16(23); 5664–78. ©2010 AACR.

Strategies to advance translational research into brain barriers

There is a paucity of therapies for most neurological disorders--from rare lysosomal storage diseases to major public health concerns such as stroke and Alzheimers disease. Advances in the targeting of drugs to the CNS are essential for the future success of neurotherapeutics; however, the delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by the blood-brain barrier, the blood-CSF barrier, or other specialised CNS barriers. These brain barriers are now recognised as a major obstacle to the treatment of most brain disorders. The challenge to deliver therapies to the CNS is formidable, and the solution will require concerted international efforts among academia, government, and industry. At a recent meeting of expert panels, essential and high-priority recommendations to propel brain barrier research forward in six topical areas were developed and these recommendations are presented here.

Chemotherapy Delivery Issues in Central Nervous System Malignancy: A Reality Check

PURPOSE This review assesses the current state of knowledge regarding preclinical and clinical pharmacology for brain tumor chemotherapy and evaluates relevant brain tumor pharmacology studies before October 2006. RESULTS Chemotherapeutic regimens in brain tumor therapy have often emerged from empirical clinical studies with retrospective pharmacologic explanations, rather than prospective trials of rational chemotherapeutic approaches. Brain tumors are largely composed of CNS metastases of systemic cancers. Primary brain tumors, such as glioblastoma multiforme or primary CNS lymphomas, are less common. Few of these tumors have well-defined optimal treatment. Brain tumors are protected from systemic chemotherapy by the blood-brain barrier (BBB) and by intrinsic properties of the tumors. Pharmacologic studies of delivery of conventional chemotherapeutics and novel therapeutics showing actual tumor concentrations and biologic effect are lacking. CONCLUSION In this article, we review drug delivery across the BBB, as well as blood-tumor and -cerebrospinal fluid (CSF) barriers, and mechanisms to increase drug delivery to CNS and CSF tumors. Because of the difficulty in treating CNS tumors, innovative treatments and alternative delivery techniques involving brain/cord capillaries, choroid plexus, and CSF are needed.

Transport of Glutamate and Other Amino Acids at the Blood-Brain Barrier

In most regions of the brain, the uptake of glutamate and other anionic excitatory amino acids from the circulation is limited by the blood-brain barrier (BBB). In most animals, the BBB is formed by the brain vascular endothelium, which contains cells that are joined by multiple bands of tight junctions. These junctions effectively close off diffusion through intercellular pores; as a result, most solutes cross the BBB either by diffusing across the lipoid endothelial cell membranes or by being transported across by specific carriers. Glutamate transport at the BBB has been studied by both in vitro cell uptake assays and in vivo perfusion methods. The results demonstrate that at physiologic plasma concentrations, glutamate flux from plasma into brain is mediated by a high affinity transport system at the BBB. Efflux from brain back into plasma appears to be driven in large part by a sodium-dependent active transport system at the capillary abluminal membrane. Glutamate concentration in brain interstitial fluid is only a fraction of that of plasma and is maintained fairly independently of small fluctuations in plasma concentration. Restricted brain passage is also observed for several excitatory glutamate analogs, including domoic acid and kynurenic acid. In summary, the BBB is one component of a regulatory system that helps maintain brain interstitial fluid glutamate concentration independently of the circulation.

Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position.

Drug interactions due to efflux transport inhibition at the blood–brain barrier (BBB) have been receiving increasing scrutiny because of the theoretical possibility of adverse central nervous system (CNS) effects identified in preclinical studies. In this review, evidence from pharmacokinetic, pharmacodynamic, imaging, pharmacogenetic, and pharmacovigilance studies, along with drug safety reports, is presented supporting a low probability of modulating transporters at the human BBB by currently marketed drugs.

Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks

Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. Results: Following systemic administration, uptake of [14C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. Conclusions: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. (Clin Cancer Res 2009;15(19):6148–57)

Brain Metastases of Breast Cancer

Central nervous system or brain metastases traditionally occur in 10-16% of metastatic breast cancer patients and are associated with a dismal prognosis. The development of brain metastases has been associated with young age, and tumors that are estrogen receptor negative, Her-2+ or of the basal phenotype. Treatment typically includes whole brain irradiation, or either stereotactic radiosurgery or surgery with whole brain radiation, resulting in an approximately 20% one year survival. The blood-brain barrier is a formidable obstacle to the delivery of chemotherapeutics to the brain. Mouse experimental metastasis model systems have been developed for brain metastasis using selected sublines of human MDA-MB-231 breast carcinoma cells. Using micron sized iron particles and MRI imaging, the fate of MDA-MB-231BR cells has been mapped: Approximately 2% of injected cells form larger macroscopic metastases, while 5% of cells remain as dormant cells in the brain. New therapies with permeability for the blood-brain barrier are needed to counteract both types of tumor cells.

Fatty acid uptake and incorporation in brain: studies with the perfusion model.

The contributions of individual components of blood to brain [14C]palmitate uptake and incorporation were studied with the in situ brain perfusion technique in the pentobarbital-anesthetized rat. With whole-blood perfusate, brain unacylated [14C]palmitate uptake was linear with time and extrapolated to zero at T=0 s of perfusion. Tracer accumulated in brain with a blood-to-brain transfer coefficient of 1.8 ± 0.1 × 10−4 mL/s/g (whole cerebral hemisphere). Incorporation into brain lipids was rapid such that ∼40% of tracer in brain at 45 s of perfusion was in cerebral phospholipids and neutral lipids. Similar rates of uptake were obtained during unacylated [14C]palmitate perfusion in whole rat plasma, serum, or artificial saline containing 2–3% albumin, suggesting that albumin has a key role in determining [14C]palmitate uptake in brain. The excellent match in brain uptake rates between whole blood and albumin-containing saline fluid suggests that the perfusion technique will be useful method for quantifying the individual contributions of blood constituents and albumin binding on brain [14C]palmitate uptake.

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique: Blood-brain barrier choline transport

Choline enters brain by saturable transport at the blood–brain barrier (BBB). In separate studies, both sodium‐dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re‐examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (Vmax= 2.4–3.1 nmol/min/g; Km = 39–42 µm) with an apparent affinity (1/Km) for choline five to ten‐fold greater than previously reported in vivo, but less than neuronal ‘high‐affinity’ brain choline transport (Km = 1–5 µm). BBB choline uptake from a sodium‐free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium‐3 inhibited brain choline uptake with a Ki (57 ± 11 µm) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The Vmax of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.

Role of Site-Specific Binding to Plasma Albumin in Drug Availability to Brain

Many studies have reported greater drug uptake into brain than that predicted based upon existing models using the free fraction (fu) of drug in arterial serum. To explain this difference, circulating plasma proteins have been suggested to interact with capillary membrane in vivo to produce a conformational change that favors net drug dissociation and elevation of fu. Albumin, the principal binding protein in plasma, has two main drug binding sites, Sudlow I and II. We tested this hypothesis using drugs that bind selectively to either site I (warfarin) or site II (ibuprofen), as well as mixed ligands that have affinity for both sites (tolbutamide and valproate). Brain uptake was determined in the presence and absence of albumin using the in situ rat brain perfusion technique. Unidirectional brain uptake transfer constants (Kin) were measured and compared with those predicted using the modified Kety-Crone-Renkin model: Kin = F(1 – e–fu × PSu/F), where F is perfusion flow and PSu is the permeability-surface area product to free drug of brain capillaries. The results demonstrated good agreement between measured and predicted Kin over a 100-fold range in perfusion fluid albumin concentration using albumin from three different species (i.e., human, bovine, and rat), as well as whole-rat serum. Kin decreased in the presence of albumin in direct proportion to perfusion fluid fu with constant PSu. The results show that brain uptake of selected Sudlow site I and II ligands matches that predicted by the modified Kety-Crone-Renkin model with no evidence for enhanced dissociation.