Overcoming the blood–brain tumor barrier for effective glioblastoma treatment

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Overcoming the blood–brain tumor barrier for effective glioblastoma treatment O. van Tellingen d,∗∗,1 , B. Yetkin-Arik c,1 , M.C. de Gooijer d,1 , P. Wesseling e,f , T. Wurdinger a,b , H.E. de Vries c,∗ a Neuro-oncology Research Group, Department of Neurosurgery, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands b Molecular Neurogenetics Unit, Departments of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA c Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands d Department of Clinical Chemistry/Preclinical Pharmacology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands e Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands f Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands

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Article history: Received 29 October 2014 Received in revised form 23 February 2015 Accepted 25 February 2015 Keywords: Glioma Blood–brain barrier Drug delivery

a b s t r a c t Gliomas are the most common primary brain tumors. Particularly in adult patients, the vast majority of gliomas belongs to the heterogeneous group of diffuse gliomas, i.e. glial tumors characterized by diffuse infiltrative growth in the preexistent brain tissue. Unfortunately, glioblastoma, the most aggressive (WHO grade IV) diffuse glioma is also by far the most frequent one. After standard treatment, the 2-year overall survival of glioblastoma patients is approximately only 25%. Advanced knowledge in the molecular pathology underlying malignant transformation has offered new handles and better treatments for several cancer types. Unfortunately, glioblastoma multiforme (GBM) patients have not yet profited as although numerous experimental drugs have been tested in clinical trials, all failed miserably. This grim prognosis for GBM is at least partly due to the lack of successful drug delivery across the blood–brain tumor barrier (BBTB). The human brain comprises over 100 billion capillaries with a total length of 400 miles, a total surface area of 20 m2 and a median inter-capillary distance of about 50 ␮m, making it the best perfused organ in the body. The BBTB encompasses existing and newly formed blood vessels that contribute to the delivery of nutrients and oxygen to the tumor and facilitate glioma cell migration to other parts of the brain. The high metabolic demands of high-grade glioma create hypoxic areas that trigger increased expression of VEGF and angiogenesis, leading to the formation of abnormal vessels and a dysfunctional BBTB. Even though the BBTB is considered ‘leaky’ in the core part of glioblastomas, in large parts of glioblastomas and, even more so, in lower grade diffuse gliomas the BBTB more closely resembles the intact blood–brain barrier (BBB) and prevents efficient passage of cancer therapeutics, including small molecules and antibodies. Thus, many drugs can still be blocked from reaching the many infiltrative glioblastoma cells that demonstrate ‘within-organ-metastasis’ away from the core part to brain areas displaying a more organized and less leaky BBTB. Hence, drug delivery in glioblastoma deserves explicit attention as otherwise new experimental therapies will continue to fail. In the current review we highlight different aspects of the BBTB in glioma patients and preclinical models and discuss the advantages and drawbacks of drug delivery approaches for the treatment of glioma patients. We provide an overview on methods to overcome the BBTB, including osmotic blood–brain barrier disruption (BBBD), bradykinin

∗ Corresponding author at: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel.: +31 20 4448077; fax: +31 20 4448081. ∗ ∗ Corresponding author at: Department of Clinical Chemistry/Preclinical Pharmacology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: +31 20 5122792; fax: +31 20 5122799. E-mail addresses: [email protected] (O. van Tellingen), [email protected] (H.E. de Vries). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.drup.2015.02.002 1368-7646/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002

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receptor-mediated BBTB opening, inhibition of multidrug efflux transporters, receptor-mediated transport systems and physiological circumvention of the BBTB. While our knowledge about the molecular biology of glioma cells is rapidly expanding and is, to some extent, already assisting us in the design of tumor-tailored therapeutics, we are still struggling to develop modalities to expose the entire tumor to such therapeutics at pharmacologically meaningful quantities. Therefore, we must expand our knowledge about the fundamentals of the BBTB as a step toward the design of practical and safe devices and approaches for enhanced drug delivery into the diseased brain area. © 2015 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

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Gliomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular composition of the BBB and implications for drug entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The blood–brain tumor barrier (BBTB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circumventing or overcoming the BBTB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Osmotic blood–brain barrier disruption (BBBD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Bradykinin receptor-mediated BBTB opening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Inhibition of drug efflux transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Exploiting receptor mediated transport system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Circumvention of the BBTB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Gliomas Gliomas account for approximately 80% of all tumors arising in brain tissue, with an incidence of about 7 per 100,000 individuals worldwide. Patients with gliomas may present with several neurological symptoms such as headaches, seizures, focal neurologic deficits, memory loss, personality changes, vomiting, and visual changes (Chandana et al., 2008; Ferguson, 2011; Wen and Kesari, 2008). According to the World Health Organization (WHO), gliomas are classified according to their cell type and malignancy grade (Louis et al., 2007). The vast majority of gliomas in adult patients are so-called diffuse gliomas, i.e. tumors that are characterized by diffuse infiltration of tumor cells in the preexistent brain tissue. However, diffuse gliomas may also occur in children. Based on the phenotype of the tumor cells diffuse gliomas are according to the WHO 2007 classification typed as astrocytic, oligodendroglial or oligoastrocytic tumors. Furthermore, a malignancy grade (grade II = low grade diffuse glioma; grade III = malignant/anaplastic diffuse glioma; grade IV = glioblastoma) is assigned to these tumors based on the presence/absence of features like brisk mitotic activity, florid microvascular proliferation and necrosis (Louis et al., 2007; Sanai et al., 2011). Glioblastomas are by far the most common and most malignant type (Nupponen and Joensuu, 2006; Wen and Kesari, 2008). Of note, WHO grade I is reserved for more circumscribed glioma variants such as pilocytic astrocytoma that occur especially in children. Despite their initially often relatively indolent nature, most lowgrade diffuse gliomas eventually progress to anaplastic glioma or glioblastoma (Nupponen and Joensuu, 2006). Glioblastomas can be further subdivided into primary and secondary glioblastoma and are characterized by marked cellular proliferation, necrosis, florid microvascular proliferation (i.e. a peculiar form of angiogenesis), resistance to apoptosis, and genomic aberrations (Furnari et al., 2007). Primary glioblastomas comprise the majority of cases (>90%), generally occur in older patients (>50 years) and are considered to be WHO grade IV from the start. In contrast, secondary glioblastomas occur in younger patients and arise from progression of a lower grade glioma (Ohgaki and Kleihues, 2007). Most lower

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grade diffuse gliomas carry a mutation in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) gene, and it was recently proposed to define primary vs secondary glioblastoma based on the IDH1/IDH2 mutation status of the tumor (Ohgaki and Kleihues, 2013). 2. Molecular composition of the BBB and implications for drug entry The human brain comprises over 100 billion capillaries with a total length of 400 miles, a total surface area of 20 m2 and a median inter-capillary distance of about 50 ␮m, making it the best perfused organ in the body (Pardridge, 2005). Proper function of the vasculature in the central nervous system (CNS) is essential for adequate brain function, not only to efficiently supply the brain with nutrients and oxygen, but also to protect the brain from potentially neurotoxic compounds. This protective blood barrier known as the blood–brain barrier (BBB), between the blood compartment and the brain is an essential prerequisite to secure correct neuronal functioning of the brain. The BBB is a cellular barrier (Fig. 1 and 2A) that regulates the ionic composition for proper synaptic signaling function, prevents macromolecules and unwanted cells from entering the brain as well as protects the CNS from neurotoxic substances and ensures brain nutrition. In essence, the BBB is formed by the specialized brain endothelial cells that exert their barrier properties through the continuous interaction with surrounding cells like astrocytes, pericytes, and perivascular macrophages, forming the so-called neurovascular unit (Abbott et al., 2006). Astrocytic endfeet cover the basal lamina of the brain capillaries and provide the cellular connection to neurons. Astrocytes play a key role in the maintenance of the barrier properties of the endothelium (Abbott et al., 2006; Alvarez et al., 2013). Pericytes cover the endothelium and contribute to the structural integrity of the BBB and the induction of barrier properties during development (Daneman et al., 2010; Obermeier et al., 2013). There is increasing evidence that astrocytes and pericytes secrete soluble developmental cues, like sonic hedgehog, retinoic acid and Wnt that control the onset of barrier properties (Alvarez

Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002

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Fig. 1. Glioblastomas consist of different regions characterized by various degrees of BBTB integrity. Heterogeneous BBTB integrity can be found throughout a glioblastoma, varying from completely compromised in bulky tumor areas (left panel) to slightly leaky in more invasive peripheral regions (middle panel) or even completely intact in sparsely invaded regions distant from the tumor bulk (right panel). Where the BBTB is compromised macromolecules (e.g. antibodies) and compounds that are normally efficiently restricted from the brain by efflux transporters can extravasate and engage glioma cells, while an intact BBTB protects isolated invaded tumor cells against efficient delivery of therapeutics. This protection has strong implications for anti-tumor treatment efficacy, especially considering the fact that these regions are unresectable and often give rise to recurrent tumors. Note: Figs. 1 and 2 have been prepared using Servier Medical Art (http://www.servier.com/Powerpoint-image-bank) under a Creative Commons Attribution 3.0 Unported License.

et al., 2011; Daneman et al., 2009; Liebner et al., 2008; Mizee and de Vries, 2013; Mizee et al., 2013). Brain endothelial cells are closely connected by intercellular tight junctions. Except for some very small or gaseous molecules (e.g. water, carbon dioxide) the tight junctions prevent brain entry of molecules via para-cellular trafficking in between the endothelial cells (Abbott et al., 2006). Tight junction associated proteins include occludin, claudin-1, claudin-5 and junctional adhesion molecules (JAMs) (Abbott et al., 2010; Wolburg and Lippoldt, 2002). Claudin-1 and occludin are linked via zonula occludens (ZO) protein complexes to tight junction associated proteins (e.g. Cingulin, Jacop, 7H6) that bind to the actin/myosin cytoskeletal system, resulting in modification of the tight junction properties. In adherens junctions that ensure the structural formation of tight junctions, VE-cadherin proteins are linked in the cytoplasm to ␣, ␤ and ␥-catenin. The sealing function of tight junctions depends not only on the presence of the proteins, but is also influenced by the organization and interaction of these proteins (Hamm et al., 2004). As a result of the tight structure of the endothelial cell layer and the lack of fenestrae, the BBB actively regulates the transendothelial passage of soluble substances from the blood to the brain and vice versa. Solutes can cross these endothelial barriers by passive diffusion, but the efficiency of this process depends on their physicochemical properties, such as electrical charge, molecular weight, and lipid solubility (Pardridge, 2005). Uptake of essential nutrients is actively regulated via carrier-mediated transport using one of the several solute carriers (e.g. SLC2/Glut-1 for glucose) or receptor mediated transport (e.g. transferrin) (Figs. 2A and 3). Solute carriers can also shuttle drugs across membranes, but their impact at the BBB is still not yet clear. For example OATP1A2 and ATP2B1 are expressed at the BBB and may be involved in uptake into and/or efflux from the brain (Konig et al., 2013; Kusuhara and Sugiyama, 2005; Shitara et al., 2013). The absence of Oatp1a/1b in mice causes a profound

reduction of the hepatic uptake of methotrexate and paclitaxel (van de Steeg et al., 2011), but effects on the brain penetration of these drugs has not been reported. In contrast, efficient efflux of unwanted compounds is achieved by the presence of a range of active (ATP Binding cassette (ABC)) efflux transporters, such as ABCB1 (P-glycoprotein) and ABCG2 (Breast Cancer Resistance Protein). ABCB1 was first discovered by its ability to confer multidrug resistance in tumor cells (Juliano and Ling, 1976). ABCB1 recognizes a remarkably wide range of substrates, including about 60% of all marketed drugs and mediates resistance by via its ability to extrude drug molecules out of the cells, thus reducing intracellular drug accumulation and cytotoxicity. It was already known since the late 1980s that ABCB1 was also expressed at the BBB (Cordon-Cardo et al., 1989), but the true functional impact of its presence became clear only after the generation of the Abcb1a knockout mouse model (Schinkel et al., 1994). Most strikingly, the absence of Abcb1a rendered ivermectin, a widely used and safe drug into a lethal neurotoxic agent. Since then numerous papers have confirmed the impact of ABCB1 on the brain penetration of a wide range of agents (Demeule et al., 2002). At the turn of the millennium, ABCG2, a drug efflux transporter that was also first discovered in tumor cells (Doyle et al., 1998) was also found to be expressed at the BBB (Cooray et al., 2002; Eisenblatter and Galla, 2002). The functionality of ABCG2 at the BBB was demonstrated using knockout (KO) mice, but only after the combined Abcb1a/b;Abcg2 (triple KO) mice were generated, its role in driving the efflux of compounds became apparent (de Vries et al., 2007). Previous work in single Abcg2-deficient mice was not convincing (Lee et al., 2005), since there is a large overlap in substrate affinity between Abcb1a/b and Abcg2. The action of Abcb1a/b at the BBB of single Abcg2-deficient mice was sufficient to expel those substrates out of the brain. The functional impact of Abcg2 at the BBB becomes most evident when comparing the brain penetration

Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002

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Fig. 2. Therapeutic brain delivery strategies that target the physiological make-up of the BBB. (A) The blood–brain barrier consists of both a physical and physiological barrier, where the physical component consists of tight junctions (TJ), gap junctions (GJ) and adherens junctions (AJ) that together restrict paracellular exchange between the blood and the brain and allow the endothelial cells to cooperate closely by providing channels for direct cytoplasmic exchange. The tight junctions are under regulation by the bradykinin receptor to maintain their stringent barrier character. The physiological aspect of the BBB aims to restrict the brain entry of potentially harmful exogenous compounds while at the same time allows for efficient uptake of essential nutrients. Brain penetration of exogenous compounds is efficiently restricted by ATP-binding cassette (ABC) transporters, a group efflux transport proteins located at the luminal endothelial cell membrane. In contrast, endothelial cells that constitute the BBB are equipped with a wide range of solute carriers (SLC), influx transporters that efficiently facilitate uptake of nutrients such as glucose (GLUT-1), amino acids (LAT-2), anions (the OATP family) and cations (the OCT family). Moreover, a wide range of receptors mediates transport through transcytosis. Among these are the transferrin receptor (TfR), insulin receptor (IR), LDL-receptor-related protein (LRP) and glutathione receptor (GSHR). (B) Strategies against various modalities of the intact BBB have been developed for improving therapeutic delivery to the brain. Elacridar inhibits ABCB1 and ABCG2, thereby reducing efflux and allowing increased brain penetration of small molecules. OX26, human insulin receptor monoclonal antibody conjugated nanoparticles (HIRMab NP), Ang1005 and 2B3-101 all target transcytosis pathways while Cereport targets tight junction stringency signaling through the bradykinin receptor.

of compounds between Abcb1a/b vs Abcb1a/b;Abcg2 deficient mice. For certain drugs, such as vemurafenib, brain penetration in Abcb1a/b; Abcg2-deficient mice increased over 10-fold compared to wild type controls (Durmus et al., 2012). Next to ABCB1 and ABCG2, there are several members of the ABCC family of transporters localized at the BBB (viz. ABCC1, ABCC2, ABCC4 and ABCC5) (Potschka et al., 2003; Soontornmalai et al., 2006; Zhang et al., 2004). These ABCC family members generally transport a range of more polar molecules, including drug conjugates and endogenous metabolites (Slot et al., 2011). However, the functional impact of these ABCC transporters has only been demonstrated for Abcc4 with the oseltamivir metabolite (RO 64-0802) (Ose et al., 2009), the camptothecin analogs (topotecan, SN38 and gimatecan) (Lin et al., 2013b) and methotrexate (Sane et al., 2014). In these latter cases, the fact that other ABC transporters at the BBB (Abcb1, Abcg2) recognize similar

substrates, can (partly) obscure the functional impact of Abcc4 at the BBB. The same may apply for Abcc1 (Mrp1). Whereas Abcb1;Abcc1 KO mice had similar brain levels of etoposide as Abcb1a/b KO (Wijnholds et al., 2000), this might be due to the affinity of etoposide for Abcg2 (Allen et al., 2003). The demonstration of functional activity of Abcc2 was hampered by the fact that the FVB mouse strain that is used for backcrosses of ABC transporter knockout genes does not express Abcc2 at the BBB (Soontornmalai et al., 2006). Apart from active efflux, metabolic degradation may play some role in reducing brain accumulation of compounds (Robillard et al., 2014), however this is still a largely unexplored area. Together, the superfamily of ABC transporters plays a key role in limiting the brain penetration of a wide range of compounds, including many of the targeted agents that may be useful to inhibit aberrant signaling pathways in glioma (Lin et al., 2013a).

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quantities of potentially effective therapeutic agents (JuilleratJeanneret, 2008; Korfel and Thiel, 2007). The BBTB is formed by brain tumor capillaries and comprises a barrier that is variably distinct from the BBB. Brain tumor capillaries may show overexpression of receptors that mediate ligand dependent drug delivery, which can be exploited to selectively enhance drug delivery to tumor tissues (Ningaraj et al., 2002; Nishio et al., 1983). The BBTB can be composed of three distinct microvessel populations, i.e. continuous and non-fenestrated capillaries (resembling normal brain capillaries, e.g. in diffuse low grade gliomas), continuous and fenestrated capillaries, and capillaries containing inter-endothelial gaps (Blasberg et al., 1987; Groothuis, 2000; Schlageter et al., 1999). Drug efflux transporters expressed at the BBB can also be found in endothelial cells at the BBTB (Aronica et al., 2005; Takamiya et al., 1997; Tanaka et al., 1994) (Figs. 2A and 3). Moreover, ABC transporters can be expressed in a (subset of) tumor cells as well and may confer chemo-resistance to glioblastomas (Lin et al., 2014) (Fig. 3), thus forming an additional hurdle toward curative treatment.

4. Circumventing or overcoming the BBTB

Fig. 3. Immunohistochemistry for ABC transporters, solute carriers, and transport receptors. Examples of immunohistochemical staining for ABC transporters, solute carriers, and transport receptors in non-neoplastic brain (NNB) and glioblastoma (GBM). See Fig. 2 for schematic depiction of these molecules. The immunohistochemical images were retrieved from www.proteinatlas.org.

During the last decades numerous strategies to improve the delivery of agents to brain tumors have been under investigation (Fig. 2B). Unfortunately, major changes in the landscape of brain tumor therapy have not yet matured from these efforts. In this section, we will briefly discuss the history, pitfalls and potential of developed methodologies.

3. The blood–brain tumor barrier (BBTB)

4.1. Osmotic blood–brain barrier disruption (BBBD)

The functioning and organization of the BBB can be altered under pathological conditions, such as multiple sclerosis, epilepsy, autoimmune deficiency syndrome (AIDS), dementia, stroke, and brain cancer (Abbott et al., 2006; de Vries et al., 2012; Obermeier et al., 2013). Importantly, alterations in the barrier evoked by tumors in the brain do not link with tumor size, tumor type, or anatomic location and is variable within any single neoplasm (Fig. 1) (Groothuis et al., 1984). In low-grade gliomas, the normal vascularization and the function of the BBTB remain mostly intact and resemble the BBB as under normal conditions (Machein et al., 1999). High-grade gliomas, however, are characterized by major alterations of the normal vascular function resulting in a disrupted, ‘leaky’ BBTB as manifested by contrast-enhanced MRI (Dhermain et al., 2010). Very recently it was shown that displacement of the astrocytic endfeet from a endothelial cell by a single invading tumor cell would already suffice to cause local breaching of the BBB (Watkins et al., 2014). However, it is unlikely that the magnitude of this local disruption is sufficient to allow drug penetration in meaningful quantities, taking into account that areas with invasion commonly do not enhance during contrast-enhanced MRI. The high metabolic demands of high-grade glioma creates hypoxic areas that trigger increased expression of Vascular Endothelial Growth Factor (VEGF) and angiogenesis, leading to the formation of abnormal vessels and a dysfunctional BBTB (Plate et al., 2012; Hardee and Zagzag, 2012). Importantly, however, the invasive potential of glioma causes widespread proliferation of high-grade glioma cells outside regions of the disrupted BBTB and inside areas of otherwise normal brain, where the function of the barrier is still (much more) intact. Typically, this includes the areas that do not show gadolinium enhancement on T1 weighted MRI (la Fougere et al., 2011). As a consequence, both in low-grade and high-grade glioma the BBB and the BBTB barrier form a major obstacle in brain tumor therapy by preventing the delivery of sufficient

The oldest work to literally breach the BBTB has been pioneered since the early 1970s by Rapoport (1970) and Rapoport et al. (1972). This strategy of so-called Blood Brain Barrier Disruption (BBBD) is based on intra-arterial infusion of a hyperosmotic solution of mannitol, causing temporary shrinkage of endothelial cells and subsequent opening of the tight junctions for several hours (Siegal et al., 2000). This time window can then be used for the intra-arterial delivery of the chemotherapeutic agents such as methotrexate or carboplatin. There are substantial technical challenges that limit the feasibility of this procedure and obstacles have been identified that jeopardize patient safety. The non-selective opening of not only the BBTB but also the BBB causes gross and uncontrolled influx of low and high molecular weight compounds and an increase of brain fluid leading to neurological toxicity, aphasia and hemiparesis (Siegal et al., 2000). On the other hand, the clinical benefit of this method for treatment of glioma has still not been established, despite more than 40 years of concentrated efforts. A randomized phase III study was announced in 2005 (Fortin et al., 2005), but no reports have appeared since then. Only in the case of primary CNS lymphoma, improved efficacy has been claimed, but these studies were all relative small and uncontrolled (Angelov et al., 2009). More recently, a phase I clinical trial has been reported on the use of BBBD in combination with bevacizumab, a monoclonal antibody directed against VEGF (Burkhardt et al., 2012). The rationale of using BBBD for the delivery of bevacizumab in glioblastoma patients remains somewhat unclear and just appears to be a very complicated modality to administer this biological agent. VEGF is mainly produced in areas of florid microvascular proliferation, where disruption of the BBTB is evident. Moreover, the bevacizumab mode of action requires long term presence in order to scavenge VEGF as is already achieved by 3-weekly IV dosing, since bevacizumab has a systemic half-life of about 2 weeks. It is therefore unlikely that the short pulse of extra

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bevacizumab that may enter during the hours of BBBD will add to this therapy.

4.2. Bradykinin receptor-mediated BBTB opening Another attempt to open up the BBTB/BBB which is more subtle, is based on the stimulation of brain endothelial receptors that induce opening of the tight junctions through utilization of compounds like bradykinin via second messengers (Raymond et al., 1986). This has led to the clinical development of RMP-7 (Cereport or labramidil), a bradykinin analog with improved systemic exposure following IV injection (Elliott et al., 1996; Inamura et al., 1994). In several animal studies and uncontrolled phase II studies RMP-7 revealed very promising results. However, a randomized, double blind, placebo controlled phase II study of RMP-7 in combination with carboplatin failed to demonstrate clinical benefit (Prados et al., 2003). A few subsequent studies on childhood gliomas also had a negative outcome (Warren et al., 2006), and further clinical development was discontinued. A potential reason for the negative results was that the dose level of RMP-7 of 300 ng/kg might have been inadequate. Further dose escalation, however, has not been attempted in follow up studies for reasons that have not been published, leaving this issue open ended. Other explanations for the negative outcome might be related to the drug scheduling, since the time lapse of BBTB/BBB opening is relatively short, or the potential insufficient gain of drug influx by opening the BBTB/BBB with RMP7. In experimental models using the most optimal scheduling in combinatorial treatment and with higher doses of RMP-7, the gain in brain tumor drug concentration was only about 2-fold (Emerich et al., 1999). Taking into account that carboplatin has no single agent activity; a mere 2-fold increase may still be inadequate to turn this combinatorial therapy into an active treatment regimen.

4.3. Inhibition of drug efflux transporters As outlined above, many drugs exhibit a profoundly enhanced brain penetration when drug efflux transporters are absent (as observed in knockout mice) (Lin et al., 2013a). Consequently, inhibition of these multidrug resistance efflux transporters by specific inhibitors seems an appealing strategy to boost drug penetration into the brain without compromising the integrity of the endothelial layer and tight junctions that might cause toxicities as seen with BBBD. Most of the clinically used ABC-transport inhibitors originate from the eighties and nineties of the previous century. At that time all major pharmaceutical companies had active programs aimed at reversing the multidrug resistance phenotype of tumors by concomitant use of chemotherapy and ABCB1 inhibitors. As a consequence, most inhibitors have been developed to target ABCB1, although later studies demonstrated that elacridar and tariquidar (both structurally related) are also ABCG2 transport inhibitors (de Bruin et al., 1999). Unfortunately, clinical trials aimed at reversing multidrug resistance in solid tumors have failed. As a consequence, companies lost their interest and further developments were halted. Plausible explanations for the lack of efficacy are that multidrug resistance is multifactorial and that tumor cells (due to their genomic instability) are “moving targets” that cannot be captured by a single modality (Stein et al., 2004). However, the concept of using these reversal agents to improve BBB drug penetration is very different from using these agents to block multidrug resistance in genomically unstable cancer cells. The objective here is to make a sanctuary site more accessible by targeting ABC transporters in genomically stable endothelial cells. Obviously, when attempting to implement this strategy there are still a few challenges to be addressed.

(A) Since most drugs are substrates of both ABCB1 and ABCG2, inhibition of both efflux transporters will be mandatory to yield adequate brain penetration. Based on this criterion, elacridar and tariquidar are currently the two only suitable options. Both have been used in clinical trials for reversing multidrug resistance. Given the lack of interest of pharmaceutical companies, it is unlikely to expect that other clinically applicable and more potent ABC-transport inhibitors will become available. (B) Inhibition of ABC transporters at the BBB is more difficult than inhibition of ABC transporters in other tissues, such as lymphocytes that are frequently used as surrogate marker (Choo et al., 2006). Comparative studies in ABC transporter KO mice demonstrate that first and second generation ABCB1 inhibitors are generally not potent enough to inhibit ABCB1 at the BBB (Choo et al., 2006; Kemper et al., 2003). These results underscore the need to use a dedicated and potent inhibitor. Repurposing ofthe-shelf drugs such as cyclosporin A will unlikely be successful. (C) Tariquidar has already been tested in humans to investigate inhibition of ABCB1 and ABCG2 transport at the BBB. In a healthy volunteer study, 150 mg tariquidar given by IV infusion did not evoke central opioid effects of loperamide, whereas it profoundly inhibited ABCB1 in lymphocytes (Kurnik et al., 2008). Tariquidar did enhance the brain penetration of verapamil in rats and humans by 11 and 2.7-fold, respectively, as assessed by positron emission tomography (PET). The lower enhancement in humans was thought to be due to incomplete inhibition of ABCB1 at the BBB (Bauer et al., 2012). In line with this assumption, a recent study using IV infusion resulting in tariquidar plasma levels of 3 ␮M demonstrated a 3.7-fold increase (Bauer et al., 2015). The potency of tariquidar to inhibit ABCB1 at the BBB appears to be less than that of elacridar (Choo et al., 2006; Kuntner et al., 2010), suggesting that the latter may be preferred. (D) The first candidate ‘commuter’ agents to be used in order to clinically test the concept of enhancing their brain penetration by inhibition of multidrug resistance efflux transporters must also be selected with care. The potency of inhibitors to inhibit efflux in part depends on transport substrate affinity of the candidate agents for ABCB1 and ABCG2. For example, elacridar was able to fully inhibit Abcb1-mediated efflux of topotecan from the brain but not Abcg2-mediated efflux (de Vries et al., 2007). Similarly, elacridar was not potent enough to fully inhibit gefitinib efflux from the brain (Agarwal et al., 2010). Currently, we do not know what level of inhibition will be required to yield clinical benefit, but initial clinical testing this concept should preferably be done using relatively weak substrates. Preclinical studies have shown promising results using elacridar in combination with dasatinib (Agarwal et al., 2012; Becher et al., 2014; Lagas et al., 2009), sorafenib (Lagas et al., 2010), sunitinib (Oberoi et al., 2013) or temozolomide and veliparib (Lin et al., 2014). Ideally, clinical development should be guided by monitoring predictive pharmacodynamic biomarkers as are present for most tyrosine kinase inhibitors. Beyond the treatment of brain tumors there may be opportunities in the treatment of drug resistant epilepsy with phenobarbital or phenytoin (Brandt et al., 2006; van Vliet et al., 2007). (E) The bioavailability (i.e. systemic levels of the inhibitor) should be adequate for inhibition of ABC-transport at the BBB. Tariquidar is available as an IV formulation, which may be good for proof-of-concept testing as plasma levels of 1000 ng/ml can be achieved (Bauer et al., 2012). However, for therapeutic purposes, an oral formulation will be more preferable. Most of the preclinical studies with elacridar have been performed in mice receiving 100 mg/kg p.o. and resulting in plasma levels of about 400–500 ng/ml for about 24 h (Kemper et al., 2003; Sane et al., 2013). Under these conditions the brain penetration

Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002

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of paclitaxel was almost similar as in Abcb1a/b knockout mice, whereas lower doses were less effective. Importantly, however, the plasma levels in patients receiving oral elacridar at dose levels between 100 and 1000 mg never exceeded 130 ng/ml (Kuppens et al., 2007). Elacridar is a poorly water soluble and membrane permeable drug and very difficult to formulate into a product with good oral bioavailability. Most likely, the conventional oral formulation as originally designed by GlaxoSmithKline is not sufficiently suitable to yield high enough plasma levels of elacridar. Consequently, better formulations of elacridar are necessary and initiatives are ongoing (Bunt and Van Tellingen, 2014; Sane et al., 2013). Given the structural similarities between elacridar and tariquidar, it is likely that development of a good oral formulation of the latter may pose the same challenge as for elacridar. In summary, for the first proof-of-concept that overcoming the BBB/BTBB by blocking drug efflux transporters will be of benefit for glioma patients there is a need for a potent inhibitor with good systemic bioavailability as well as for a ‘commuter’ agent with moderate affinity for these efflux transporters. If successful, further optimization is warranted to explore the potential of enhancing brain penetration under more stringent conditions. 4.4. Exploiting receptor mediated transport system Another strategy to enhance brain penetration without disrupting the tight junctions and integrity of the endothelial layer is to employ receptor-mediated uptake mechanisms. Toward this end, a shuttling factor that targets a receptor (e.g. insulin or transferrin receptor) at the BBB is coupled to a drug (e.g. paclitaxel) or a vehicle (e.g. liposome) containing a drug or radiopharmaceutical (Fig. 2B). These are classic technology platforms that allow the shuttling of a range of drugs. Several receptor mediated uptake systems have been described and tested for enhancing the brain uptake of drugs and radiopharmaceuticals as reviewed by Pardridge (2012). We will focus here on two of these strategies that have already yielded products that reached clinical evaluation. The first is GRN1005 (formerly ANG1005) developed by AngioChem, and is a conjugate of paclitaxel and the angiopep2 peptide that targets the lipoprotein receptor-related protein 1 (LRP1) and crosses the BBB by transcytosis (Drappatz et al., 2013). Although this is of course still in the early days, it is encouraging that responses have been observed in Phase I study. Currently, 3 phase II clinical trials for glioma (Clintrials.gov: NCT01967810) and breast cancer brain metastasis (NCT02048059 and NCT01480583) are accruing. The same angiopep-2 peptide has also been coupled to doxorubicin (ANG1997) and etoposide (ANG1009) (Che et al., 2010) or anti-HER2 mAb (ANG4043) (Lachowicz et al., 2013), demonstrating the versatility of this approach, but these have so far only been tested in preclinical studies. The other clinically advanced candidate is 2B3-101 (Clintrial.gov ID: NCT01386580, NCT01818713), which is a PEGylated liposome that is conjugated with glutathione (GSH). Preliminary results are also encouraging (Gaillard et al., 2014), but similar to GRN1005, it is far too early to draw any conclusion about the usefulness of this therapeutic approach. Unfortunately, currently there is not an easy surrogate marker to demonstrate better drug delivery to the brain and/or brain tumor. PEGylated liposomes have a long circulation half-life in blood. Consequently, plasma levels of the (encapsulated) drug are very high relative to the actual levels that have been attained in the brain and results of measurement of brain homogenates are flawed by admixing of blood in the tissue specimen. A better alternative would be measurement in brain interstitial

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fluid via a microdialysis probe. However, technical problems (adsorption to the membrane) markedly decrease the accuracy of this approach. Recently, a cerebral open flow microperfusion method has been presented and used for 2B3-101 vs untargeted PEGylated liposomes and demonstrated a 4.7-fold increase in brain interstitial fluid doxorubicin levels (Birngruber et al., 2014). Very recently an interesting paper appeared, demonstrating that the docosahexanoic transporter Mfsd2a acts by suppressing transcytosis in CNS endothelial cells (Ben-Zvi et al., 2014). Interfering with its function or expression may enhance transcytosis and thereby improve drug delivery via this route. 4.5. Circumvention of the BBTB An apparently straightforward approach to overcome the BBTB is by delivery of therapeutic agents directly into the brain (tumor) parenchyma or into the resection cavity of glioma patients. Intracavitary drug injection, with the use of an intraventricular/intracavitary system such as an ommaya reservoir, allows for such direct delivery of chemotherapeutic agents to the tumor cavity. Injection occurs percutaneously into the subcuteaneous reservoir and the drug is subsequently transferred through the skull into the tumor (cavity) by manual compression of the reservoir. The distribution of the drug is determined by the rate and duration of the injection and by diffusion (Allhenn et al., 2012; Bidros et al., 2010; Jacobs et al., 1981; Tomita, 1991). This method has been applied in various clinical trials for local delivery of drugs such as methotrexate and nitrosourea (Allhenn et al., 2012; Bidros et al., 2010). However, insufficient drug delivery, infections, neurotoxicity, and catheter obstruction has prevented successful application of this method in a routine setting (Bidros et al., 2010; Tomita, 1991). Another direct local delivery approach is the use of gliadel wafers, which are biodegradable polymer wafers loaded with a chemotherapeutic agent implanted into the brain or tumor cavity at the time of surgery (Brem et al., 1995; Cao and Schoichet, 1999; Groothuis, 2000; Westphal et al., 2003). The biodegradable wafers, 1.45-cm diameter discs that contains 3.85% carmustine (BCNU), provide continuous delivery of therapeutic molecules to the tumor with ideally some control over the release and the spatial distribution of the drug in the course of time (Avgeropoulos and Batchelor, 1999; Korfel and Thiel, 2007). The drug is released from the wafer over a limited period of only two to three weeks and is delivered via diffusion from the source into surrounding brain tissue to produce a local anti-neoplastic effect (Valtonen et al., 1997). In 1996, the gliadel wafer was approved by the US Food and Drug Administration (FDA) as the first FDA-approved local treatment for recurrent and newly diagnosed glioblastoma (Buonerba et al., 2011). However, the limitations of this method are significant and include the requirement of surgical placement, increased risk of local neurotoxicity, and local complications such as edema and infections (Bidros and Vogelbaum, 2009; Korfel and Thiel, 2007). Moreover, the rather diffuse distribution of the drug may well result in insufficient therapeutic drug concentrations at the site of more peripherally located infiltrative glioma cells. Indeed, so far all patients treated with this approach showed glioma progression (Buonerba et al., 2011; Fung et al., 1998). A technique considered more promising is convection enhanced delivery (CED) (Allhenn et al., 2012; Barua et al., 2014; Bidros et al., 2010; Buonerba et al., 2011; Saito and Tominaga, 2012). CED involves the direct and continuous injection of a therapeutic agent under positive pressure using stereotactically placed intraparenchymal microcatheters, thereby allowing the passage of molecules, regardless of charge and size, into any section of the brain (Lonser et al., 2007; Westphal and Lamszus, 2011; Yamashita et al., 2007). At the tip of the infusion catheter a pressure

Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002

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gradient creates flow of the therapeutic agents through the interstitial spaces of the brain (Jordan and Maier-Hauff, 2007; Korfel and Thiel, 2007; Westphal and Lamszus, 2011). Despite the advantages in terms of drug distribution throughout the brain as compared to ommaya reservoirs and wafers and compared to systemic delivery methods, CED remains experimental and several practical challenges need to be resolved, including determining optimal infusion rate and volume, anatomical prerequisites for the infusion site, and approach-related problems such as backflow along the tract (Bidros et al., 2010; Kesari, 2011; Westphal and Lamszus, 2011). Recent preclinical efforts using CED in glioma models include the use of radiolabeled monoclonal antibodies (Luther et al., 2014), etoposide (Sonabend et al., 2014), carmustine (Sewing et al., 2014), carboplatin (Yang et al., 2014) or an ATM inhibitor (Vecchio et al., 2014) in combination with radiotherapy, nanoparticles (Bernal et al., 2014), nanoliposomal formulations with irinotecan (Chen et al., 2013) or cisplatin (Huo et al., 2012), and therapeutic stem cells (Duebgen et al., 2014). Neuro-oncological clinical trials with CED have evaluated a wide range of potential therapeutic agents, including conventional chemotherapies such as paclitaxel (Lidar et al., 2004), topotecan (Bruce et al., 2011); (Anderson et al., 2013), and nimustine (Saito et al., 2011), targeted toxins including IL13PE38QRR (Kunwar et al., 2010; Mueller et al., 2011; Sampson et al., 2010) and IL4-PE38 (Rand et al., 2000; Shimamura et al., 2006), TP-38 (Rainov and Heidecke, 2004; Sampson et al., 2005), oligonucleotides (Carpentier et al., 2010), and TGF-␤2 inhibitors (Bogdahn et al., 2011). Unfortunately, the results of all these trials were rather disappointing, which is largely attributed to poor drug distribution to more peripheral areas of diffuse gliomas and drug reflux, resulting in side effects and subtherapeutic drug concentrations within the tumor target cells (Mueller et al., 2011; Kunwar et al., 2010; Sampson et al., 2010). Another major problem in many of these early clinical studies is the lack of visualization of the distribution of the infused drug and unacceptable device-related adverse events. Clinical and preclinical imaging studies are ongoing to further optimize drug distribution via CED (Chittiboina et al., 2014; Suzuki et al., 2014). In order to obtain efficient delivery of the drugs via CED, it is essential to optimize the CED catheter technology and use therapeutic agents with molecular characteristics suitable for CED, allowing for widespread drug distribution (Barua et al., 2014; Saito and Tominaga, 2012; Bidros et al., 2010; Buonerba et al., 2011). An additional point of consideration is the effect on CED flow and drug distribution of anatomical cavities in/around the CNS (ventricles, subarachnoid space) and of artificial cavities left after surgical resection of the bulk of the glioblastoma tissue. The issue of suboptimal CED due to resection cavities may be non-existing in the highly malignant diffuse intrinsic pontine gliomas (DIPGs) – since in the brainstem bulk resection of such gliomas simply cannot be performed – hence DIPGs may provide a more suited tumor type for the evaluation of CED. It is unlikely that by current CED approaches all infiltrative glioblastoma cells will be reached, also not in the case of brainstem gliomas, and glioblastoma recurrences at local and more distal sites can still be expected to occur in most – if not all – patients with glioma. In order to reach more glioma cells, clinical trials are ongoing making use of tumor-selective self-replicative oncolytic viruses (Chiocca et al., 2004; Forsyth et al., 2008; Geletneky et al., 2012; Kicielinski et al., 2014; Markert et al., 2014), and several oncolytic viruses have been evaluated preclinically in orthotopic glioma models (Allen et al., 2013; Idema et al., 2011; Lun et al., 2010; Muik et al., 2014; Verheije et al., 2009; White et al., 2011). This may allow for local amplification and spread after the diffuse delivery of the therapeutic agent via CED, and possibly result in a continued attack of the cancer cells. Clearly, further research is warranted toward the use of CED in combination with self-amplifying therapeutic agents.

5. Future directions Effective treatment of glioma is hampered by the presence of the BBTB. Especially in areas where the BBTB more closely resembles the BBB, anti-cancer drugs are denied access to the CNS and the tumor cells that reside therein. In this review, we presented a comprehensive synopsis of all the – also clinically applied – approaches used to date to enhance the permeability of the BBTB. While our knowledge about the molecular biology of glioma cells is rapidly expanding and is, to some extent, already assisting us in the design of tumor-tailored therapeutics, we are still struggling to develop modalities to expose the entire tumor to such therapeutics at pharmacologically meaningful quantities. Therefore, we need to expand our knowledge about the fundamentals of the BBTB as a step toward the design of practical and safe devices and approaches for improved drug delivery into the diseased brain area. In this respect, the recent discovery of the impact of Mfsd2a on receptor-mediated transcytosis is a remarkable example. The predictive value of preclinical testing in in vivo models also needs to be improved by implementing animal models that more faithfully recapitulate the BBTB as observed in glioma patients. As we stand today, some potentially useful technologies based on receptor-mediated transcytosis are currently under clinical evaluation and, when successful, will provide handles for further improvements. Ongoing research will hopefully also lead to the identification of other BBTB selective surface proteins that can be used as receptors for transcytosis or as selective recognition sites that enable targeting of therapeutics to and through the BBTB. Inhibition of drug efflux transporters at the BBTB is also an approach that warrants further exploration in clinical trials. Importantly for treatment of brain tumors, no matter how well the target is selected, there is no benefit if it is not reached. Conflict of interest statement Olaf van Tellingen is co-inventor on a patent application (Bunt and Van Tellingen, 2014) dealing with development of an improved oral formulation for elacridar. Acknowledgements This work was supported by the VIDI fellowship 91711366 (T.W.) from the Dutch Organization of Scientific Research (NWO), European Research Council Starting Researchers Grant 336540 (T.W), and Stichting Stophersentumoren.nl (T.W., O.v.T.). References Abbott, N.J., Patabendige, A.A., Dolman, D.E., Yusof, S.R., Begley, D.J., 2010. Structure and function of the blood–brain barrier. Neurobiol. Dis. 37, 13–25. Abbott, N.J., Ronnback, L., Hansson, E., 2006. Astrocyte–endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53. Agarwal, S., Mittapalli, R.K., Zellmer, D.M., Gallardo, J.L., Donelson, R., Seiler, C., Decker, S.A., Santacruz, K.S., Pokorny, J.L., Sarkaria, J.N., Elmquist, W.F., Ohlfest, J.R., 2012. Active efflux of Dasatinib from the brain limits efficacy against murine glioblastoma: broad implications for the clinical use of molecularly targeted agents. Mol. Cancer Ther. 11, 2183–2192. Agarwal, S., Sane, R., Gallardo, J.L., Ohlfest, J.R., Elmquist, W.F., 2010. Distribution of gefitinib to the brain is limited by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)-mediated active efflux. J. Pharmacol. Exp. Ther. 334, 147–155. Allen, C., Opyrchal, M., Aderca, I., Schroeder, M.A., Sarkaria, J.N., Domingo, E., Federspiel, M.J., Galanis, E., 2013. Oncolytic measles virus strains have significant antitumor activity against glioma stem cells. Gene Ther. 20, 444–449. Allen, J.D., Van Dort, S.C., Buitelaar, M., van Tellingen, O., Schinkel, A.H., 2003. Mouse breast cancer resistance protein (Bcrp1/Abcg2) mediates etoposide resistance and transport, but etoposide oral availability is limited primarily by P-glycoprotein. Cancer Res. 63, 1339–1344. Allhenn, D., Boushehri, M.A., Lamprecht, A., 2012. Drug delivery strategies for the treatment of malignant gliomas. Int. J. Pharm. 436, 299–310.

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Please cite this article in press as: van Tellingen, O., et al., Overcoming the blood–brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. (2015), http://dx.doi.org/10.1016/j.drup.2015.02.002