Systemic drug delivery to the brain is a difficult challenge for modern drug development.
For an effective treatment of diseases in the CNS drugs have to overcome several barriers including the blood-brain barrier and the blood-cerebrospinal fluid (CSF) barrier. If the target of intracranial drug delivery is a CNS tumor an additional barrier, the blood-tumor barrier has to be considered.
1.1.1 The Blood-Brain Barrier
The general concept of a restriction on the passage of dissolved substances out of the blood into the brain was first postulated by Paul Ehrlich (Ehrlich 1902). Edwin Gold-mann, a student of Paul Ehrlich, injected the dye trypan blue into the CSF and the dye stained only the brain but not the other organs (Goldmann 1909). This experiment together with the studies of Romanowsky, who used Prussians’ blue as a reagent in the late 1890s (Brightman 1992), established the concept of the blood-brain barrier (BBB).
The cytoarchitecture of the BBB was discovered in the late 1960s by means of electron microscopic studies (Miller 2002).
The BBB is build up by the endothelial cells of the brain capillaries (Fig. 1.1). Astro-cytes, microglial cells, pericytes and nerve endings surrounding the capillaries are consid-ered to be essential for the differentiation of the endothelial cells and the maintenance of
2 Introduction the barrier (Abbott 2002, Brightman 1992, Rubin and Staddon 1999). Astrocytes are the structural frame of the neurons and their adjoining foot processes fully encapsulate the capillaries (Lo et al. 2001). Furthermore, they are necessary for the expression of vari-ous growth factors and transport systems such as the glucose transporter GLUT1 (Janzer and Raff 1987). Microglia and pericytes derived from mononuclear blood macrophages en-hance the BBB function and are conducive to modulatory signaling (Zenker et al. 2003).
Axonal endings that are closely abutted to the endothelial cells, are considered to be important for BBB permeability (Rennels et al. 1983).
pericyte
endothelial cell
extracellular matrix axonal ending
astrocytic foot process microglial cell
tight junction
Figure 1.1: Schematic diagram of the cells forming the BBB. Endothelial cells of brain capil-laries are sealed by tight junctions. These cells are surrounded by pericytes and foot processes of astrocytes responsible for the structural integrity of the barrier.
The microglial cells are part of the immune system due to their descent from macrophages. Axonal endings provide vasoactive neurotransmitters which are im-portant for BBB permeability (from Begley (2004)).
Brain capillaries have a total length of more than 600 kilometers through the human brain, a total surface area of 12 m2 (Misra et al. 2003, Miller 2002) and are much smaller in diameter as well as thinner walled compared to vessels in other organs. The typical characteristics of blood vessels such as intercellular clefts, pinocytosis and fenestrae are not found in brain capillaries. The endothelial cells form intercellular tight junctions (zona occludes) that completely seal the capillaries and close the paracellular pathway
1.1 Barriers inhibiting drug delivery to the brain 3 (Brightman and Reese 1969, Saunders et al. 1999, Fenstermacher 2001). Several trans-membrane proteins, particulary occludin and claudin, are responsible for the formation of the junctions by complex interactions with each other (Kniesel and Wolburg 2000). The high seclusiveness of the brain to the other organs and the blood circulation results in an extremely high trans-endothelial electrical resistance of 1500 to 2000 Ω·cm2 (Butt et al.
1990). Moreover, the brain capillaries are identified by limited paracellular transport due to the low endocytic activity and the absence of fenestration (Kemper et al. 2004a).
All these properties of the endothelial cells contribute to a strongly restricted per-meability of the BBB, which is required to protect the brain against foreign and toxic substances as well as neurotransmitters and hormones (van Asperen et al. 1997). Ad-ditionally, the relative impermeability supports the maintenance of a constant internal environment, which is very important for proper neuronal function in the brain. Also, the composition of the resulting extracellular fluid of the brain parenchyma can be precisely controlled (Begley 2004). Due to the limited access of substances to the brain, only small lipid soluble compounds can cross the BBB by passive diffusion. To enable the uptake of essential polar substances into the brain, a lot of different transport proteins are ex-pressed at the BBB (Begley 2003). These active transporters include carriers for glucose and amino acids as well as receptor mediated systems for certain peptides such as insulin or transferrin (Begley 1996). Fig. 1.2 shows different possible pathways through the BBB.
Apart from the transporters that are responsible for substance uptake into the brain, two specific mechanisms exist at the BBB. which are concerned with the protection of the brain. In order to degrade unwanted crossing substances the endothelial cells con-tain a large number of degrading enzymes and a high density of mitochondria, which are metabolically highly active organelles (Misra et al. 2003, Brownlees and Williams 1993). The high ability of the BBB to detoxify and transform compounds has been un-derestimated in the past. Furthermore, active efflux transport proteins are expressed at high concentrations at the luminal or basolateral membrane of the endothelial cells such as ABCB1 (p-glycoprotein 170), ABCC1 (MRP1) and ABCG2 (BCRP) and are able to transport a broad variety of compounds against a concentration gradient from the endothelial cytoplasm back to the lumen of the brain capillaries (Ramakrishnan 2003).
These efflux transporters are described in detail in chapter 1.3.
4 Introduction
Figure 1.2: Possible routes for transport across the BBB. (A) Cells, particularly leukocytes, cross the BBB adjacent to tight junctions or directly. (B) The most common pathway for compounds is passive diffusion. (C) However, passively transported substances may be carried out by active efflux pumps. (D) Carrier mediated influx is used by essential polar solutes such as glucose. (E) Macromolecules, e.g. insulin, are transported by specific receptors. (F) Alternatively, adsorptive mediated trans-cytosis may occur induced by negatively charged macromolecules (from Begley (2004) with modifications).
1.1.2 The Blood-CSF Barrier
The most important function of the choroid plexus is the secretion of CSF. To produce this fluid, a broad variety of nutrients and other blood borne solutes are necessary. Since, however, molecules can be exchanged between the CSF and the interstitial fluid of the brain parenchyma (Misra et al. 2003), the uptake of these solutes into the CSF has to be controlled in order to restrict the penetration of cytotoxic agents or other unwanted substances into the brain. This is done by the blood-CSF barrier (BCB) which is formed by the epithelia of the choroid plexus and the circumventricular organs (CVO) (Wolburg et al. 2001). Furthermore, the arachnoid membrane is also involved in the function of the
1.1 Barriers inhibiting drug delivery to the brain 5 BCB (Siegal 2001). The arachnoid membrane consists of a double layer of ependymal cells between dura and pia mater. Tight junctions between the ependymal cells seal the arachnoid membrane against the paracellular pathway. Also, the epithelial cells of the choroid plexus, arranged in a close sheet, form tight junctions to prevent macromolecular diffusion into further brain regions. However, these epithelial-like cells show a lower resistance of approximately 200 Ω·cm2 compared to the endothelial cells of the BBB (Misra et al. 2003). Moreover, paracellular diffusion is impeded at the CVO due to the occurrence of tight junctions between the ependymal cells surrounding the CVO. To enable the passage of peptides, ions and further nutrients from blood into the CSF, the capillaries of the choroid plexus and the CVO are fenestrated and non-continuous. Due to the sealed paracellular pathways at the surrounding epithelia, exchange of molecules can only occur in a small restricted area of the extracellular fluid immediately around the CVO. Within this limited volume, the activity of dendritic processes and neuron receptors can be influenced by blood borne compounds leading to certain neural impulses in distant brain areas (Begley 2004).
However, the surface area of the BCB is approximately 1,000 fold smaller compared to the BBB surface area (Pardridge 1997). Hence, drug entry to the brain via the BCB plays a secondary role in brain drug uptake (Rautio and Chikhale 2004).
1.1.3 The Blood-Tumor-Barrier
Although in brain tumors the BBB is at least partly disrupted, other barriers together with the physiological conditions in the brain tumor tissue such as abnormal blood capil-laries, hamper effective drug delivery to the tumor. In contrast to brain capilcapil-laries, the blood capillaries within different regions of the tumor tissue show significantly different morphology (Schlageter et al. 1999, Siegal 2001). These changes include alterations in the tight junction structure and the irregular appearance of endothelial cells with either many fenestrations, increased pinocytosis or a totally irregular basal membrane (Bart et al. 2000). The inconsistent spatial density of the capillaries together with the decreased vascular surface compared to the tumor volume contribute to an insufficient brain tumor drug delivery. Furthermore, tumor blood capillaries are sometimes leaky, leading to an
6 Introduction accumulation of interstitial fluid and hence, an increase of the interstitial tumor pressure (Jain 1994). The high intratumoral pressure limits drug penetration into the tumor tissue and could effect the drug permeability of capillaries in tumor adjacent regions of normal brain, resulting in low extratumoral interstitial drug concentrations (Cornford et al. 1982).
The term blood-tumor barrier includes all aforementioned aspects of drug delivery to brain tumors.