Our data support the hypothesis that the diseased BBB acts as a metabolic barrier, possibly confounding the pharmacokinetics of central nervous system (CNS) drugs. The results obtained from a relatively small cohort of patients suggest the presence of CYP enzymes at the diseased BBB, including endothelium derived from epileptics. We also discovered that hemodynamic conditions play an important role in regulating CYP expression and function. Cerebrovascular drug metabolisms could alter the pharmacokinetics of CNS drugs and possibly contribute to the multifaceted drug-resistant phenotype observed in refractory epilepsy.
CYPs at the human brain endothelium
Liver and gut cells ensure the metabolic transformation of xenobiotics and endogenous molecules (Martignoni et al., 2006
). The data presented herein represent a significant departure from the concept of systemic drug metabolism, rather suggesting a local cerebrovascular drug transformation process. In the past, enzymes that are involved in hepatic drug metabolism have been mostly studied in the choroid plexus and leptomeninges (Ghersi-Egea et al., 1993
). Our data reveal CYP expression in the diseased human brain endothelium (drug-resistant epileptic and aneurism-derived brain entothelial cells). Specifically, an abnormal pattern of CYP expression in the epileptic brain could influence the pharmacokinetics of AEDs. Recent evidence revealed the presence of CYP enzymes in a human brain–derived cell line (hCMEC/D3) and human isolated microvessels (Dauchy et al., 2008
). Accordingly, our data obtained using freshly isolated endothelium showed the presence of CYP1A1, 1B1, 2B6, 2E1, and 2J2 genes (Dauchy et al., 2008
). In accordance with previous findings (Dauchy et al., 2009
), we generally measured higher CYP transcript levels in HUVEC compared to control brain endothelial cells (HBMEC).
We found levels of CYP3A4, CYPC9, CYP2A6, and CYP2J2 mRNA encoding for enzymes that are known to be responsible for the metabolism of AEDs ( and ). We specifically confirmed the upregulation of CYP3A4 protein at the epileptic brain endothelium.
Effect of shear stress on CYP regulation and function
Physiologic shear stress affects brain endothelial cell differentiation, tight-junction formation, and regulates the cell cycle causing mitotic arrest (Arisaka et al., 1995
; Desai et al., 2002
). The DIV model used in our experiments generates shear stress resembling physiologic conditions (Stanness et al., 1996
). Under exposure to flow, CYP and drug transporter mRNA ratios (shear/no-shear) were greater than 1 (), indicating the regulatory effect of shearing forces on transcription (Egnell et al., 2003
). Previous findings have demonstrated that CYP1A1 and 1B1 mRNA levels are induced in control nonbrain endothelial cells under shear conditions (Eskin et al., 2004
). The dissimilar effect provoked by shear stress in control and “epileptic” brain endothelial cells needs to be further clarified. Several variables such as cellular proliferation/differentiation and survival are involved when considering “diseased” endothelium (Desai et al., 2002
; Han et al., 2008
). We are also aware of the possible variations associated with the use of freshly isolated brain cells from patients. Nevertheless, human-based investigations provide valuable data to be compared with those obtained using animal models or cell lines.
In the epileptic brain, local brain hyperperfusion is a common ictal event. It is possible that hemodynamic changes could be involved in the expression of CYPs and drug transporter proteins at the epileptic BBB. It is important to understand under what circumstances the effect of flow becomes operant. It is not farfetched to suggest a dynamic model of brain drug resistance. The changes in cerebral blood perfusion in response to the ictal-interictal cycle acting in the diseased brain could shape the profile of endothelial expression of several proteins, including CYP enzymes and multidrug transporters.
We have previously demonstrated that the flow-based DIV-BBB recapitulates the physiologic permeability properties of the BBB in vivo and is also capable of mimicking a drug-resistant phenotype (Cucullo et al., 2007
). We now propose that the DIV-BBB also reproduces the metabolic barrier properties of the human epileptic BBB (). Our results suggest that in vitro modeling of CNS pharmacodynamics requires appropriate models (such as those based on flow) and realistic cell types (as those taken from specific brain pathologies).
Our result also showed that preventive exposure to shear stress precludes any further induction of CYP expression (). This highlights the importance of culturing conditions (e.g., those mimicking the physiologic parameters) when analyzing the pattern of gene and protein expression in brain-derived endothelial cells. The effect of shear stress exposure was specific for endothelial cells. Hepatocytes exposed to laminar flow did not significantly change their expression of CYP3A4 (Fig. S3
). However, the relative contribution of variables associated with the epileptic condition (e.g., underlying pathology, hemodynamic changes, AED regimen, and seizure activity) in determining CYP/MDR overexpression needs to be further clarified. The possibility exists that EPI-EC deriving from patients treated with CBZ displays higher expression of CYP due to drug induction. However, within our cohort of patients with drug-resistant epilepsy, only three patients received CBZ (Table S1
), whereas other AEDs were taken in combination.
Our HPLC data demonstrate that endothelial cells are capable of drug metabolism. In particular, EPI-EC displayed metabolic activity identical to that of cultured hepatocytes, which was significantly elevated compared to control endothelium ( and ). Concomitant to CYP3A4 overexpression, exposure to shear stress increased the metabolic potency of endothelial cells ().
BBB CYP3A4 expression in the drug-resistant epileptic brain
Over the last decade, it has become evident that the epileptic brain has a tendency to overexpress a broad spectrum of multidrug transporter proteins (Dombrowski et al., 2001
; Abbott et al., 2002
; Aronica et al., 2004
; Marchi et al., 2004
; Loscher & Potschka, 2005
; Aronica & Gorter, 2007
; Janigro et al., 2007
; Loscher, 2007
). Immunohistochemical data revealed the presence of CYP3A4 in blood vessels, in particular in those brain areas characterized by reactive gliosis (). We are aware that one of the limitations of our study is the lack of normal brain tissue. Because of practical difficulties, control brain tissue was provided by histologically normal neocortex adjacent to the hippocampus with sclerosis. In such cases, control tissues are thus of the same age and have been exposed to the same environmental factors or drugs as has diseased tissue (Palmini et al., 2004
; Sen et al., 2007
). Further studies are required to confirm overexpression of CYP enzymes compared to “more adequate” control human brains. For instance, recent evidence has suggested an increased vascularization at the epileptic foci
, underscoring the complexity of the pathophysiologic events characterizing the epileptic brain (Rigau et al., 2007
P-Glycoprotein, multidrug resistance–associated proteins (MRPs), and CYP3A4 together constitute a highly efficient barrier for many drugs (Schuetz et al., 2000
; Yasuda et al., 2002
; Pal & Mitra, 2006
). Our data have demonstrated that the epileptic BBB expresses both multi-drug transporter proteins and CYP enzymes. There is a striking overlap in CYP3A4 and P-glycoprotein inducers, substrates, and inhibitors (Yasuda et al., 2002
; Pal & Mitra, 2006
). Both MDR1 and CYP3A4 are under the control of the pregnane X receptor (PXR), a nuclear receptor family regulating a number of enzymes and transporters in mammals (Ma et al., 2008
). One captivating hypothesis is that expression of CYP enzymes and drug transporters could be involved in cellular detoxification and promote a process whereby cells are allowed to survive in an otherwise hostile environment (Marroni et al., 2003
; Marchi et al., 2004
In conclusion, we demonstrated the expression and function of metabolic enzymes by the diseased endothelial cells at the BBB. Our results also underscore the effect of hemodynamic forces on cerebrovascular gene expression, with emphasis on BBB drug metabolism and transport. Further investigations are needed to elucidate the significance of these findings, specifically to refractory forms of epilepsy, and whether a “metabolic” vascular barrier influences AED brain access.