Targeting the Blood-brain Barrier with a Non-canonical Iron-mimicry Mechanism

Treatment of central nervous system (CNS) diseases is limited by the blood-brain barrier (BBB), a selective vascular interface restricting passage of most molecules from blood into brain. Specific transport systems have evolved allowing circulating polar molecules to cross the BBB and gain access to the brain parenchyma. However, to date, few ligands exploiting such systems have proven clinically viable in the setting of CNS diseases. We reasoned that combinatorial phage-display screenings in vivo would yield peptides capable of crossing the BBB and allow for the development of ligand-directed targeting strategies of the brain. Here we show the identification of a peptide mediating systemic targeting to the normal brain and to an orthotopic human glioma model. We demonstrate that this peptide functionally mimics iron through an allosteric mechanism and that a non-canonical association of (i) transferrin, (ii) the iron-mimic ligand motif, and (iii) transferrin receptor mediates binding and transport of particles across the BBB. We also show that in orthotopic human glioma xenografts, a combination of transferrin receptor over-expression plus extended vascular permeability and ligand retention result in remarkable brain tumor targeting. Moreover, such tumor targeting attributes enables Herpes simplex virus thymidine kinase-mediated gene therapy of intracranial tumors for molecular genetic imaging and suicide gene delivery with ganciclovir. Finally, we expand our data by analyzing a large panel of primary CNS tumors through comprehensive tissue microarrays. Together, our approach and results provide a translational avenue for the detection and treatment of brain tumors.

Transport of prion protein across the blood-brain barrier.

The cellular form of the prion protein (PrP(c)) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP(c) is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP(c) between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP(c) can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP(c) crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP(c) was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP(c) entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP(c) has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP(c) function and prion replication.

Quantification of the spatiotemporal microstructural organization of the human brain association, projection and commissural pathways across the lifespan using diffusion tensor tractography.

Using diffusion tensor tractography, we quantified the microstructural changes in the association, projection, and commissural compact white matter pathways of the human brain over the lifespan in a cohort of healthy right-handed children and adults aged 6-68 years. In both males and females, the diffusion tensor radial diffusivity of the bilateral arcuate fasciculus, inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, uncinate fasciculus, corticospinal, somatosensory tracts, and the corpus callosum followed a U-curve with advancing age; fractional anisotropy in the same pathways followed an inverted U-curve. Our study provides useful baseline data for the interpretation of data collected from patients.

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

Development and validation of the Neuro Symptom Inventory in the primary brain tumor patient population

Symptoms has been shown to predict quality of life, treatment course and survival in solid tumor patients. Currently, no instrument exists that measures both cancer-related symptoms and the neurologic symptoms that are unique to persons with primary brain tumors (PBT). The aim of this study was to develop and validate an instrument to measure symptoms in patients who have PBT. A conceptual analysis of symptoms and symptom theories led to defining the symptoms experience as the perception of the frequency, intensity, distress, and meaning that occurs as symptoms are produced, perceived, and expressed. The M.D. Anderson Symptom Inventory (MDASI) measures both symptoms and how they interfere with daily functioning in patients with cancer, which is similar to the situational meaning defined in the analysis. A list of symptoms pertinent to the PBT population was added to the core MDASI and reviewed by a group of experts for validity. As a result, 18 items were added to the core MDASI (the MDASI-BT) for the next phase of instrument development, establishing validity and reliability through a descriptive, cross-sectional approach with PBT patients. Data were collected with a patient completed demographic data sheet, an investigator completed clinician checklist, and the MDASI-BT. Analysis evaluated the reliability and validity of the MDASI-BT in PBT patients. Data were obtained from 201 patients. The number of items was reduced to 22 by evaluation of symptom severity as well as cluster analysis. Regression analysis showed more than half (56%) of the variability in symptom severity was explained by the brain tumor module items. Factor analysis confirmed that the 22 item MDASI-BT measured six underlying constructs: (a) affective; (b) cognitive; (c) focal neurologic deficits; (d) constitutional symptoms; (e) treatment-related symptoms; and (f) gastrointestinal symptoms. The MDASI-BT was sensitive to disease severity and if the patient was hospitalized. The MDASI-BT is the first instrument to measure symptoms in PBT patients that has demonstrated reliability and validity. It is the first step in a program of research to evaluate the occurrence of symptoms and plan and evaluate interventions for PBT patients. ^

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

Characterization of the interaction between local cerebral metabolic rate for glucose and acid -base index in ischemic rat brain employing a double-isotope methodology

The association between increases in cerebral glucose metabolism and the development of acidosis is largely inferential, based on reports linking hyperglycemia with poor neurological outcome, lactate accumulation, and the severity of acidosis. We measured local cerebral metabolic rate for glucose (lCMRglc) and an index of brain pH--the acid-base index (ABI)--concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using ($\sp{14}$C) 2-deoxyglucose and ($\sp{14}$C) dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to tamponade-induced middle cerebral occlusion showed areas of normal, depressed and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after two hours of ischemia. Regions of normal glucose metabolic rate showed normal ABI (pH $\pm$ SD = 6.97 $\pm$ 0.09), regions of depressed lCMRglc showed severe acidosis (6.69 $\pm$ 0.14), and regions of elevated lCMRglc showed moderate acidosis (6.88 $\pm$ 0.10), all significantly different at the.00125 level as shown by analysis of variance. Moderate acidosis in regions of increased lCMRglc suggests that anaerobic glycolysis causes excess protons to be generated by the uncoupling of ATP synthesis and hydrolysis. ^