Blood-brain Barrier In Mice Research Articles

Lack of brain‐to‐blood efflux transport activity of low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) for amyloid‐β peptide(1–40) in mouse: involvement of an LRP‐1‐independent pathway

The contribution of low-density lipoprotein receptor-related protein-1 (LRP-1) to the brain-to-blood amyloid-beta peptide (A beta) efflux transport across the blood-brain barrier (BBB) remains controversial. The purpose of the present study was to clarify whether or not LRP-1 plays a role in efflux transport of A beta at the BBB. After microinjection of [(125)I]activated alpha2-macroglobulin (alpha 2M), a typical LRP-1 ligand, into mouse secondary somatosensory cortex region under ketamine-xylazine anesthesia, residual radioactivity was not significantly decreased up to 90 min, whereas after microinjection of [(125)I]human A beta(1-40) [hA beta(1-40)], the residual radioactivity decreased time-dependently. Co-administration of receptor-associated protein, an inhibitor of LRP-1, did not influence [(125)I]hA beta(1-40) elimination from mouse brain, suggesting that members of the LDL receptor gene family, including LRP-1, do not contribute to hA beta(1-40) elimination from mouse brain across the BBB. There was no significant difference between the uptakes of [(125)I]activated alpha 2M and [(14)C]inulin by mouse brain slices, suggesting that activated alpha 2M was not significantly bound to and/or taken up by parenchymal cells. In conclusion, our results show that LRP-1 does not play a significant role in the brain-to-blood efflux transport of A beta(1-40) at the mouse BBB, but an unidentified transporter(s) appears to be involved.

Involvement of Multidrug Resistance-Associated Protein 4 in Efflux Transport of Prostaglandin E2 across Mouse Blood-Brain Barrier and Its Inhibition by Intravenous Administration of Cephalosporins

Prostaglandin E(2) (PGE(2)) acts as a modulator of synaptic signaling and excitability in the brain. Because PGE(2) is barely inactivated enzymatically in adult brain, its brain level is considered to be controlled by efflux transport across the blood-brain barrier (BBB). The purpose of the present study was to clarify the efflux transport of PGE(2) at the BBB and the interaction of various drugs with this process. [(3)H]PGE(2) was eliminated from brain across the BBB with a half-life of 16.3 min, and the elimination was inhibited by 3 mM unlabeled PGE(2). Multidrug resistance-associated protein 4 (MRP4/ABCC4) was reported to be localized at the luminal membrane of the BBB. MRP4-expressing membrane vesicles showed significant uptake of [(3)H]PGE(2) and the uptake was inhibited by cefmetazole with an IC(50) value of 10.2 microM. At the concentration of 20 microM, several drugs, including cefazolin, cefotaxime, ceftriaxone, and ketoprofen, significantly inhibited [(3)H]PGE(2) uptake into MRP4-expressing membrane vesicles. Using the brain efflux index method, preadministration of cefmetazole, cefazolin, ceftriaxone, and cefotaxime was found to inhibit [(3)H]PGE(2) efflux from brain across the BBB. Furthermore, intravenous administration of the cefmetazole dose dependently reduced [(3)H]PGE(2) elimination across the BBB (ID(50) = 120 mg/kg). These results indicate that PGE(2) is eliminated from the brain by MRP4-mediated efflux transport at the BBB, and peripheral administration of cefmetazole decreases the efflux transport of PGE(2) at the BBB; this interaction may influence brain function.

Reduction of L-Type Amino Acid Transporter 1 mRNA Expression in Brain Capillaries in a Mouse Model of Parkinson's Disease

The blood-brain barrier (BBB) expresses transporters that influence both dopaminergic neuronal function and drug therapy for Parkinson's disease (PD). The purpose of the present study was to clarify changes of transporter mRNA expression at the BBB in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of PD, in order to understand the pathophysiological role of BBB transport function in PD. At 7 d after MPTP treatment, mice showed a motor deficit and a loss of dopaminergic neurons. At the same time, L-type amino acid transporter 1 (LAT1) mRNA expression in the brain capillary fraction of the MPTP-treated mice was significantly reduced by 62.6% compared with saline-treated mice, while no significant change was observed in the expression of glucose transporter 1, creatine transporter 1, taurine transporter, organic cation transporter 2, serotonin transporter, norepinephrine transporter and dopamine transporter. LAT1 mRNA expression in whole brain was not affected at 1, 3 and 5 d after the treatment, but was reduced by 46.3% at 7 d. LAT1 mediates the transport of large neutral amino acids, including tyrosine, as well as the PD-therapeutic drug levodopa, across the BBB. Our findings indicate that decreased LAT1 expression at the BBB in PD patients may adversely affect amino acid supply from the circulating blood and levodopa distribution into the brain.

Quinoline‐n‐butylcyanoacrylate‐based nanoparticles for brain targeting for the diagnosis of Alzheimer's disease

A survey of research activity on nanoparticles (NPs) based on polymeric devices that could cross the blood-brain barrier (BBB) is given along with the presentation of our own data on the development of NPs of n-butyl-2-cyanoacrylate (BCA) for brain delivery to aid the early diagnosis of Alzheimer's disease (AD), a neurodegenerative disorder of the elderly people, the most prevalent form of dementia. Typical data are presented on in vivo detection of amyloid peptides (A beta) (amyloid plaques) that are used as targets for developing the biological markers for the diagnosis of AD. In order to develop efficient in vivo probes, polymeric n-butyl-2-cyanoacrylate (PBCA) NPs have been prepared and encapsulated with the radio-labeled amyloid affinity drug (125)I-clioquinol (CQ, 5-chloro-7-iodo-8-hydroxyquinoline) to improve the transport to brain and amyloid plaque retention of (125)I-CQ using the NPs of PBCA. The (125)I-CQ discriminately binds to the AD post-mortem brain tissue homogenates versus control. (125)I-CQ-PBCA NPs labeled the A beta plaques from the AD human post-mortem frontal cortical sections on paraffin-fixed slides. Storage phosphor imaging verified preferential uptake by AD brain sections compared to cortical control sections. The (125)I-CQ-PBCA NPs crossed the BBB in wild type mouse, giving an increased brain uptake measured in terms of % ID/g i.e., injected dose compared to (125)I-CQ. Brain retention of (125)I-CQ-PBCA NPs was significantly increased in the AD transgenic mice (APP/PS1) and in mice injected with aggregated A beta 42 peptide versus age-matched wild type controls. The results of this study are verified by in vivo storage phosphor imaging and validated by histopathological staining of plaques and select metal ions, viz. Fe(2+) and Cu(2+). The (125)I-CQ-PBCA NPs had more efficient brain entry and rapid clearance in normal mice and enhanced the retention in AD mouse brain demonstrating the ideal in vivo imaging characteristics. The (125)I-CQ-PBCA NPs exhibited specificity for A beta plaques both in vitro and in vivo. This combination offered radio-iodinated CQ-PBCA NPs as the promising delivery vehicle for in vivo single photon emission tomography (SPECT) ((123)I) or PET ((124)I) amyloid imaging agent. The importance of the topic in relation to brain delivery and other similar type of work published in this area are covered to highlight the importance of this research to medical disciplines.

Ibogaine labeling with 99mTc-tricarbonyl: Synthesis and transport at the mouse blood–brain barrier

The 99mTc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most 99mTc-tricarbonyl-labeled agents have difficulty crossing the blood–brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with 99mTc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [99mTc(CO)3-ibogaine]+ and 99mTc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [99mTc(CO)3-ibogaine]+ entered the brain (Kin) poorly (0.18 µL/g/s), at about the same rate as 99mTc-tricarbonyl (0.16 µL/g/s) and [99mTc-sestamibi]+ (0.10 µL/g/s). The CNS tracer [99mTc-HMPAO]0 entered the brain ∼70-times higher than [99mTc(CO)3-ibogaine]+. In vitro studies revealed that neither [99mTc(CO)3-ibogaine]+ nor 99mTc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [99mTc(OH2)3(CO)3]+ ∼3-fold. Thus, ibogaine directly labeled with 99mTc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential.

Peroxisome Proliferator-Activated Receptor-α Activation Protects Brain Capillary Endothelial Cells from Oxygen-Glucose Deprivation-Induced Hyperpermeability in the Blood-Brain Barrier

That promising neuroprotectants failed to demonstrate benefit against stroke highlights the great difficulties to translate preclinical pharmacological effects in clinical outcomes. Part of this hurdle implies the complex response to injury of the neurovascular unit increasing the cerebrovascular permeability at the level of the blood-brain barrier (BBB). Previous studies reported neuroprotection in animal models upon activation of the nuclear receptor PPARalpha(peroxisome proliferator-activated receptor)alpha, but the cellular targets at the BBB level remain largely unexplored. Here, to study whether PPAR-alpha activation acts on BBB permeability, we adapted a mouse BBB cell model to ischaemic conditions at the stage of occlusion defined in vitro as oxygen-glucose deprivation (OGD). This model consists of a co-culture of brain capillary endothelial cells (ECs) on a filter insert placed upon a rat glial cell culture. The EC monolayer permeability increase induced by 4 h of OGD was significantly restricted after treatment with the PPAR-alpha agonist fenofibric acid (FA) 24 h before or at the onset of OGD. Treatments of separated ECs or glial cells showed that this protective effect was conferred by BBB ECs but not glial cells. Furthermore, co-cultures with ECs from PPAR-alpha-deficient mice revealed that FA had no effect on OGD-induced hyperpermeability. No transcriptional modulation of classical PPAR-alpha target genes such as SOD, ICAM-1, VCAM-1, ACO, CPT-1, PDK-4 or ET-1 was observed in wild type mouse ECs. In conclusion, these results suggest that part of the preventive PPAR-alpha-mediated protection may occur via BBB ECs by limiting hyperpermeability.

P-glycoprotein deficient mouse in situ blood–brain barrier permeability and its prediction using an in combo PAMPA model

The purpose of the study was to assess the permeability of mouse blood–brain barrier (BBB) to a diverse set of compounds in the absence of P-glycoprotein (Pgp) mediated efflux, to predict it using an in combo PAMPA model, and to explore its role in brain penetration classification (BPC). The initial brain uptake ( K in) of 19 compounds in both wild-type and Pgp mutant [mdr1a(−/−)] CF-1 mice was determined by the in situ brain perfusion technique. PAMPA measurements were performed, and the values were used to develop an in combo model, including Abraham descriptors. Published rodent K in values were used to enhance the dataset and validate the model. The model predicted 92% of the variance of the training set permeability. In all, 182 K in values were considered in this study, spanning four log orders of magnitude and where Pgp decreased brain uptake by as much as 14-fold. The calculated permeability-surface area (PS) values along with literature reported brain tissue binding were used to group molecules in terms of their brain penetration classification. The in situ BBB permeability can be predicted by the in combo PAMPA model to a satisfactory degree, and can be used as a lower-cost, high throughput first-pass screening method for BBB passive permeability.

Clonidine Transport at the Mouse Blood—Brain Barrier by a New H+ Antiporter that Interacts with Addictive Drugs

Identifying drug transporters and their in vivo significance will help to explain why some central nervous system (CNS) drugs cross the blood-brain barrier (BBB) and reach the brain parenchyma. We characterized the transport of the drug clonidine at the luminal BBB by in situ mouse brain perfusion. Clonidine influx was saturable, followed by Michaelis-Menten kinetics (K(m)=0.62 mmol/L, V(max)=1.76 nmol/sec per g at pH 7.40), and was insensitive to both sodium and trans-membrane potential. In vivo manipulation of intracellular and/or extracellular pH and trans-stimulation showed that clonidine was transported by an H+-coupled antiporter regulated by both proton and clonidine gradients, and that diphenhydramine was also a substrate. Organic cation transporters (Oct1-3), P-gp, and Bcrp did not alter clonidine transport at the BBB in knockout mice. Secondary or tertiary amine CNS compounds such as oxycodone, morphine, diacetylmorphine, methylenedioxyamphetamine (MDMA), cocaine, and nicotine inhibited clonidine transport. However, cationic compounds that interact with choline, Mate, Octn, and Pmat transporters did not. This suggests that clonidine is transported at the luminal mouse BBB by a new H+-coupled reversible antiporter.

Diffusion of docosahexaenoic and eicosapentaenoic acids through the blood–brain barrier: An in situ cerebral perfusion study

Docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are n-3 polyunsaturated fatty acids with a therapeutic potential for CNS diseases. Here, using an in situ brain perfusion technique in mice, we show that [ 14C]-DHA and [ 14C]-EPA readily cross the mouse blood–brain barrier (BBB) with brain transport coefficients (Clup) of 48 ± 3 μl g −1 s −1 and 52 ± 4 μl g −1 s −1, respectively. Mechanical capillary depletion of brain homogenates showed that less than 10% of [ 14C]-DHA or [ 14C]-EPA remained in endothelial cells of the brain vasculature, demonstrating that both molecules fully crossed the BBB. Addition of bovine serum albumin decreased the Clup of [ 14C]-DHA to 0.6 ± 0.3 μl g −1 s −1, indicating that binding to albumin reduced importantly, but not totally, the passage of DHA through the BBB. The Clup of [ 14C]-DHA or [ 14C]-EPA was not saturable at concentration up to 100 μM, suggesting that these compounds crossed the BBB by simple diffusion. However, long-term high-DHA dietary consumption reduced the Clup of [ 14C]-DHA to 33 ± 6 μl g −1 s −1 (−20%, p < 0.01). These results confirm that the brain uptake of DHA or EPA perfused with a physiological buffer is comparable to highly diffusible drugs like diazepam, and can be modulated by albumin binding and chronic dietary DHA intake.

Cutting Edge: Natalizumab Blocks Adhesion but Not Initial Contact of Human T Cells to the Blood-Brain Barrier In Vivo in an Animal Model of Multiple Sclerosis

The humanized anti-alpha(4) integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the alpha(4) integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As alpha(4) integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-alpha-induced inflammation. Our data demonstrate that alpha(4) integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis.

Functional Blocking Monoclonal Antibodies against IL-12p40 Homodimer Inhibit Adoptive Transfer of Experimental Allergic Encephalomyelitis

IL-12p70 (p40:p35) and IL-23 (p40:p19) are bioactive cytokines and their role in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, are becoming clear. On the other hand, the IL-12p40 homodimer (p40(2)) was considered as an inactive or inhibitory molecule and its functions are poorly understood. To facilitate the studies on p40(2), we have recently generated neutralizing mAb against mouse p40(2). The present study demonstrates the effectiveness of p40(2) mAb in treating the disease process of relapsing-remitting EAE in female SJL/J mice. The p40(2) mAb ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological and blood-brain barrier (BBB) and blood-spinal cord barrier (BSB) permeability studies reveal that p40(2) mAb effectively inhibited the infiltration of mononuclear cells into brain and spinal cord and improved the integrity of BBB and BSB in EAE mice. Consequently, p40(2) mAb also suppressed the expression of proinflammatory molecules, normalized the expression of myelin genes, and blocked demyelination in the CNS of EAE mice. On the other hand, recombinant mouse p40(2) increased the infiltration of mononuclear cells into the CNS, enhanced the permeability through BBB and BSB, stimulated CNS expression of proinflammatory molecules, and aggravated the disease process of EAE. Taken together, our results suggest that p40(2) participates in the pathogenesis of EAE and that neutralization of p40(2) may be beneficial in multiple sclerosis patients.

Changes in dipole membrane potential at the mouse blood–brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2

Cationic (99m)Tc-agents like (99m)Tc-hexakis-2-methoxyisobutyl isonitrile ((99m)Tc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged (99m)Tc-compounds. The mechanism by which cationic (99m)Tc-agents are transported across the blood-brain barrier (BBB) remains unclear. We explored (99m)Tc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced (99m)Tc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased (99m)Tc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased (99m)Tc-MIBI transport, but tetraethylammonium did not. (99m)Tc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of (99m)Tc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting (99m)Tc-MIBI permeability.

ApoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.

Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood–brain barrier delivery in the mouse

Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.

Endothelial Protein C Receptor-Assisted Transport of Activated Protein C across the Mouse Blood—Brain Barrier

Activated protein C (APC), a serine-protease with anticoagulant, anti-inflammatory, and cytoprotective activities, is neuroprotective and holds potential to treat different neurologic disorders. It is unknown whether APC crosses the blood-brain barrier (BBB) to reach its therapeutic targets in the brain. By using a brain vascular perfusion technique, we show that (125)I-labeled plasma-derived mouse APC enters the brain from cerebrovascular circulation by a concentration-dependent mechanism. The permeability surface area product of (125)I-APC (0.1 nmol/L) in different forebrain regions ranged from 3.11 to 4.13 microL/min/g brain. This was approximately 80- to 110-fold greater than for (14)C-inulin, a simultaneously infused reference tracer. The K(m) value for APC BBB cortical transport was 1.6+/-0.2 nmol/L. Recombinant APC variants with reduced anticoagulant activity, 5A-APC and 3K3A-APC, but not protein C, exhibited high affinity for the APC BBB transport system. Blockade of APC-binding site on endothelial protein C receptor (EPCR), but not blockade of its protease-activated receptor-1 (PAR1) catalytic site, inhibited by >85% APC entry into the brain. APC brain uptake was reduced by 64% in severely deficient EPCR mice, but not in PAR1 null mice. These data suggest that APC and its variants with reduced anticoagulant activity cross the BBB via EPCR-mediated saturable transport.

RNAi‐mediated reversible opening of the blood‐brain barrier

The blood-brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier. Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin-5 to the endothelial cells of the BBB in mice. We have shown a significant decrease in claudin-5 mRNA levels 24 and 48 hours post-delivery of siRNA, with levels of protein expression decreasing up to 48 hours post-injection compared to uninjected, phosphate-buffered saline (PBS)-injected and non-targeting siRNA-injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size-selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin-releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post-injection of siRNA targeting claudin-5 significantly modifies behavioural output. These data demonstrate that it is now possible to transiently and size-selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases.

Human Immunodeficiency Virus Type 1 Infection Increases the In Vivo Capacity of Peripheral Monocytes To Cross the Blood-Brain Barrier into the Brain and the In Vivo Sensitivity of the Blood-Brain Barrier to Disruption by Lipopolysaccharide

Human immunodeficiency virus type 1 (HIV-1), introduced into the brain by HIV-1-infected monocytes which migrate across the blood-brain barrier (BBB), infects resident macrophages and microglia and initiates a process that causes HIV-1-associated neurocognitive disorders. The mechanism by which HIV-1 infection circumvents the BBB-restricted passage of systemic leukocytes into the brain and disrupts the integrity of the BBB is not known. Circulating lipopolysaccharide (LPS), which can compromise the integrity of the BBB, is significantly increased in HIV-1-infected individuals. We hypothesized that HIV-1 infection increases monocyte capacity to migrate across the BBB, which is further facilitated by a compromise of BBB integrity mediated by the increased systemic LPS levels present in HIV-1-infected individuals. To investigate this possibility, we examined the in vivo BBB migration of monocytes derived from our novel mouse model, JR-CSF/EYFP mice, which are transgenic for both a long terminal repeat-regulated full-length infectious HIV-1 provirus and ROSA-26-regulated enhanced yellow fluorescent protein. We demonstrated that JR-CSF/EYFP mouse monocytes displayed an increased capacity to enter the brain by crossing either an intact BBB or a BBB whose integrity was partially compromised by systemic LPS. We also demonstrated that the JR-CSF mouse BBB was more susceptible to disruption by systemic LPS than the control wild-type mouse BBB. These results demonstrated that HIV-1 infection increased the ability of monocytes to enter the brain and increased the sensitivity of the BBB to disruption by systemic LPS, which is elevated in HIV-1-infected individuals. These mice represent a new in vivo system for studying the mechanism by which HIV-1-infected monocytes migrate into the brain.

Patupilone for the treatment of recurrent/progressive brain metastases in patients (pts) with non-small cell lung cancer (NSCLC): An open-label phase II study

2033 Background: Preclinical data have shown that patupilone, a novel microtubule stabilizer, crosses the intact blood brain barrier in mice, rats and dogs and has antitumor activity in brain tumor models. This study evaluates patupilone's efficacy in NSCLC pts with brain metastases and progression after chemotherapy, surgery and/or radiation to the brain. Methods: This open-label, single-arm, multicenter, phase II, Simon 2-stage (St) study (25 pts per St) uses a multinomial design (response (R), early progression (EP)). Primary efficacy endpoints were a combination of EP (PD or death before cycle 1 Day 21) and R (progression free at 9 wks). Criteria to proceed to St 2 were >3 R and <11 EP in 25 pts. Eligible pts had confirmed NSCLC and ≥1 recurrent, measurable intracranial lesion ≥2 cm. Patupilone was given as 10 mg/m2 in a single 20-min IV infusion every 3 wks. Results: So far, 25 pts in St I and 2 pts in St II (median age 62 yrs, range 40–74, 59% male) have received 78 cycles (median 2 cycles). All pts received prior radiotherapy and 10/27 (37%) had prior chemotherapy for the brain. Karnofsky status in 25/27 ≥70% and in 2/27 <70%. Common G1/2 adverse events (AEs) were as follows: diarrhea 59%, nausea 19%, fatigue and peripheral neuropathy, each 11%. There were 21 G3 AEs (44%); 10 were related to study drug (diarrhea in 6). Eight G4 AEs (19%) were not related and 1 G4 AE (colitis) was suspected. Investigator reported efficacy data from St 1 (n=25) showed 8 responders (median duration of response 192 days, range 191–273); 7 had stable disease, 9 EP and assessment was unknown in 1 pt. Investigator reported median progression free survival is 1.2 mos. Three of the responding patients continued ≥8 cycles. Conclusions: The study met the statistical criteria to proceed to St 2. Proof of concept of patupilone activity in human brain metastases has been reached. These results warrant further investigation of patupilone as a treatment for CNS metastases from NSCLC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Novartis Novartis AstraZeneca, Aventis, Eli Lilly, Genentech™ BioOncology, Pfizer Merck, Novartis

Starvation and Triglycerides Reverse the Obesity-Induced Impairment of Insulin Transport at the Blood-Brain Barrier

Insulin in the brain acts as a satiety factor, reduces appetite, and decreases body mass. Altered sensing by brain of insulin may be a leading cause of weight gain and insulin resistance. A decrease in the transport across the blood-brain barrier (BBB) of insulin may induce brain insulin resistance by inducing obesity. We here report that transport of iv administrated insulin across the BBB of obese mice, as measured by multiple-time regression analysis, was significantly lower than that in thin adult mice. The reduction in obese mice was reversed by starvation for 48 h. There were no differences in insulin transport rates across the BBB of obese, thin, or starved obese mice when studied by the brain perfusion model, demonstrating that BBB transport of insulin is modulated by circulating factors. In the brain perfusion study, the triglyceride triolein significantly increased the brain uptake of insulin, an effect opposite to that on leptin transport, in starved obese mice. Thus, circulating triglycerides are one of the systemic modulators for the transport of insulin across the BBB.

In Vivo Evaluation of the Uptake of [123I]FIAU, [123I]IVFRU and [123I]IVFAU by Normal Mouse Brain: Potential For Noninvasive Assessment of HSV-1 Thymidine Kinase Gene Expression in Gliomas

Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [124I]IVFRU and [124I]IVFAU in tumor-bearing mice are warranted.