Efficacy and Safety of Etrasimod in Patients with Moderately to Severely Active Isolated Proctitis: Results From the Phase 3 ELEVATE UC Clinical Programme.

BACKGROUND AND AIMS: Pivotal trials in ulcerative colitis have historically excluded patients with isolated proctitis. Etrasimod is an oral, oncedaily, selective sphingosine 1phosphate1,4,5 receptor modulator for the treatment of moderately to severely active ulcerative colitis. This post hoc analysis assessed efficacy and safety of etrasimod 2 mg once daily in patients with isolated proctitis (centrally read) from the phase 3 ELEVATE UC 52 and ELEVATE UC 12 trials. METHODS: Patients, including those with isolated proctitis (<10 cm rectal involvement) who met all other inclusion criteria in ELEVATE UC 52 and ELEVATE UC 12, were randomised 2:1 to receive etrasimod or placebo. Primary, secondary and other identified efficacy endpoints and safety were assessed. RESULTS: We analysed data from 64 and 723 patients at Week 12 (both trials pooled), and 36 and 397 patients at Week 52 (ELEVATE UC 52 only) with isolated proctitis and more extensive colitis (≥10 cm rectal involvement), respectively. Patients with isolated proctitis receiving etrasimod demonstrated significant improvements versus placebo, including clinical remission rates at Weeks 12 (42.9% vs 13.6%) and 52 (44.4% vs 11.1%), endoscopic improvement (52.4% vs 22.7%) at Week 12 and bowel urgency numerical rating scale score at Week 12 (all p<0.01). Generally similar trends were observed in patients with more extensive colitis. Safety was consistent across subgroups, with no new findings. CONCLUSIONS: Etrasimod demonstrated significant improvements versus placebo in patients with isolated proctitis, and those with more extensive disease, in most efficacy endpoints at Week 12 and 52.

The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence.

The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. We showed that astrocytes secrete Sonic hedgehog and that BBB ECs express Hedgehog (Hh) receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacological inhibition and genetic inactivation of the Hh signaling pathway in ECs, we also demonstrated a critical role of the Hh pathway in promoting the immune quiescence of BBB ECs by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Overall, the Hh pathway provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis.

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system.

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

The blood-brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells.

Trafficking of antigen-presenting cells into the CNS is essential for lymphocyte reactivation within the CNS compartment. Although perivascular dendritic cells found in inflammatory lesions are reported to polarize naive CD4+ T lymphocytes into interleukin-17-secreting-cells, the origin of those antigen-presenting cells remains controversial. We demonstrate that a subset of CD14+ monocytes migrate across the inflamed human blood-brain barrier (BBB) and differentiate into CD83+CD209+ dendritic cells under the influence of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor. We also demonstrate that these dendritic cells secrete interleukin-12p70, transforming growth factor-beta and interleukin-6 and promote the proliferation and expansion of distinct populations of interferon-gamma-secreting Th1 and interleukin-17-secreting Th17 CD4+ T lymphocytes. We further confirmed the abundance of such dendritic cells in situ, closely associated with microvascular BBB-endothelial cells within acute multiple sclerosis lesions, as well as a significant number of CD4+ interleukin-17+ T lymphocytes in the perivascular infiltrate. Our data support the notion that functional perivascular myeloid CNS dendritic cells arise as a consequence of migration of CD14+ monocytes across the human BBB, through the concerted actions of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor.

Angiotensin II controls occludin function and is required for blood brain barrier maintenance: relevance to multiple sclerosis.

The blood-brain barrier (BBB) restricts molecular and cellular trafficking between the blood and the CNS. Although astrocytes are known to control BBB permeability, the molecular determinants of this effect remain unknown. We show that angiotensinogen (AGT) produced and secreted by astrocytes is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT1) expressed by BBB endothelial cells (ECs). Activation of AT1 restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knock-out animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, compared with wild-type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knock-out animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent, in vitro, on the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions.

Death receptor expression and function at the human blood brain barrier.

The blood brain barrier (BBB) is composed of specialized endothelial cells tightly anastomosed to one another and surrounded by a thick extracellular matrix, the basement membrane. Together these components restrict the diffusion of cells and molecules from the periphery into the central nervous system (CNS), providing immune privilege and homeostasis. Dysregulation of the BBB and trans-endothelial migration of immune cells are amongst the earliest CNS changes partaking in lesion formation in multiple sclerosis (MS). Death receptors are members of the tumor necrosis factor receptor (TNFR) super-family. They are expressed on a variety of tissues including endothelium, but the consequence of their triggering appears to be cell type specific. In this study, we describe the expression of death receptors TNFR1, Fas and DR5 on primary cultures of human BBB-derived endothelial cells (ECs), as well as the effects of receptor activation on human brain endothelial cell (HBEC) function. We show that HBECs are resistant to cell death mediated via TNFalpha, FasL and TRAIL and that neither receptor ligation induces cellular proliferation of HBECs. TNFR1 ligation induces NFkappaB activation and the upregulation of chemokines MCP-1 and IL-8, as well as adhesion molecules ICAM-1 and VCAM-1, while Fas and DR5 triggering activate the extracellular signal regulated kinases-1 and -2 (Erk 1/2, p42/44 MAPK) inducing the release of matrix metalloproteinase 9 (MMP9) by BBB-derived ECs.

Expression of the ATP-binding cassette membrane transporter, ABCG2, in human and rodent brain microvessel endothelial and glial cell culture systems.

PURPOSE: The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments. MATERIALS AND METHODS: RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells. RESULTS: RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems. CONCLUSIONS: Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

Statins reduce human blood-brain barrier permeability and restrict leukocyte migration: relevance to multiple sclerosis.

OBJECTIVE: Dysregulation of the blood-brain barrier (BBB) and transendothelial migration of immune cells are among the earliest central nervous system changes partaking in lesion formation in both multiple sclerosis (MS) and its early clinical form, the clinically isolated syndrome. Evidence for the anti-inflammatory effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors within the central nervous system arose from studies demonstrating that statins improve clinical signs in the animal model of MS and reduce the number of gadolinium-enhancing lesions in MS. METHODS: We sought to describe the impact of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor treatment on the physiology and immunology of human BBB-derived endothelial cells (ECs). RESULTS: We demonstrate that lovastatin and simvastatin induce a 50 to 60% reduction in the diffusion rates of bovine serum albumin and [(14)C]-sucrose across human BBB-ECs in vitro through abrogation of isoprenylation processes, but independent of the expression of the tight junction molecules occludin, VE-cadherin, JAM-1, zonula occluden-1, and zonula occluden-2. Simvastatin and lovastatin were equipotent in reducing BBB permeability in vitro, with median effective concentration (EC(50)) of 9.5 x 10(-8) and 1.0 x 10(-7)M, respectively. We further demonstrate that lovastatin and simvastatin treatment of BBB-ECs significantly restricts the migration of clinically isolated syndrome-derived and MS-derived monocytes and lymphocytes across the human BBB in vitro, through a specific reduction in the secretion of the chemokines monocyte chemotactic protein-1/CCL2 and interferon-gamma-inducible protein-10/CXCL10 by BBB-ECs. INTERPRETATION: Our data parallel the previously reported magnetic resonance imaging-based radiological findings and suggest an effect of statins that could be beneficial in early MS, restricting the diffusion of molecular tracers and the migration of immune cells across the human BBB.

Impact of the coxsackie and adenovirus receptor (CAR) on glioma cell growth and invasion: requirement for the C-terminal domain.

Expression of the coxsackie and adenovirus receptor (CAR) is downregulated in malignant glioma cell lines and is barely detectable in high-grade primary astrocytoma (glioblastoma multiforme). We determined the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87-MG, which does not express any CAR. Although retrovirally mediated expression of full-length CAR in U87-MG cells did not affect monolayer growth in vitro, it did reduce glioma cell invasion in a 3-dimensional spheroid model. Furthermore, in xenograft experiments, intracerebral implantation of glioma cells expressing full-length CAR resulted in tumors with a significantly reduced volume compared to tumors generated by control vector-transduced U87-MG cells. In contrast, U87-MG cells expressing transmembrane CAR with a deletion of the entire cytoplasmic domain (except for the first 2 intracellular juxtamembrane cysteine amino acids) had rates of invasion and tumor growth that were similar to those of the control cells. This difference in behavior between the 2 forms of CAR was not due to improper cell surface localization of the cytoplasmically deleted CAR as determined by comparable immunostaining of unpermeabilized cells, equivalent adenoviral transduction of the cells and similar extent of fractionation into lipid-rich domains. Taken together, these results suggest that the decrease or loss of CAR expression in malignant glioma may confer a selective advantage in growth and invasion to these tumors.

Resistance of human adult oligodendrocytes to AMPA/kainate receptor-mediated glutamate injury.

Multiple sclerosis is an inflammatory disease of the CNS leading to the destruction of oligodendrocytes (OLs), myelin sheaths and axons. The mediators of tissue injury remain unknown. Glutamate, which can be released by activated immune cells or produced within the CNS, has been implicated as a potential mediator of tissue injury in multiple sclerosis. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainate are highly toxic when added to rodent OL cultures. Using OLs derived from human adult surgical specimens, we investigated AMPA/kainate receptor expression and the effects of receptor stimulation on the viability of human OLs. We find that human adult OLs in vitro express low levels of ionotropic glutamate receptors and are resistant to excitotoxicity mediated by high and sustained doses of AMPA or kainate, even when receptor desensitization is blocked. In contrast, rat OLs show strong AMPA receptor expression and are susceptible to excitotoxicity, as previously demonstrated. Furthermore, we show in human brain sections that OLs do not express AMPA receptors in situ and that glial expression of AMPA receptors is limited to astrocytes. The apparent lack of glutamate receptor expression on human OLs and their resistance to AMPA/kainate toxicity should be considered when postulating mechanisms of tissue injury in multiple sclerosis.

Oligodendrocyte injury in multiple sclerosis: a role for p53.

Multiple sclerosis (MS) is a neurological disorder characterized by myelin destruction and a variable degree of oligodendrocyte death. We have previously shown that overexpression of the transcription factor p53 can induce oligodendrocyte apoptosis. We investigated the mechanism of p53-induced apoptosis using primary cultures of central nervous system-derived adult human oligodendrocytes. Adenovirus-mediated p53 overexpression resulted in up-regulation of the death receptors Fas, DR4 and DR5 with subsequent caspase-mediated apoptosis of the oligodendrocytes. The oligodendrocytes were protected from p53-induced cell death by blocking signaling through Fas and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. Although lower levels of p53 did not induce apoptosis, the increase in death receptor expression was sufficient to render the oligodendrocytes susceptible to apoptosis in the presence of exogenous Fas ligand and TRAIL. These ligands are present in the inflammatory milieu of active MS lesions. In situ analysis of active MS lesions revealed increased p53 expression in oligodendrocytes in lesions that featured oligodendrocyte apoptosis and cell loss. Our data provide evidence for a novel role for p53 in the pathogenesis of MS.

Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia.

We assessed the effects of soluble molecules (supernatants) produced by pro- (Th1) and anti- (Th2) inflammatory T-cell lines on the capacity of adult human CNS-derived microglia to express or produce selected cell surface and soluble molecules that regulate immune reactivity or impact on tissue protection/repair within the CNS. Treatment of microglia with supernatants from allo-antigen and myelin basic protein-specific Th1 cell lines augmented expression of cell surface molecules MHC class II, CD80, CD86, CD40, and CD54, enhanced the functional antigen-presenting cell capacity of microglia in a mixed lymphocyte reaction, and increased cytokine/chemokine secretion (TNFalpha, IL-6, and CXCL10/IP-10). These Th1-induced effects were not reproduced by interferon-gamma (IFNgamma) alone and were only incompletely blocked by anti-IFNgamma antibody. Th2 cell supernatant treatments did not alter costimulatory/adhesion molecule expression or induce cytokine/chemokine production by microglia. Th2 treatment, furthermore, failed to reduce the induction observed in response to Th1 supernatants. Neither Th1 nor Th2 supernatants induced production of the neurotrophin molecules, nerve growth factor, or brain-derived neurotrophic factor. Our results suggest that soluble molecules released by Th1 and not Th2 cells that infiltrate the CNS can stimulate resident microglia to acquire enhanced effector and accessory cell functions; the Th1-induced effects were not downregulated by Th2 supernatant-mediated bystander suppression.

Human brain endothelial cells supply support for monocyte immunoregulatory functions.

Blood-derived monocytic cells comprise a significant component of most inflammatory responses that occur in the CNS. We utilized human brain-derived endothelial cells (HBECs) coated membranes in Boyden chambers to assess immune function related properties of human blood-derived monocytes following interaction with HBECs. Monocytes in contact with HBECs maintained functional antigen-presenting capacity and chemokine/cytokine production in contrast to monocytes that migrated through the HBEC barrier. These results indicate that HBECs, although themselves incapable of serving as competent antigen-presenting cells during the course of inflammatory CNS disorders, supply support needed for infiltrating perivascular monocytes to maintain their functions. Monocyte migration across HBECs was inhibited by interferon-beta.