Carolyn A. Foster

Brain Penetration of the Oral Immunomodulatory Drug FTY720 and Its Phosphorylation in the Central Nervous System during Experimental Autoimmune Encephalomyelitis: Consequences for Mode of Action in Multiple Sclerosis

FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.

FTY720 sustains and restores neuronal function in the DA rat model of MOG-induced experimental autoimmune encephalomyelitis

FTY720 (fingolimod) is an oral sphingosine 1-phosphate (S1P) receptor modulator under development for the treatment of multiple sclerosis (MS). To elucidate its effects in the central nervous system (CNS), we compared functional parameters of nerve conductance in the DA rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) after preventive and therapeutic treatment. We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms, neuropathology, and disturbances to visual and somatosensory evoked potentials (VEP, SEP). Moreover, therapeutic treatment from day 25 to 45 markedly reversed paralysis in established EAE and normalized the electrophysiological responses, correlating with decreased demyelination in the brain and spinal cord. The effectiveness of FTY720 in this model is likely due to several contributing factors. Evidence thus far supports its role in the reduction of inflammation and preservation of blood-brain-barrier integrity. FTY720 may also act via S1P receptors in glial cells to promote endogenous repair mechanisms that complement its immunomodulatory action.

FTY720 Rescue Therapy in the Dark Agouti Rat Model of Experimental Autoimmune Encephalomyelitis: Expression of Central Nervous System Genes and Reversal of Blood‐Brain‐Barrier Damage

FTY720 (fingolimod) is an oral sphingosine‐1 phosphate (S1P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP‐9 and increased its counterregulator—tissue inhibitor of metalloproteinase, TIMP‐1—resulting in a proteolytic balance that favors preservation of blood‐brain‐barrier (BBB) integrity. Furthermore, FTY720 reduced S1P lyase that increases the S1P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S1P receptors on resident brain cells. This is accompanied by an increase in S1P1 and S1P5 in contrast with the attenuation of S1P3 and S1P4. Late‐stage rescue therapy with FTY720, even up to 1 month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.

Predictability of FTY720 efficacy in experimental autoimmune encephalomyelitis by in vivo macrophage tracking: Clinical implications for ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging

To examine the efficacy of FTY720 as a new agent to reduce inflammatory activity in an animal model of multiple sclerosis (MS) by in vivo macrophage tracking.

Characterization of the Potent and Highly Selective A2A Receptor Antagonists Preladenant and SCH 412348 [7-[2-[4-2,4-Difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in Rodent Models of Movement Disorders and Depression

The adenosine A2A receptor has been implicated in the underlying biology of various neurological and psychiatric disorders, including Parkinsons disease (PD) and depression. Preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] are potent competitive antagonists of the human A2A receptor (Ki = 1.1 and 0.6 nM, respectively) and have >1000-fold selectivity over all other adenosine receptors, making these compounds the most selective A2A receptor antagonists reported to date. Both compounds attenuate hypolocomotion induced by the A2A receptor agonist CGS-21680 [2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine], suggesting that they inhibit A2A receptor activity in vivo. Their high degree of selectivity and robust in vivo activity make preladenant and SCH 412348 useful tools to investigate the role of the A2A receptor system in animal models of PD and depression. Oral administration of preladenant and SCH 412348 (0.1–1 mg/kg) to rats potentiated 3,4-dihydroxy-l-phenylalanine (l-Dopa)-induced contralateral rotations after 6-hydroxydopamine lesions in the medial forebrain bundle and potently attenuated the cataleptic effects of haloperidol. Preladenant (1 mg/kg) inhibited l-Dopa-induced behavioral sensitization after repeated daily administration, which suggests a reduced risk of the development of dyskinesias. Finally, preladenant and SCH 412348 exhibited antidepressant-like profiles in models of behavioral despair, namely the mouse tail suspension test and the mouse and rat forced swim test. These studies demonstrate that preladenant and SCH 412348 are potent and selective A2A receptor antagonists and provide further evidence of the potential therapeutic benefits of A2A receptor inhibition in PD (with reduced risk of dyskinesias) and depression (one of the primary nonmotor symptoms of PD).

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61β. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

The 3D-structure of a natural inhibitor of cell adhesion molecule expression

The three‐dimensional structure of cyclopeptolide HUN‐7293, a naturally‐occurring inhibitor of cell adhesion molecule expression, has been determined from nuclear magnetic resonance data recorded in solution and from X‐ray diffraction analysis of single crystals. The backbone conformation of HUN‐7293 is characterized by two cis‐peptide bonds in both the solution and crystalline state. Differences between the solution and crystal structure are visible for the orientation of some side chains and the strength of two transannular hydrogen bonds. Such structural information helps to provide insight into the molecular architecture of HUN‐7293 on the atomic level and opens the way for structure‐based modifications of this novel inhibitor of cell adhesion molecule expression.

Ascomycete derivative to MS therapeutic: S1P receptor modulator FTY720

Fingolimod (FTY720) represents the first in a new class of immune-modulators whose target is sphingosine-1-phosphate (S1P) receptors. It was first identified by researchers at Kyoto University and Yoshitomi Pharmaceutical as a chemical derivative of the ascomycete metabolite ISP-1 (myriocin). Unlike its natural product parent, FTY720 does not interfere with sphingolipid biosynthesis. Instead, its best characterized mechanism of action upon in vivo phosphorylation, leading to the active principle FTY720-P, is the rapid and reversible inhibition of lymphocyte egress from peripheral lymph nodes. As a consequence of S1P1 receptor internalization, tissue-damaging T-cells can not recirculate and infiltrate sites of inflammation such as the central nervous system (CNS). Furthermore, FTY720-P modulation of S1P receptor signaling also enhances endothelial barrier function. Due to its mode of action, FTY720 effectively prevents transplant rejection and is active in various autoimmune disease models. The most striking efficacy is in the multiple sclerosis (MS) model of experimental autoimmune encephalomyelitis, which has now been confirmed in the clinic. FTY720 demonstrated promising results in Phase II trials and recently entered Phase III in patients with relapsing MS. Emerging evidence suggests that its efficacy in the CNS extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain-barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function. FTY720 may represent a potent new therapeutic modality in MS, combined with the benefit of oral administration.

Inhibitors of vascular cell adhesion molecule-1 expression

Leukocyte extravasation, a complex multistep process involving the selective recruitment of white blood cell subpopulations to specific tissues, is finely regulated to a large extent by inducible adhesion molecules on the endothelium. Secreted mediators such as tumour necrosis factor alpha (TNF-α) transcriptionally activate endothelial cells at sites of injury or inflammation, resulting in the localised de novo synthesis of cell surface proteins such as vascular cell adhesion molecule-1 (VCAM-1). Circulating leukocytes bearing the VCAM1 ligand, namely the integrin very late antigen4 (VLA-4), then bind to their corresponding adhesion receptor that is newly expressed on the luminal surface of postcapillary venules. Preclinical data strongly suggest that the VCAM-1/VLA-4 adhesion pathway significantly contributes to the pathogenesis of numerous diseases including asthma, atherosclerosis, inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis. VCAM-1 thus represents an attractive target for drug intervention due to its central role in sequential events leading to the transendothelial migration of leukocytes (e.g., T and B lymphocytes, eosinophils, monocytes). This article focuses on patents claiming inhibition of VCAM-1 gene and protein expression, primarily covering antioxidants, antisense oligonucleotides, cyclic depsipeptides of the HUN-7293 type and small heterocyclic compounds.

Synthesis and evaluation of Aza HUN-7293

The aza analogue of the cyclic heptadepsipeptide HUN-7293 (1), which is a potent naturally occurring inhibitor of inducible cell adhesion molecule expression, and its C2(3) (MLEU3 C2) epimer were prepared via solution-phase synthesis. Biological evaluations of these two compounds as inhibitors of cell adhesion molecules expression are detailed.