Objective Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system. Circulating autoantibodies (NMO-IgG) against astrocyte water channel aquaporin-4 (AQP4) cause complement- and cell-mediated astrocyte damage with consequent neuroinflammation and demyelination. Current NMO therapies, which have limited efficacy, include immunosuppression and plasma exchange. The objective of this study was to develop a potential new NMO therapy based on blocking of pathogenic NMO-IgG to its target, AQP4. Methods We generated non-pathogenic recombinant monoclonal anti-AQP4 antibodies that selectively block NMO-IgG binding to AQP4. These antibodies comprise a tight-binding anti-AQP4 Fab and a mutated Fc that lacks functionality for complement- and cell-mediated cytotoxicity. The efficacy of the blocking antibodies was studied using cell culture, spinal cord slice and in vivo mouse models of NMO. Results In AQP4-expressing cell cultures, the non-pathogenic competing antibodies blocked binding of NMO-IgG in human sera, reducing to near zero complement- and cell-mediated cytotoxicity. The antibodies prevented the development of NMO lesions in an ex vivo spinal cord slice model of NMO and in an in vivo mouse model, without causing cytotoxicity. Interpretation Our results provide proof-of-concept for therapy of NMO with blocking antibodies. The broad efficacy of antibody inhibition is likely due to steric competition because of its large physical size compared to AQP4. Blocker therapy to prevent binding of pathogenic autoantibodies to their targets may be useful for treatment of other autoimmune diseases as well.

Background: Complement-dependent cytotoxicity (CDC) plays a central role in neuromyelitis optica (NMO), in which NMO autoantibodies (NMO-IgG) bind to AQP4 on astrocytes. Results: NMO-IgG produced CDC only when AQP4 was assembled in orthogonal arrays of particles (OAPs). Conclusion: AQP4 assembly in OAPs is required for CDC by a mechanism involving multivalent C1q binding. Significance: Our results establish a new mechanism of OAP-dependent pathogenesis in NMO and suggest a novel therapeutic strategy.

Crofelemer, a purified proanthocyanidin oligomer extracted from the bark latex of Croton lechleri, is in clinical trials for secretory diarrheas of various etiologies. We investigated the antisecretory mechanism of crofelemer by determining its effect on the major apical membrane transport and signaling processes involved in intestinal fluid transport. Using cell lines and measurement procedures to isolate the effects on individual membrane transport proteins, crofelemer at 50 M had little or no effect on the activity of epithelial Na ϩ or K ϩ channels or on cAMP or calcium signaling. Crofelemer inhibited the cystic fibrosis transmembrane regulator (CFTR) Cl Ϫ channel with maximum inhibition of ϳ60% and an IC 50 ϳ7 M. Crofelemer action at an extracellular site on CFTR produced voltage-independent block with stabilization of the channel closed state. Crofelemer did not affect the potency of glycine hydrazide or thiazolidinone CFTR inhibitors. Crofelemer action resisted washout, with Ͻ50% reversal of CFTR inhibition after 4 h. Crofelemer was also found to strongly inhibit the intestinal calcium-activated Cl Ϫ channel TMEM16A by a voltage-independent inhibition mechanism with maximum inhibition Ͼ90% and IC 50 ϳ6.5 M. The dual inhibitory action of crofelemer on two structurally unrelated prosecretory intestinal Cl Ϫ channels may account for its intestinal antisecretory activity.

Aquaporin-4 (AQP4) is a water transporting protein expressed at the plasma membrane of astrocytes throughout the central nervous system (CNS). Analysis of AQP4 knockout mice has suggested its broad involvement in brain water balance, neuroexcitation, glial scarring, neuroinflammation, and even neurodegenerative and neuropsychiatric disorders. Broad clinical utility of AQP4 modulators has been speculated. Area covered: This review covers the biology of AQP4, evidence for its roles in normal CNS function and neurological disorders, and progress in AQP4 drug discovery. Expert opinion: Critical examination of available data reduces the lengthy potential applications list to AQP4 inhibitors for early therapy of ischemic stroke and perhaps for reduction of glial scarring following CNS injury. Major challenges in identification and clinical development of AQP4 inhibitors include the apparent poor druggability of AQPs, the many homologous AQP isoforms with broad tissue distribution and functions, technical issues with water transport assays, predicted undesired CNS and non-CNS actions, and the need for high blood-brain barrier permeation. To date, despite considerable effort, validated small-molecule AQP4 inhibitors have not been advanced. However, a biologic ('aquaporumab') is in development for neuromyelitis optica, an autoimmune inflammatory demyelinating disease where CNS pathology is initiated by binding of anti-AQP4 autoantibodies to astrocyte AQP4.

We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (Tradtrantip et al., J. Med. Chem. 52, 6447–6455, 2009). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC50 ~ 90 nM), we synthesized 16 PPQ analogs and 11 BPO analogs. The analogs were efficiently synthesized in 5–6 steps and 11–61 % overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC50 ~ 8 nM, and, compared to 8, had >10-fold greater metabolic stability and much greater polarity / aqueous solubility. In an embryonic kidney culture model of PKD 42 prevented cyst growth with IC50 ~ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for anti-secretory therapy of PKD.