The aquaporin-4 inhibitor AER-271 blocks acute cerebral edema and improves early outcome in a pediatric model of asphyxial cardiac arrest.

BACKGROUND: Cerebral edema after cardiac arrest (CA) is associated with increased mortality and unfavorable outcome in children and adults. Aquaporin-4 mediates cerebral water movement and its absence in models of ischemia improves outcome. We investigated early and selective pharmacologic inhibition of aquaporin-4 in a clinically relevant asphyxial CA model in immature rats in a threshold CA insult that produces primarily cytotoxic edema in the absence of blood-brain barrier permeability. METHODS: Postnatal day 16-18 Sprague-Dawley rats were studied in our established 9-min asphyxial CA model. Rats were randomized to aquaporin-4 inhibitor (AER-271) vs vehicle treatment, initiated at return of spontaneous circulation. Cerebral edema (% brain water) was the primary outcome with secondary assessments of the Neurologic Deficit Score (NDS), hippocampal neuronal death, and neuroinflammation. RESULTS: Treatment with AER-271 ameliorated early cerebral edema measured at 3 h after CA vs vehicle treated rats. This treatment also attenuated early NDS. In contrast to rats treated with vehicle after CA, rats treated with AER-271 did not develop significant neuronal death or neuroinflammation as compared to sham. CONCLUSION: Early post-resuscitation aquaporin-4 inhibition blocks the development of early cerebral edema, reduces early neurologic deficit, and blunts neuronal death and neuroinflammation post-CA.

Aquaporin 4 blockade improves survival of murine heart allografts subjected to prolonged cold ischemia.

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia-reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC-mismatched murine cardiac allografts subjected to 8 hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short-time posttransplantation improves the viability of donor graft cells, diminishes donor-reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte-associated antigen 4-Ig in prolonging survival of 8-hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor-reactive T cells. These results identify AQPs as a promising target for diminishing donor-specific alloreactivity and improving the survival of high-risk organ transplants.

Water channel aquaporin 4 is required for T cell receptor mediated lymphocyte activation.

Aquaporins are a family of ubiquitously expressed transmembrane water channels implicated in a broad range of physiological functions. We have previously reported that aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. Using either genetic deletion or small molecule inhibitor, we show that AQP4 supports T cell receptor mediated activation of both mouse and human T cells. Intact AQP4 is required for optimal T cell receptor (TCR)-related signaling events, including nuclear translocation of transcription factors and phosphorylation of proximal TCR signaling molecules. AQP4 deficiency or inhibition impairs actin cytoskeleton rearrangements following TCR crosslinking, causing inferior TCR polarization and a loss of TCR signaling. Our findings reveal a novel function of AQP4 in T lymphocytes and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation.

Aquaporin 4 inhibition alters chemokine receptor expression and T cell trafficking.

Aquaporins (AQPs) are water channels that mediate a variety of biological processes. However, their role in the immune system is poorly understood. We recently reported that AQP4 is expressed by naïve and memory T cells and that AQP4 blockade with a small molecule inhibitor prolongs murine heart allograft survival at least partially through diminishing T cell activation, proliferation and trafficking. The goal of this study was to determine how AQP4 function impacts T cells in the absence of antigen stimulation. AQP4 inhibition transiently reduced the number of circulating CD4+ and CD8+ T cells in naïve non-transplanted mice in the absence of systemic T cell depletion. Adoptive transfer studies demonstrated T cell intrinsic effect of AQP4 inhibition. AQP4 blockade altered T cell gene and protein expression of chemokine receptors S1PR1 and CCR7, and their master regulator KLF-2, and reduced chemotaxis toward S1P and CCL21. Consistent with the in vitro data, in vivo AQP4 inhibition reduced T lymphocyte numbers in the lymph nodes with simultaneous accumulation in the liver. Our findings indicate that blocking AQP4 reversibly alters T lymphocyte trafficking pattern. This information can be explored for the treatment of undesirable immune responses in transplant recipients or in patients with autoimmune diseases.

Functionalized Phenylbenzamides Inhibit Aquaporin-4 Reducing Cerebral Edema and Improving Outcome in Two Models of CNS Injury.

Cerebral edema in ischemic stroke can lead to increased intracranial pressure, reduced cerebral blood flow and neuronal death. Unfortunately, current therapies for cerebral edema are either ineffective or highly invasive. During the development of cytotoxic and subsequent ionic cerebral edema water enters the brain by moving across an intact blood brain barrier and through aquaporin-4 (AQP4) at astrocyte endfeet. Using AQP4-expressing cells, we screened small molecule libraries for inhibitors that reduce AQP4-mediated water permeability. Additional functional assays were used to validate AQP4 inhibition and identified a promising structural series for medicinal chemistry. These efforts improved potency and revealed a compound we designated AER-270, N-[3,5-bis (trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide. AER-270 and a prodrug with enhanced solubility, AER-271 2-{[3,5-Bis(trifluoromethyl) phenyl]carbamoyl}-4-chlorophenyl dihydrogen phosphate, improved neurological outcome and reduced swelling in two models of CNS injury complicated by cerebral edema: water intoxication and ischemic stroke modeled by middle cerebral artery occlusion.