Inhibiting Plasma Kallikrein for Hereditary Angioedema Prophylaxis.

BACKGROUND: Hereditary angioedema with C1 inhibitor deficiency is characterized by recurrent, unpredictable swelling episodes caused by uncontrolled plasma kallikrein generation and excessive bradykinin release resulting from cleavage of high-molecular-weight kininogen. Lanadelumab (DX-2930) is a new kallikrein inhibitor with the potential for prophylactic treatment of hereditary angioedema with C1 inhibitor deficiency. METHODS: We conducted a phase 1b, multicenter, double-blind, placebo-controlled, multiple-ascending-dose trial. Patients with hereditary angioedema with C1 inhibitor deficiency were randomly assigned in a 2:1 ratio to receive either lanadelumab (24 patients) or placebo (13 patients), in two administrations 14 days apart. Patients assigned to lanadelumab were enrolled in sequential dose groups: total dose of 30 mg (4 patients), 100 mg (4 patients), 300 mg (5 patients), or 400 mg (11 patients). The pharmacodynamic profile of lanadelumab was assessed by measurement of plasma levels of cleaved high-molecular-weight kininogen, and efficacy was assessed by the rate of attacks of angioedema during a prespecified period (day 8 to day 50) in the 300-mg and 400-mg groups as compared with the placebo group. RESULTS: No discontinuations occurred because of adverse events, serious adverse events, or deaths in patients who received lanadelumab. The most common adverse events that emerged during treatment were attacks of angioedema, injection-site pain, and headache. Dose-proportional increases in serum concentrations of lanadelumab were observed; the mean elimination half-life was approximately 2 weeks. Lanadelumab at a dose of 300 mg or 400 mg reduced cleavage of high-molecular-weight kininogen in plasma from patients with hereditary angioedema with C1 inhibitor deficiency to levels approaching that from patients without the disorder. From day 8 to day 50, the 300-mg and 400-mg groups had 100% and 88% fewer attacks, respectively, than the placebo group. All patients in the 300-mg group and 82% (9 of 11) in the 400-mg group were attack-free, as compared with 27% (3 of 11) in the placebo group. CONCLUSIONS: In this small trial, administration of lanadelumab to patients with hereditary angioedema with C1 inhibitor deficiency reduced cleavage of high-molecular-weight kininogen and attacks of angioedema. (Funded by Dyax; ClinicalTrials.gov number, NCT02093923 .).

A phase 1 study investigating DX-2930 in healthy subjects.

BACKGROUND: DX-2930 is a human monoclonal antibody inhibitor of plasma kallikrein under investigation for long-term prophylaxis of hereditary angioedema. OBJECTIVE: To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of DX-2930 in healthy subjects. METHODS: A single-center, double-blinded study was performed in 32 healthy subjects randomized 3:1 to receive a single subcutaneous administration of DX-2930 or placebo within 1 of 4 sequential, ascending dose cohorts (n = 8 each): 0.1, 0.3, 1.0, or 3.0 mg/kg. RESULTS: No dose-limiting toxicity was observed. Headache was the most commonly reported treatment emergent adverse event (AE), occurring at a rate of 25% in the DX-2930- and placebo-treated groups; none were severe and all resolved. There were no serious AEs, discontinuations owing to an AE, or deaths. Two subjects had a severe AE reported as related to treatment by the blinded investigator; the 2 AEs were asymptomatic creatinine phosphokinase elevations of 902 U/L in 1 subject receiving 0.1 mg/kg DX-2930 and 1,967 U/L in 1 subject receiving placebo. For the 0.1-, 0.3-, 1.0-, and 3.0-mg/kg dose groups, respectively, mean maximum plasma concentrations were 0.6, 1.4, 5.6, and 14.5 µg/mL and mean elimination half-lives were 20.6, 16.8, 17.6, and 21.2 days. Exploratory biomarker assays, involving ex vivo activation of the kallikrein pathway, showed dose- and time-dependent inhibition of plasma kallikrein, with evidence of sustained bioactivity consistent with the pharmacokinetics profile. CONCLUSION: A single administration of DX-2930 in healthy subjects up to doses of 3.0 mg/kg was well tolerated without dose-limiting toxicity. Pharmacokinetic and pharmacodynamic data provide evidence for a long-acting biological effect relevant to long-term prophylaxis for hereditary angioedema with C1-inhibitor deficiency. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01923207.

Increased cytokine and chemokine gene expression in the CNS of mice during heat stroke recovery.

Heat stroke (HS) is characterized by a systemic inflammatory response syndrome (SIRS) consisting of profound core temperature (Tc) changes in mice. Encephalopathy is common at HS collapse, but inflammatory changes occurring in the brain during the SIRS remain unidentified. We determined the association between inflammatory gene expression changes in the brain with Tc disturbances during HS recovery in mice. Gene expression changes of heat shock protein (HSP)72, heme oxygenase (hmox1), cytokines (IL-1ß, IL-6, TNF-α), cyclooxygenase enzymes (COX-1, COX-2), chemokines (MCP-1, MIP-1α, MIP-1ß, CX3CR1), and glia activation markers (CD14, aif1, vimentin) were examined in the hypothalamus (HY) and hippocampus (HC) of control (Tc ∼ 36.0°C) and HS mice at Tc,Max (42.7°C), hypothermia depth (HD; 29.3 ± 0.4°C), and fever (37.8 ± 0.3°C). HSP72 (HY

Activation of the infralimbic cortex in a fear context enhances extinction learning.

Activation of the infralimbic region (IL) of the medial prefrontal cortex (mPFC) reduces conditioned fear in a variety of situations, and the IL is thought to play an important role in the extinction of conditioned fear. Here we report a series of experiments using contextual fear conditioning in which the IL is activated with the GABAa antagonist picrotoxin (Ptx) during a single extinction session in the fear context. We investigate the impact of this manipulation on subsequent extinction sessions in which Ptx is no longer present. First, we demonstrate that a single treatment with intra-IL Ptx administered in a conditioned fear context greatly accelerates the rate of extinction on the following days. Importantly, IL-Ptx also enhances extinction to a different fear context than the one in which IL-Ptx was administered. Thus, IL-Ptx primes extinction learning regardless of the fear context in which the IL was initially activated. Second, activation of the IL must occur in conjunction with a fear context in order to enhance extinction; the extinction enhancing effect is not observable if IL-Ptx is administered in a neutral context. Finally, this extinction enhancing effect is specific to the IL for it does not occur if Ptx is injected into the prelimbic region (PL) of the mPFC. The results indicate a novel persisting control of fear induced by activation of the IL and suggest that IL activation induces changes in extinction-related circuitry that prime extinction learning.

IL-1RA blocks E. coli-induced suppression of Arc and long-term memory in aged F344xBN F1 rats.

In normal aging, a peripheral immune challenge induces a sensitized and protracted neuroinflammatory response in parallel with long-term memory (LTM) impairments. Pro-inflammatory mediators of neuroinflammation impair LTM, synaptic plasticity and LTP. The immediate early gene Arc is considered a critical protein regulating LTM and synaptic plasticity. The present investigation examined whether (1) a peripheral Escherichia coli infection suppresses hippocampal Arc expression, and (2) central pro-inflammatory cytokines (IL-1beta and IL-6) mediate the effects of peripheral E. coli infection on Arc and LTM. In 24 months F344xBN F1 rats, E. coli infection suppressed basal Arc gene expression as well as contextual fear conditioning-induced Arc expression. E. coli treatment failed to alter either basal or conditioning-induced c-Fos expression. At 24h post-infection, intra-cisterna magna (ICM) treatment with the anti-inflammatory cytokine IL-1RA blocked the E. coli-induced suppression of hippocampal Arc and increases in IL-6 protein. At 4-day post-infection, IL-1RA blocked the E. coli-induced LTM impairments and increases in IL-6 protein. The present results suggest that central pro-inflammatory cytokines play a salient role in the suppression of Arc and impairments of LTM by a peripheral immune challenge in older animals.

Hippocampal and extrahippocampal systems compete for control of contextual fear: role of ventral subiculum and amygdala.

Two neural systems, a hippocampal system and an extrahippocampal system compete for control over contextual fear, and the hippocampal system normally dominates. Our experiments reveal that output provided by the ventral subiculum is critical for the hippocampal system to win this competition. Bilateral electrolytic lesions of the ventral subiculum after conditioning, but not before conditioning, impaired contextual fear conditioning. Reversibly inactivating this region by bilateral injections of muscimol produced the same results-no impairment when the injection occurred prior to conditioning but a significant impairment when this region was inactivated after conditioning. Thus, the extrahippocampal system can support contextual fear conditioning if the ventral subiculum is disabled before conditioning but not if it is disabled after conditioning. Our experiments also reveal that the basolateral region of the amygdala (BLA) is where the two systems compete for associative control of the fear system. To test this hypothesis we reasoned that the extrahippocampal system would also acquire associative control over the fear system, even if the hippocampal system were functional, if the basal level of plasticity potential in the BLA could be increased. We did this by injecting the D1 dopamine agonist, SKF82958, into the BLA just prior to conditioning. This treatment resulted in a significant increase in freezing when the ventral subiculum was disabled prior to the test. These results are discussed in relationship to the idea that D1 agonists increase plasticity potential by increasing the pool of available extrasynaptic GluR1 receptors in the population of neurons supporting acquired fear.

Context preexposure prevents forgetting of a contextual fear memory: implication for regional changes in brain activation patterns associated with recent and remote memory tests.

Contextual fear conditioning was maintained over a 15-day retention interval suggesting no forgetting of the conditioning experience. However, a more subtle generalization test revealed that, as the retention interval increased, rats showed enhanced generalized fear to an altered context. Preexposure to the training context prior to conditioning, however, prevented this enhanced generalized fear from developing. These results support the hypothesis that the memory representation of the context degrades as the memory ages and is responsible for enhanced generalization. The implications of these results for systems consolidation versus forgetting interpretations of regional changes in neural activation patterns that occur as memories age are discussed.

Characterization of the temporo-spatial effects of chronic bilateral intrahippocampal cannulae on interleukin-1beta.

The implantation of a foreign object in the brain produces an acute neuroinflammatory state in which glia (astrocytes and microglia) may remain chronically activated in response to the inert foreign object. Activated glia can exhibit a sensitized pro-inflammatory response to immunogenic stimuli. This may be relevant to intracranial cannula implantation, which is commonly used to administer substances directly into the brain. If intracranial cannulation activates glia, a subsequent neuroinflammatory stimulus might induce a potentiated pro-inflammatory response, thereby introducing a potential experimental confound. We tested the temporal and spatial responses of interleukin-1beta (IL-1beta) to an acute immune challenge produced by lipopolysaccharide (LPS) in animals with chronic bilateral intrahippocampal cannulae implants (stainless steel). Cannulation increased the gene expression of the microglia activation antigens MHC II and CD11b, but not the astrocyte antigen GFAP. Moreover, this activation was temporally and spatially dependent. In addition, IL-1beta mRNA, but not IL-1beta protein, was significantly elevated in cannulated animals. Administration of LPS, however, significantly potentiated the brain IL-1beta response in cannulated animals, but not in stab wounded or naïve animals. This IL-1beta response was also temporo-spatially dependent. Thus, the pro-inflammatory sequelae of intracranial cannulation should be considered when designing studies of neuroinflammatory processes.

Neonatal infection-induced memory impairment after lipopolysaccharide in adulthood is prevented via caspase-1 inhibition.

We have reported that neonatal infection leads to memory impairment after an immune challenge in adulthood. Here we explored whether events occurring as a result of early infection alter the response to a subsequent immune challenge in adult rats, which may then impair memory. In experiment 1, peripheral infection with Escherichia coli on postnatal day 4 increased cytokines and corticosterone in the periphery, and cytokine and microglial cell marker gene expression in the hippocampus of neonate pups. Next, rats treated neonatally with E. coli or PBS were injected in adulthood with lipopolysaccharide (LPS) or saline and killed 1-24 h later. Microglial cell marker mRNA was elevated in hippocampus in saline controls infected as neonates. Furthermore, LPS induced a greater increase in glial cell marker mRNA in hippocampus of neonatally infected rats, and this increase remained elevated at 24 h versus controls. After LPS, neonatally infected rats exhibited faster increases in interleukin-1beta (IL-1beta) within the hippocampus and cortex and a prolonged response within the cortex. There were no group differences in peripheral cytokines or corticosterone. In experiment 2, rats treated neonatally with E. coli or PBS received as adults either saline or a centrally administered caspase-1 inhibitor, which specifically prevents the synthesis of IL-1beta, 1 h before a learning event and subsequent LPS challenge. Caspase-1 inhibition completely prevented LPS-induced memory impairment in neonatally infected rats. These data implicate IL-1beta in the set of immune/inflammatory events that occur in the brain as a result of neonatal infection, which likely contribute to cognitive alterations in adulthood.

Tracking the Time-Dependent Role of the Hippocampus in Memory Recall Using DREADDs.

The hippocampus is critical for the storage of new autobiographical experiences as memories. Following an initial encoding stage in the hippocampus, memories undergo a process of systems-level consolidation, which leads to greater stability through time and an increased reliance on neocortical areas for retrieval. The extent to which the retrieval of these consolidated memories still requires the hippocampus is unclear, as both spared and severely degraded remote memory recall have been reported following post-training hippocampal lesions. One difficulty in definitively addressing the role of the hippocampus in remote memory retrieval is the precision with which the entire volume of the hippocampal region can be inactivated. To address this issue, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), a chemical-genetic tool capable of highly specific neuronal manipulation over large volumes of brain tissue. We find that remote (>7 weeks after acquisition), but not recent (1-2 days after acquisition) contextual fear memories can be recalled after injection of the DREADD agonist (CNO) in animals expressing the inhibitory DREADD in the entire hippocampus. Our data demonstrate a time-dependent role of the hippocampus in memory retrieval, supporting the standard model of systems consolidation.

Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats.

Mirror-image allodynia is a mysterious phenomenon that occurs in association with many clinical pain syndromes. Allodynia refers to pain in response to light touch/pressure stimuli, which normally are perceived as innocuous. Mirror-image allodynia arises from the healthy body region contralateral to the actual site of trauma/inflammation. Virtually nothing is known about the mechanisms underlying such pain. A recently developed animal model of inflammatory neuropathy reliably produces mirror-image allodynia, thus allowing this pain phenomenon to be analyzed. In this sciatic inflammatory neuropathy (SIN) model, decreased response threshold to tactile stimuli (mechanical allodynia) develops in rats after microinjection of immune activators around one healthy sciatic nerve at mid-thigh level. Low level immune activation produces unilateral allodynia ipsilateral to the site of sciatic inflammation; more intense immune activation produces bilateral (ipsilateral + mirror image) allodynia. The present studies demonstrate that both ipsilateral and mirror-image SIN-induced allodynias are (1) reversed by intrathecal (peri-spinal) delivery of fluorocitrate, a glial metabolic inhibitor; (2) prevented and reversed by intrathecal CNI-1493, an inhibitor of p38 mitogen-activated kinases implicated in proinflammatory cytokine production and signaling; and (3) prevented or reversed by intrathecal proinflammatory cytokine antagonists specific for interleukin-1, tumor necrosis factor, or interleukin-6. Reversal of ipsilateral and mirror-image allodynias was rapid and complete even when SIN was maintained constantly for 2 weeks before proinflammatory cytokine antagonist administration. These results provide the first evidence that ipsilateral and mirror-image inflammatory neuropathy pain are created both acutely and chronically through glial and proinflammatory cytokine actions.

Characterization of anaphylaxis after ecallantide treatment of hereditary angioedema attacks.

BACKGROUND: Ecallantide is a human plasma kallikrein inhibitor indicated for treatment of acute attacks of hereditary angioedema for patients 12 years of age and older. Ecallantide is produced in Pichia pastoris yeast cells by recombinant DNA technology. Use of ecallantide has been associated with a risk of hypersensitivity reactions, including anaphylaxis. OBJECTIVE: The objective of this detailed retrospective data review was to characterize anaphylaxis cases within the ecallantide clinical trials database. METHODS: Potential cases of hypersensitivity reactions in the ecallantide clinical development program were identified by examining reported adverse events. The National Institute of Allergy and Infectious Disease criteria were used to identify those events that were consistent with anaphylaxis; these cases were then reviewed in detail. Results from investigational antibody testing also were examined. RESULTS: Among patients who received subcutaneous ecallantide (n = 230 patients; 1045 doses of 30 mg ecallantide), 8 patients (3.5%) had reactions that met the National Institute of Allergy and Infectious Disease criteria for anaphylaxis; none occurred on first exposure to the drug. All 8 reactions had symptom onset within 1 hour of exposure and cutaneous manifestations commonly observed in type I hypersensitivity reactions. All the reactions responded to standard management of type I hypersensitivity reactions and resolved without fatal outcomes. IgE antibody testing to ecallantide or P pastoris was not consistently positive in patients who experienced apparent type I hypersensitivity reactions. CONCLUSION: Anaphylaxis episodes after subcutaneous ecallantide exposure have clinical features suggestive of type I hypersensitivity reactions. However, anti-ecallantide or anti-P pastoris IgE antibody status was not found to be reliably associated with anaphylaxis.

Fully human monoclonal antibody inhibitors of the neonatal fc receptor reduce circulating IgG in non-human primates.

The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.

HIV-1 gp120 stimulates proinflammatory cytokine-mediated pain facilitation via activation of nitric oxide synthase-I (nNOS).

It has become clear that spinal cord glia (microglia and astrocytes) importantly contribute to the creation of exaggerated pain responses. One model used to study this is peri-spinal (intrathecal, i.t.) administration of gp120, an envelope protein of HIV-1 known to activate glia. Previous studies demonstrated that i.t. gp120 produces pain facilitation via the release of glial proinflammatory cytokines. The present series of studies tested whether spinal nitric oxide (NO) contributes to i.t. gp120-induced mechanical allodynia and, if so, what effect NO has on spinal proinflammatory cytokines. gp120 stimulation of acutely isolated lumbar dorsal spinal cords released NO as well as proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta (IL1), interleukin-6 (IL6)), thus identifying NO as a candidate mediator of gp120-induced behavioral effects. Behaviorally, identical effects were observed when gp120-induced mechanical allodynia was challenged by i.t. pre-treatment with either a broad-spectrum nitric oxide synthase (NOS) inhibitor (L-NAME) or 7-NINA, a selective inhibitor of NOS type-I (nNOS). Both abolished gp120-induced mechanical allodynia. While the literature pre-dominantly documents that proinflammatory cytokines stimulate the production of NO rather than the reverse, here we show that gp120-induced NO increases proinflammatory cytokine mRNA levels (RT-PCR) and both protein expression and protein release (serial ELISA). Furthermore, gp120 increases mRNA for IL1 converting enzyme and matrix metalloproteinase-9, enzymes responsible for activation and release of proinflammatory cytokines.

Context memories and reactivation: constraints on the reconsolidation hypothesis.

Recent evidence suggests that the maintenance of a reactivated contextual-fear memory requires a protein-synthesis-dependent reconsolidation process in the hippocampus (K. Nader, 2003). In contrast, the authors report a systematic set of experiments that failed to find evidence that the rat's reactivated memory for context becomes labile and requires a new protein to restabilize. Although injecting the protein-synthesis inhibitor into the dorsal hippocampus or intracerebroventricularly following the reactivation of the context memory had no effect, these same treatments did impair the initial consolidation of the context memory and the consolidation of a contextual-fear memory. These results suggest that there may be important constraints determining when a reactivated memory requires reconsolidation. The authors offer 2 hypotheses about the nature of these constraints.

mRNA up-regulation of MHC II and pivotal pro-inflammatory genes in normal brain aging.

In normal brain aging, CNS resident macrophages exhibit increased expression of major histocompatibility complex (MHC) II expression. However, the transcriptional basis for this observation has not been clarified nor have age-related alterations in pivotal pro-inflammatory genes been characterized. Age-related mRNA alterations in MHC II, MHC II accessory molecules and several pro-inflammatory mediators were measured in older (24 months) and younger (3 months) male F344xBN F1 rats. Real time RT-PCR was utilized to measure steady state mRNA levels in hippocampus. Older as compared to younger animals exhibited increased mRNA levels of MHC II, CD86, CIITA and IFN-gamma. Furthermore, IL-10 and CD200 mRNA, molecules that down-regulate macrophage activation, was decreased in older animals. The present results indicate that normal brain aging is characterized by a shift towards a pro-inflammatory microenvironment in the CNS.

Peripheral infection and aging interact to impair hippocampal memory consolidation.

We report that a peripheral injection of Escherichia coli produces both anterograde and retrograde amnesia in 24 month old, but not 3 month old rats for memories that depend on the hippocampus, that is, memory of context, contextual fear, and place learning. The anterograde effect was restricted to measures of long-term memory. Short-term memory was not affected, nor did E. coli produce amnesia for auditory-cue fear conditioning. There were no age related effects on memory in vehicle-treated rats. In addition to these age-related cognitive effects of E.coli, we report that it produced a marked increased in IL-1beta levels in the hippocampus, but not in parietal cortex or serum. These findings support the hypothesis that age is a vulnerability factor that increases the likelihood that an immune challenge will produce a cognitive impairment. It is possible that this cognitive vulnerability is mediated by age-related changes in the glial environment that results in an exaggerated brain pro-inflammatory response to infection.

Further characterization of high mobility group box 1 (HMGB1) as a proinflammatory cytokine: central nervous system effects.

High mobility group box 1 (HMGB1), an abundant, highly conserved cellular protein, is widely known as a nuclear DNA-binding protein. HMGB1 has been recently implicated as a proinflammatory cytokine because of its role as a late mediator of endotoxin lethality and ability to stimulate release of proinflammatory cytokines from monocytes. Production of central cytokines is a critical step in the pathway by which endotoxin and peripheral proinflammatory cytokines, including interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF), produce sickness behaviors and fever. Intracerebroventricular (ICV) administration of HMGB1 has been shown to increase TNF expression in mouse brain and induce aphagia and taste aversion. Here we show that ICV injections of HMGB1 induce fever and hypothalamic IL-1 in rats. Furthermore, we show that intrathecal administration of HMGB1 produces mechanical allodynia (lowering of the response threshold to calibrated stimuli). Finally, while endotoxin (lipopolysaccharide, LPS) administration elevates IL-1 and TNF mRNA in various brain regions, HMGB1 mRNA is unchanged. It remains possible that HMGB1 protein is released in brain in response to LPS. Nonetheless, these data suggest that HMGB1 may play a role as an endogenous pyrogen and support the concept that HMGB1 has proinflammatory characteristics within the central nervous system.