Conjunctivitis in dupilumab clinical trials.

BACKGROUND: Dupilumab blocks the shared receptor component for interleukin (IL)-4 and IL-13. It is approved in the U.S.A. for patients aged ≥ 12 years with moderate-to-severe atopic dermatitis (AD) uncontrolled by topical prescription medicines or who cannot use topical medicines, for patients in Japan whose AD is uncontrolled with existing therapies, for patients with moderate-to-severe AD in Europe who are candidates for systemic therapy and for patients aged ≥ 12 years for maintenance treatment of moderate-to-severe asthma uncontrolled with their current medicines. AD trials have reported increased incidence of conjunctivitis for dupilumab vs. placebo. OBJECTIVES: To characterize further the occurrence and risk factors of conjunctivitis in dupilumab clinical trials. METHODS: We evaluated randomized placebo-controlled trials of dupilumab in AD (n = 2629), asthma (n = 2876), chronic rhinosinusitis with nasal polyps (CRSwNP) (n = 60) and eosinophilic oesophagitis (EoE) (n = 47). RESULTS: In most AD trials, dupilumab-treated patients had higher conjunctivitis incidence than placebo controls. Higher baseline AD severity and previous history of conjunctivitis were associated with increased conjunctivitis incidence. Conjunctivitis was mostly mild to moderate. Most cases recovered or resolved during the treatment period; two patients permanently discontinued dupilumab due to conjunctivitis or keratitis. Common treatments included ophthalmic corticosteroids, antibiotics, and antihistamines or mast cell stabilizers. Most cases were diagnosed by the investigators. In asthma and CRSwNP trials, the incidence of conjunctivitis was lower for both dupilumab and placebo than in AD trials; dupilumab did not increase the incidence compared with placebo. In the EoE trial, no patients had conjunctivitis. CONCLUSIONS: Conjunctivitis was more frequent with dupilumab treatment in most AD trials. In dupilumab trials in other type 2 diseases, incidence of conjunctivitis was overall very low, and was similar for dupilumab and placebo. In AD, the incidence of conjunctivitis was associated with AD severity and prior history of conjunctivitis. The aetiology and treatment of conjunctivitis in dupilumab-treated patients require further study. What's already known about this topic? Ocular disorders, including allergic conjunctivitis, are common in patients with atopic dermatitis (AD). In most dupilumab AD trials, dupilumab-treated patients had higher conjunctivitis incidence than those receiving placebo. Most cases were mild to moderate and recovered or were recovering during study treatment; study treatment discontinuation due to conjunctivitis was rare. Conjunctivitis incidence was very low and similar for dupilumab and placebo in clinical trials in asthma, chronic rhinosinusitis with nasal polyps and eosinophilic oesophagitis. What does this study add? This analysis confirms and extends the results of the individual clinical trials. Baseline disease-related factors, including AD severity, prior conjunctivitis history and certain biomarkers (thymus and activation-regulated chemokine, IgE, eosinophils), were associated with increased incidence of conjunctivitis. Patients who responded well to dupilumab had reduced incidence of conjunctivitis. Further study is needed to elucidate the aetiology and treatment of conjunctivitis in dupilumab-treated patients with AD.

Dupilumab provides important clinical benefits to patients with atopic dermatitis who do not achieve clear or almost clear skin according to the Investigator's Global Assessment: a pooled analysis of data from two phase III trials.

BACKGROUND: In the U.S.A., an Investigator's Global Assessment (IGA) score of ≤ 1 (clear or almost clear skin) has been the standard measure in regulatory outcomes for registration clinical trials in atopic dermatitis (AD), including those supporting the recent approval of dupilumab. OBJECTIVES: To evaluate the treatment effect of dupilumab in patients with IGA > 1 at the end of treatment, using other validated outcome measures for AD signs, symptoms and quality of life. METHODS: LIBERTY AD SOLO 1 and 2 were two 16-week, randomized, double-blind trials enrolling adult patients with moderate-to-severe AD (IGA ≥ 3) inadequately controlled with topical treatment. We performed a post hoc analysis in patients receiving dupilumab 300 mg every 2 weeks (q2w) or placebo. Outcome measures in patients with IGA > 1 included Eczema Area and Severity Index (EASI), pruritus numerical rating scale (NRS), affected body surface area (BSA), Patient-Oriented Eczema Measure (POEM) and Dermatology Life Quality Index (DLQI). The trials were registered at ClinicalTrials.gov: NCT02277743 and NCT02277769. RESULTS: At week 16, 278 of 449 dupilumab q2w-treated patients (median age 36·0 years) and 396 of 443 placebo-treated patients had IGA > 1. Among patients with IGA > 1 at week 16, dupilumab significantly improved several outcome measures compared with placebo: EASI (-48·9% vs. -11·3%, P < 0·001), pruritus NRS (-35·2% vs. -9·1%, P < 0·001), affected BSA (-23·1% vs. -4·5%, P < 0·001), POEM score ≥ 4-point improvement (57·4% vs. 21·0%, P < 0·001) and DLQI score ≥ 4-point improvement (59·3% vs. 24·4%, P < 0·001). CONCLUSIONS: In patients with IGA > 1 at week 16, dupilumab induced statistically significant benefits in multiple validated outcome measures compared with placebo. The IGA ≤ 1 end point significantly underestimates clinically relevant dupilumab treatment effects.

Cultured rat microglia express C1q and receptor for C1q: implications for amyloid effects on microglia.

Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process-bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds beta 1-42, the major constituent of senile plaques, and since beta 1-42 is toxic to microglia in vitro, we asked if preincubation of beta 1-42 with C1q alters either metabolic indices of amyloid-induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that pre-incubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to beta 1-42 on beta 1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-gamma. While these data exclude a role for the C1q receptor in beta 1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-gamma would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques.

Differential regulation by beta-amyloid peptides of intracellular free Ca2+ concentration in cultured rat microglia.

We have previously shown that exposure to beta-amyloid peptides alters microglial activity and viability. It is thought that beta-amyloid peptides induce toxicity in neuronal cultures by destabilizing Ca2+ homeostasis. To investigate the effects of beta-amyloid peptides on intracellular free Ca2+ concentration ([Ca2+]i) in cultured microglia, we used Fura-2 imaging. Exposure to 25 microM beta-amyloid-(25-35) induced increases in 2+]i within 1 h. In contrast, exposure to 25 microM beta-amyloid-(1-42), the full-length homolog to the beta-amyloid protein deposited in plaques, does not, over the same time period. However, the average [Ca2+]i of microglia is increased by a 6 h exposure to beta-amyloid-(1-42). Thus, beta-amyloid-(25-35) can alter [Ca2+]i in microglia on a different time scale than beta-amyloid-(1-42), indicating a specificity in the response of these cells as compared to neurons.

beta-Amyloid peptides induce degeneration of cultured rat microglia.

Microglia are often associated with senile plaques, a primary pathological hallmark of Alzheimer's disease (AD) that consists largely of insoluble deposits of beta-amyloid (A beta) protein. Synthetic A beta peptides have been shown to induce neurite dystrophy and neuronal death in vitro when the peptides are assembled into aggregates. We now report that assembled A beta peptides induce morphological evidence of degeneration in process-bearing microglia in vitro, as well as metabolic dysfunction in microglial cultures, but a non-assembling scrambled sequence A beta peptide does not.

Carbachol increases intracellular free calcium in cultured rat microglia.

Microglia are resident macrophages in the CNS and have been shown to exhibit immune system responses common to other macrophages, including phagocytosis, secretion of superoxide anions, and secretion of regulatory and trophic factors such as interleukin-1. Phagocytosis and oxidative burst by macrophages are often reported to be preceded by an increase in cytosolic free calcium. In addition, a variety of compounds, including neuroactive peptides, have been shown to elicit such calcium responses in various macrophage preparations. The results presented demonstrate that cultured rat microglia respond to exposure to carbachol with an increase in intracellular free calcium which is atropine-sensitive and the result of the release of calcium from intracellular stores. Norepinephrine also induced increases in free calcium, whereas the metabotropic glutamate agonist 1S,3R-ACPD, serotonin, adenosine and ATP did not. These results suggest that microglia can respond to select neurotransmitters, and that there may exist a signaling loop between neurons and microglia. Furthermore, since cholinergic fibers have been shown to infiltrate neuritic plaques in Alzheimer's disease (AD) and microglia have been reported to be activated in plaques, these results suggest that interactions between select neurotransmitters and microglia may play a key role in neurodegenerative diseases.

Voltage-gated currents expressed by rat microglia in culture.

Using the whole-cell patch-clamp technique, at least three types of voltage-gated currents expressed by cultured rat microglia were identified: an inward rectifier K+ current, a delayed rectifier K+ current (IK), and a Na+ current activated by depolarization. The inward rectifier conductance was activated by hyperpolarization to potentials more negative than -80 mV, depended on the external K+ concentration, and declined over time during whole cell recording, as the cell was internally dialyzed. The delayed rectifier current was activated by depolarization to potentials more positive than -40 mV and the rates of activation and deactivation showed a voltage-dependence similar to such currents seen in other preparations. An inward current possibly carried by Na+ was seen in a small percentage of cells. Recordings had been made from two morphological cell types, namely process-bearing ("ramified") and non-process-bearing ("ameboid"). Each of these currents was present in microglia of both morphological types. However, microglial morphology, which is thought to represent different states of activation, was significantly related to the types of combinations of currents expressed in a given cell.

Effects of tetrahydroaminoacridine (THA) on functional recovery after sequential lesion of the entorhinal cortex.

Unilateral lesions of rat entorhinal cortex produce a transitory performance deficit on spatial learning tasks, such as reinforced alternation in a T-maze. Tetrahydroaminoacridine (THA), a cholinesterase inhibitor, was administered to determine its effects on behavioral recovery using a reinforced alternation task in a T-maze. Rate of recovery after unilateral entorhinal lesion was not affected by a low dose of THA (0.05 mg/kg), while a higher dose (5.0 mg/kg) impaired recovery. Behavioral recovery was subsequently evaluated in the same rats following lesions to the contralateral entorhinal cortex. Serial bilateral lesions of the entorhinal cortex are known to produce a prolonged performance deficit on the alternation task. The 0.05 mg/kg THA group exhibited an intermediate rate of recovery, between the undamaged control group and bilateral lesion-saline injected groups. The group receiving 5.0 mg/kg of THA after bilateral lesion did not differ from the bilateral lesion-saline group. The failure of THA to significantly improve functional recovery in rats with lesions of the entorhinal cortex indicates that the compound may have limited applicability in treating human neurodegenerative disorders such as Alzheimer's disease.

Hypothalamic effects on medullary reticular activation of deep back muscle EMG.

Medullary reticular stimulation can activate deep back muscle EMG in urethane-anesthetized female rats. Midbrain central gray stimulation can facilitate brainstem reticular control over deep back muscles. Since these deep back muscles lateral longissimus (LL) and medial longissimus (ML) execute the vertebral dorsiflexion of lordosis behavior, and since the motor control hierarchy sketched above parallels lordosis behavior circuitry, we tested the hypothesis that medial hypothalamic lesions (which, in behavioral experiments, decrease lordosis) can also reduce medullary reticular activation of deep back muscle EMG. Urethane-anesthetized rats were tested systematically for amplitude of lateral longissimus (LL) and medial longissimus (ML) EMG responses to electrical stimulus trains applied to the nucleus gigantocellularis (NGC) of the medullary reticular formation, before and after electrolytic lesions of the ventromedial hypothalamus (n = 18) or control sites (n = 30). Bilateral ventromedial hypothalamic lesions were able to greatly reduce EMG responses in LL and ML, often with a time course similar to previous lordosis behavioral results. Surprisingly, lesions at the anterior ventromedial nucleus pole were particularly effective, and may reflect importance of intraventromedial local neurons. Although, on the average, various control lesions were less effective, the ventromedial hypothalamic effect was not unique. For example, it was possible to see an EMG decrease following lesions of the dorsomedial thalamus. Nevertheless, EMG loss was not well correlated with changes in the cortical EEG, and thus does not appear to be a simple consequence of changes in "arousal." In conclusion, it appears that ventromedial hypothalamic neurons can affect medullary reticular control of back muscle EMG, but must share this role with other forebrain elements.

Steroid hormone effects on picrotoxin-induced seizures in female and male rats.

Picrotoxin (1 mg/kg, i.p.), evoked a single generalized seizure in 75% of ovariectomized rats. Pretreatment of matched pairs with silastic implants containing 100% estradiol had an anticonvulsant effect; it protected all rats against such seizures. Implants containing 10% estradiol in cholesterol were less effective in protecting against picrotoxin-induced seizures. With 2 mg/kg picrotoxin, 85% of the seizure-affected ovariectomized controls had multiple seizures. The incidence of seizures and the ratio of single to multiple seizures induced by the higher dose of picrotoxin were unaffected by estradiol silastic implants, intraperitoneal injections of progesterone (0.5 mg, 4-5 h before convulsant) or the combination of both hormones. At the 2 mg/kg dose, 8/8 intact males had no seizures while all paired ovariectomized females had seizures. By contrast, the incidence of seizures in pairs of gonadectomized males and females did not differ. Testosterone treatment improved the ratio of single to multiple seizures in males but not in females. Males had statistically fewer multiple seizures than did females after testosterone treatment. The distribution of latencies to a single seizure is statistically different from the distribution of latencies to the first of multiple seizures irrespective of dose, sex and hormone treatment. This suggests that the population of rats responding with a single seizure at the higher dose of picrotoxin have a higher threshold for acquiring multiple seizures and that testosterone predisposes males but not females to this population.