Assessment of rebound and relapse following ecallantide treatment for acute attacks of hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a rare genetic disease characterized by unpredictable and recurring attacks of angioedema. This study assessed potential attack rebound and relapse following treatment with ecallantide, a plasma kallikrein inhibitor approved for HAE attack treatment. METHODS: Results were integrated from 2 double-blind, placebo-controlled studies of ecallantide treatment for HAE: EDEMA3-DB and EDEMA4. Symptoms were assessed by treatment outcome score (TOS), mean symptom complex severity (MSCS) score, and global response. Patients with improvement at 4 h post-dosing in all three measures followed by any sign of worsening at 24 h were considered to show potential rebound if worsening was beyond baseline or potential relapse if not beyond baseline. Likeliness of rebound or relapse was determined by the number of measures showing worsening and the magnitude of worsening. Patients receiving placebo who met the criteria for rebound/relapse were evaluated for descriptive comparison only. RESULTS: Significantly more ecallantide-treated patients (42 of 70) compared to placebo (26 of 71) showed improvement in three measures at 4 h and were thus eligible for rebound/relapse (P = 0.006). Of the nine ecallantide-treated patients with signs of worsening at 24 h, none were likely rebound, one was assessed as possible rebound, one as likely relapse, and two as possible relapse. No patient with potential rebound/relapse experienced new symptoms after dosing. Medical intervention was required in one ecallantide-treated patient. CONCLUSION: Ecallantide was efficacious for treating acute HAE attacks. Relapse was observed in a small proportion of patients, and there was little evidence of rebound.

Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

Efficacy and safety of pramipexole in restless legs syndrome.

OBJECTIVE: To evaluate the efficacy and safety of pramipexole in patients with moderate to severe restless legs syndrome (RLS) METHODS: The authors conducted a 12-week, double-blind, randomized, placebo-controlled trial of fixed doses of pramipexole (0.25, 0.50, and 0.75 mg/day). Patients (N = 344) were up-titrated to their randomized dose over 3 weeks. The primary efficacy endpoints were patient ratings of symptom severity on the International RLS Study Group Rating Scale (IRLS) and clinician ratings of improvement on the Clinical Global Impressions-Improvement (CGI-I) scale. Secondary efficacy endpoints included visual analogue ratings of sleep and quality of life (QOL) RESULTS: By both primary measures, pramipexole was superior to placebo. For IRLS, the adjusted mean (SE) change from baseline to week 12 was -9.3 (1.0) for placebo, -12.8 (1.0) for 0.25 mg/day, -13.8 (1.0) for 0.50 mg/day, and -14.0 (1.0) for 0.75 mg/day (all p < 0.01). Similarly, pramipexole increased the percentage of patients with a CGI-I rating of "very much improved" or "much improved" at the end of the trial (51.2% for placebo and 74.7%, 67.9%, and 72.9% for pramipexole; all p < 0.05). Pramipexole significantly improved ratings of symptom severity, day and night, and also ratings of sleep satisfaction and QOL. Pramipexole was well tolerated: The most frequent adverse events with higher occurrence in the pramipexole group were nausea (19.0% vs 4.7%) and somnolence (10.1% vs 4.7%) CONCLUSION: As rated by patients and by clinicians, pramipexole was efficacious and safe in reducing the symptoms of restless legs syndrome.

Cerebral-abdominal interganglionic coordinating neurons in Aplysia.

Three cerebral-abdominal interneurons (CAIs), CC2, CC3, and CC7, were identified in the cerebral ganglion C cluster. The cells send their axons to the abdominal ganglion via the pleural-abdominal connective. CC2 and CC3 are bilaterally symmetrical cells, whereas CC7 is a unilateral cell. CC3 is immunopositive for serotonin and may be the same cell (CB-1) previously described as located in the B cluster rather than the C cluster. We suggest that the full designation of CC3, be CC3(CB-1). All three cells respond to feeding-related inputs. Each CAI has a monosynaptic connection to at least one abdominal ganglion neuron involved in the control of various nonsomatic organs. The CAIs also exert widespread polysynaptic actions in the abdominal and head ganglia. The results suggest that the CAIs may act as interneurons that coordinate visceral responses mediated by the abdominal ganglion, with behaviors such as feeding and head withdrawal, that are controlled by neurons located in the head ganglia of the animal.

New millennium bronchodilators for asthma: single-isomer beta agonists.

Racemic beta2 agonists are composed of a 50:50 mixture of R and S isomers. The R isomer exhibits virtually all the bronchodilation, whereas the S isomers are generally considered inert. However, (S)-albuterol was shown to enhance bronchial reactivity to methacholine, eosinophil activation, and histamine-induced influx of fluid, proteins, and neutrophils into the airspaces. Actions such as these may compress the potency and foreshorten the duration of (R)-albuterol. Accordingly, pure (R)-albuterol provides bronchodilation at lower doses than racemate, allowing for fewer beta-adrenergic-mediated side effects. In addition, differential metabolism may allow for the progressive accumulation of (S)-albuterol. This logic is applicable to long-acting beta2 agonists: the therapeutically active (R,R)-formoterol is currently being developed in the United States, and preliminary results suggest rapid improvements in FEV1 with up to 24-hour duration of action. These combined observations with the R isomers of beta2 agonists suggest that potential improvements in therapeutic indices can be achieved with isomerically pure versions of existing racemic drugs.

Evidence that hemolymph glucose in Aplysia californica is regulated but does not affect feeding behavior.

Hemolymph glucose increased following a meal of a commercially available dried seaweed (laver) in Aplysia californica (Aplysia). Glucose injected into the hemocoel did not affect meal size, bite latencies, swallowing rate, or 24-hr food intake. The authors found that injection of a homogenate of nerves containing a putative Aplysia insulin-like substance decreased hemolymph glucose. The nerve homogenate, however, did not affect feeding behavior. Injection of 2-deoxy-D-glucose was found to increase hemolymph glucose, an indication of gluco-privation, but instead of increasing feeding it either had no effect or, at high doses, debilitated animals and interfered with feeding. These studies suggest that glucose may be physiologically regulated in Aplysia, but it does not appear to play a role in the control of feeding behavior.

A multicenter comparison of the ocular efficacy and safety of diclofenac 0.1% solution with that of ketorolac 0.5% solution in patients with acute seasonal allergic conjunctivitis.

Only one of several available ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) is currently FDA approved for use in acute seasonal allergic conjunctivitis (SAC). Sixty patients with SAC and moderate itching and bulbar conjunctival injection were enrolled in a multicenter, randomized, double-masked, parallel-group trial comparing diclofenac sodium (DS) with ketorolac tromethamine (KT). Patients instilled 1 drop four times daily while awake for 14 days. Ocular signs and symptoms were evaluated at one and two weeks. The primary efficacy variables were itching and bulbar conjunctival injection. For both treatments, the ocular allergy sign and symptom scores were comparable at baseline. Both treatments evaluated in this study were well tolerated. Significant clinical and statistical reductions from baseline were observed in the primary efficacy variables. Treatment group differences were observed for the pain/soreness score with an advantage observed for the DS group at 30 minutes and at day 7. Our conclusion is that diclofenac sodium and ketorolac tromethamine acted similarly to reduce the ocular signs and symptoms associated with acute seasonal allergic conjunctivitis. There was a statistically significant advantage for the DS group to be free of symptoms at the day 7 visit as compared to the KT group (20.7% vs. 3.2%).

Evaluation of diclofenac sodium 0.1% ophthalmic solution in the treatment of ocular symptoms after bilateral radial keratotomy.

PURPOSE: Patients frequently have ocular pain, photophobia, foreign-body sensation, and burning/stinging after radial keratotomy. This study was a prospective, randomized, double-masked, multicenter, fellow-eye comparison of diclofenac sodium (Voltaren Ophthalmic, 0.1% solution) and placebo for controlling these ocular symptoms after bilateral radial keratotomy. METHODS: Patients who were pain free in both eyes before surgery were randomly assigned to treatment with diclofenac sodium in one eye and placebo in the other. One drop of each masked trial medication was administered 30-60 min before surgery, 5 min and 6 h after surgery, at bedtime on the day of surgery, and four times daily for 2 additional days. Patients evaluated ocular symptoms in each eye 0.5, 1, 2, 4, 6, 24, and 48 h after surgery and provided a global evaluation 6, 24, and 48 h after surgery. For each assessment, the difference in scores between eyes was analyzed by using a paired t test. RESULTS: Diclofenac sodium was significantly (p < 0.001) superior to placebo in controlling each ocular symptom at each interval after surgery and for patient global assessments 6, 24, and 48 h after surgery. CONCLUSION: Diclofenac sodium 0.1% ophthalmic solution is clinically effective in controlling adverse ocular symptoms occurring after bilateral radial keratotomy.

The role of K+ currents in frequency-dependent spike broadening in Aplysia R20 neurons: a dynamic-clamp analysis.

The R20 neurons of Aplysia exhibit frequency-dependent spike broadening. Previously, we had used two-electrode voltage clamp to examine the mechanisms of this spike broadening (Ma and Koester, 1995). We identified three K+ currents that mediate action-potential repolarization: a transient A-type K+ current (I(Adepol)), a delayed rectifier current (IK-V), and a Ca(2+)-sensitive K+ current(IK-CA). A major constraint in that study was the lack of completely selective blockers for I(Adepol) and I(K-V), resulting in an inability to assess directly the effects of their activation and inactivation on spike broadening. In the present study, the dynamic-clamp technique, which employs computer simulation to inject biologically realistic currents into a cell under current-clamp conditions (Sharp et al., 1993a,b), was used either to block I(Adepol) or I(K-V) or to modify their inactivation properties. The data in this paper, together with earlier results, lead to the following hypothesis for the mechanism of spike broadening in the R20 cells. As the spike train progresses, the primary responsibility for spike repolarization gradually shifts from I(Adepol) to I(K-V) to I(K-Ca). This sequence can be explained on the basis of the relative rates of activation and inactivation of each current with respect to the constantly changing spike durations, the cumulative inactivation of I(Adepol) and I(K-V), and the progressive potentiation of I(K-Ca). Positive feedback interactions between spike broadening and inactivation contribute to the cumulative inactivation of both I(Adepol) and I(K-V). The data also illustrate that when two or more currents have similar driving forces and partially overlapping activation characteristics, selectively blocking one current under current-clamp conditions can lead to a significant underestimate of its normal physiological importance.

Consequences and mechanisms of spike broadening of R20 cells in Aplysia californica.

We studied frequency-dependent spike broadening in the two electrically coupled R20 neurons in the abdominal ganglion of Aplysia. The peptidergic R20 cells excite the R25/L25 interneurons (which trigger respiratory pumping) and inhibit the RB cells. When fired at 1-10 Hz, the duration of the falling phase of the action potential in R20 neurons increases 2-10 fold during a spike train. Spike broadening recorded from the somata of the R20 cells affected synaptic transmission to nearby follower cells. Chemically mediated synaptic output was reduced by approximately 50% when recorded trains of nonbroadened action potentials were used as command signals for a voltage-clamped R20 cell. Electrotonic EPSPs between the R20 cells, which normally facilitated by two- to fourfold during a high frequency spike train, showed no facilitation when spike broadening was prevented under voltage-clamp control. To examine the mechanism of frequency-dependent spike broadening, we applied two-electrode voltage-clamp and pharmacological techniques to the somata of R20 cells. Several voltage-gated ionic currents were isolated, including INa, a multicomponent ICa, and three K+ currents--a high threshold, fast transient A-type K+ current (IAdepol), a delayed rectifier K+ current (IK-V), and a Ca(2+)-sensitive K+ current (IK-Ca), made up of two components. The influences of different currents on spike broadening were determined by using the recorded train of gradually broadening action potentials as the command for the voltage clamp. We found the following. (1) IAdepol is the major outward current that contributes to repolarization of nonbroadened spikes. It undergoes pronounced cumulative inactivation that is a critical determinant of spike broadening. (2) Activity-dependent changes in IK-V, IK-Ca, and ICa have complex effects on the kinetics and extent of broadening. (3) The time integral of ICa during individual action potentials increases approximately threefold during spike broadening.

A nursing career leadership program.

A three-level leadership track to parallel the clinical career ladder focuses on communication and interpersonal skills, professional, clinical and leadership experience, continuing education and leadership training. The program's success has led to career ladders in respiratory, physical and occupational therapy.

Corneal wound healing strength with topical antiinflammatory drugs.

The objective of this study was to compare the force required to separate corneal wounds after topical applications of nonsteroidal antiinflammatory drugs (NSAIDs) or corticosteroids. Bilateral central 8-mm long corneal full-thickness incisions in 50 NZW rabbits were closed with five interrupted 10-0 nylon sutures. There were four paired-eye groups: (a) control/control, (b) control/diclofenac sodium (0.1%), (c) control/flurbiprofen sodium (0.03%), and (d) control/prednisolone acetate (1%) treated six times per day for 7 or 21 days. The wound strength was measured by determining the force necessary to separate the incision along its length. The eyes did not differ statistically from their contralateral eye for each group except control/diclofenac (7.98 g/12.32 g) and control/flurbiprofen (6.96 g/11.67 g) at 21 days. The strongest scars occurred after treatment with diclofenac and flurbiprofen, which were similar (p = 0.74). The weakest wounds for each time period were with prednisolone (1.74 g/3.21 g). The diclofenac and flurbiprofen were stronger than prednisolone-treated eyes at 7 days (p = 0.028 and p = 0.023, respectively) and at 21 days (p < 0.001). The bilateral controls were stronger than the prednisolone controls (p = 0.008 at 7 days and p = 0.001 at 21 days). Steroid treatment caused weaker corneal wound scars than did the NSAIDs. Unilateral steroid treatment adversely affected their untreated contralateral eyes. The NSAID-treated wounds were the strongest and stronger than their contralateral control eyes.

The serine-rich Entamoeba histolytica protein is a phosphorylated membrane protein containing O-linked terminal N-acetylglucosamine residues.

Previously, we described the isolation of a cDNA clone and the gene encoding a protective antigen of the protozoan parasite Entamoeba histolytica, the serine-rich Entamoeba histolytica protein (SREHP). The derived amino acid sequence of the SREHP cDNA clone was remarkable for a high serine content (52/233 amino acids), a putative signal sequence, multiple hydrophilic dodecapeptide and octapeptide tandem repeats, and a hydrophobic C-terminal putative membrane-spanning region. Here, we show that SREHP is modified by the addition of phosphate at serine residues, O-linked terminal N-acetylglucosamine residues, and by acylation. When the SREHP gene is expressed in baculovirus transformed Sf-9 cells, the product is also phosphorylated and glycosylated and is localized to the plasma membrane of the insect cells. The native SREHP molecule also serves as a potent chemoattractant for amebic trophozoites. The data presented here suggest that SREHP is a unique membrane protein with phosphorylation and glycosylation patterns usually associated with nuclear or cytoplasmic proteins.

Safety and efficacy of diclofenac sodium 0.1% ophthalmic solution in acute seasonal allergic conjunctivitis.

A randomized clinical trial was conducted to compare diclofenac sodium 0.1% ophthalmic solution to placebo in relieving ocular signs and symptoms in patients with acute seasonal allergic conjunctivitis. Twenty patients (10 per treatment) qualified for this two week, double-masked study with moderate itching, bulbar conjunctival injection and a positive skin test. Diclofenac was statistically and clinically superior in the physician's global evaluation (p = 0.03) and the primary composite score [itching + bulbar/palpebral conjunctival injection (p = 0.037)] after two weeks of treatment. Four patients experienced some transient ocular burning/stinging with diclofenac. Diclofenac sodium appears to be effective for relieving the ocular signs and symptoms associated with acute seasonal allergic conjunctivitis.

Control of the cardiovascular system of Aplysia by identified neurons.

The neural network that controls the cardiovascular system of Aplysia adapts cardiovascular function to a variety of different physiological and behavioral situations. It (1) coordinates the cardiovascular system with the renal and respiratory systems; (2) modifies both systemic and regional blood flow during food-elicited arousal and feeding; and (3) changes the tension of longitudinal vascular muscle to adapt the arterial tree to changes in body shape. Indirect evidence suggests that the cardiovascular control circuit may also play a role in maintaining homeostasis during egg laying. Several putative neurotransmitters, including acetylcholine, serotonin, R15 alpha 1 and R15 alpha 2 peptides, have been localized to identified neurons in this circuit.

The morphology, innervation and neural control of the anterior arterial system of Aplysia californica.

The morphology, innervation, and neural control of the anterior arterial system of Aplysia californica were investigated. Immunocytochemical and histochemical techniques generated positive reactions in the anterior arterial system for several neuroactive substances, including SCPB, FMRFamide, R15 alpha 1 peptide, dopamine and serotonin. Three neurons were found to innervate the rostral portions of the anterior arterial tree. One is the identified peptidergic neuron R15 in the abdominal ganglion, and the other two are a pair of previously unidentified neurons, one in each pedal ganglion, named pedal arterial shorteners (PAS). The endogeneously bursting neuron R15 was found to innervate the proximal anterior aorta. It also innervates a branch of the distal anterior aorta, the left pedal-parapodial artery. Activity in R15 causes constriction of the left pedal-parapodial artery. This effect is presumed to direct hemolymph towards the genital groove and penis on the right side in vivo. This vasoconstrictor action of R15 is mimicked by the R15 alpha 1 peptide. The PAS neuron pair causes longitudinal contraction of the rostral anterior aorta and the pedal-parapodial arteries. In vivo, the pair is active during behaviors involving head withdrawal and turning. By adjusting the length of the arteries during postural changes, the PAS neurons may prevent disturbances in blood flow due to bending or kinking of the arterial walls.

R15 alpha 1 and R 15 alpha 2 peptides from Aplysia: comparison of bioactivity, distribution, and function of two peptides generated by alternative splicing.

The mRNA precursor encoded by the R15 gene is alternatively spliced in different neurons to form two related variants, R15-1 and R15-2 mRNA. One of the peptides encoded by the R15-2 mRNA, the R15 alpha 1 peptide, is expressed in the endogenously bursting neuron R15 and mediates some of its central and peripheral synaptic actions. In this study we found that the R15 alpha 2 peptide, which is encoded by the R15-1 mRNA, is synthesized in other neurons in the abdominal ganglion and is also bioactive. The R15 alpha 1 and R15 alpha 2 peptides were found to exert many similar actions on the cardiovascular, digestive, respiratory, and reproductive systems. However, the differences between many of the pharmacological effects of the R15 alpha 1 and R15 alpha 2 peptides indicate that alternative splicing in this system results in two functionally different peptides. Widespread immunoreactivity was found for an antibody directed against the R15 alpha 2 peptide, both in the central nervous system and the periphery. But because of the shared sequence with the R15 alpha 1 peptide, the antibody cross-reacts with the R15 alpha 1 peptide. To distinguish immunocytochemically between the two peptides, we also raised a second antibody that recognizes only the R15 alpha 1 peptide. This antibody labeled the cell body of only one neuron in the central nervous system, R15, although widespread immunoreactivity was found in axons and varicosities in the periphery.

Synaptic actions of identified peptidergic neuron R15 in Aplysia. I. Activation of respiratory pumping.

The purpose of the study described in this and the following two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 increases the frequency of bursting in the R25/L25 network that triggers respiratory pumping. This excitatory modulatory effect appears to be mediated by R15 alpha 1 peptide. R15 activates both strong and weak modes of respiratory pumping. In contrast, the two R20 cells, which are thought to use the neuropeptides SCPA and SCPB as transmitters, elicit only strong episodes of respiratory pumping. The synaptic actions of R15 also differ from those of the R20 cells in being longer lasting and in exhibiting profound desensitization. Chronic recording of R15 activity in vivo indicates that it does not burst spontaneously in the intact animal, so the synaptic actions of R15 are not chronically desensitized. The neuroendocrine bag cells, which initiate egg laying, had been shown by others to excite R15 and the R25/L25 network that triggers respiratory pumping. Our data indicate that the excitatory effects of the bag cells on the R25/L25 cells are mediated in part by R15.

Synaptic actions of identified peptidergic neuron R15 in Aplysia. III. Activation of the large hermaphroditic duct.

The purpose of the study described in this and the preceding two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 activates anterograde peristaltic movements in the segment of the large hermaphroditic duct through which eggs move during egg-laying behavior. This action is mimicked by R15 alpha 1 peptide, a putative transmitter of R15. The neuroendocrine bag cells, which initiate egg laying when they fire in a population burst, have been shown by others to excite R15. Our data suggest that R15 mediates excitatory effects of the bag cells on the large hermaphroditic duct. Taken with the results of the two companion papers, these data support the hypothesis that R15 integrates various aspects of egg-laying behavior. The desensitization of R15's postsynaptic actions may complement the long-lasting refractoriness of the bag cells described by others, with both effects contributing to the episodic nature of egg laying.