Safety and clinical effect of i.v. infusion of cyclopropyl-methoxycarbonyl etomidate (ABP-700), a soft analogue of etomidate, in healthy subjects.

BACKGROUND: Cyclopropyl-methoxycarbonyl metomidate, or ABP-700, is a second generation analogue of etomidate, developed to retain etomidate's beneficial haemodynamic and respiratory profile but diminishing its suppression of the adrenocortical axis. The objective of this study was to characterise the safety and efficacy of 30-min continuous infusions of ABP-700, and to assess its effect on haemodynamics and the adrenocortical response in healthy human volunteers. METHODS: Five cohorts involving 40 subjects received increasing infusion doses of ABP-700, propofol 60 µg kg-1 min-1 or placebo. Safety was evaluated through adverse event (AE) monitoring, safety laboratory tests, and arterial blood gasses. Haemodynamic and respiratory stability were assessed by continuous monitoring. Adrenocortical function was analysed by adrenocorticotropic hormone (ACTH) stimulation tests. Clinical effect was measured using the modified observer's assessment of alertness/sedation (MOAA/S) and continuous bispectral index monitoring. RESULTS: No serious AEs were reported. Haemodynamic and respiratory effects included mild dose-dependent tachycardia, slightly elevated blood pressure, and no centrally mediated apnoea. Upon stimulation with ACTH, no adrenocortical depression was observed in any subject. Involuntary muscle movements (IMM) were reported, which were more extensive with higher dosing regimens. Higher dosages of ABP-700 were associated with deeper sedation and increased likelihood of sedation. Time to onset of clinical effect was variable throughout the cohorts and recovery was swift. CONCLUSIONS: Infusions of ABP-700 showed a dose-dependent hypnotic effect, and did not cause severe hypotension, severe respiratory depression, or adrenocortical suppression. The presentation and nature of IMM is a matter of concern. CLINICAL TRIAL REGISTRATION: NTR4735.

Agrin is a differentiation-inducing "stop signal" for motoneurons in vitro.

Proteins of the synaptic basal lamina are important in directing the differentiation of motor nerve terminals. One synaptic basal lamina protein, agrin, which influences postsynaptic muscle differentiation, has been suggested to influence nerve terminals as well. To test this hypothesis, we cocultured chick ciliary ganglion neurons with agrin-expressing CHO cells. Ciliary ganglion neurons, but not sensory neurons, adhered five times as well to agrin-expressing cells as to untransfected cells. Further, ciliary ganglion neurites were growth inhibited upon contact with agrin-expressing cells. Finally, the synaptic vesicle protein synaptotagmin became concentrated at contacts between ciliary ganglion neurites and agrin-expressing cells. These activities were shared by neuronal and muscle-derived agrin isoforms, consistent with the hypothesis that muscle agrin may influence the presynaptic axon. Our results suggest that agrin influences the growth and differentiation of motoneurons in vivo.

Lipid rafts are involved in C95 (4,8) agrin fragment-induced acetylcholine receptor clustering.

During development of the neuromuscular junction, high densities of acetylcholine receptors accumulate beneath the overlying nerve terminal. A defining feature of mature synapses is the sharp demarcation of acetylcholine receptor density, which is approximately 1000-fold higher in the postsynaptic as compared with the contiguous extrasynaptic muscle membrane. These high densities of receptors accumulate by at least four mechanisms, re-distribution of existing surface receptors, local synthesis of new receptors, decreased turnover of synaptic receptors, and limitation of diffusion of sub-neural, aggregated receptors. The limitation of receptor diffusion within the membrane is likely in part due to the anchoring of acetylcholine receptor complexes to components of the cytoskeleton. Here we have tested the idea that lipid rafts--mobile, cholesterol enriched microdomains within the lipid bilayer--are another mechanism by which acetylcholine receptors are clustered in the postsynaptic apparatus. Using mouse C2C12 cells, a muscle cell line, we show that a carboxy terminal 95 amino acid fragment [C95 (4,8)] of the extracellular matrix molecule agrin that is essential for nerve-induced postsynaptic differentiation, promotes the redistribution of acetylcholine receptors into lipid rafts. Disruption of lipid rafts before agrin treatment largely inhibits de novo agrin-induced acetylcholine receptor clustering. Moreover, mature acetylcholine receptor clusters are destabilized if lipid rafts are disrupted. These results show that lipid rafts are important in both the initial clustering and later stabilization of agrin-induced acetylcholine receptor clusters and also suggest that lipid rafts may contribute to the postsynaptic localization of acetylcholine receptors in vivo.

Pilocarpine spray: an alternative delivery method.

To determine if pilocarpine 4% produces miosis when delivered via a spray, eleven eyes of ten patients undergoing a laser peripheral iridotomy for occludable angles were pretreated with one application of pilocarpine spray to closed eyelids. Standardized pupil photographs for pupil diameter measurements were taken before and thirty minutes after the spray. Contralateral eyes served as controls (9 eyes) All treated eyes achieved miosis. The mean pupil diameter in the treated group was 4.1+/-0.9 mm before the spray and 1.5+/-0.3 mm after the spray (p < 0.0001). The average change in pupil diameter in the treated group was 2.6+/-0.9 mm compared to 0.0+/-0.2 mm in the control eyes (p < 0.0001). Pilocarpine spray is an effective delivery modality for intraocular miosis.

Tenon's cyst formation after trabeculectomy with mitomycin C.

One hundred eyes of 100 patients in whom Tenon's cysts (TCs) developed in the early postoperative period following trabeculectomy with mitomycin C were retrospectively studied to determine their incidence, possible risk factors associated with their development, and the clinical course of the patients in whom they occurred. The incidence of TC formation was 29/100 (29%). Significantly more cysts developed in the men than in the women. There were no significant differences between those patients in whom TCs developed and those in whom they did not in terms of age, race, type of preoperative medications, mean preoperative intraocular pressure (IOP), prior argon laser trabeculoplasty, prior trabeculectomy, prior Tenon's cyst formation, or prior cataract surgery. The 29 patients in whom a Tenon's cyst developed had a mean follow up of 22.0 +/- 12.9 weeks, with a mean IOP at the last examination of 14.2 +/- 6.3 mm Hg (as compared with 20.7 +/- 8.3 mm Hg at the time of diagnosis). One patient required a repeat trabeculectomy with mitomycin C. Two patients required bleb needling, subsequent TC excision, and eventual seton placement. Of the remaining 26, 14 (54%) required no glaucoma medication for IOP control, 11 (42%) required one, and 1 (4%) required two.

Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro.

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.

Control of intraocular pressure elevations after argon laser trabeculoplasty: comparison of brimonidine 0.2% to apraclonidine 1.0%.

OBJECTIVE: To determine whether brimonidine 0.2% can control intraocular pressure (IOP) spikes as well as apraclonidine 1.0% can in those patients undergoing argon laser trabeculoplasty (ALT). DESIGN: Prospective, randomized, double-masked, clinical trial. PARTICIPANTS: A total of 56 eyes of 41 patients with open-angle glaucoma or ocular hypertension were entered in the study; 46 eyes of 41 patients were eventually used for the final analysis. INTERVENTION: Patients were randomized to receive either brimonidine 0.2% or apraclonidine 1.0% before and after 360 degrees ALT. Both patient and physician were masked as to which agent each patient received. MAIN OUTCOME MEASURES: Intraocular pressure measurements were recorded before surgery and at 1, 2, and 4 hours after surgery. The difference between the preoperative IOP (baseline) and the highest recorded postoperative IOP was recorded as the maximum IOP change. The mean of the maximum IOP change for each group was analyzed using a two-sample, one-tailed t test. RESULTS: The mean of the maximum IOP change in the brimonidine 0.2% group was -2.6+/-3.6 mmHg, and the mean for the apraclonidine 1.0% group was -2.3+/-3.7 mmHg (P = 0.8). No patient had a pressure spike greater than 10 mmHg. CONCLUSIONS: Brimonidine 0.2% appears to be as effective as apraclonidine 1.0% in preventing IOP spikes after argon laser trabeculoplasty.

Target contact regulates expression of synaptotagmin genes in spinal motor neurons in vivo.

During neuromuscular development, neuronal contact with peripheral targets is associated with an increase in synaptic vesicle protein (SVP) gene expression, suggesting that target contact and upregulation of SVP genes are causally related. To test this idea, we analyzed the developmental expression pattern of synaptotagmin (syt) mRNAs in the chick lateral motor column (LMC) using in situ hybridization. Syt I mRNA in the LMC is upregulated from Embryonic Day 4.5 (E4.5) to E5.5, coincident with the time these neurons begin to make contact with their muscle targets. In contrast, levels of mRNA for neurofilament do not change during this time. Extirpation of the limb bud prior to motor axon outgrowth eliminates the increase in syt I mRNA ipsilaterally. Later in development, there is a switch in syt isoform abundance in the LMC, with syt II mRNA being upregulated between E15 and E20 and syt I mRNA being downregulated. Our results suggest that contact with targets upregulates syt I gene expression during neuromuscular synapse formation in vivo, and that a later stage of synaptic maturation involves changes in SVP isoform abundance.

Heat shock protein 27: developmental regulation and expression after peripheral nerve injury.

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.