A pilot study of high-dose short duration daptomycin for the treatment of patients with complicated skin and skin structure infections caused by gram-positive bacteria.

BACKGROUND: Methicillin-susceptible and -resistant (MRSA) Staphylococcus aureus are significant causes of complicated skin and skin structure infections (cSSSI). The bactericidal antibiotic daptomycin is approved for gram-positive cSSSI at 4 mg/kg/day for 7-14 days, but the optimal dose level and duration of therapy have not been firmly established. This pilot study evaluated the efficacy and safety of daptomycin at 10 mg/kg every 24 h for 4 days [high-dose short duration (HDSD) regimen] vs. standard of care therapy with vancomycin or semi-synthetic penicillin for the treatment of cSSSI. METHODS: This was a semi-single blind, randomised, multicentre, comparative trial. The primary efficacy end-point was the clinical response 7-14 days posttherapy. RESULTS: One hundred patients were randomised; 48 in each arm were treated. The treatment groups were well balanced with respect to demographics, comorbidities and the type of infection (75% because of MRSA). Overall, clinical success rates were 75.0% (36/48) for daptomycin and 87.5% (42/48) for comparator (95% confidence interval for the difference: -27.9, 2.9). The median duration of comparator therapy was 8 days. Two comparator patients and no daptomycin patients experienced treatment-related serious adverse events requiring hospitalisation. CONCLUSION: We found that the HDSD regimen had a safety profile similar to that seen in previous studies. Although the differences were not statistically significant, clinical success rates for comparator were higher than for daptomycin. In post hoc analyses HDSD daptomycin performed better in some subgroups (e.g. outpatients) than in others (e.g. certain MRSA infections). These observations require confirmation in larger trials.

The application of in vitro data in the derivation of the acceptable daily intake of food additives.

The acceptable daily intake (ADI) for food additives is commonly derived from the NOAEL (no-observed-adverse-effect level) in long-term animal in vivo studies. To derive an ADI a safety or uncertainty factor (commonly 100) is applied to the NOAEL in the most sensitive test species. The 100-fold safety factor is considered to be the product of both species and inter-individual differences in toxicokinetics and toxicodynamics. Although in vitro data have previously been considered during the risk assessment of food additives, they have generally had no direct influence on the calculation of ADI values. In this review 18 food additives are evaluated for the availability of in vitro toxicity data which might be used for the derivation of a specific data-derived uncertainty factor. For the majority of the food additives reviewed, additional in vitro tests have been conducted which supplement and support the short- and long-term in vivo toxicity studies. However, it was recognized that these in vitro studies could not be used in isolation to derive an ADI; only when sufficient in vivo mechanistic data are available can such information be used in a regulatory context. Additional short-term studies are proposed for the food additives which, if conducted, would provide data that could then be used for the calculation of data-derived uncertainty factors.

Neuroprotective use-dependent blockers of Na+ and Ca2+ channels controlling presynaptic release of glutamate.

We have originated a family of N,N'-disubstituted guanidines that block the voltage-activated Ca2+ and Na+ channels governing glutamate release. These compounds, CNS 1237 (N-acenaphthyl-N'-methoxynaphthyl guanidine) and its analogues, are "use dependent" in their ability to attenaute neurotransmitter release: they block glutamate release with greater efficacy under conditions of persistent or repetitive depolarization, as would be encountered under pathophysiological circumstances, relative to their ability to block glutamate release elicited by brief, transient depolarizations more characteristic of normal physiological release events in nonischemic brain. Using electrophysiological and rapid kinetic methods, we have differentiated the use-dependent block of the relevant Na+ and Ca2+ channels governing neurotransmitter release from the mechanism of channel antagonism exhibited by, respectively, the substituted guanidine Na+ channel blocker tetrodotoxin (TTX) and venom peptide Ca2+ antagonists. To characterize use-dependent Na+ channel block by CNS 1237, we have employed whole-cell voltage-clamp recordings from a Chinese hamster ovary (CHO) cell line expressing cloned mammalian type II Na+ channels. These experiments demonstrated that, in contrast to the actions of TTX under the same conditions, the potency of Na+ channel block by CNS 1237 is greatly enhanced by depolarizing stimuli in a frequency-dependent manner. Ca2+ channel-activated glutamate release from brain nerve terminal preparations was measured with approximately 300 msec time resolution over a 5-second period of high K(+)-depolarization, using a rapid superfusion technique. CNS 1237 and analogues, at 1-3 microM, accelerated the decay of glutamate release by 40-70%, reflecting depolarization-induced enhancement of block. In contrast, blockade of glutamate release by the Ca2+ channel antagonist peptide toxins omega-aga IV-A (from spider venom) and omega-conotoxin M-VII-C (from cone snail venom) exhibited "reverse-use-dependence:" at concentrations of 0.3 microM, which blocked the initial amplitude of glutamate release by 40-60%, the decay time constant for glutamate release was significantly increased, indicating depolarization-induced relief of block. These findings establish that CNS 1237 and other members of this compound series are use-dependent blockers of the voltage-activated ion channels governing glutamate release. Studies of CNS 1237 in the rat middle cerebral artery occlusion (MCAO) focal stroke model have indicated infarct size reduction comparable to that observed by the same investigators for the glutamate release blocker (BW 619C89 (Burroughs-Wellcome, now in clinical development). Maximal infarct size reduction is achieved with a 3-mg/kg bolus followed by a 4-hour infusion of 0.75 mg/kg/hr.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro neuroprotection by substituted guanidines with varying affinities for the N-methyl-D-aspartate receptor ionophore and for sigma sites.

Radioligand binding techniques were used to determine the affinity of a series of substituted guanidine derivatives for 1) the binding site within the ion channel of the N-methyl-D-aspartate (NMDA) receptor, as defined by displacement of MK-801 ([3H]dizocilpine) and 2) sigma sites as defined by displacement of [3H]N,N'-di-(o-tolyl)guanidine. The goal was to find ligands with high affinity and selectivity for the NMDA receptor ion-channel site. The neuroprotective activity of these compounds was assessed by their ability to protect cortical neurons from injury caused by a 5-min exposure to 500 microM glutamate in vitro. Release of lactate dehydrogenase into the culture medium by damaged neurons was used as an index of neuronal injury. The 14 compounds tested had IC50 values ranging from 37.3 nM to 12.7 microM for the NMDA receptor ion-channel site and from 8.3 nM to 7.25 microM for sigma sites. Affinity for the ion-channel site was improved by unsymmetrical substitutions on the guanidine moiety. All compounds in the series protected cortical neurons against glutamate toxicity, with EC50 values (concentration affording 50% protection) ranging from 0.38 to 28.25 microM. The neuroprotective effect of each compound was positively correlated with its ion-channel site affinity (r = 0.94); no correlation between neuroprotective efficacy and sigma site binding affinity was found (r V -0.13) establishing clearly that neuroprotection in this assay was linked to NMDA antagonist properties.

Effects of a novel NMDA antagonist on experimental stroke rapidly and quantitatively assessed by diffusion-weighted MRI.

We employed diffusion-weighted MRI (DWI) to identify regions of focal brain ischemia during the first 3 hours after permanent occlusion of the middle cerebral artery in rats. Using DWI as early as 30 minutes after the onset of ischemia, it was possible to identify the areas of brain destined to progress to infarction over the next 24 hours in untreated animals, as demonstrated by postmortem evaluation. DWI studies revealed the cerebroprotective effects of a noncompetitive N-methyl-D-aspartate receptor antagonist, CNS 1102, administered 15 minutes postocclusion, both on the cortical and caudoputaminal regions during the initial 3 hours of ischemia. Although the treatment effect lessened over the next 21 hours in a few animals with lower plasma drug levels at 3 hours, postmortem studies demonstrated a 66% reduction in the total volume of infarcted tissue with the treatment and confirmed the DWI results. T2-weighted MRI obtained at similar times revealed little or no abnormality. These results suggest that DWI provides a sensitive in vivo measure of focal cerebral ischemic injury and can assess the beneficial effects of cytoprotective therapy. DWI may be useful in the early evaluation of human stroke patients and in monitoring the effects of cerebroprotective therapies in the clinical setting.

New CNS-specific calcium antagonists.

Ischemic insults to the brain in stroke or traumatic brain injury produce excessive release of glutamate from depolarized nerve terminals. This excessive glutamate release in turn stimulates massive calcium entry into nerve cells, activating a biochemical cascade that results in cell death. A major pathway of calcium entry into depolarized nerve cells is through voltage-sensitive, high threshold calcium channels. A large fraction of this calcium entry is mediated through "R-type" calcium channels, channels resistant to blockage by dihydropyridine calcium antagonists such as nimodipine. A newly discovered compound derived from spider venom, CNS 2103, antagonizes both R-type channels and dihydropyridine-sensitive ("L-type") calcium channels. This broad spectrum of action, coupled with selectivity for calcium channels over other classes of voltage-sensitive and ligand-gated ion channels, makes CNS 2103 an interesting lead for development of drugs to treat ischemic brain injury. Activation of presynaptic ("N-type") calcium channels in nerve terminals is a primary cause of excessive neurotransmitter release in brain ischemia. Prevention of glutamate release by blockade of N-type channels in glutamatergic nerve terminals may, at an early stage in the pathophysiological cascade, abort the process leading to nerve cell death. Cambridge NeuroScience has developed a novel rapid kinetic approach for monitoring glutamate release from brain nerve terminals in vitro, and this has led to CNS 1145, a substituted guanidine that selectively blocks a kinetic component of calcium-dependent glutamate release mediated by persistent depolarization. Additional evidence suggests that CNS 1145 antagonizes presynaptic N-type calcium channels, and this may account at least in part for its ability to block glutamate release.

Dopamine modulates the kinetics of ion channels gated by excitatory amino acids in retinal horizontal cells.

Upon exposure to dopamine, cultured teleost retinal horizontal cells become more responsive to the putative photoreceptor neurotransmitter L-glutamate and to its analog kainate. We have recorded unitary and whole-cell currents to determine the mechanism by which dopamine enhances ion channels activated by these agents. In single-channel recordings from cell-attached patches with agonist in the patch pipette, the frequency of 5- to 10-pS unitary events, but not their amplitude, increased by as much as 150% after application of dopamine to the rest of the cell. The duration of channel openings also increased somewhat, by 20-30%. In whole-cell experiments, agonists with and without dopamine were applied to voltage-clamped horizontal cells by slow superfusion. Analysis of whole-cell current variance as a function of mean current indicated that dopamine increased the probability of channel opening for a give agonist concentration without changing the amount of current passed by an individual channel. For kainate, noise analysis additionally demonstrated that dopamine did not alter the number of functional channels. Dopamine also increased a slow spectral component of whole-cell currents elicited by kainate or glutamate, suggesting a change in the open-time kinetics of the channels. This effect was more pronounced for currents induced by glutamate than for those induced by kainate. We conclude that dopamine potentiates the activity of horizontal cell glutamate receptors by altering the kinetics of the ion channel to favor the open state.

Horizontal cell gap junctions: single-channel conductance and modulation by dopamine.

Horizontal cells form an electrically coupled network for the transmission of inhibitory signals in the outer retina. In teleosts, horizontal cell coupling is modulated by the neurotransmitter dopamine. Using voltage-clamped pairs of teleost horizontal cells, we have examined the effects of dopamine on the conductance and gating properties of the cell-to-cell channels that mediate electrical synaptic transmission. Variance analysis of the junctional current noise showed that dopamine substantially reduced the open probability of gap junction channels, from 0.75 to 0.14. Direct observation of unitary junctional gating events in poorly coupled cell pairs indicated that these channels have a unitary conductance of 50-60 pS. The elementary conductance of channels in cell pairs treated with dopamine (48.7 +/- 6.6 pS) was statistically indistinguishable from channels in untreated cells (53.2 +/- 7.2 pS). Uncoupling with octanol also yielded a similar unitary conductance (61.1 +/- 11.1 pS). Our results suggest that dopamine reduces the open probability of gap junctional channels by decreasing their open duration.

Responses of isolated white perch horizontal cells to changes in the concentration of photoreceptor transmitter agonists.

Current and voltage responses elicited by increasing or decreasing the concentration of L-glutamate or its analog kainate around isolated cone horizontal cells were measured with patch pipettes using the whole cell recording configuration. Application of these photoreceptor transmitter agonists induced inward currents in voltage-clamp experiments (for negative holding potentials) and depolarizing responses in current-clamp experiments. Continuous exposure to either drug produced inward currents which were maintained for as long as superfusion with the drugs continued. Reducing the concentration of the agonists by pressure ejection of pulses of drug-free Ringer's solution onto the cells completely turned off the drug-induced currents. Under current-clamp conditions, pulses of control Ringer's elicited hyperpolarizing responses of large amplitude (40-80 mV). The data demonstrate the ability to simulate in vitro the horizontal cell's photoresponses and thus support the use of cultured cells as a model system for studying horizontal cell physiology and pharmacology.

Enhancement of kainate-gated currents in retinal horizontal cells by cyclic AMP-dependent protein kinase.

Dopamine, acting via cyclic adenosine 3':5'-monophosphate (cAMP), has been shown to enhance a kainate-gated ionic conductance in white perch retinal horizontal cells in vitro. To determine whether this effect involves stimulation of a protein kinase, kainate-gated currents were observed in cultured horizontal cells that were dialyzed with the catalytic subunit of cAMP-dependent protein kinase. Intracellular application of catalytic subunit or cAMP, but not heat-inactivated catalytic subunit, caused significant enhancement of the kainate-evoked currents. These results suggest that kainate-gated channels in horizontal cells may be modified by a phosphorylation event.

Local superfusion modifies the inward rectifying potassium conductance of isolated retinal horizontal cells.

1. Horizontal cells were enzymatically and mechanically dissociated from the white perch (Roccus americana) retina and voltage clamped using patch electrodes. Steady-state current-voltage (I-V) relationships of solitary horizontal cells were determined by changing the membrane potential in a rampwise fashion. 2. The I-V curve of cells bathed in normal Ringer solution exhibited a large conductance increase at negative membrane potentials. This conductance activated near the K+ equilibrium potential, had no clear reversal potential, was enhanced by raising the extracellular concentration of K+, and was suppressed by external Cs+. These properties identify the conductance as the inward (anomalous) rectifier. 3. Continuous superfusion of the cells' local environment with drug-free Ringer reduced the magnitude of the inward rectifier current and shifted its activation point to more negative potentials. This effect developed over approximately 30 s, lasted as long as superfusion continued and was reversible upon cessation of superfusion. 4. Pressure ejection of drug-free Ringer solution onto cells bathed in the identical solution also reduced the magnitude of the inward rectifier current, although the effects were more rapid and more transient than those exerted by superfusion. Pressure ejection had little effect when cells were simultaneously superfused with Ringer, suggesting a common mode of action on the inward rectifier. 5. In the absence of superfusion, pressure ejection of Ringer containing 200 microM L-glutamate had a biphasic effect on membrane conductance. At potentials above -60 mV, glutamate caused a conductance increase with a reversal potential near +10 mV. At potentials below -60 mV, glutamate caused a conductance decrease whose reversal potential could not reliably be determined. The latter effect was similar to the suppression of the inward rectifier by application of Ringer alone, suggesting that it may represent an artifact of pressure ejection rather than a direct effect of glutamate. 6. In support of this interpretation, we found that pressure ejection of glutamate in the presence of external Cs+ (which blocks the inward rectifier) or during local superfusion with Ringer (which prevents attenuation of the inward rectifier by pressure ejection) did not cause a conductance decrease at negative potentials. Under these conditions, glutamate caused primarily a conductance increase with a reversal potential near +10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of vertebrate eye movements following intravitreal drug injections. I. Blockade of retinal ON-cells by 2-amino-4-phosphonobutyrate eliminates optokinetic nystagmus.

1. Horizontal optokinetic nystagmus (OKN) was examined in alert rabbits and cats following intravitreal injection of 2-amino-4-phosphonobutyrate (APB), an agent which selectively blocks the light-responsiveness of retinal ON-cells while having little effect on OFF-cells. The retinal actions of APB were assessed independently by electroretinography. 2. In five rabbits, doses of APB sufficient to eliminate the b-wave of the electroretinogram reduced drastically the ability of the injected eye to drive OKN at all stimulus speeds tested (1-96 degrees/s). Impairment of OKN was apparent within minutes of the injection, remained maximal for several hours, and recovered completely in 1-7 days. OKN in response to stimulation of the uninjected eye alone remained qualitatively and quantitatively normal. 3. Following administration of APB, OKN in response to binocular stimulation displayed a directional asymmetry. Stimuli moving in the preferred (temporal-to-nasal) direction for the uninjected eye became more effective than stimuli moving in the opposite direction, indicating that the injected eye could no longer contribute to binocular OKN. 4. When rabbits viewed stationary stimuli through the APB-treated eye alone, episodes of slow (less than 1 degrees/s) ocular drift were observed, similar to the positional instability seen when rabbits are placed in darkness or when the retinal image is stablized artifically (12). 5. APB had little effect on OKN in normal cats. In two cats that had previously received large lesions of the visual cortex, however, APB eliminated the ability of the injected eye to drive monocular OKN. The extent of the impairment was similar to that seen in rabbits. Because the cortex is thought to contribute more to OKN in cats than in rabbits, this result suggests that the optokinetic pathways disrupted by APB project subcortically. 6. This study demonstrates that the integrity of retinal ON-cells is required to sustain normal OKN. The results are consistent with additional anatomic and physiological evidence suggesting that a particular subclass of retinal ganglion cells, the ON-direction-selective cells, may provide a crucial source of visual input to central optokinetic pathways.

Analysis of vertebrate eye movements following intravitreal drug injections. II. Spontaneous nystagmus induced by picrotoxin is mediated subcortically.

1. Eye movements were observed following an injection of picrotoxin, a GABA antagonist, into the vitreous of one eye. A spontaneous nystagmus was observed in cats, rabbits, and turtles, even in total darkness, with slow-phase eye movements in the temporal-to-nasal direction for the injected eye. 2. During visual stimulation by a horizontal drifting pattern, injected eyes moved in the temporal-to-nasal direction, irrespective of stimulus direction. In cats, however, the nystagmus was usually slower when the injected eye viewed nasal-to-temporal motion (opposite to the direction of the spontaneous nystagmus). The spontaneous nystagmus could be halted or even reversed by allowing cats to view motion opposite to the direction of the nystagmus with the uninjected eye alone. The nystagmus could not be overridden in this fashion in rabbits or turtles. 3. The nystagmus induced by picrotoxin could also be modified by vestibular stimulation. When cats were placed on their sides, the spontaneous horizontal nystagmus often decreased and spontaneous vertical nystagmus with upward slow phase movements occurred. During sinusoidal horizontal vestibular stimulation, the horizontal nystagmus due to picrotoxin added to the vestibuloocular reflex as a velocity offset in the temporal-to-nasal direction. 4. Following bilateral ablation of the cat visual cortex, picrotoxin's effect became even more pronounced than before the ablation. Therefore, at least some picrotoxin-sensitive cells can use subcortical pathways, perhaps to the accessory optic nuclei. The visual cortex, which also processes directional information, may be able to compensate for changes in retinal processing induced by picrotoxin in intact animals. 5. This study demonstrates the importance of retinal GABA in the control of eye stability. As GABA is known to be responsible for null direction inhibition of directionally sensitive retinal ganglion cells, these results suggest that the output of these cells may be critical for the normal functioning of central optokinetic pathways, even in the absence of visual cortex.

Dopamine enhances excitatory amino acid-gated conductances in cultured retinal horizontal cells.

In the teleost retina, cone horizontal cells receive extensive innervation from dopaminergic interplexiform cells, and possess dopamine receptors whose activation stimulates adenylate cyclase. Exogenously applied dopamine modifies several aspects of horizontal cell activity in the intact retina, including the responsiveness of these neurons to light and the strength of electrical coupling between them. We have used whole-cell voltage clamp methods to examine whether dopamine can alter the light-responsiveness of horizontal cells by changing their sensitivity to the neurotransmitter released by the photoreceptors. We report that dopamine and cyclic AMP, although having little direct effect on resting membrane conductance, greatly enhance ionic conductances gated by kainate, an agonist of the transmitter released by the photoreceptors, and by L-glutamate, the agent proposed to be the photoreceptor transmitter. Our results provide the first direct evidence for dopaminergic regulation of excitatory amino-acid neurotransmission in the vertebrate nervous system and suggest a possible mechanism to explain the reduction in the responsiveness of horizontal cells observed when retinas are treated with dopamine.

The contribution of on-bipolar cells to the electroretinogram of rabbits and monkeys. A study using 2-amino-4-phosphonobutyrate (APB).

We have obtained electroretinograms (ERGs) from rabbits and macaque monkeys after vitreal administration of 2-amino-4-phosphonobutyrate (APB), which selectively blocks light-responsiveness in retinal on-bipolar cells. Microelectrode recordings from central visual structures provided an independent measure of the drug effects. In rabbits, APB blocked the b-wave, a sustained corneal-positive potential, and a transient corneal-negative off-response. In monkeys, APB abolished the b-wave but had little or no effect on the d-wave. The d-wave could be eliminated, however, by the use of broadband blue stimuli. The results are discussed in light of recent hypotheses about the cellular origins of the ERG.

Response properties of cells in rabbit's lateral geniculate nucleus during reversible blockade of retinal on-center channel.

In the vertebrate retina, visual information is segregated into an on channel excited by light increment and a complementary off channel excited by light decrement. We used 2-amino-4-phosphonobutyric acid (APB), which selectively blocks the on channel in the retina (29), to determine the contributions of the on and off pathways to response properties of neurons in the lateral geniculate nucleus (LGN) of anesthetized, paralyzed rabbits. Visually evoked responses were recorded from 46 single cells in the LGN before, during, and after vitreal perfusion with 200-1,000 microM APB. APB reversibly blocked responses of on uniform-field cells and on center concentric-field cells to stationary, flashing spots of light. Responses to off uniform-field cells and off-center concentric-field cells were largely unaffected. APB did not differentially affect responses elicited from the receptive-field centers, as opposed to the surrounds, of on-center concentric-field cells. This finding suggests that these cells are driven exclusively by the on retinal channel and that the center-surround organization of their receptive fields does not result from a convergence of the on and off pathways. We studied a small number of cells that were selective for stimulus direction or motion. In each case, APB eliminated the cell's response to a moving light edge. The surviving response to a moving dark edge retained its original direction or motion preference, suggesting that these response properties do not depend critically on interactions between the on and the off pathways. The findings obtained in the rabbit are reminiscent of the results of similar investigations in the cat (10, 11) and the monkey (25). Taken together, they indicate that in the LGN of several vertebrate species there is a precise segregation vertebrate species there is a precise segregation of on and off information, at least for some functional classes of cells. The combination of on and off information does not seem to play a major role in establishing the response properties observed at this level in the visual system.

Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry.

In experiments that use horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) for tracing neural connections, the activity of tissue-bound enzyme as well as the stability of the resultant reaction product are influenced by the duration of storage, the composition of the storage medium, the type of counterstaining and even the details of histological dehydration. Furthermore, the conditions for preserving HRP activity are very different from those necessary for preserving the stability of the tetramethyl benzidine (TMB) reaction product. Thus, tissue-bound HRP activity is stable at a neutral pH, while a much lower pH, around 3.3, is required for preserving the stability of the TMB reaction product. Recent evidence indicates that the stabilization bath in sodium nitroferricyanide that was previously recommended is not necessary. However, gradual dehydration of mounted sections is essential for long-term stability. Excessive counterstaining and excessive dehydration interfere with the detection of reaction product. These considerations are pertinent to experiments using free HRP as well as to those where the enzyme has been conjugated to wheat germ agglutinin.