Comparative in vivo bioequivalence and in vitro dissolution of two cyclosporin A soft gelatin capsule formulations.

OBJECTIVE: A study was conducted to establish the bioequivalence between a newly developed cyclosporin A (CsA) oral formulation, Deximune soft-gelatin capsules (Dexcel Ltd.) and Sandimmune Neoral (Novartis Inc.). MATERIALS AND METHODS: The clinical investigation was designed as a randomized, open-labeled, two-period, two-treatment crossover study, in 24 healthy fasted male volunteers. The subjects were administered a single 200 mg CsA dose of either formulation. Serial venous blood samples were obtained over 24 hours after each administration to measure CsA in whole blood by a specific TDx-immunoassay. In addition, the comparative drug release rate was assessed using a dissolution apparatus test according to the USP-24 method. RESULTS: For both treatments, a mean maximum blood concentration (Cmax) of approximately 1,200 ng/ml was obtained at about 1.6 hours (tmax) after administration and the geometric mean of the area under the blood concentration-time curve (AUC) both for test and reference was approximately 4,900 ng x h/ml. Bioequivalence was conclusively demonstrated for both rate (Cmax and tmax) and extent (AUC) of CsA absorption, between the two treatments. Moreover, the CsA blood concentration measurement at 2 hours after administration (C2), demonstrated equivalent results between the two products. The point estimates and their 90% confidence intervals were within the respective equivalence ranges for the pharmacokinetic parameters and were included in the range for drugs with a narrow therapeutic index. The comparative dissolution test for both formulations showed an in vitro release rate of more than 90% within 15 minutes. CONCLUSIONS: Based on the results, the two oral CsA formulations compared are bioequivalent and can be interchanged without need for dosage adjustment.

Pharmacokinetics of vancomycin administered as prophylaxis before cardiac surgery.

Vancomycin concentrations in serum, tissues, and sternum, administered as prophylaxis to patients during coronary artery bypass surgery, were measured. Vancomycin (15 mg/kg) was administered to 15 patients 1 hour before skin incision. Blood, tissue, and sternum samples were collected before, during, and after bypass. The concentration in serum at the end of infusion was 55.1 +/- 22.8 microg/mL, the mean elimination half-life was 9 +/- 4 hours, the areas under the concentration-time curve (AUC) from 0 to 12 hours and from 0 to infinity were 90.6 +/- 25.1 and 289.7 +/- 86.5 microg/h per mL, respectively, the mean residence time (MRT) was 11.9 +/- 5.0 hours, the mean volume of distribution was 51.1 +/- 12.2 L, and the total clearance was 78.3 +/- 32.6 mL/min. Vancomycin concentrations in serum, tissues, and sternum during the operation were greater than the MIC90 for most staphylococci and ranged from 16 to 55 microg/mL in serum and from 4 to 39 microg/g in sternum and tissues.

The role of the pedunculopontine tegmental nucleus in relation to conditioned motor performance in the cat. I. Context-dependent and reinforcement-related single unit activity.

The activity of the pedunculopontine tegmental nucleus (PPTg) neurons was recorded in three unrestrained cats operantly conditioned to perform a lever-release movement. The movement had to be initiated either rapidly after a (click) stimulus in a simple reaction-time paradigm or had to be delayed after the same stimulus in trials identified by a tone cue. Successful trials were rewarded by a food pellet. A total of 107 neurons were recorded with microelectrodes. Brief spike neurons (mean duration: 0.7 ms) and broad spike neurons (mean duration: 2 ms) presumed to be cholinergic were detected. Of the 73 neurons localized in the PPTg area, 53 had brief spikes and 20 broad spikes. Changes in activity most commonly occurred very early after the stimulus or during the reinforcement process. Most neurons with brief spikes exhibited very early excitation after the stimulus and reinforcement-related activity. These neurons had a mean activity of 23.7 impulses/s in the period preceding the stimulus. The onset of activation after the stimulus had a latency of 8.6+/-6.9 ms (mean+/-SD), with a range of 4-35 ms. In trials where the movement had to be delayed after the stimulus, the early activation disappeared or was considerably reduced, showing that it was context-dependent. A small proportion of neurons with brief spikes initially decreased activity after the stimulus, but with a latency >9 ms. All the neurons with broad spikes, except one, had reinforcement-related activity. Half of them showed exclusively reinforcement-related activity, the other half also early activation after the stimulus. These neurons were about half as active in the period preceding the stimulus occurrence than the neurons with brief spikes. The early context-dependent activation is discussed in relation to the excitatory projection of PPTg neurons on the subthalamic nucleus. The reinforcement-related activity, preferentially evidenced in broad spike neurons presumed to be cholinergic, is speculated to be associated with cholinergic projection of PPTg neurons to the dopaminergic neurons of the substantia nigra. Finally, the role of PPTg in the ongoing control of motor performance and reinforcement processes is discussed in relation to the basal ganglia circuitry.

The role of the pedunculopontine tegmental nucleus in relation to conditioned motor performance in the cat. II. Effects of reversible inactivation by intracerebral microinjections.

The effects of reversible pharmacological manipulation of the neuronal activity in the pedunculopontine tegmental nucleus (PPTg) on the performance of a conditioned movement was studied in two freely moving cats. The microinjections were given in regions where, in the same subjects, we had previously identified neurons with context-dependent early activity after a trigger stimulus and with reinforcement-related activity. The subjects were conditioned to perform a forelimb-flexion movement controlled by a simple reaction-time task. In addition, one subject was trained to execute the same flexion movement, but delayed after the trigger stimulus. Food pellets were used as the reinforcer. Lidocaine injections (1 microl of 2% solution, injected over a 6-min period) induced a transient arrest of performance within minutes. The cessation of performance could be preceded by behavioral signs such as meowing, attempt to escape from the experimental booth, licking, or stereotyped posture. No rotational behavior could be observed. The effects of lidocaine could be mimicked in one subject by an extinction procedure. Muscimol injections (two injections of 0.2 microg in 1 microl, tested in one subject) also induced arrest of performance, but the return to pre-injection level of performance could not be obtained within the time of the test session. The quantitative analysis of reaction times and of inter-trial intervals showed that altering PPTg activity affected inter-trial intervals, but only slightly affected the reaction times. It is speculated that the PPTg is involved in the reinforcement process related to selecting the appropriate motor program.

A modified HPLC method for the determination of vancomycin in plasma and tissues and comparison to FPIA (TDX).

A modified high performance liquid chromatography (HPLC) method for the quantification of vancomycin levels in plasma and tissues is described. The method uses solid phase extraction (SPE) of vancomycin from the samples and reversed phase HPLC with UV detection. The method was fully validated in terms of recovery, linearity, selectivity and various stability conditions. Vancomycin was determined in plasma samples obtained from 15 patients undergoing cardiopulmonary bypass, before and repeatedly during 12 h after drug administration. The vancomycin levels in plasma were measured by HPLC and by fluorescence polarization immunoassay (FPIA) (TDX). The following correlation was found: TDX = 0.84 HPLC + 1.04. The mean vancomycin levels in skin, fat, atrium, pericardium and sternum, before and after bypass, are reported.

Correlations between activity of pallidal neurons and motor parameters.

The relationships between the activity of 87 pallidal neurons and parameters characterizing motor performance were analysed quantitatively in six unrestrained cats performing a forelimb flexion movement controlled by a reaction-time paradigm. Three motor variables were considered: the static force exerted when the trigger stimulus occurred, the latency of the force change initiating the liver-release movement and the velocity of the force change. For all the cats, the latency of the change in force was the prevalent parameter in determining the movement onset occurrence. The single-unit activity was quantified on a trial-by-trial basis by measuring the mean firing rate in windows of fixed duration. Both normal (total) and partial linear correlation coefficients were used. Partial correlation coefficients were calculated between the mean firing rate during selected windows and each of the motor parameters. A total of 17% and 9% of all partial correlation coefficients tested (n = 1566) was found to be significantly different from zero, respectively with p < 0.05 and p < 0.01. The mean percentages of explained variation, measured by the square of the correlation coefficient, were 17% (p < 0.05) and 21% (p < 0.01). The 46 pallidal neurons presenting an increase in discharge after the trigger stimulus could be classified in homogeneous subgroups with respect to the signs of the correlations. Representations of the motor programme underlying reaction-time performance evidenced at the pallidal level further substantiate a specific involvement of the basal ganglia in the latency of motor triggering.

Activity of neurons of the subthalamic nucleus in relation to motor performance in the cat.

The activity of subthalamic nucleus neurons related to motor performance was studied in three unrestrained cats operantly conditioned to perform a lever-release movement. The movement was initiated either rapidly after the trigger stimulus (a brief sound) in a simple reaction-time paradigm or after a delay in trials identified by a tone cue. These paradigms were randomly presented. The activity of 171 neurons was recorded in the contralateral and in the ipsilateral subthalamic nucleus, with respect to the performing limb. The mean spontaneous activity of cells in the ipsilateral side (18.5 +/- 13.8 imp/s, mean +/- SD) was higher than that in the contralateral side (8.5 +/- 8.1 imp/s). A total of 145 cells (85%) presented significant changes in activity in relation to the lever-release movement (task-related cells). The remaining 26 cells were either related to other events of the task (n = 15; lever-press or reinforcement occurrence) or not related at all to the task performance (n = 11). The majority of changes of activity of task-related cells were initial increases in discharge, which started on average, 127 ms before movement onset and lasted several hundreds of milliseconds. These increases in discharge were more frequent in the contralateral side (75 of 80 task-related cells, 94%) than in the ipsilateral side (43 of 65 task-related cells, 66%). The changes in activity, either increases or decreases, occurred early after the trigger stimulus, since 62% of them had a latency of less than 100 ms. Although the mean latency of initial increases was rather similar in both sides (97 ms contralateral versus 104 ms ipsilateral), the contralateral side was characterized by a high proportion of very early responses (less than 20 ms). For most neurons, the early changes in activity described above were absent after the trigger stimulus in the delayed condition. For certain neurons, the changes in activity prior to movement were different in reaction-time condition and in delayed condition, showing that the pattern of activity preceding movement might depend on the temporal requirements for motor initiation. The results suggest that a significant proportion of subthalamic cells are involved in the preparation and the initiation phases of the lever-release movement studied, although other hypotheses (e.g. stimulus-related responses) cannot be definitely ruled out. The timings and patterns of the changes in activity observed in the subthalamic nucleus in the present study, and in the pallidal complex previously, cannot be explained easily by the classical scheme where the external pallidum inhibits the subthalamic nucleus. The results suggest rather that the subthalamic nucleus, driven by a yet-to-be-determined excitatory input, exerts an excitatory influence on the pallidum and plays a crucial role in the control of the basal ganglia output neurons.

The role of putamen and pallidum in motor initiation in the cat. I. Timing of movement-related single-unit activity.

The participation of basal ganglia in motor initiation was studied in six cats operantly trained to perform a ballistic flexion movement, triggered after a brief sound in a simple reaction time condition or delayed after the same sound in the presence of a tone cue. The activity of 356 neurons was recorded in the putamen and in the pallidum (globus pallidus and entopeduncular nucleus). A total of 19.4% of the neurons were not related to the conditioned flexion movement: they were either unrelated to the task (10.1%) or related to other periods of the motor performance such as trial beginning or reward delivery (9.3%). About 60% of the remaining neurons--defined as task-related--exhibited changes in firing rate that occurred, in the reaction time condition, less than 100 ms after the go signal and therefore began prior to movement onset. For most neurons, in the delayed condition, these early changes were absent, suggesting that their occurrence in the reaction time condition was not a sensory response but rather was related to the movement initiation. In addition, for many neurons these changes shifted in time, remaining time-locked to the movement. Correlations between these early changes in activity and motor parameters were demonstrated, suggesting that these changes were movement-related. For most neurons the firing levels observed during intertrial intervals and during foreperiod were similar. The mean discharge rate during the foreperiod was 19.2 impulses/s. Three patterns of activity were observed before movement: increases in discharge rate (61% of task-related neurons), transient decreases followed by increases (11%), or prolonged decreases (28%). Only minor differences were found between the characteristics of the populations of neurons recorded in the three sites under study: on average the neurons recorded in the globus pallidus were more active than the neurons recorded in the putamen or in the entopeduncular nucleus. The fact that, for certain neurons, the changes of activity prior to movement were different in reaction time condition and in delayed condition showed that the pattern of activity preceding movement might depend on the temporal requirements for motor initiation. Taken together with the motor effects obtained in the same task following GABA-receptor activation with muscimol microinjections in these structures, the present results suggest that putamen and pallidal neurons participate in the initiation of the conditioned movement under study.

GABA-receptor activation in the globus pallidus and entopeduncular nucleus: opposite effects on reaction time performance in the cat.

The possible role of GABAergic mechanisms in the control of the basal ganglia output structures, the globus pallidus (GP) and the entopeduncular nucleus (EP), was studied in cats performing a conditioned flexion movement triggered by an auditory stimulus. The effects of discrete unilateral microinjections of low doses of the GABAA receptor agonist (muscimol 5-100 ng/0.5 microliter) and antagonist (bicuculline methiodide 25-150 ng/0.5 microliter) in the GP and the EP were tested on the motor performance of eight animals trained to release a lever in a simple reaction time (RT) schedule after an auditory stimulus. Control injections in neighboring structures did not induce any effect except with five- to tenfold higher doses in the closest injection sites. The dose of 20 ng muscimol injected into the ventral and medial part of the GP produced an arrest of the performance after a few unsuccessful trials (over the RT reinforcement limit of 500 ms), while muscimol injected in sites located in the lateral GP resulted in a dose-dependent lengthening in RTs, with a concomitant increase in the force change latency. In most of the subjects, the force exerted on the lever was higher after muscimol than after vehicle injection. Force change velocity was then significantly increased. In contrast, muscimol injected in the ventral and rostral region of the EP produced a decrease in RTs or a complete cessation of responding after a high number of anticipatory responses (release of the lever before the trigger stimulus). No significant changes in the force change latency could be observed while there was a non-significant tendency for the force levels to be lowered. Bicuculline injections in the EP were found to increase RTs with a concomitant increase in force change latency and a slowness of velocity, while no significant effect was observed following injections in the GP. These results suggest that a balance between GABAergic activity in the two output nuclei of the basal ganglia, the GP and the EP, is crucial for the correct initiation and execution of the conditioned motor task.

Use of fractal geometry to determine effects of surface morphology on drug dissolution.

We extend and modify the Noyes-Whitney equation and the Hixon-Crowell cube root law to include surface roughness effects on the dissolution rate of drugs. The problem is treated theoretically in terms of the fractal reaction dimension, DR, of the effective surface which undergoes dissolution. Effects of changes in DR on the dissolution rate are identified and explained.

Fractal analysis of size effects and surface morphology effects in catalysis and electrocatalysis.

Fractal geometry tools are used in order to analyze several related problems in surface science, catalysis, and electrocatalysis. The effects of complex morphologies of adsorbents, catalysts, and electrodes on various molecular processes with these materials are determined both theoretically and experimentally. It is shown that fractal geometry provides a convenient and natural tool for the elucidation of geometry-performance relations in heterogeneous chemistry. Issues covered are particle size effects in physisorption and chemisorption; morphology effects on a variety of catalytic processes with unsupported catalysts (including coal liquefaction, alkene polymerizations, oxidations, dehydrogenations, and esterifications); surface accessibility effects on molecular interactions in an Eley-Rideal mechanism; surface patterning effects on concentration profiles near the surface; and electrode-morphology effects on a variety of electrochemical and electrocatalytic processes. The domains of applicability of the fractal approach to these problems is discussed.

Changes in motor performance and rubral single unit activity in cats after microinjections of serotonin into the red nucleus area.

The serotonergic control exerted on the red nucleus (RN) was studied in unrestrained cats during the performance of a simple reaction time task which consisted of releasing a lever in response to an auditory go-signal. The effects of microinjections of serotonin-oxalate salt into the rubral area on the motor activity and on the firing of neurons recorded concomitantly in the red nucleus were investigated. Injections of serotonin (5-HT) (200-400 ng) into the red nucleus or its dorsal border induced subtle alterations in the conditioned motor performances but had no major effects on the spontaneous motor behavior. The changes in the conditioned motor output (an increase in the static force exerted on the lever and a speeding up of the lever release) are reminiscent of the facilitatory influence of serotonin on various motor reflexes previously reported. Changes in the neuronal activity were observed concomitantly with the effects on the motor output: 5-HT either enhanced or reduced the firing rate of the rubral neurons. These effects were apparently dependent on the discharge pattern of the neurons during the static motor activity. The results suggest that the serotonergic input to the red nucleus may participate in motor control by exerting a dual modulatory action on the activity of rubral neurons.

GABAergic control of rubral single unit activity during a reaction time task.

The activity of 61 rubral neurones was recorded in association with microinjections of GABA, muscimol, bicuculline-methiodide or saline, in cat Red Nucleus area, during the performance of a reaction time task. The depressing action of GABA and muscimol on the firing of most neurones (17/23) suggests that, in a behavioural situation, an inhibitory GABAergic control can be exerted on rubral neurones discharging with different patterns during the reaction time task. The motor slowing down induced by GABA and muscimol is in agreement with a general reduction of the rubral output. Injections of bicuculline, whose antagonistic effects on GABA transmission are well established in the Red Nucleus, had various consequences on the firing of rubral neurones: 1) the decreases of activity related to the reaction time task were never suppressed, suggesting that these task-related inactivations are probably not mediated by GABA A receptors; 2) an enhancement of the tonic and phasic discharges was found for 1/4 of the neurones (7/29), which were either activated or not modulated in relation to the reaction time task, suggesting that a sustained GABA A-mediated inhibition, blocked by bicuculline, could be exerted on these neurones; 3) a reduction of the tonic and phasic discharges was observed for other neurones (15/29), which were either activated, inactivated or not modulated in relation to the reaction time task, suggesting that the activity of these neurones could be controlled by inhibitory processes not mediated by GABA A receptors, possibly enhanced or released by bicuculline. The delay in motor triggering induced by bicuculline could be related to the disruption of the pattern or rubral output during the reaction time task, as a result of the opposite changes affecting the firing of rubral neurones. A well-balanced GABAergic activity appears to be critical in the control of rubral firing during the performance of the reaction time task.

GABAergic mechanisms in the cat red nucleus: effects of intracerebral microinjections of muscimol or bicuculline on a conditioned motor task.

Interneurons in the Red Nucleus (RN) are known to be under cortical control and to exert an inhibitory action, mediated by GABAergic mechanisms, on the main output towards the spinal cord. The effects of discrete injections of a GABA receptor agonist (muscimol) or an antagonist (bicuculline) in the Red Nucleus were tested on a motor task performed by seven cats. The subjects were trained to release a lever with a flexion movement of the forelimb controlled by a reaction time (RT) paradigm. Muscimol as well as bicuculline increased RTs in a dose-dependent manner at doses below 100 ng. However the parameters of the force exerted on the lever were differentially altered by the two drugs. Muscimol increased RTs by slowing down the force change preceding movement as well as slightly delaying its latency. While bicuculline increased drastically the force change latency. It could also speed up the force change velocity for low doses. At higher doses (up to 500 ng) both drugs produced an arrest of the performance either associated with anxiety signs (bicuculline) or dystonic movements of the head followed by body rotations (muscimol). The strong motor impairments as well as the disruption of the conditioned performances following muscimol or bicuculline microinjection in the RN suggest an important functional role for GABAergic interneurons. Under the control of cortical afferences they can modulate rubrospinal activity and participate in the triggering of a conditioned movement.

Cat red nucleus activity preceding movement depends on initiation conditions.

The activity of 98 Red Nucleus neurons was recorded in 3 cats operantly conditioned to perform a ballistic forelimb flexion movement triggered after a brief sound in a simple Reaction Time condition, or Delayed after the same sound in the presence of a tone cue. Fifty-eight task related neurons presented changes of activity in either one or both conditions. Forty-four of them were studied quantitatively and classified in 3 categories: 1) only 16% of the units presented similar changes of firing preceding the triggered or delayed movement; 2) most units (55%) presented different changes of activity in the two conditions: in the Delayed condition, the activation occurred earlier before the movement, and/or the change in magnitude was reduced or the pattern of activity was modified; 3) moreover, for 29% of the units, the change of activity observed before movement in the Reaction Time condition was severely reduced or even absent in the Delayed condition. For some of these neurons a building-up of activity was observed very early in the Reaction Time condition, during the preparatory period, well before the occurrence of the conditioned stimulus. These results show that the Red Nucleus activity preceding a movement is clearly dependent on its initiation conditions. The distinct patterns of unit firing observed in the Reaction Time condition and in the Delayed condition are tentatively related to the different preparation and initiation constraints determined by the behavioral conditions.

Participation of the red nucleus in motor initiation: unit recording and cooling in cats.

Cats were trained to release (Go) or not to release (No-go) a lever after a brief auditory signal, depending on the presence of an additional tone (No-go cue). Unit recording and cooling were made in the red nucleus (RN) contralateral to the performing limb. Three major results were found: (1) in the Go condition, we observed phasic increases of rubral firing, with a constant latency after the auditory signal and with an amplitude correlated to the latency of motor triggering (i.e. reaction time, RT); the tonic activity preceding the auditory signal could also be correlated to the RTs for some units; (2) in the No-go condition, there was no phasic increase of rubral firing after the auditory signal; the tonic activity during the presentation of the No-go cue was markedly decreased compared to the Go trials; (3) cooling of the RN increased the RTs and could also block the motor triggering. These results suggest that the RN is involved in setting and triggering a conditioned motor response according to sensory cues.

Activity of caudate nucleus neurons in cat performing a reaction time task.

During a reaction time task, single units were recorded in the caudate nucleus of freely moving cats. Neuronal changes of activity were related to CS, to initiation of movement or to reinforcement. It is suggested that these changes of activity are involved in a process in which informations on the CS and on the on-going movement are associated with information on the occurrence of reinforcement.

Cat red nucleus changes of activity during the motor initiation in a reaction time task.

Unit recordings were made in the posterior red nucleus of 4 freely moving cats, performing a conditioned forelimb flexion in response to a sound, in a simple reaction time (RT) paradigm. More than 50% of the recorded neurons (154) were found to be task-related, with marked changes of firing (mostly increases) time-locked with the go-signal (mean latency: 40.8 ms +/- 12 ms SD) and frequency-correlated with the duration of the RT. These RT-correlated changes of firing disappeared or were markedly decreased when the movement was not triggered. Most of the task-related neurons also displayed late changes of firing during the force change, time-locked with the movement. Furthermore, a clear relation was observed between the rubral firing before and after the go-signal and the sensorimotor field of the recorded neuron: the neurons preferentially related to limb or trunk movements discharged with the highest frequency throughout the conditioned sequence and exhibited the best RT-correlated increases of firing during the movement initiation. In contrast, rubral neurons apparently related to head-movements generally discharged with low frequency and commonly exhibited decreases of firing in the initiation period. The changes of rubral firing prior to the motor activity, correlated to the RTs, suggest a command function for the RN, in the triggering of the conditioned motor response.