Feasibility and safety of vagal stimulation in monkey model.

The feasibility, safety, and preliminary effects of chronic vagal stimulation were studied in an aluminagel monkey model. Pilot studies to perfect the equipment, determine stimulation thresholds, and insure the comfort and safety of the animals preceded this study. Four monkeys were equipped with an indwelling, 2-electrode cuff (titanium bands spaced 7 mm apart; silicone encased; 1.5 cm total length) in contact around the right vagus nerve; avoidance of the cardiac branch was confirmed by electrocardiograms. After postsurgical recovery, the intact and awake animals received constant-current stimulation (5 mA; 83 Hz, 143 Hz, or 50-250 Hz randomly; 0.5-ms pulse width) at the onset of every spontaneous seizure for the duration of the seizure or every 3 h for 40 s if stimulation had not occurred in the preceding hour. Stimulation periods of 2-6 weeks, with differing levels of stimulation, were preceded and followed by at least a 2-week baseline period of no stimulation. During the stimulation periods, the seizure rate decreased to zero in two monkeys and the interseizure intervals became invariable in the remaining two monkeys. These effects carried over temporarily into the poststimulation baseline periods. Vagal stimulation had no consistent effects on seizure severity or EEG interictal spikes. Histological studies of six vagus nerves were unable to separate electrode cuff damage from any direct effects stimulation may have had on the nerves. Although it appears that chronic vagal stimulation is feasible and that epileptogenic processes are influenced, the safety and efficacy of the procedure are still in question.

Slow-speed EEG for chronic monitoring of clinical seizures in monkey model.

A method for utilizing slow-speed EEG (1/4 mm sec) to detect and quantify gross-motor clinical seizures in a chronic monkey model is described. The technique is particularly useful in the detection of small clinical seizures that may occur in studies of drug efficacy. It estimates the reams of paper generated by standard EEG recording (30 mm/sec) that heretofore precluded easy data collation and thereby essentially prevented continuous monitoring of EEG paroxysms. (This method, however, does not allow the detection of single or non-clumped interictal spikes.) The headplug and recording procedures employed in our laboratory are detailed. The technique can be used alone or, for greater precision, in conjunction with either the recording of motor-activity envelopes or a videotape seizure-confirmation system, or both (Lockard and Barensten, 1967; Lockard et al., 1976c). Unlike our motor-activity and closed-circuit TV method for detecting clinical seizures, the technique of slow-speed EEG could be easily employed in other laboratories already equipped with EEG polygraphs. This method also permits the simultaneous recording of slow-speed and standard-speed EEGs (on separate polygraphs) to facilitate periodically the discrimination between artifacts and clinical seizures.

Cinromide's metabolite in monkey model: gastric administration and seizure control.

In a previous study (Lockard et al., 1979) Cinromide (3 bromo-N-ethylcinnamamide), an experimental anticonvulsant (Burroughs-Wellcome Pharmaceutical Co.), was given a preliminary evaluation. Since that research was concerned primarily with EEG paroxysms, the present study was conducted to address drug efficacy in terms of clinical seizures. Cinromide's major metabolite (3-bromocinnamamide, BC) was the main focus. Eight alumina-gel monkeys were given by gastric bolus every 6 hr for 10 days (Phase I) either the solvent alone (Tween 80), Cinromide (BEC), or its synthetic metabolite (SBC). Subsequently (Phase II), four animals were given BEC or SBC by chronic gastric infusion for 20 days. In both phases Cinromide's metabolite (either via BEC or especially SBC) was effective in half of the animals in reducing seizure frequency and/or duration at plasma levels above 5 mcg/m. The data suggest that the drug's efficacy is individually specific. Another species of Cinromide metabolism, 3-bromocinnamic acid, is also discussed.

Clonazepam in a focal-motor monkey model: efficacy, tolerance, toxicity, withdrawal, and management.

Since the clinical data have been equivocal in regard to the effects of clonazepam (CZP) in focal-motor seizures, an alumina gel monkey model was used to evaluate quantitatively its efficacy with respect to this seizure category. The insolubility of CZP and its short biological half-life in monkey necessitated its evaluation in the model via constant-rate intravenous administration in a solution of polyethylene glycol 400 (PEG). Two groups of monkeys were given CZP in PEG (N = 6) or a PEG solution alone as a control compound (N = 5) for 6 weeks; these treatments were bordered at both ends by 3 weeks of treatment with saline only in order to establish a baseline. CZP was administered at a concentration sufficient to achieve a plasma level of 30 ng/ml in drug step I (3 weeks) and at least double that level in drug step II (3 weeks). As a solute for CZP, and when given by itself, PEG was always administered at a concentration of 35%. The results indicate that CZP is effective for focal-motor seizures and secondarily generalized tonic-clonic seizures, particularly when its concentration in plasma is higher than 60 ng/ml. Withdrawal seizures were evident on cessation of CZP administration. CZP appears to be a useful broad-spectrum anticonvulsant when managed carefully. An unexpected finding was the irreversibility of the pharmacological effect of PEG. Cessation of PEG administration significantly reduced seizure frequency in subsequent weeks to a level below the initial baseline level.

Experimental anticonvulsant cinromide in monkey model: preliminary efficacy.

Cinromide (3 brono-N-ethylcinnamide), an experimental anticonvulsant (Burroughs-Wellcome Pharmaceutical Co.), was given a preliminary evaluation in our alumina-gel monkey model. The parent drug has a biological half-life in monkey of 1-2 hr and its active metabolite, 3-bromocinnamide, a half-life of 4-6 hr. In phase 1, 6 chronically epileptic monkeys, with focal motor and secondarily generalized tonic-clonic seizures, received the drug in a vehicle of 65% polyethylene glycol 400 (PEG) by constant-rate intravenous infusion followed by baseline days of saline only and PEG only. Three different concentrations of Cinromide (12, 24, and 36 mg/ml/hr) were administered, respectively, to achieve mean steady state plasma levels of approximately 5, 10 and 20 micrograms/ml of the metabolite (0.5 to 5.0 micrograms/ml of the parent drug). In phase 2, Cinromide was administered for 7 days at the middle concentration to all monkeys. Baseline periods similar to those of phase 1 were used as controls. The data tentatively suggest that Cinromide is efficacious in the monkey model at a plasma concentration range of 7-14 micrograms/ml of the metabolite. With the exception of one animal, no secondarily generalized seizures were exhibited during drug administration (but were evident in the baseline periods), and EEG bursting decreased significantly in several monkeys. Minimal side effects were manifested at these plasma levels but withdrawal seizures were evinced with cessation of the drug. Further evaluation of Cinromide by gastric administration in our animal model is planned.

Cerebellar stimulation in alumina-gel monkey model: inverse relationship between clinical seizures and EEG interictal bursts.

The efficacy of cerebellar stimulation was addressed in a chronic monkey model (N = 12) of spontaneous focal motor and secondarily generalized seizures using 24 hr seizure frequency monitoring and all-night EEG recording. The anterior cerebellar vermis was stimulated employing parameters similar to those used in man, 10 Hz, 1 msec pulses, 10 min on, 10 off, at an average current of 2.0 mA. Six weeks pre- and post-base-line periods were compared to a stimulation period of the same length. The results contribute to a clarification of conflicting findings of previous researchers by revealing an inverse relationship between seizure frequency and interictal EEG bursts during the weeks of stimulation. Seizure frequency increased significantly and interictal bursts decreased. Both of these effects (especially the former) were evident in the post-stimulation period, but for different reasons than hypothesized for the period of stimulation. Whereas the therapeutic value of cerebellar stimulation on seizures may be in question, its utilization in the study of mechanisms of epilepsy may be warranted.

Carbamazepine revisited in a monkey model.

In a previous study on carbamazepine (Lockard et al., 1974), the problem of its low bioavailability in solid form and its short half-life in monkey were addressed. The present research was designed to evaluate carbamazepine under constant-rate intravenous infusion in our alumina-gel monkey model. Since carbamazepine is insoluble in an aqueous solution, polyethylene glycol 400 was used as the vehicle for administration of this drug to a group of 8 epileptic monkeys. The attenuation of seizures by carbamazepine was not statistically significant since the serum levels of carbamazepine after enzyme induction were less than 2.0 micrograms/ml. This study (a) illustrates that some problems in drug evaluation may be insoluble with our present technology even though we are cognizant of them; (b) makes explicit the fact that the efficacy of carbamazepine is a function of adequate serum levels; (c) demonstrates endogenous oscillations of carbamazepine serum concentrations; and (d) reports simultaneous serum levels of carbamazepine and its 10--11 epoxide in the monkey model.

Efficacy and toxicity of the solvent polyethylene glycol 400 in monkey model.

Several antiepileptic drugs, such as carbamazepine and clonazepam, have low bioavailability in solid form and are insoluble in an aqueous solution. Alcohol solvents are often employed as vehicles when these drugs are studied in animal models. Secondary and particularly tertiary alcohols are suspected of some anticonvulsant activity. The present research evaluated the possibility that polyethylene glycol 400 (PEG 400) might be efficacious, toxic, or both. Monkeys (N = 11) rendered epileptic by aluminum-hydroxide were administered PEG 400 by constant rate (1 ml/hr) intravenous infusion for 3--4 weeks, preceded and followed by several weeks of baseline. At a concentration of 60%, PEG 400 significantly reduced seizure frequency, but also exhibited severe side effects. These findings suggest that experimental testing of anticonvulsants may be compromised when this or similar solvents are used chronically.

Time-dependent kinetics III: diurnal oscillations in steady-state plasma valproic acid levels in rhesus monkeys.

Valproic acid was administered by constant rate intravenous infusion to catheterized chained rhesus monkeys for 8-10 weeks under controlled environmental conditions. Steady-state plasma levels were monitored at 2-hr intervals for 26 hr (10 am-12 noon on the following day), 1 day/week for 6 weeks. Individual steady-state plasma concentration-time plots exhibited the following characteristics. During Period A (10 am-6 pm), plasma levels remained stable or decreased. During Period B (6 pm-6 am), plasma levels increased, reached a maximum, and remained markedly higher than during Period A. The maximum concentrations were 40-140% higher than the observed minimum concentrations. During Period C (6 am-noon), plasma levels tended to decline from the maximum concentrations achieved in Period B. In most cases, plasma concentrations at 10 am and 12 noon of the 2nd experimental day fell within 10% of their respective values on the previous day. The mean (+/- SD) periods obtained by cross-correlation analysis of individual plasma concentration-time plots were 30.7 (+/- 3.7) and 22.8 (+/- 3.6) hr for Animals 903 and 923, respectively. The corresponding mean (+/- SD) amplitudes were 27.3 (+/- 12.6) and 17.4 (+/- 2.3)%. A circadian rhythm in total body clearance was hypothesized, and its pharmacokinetic implications are discussed.

Diurnal variation of valproic acid plasma levels and day-night reversal in monkey.

Four normal monkeys each equipped with an EEG plug and two indwelling catheters for drug infusion and sampling, respectively, were administered valproic acid (VPA) before and after a 12-hr light, 12-hr dark phase shift. Before day-night reversal, diurnal oscillations of VPA plasma levels under steady-state intravenous constant-rate infusions were 30-50%, with maximum concentrations during the dark phase of the cycle. After reversal, maximum VPA plasma concentrations tended to follow the dark phase shift. The correlation was not perfect, nor was the sleep cycle completely reversed since the animals slept less after the phase shift. Possible mechanisms of the diurnal plasma level fluctuations and the importance of oscillations of this magnitude to clinical drug regimens are discussed.

Prophylaxis with diphenylhydantoin and phenobarbital and alumina-gel monkey model. I. Twelve months of treatment: seizure, EEG, blood, and behavioral data.

Utilizing an alumina-gel epileptic monkey model, with instrumentation for continuous monitoring of all overt, spontaneous motor seizures, the efficacy of pharmacologic prophylactic treatment of posttraumatic epilepsy was explored. The alumina-gel model provides a relatively standardized brain trauma from monkey to monkey, resulting in virtually complete assurance that all animals will manifest, in time, electrical and clinical seizures if not treated. Thirteen rhesus monkeys were divided into two groups of 8 drug-treated and 5 placebo animals, respectively. Administration of diphenylhydantoin and phenobarbital in a combined regimen commenced within 48 hr of the alumina-gel injections. After 1 year the monkeys were withdrawn from either their drugs or placebo and followed for a subsequent 4 month period. The data for the first 12-month period indicate that anticonvulsant treatment of potentially epileptic monkeys decreased both the frequency and severity of seizures they would have had without treatment. All animals manifested an electrical focus and overt seizures, but the drug monkeys had only partial seizures whereas the placebo monkeys exhibited secondarily generalized tonic-clonic seizures. The follow-up, no-treatment data of 4 months are reported in the following paper.

Prophylaxis with diphenylhydantoin and phenobarbital in alumina-gel monkey model. II. Fourth-month follow-up period: seizure, EEG, blood and behavioral data.

This study, a 4-month follow-up period of a 12-month treatment study by the present authors, was concerned with the permanent effects of treatment with diphenylhydantoin and phenobarbital in the alumina-gel monkey model. Whereas the 8 drug animals during withdrawal increased their seizure frequency, duration, and severity, those 4 animals having received 120 mg/kg/day DPH in weeks 6-12 had one-half the number of seizures of the 4 placebo monkeys in the follow-up period. The other 4 drug animals who had continued to receive 60 mg/kg/day DPH during those weeks had two to four times the number of seizures of the placebo group during posttreatment. (All drug monkeys received 80 mg/kg/day of DPH from weeks 13-52 and 6 mg/kg/day of phenobarbital throughout the 12-month treatment period). The results reaffirm the problems of drug withdrawal and the importance of altering seizure mechanisms with sufficiently high doses of efficacious anticonvulsants rather than merely treating epileptic manifestations at lower doses.