Reversal of Behavioral Effects of Pentylenetetrazol by the Neuroactive Steroid Ganaxolone 1

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1 /98/ $00.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 3 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 284: , 1998 Reversal of Behavioral Effects of Pentylenetetrazol by the Neuroactive Steroid Ganaxolone 1 MARJOLEIN BEEKMAN 2, JESSE T. UNGARD, MACIEJ GASIOR 3, RICHARD B. CARTER, DURK DIJKSTRA 2, STEVEN R. GOLDBERG and JEFFREY M. WITKIN Drug Development Group (J.T.U., M.G., S.R.G., J.M.W.), Preclinical Pharmacology Laboratory, Addiction Research Center, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland and Department of Medicinal Chemistry (M.B., D.D.), University Centre of Pharmacy, Ant. Deusinglaan 1, The Netherlands and Department of Pharmacology (R.B.C.), CoCensys Inc., Irvine, California Accepted for publication November 10, 1997 This paper is available online at ABSTRACT Neuroactive steroids are naturally occurring or synthetically derived compounds many of which have anticonvulsant, anesthetic, anxiolytic, analgesic or hypnotic properties. The major site of neuronal activity appears to be with a specific steroidsensitive site on the -aminobutyric acid A receptor/chloride ionophore complex. Ganaxolone (3 -hydroxy-3 -methyl-5 pregnan-20-one) is a synthetic neuroactive steroid protected from metabolic attack of the 3 position. Ganaxolone is an efficacious anticonvulsant agent in a variety of acute seizure models, as well as in electrical and chemical kindling models, and is currently under Phase II clinical investigation for epilepsy. A prior observation that ganaxolone appeared to reverse the marked behavioral changes induced by the convulsant pentylenetetrazol (PTZ) was systematically examined in the present study. A model to quantify PTZ-induced behaviors is described and used to evaluate ganaxolone in comparison with the anticonvulsants valproate, ethosuximide, clonazepam, diazepam and phenobarbital. All compounds were compared using dose equivalents based on their respective ED 50 values in NAS are steroidal compounds that alter excitability in the CNS. They occur naturally as metabolites of the hormones progesterone and desoxycorticosterone or are synthetically derived compounds such as the anesthetic alphaxalone (Paul and Purdy, 1992; Lambert et al., 1995). NAS are fast-acting (Selye et al., 1941) and appear to exert their neuronal effects Received for publication April 29, Animals used in these studies were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals as promulgated by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. 2 Partly supported by the Netherlands Organization for Scientific Research (NWO). 3 A Visiting Fellow in the NIH Visiting Program granted from Fogarty International Center, Bethesda, MD. Dr. Maciej Gasior s permanent affiliation: Department of Pharmacology, Medical University School, Lublin, Poland. preventing convulsions induced by 70 mg/kg PTZ. The ED 50 and lower doses of ganaxolone prevented the observed behavioral effects of PTZ as well as its depressant effects on locomotor activity and rearing of mice. In contrast, the other anticonvulsants, if effective, were much less potent. Strikingly, most of the other anticonvulsants were incapable of preventing all the behavioral effects of PTZ. Only phenobarbital prevented all the behavioral effects of PTZ and only at doses 4 to 8 times the anticonvulsant ED 50. Rather than normalizing behavior as ganaxolone did, however, phenobarbital resulted in supranormal behavioral responses (e.g., increases in activity). Repeated administration of PTZ did not decrease the protective efficacy of ganaxolone. The results document the unique pharmacological profile of ganaxolone and suggest additional potential benefits from its use as an antiepileptic. Furthermore, because behavioral effects of PTZ have been used to model anxiety and anxiety associated with withdrawal from drugs of abuse, ganaxolone may find additional therapeutic application in those areas. through a nongenomic pathway that is independent of membrane perturbation and intracellular receptors (Gee et al., 1988; Lambert et al., 1995). It is now generally accepted that many NAS work by a selective interaction with the GABA A receptor, as was first demonstrated for alphaxalone (Harrison and Simmonds, 1984). NAS can either antagonize the Cl conductance, as for example with the sulfate ester of pregnenolone, possibly by acting on the picrotoxin site (Harrison et al., 1987; Majewska and Schwartz, 1987; Majewska, 1992; Mienville and Vicini, 1989; Wu et al., 1990), or potentiate the Cl influx (Morrow et al., 1987, 1988, 1989; Olsen et al., 1988), by increasing the probability that the Cl channel will enter an open state of long duration (Barker et al., 1987; Twyman and MacDonald, 1992) as well as increasing the frequency of channel openings (Twyman and MacDonald, 1992). The GABA-potentiating NAS are thought to act through a steroid site on the GABA A receptor that is distinct ABBREVIATIONS: ED, effective dose; GABA, -aminobutyric acid; NAS, neuroactive steroids; PTZ, pentylenetetrazol; TBPS, t-butylbiclyclophosphoorothionate. 868

2 1998 Behavioral Effects of Ganaxolone 869 from both the benzodiazepine (Harrison and Simmonds, 1984; Cottrell et al., 1987b) and barbiturate sites (Cottrell et al., 1987b; Gee et al., 1987, 1988; Peters et al., 1988; Turner et al., 1989). These GABA-potentiating NAS form an interesting class of compounds with potential clinical use as anesthetics, anticonvulsants, anxiolytics, hypnotics and analgesics (for reviews on NAS, see Simmonds, 1991; Majewska, 1992; Paul and Purdy, 1992; Gee et al., 1995; Lambert et al., 1995). Among the most potent ligands in this class is allopregnanolone (3 -hydroxy-5 -pregnan-20-one), a major metabolite of progesterone. Allopregnanolone has been shown to protect against convulsions induced by PTZ, bicuculline, picrotoxin, nicotine, strychnine and cocaine (Belelli et al., 1989; Luntz-Leybman et al., 1990; Kokate et al., 1994; Gasior et al., 1997a). However, allopregnanolone has poor oral availability, presumably because of metabolism at the 3 -hydroxy position. To improve bioavailability, the 3 -methyl analog has been synthesized. This compound, ganaxolone, is a potent and efficacious anticonvulsant agent with predicted utility in the control of generalized absence and partial seizures (Carter et al., 1997) as well as for convulsions due to cocaine poisoning (Gasior et al., 1997a). Ganaxolone is now undergoing Phase II clinical trials. In addition to preventing seizures induced by acute chemical challenge, ganaxolone blocks the development and expression of electrical and chemical kindling (Carter et al., 1997; Gasior et al., 1997a, b). Ganaxolone not only was effective in preventing seizure kindling but also appeared to block the behavioral sequella resulting from PTZ administration. Diazepam and valproate, in contrast, were not as potent or efficacious in altering seizure kindling and did not appear to affect the behavioral changes induced by PTZ challenge. In order to quantify these observations, we have developed a model to describe behavior in mice after the administration of PTZ and to compare the effects of ganaxolone on this behavior to those of other, clinically used anticonvulsants with different mechanisms of action. The results of these studies document both the quantitative and the qualitative superiority of ganaxolone in preventing the behavioral sequella engendered by high doses of PTZ given acutely or repeatedly. Material and Methods Subjects and treatments. Male Swiss Webster mice (Taconic Farms, Germantown, NY), weighing 30 to 50 g, resided in groups of six within a temperature-controlled vivarium on a 12-h light-dark cycle. The experiments were conducted during the light phase. All mice were naive and were utilized only once except in the repeateddosing experiments. Animals received a dose of one of the test compounds s.c., followed by an i.p. injection of PTZ. Ganaxolone was given 15 min before PTZ, and the other compounds were given 30 min before. Pretreatment times were based on efficacy against the acute convulsant effects of PTZ (Gasior et al., 1997a). The doses that were tested are multiples of the ED 50 values of the anticonvulsants in preventing convulsions induced by the acute administration of 70 mg/kg PTZ (Gasior et al., 1997a). Depending on the effect of 1 ED 50, subsequent doses were either increased or decreased. To determine the effects of the compounds alone, a dose of 1 ED 50 and the highest dose or doses tested were also given without PTZ. Eight animals participated in each treatment. Immediately after the PTZ injection, the animals were put separately into Digiscan activity monitors (Omnitech Electronics, Columbia, OH) with a surface area of cm and with photoelectric detectors placed 2.5 cm apart along the perimeter and 10 cm (vertical activity) above the floor. Data on the total distance traveled and vertical activity were collected at 10-min intervals for 30 min. During the recording of the locomotor activity, the animals were observed for 2-min periods at t 5 min (immediately after all members of the group received injections), t 15 min and t 30 min after the administration of PTZ. Mice were observed during these periods for the presence or absence of the following postures and/or behaviors: 1) walking, 2) rearing, 3) grooming, 4) sleeping (defined as a posture in which the mice were curled up with the top of their head on the floor), 5) sitting in a corner with the tail curled around the body so that the tip of the tail was under the nose, 6) sitting or lying, not necessarily in a corner, without moving at least once every 15 s, 7) lying in a way that at least one of the back limbs was clearly visible, 8) the occurrence of hiccups, or small twitches of the body, 9) the tail being in line with the rest of the body, 10) sitting or lying with the nose facing the corner and 11) sitting or lying with the tail pressed along a wall of the locomotor cage. A pilot study indicated that behaviors 1 to 5 were characteristic of saline-treated mice, whereas behaviors 6 to 11 characterized PTZ-treated mice whether or not PTZ led to clonic convulsions. In addition, we determined whether the results were influenced by repeated administration of drugs over the time period in which seizure kindling develops. In this study, the anticonvulsant ED 50 dose of ganaxolone was compared with that of diazepam. Here, the same animals were used on days 1, 3, 5 and 8. On these days the animals were injected with ganaxolone, diazepam or vehicle s.c., 15 min (30 min in the case of diazepam) before an i.p. injection of PTZ or vehicle. The animals were immediately placed in the activity monitors, and data were obtained every 10 min for a 50-min period. At t 5, t 15, t 30 and t 45 min, the mice were observed for a 2-min period for the presence or absence of the behaviors noted above and for the occurrence of clonic convulsions (rapid clonic forelimb and hindlimb movement lasting continuously at least 5 s each, with accompanying loss of righting response). Drugs. PTZ (Sigma Chemical Co., St. Louis, MO) was dissolved in sterile saline. Ganaxolone (CCD , CoCensys Inc. Irvine, CA) was dissolved in 40% w/v hydroxypropyl- -cyclodextrin (Research Biochemicals International, Natick, MA) in saline. Diazepam (Hoffmann LaRoche, Nutley, NJ) and clonazepam (Sigma) were dissolved in 20% v/v propylene glycol (Sigma) in saline. Phenobarbital sodium (Ruger Chemical Co., New York, NY) was dissolved in sterile saline. Ethosuximide (Research Biochemicals) was dissolved in sterile water, with a minimal amount of Tween 80. Valproic acid (Sigma) was dissolved in sterile saline. The appropriate drug vehicles were used in control experiments for evaluation of the effects of the different drugs used in this study. Mild heating, stirring and sonication aided the dissolution of the compounds into solution. Drug doses are expressed as the drug forms noted. Injection volumes were 0.01 ml/g b.wt. PTZ was administered in a dose of 45 mg/kg, the same dose that was used in the chemical kindling experiments in which the initial observations on the anti-ptz-like behavioral effects of ganaxolone were made (Gasior et al., 1997b). The other drugs were used in multiples of the anticonvulsant ED 50 value, as determined by Gasior et al. (1997a) or as experimentally determined in the present study against a higher dose of PTZ. The anticonvulsant ED 50 is the dose that protected 50% of animals from clonic convulsions induced by 70 mg/kg PTZ. Using anticonvulsant potency as a basis for comparing, dose to dose, these drugs against the behavioral effects of PTZ, we uncovered a potential dissociation, as suspected, between the anticonvulsant effects and the anti-ptz-like behavioral effects of these drugs. Data analysis. Every animal in each observation period was characterized as either saline-like, PTZ-like or mixed according to the following criteria. Animals that exhibited one or more of

3 870 Beekman et al. Vol. 284 TABLE 1 Analysis of individual behavioral components of PTZ-induced behavioral changes as a function of drug treatment The numbers of animals (mean S.E.M.) that displayed specific behaviors in one of the four observational periods were summed to yield a total theoretical maximal score of 32. Behaviors 1 to 5 represent saline-like behaviors, and behaviors 6 to 11 represent PTZ-like behaviors. Ganaxolone and diazepam were given as pretreatments 15 min and 30 min, respectively, before the administration of 45 mg/kg PTZ. Drugs were administered in doses equivalent to their ED 50 values for protection against convulsions engendered by 70 mg/kg PTZ. * indicates a significant difference (P.05; Dunnett s one-tailed test) from PTZ alone. Saline-like Features Vehicle Vehicle Vehicle PTZ Ganaxolone PTZ Diazepam PTZ 1 Walking * * Rearing * * Grooming * * Sleeping Tail curled PTZ-like Features 6 Motionlessness * * Hindlimbs visible * * * 8 Hiccups Tail straight * * * 10 Nose to corner * * Tail along wall * * * behaviors 1 to 5 and none of behaviors 6 to 11 (table 1) were considered saline-like, and animals exhibiting two or more of behaviors 6 to 11 and none of behaviors 1 to 5 were defined as PTZ-like. Animals that exhibited behaviors 1 to 5 and behaviors 6 to 11 or exhibited only one of the PTZ-like characteristics were considered mixed. The number of animals that were PTZ-like was compared with the number of PTZ-like animals in a control experiment (vehicle PTZ) using the Fisher exact probability test. Dose-effect curves and ED 50 values with associated 95% CL were analyzed by the methods of Litchfield and Wilcoxon (1949). The locomotor data (horizontal distance traveled, in centimeters, and counts of vertical activity) were summed per mouse, starting at 10 min after placement in the activity monitors. Means and S.E.M. were derived from these sums per treatment per day. Analysis of variance was used to determine whether the effects were dose-dependent. Dunnett s comparison of treatments with a standard was used to show the significance of differences between the drug doses and the PTZ vehicle control standard. Occasional comparisons with a vehicle vehicle control group were made by a one-tailed t test. ED 50 values and 95% CL were determined by linear regression. For the kindling experiment, one-tailed t tests were used to compare drug and control data for the same time interval or day. The difference in occurrence of clonic convulsions was tested with Fisher s exact probability test. For all statistical comparisons, results with P.05 were considered not to be significant. Results PTZ (45 mg/kg) had a profound influence on behavior. The saline-like and PTZ-like behaviors observed are summarized in table 1. PTZ-treated mice were observed to lie in the cage without moving, their hind body was flattened so that their limbs were visible and they did not curl up their tails as control mice did. PTZ-treated mice also tended to make more contact with the floor and wall than did control mice. Saline controls moved about and explored the cage by sniffing and rearing, and they would groom or sit in a corner with their tails curled around their bodies. Occasionally one fell asleep. The saline control mice sat when stationary rather than being prone like the PTZ-treated mice. The position of the mice within the locomotor arena also differed depending on whether saline or PTZ was administered. Saline control mice sat in a corner facing the center of the cage during periods of rest, whereas the PTZ-treated mice faced the wall or corner or sometimes remained prone in the middle of the cage. Figure 1 shows the results of the last observational period (at 30 min). All animals that had received PTZ were characterized as PTZ-like (unconnected filled circles), whereas none of the vehicle controls (unconnected open circles) were characterized as PTZ-like. Similar results were observed for the other observational periods (at 5 and 15 min). PTZ when given alone also produced clonic convulsions in 33% of the mice tested. All of the doses of the anticonvulsants that we evaluated, conferred complete protection against convulsive episodes. Of the compounds tested, ganaxolone was the most potent in preventing PTZ-like behavior: all doses tested, at or below the ED 50 value (doses on abscissa of figs. 1 3 are in multiples of the ED 50 for blocking convulsions induced by 70 mg/kg PTZ), significantly decreased the percentage of mice exhibiting PTZ-like behaviors. For the highest dose of ganaxolone tested (1 ED 50 ), the percentage of PTZ-like animals was significantly lower than for PTZ-treated mice throughout the experiment. At the lower concentrations, more animals were initially scored as PTZ-like, but within 15 min their behavior normalized and they were scored as saline-like for the remainder of the experimental session (time course data not shown). Fifty percent reversal was reached at a dose of 0.16 ED 50 (table 2). When the highest dose of ganaxolone was given without PTZ, it had no influence on the saline-like behavior (table 3). Valproate significantly reduced PTZ-like signs at 2 ED 50, whereas neither 1 ED 50 nor 4 ED 50 reversed the PTZ-like behavior. Because of the nonlinear nature of the dose-effect curve, it was not possible to calculate an ED 50 for the behavior-reversing effects of valproate. Even though not all the animals were characterized as PTZ-like at 2 ED 50, the others were not all saline-like: their behavior alternated between lying down and periods of walking and rearing. At 4 ED 50, the mice did not display these reversals, and behavior remained PTZ-like. This dose of valproate given alone also made the animals look PTZ-like in terms of the behavioral ratings (table 3). A similar picture was seen for ethosuximide: whereas 1 ED 50 had no significant effect on the number of PTZ-like animals, 4 and 6 ED 50 did. However, as with valproate, the highest dose was less effective, and effects of ethosuximide itself started to become evident. Although they exhibited no changes that registered on the PTZ behavioral rating scale, these mice repeatedly scratched, turned in cir-

4 1998 Behavioral Effects of Ganaxolone 871 cles or fell while walking when the highest dose of ethosuximide was given alone or in conjunction with PTZ. The ED 50 for reversing the behavioral effects of PTZ was 1.98 ED 50 (table 2). Neither clonazepam nor diazepam resulted in significant reductions in PTZ-like signs until 8 ED 50. At this dose, clonazepam did not produce significant effects itself, but the animals switched between PTZ-like and saline-like behaviors. Diazepam was also tested at a dose of 16 ED 50, which yielded significant improvement but less than with 8 ED 50. In the highest dose, however, diazepam itself started to have sedating effects that manifested as non-saline-like sleeping (nonsignificant changes, table 3). The ED 50 values for the behavior-reversing effects were 3.07 ED 50 for clonazepam and 6.62 ED 50 for diazepam (table 2). Phenobarbital displayed a steep dose-effect curve with 50% effect at 1.50 ED 50 (table 2), in which 4 and 8 ED 50 resulted in 0% PTZ-like animals. At these doses, the mice reared and ambulated frequently but hardly groomed. Phenobarbital given alone did not affect observed behavior (table 3). Differences in the anti-ptz effects of ganaxolone and diazepam are directly compared in table 1 for the individual behaviors that make up the PTZ behavioral rating scale. Fig. 1. Percentage of mice exhibiting PTZ-like behavioral signs with different doses of ganaxolone and comparison anticonvulsants at 30 min after injection of PTZ (F) or vehicle (E). Doses are expressed as multiples of the ED 50 for reversing clonic convulsions induced by 70 mg/kg PTZ (Gasior et al., 1997a). These ED 50 values are 3.45 mg/kg for ganaxolone, 241 mg/kg for valproate, 95 mg/kg for ethosuximide, 0.14 mg/kg for clonazepam, 0.26 mg/kg for diazepam and 6 mg/kg for phenobarbital. * indicates significant difference from control level (vehicle PTZ), according to Fisher s exact probability test with P.05. These data indicate that even for specific behaviors that are altered by PTZ, ganaxolone produced a more effective normalization of behavior toward vehicle control levels than did diazepam. Again, this difference in behavioral efficacy was demonstrated by comparing doses of these two drugs that were equally efficacious in blocking the convulsant effects of PTZ (ED 50 values). PTZ also significantly reduced locomotor activity (fig. 2). As with the direct behavioral observations (fig. 1), ganaxolone was the most potent compound tested in reversing the reductions in horizontal activity induced by PTZ. Complete reversal was achieved at 1 ED 50. The ED 50 for this effect was 0.38 ED 50. Valproate only marginally reversed the PTZ-induced reductions in horizontal locomotor activity at 2 ED 50. This dose given alone did not alter the horizontal distance traveled from vehicle control values (table 3). At 4 ED 50, valproate itself suppressed distance traveled (table 3) and did not reverse the PTZ-effect. Ethosuximide reversed the PTZinduced decrease in locomotion, but in higher doses than ganaxolone; this effect was seen at 4 and 6 ED 50. The ED 50 of ethosuximide for this effect was 2.71 ED 50 (table 2). The locomotor-depressant effects of PTZ were also reversed

5 872 Beekman et al. Vol. 284 by clonazepam at a high dose (8 ED 50 ). However, locomotion was elevated above the level of vehicle controls, as was the case when this concentration was administered alone (table 3). The ED 50 of clonazepam for reversing the effects of PTZ was 2.62 ED 50 (table 2). Diazepam brought the distance traveled to the vehicle control level in doses of 8 and 16 ED 50. A dose of 4.56 ED 50 diazepam was predicted to be the dose that would yield 50% protection (table 2). Phenobarbital also reversed the decreases in activity induced by PTZ, with doses of 4 and 8 ED 50 (50% effect was 1.89 ED 50 ), but as with clonazepam, activity at the highest dose was greater than that of vehicle controls (note the difference in scale in fig. 2). Effects of the anticonvulsants on vertical activity are shown in figure 3. Most striking is the inability of most drugs to reverse the PTZ-induced depression of vertical activity. Only ganaxolone, clonazepam and phenobarbital reduced this behavioral effect of PTZ; with ganaxolone demonstrating the greatest potency. The effective dose of phenobarbital (8 ED 50 ) produced effects that were greater than those of vehicle-treated mice. ED 50 values for reversing the vertical activity deficit engendered by PTZ could be calculated only for Fig. 2. Horizontal distance traveled, in centimeters, for different doses of anticonvulsants, summed from 10 to 30 min after PTZ or vehicle injection. Error bars represent S.E.M. *indicates significant difference from vehicle PTZ control, determined by Dunnett s one-tailed test, P.05. # indicates significant difference from vehicle vehicle control, indicated by a one-tailed t- test, P.05. n 8. Other details are as described in fig. 1 caption. ganaxolone and phenobarbital. These values were 0.56 ED 50 and 2.12 ED 50, respectively (table 2). None of the compounds at a dose of 1 ED 50 produced significant changes in vertical activity when given alone. Higher doses of valproate, ethosuximide, clonazepam and diazepam all significantly reduced vertical activity counts when given alone (table 3). The effects of repeated administration of ganaxolone and diazepam are shown in figure 4. The top left panel in figure 4 shows the percentage of PTZ-like animals for controls (circles) and for diazepam (triangles) and ganaxolone (squares) treatments. Because there was little difference over the 4 days, the data were summarized and plotted as a function of time. Both control groups remained constant throughout the experiment at a level of 0 0% for the vehicle control and % for the PTZ control. Diazepam did not reverse the effect of PTZ within this time frame; the mean percentage of PTZ-like animals, %, did not differ from PTZ controls. In contrast, ganaxolone showed a time-dependent effect; at all times the percentage of PTZ-like animals was significantly lower than without the drug, and only the level at the first observational period was significantly different

6 1998 Behavioral Effects of Ganaxolone 873 Fig. 3. Vertical activity, in counts, for different doses of anticonvulsants, summed from 10 to 30 min after PTZ or vehicle injection. Other details are as described in fig. 2 caption. TABLE 2 ED 50 values for reversing PTZ-induced behaviors ED 50 values (95% CL in parentheses) (n 8/dose) for reversing PTZ-induced behavior, both in milligrams per kilogram and as multiples of the ED 50 for preventing convulsions in acute experiments against 70 mg/kg PTZ, as determined by or according to Gasior et al. (1997a) (see caption to fig. 1). The 50% effect is reached when the percentage of animals, distance or activity is half of the difference between effects of vehicle vehicle and PTZ vehicle. Dashes in the table indicate that the ED 50 could not be calculated. Observed Behavior Total Distance Vertical Activity mg/kg part of ED 50 mg/kg part of ED 50 mg/kg part of ED 50 Ganaxolone ( ) ( ) ( ) ( ) ( ) ( ) Valproate Ethosuximide ( ) ( ) ( ) ( ) Clonazepam ( ) ( ) ( ) ( ) Diazepam ( ) ( ) ( ) ( ) Phenobarbital ( ) ( ) ( ) ( ) ( ) ( ) from saline (mean %). This difference decreased, and in the final period, ganaxolone reached maximal efficacy with a level of 0 0%. The top right panel of figure 4 shows the horizontal distance traveled for the 4 experimental days. There were no marked trends in the data (means were for vehicle controls, for PTZ controls, for ganaxolone-treated animals and in

7 874 Beekman et al. Vol. 284 TABLE 3 Control data The percentage of mice exhibiting PTZ-like behaviors at t 30 min, the mean horizontal distance S.E.M. and the mean vertical activity S.E.M. for the drugs given without PTZ (n 8). Doses are in multiples of the anticonvulsant ED 50 (see caption to fig. 1). * indicates a significant difference from vehicle control (P.05) as calculated by Dunnett s two-tailed test. % PTZ-like Animals Horizontal Distance Vertical Activity Control ED ganaxolone 1 ED 50 valproate ED 50 valproate * 4 ED 50 valproate 100* * * 1 ED ethosuximide 6 ED * ethosuximide 1 ED clonazepam 8 ED * * clonazepam 1 ED 50 diazepam ED 50 diazepam * 16 ED * diazepam 1 ED phenobarbital 8 ED 50 phenobarbital * the case of diazepam treatment). There was a significant difference between the effects of ganaxolone PTZ and PTZ alone, whereas there was no significant effect of diazepam. Activity levels were somewhat higher, compared with vehicle control, when ganaxolone was given with PTZ, and on days 2 and 4 this difference was significant. Ganaxolone also reversed the effects of PTZ on vertical activity over successive days (fig. 4, bottom left panel), as was the case with distance traveled. The vertical activity of ganaxolone-treated mice was higher than for vehicle controls on day 2. Diazepam, in contrast, had no significant influence on vertical activity in PTZ-treated mice. Even though behavior did not change markedly over time, the bottom right panel of figure 4 shows that the convulsant effects of PTZ increased from 33% on day 1 to 83% on day 4 (filled circles). Although both ganaxolone and diazepam completely prevented PTZ-induced convulsions on day 1, diazepam was less effective in controlling seizures over repeated tests. Discussion The present report provides quantitative observations in support of the conclusion that the neuroactive steroid ganaxolone is superior to a host of standard antiepileptic agents in controlling the behavioral disturbances that result from both acute PTZ administration and a regimen of PTZ that induces seizure kindling. These findings support previous nonsystematic visual observations of behavior and substantiate the superior potency and efficacy of ganaxolone in blocking the development of PTZ-kindled seizures (Gasior et al., 1997b). Ganaxolone was more potent than the other antiepileptic drugs studied, with potency differences that were 4-fold and greater. Ganaxolone also displayed greater efficacy than the other compounds studied. In this respect, ganaxolone was the only compound that completely prevented all of the behavioral signs of PTZ intoxication. For example, although diazepam at high enough doses reversed PTZ-like signs and the decreases in horizontal activity, diazepam was ineffective in reversing the decreases in vertical activity induced by PTZ. Phenobarbital (with reduced potency) also prevented all of the behavioral sequella of PTZ administration; however, reversal was accompanied by effects that were different from vehicle controls. Ganaxolone, in contrast, normalized behavior, returning PTZ-induced behavior to vehicle control levels. Phenobarbital, and to a lesser extent clonazepam, produced a reversal that overshot control levels. Differences in pharmacokinetic variables cannot account for the differential effects of the drugs reported here. All of the compounds are 100% effective in preventing the clonic seizures produced by PTZ in this strain of mice (Gasior et al., 1997a and present study), and the doses used in the present study were based on their respective anticonvulsant ED 50 values. Because the doses of all of the drugs tested are comparable in terms of anticonvulsant efficacy, the present findings indicate that there are striking differences in the potencies and efficacies of anticonvulsants to exert anticonvulsant vs. behavioral protection against PTZ. As far as we know, this differential potency and efficacy relationship has not been previously investigated. Before different mechanisms of action are proposed to explain these relationships, additional factors must first be systematically eliminated. The ability to block behavioral effects of PTZ may be related to the specific behavioral effects of the anticonvulsants alone. Potencies to block physostigmine-induced decreases in food-maintained responding, for example, are positively correlated with drug potencies to decrease responding on their own (Genovese et al., 1990). In the present study, behavioral effects of the anticonvulsants did not appear to contribute substantially to the prevention of PTZ-induced behavioral effects. Neither ganaxolone nor phenobarbital, given alone, produced significant changes from vehicle controls under the PTZ behavioral rating scale, and all of the drugs at some doses prevented the PTZ-induced behavioral signs. Although valproate, diazepam and clonazepam all produced some observed changes in behavioral ratings when given alone at higher doses, they were all (except the highest dose of valproate) capable of preventing PTZ-induced behavioral signs. Likewise, for the most part, the highest doses of the anticonvulsants did not affect locomotor activity, and yet all of the drugs significantly prevented PTZ-induced depressions in activity. Clonazepam, on the other hand, increased activity when given alone at 8 ED 50 ; this was also the only dose that reversed effects of PTZ. NAS have been reported previously to increase locomotor activity (e.g., Wieland et al., 1995), but they did not produce increases at the doses studied here (present study and Gasior et al., 1997a). For vertical activity, only ganaxolone, clonazepam and phenobarbital prevented the PTZ-induced decreases. Of these three drugs, only clonazepam decreased vertical activity when given alone. Thus the preponderance of the data do not link the behavioral effects of the anticonvulsants alone with their efficacies in reversing behavioral effects of PTZ. Nonetheless, it is possible that combinations of specific anticonvulsants with PTZ may uncover behavioral effects not observed with the drugs alone that may be incompatible with, or otherwise interfere with, the PTZ blockade. Many anticonvulsant agents exert several of the same be-

8 1998 Behavioral Effects of Ganaxolone 875 Fig. 4. Effects of vehicle alone, PTZ alone (45 mg/kg) and PTZ in conjunction with either diazepam or ganaxolone when given for four experimental sessions. E: vehicle vehicle; F: vehicle PTZ; : ganaxolone (3.45 mg/kg) PTZ; Œ: diazepam (0.26 mg/kg) PTZ. Doses of ganaxolone and diazepam represent ED 50 doses for protection against acutely administered PTZ (70 mg/kg, Gasior et al., 1997a). * represents significant (P.05) difference from PTZ alone, according to Fisher s exact probability test for the percentage of PTZ-like mice and convulsions; one-tailed t tests were used for the locomotor activity data. Top left) Time course of percentage of mice exhibiting PTZ-like behavioral effects, averaged over the 4 days of the experiment. Top right) Mean ( S.E.M.) horizontal distance traveled, in centimeters, for the 4 treatment days, summed from 10 to 50 min (n 8). Bottom left) Mean ( S.E.M.) vertical activity in counts for the 4 treatment days, summed from 10 to 50 min (n 8). Bottom right) Percentage of clonic convulsions for the 4 treatment days. havioral effects. Thus benzodiazepines, barbiturates, valproate and the NAS demonstrate anxiolytic efficacy in preclinical models (Vellucci and Webster, 1984; Crawley et al., 1986; Wieland et al., 1995; Britton et al., 1991). The effects of ganaxolone reported here suggest its anxiolytic activity, which should be confirmed in other animal models of anxiety. PTZ has been used to model anxiety in rodents (Lal and Sherman, 1980; Lal and Emmett-Oglesby, 1983; Lal and Fielding, 1985; Giusti et al., 1991). PTZ, in subconvulsant doses, produces anxiety in humans (Rodin and Calhoun, 1970), and the presumed anxiogenic effects of PTZ are responsive to treatment with valproate, diazepam and other anxiolytic compounds (Sherman and Lal, 1980; Lal and Fielding, 1985; Giusti et al., 1991). The reported lack of efficacy of ethosuximide may be due to the low doses tested (Lal et al., 1980). NAS also appear to share discriminative stimulus effects with benzodiazepines and barbiturates, an effect that has been used to predict the subjective effects of drugs in humans (Holtzman, 1990; Preston and Bigelow, 1991; Kamien, 1993). NAS substitute for the training drug in rats trained to discriminate pentobarbital, diazepam, midazolam or ethanol from vehicle (Ator et al., 1993; Deutsch and Mastropaolo, 1993). Clearly, these common behavioral actions appear insufficient to account for the unique pharmacological effects of ganaxolone that are revealed here. Now that pharmacokinetics and behavioral effects of the drugs alone have been ruled out as major factors in the differential potencies and efficacies observed here, the conclusion that the ability to alter convulsions and behavioral effects of PTZ may be mediated by different mechanisms becomes more tenable. What, then, is the nature of these differential mechanisms? The drugs used in this study form a heterogenous group of compounds. The drugs have different clinical profiles as well as marked differences in their pharmacology. Clinically, ethosuximide is primarily used in the treatment of generalized absence seizures, the benzodiazepines are most effective for myoclonic convulsions, phenobarbital can be used for myoclonic convulsions and in generalized tonic-clonic and partial seizures and valproate has clinical significance for generalized tonic-clonic, absence, myoclonic and partial seizures (MacDonald and Meldrum, 1989). Preclinical indications have suggested efficacy for ganaxolone in the management of generalized absence as well as simple and complex partial seizures (Carter et al., 1997; Gasior et al., 1997b). Thus the common clinical or suggested clinical utility of phenobarbital, valproate and ganaxolone does not predict efficacy against behavioral effects of PTZ. The pharmacological profiles of the compounds are diverse as well. Ethosuximide acts on T calcium channels and valproate on sodium channels. Diazepam and clonazepam interact with the benzodiazepine site of the GABA A receptor as well as with sodium channels, whereas phenobarbital acts on the barbiturate site and on sodium channels (MacDonald and Meldrum, 1989). The anticonvulsant profile of ganaxolone, though different in some respects, closely resembles that of valproate (Carter et al., 1997) and clonazepam (Kokate et al., 1994). Nonetheless, neither valproate nor clonazepam demonstrated the degree of protection against behavioral effects of PTZ (present study) or against PTZ-kindled seizures (Gasior et al., 1997b) that is demonstrated by ganaxolone. Ganaxolone is unique in modulating GABA transmission by allosteric modulation of a steroid-sensitive site on the GABA A receptor complex with only weak interactions at other known anticonvulsant sites of action (Morrow et al., 1987; Lambert et al., 1995; Carter et al., 1997). The specific modulation of GABA through the steroid binding site may confer on neuroactive steroids and endogenous steroids the capacity to function as efficacious inhibitors of both epileptogenic and behavioral effects induced by PTZ with comparable potency. The novel anticonvulsant properties of NAS have previously been documented (Gasior et al., 1997a, b). In addition, ganaxolone exhibited a reduced propensity to affect cognitive function, and to produce in its motor-intoxicating interactions with ethanol, compared with valproate (R. B. Carter unpublished observations). The possibility that such

9 876 Beekman et al. Vol. 284 protection also functions against other convulsant and behaviorally disrupting stimuli cannot be overlooked. Ganaxolone was the only drug that potently and completely reversed the full range of behavioral effects of PTZ without producing an overshoot in control values. Although much less potent than ganaxolone, and although it did not fully restore behavior to normal levels, phenobarbital was the drug that most closely mirrored ganaxolone in its anti- PTZ effects. Thus only ganaxolone and phenobarbital dosedependently prevented all of the PTZ-induced behaviors and did so without causing concomitant motor toxicity such as uncoordinated walking or circling. Similarities between NAS and barbiturates have been observed previously. Majewska et al. (1986) reported barbiturate-like behavioral, biochemical and electrophysiological properties of NAS. Like barbiturates, NAS displace TBPS binding, enhance binding of muscimol and flunitrazepam and can also directly influence Cl flux, albeit at high concentrations (Barker et al., 1987; Cottrell et al., 1987a; Peters et al., 1988; Carter et al., 1997). The unique potency and efficacy of ganaxolone against PTZ-induced behaviors have several implications. First, these antibehavioral effects of PTZ appear to be predictive of the antikindling potency and efficacy of ganaxolone. Ganaxolone demonstrates superior potency and efficacy, compared with valproate and diazepam, in blocking the development of PTZ-kindled seizures (Gasior et al., 1997b). Second, ganaxolone may provide additional benefit in the treatment of epilepsy by controlling anxiety, mood changes and other behavioral alterations associated with preseizure activity. Finally, ganaxolone or other NAS may represent a novel treatment approach to the clinical control of anxiety disorders. Because withdrawal from a host of drugs of abuse engenders an anxiety-like state that can be mimicked by PTZ administration (Emmett-Oglesby et al., 1990), ganaxolone and other NAS may be of value in the treatment of this phase of drug addictions. References Ator NA, Grant KA, Purdy RH, Paul SM and Griffiths RR (1993) Drug discrimination analysis of endogenous neuroactive steroids in rats. Eur J Pharmacol 241: Barker JL, Harrison NL, Lange GD and Owen DG (1987) Potentiation of -aminobutyric-acid-activated chloride conductance by a steroid anaesthetic in cultured rat spinal neurones. J Physiol 386: Belelli D, Bolger MB and Gee KW (1989) Anticonvulsant profile of the progesterone metabolite 5-pregnan-3-ol-20-one. Eur J Pharmacol 166: Britton KT, Page M, Baldwin H and Koob GF (1991) Anxiolytic activity of steroid anesthetic alphaxalone. J Pharmacol Exp Ther 258: Carter RB, Wood PL, Wieland S, Hawkinson JE, Belelli D, Lambert JJ, White HS, Wolf HH, Mirsadeghi S, Tahir H, Bolger MB, Lan NC and Gee KW (1997) Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3-hydroxy-3-methyl-5-pregnan-20-one), a selective high-affinity steroid modulator of the GABA A receptor. J Pharmacol Exp Ther 280: Cottrell GA, Lambert JJ and Mistry DK (1987a) Alphaxalone potentiates GABA and activates GABA A receptor of mouse spinal neurones in culture. J Physiol 382: 132P. Cottrell GA, Lambert JJ and Peters JA (1987b) Modulation of GABA A receptor activity by alphaxalone. Br J Pharmacol 90: Crawley JN, Glowa JR, Majewska MD and Paul SM (1986) Anxiolytic activity of an endogenous adrenal steroid. Brain Res 398: Deutsch SI and Mastropaolo J (1993) Discriminative stimulus properties of midazolam are shared by a GABA-receptor positive steroid. Pharmacol Biochem Behav 46: Emmett-Oglesby MW, Mathis DA, Moon RTY and Lal H (1990) Animal models of drug withdrawal symptoms. Psychopharmacology 101: Gasior M, Carter RB, Goldberg SR and Witkin JM (1997a) Anticonvulsant and behavioral effects of neuroactive steroids alone and in conjunction with diazepam. J Pharmacol Exp Ther 282: Gasior M, Carter RB, Beekman M, Goldberg SR and Witkin JM (1997b) Neuroactive steroids as a treatment for epilepsy. Current status and future directions. Acta Neurobiologiae Experimentalis 57(suppl.):76. Gee KW, Bolger MB, Brinton RE, Coirini H and McEwen BS (1988) Steroid modulation of the chloride ionophore in rat brain: Structure-activity requirements, regional dependence and mechanism of action. J Pharmacol Exp Ther 246: Gee KW, Chang W, Brinton RE and McEwen BS (1987) GABA-dependent modulation of the Cl ionophore by steroids in rat brain. Eur J Pharmacol 136: Gee KW, McCauley L and Lan NC (1995) A putative receptor for neurosteroids on the GABA A receptor complex: The pharmacological properties and therapeutic potential of epalons. Crit Rev Neurobiol 9: Genovese RF, Elsmore TF and Witkin JM (1990) Relationship of the behavioral effects of aprophen, atropine and scopolamine to antagonism of the behavioral effects of physostigmine. Pharmacol Biochem Behav 37: Giusti P, Guidetti G, Costa E and Guidotti A (1991) The preferential antagonism of pentylenetetrazole proconflict responses differentiates a class of anxiolytic benzodiazepines with potential antipanic action. J Pharmacol Exp Ther 257: Harrison NL, Majewska MD, Harrington JW and Barker JL (1987) Structureactivity relationships for steroid interaction with the -aminobutyric acid A receptor complex. J Pharmacol Exp Ther 241: Harrison NL and Simmonds MA (1984) Modulation of GABA receptor complex by a steroid anaesthetic. Brain Res 323: Holtzman SG (1990) Discriminative stimulus effects of drugs: Relationship to potential for abuse. In Modern Methods in Pharmacology, Testing and Evaluation of Drugs of Abuse (vol 6), (Adler MW and Cowan A, eds) pp Wiley-Liss Inc., New York. Kamien JB, Bickel WK, Hughes JR, Higgins ST and Smith B (1993) Drug discrimination by humans compared to nonhumans: Current status and future directions. Psychopharmacology 111: Kokate TG, Svensson BE and Rogawski MA (1994) Anticonvulsant activity of neurosteroids: Correlation with -aminobutyric acid-evoked chloride current potentiation. J Pharmacol Exp Ther 270: Lal H and Emmett-Oglesby MW (1983) Behavioral analogs of anxiety: Animal models. Neuropharmacology 22: Lal H and Fielding S (1985) Antagonism of discriminative stimuli produced by anxiogenic drugs as a novel approach to bioassay of anxiolytics. Drug Dev Res 4:3 21. Lal H and Sherman GT (1980) Interoceptive discriminative stimuli in the development of CNS drugs and a case of an animal model of anxiety. Ann Rep Med Chem 15: Lal H, Sherman GT, Fielding S, Dunn R, Kruse and Theurer K (1980) Evidence that GABA mechanisms mediate the anxiolytic action of benzodiazepines: A study with valproic acid. Neuropharmacology 19: Lambert JJ, Peters JA and Cottrell GA (1987) Actions of synthetic and endogenous steroids on the GABA A receptor. Trends Pharmacol Sci 8: Lambert JJ, Belelli D, Hill-Venning C and Peters JA (1995) Neurosteroids and GABA A receptor function. Trends Pharmacol Sci 16: Litchfield JT and Wilcoxon F (1949) A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 95: Luntz-Leybman V, Freund RK and Collins AC (1990) 5-Pregnan-3-ol-20-one blocks nicotine-induced seizures and enhances paired-pulse inhibition. Eur J Pharmacol 185: MacDonald RL and Meldrum BS (1989) General principles. Principles of antiepileptic drug action. In Antiepileptic Drugs, 3rd ed, Levy R, Mattson R, Meldrum B, Penry JK and Dreifuss FE, eds) pp New York, Raven Press, Ltd. Majewska MD (1992) Neurosteroids: Endogenous bimodal modulators of the GABA A receptor. Mechanism of action and physiological significance. Prog Neurobiol 38: Majewska MD, Harrison NL, Schwartz RD, Barker JL and Paul SM (1986) Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232: Majewska MD and Schwartz RD (1987) Pregnenolone-sulfate: An endogenous antagonist of the -aminobutyric acid receptor complex in brain? Brain Res 404: Mienville J and Vicini S (1989) Pregnenolone sulfate antagonizes GABA A receptormediated currents via a reduction of channel opening frequency. Brain Res 489: Morrow AL, Pace JR, Purdy RH and Paul SM (1989) Characterization of steroid interactions with -aminobutyric acid receptor-gated chloride ion channels: Evidence for multiple steroid recognition sites. Mol Pharmacol 37: Morrow AL, Suzdak PD and Paul SM (1987) Steroid hormone metabolites potentiate GABA receptor-mediated chloride ion flux with nanomolar potency. Eur J Pharmacol 142: Morrow AL, Suzdak PD and Paul SM (1988) Benzodiazepine, barbiturate, ethanol and hypnotic steroid hormone modulation of GABA-mediated chloride ion transport in rat brain synaptoneurosomes. In Chloride Channels and Their Modulation by Neurotransmitters and Drugs, vol 45, (Biggio G and Costa E, eds) pp , Raven Press, New York. Olsen RW, Yang SS-J and Ransom RW (1988) GABA-stimulated 36 Cl flux in brain slices as an assay for modulation by CNS depressant drugs. In Chloride Channels and Their Modulation by Neurotransmitters and Drugs, vol 45, (Biggio G and Costa E, eds) pp , Raven Press, New York. Paul SM and Purdy RH (1992) Neuroactive steroids. FASEB J 6: Peters JA, Kirkness EF, Callachan H, Lambert JJ and Turner AJ (1988) Modulation of the GABA A receptor by depressant barbiturates and pregnane steroids. Br J Pharmacol 94: Preston KL and Bigelow GE (1991) Subjective and discriminative effects of drugs. Behav Pharmacol 2: Rodin EA and Calhoun HD (1970) Metrazol tolerance in a normal volunteer population. J Psychiat Res 13: Selye H (1941) Anesthetic effect of steroid hormones. Proc Soc Exp Biol Med 46: