Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
FULL-LENGTH ORIGINAL RESEARCH
Pharmacodynamic and pharmacokinetic interactions between common antiepileptic drugs and acetone, the chief anticonvulsant ketone body elevated in the ketogenic diet in mice *Iwona Zarnowska, *yJarogniew J. Luszczki, zTomasz Zarnowski, xGrzegorz Buszewicz, xRoman Madro, *yStanislaw J. Czuczwar, and {Maciej Gasior *Department of Pathophysiology, Medical University, Lublin, Poland; yDepartment of Physiopathology, Institute of Agricultural Medicine, Lublin, Poland; zTadeusz Krwawicz Chair of Ophthalmology and First Eye Hospital, Medical University, Lublin, Poland; xDepartment of Forensic Medicine, Medical University, Lublin, Poland; and {CNS Biology, Cephalon Inc., West Chester, PA, U.S.A.
SUMMARY Purpose: Acetone is the principal ketone body elevated in the ketogenic diet (KD), with demonstrated robust anticonvulsant properties across a variety of seizure tests and models of epilepsy. Because the majority of patients continue to receive antiepileptic drugs (AEDs) during KD treatment, interactions between acetone and AEDs may have important clinical implications. Therefore, we investigated whether acetone could affect the anticonvulsant activity and pharmacokinetic properties of several AEDs against maximal electroshock (MES)–induced seizures in mice. Methods: Effects of acetone given in subthreshold doses were tested on the anticonvulsant effects of carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), topiramate (TPM) and valproate (VPA) against MES-induced seizures in mice. In addition, acute adverse effects of acetone–AEDs
The high fat/low carbohydrate and protein diet (ketogenic diet [KD]) provides three-fourths of drug-resistant epileptic patients with substantial improvement (Freeman
Accepted August 13, 2008; Early View publication October 24, 2008. Address correspondence to Maciej Gasior, CNS Biology, Cephalon Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A. E-mail:
[email protected] Wiley Periodicals, Inc. ª 2008 International League Against Epilepsy
combinations were assessed in the chimney test (motor performance) and passive avoidance task (long-term memory). Pharmacokinetic interactions between acetone and AEDs were also studied in the mouse brain tissue. Results: Acetone (5 or 7.5 mmol/kg, intraperitoneally [i.p.]) enhanced the anticonvulsant activity of CBZ, LTG, PB, and VPA against MES-induced seizures; effects of OXC, PHT, and TPM were not changed. Acetone (7.5 mmol/kg) did not enhance the acute adverse-effect profiles of the studied AEDs. Acetone (5 or 7.5 mmol/kg, i.p.) did not affect total brain concentrations of the studied AEDs. In contrast, VPA, CBZ, LTG, OXC, and TPM significantly decreased the concentration of free acetone in the brain; PB and PHT had no effect. Conclusions: Acetone enhances the anticonvulsant effects of several AEDs such as VPA, CBZ, LTG, and PB without affecting their pharmacokinetic and side-effect profiles. KEY WORDS: Acetone, Ketogenic diet, Epilepsy, Antiepileptic drugs.
et al., 1998, 2007; Keene, 2006). Although the KD has been in clinical use for much longer than many other treatments for epilepsy, its mechanism of action remains speculative and a subject of extensive preclinical and clinical research (Freeman et al., 2006; Hartman et al., 2007). There is also accumulating evidence that therapeutic effects of KD may extend beyond epilepsy over a larger group of neuropsychiatric disorders associated with progressive neuronal loss (Gasior et al., 2006).
1132
1133 Interactions of Acetone with AEDs in the MES Test in Mice Consumption of the KD results in overproduction and accumulation of so-called ketone bodies (i.e., b-hydroxybutyrate, acetoacetate, and acetone), which become the main source of energy in the absence of carbohydrates (Withrow, 1980; Seymour et al., 1999; Stafstrom & Bough, 2003). Development and maintenance of ketosis are paramount during the KD and raising levels of ketone bodies have been among many factors contributing to the therapeutic efficacy of the KD (Huttenlocher, 1976; Gilbert et al., 2000; Stafstrom & Bough, 2003; Bough & Rho, 2007; Hartman et al., 2007; Ma et al., 2007; Henderson, 2008). Preclinical data support a role of, at least some, ketone bodies in seizure suppression produced by the KD. Anticonvulsant properties of acetone were demonstrated as early as in 1930 (Keith, 1931) and soon were confirmed by others (Driver, 1947; Chu et al., 1950; Jenney & Pfeiffer, 1958). Likewise, several morerecent studies affirmed efficacy of acetone against seizures in a broad range of seizure tests and models of epilepsy in rodents including seizures induced by maximal electroshock (MES), 6-Hz electrical stimulation, amygdala kindling, AY-9944, 4-aminopyridine, pentylenetetrazole, and audiogenic stimulation (Likhodii & Burnham, 2002; Rho et al., 2002; Likhodii et al., 2003; Yankura et al., 2006; Gasior et al., 2007; Hartman et al., 2008). In contrast to acetone, there is less evidence for the anticonvulsant properties of b-hydroxybutyrate and acetoacetate (Thio et al., 2000; Likhodii & Burnham, 2002; Rho et al., 2002). Although epileptic patients on the KD usually continue receiving multiple antiepileptic drugs (AEDs) (Freeman et al., 1998; Dahlin et al., 2006), interactions between the two treatment modalities have not been studied extensively (Bough & Eagles, 2001; Lyczkowski et al., 2005). Therefore, the aim of the present study was to quantitatively evaluate the influence of acetone as the only ketone body with proven anticonvulsant properties on the anticonvulsant efficacy of several AEDs used in humans such as valproate (VPA), carbamazepine (CBZ), phenobarbital (PB), phenytoin (PHT), lamotrigine (LTG), oxcarbazepine (OXC), and topiramate (TPM) in MES-induced seizures in mice. In addition, behavioral and pharmacokinetic interactions of selected combinations of acetone and AEDs were evaluated. This study revealed that acetone in doses devoid of any anticonvulsant efficacy per se significantly potentiated anticonvulsant effects of some AEDs (CBZ, LTG, PB, and VPA) without affecting their pharmacokinetic and behavioral properties.
Materials and Methods Animals Adult male Swiss mice weighing 20–25 g were kept in an environmentally controlled vivarium (temperature and relative humidity, 21 € 1C and 55 € 3%, respectively)
operating under a natural light–dark cycle. The animals were housed in colony cages with free access to food (chow pellets) and tap water. Only experimentally naive mice were used and experimental groups consisted of, at least, eight mice per group. All tests were performed between 0900 and 1200 h. Procedures involving animals and their care were conducted in accordance with the European Communities Council Directive of 24th November 1986 (86/609/EEC) and Polish legislation on animal use in biomedical experiments. The experimental protocols and procedures listed in the following were approved by the First Local Ethics Committee in Lublin and conformed to the Guide for the Care and Use of Laboratory Animals (http://www.nap.edu/readingroom/books/labrats). Drugs The following AEDs were used: CBZ (Polfa, Starogard, Poland), PB (Polfa, Krakow, Poland), PHT (Polfa, Warszawa, Poland), LTG (Lamictal; Glaxo Wellcome, Kent, UK), OXC (Trileptal; Novartis Pharma AG, Basel, Switzerland), TPM (Topamax; Cilag AG, Schaffhausen, Switzerland), and VPA (kindly donated by ICN Polfa, Rzeszow, Poland). All AEDs, except for VPA, were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, U.S.A.) in sterile saline, whereas VPA was dissolved in sterile saline. Acetone (POCH, Warszawa, Poland) was dissolved in sterile saline to the respective concentrations. All drugs were administered intraperitoneally (i.p.) in a volume of 10 ml/kg body weight. Doses of AEDs were expressed as mg/kg body weight; doses of acetone were expressed as mmol/kg body weight. The AEDs were administered as follows: PHT at 120 min; PB, LTG, and TPM at 60 min; and CBZ, OXC, and VPA at 30 min before seizure tests and other behavioral and pharmacokinetic studies. Acetone was administered 30 min before testing. The selected pretreatment times for the AEDs and acetone correspond to their peak anticonvulsant activity based on the available literature and our previous experiments (Luszczki et al., 2003, 2006b, 2007b; Gasior et al., 2007). Electroconvulsions Electroconvulsions were induced by applying an alternating current (50 Hz; 500 V) via ear-clip electrodes from a rodent shocker generator (type 221; Hugo Sachs Elektronik, Freiburg, Germany). The stimulus duration was 0.2 s. Tonic hind-limb extension was taken as the endpoint. This apparatus was used to induce seizures in two methodologically different experimental approaches: maximal electroshock seizure threshold (MEST) test and MES seizure test (Lçscher et al., 1991). The MEST test The MEST test was first used to assess the anticonvulsant effects of acetone administered alone. In this test, Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
1134 I. Zarnowska et al. separate groups of mice, each consisting of at least eight animals, were challenged with currents of varying intensities until data were collected with at least three current intensities at which close to 0%, 50%, and 100% of animals exhibited the endpoint. After establishing the current intensity-effect curve (i.e., current intensity in mA vs. percentage of mice convulsing) for each dose of acetone tested, the electroconvulsive threshold was calculated according to a log-probit method by Litchfield and Wilcoxon (1949). The electroconvulsive threshold was expressed as the median current strength value (CS50) in mA predicted to produce tonic hind-limb extension in 50% of the animals tested. This experimental procedure was performed for various increasing doses of acetone (i.e., 5, 7.5, and 10 mmol/kg), until the threshold for electroconvulsions of acetone-injected animals was statistically different from that of the control animals. Only doses of acetone that did not significantly affect the seizure threshold in the MEST test were selected for testing in combination with AEDs in the MES test (see subsequent text). This experimental approach has been described in more detail in our earlier studies (Luszczki & Czuczwar, 2004, 2005). MES test In the MES test, mice were challenged with a current of the fixed intensity (25 mA) that was 4–5-fold higher than the CS50 value in vehicle-treated control mice (Lçscher et al., 1991). These parameters of stimulation (so-called MES) typically result in all mice responding with tonus immediately after stimulation. The AEDs administered alone and in their combination with acetone were tested for their ability to increase the number of animals not responding with tonus (i.e., protected from tonus) after stimulation. Again, at least three groups of mice, each consisting of at least eight animals and treated with a different dose of the drug alone or in combination with acetone, were challenged to collect data where close to 0%, 50%, and 100% of animals were protected from tonic seizures. After constructing a dose–effect curve (i.e., dose in mg/kg vs. percentage of mice protected), the protective median effective dose (ED50) value of the drug tested was calculated according to a log-probit method (Litchfield & Wilcoxon, 1949). Each ED50 value represented a dose of the test drug (in mg/kg) predicted to protect 50% of mice tested against MES-induced extension of the hind limbs. Measurement of total brain AED concentrations Total brain concentrations of the selected AEDs were measured at doses corresponding to their ED50 values in combination with acetone determined in the MES test. Specifically, mice pretreated with a given AED alone or in combination with acetone were decapitated and the whole brain was collected, weighed, and Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
homogenized using Abbott buffer (1:2 weight/volume; Abbott Laboratories, North Chicago, IL, U.S.A.) in an Ultra-Turrax T8 homogenizer (IKA-Werke, Staufen, Germany). The homogenates were then centrifuged at 10,000 g for 10 min and the supernatant samples of 100 ll were collected and then analyzed for AED content. Total brain concentrations of CBZ, PB, and VPA were measured by a fluorescence polarization immunoassay (FPIA) using an analyzer (Abbott TDx) and manufacturer-supplied reagent kits (Abbott Laboratories). In contrast, total brain concentration of LTG was measured by high-performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization mass spectrometer (HPLC-APCI-MS) Surveyor—LCQ Advantage MAX system (Thermo Electron Corp., San Jose, CA, USA). The chromatographic system worked under control of X’Calibur software (Thermo Electron Corp.). HPLC column LiChroCART Purospher STAR RP-18e, 125 · 3 mm id (Merck, Darmstadt, Germany) with precolumn was used at 40C. The gradient mobile phase was: acetonitrile (25 mm ammonium formate buffer pH = 4.5; 0–100% in 45 min), flow rate 0.4 ml/min. LTG concentration was calculated on the basis of calibrating curves performed for brain homogenates. Supernatant samples of 300 ll were mixed with internal standard solution containing 500 ng/ml oxazepam-D5 and 300 ll of carbonate buffer (0.5 m, pH = 9). Extraction was carried out by 3 ml of anhydrous ethyl acetate. The organic layer was evaporated to dryness under a nitrogen stream and redissolved in 60 ll of the mobile phase; samples of 10 ll were then injected into the chromatograph. The mass spectrometer was operated in full scan (90– 650 m/z range) positive ions mode; 256.2 m/z for LTG and 292.2 m/z for internal standard were monitored. At least eight animals were used per each treatment group; and brain concentrations of AEDs were expressed as group means € SD in lg/ml of brain supernatant. Measurement of total brain acetone concentration The measurement of total brain concentration of acetone was performed at the selected doses of acetone used in combination with AEDs corresponding to their ED50 values determined in the MES test. Preparation of brain homogenates for the detection of acetone concentrations was identical as that described earlier for the measurement of total AED concentrations. Free acetone concentration was determined by the head-space technique using a gas chromatograph, Fisons 8160 (Fisons, Milan, Italy) with 2xFID detectors, autosampler HS-2000 (Thermo Finnigan, Milan, Italy) and dual capillary column connected by a Y-splitter: Restek BAC-1 and BAC-2 (0.52 mm ID; 30 m). The chromatographic analysis was performed using the carrier gas— helium 12.7 cm3/min and constant temperature 40C.
1135 Interactions of Acetone with AEDs in the MES Test in Mice Brain supernatants of 0.1 ml were added to 0.1 ml of the internal standard (tert-butanol 0.1 g/L in water) and subsequently, the mixture was placed into the 10-ml headspace vials. After 6 min of incubation at 60C, 0.75 ml of the volatile phase was subjected to the chromatographic analysis. At least eight animals were used per each treatment group; and brain acetone concentration was expressed as means € SD in mmol/ml of brain supernatant. Chimney test The effects of AEDs alone or in combination with acetone (at a constant dose of 7.5 mmol/kg) were assessed on motor coordination in the chimney test as originally described by Boissier et al. (1960). In this test, animals had to climb backward within a vertical plastic tube (3 cm inner diameter, 25 cm length). Typically all vehicletreated and experimentally naive mice climb backward up the tube in less than 30 s without any prior training. Motor coordination impairment was measured by the inability of mice to complete this task within 60 s. Results were calculated as the percentage of animals failing to perform the test. Step-through passive avoidance task The effects of acetone (at a constant dose of 7.5 mmol/ kg) in combination with AEDs (at doses corresponding to their ED50 values in the MES test) were assessed on longterm memory in the step-through passive avoidance task in mice (Venault et al., 1986). The tests consisted of two trials separated by 24 h. Each animal was administered with a combination of acetone and AED before trial 1 after the same pretreatment times used in the MES test. After treatment, animals were kept in their home cages until testing. Then, the mice were placed in an illuminated box (10 cm · 13 cm and 15 cm in height) that was connected to a dark box (25 cm · 20 cm and in 15 cm height). The dark box was equipped with an electric grid floor. Entrance of animals to the dark box was punished by an electric foot shock (0.6 mA for 2 s). In trial 1, mice were allowed 60 s to enter the dark box. Upon entry (all four paws in the dark box), the electric shock was delivered. Then the mice were removed and placed back into their home cages until the next day; mice that did not enter the dark compartment within 60 s were excluded from the experiment. The mice were placed into the illuminated box again 24 h later and allowed 180 s to enter the dark compartment. The median latency with 25th and 75th percentiles to enter the dark box during trial 2 was subsequently calculated for each group of mice tested. Statistics Both CS50 and ED50 values were calculated using the log-probit method (Litchfield & Wilcoxon, 1949), followed by the method transforming 95% confidence
limits (CLs) to standard error (Luszczki et al., 2003, 2006b). Statistical analysis of data from the MEST and MES tests was performed with one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for multiple comparisons. Total brain concentrations of AEDs and acetone administered alone or in combination were statistically analyzed using the unpaired Student’s t test or one-way ANOVA followed by the post hoc Bonferroni t test for multiple comparisons. The results from the passive avoidance test were statistically analyzed with Kruskal-Wallis nonparametric ANOVA test. The results from the chimney test were analyzed with the Fisher’s exact probability test. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, U.S.A.). Differences among values were considered statistically significant if p < 0.05.
Results Effect of acetone on the threshold for electroconvulsions in the MEST test in mice Acetone at the dose of 10 mmol/kg significantly elevated the threshold for electroconvulsions as evidenced by a significant (71%) increase in CS50 value from 7.00– 11.95 mA (p < 0.01; Table 1). Acetone at lower doses of 5 and 7.5 mmol/kg had no significant effect on the threshold for electroconvulsions in mice (Table 1). Effect of acetone on the anticonvulsant effects of various AEDs in the MES test Acetone administered alone at doses ranging between 16 and 40 mmol/kg protected the experimental animals against MES-induced tonic seizures with an ED50 of 26.3 (19.8–34.9) mmol/kg. Acetone administered at subthreshold doses of 5 and 7.5 mmol/kg (i.e., at doses that did not significantly affect the threshold for electroconvulsions per se) considerably increased the anticonvulsant potency of VPA against MES-induced tonic seizures (Fig. 1) by reducing the ED50 value of VPA from 253.5 mg/kg (control) to 194.6 mg/kg (23% decrease; p < 0.01), and 131.9 mg/kg (48% decrease; p < 0.001), respectively (Table 2). Acetone in the dose of 2.5 mmol/kg had no effect on the anticonvulsant potency of VPA in the MES test in mice (Table 2). Acetone had a similar effect on the anticonvulsant potencies of CBZ, LTG, and PB (Fig. 1). In the case of CBZ, acetone at the dose of 7.5 mmol/kg significantly reduced the ED50 value of CBZ from 11.62 to 6.26 mmol/kg (46%; p < 0.01; Table 2). Likewise, acetone at the dose of 7.5 mmol/kg significantly increased the anticonvulsant potency of LTG by reducing its ED50 from 4.82 to 2.93 mg/kg (28% decrease; p < 0.05; Table 2). With respect to PB, acetone at the dose of 7.5 mmol/kg significantly increased its anticonvulsant potency as reflected by Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
1136 I. Zarnowska et al. Table 1. Effect of acetone on the electroconvulsive threshold in the maximal electroshock seizure threshold (MEST) test in mice Treatment (mmol/kg)
CS50 (mA)
Vehicle 7.00 (5.84–8.38) Acetone (5) 7.90 (6.41–9.74) Acetone (7.5) 9.88 (8.29–11.76) Acetone (10) 11.95 (10.02–14.26)** F (3;68) = 5.792, p = 0.0014
n
SE
STI (%)
16 24 16 16
0.64 0.85 0.88 1.08
– 13 41 71
Results are presented as median current strengths (CS50 in mA; with 95% confidence limits in parentheses) required to produce tonic hind-limb extension in 50% of animals tested. The CS50 values were calculated using the log-probit method (Litchfield & Wilcoxon, 1949), followed by the method transforming 95% confidence limits to SE (Luszczki et al., 2003, 2006b). Acetone was administered i.p. 30 min before the test. Statistical analysis of the data was performed with one-way ANOVA followed by the post hoc Tukey-Kramer test for multiple comparisons. n, number of animals at those current strengths, whose convulsant effects ranged between 16% and 84%; SE, standard error of CS50; STI, % of seizure threshold increase as compared to control (vehicle-treated) animals; F, F-statistics from one-way ANOVA; p, probability. **p < 0.01 vs. vehicle-treated animals (Tukey-Kramer test).
a significant decrease of its ED50 from 27.4 to 17.7 mg/kg (35% decrease; p < 0.05; Table 2). However, acetone administered i.p. at lower doses of 2.5 and 5 mmol/kg had no significant effect on the anticonvulsant activities of CBZ, LTG, and PB in the MES test in mice (Table 2). In contrast, acetone administered i.p. at the dose of 7.5 mmol/kg had no significant effect on the anticonvulsant potency of OXC, PHT, and TPM in the MES test (Table 3). Effect of acetone on brain concentrations of AEDs The total brain concentrations of CBZ (6.3 mg/kg), PB (17.7 mg/kg), VPA (192.6 mg/kg), and LTG (2.9 mg/kg) administered alone at doses corresponding to their ED50 values in the MEST test did not differ significantly from that determined for the combination of these drugs with acetone (5 or 7.5 mmol/kg) (Table 4). Because acetone 7.5 mmol/kg did not significantly affect the anticonvulsant potency of OXC, PHT, and TPM in the MES test, total brain concentrations of these AEDs were not measured in this study. Effect of AEDs on brain concentration of acetone Several AEDs affected the total brain concentrations of acetone (Table 5). Specifically, VPA administered in combination with acetone (5 mmol/kg) significantly reduced (by 44%) its total brain concentration (p < 0.001; Table 5). Similar significant reductions of the total Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
Figure 1. Dose–effect function of valproic acid, carbamazepine, lamotrigine, and phenobarbital alone (yellow boxes) and in combination with 7.5 mmol/kg acetone (red boxes) against maximal electroshock (MES)–induced seizures. Each data point represents the percentage of mice protected (n = 16–32/data point); doses of AEDs (in mg/kg) on abscissa. Sigmoidal curves are the result of a least-squares fit of dose–response function for each AED alone and in combination with acetone. Points of intersections with the dashed line at 50% correspond to ED50 values of AEDs. See Table 2 for calculated the ED50 values of AEDs and other details. Epilepsia ILAE
brain concentrations of acetone (7.5 mmol/kg) were produced by CBZ (by 17%; p < 0.01), LTG (by 38%; p < 0.001), OXC (by 28%; p < 0.001), and TPM (by 29%; p < 0.001; Table 5). In contrast, administration of PHT and PB had no effect on the total brain concentrations of acetone. Effect of acetone on the acute adverse effects of AEDs in the passive avoidance task and chimney test The control group that received vehicles instead of AED plus acetone showed a maximal 180-s retention time in the passive avoidance task and no motor impairment in the chimney test. Acetone alone in the dose of 7.5 mmol/ kg (i.p., 30 min prior to the test) had no effect on retention time in the passive avoidance task (180-s retention time)
1137 Interactions of Acetone with AEDs in the MES Test in Mice Table 2. Effect of acetone on the protective action of carbamazepine (CBZ), lamotrigine (LTG), phenobarbital (PB), and valproate (VPA) in the maximal electroshock (MES) test Treatment (mmol/kg)
ED50 (mg/kg)
CBZ + vehicle 11.62 (9.20–14.66) CBZ + acetone (5.0) 9.98 (8.58–11.59) CBZ + acetone (7.5) 6.26 (4.24–9.25)** F (2;77) = 5.033, p = 0.0088 LTG + vehicle 4.82 (3.86–6.02) LTG + acetone (5.0) 4.83 (3.53–6.61) LTG + acetone (7.5) 2.93 (1.1–4.08)* F (2;61) = 3.739, p = 0.0294 PB + vehicle 27.41 (22.65–33.18) PB + acetone (2.5) 23.47 (19.16–28.75) PB + acetone (5.0) 18.39 (13.67–24.75) PB + acetone (7.5) 17.72 (14.48–21.70)* F (3;84) = 3.080, p = 0.0318 VPA + vehicle 253.5 (235.0–273.5) VPA + acetone (2.5) 233.4 (214.6–253.8) VPA + acetone (5.0) 194.6 (176.8–214.2)** VPA + acetone (7.5) 131.9 (110.4–157.6)*** F (3;76) = 21.25, p < 0.0001
n
SE
DR (%)
32 24 24
1.38 0.76 1.25
– 14 46
24 16 24
0.54 0.77 0.50
– – 28
16 24 24 24
2.67 2.43 2.79 1.83
– 14 33 35
16 32 16 16
9.81 9.96 9.53 11.97
– 8 23 48
Results are presented as median effective doses (ED50 in mg/ kg; with 95% confidence limits in parentheses) predicted to protect 50% of animals tested against MES-induced seizures. Acetone, CBZ, and VPA were administered i.p. 30 min before the MES test; LTG and PB were administered 60 min before the MES test. DR – % of dose reduction as compared to control (an AED + vehicle-treated) animals. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the respective control group (an AED + vehicle-treated animals). See Table 1 for other details.
Table 3. Effects of acetone on the protective action of oxcarbazepine (OXC), phenytoin (PHT), and topiramate (TPM) in the maximal electroshock (MES) test Treatment (mmol/kg)
ED50 (mg/kg)
n
SE
DR (%)
OXC + vehicle OXC + acetone (7.5) PHT + vehicle PHT + acetone (7.5) TPM + vehicle TPM + acetone (7.5)
10.77 (9.07–12.79) 8.44 (6.94–10.26) 11.41 (9.40–13.86) 9.98 (8.58–11.61) 58.48 (47.61–71.84) 36.17 (22.20–58.91)
16 16 24 16 40 32
0.95 0.84 1.13 0.77 6.14 9.00
– 22 – 13 – 38
Results are presented as median effective doses of OXC, PHT, and TPM against MES-induced seizures. Acetone, OXC, PHT, and TPM were administered i.p., as follows: PHT at 120 min, TPM at 60 min, OXC and acetone at 30 min prior to the MES-induced seizures. DR – % of dose reduction as compared to control (an AED + vehicle-treated) animals. See Table 1 for other details.
Table 4. Effects of acetone on the total brain concentrations of AEDs Treatment (mg/kg) + (mmol/kg) CBZ (6.3) + vehicle CBZ (6.3) + acetone (7.5) LTG (2.9) + vehicle LTG (2.9) + acetone (7.5) PB (17.7) + vehicle PB (17.7) + acetone (7.5) VPA (192.6) + vehicle VPA (192.6) + acetone (5.0)
Brain concentrations (lg/ml) 1.70 1.81 0.53 0.56 5.63 5.72 59.44 65.68
± ± ± ± ± ± ± ±
CI (%)
0.53 0.53 0.05 0.19 0.56 0.57 7.58 7.68
– 6 – 6 – 2 – 10
Data are presented as means ± SD of at least eight determinations. Statistical evaluation of the data was performed by means of unpaired Student’s t-test. CI, % increase in AED concentration as compared to control (an AED + vehicle-treated) animals; CBZ, carbamazepine; LTG, lamotrigine; PB, phenobarbital; VPA, valproate. For more details see Table 2.
Table 5. Effect of AEDs on the total brain concentrations of acetone in mice Treatment (mmol/kg) + (mg/kg) Acetone (5.0) Acetone (5.0) Acetone (7.5) Acetone (7.5) Acetone (7.5) Acetone (7.5) Acetone (7.5) Acetone (7.5) Acetone (7.5)
+ + + + + + + + +
vehicle VPA (195) vehicle CBZ (6.3) PHT (10.0) PB (17.7) LTG (2.9) OXC (8.4) TPM (36.2)
Brain concentration (mmol/L) 0.077 0.043 0.151 0.125 0.132 0.138 0.094 0.109 0.108
± ± ± ± ± ± ± ± ±
0.006 0.009*** 0.015 0.016** 0.015 0.017 0.016*** 0.017*** 0.013***
CR (%) – 44 – 17 13 9 38 28 29
Data are presented as means (in mmol/L of brain supernatant) ± SD of eight determinations. The AEDs were administered at doses corresponding to their ED50 values in combination with acetone (5 or 7.5 mmol/kg) in the MES test in mice. Statistical analysis was performed either with Student’s t test (data for acetone 5 mmol/kg) or with one-way ANOVA followed by the post hoc Bonferroni t test (data for acetone 7.5 mmol/kg). CR, % reduction of acetone concentration, as compared to control (acetone + vehicle-treated) animals. **p < 0.01 and ***p < 0.001 versus the respective control group (acetone + vehicle-treated animals). For more details see Table 4.
and on motor coordination in the chimney test (0% motor impairment). When AEDs were tested in doses corresponding to their ED50 values in the MES test, coadministration of acetone (7.5 mmol/kg) did not significantly alter the retention time in the passive avoidance test (retentions ranged from 153–180 s; p > 0.05 vs. control) and motor performance in the chimney test (% motor impairment ranged from 0% to 25%; p > 0.05 vs. control). Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
1138 I. Zarnowska et al.
Discussion The present study provides several new findings relevant to the anticonvulsant efficacy of acetone and its interactions with common AEDs. First, we demonstrated the anticonvulsant efficacy of acetone in the MEST and MES seizure tests in mice. Second, we revealed that acetone significantly potentiated the anticonvulsant effects of CBZ, PB, LTG, and VPA in the MES test in mice. Third, these interactions were likely to be of pharmacodynamic origin rather than pharmacokinetic in terms of concentrations of AED. Finally, combinations of acetone and AEDs were not associated with increased adverse effects on motor coordination and long-term memory regardless of the outcome of those combinations on seizure parameters. Anticonvulsant efficacy of exogenously administered acetone against MES-induced seizures has already been demonstrated in rats (Kohli et al., 1967; Likhodii et al., 2003). In those studies, acetone dose dependently and significantly protected the animals against tonic seizures induced by MES, with ED50 values ranging from 3.8– 6.6 mmol/kg. At higher doses, acetone also induced motor impairment with its median toxic dose (TD50) values ranging from 28.4–42.2 mmol/kg. This separation between acetone’s untoward and beneficial effects resulted in therapeutic index (TI = TD50/ED50) values ranging from approximately 4.3–11.1. There are no reports of acetone’s efficacy in the MEST test in rats. However, it is likely that acetone also would be efficacious in the MEST test and even more potent than in the MES test, since the current intensity applied to induce seizures and potencies of effective treatments are inversely correlated in tests where seizures are induced by an electric shock (Lçscher et al., 1991). The present study extends these finding by demonstrating efficacy of acetone in both the MEST and MES tests in mice. As could be predicted (Lçscher et al., 1991), the anticonvulsant potency of acetone in the MEST test (first effective dose 10 mmol/kg) was lower (approximately 1.6-fold) than in the MES test (first effective dose 16 mmol/kg and an ED50 value of 26.3 mmol/kg). On the basis of a recent estimation of acetone’s TD50 value (45.6 mmol/kg) in the inverted-screen test in mice (Gasior et al., 2007), acetone’s therapeutic window would be 2.9 and 1.7 in the MEST and MES tests in mice, respectively. Of note, the anticonvulsant potency and TI for acetone in the MES test appear lower in mice than in rats. Perhaps this is the reason that rats better than mice tolerate the KD and respond better to its anticonvulsant effects when measured in different seizure tests (Uhlemann & Neims, 1972; Appleton & De Vivo, 1973, 1974; Bough & Eagles, 2001; Bough et al., 2002; Thavendiranathan et al., 2003). However, a recent finding of a very robust anticonvulsant effect produced by the KD when assessed in a 6-Hz seizure test in mice may challenge this conclusion (Hartman et al., 2008). Epilepsia, 50(5):1132–1140, 2009 doi: 10.1111/j.1528-1167.2008.01864.x
Rational polytherapy is a common approach to treat drug-resistant forms of epilepsy (Schmidt, 1996; Deckers et al., 2000; Kwan & Brodie, 2006). By combining drugs with different or even similar pharmacologic actions, polytherapy allows for better therapeutic efficacy at lower and, thus, less toxic doses. This phenomenon is also often observed and studied under experimental conditions where one anticonvulsant agent in doses ineffective per se would potentiate anticonvulsant effects of other compounds or AEDs (Luszczki et al., 2006a, 2007a, 2008a,b). The present study shows that acetone behaves similarly to many classical and novel AEDs when administered in combinations. Specifically, acetone potentiated anticonvulsant effects of several AEDs by increasing their protective potencies against MES-induced seizures. For example, VPA in combination with acetone showed nearly 50% higher potency than when given alone. Our pharmacokinetic studies revealed that the potentiation of the anticonvulsant effects of several AEDs in the MES test were not caused by increased brain exposures to AEDs and/or acetone. Specifically, acetone had no impact on the total concentrations of AEDs in the brain. Of note, nearly all AEDs, in fact, decreased brain levels of acetone; PB and PHT were the exception and had no effect. The latter interaction strengthens the conclusion that interactions of acetone and AEDs in the MES test were pharmacodynamic in nature and that acetone alone was unlikely to have any protective efficacy per se in the MES test that could contribute to its synergistic interaction with some AEDs. Given the lack of evidence of any particular pharmacologic action of acetone, one could predict that acetone should similarly affect the anticonvulsant properties of all AEDs studied. This, however, was not the case and, instead, there was rather a complex pattern of pharmacodynamic–pharmacokinetic interactions between acetone and AEDs in the present study. For example, the anticonvulsant action of PB was significantly enhanced by acetone in the MES test, and there was no change in the brain levels of acetone caused by PB. In the case of CBZ, LTG, and VPA, however, their anticonvulsant potencies were also enhanced by acetone and it was accompanied by significant reductions of acetone’s brain levels. On the other hand, acetone did not affect the anticonvulsant potency of PHT and PHT did not alter the brain concentrations of acetone. Acetone did not affect anticonvulsant activities of OXC and TPM, whereas both AEDs significantly reduced acetone’s brain concentration. Further complicating the matter is the fact all AEDs studied represent a group of agents with many distinctive, and, in some cases, overlapping pharmacologic actions and properties (Rogawski & Lçscher, 2004). Therefore, no unequivocal conclusions and hypothesis can be formulated and more advanced neurochemical and electrophysiologic studies are required to
1139 Interactions of Acetone with AEDs in the MES Test in Mice elucidate the exact nature of the interactions between acetone and these AEDs. Finally, the present report bears some relevance beyond simple drug–drug interactions. First, there is an increasing interest in developing compounds affecting glucose metabolism, including compounds structurally similar to acetone, as potential treatments of neurologic disorders associated with neuronal hyperexcitability and/or death (Smith et al., 2005; Stafstrom et al., 2005; Garriga-Canut et al., 2006; Lian et al., 2007). The present paper provides the first preclinical proof-of-concept evidence that add-on therapies with such compounds might produce better therapeutic efficacy with fewer side effects. Second, since acetone is the chief ketone body elevated in the KD, its ability to potentiate the anticonvulsant effects of several AEDs may have some relevance to the efficacy of the KD in refractory epilepsy. Of note is that the brain levels of acetone attained after peripheral injections in the present study (0.08–0.15 mm) were comparable to the cerebral concentrations of acetone (0.7 € 0.2 mm) measured by a proton magnetic resonance spectroscopy in epileptic children treated with the KD (Seymour et al., 1999). Therefore, the ability of acetone to potentiate the effects of AEDs could translate into better seizure control in patients who remain on concomitant KD and pharmacologic therapies. This assumption is supported by the recent demonstration of the synergistic interaction between VPA and the KD in protecting against pentylenetetrazole-induced seizures in rats (Bough & Eagles, 2001). Third, it is also possible that the combination of certain AEDs and acetone produces a qualitatively different treatment entity with pharmacologic properties different from those produced by each element alone. Finally, our study also suggests that there may be AEDs with better propensity (e.g., VPA) to interact with acetone then other AEDs (e.g., PHT) and, therefore, the final therapeutic success may depend upon the specific adjunct AED in patients on the KD. Clearly, further experimental studies are warranted to elucidate mechanisms and benefits of the combined treatment of acetone and AEDs.
Acknowledgments The authors gratefully acknowledge K. Tutaj (Department of Forensic Medicine, Medical University of Lublin, Poland) for assisting in pharmacokinetic studies with lamotrigine. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidances. Conflict of interest: There are no conflicts of interest to report.
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