FULL-LENGTH ORIGINAL RESEARCH
Enantioselective pharmacodynamic and pharmacokinetic analysis of two chiral CNS-active carbamate derivatives of valproic acid *1Tawfeeq Shekh-Ahmad, *1Hafiz Mawasi, †John H. McDonough, ‡Richard H. Finnell, ‡Bogdan J. Wlodarczyk, *Eylon Yavin, and *§Meir Bialer Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
SUMMARY
Tawfeeq ShekhAhmad is an advanced PhD student at the Hebrew University who performs epilepsy research.
Objective: 2-Ethyl-3-methylbutyl-carbamate (EMC) and 2-isopropylpentyl-carbamate (IPC) are among the most potent anticonvulsant carbamate derivatives of valproic acid. EMC and IPC are chiral compounds. Consequently, the aim of the current study was to comparatively evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticonvulsant activity) profile of EMC and IPC individual enantiomers. Methods: The anticonvulsant activity of EMC and IPC individual enantiomers was evaluated in several anticonvulsant rodent models including maximal electroshock (MES), 6 Hz psychomotor, subcutaneous (pentylenetetrazole) (scMet), and the pilocarpineinduced and soman-induced status epilepticus (SE). The PK–PD relationship of EMC and IPC individual enantiomers was evaluated following intraperitoneal administration (50 mg/kg) to rats. Induction of neural tube defects (NTDs) was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. Results: In mice and rats, (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the MES and scMet tests, whereas in the 6 Hz test, racemic EMC was more potent than its two individual enantiomers. Racemic EMC exhibited a potent activity in the soman-induced SE model when administered 5 and 20 min after seizure onset with median effective dose (ED50) values of 33 and 48 mg/kg, respectively. (2R)IPC and (2S)-IPC exhibited ED50 values similar to those of racemic IPC in the mouse and rat MES and scMet models. (2R)-IPC had similar ED50 values on the 6 Hz tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpine-induced SE model when given 30 min after seizure onset. Racemic EMC and IPC and their enantiomers had similar clearance (3.8–5.5 L/h/kg) and short half-life (20 old and new antiepileptic drugs (AEDs), approximately 30% of the patients with epilepsy are not seizure free despite receiving AED therapy.2 Many carbamate compounds have demonstrated potential therapeutic uses. The discovery in the 1960s that the old dicarbamate anxiolytic drug meprobamate (Fig. 1) also possessed anticonvulsant activity, prompted the design and evaluation of several anticonvulsant carbamates.3 Structure–activity relationship (SAR) studies of carbamates showed that derivatives with two alkyl groups attached at C-2 possess stronger muscle paralyzing activity, whereas the presence of a phenyl group at the two position enhances anticonvulsant activity.4 Felbamate (Fig. 1), 2-phenyl-1,3-propanediol dicarbamate, lacked anxiolytic properties despite its structural similarity to meprobamate, yet it exhibited a broad anticonvulsant activity.5 Felbamate was approved by the U.S. Food and Drug Administration (FDA) in 1993 as a new AED for monotherapy with great expectations.6 However, it is seldom used in clinical practice because of the fatal aplastic anemia and hepatotoxicity associated with its use.7 Another anticonvulsant carbamate that reached phase III clinical trials was carisbamate (Fig. 1); however, its regulatory dossier (application) to the FDA and European Medicines Agency (EMA) was withdrawn because of lack of consistent efficacy across a clinically relevant dose range.8,9 Hen et al.10 designed and comparatively evaluated the anticonvulsant activity of a series of 19 branched alkyl and aryl carbamates, many of which were valproic acid (VPA) derivatives. Despite the close structural features of the investigated carbamates, only a couple of compounds were active in the maximal electroshock (MES) and the pilocarpine-induced status epilepticus (SE) models.10 Among the most active compounds were: 2-ethyl-3-methylbutyl carbamate (EMC; Fig. 2) and 2-isopropylpentyl carbamate (IPC; Fig. 2). EMC and IPC are chiral compounds possessing one stereogenic center in their chemical structure. Consequently, the aims of the current study were to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticon-
vulsant activity) profile as well as the PK–PD relationship of EMC and IPC individual enantiomers in comparison to their respective racemates.
Materials and Methods Chemicals and reagents See Supporting Information Data S1. Biologic testing/anticonvulsant activity Pharmacokinetic studies Analysis of EMC, IPC, and their individual enantiomers in plasma. Plasma concentrations of each compound were quantified by a gas chromatograph-mass spectrometer (GC-MS) assay. The GC-MS analysis was performed on a Hewlett Packard (HP) 5890 Series II GC apparatus (Hewlett Packard, Palo Alto, CA, U.S.A.) equipped with an HP5989A single quadruple mass spectrometer operating in electron impact (EI) mode, an HP7673 autosampler, an HP MS-DOS Chemstation, and an HP-5MS capillary column (0.25 lm 9 15 m 9 0.25 mm). Plasma (200 ll) was added to the test tubes, followed by 25 ll of methanol and 25 ll of internal standard solution a-fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide (aF-TMCD) 250 lg/ml in methanol)11 and the tubes were thoroughly vortexed. Chloroform (2 ml) was used for the extraction of the compounds. The dry residues obtained after evaporation of 1.8 ml chloroform were reconstituted with 60 ll methanol, of which 1 ll was injected into the GC-MS apparatus. The temperature program was as follows: injector temperature, 200°C; initial temperature, 50°C for 6 min; gradient of 20°C/min until 140°C; gradient of 10°C until 300°C; and hold time, 3 min. The MS parameters were set as follows: source temperature, 200°C; transfer line, 280°C; positive ion monitoring, electron ionization-mass spectrometry (EI-MS) (70 eV). The pressure of the carrier gas, helium, was set at 5 psi. For EI analysis, the ionization energy was 70 eV with a source pressure of 10 6 Torr. Retention times
Figure 1. Chemical structure of meprobamate, felbamate, and carisbamate. Meprobamate: [2-(carbamoyloxymethyl)-2-methylpentyl] carbamate; felbamate: (3-carbamoyloxy-2-phenylpropyl) carbamate; and carisbamate: (S)-2-O-carbamoyl-1-ochlorophenyl-ethanol. Epilepsia ILAE
Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1946 T. Shekh-Ahmad et al.
Figure 2. Chemical structure of 2-ethyl-3methylbutyl-carbamate (EMC) and 2isopropylpentyl-carbamate (IPC) (and their individual enantiomers). Epilepsia ILAE
of EMC, IPC, and internal standard were 11.6, 12.6, and 10.6 min, respectively. Calibration curves were constructed for each analytic run and were linear on the concentration range between 0.5 and 50 lg/ml. Calculation of pharmacokinetic parameters. The PK parameters of each compound were calculated by noncompartmental analysis based on statistical moment theory using PK software Phoenix Winnonlin Tripos L.P. (Pharsight co., Mountain View, CA, U.S.A.).12 The terminal halflife was calculated as 0.693/kz, where kz is the linear terminal slope of the log drug plasma concentration (C) versus time (t) curve. The plasma exposure or area under the C versus t curve (AUC) from zero to infinity was calculated by using the trapezoidal rule with extrapolation to infinity. The mean residence time (MRT) was calculated from the quotient AUMC/AUC, where AUMC is the area under the C 9 t product versus t (first moment-time) curve from zero to infinity. The apparent (total) clearance (CL/F) was calculated from the quotient of Dose/AUC, with F being the absolute bioavailability of the drug after intraperitoneal administration. The apparent volume of distribution (V/F) was calculated from the quotient of CL and kz. The peak plasma concentration (Cmax) and the time to reach Cmax (tmax) were determined by visual inspection. Animals and test substances used for seizure testing. Male albino CF1 mice (18–25 g; Charles River, Portage, MI, U.S.A.) and male albino Sprague-Dawley rats13–15 (100–150 g; Charles River, Wilmington, MA, U.S.A.) were Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
used as experimental animals. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC)–accredited temperature and humidity controlled facility and maintained on a standard 12 h:12 h light–dark (lights on at 06:00 a.m.) cycle with free access to standard laboratory chow Prolab RMH 3000 (Charles River, Wilmington, MA, USA) and water ad libitum. All animal experiments were performed in accordance with the guidelines set by National Institutes of Health and the University of Utah Institutional Animal Care and Use Committee (IACUC). All animals were allowed free access to both food and water, except when they were removed from their cages for the experimental procedure. Except for the kindling studies, animals were used once. All animals were euthanized in accordance with the Institute of Laboratory Resources policies on the humane care of laboratory animals. Each of the EMC and IPC enantiomers was administered (i.p. or p.o.) in 0.5% methylcellulose in a volume of 0.04 ml/10 g body weight in rats. Anticonvulsant tests. In vivo anticonvulsant activity was established by both electroconvulsant and chemoconvulsant seizure tests, which have been described previously.14,15 The electrical tests used were the MES and the 6 Hz seizure test. The chemical test was the subcutaneous pentylenetetrazole (scMet). MES test and 6 Hz test. For the MES and 6 Hz tests, a drop of anesthetic/electrolyte solution (0.5% tetracaine
1947 VPA Carbamate Derivatives hydrochloride in 0.9% saline) was applied to the eyes of each animal prior to placement of the corneal electrodes. The electrical stimulus in the MES test was 50 mA, 60 Hz, for mice and 150 mA, 60 Hz, for rats delivered for 0.2 s by an apparatus similar to that originally described by Woodbury and Davenport.16 Abolition of the hind leg tonic extensor component of the seizure was used as the endpoint. The ability of the test substance to prevent seizures induced by 6 Hz corneal stimulation (32 and 44 mA, 3 s duration) in mice was evaluated at a convulsive current that evokes a seizure in 97% of the population tested (CC97). Seizures of 6 Hz are characterized by a minimal clonic phase that is followed by stereotyped, automatistic behaviors described originally as being similar to the aura of human patients with partial seizures.17 Animals not displaying this behavior were considered protected. scMet test. In this test, a pentylenetetrazole convulsive dose (CD97) is injected subcutaneously to mice (85 mg/kg) and to rats (56 mg/kg). The animals are isolated and are observed for the presence and absence of clonic spasms that persist for at least 5 s. When clonic seizures are not observed, the test compound is considered effective in raising the seizure threshold. Lamotrigine-resistant amygdala kindled rat. Prior to the first kindling stimulation, rats were randomized into two groups, that is, control (vehicle) and IPC- treated. Rats in the vehicle-treated control group received (0.5%, W/V) methylcellulose, i.p., daily before the amygdala stimulation until they were fully kindled. Rats in the IPC-treated group received lamotrigine (LTG; 5 mg/kg, i.p.) 1 h before each amygdala stimulation. Amygdala stimulation and drug treatment were discontinued once rats on both groups were fully kindled. Two days following the last kindling, both groups were challenged with a higher dose of LTG (15 mg/kg, i.p.) given to fully kindled rats. The seizure score and afterdischarge duration (ADD) were recorded for each rat. The average and standard error of the mean (SEM) of the seizure scores and ADD are noted, as are the number of animals protected from seizure (defined as a seizure score < 3).18 Soman-induced SE. An established rodent model of nerve agent–induced status epilepticus (SE) was used; a rat HI-6 pretreatment model.19,20 The model utilized a pretreatment and adjunctive drugs that counter the acute immediate lethal effects of the nerve agent without inhibiting the development of SE. In this model the challenge dose of soman is sufficient to elicit SE in all animals within 5–8 min following the soman challenge. Subjects: Male Sprague-Dawley rats (Crl:DCBR VAF/ Plus) from Charles River Labs, weighing 250–300 g upon receipt, served as subjects. The animals were housed individually in temperature-controlled (21 2°C) and humidity-controlled (50 10%) quarters and maintained
on a 12-h light–dark full spectrum lighting cycle with lights on at 06:00 a.m. Laboratory rat chow and tap water were freely available. Each animal was anesthetized with isoflurane (5% induction; 3–1.5% maintenance, with oxygen) and placed in a stereotaxic device. Two stainless steel screws were placed in the skull bilaterally midway between bregma and lambda and approximately 3 mm lateral to the midline. A third screw was placed over the cerebellum. The screws were connected to a miniature connector with wires and the screws; wires and connector were then anchored to the skull with dental cement. The incision was sutured; the animal was removed from the frame, given the analgesic buprenorphine HCl (0.03 mg/kg, s.c.), and placed on a warming pad for at least 30 min before being returned to the animal quarters. Approximately 7 days elapsed between surgery and experimentation. The animals were typically tested in groups of eight and were randomized among treatment cohorts each test day. The animals were weighed, placed in individual recording chambers, and connected to the recording apparatus. EEG signals were recorded using CDE 1902 amplifiers and displayed on a computer running Spike2 software (Cambridge Electronic Design, Ltd., Cambridge, United Kingdom). Baseline EEG was recorded for at least 20 min. The animals were then pretreated with 125 mg/kg, i.p., of the oxime HI6 to prevent the rapid lethal effects of the soman challenge. Thirty minutes after pretreatment the rats were challenged with 180 lg/kg, s.c., soman (1.6 9 Median lethal dose (LD50) and 1 min later treated with 2.0 mg/kg, i.m., atropine methyl nitrate to inhibit peripheral secretions. The rats were then closely monitored both visually and on electroencephalography (EEG) for seizure onset, which was operationally defined as the appearance of >10 s of continuous rhythmic high-amplitude spikes or sharp waves that were at least twice the baseline amplitude accompanied by a rhythmic bilateral flicking of the ears, facial clonus, and possibly forepaw clonus. The rats received standard medical countermeasures (0.1 mg/kg atropine sulfate + 25 mg/kg 2-pyridine aldoxime methyl chloride (2-PAM Cl admixed to deliver 0.5 ml/kg, i.m., and 0.4 mg/kg, i.m., diazepam) at 5 or 20 min after seizure onset, and then were immediately given a dose (18–100 mg/kg, i.p.) of EMC dissolved in multisol (a solution of propylene glycol, alcohol, and water for injection: 5:1:4). These standard medical countermeasures (atropine, 2-PAM, and in the rat model, diazepam), at the doses and times used, are insufficient by themselves to terminate soman-induced seizures in this model. The rats were monitored for at least 5 h postexposure and then returned to the animal housing room. Twenty-four hours after the exposure, surviving animals were weighed and the EEG again recorded for at least 30 min. Evaluation and categorization of the EEG response by an individual animal to treatment was performed by a technician and an investigator, both well-experienced with the appearance of nerve Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1948 T. Shekh-Ahmad et al. agent–induced EEG seizure activity. The overall rating and timing of different events required consensus between both individuals, who were aware of the treatment conditions of an individual animal. To be rated as having the seizure terminated, all spiking and/or rhythmic waves had to stop, and the EEG had to remain normal for at least 60 min. For each animal in which the seizure was terminated, the latency to seizure termination was measured as the time from when the animal received EMC to the last observable epileptiform event in the EEG recording. Minimal behavioral toxicity tests. Minimal toxicity was identified in rats as minimal motor impairment (MMI) as determined by overt evidence of ataxia, abnormal gait, and stance.
Results Chemistry The general synthesis of the two individual stereoisomers of EMC [(2R)-EMC and (2S)-EMC] and IPC [(2R)-IPC and (2S)-IPC] is depicted in Scheme S1 and detailed in the experimental section (Data S1). The first step of the synthesis was to conjugate the starting material (isovaleric acid) to the chiral auxiliary [(1S,2S)- or (1R,2R)- pseudoephedrine] followed by alkylation (ethyl iodide for EMC; propyl iodide for IPC). Furthermore, the pseudoephedrine was removed by acidic hydrolysis. The enantioselective acid was hydrogenated using LiAlH4, and finally, the alcohol was converted to the desired compound (carbamate) by sodium isocyanate. The synthesized products were purified by crystallization. 1H-NMR (nuclear magnetic resonance) spectra of the synthesized compounds were measured in dimethyl sulfoxide (DMSO) using TMS as an internal standard. Elemental analyses were performed for all the synthesized compounds. Time to peak effects (TPEs) of EMC and IPC stereoisomers and determination of their median effective (ED50) or behavioral toxic dose (TD50) All quantitative in vivo anticonvulsant/behavioral toxicity studies were conducted at TPE previously determined in a qualitative analysis. The TPE of EMC and IPC enantiomers was 0.25 h following intraperitoneal and oral administration. Groups of 4–8 mice or rats were tested with various doses until at least two points were established between the limits of 100% protection or minimal toxicity and 0% protection or minimal toxicity. The dose of drug required to produce the desired endpoint in 50% of animals (ED50 or TD50) in each test, the 95% confidence interval, the slope of the regression line, and the standard error of the mean (SEM) of the slope were then calculated by a computer program based on the method described by Finney.21 Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
Anticonvulsant efficacy of EMC and IPC enantiomers in seizure and epilepsy rodent models The anticonvulsant activity of the individual EMC and IPC enantiomers (in comparison to racemate) is depicted in Tables 1 and 2, respectively. In mice (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the scMet and 6 Hz tests, whereas in the 6 Hz test EMC (racemate) was more potent than its two individual enantiomers. In the rat MES and scMet tests, (2S)-EMC exhibited anticonvulsant activity similar to that of the racemic EMC following intraperitoneal and oral administration. However, the rat-i.p.-ED50 values were two to three times more potent than those obtained orally and the neurotoxicity-TD50 values were lower following oral administration. Our experience with central nervous system–active VPA derivatives (e.g., valnoctamide) is that their neurotoxicity-TD50 values are less toxic when they are given orally due to the slower oral absorption compared to intraperitoneal administration.11 (2R)-IPC and (2S)-IPC exhibited anticonvulsant-ED50 values similar to that of racemic IPC in the mice and rat (i.p.) MES and scMet tests. (2R)-IPC had similar ED50 values at the 6 Hz–32 mA and 6 Hz–44 mA tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpineinduced SE model. There was also no significant difference in the neurotoxicity-TD50 values of IPC individual enantiomers and the racemate. IPC anticonvulsant efficacy in the lamotrigine-resistant amygdala kindled rat model When tested at 40 mg/kg, IPC displayed full protection (8/8) and its seizure score was 1.38/5 (5/5 for the control group), and the ADD duration for the IPC was 50% shorter compared to the control group (48 vs. 91 s). Effect of EMC (racemate) on soman-induced SE Racemic EMC exhibited a potent activity in the somaninduced SE model when administered 5 min after the onset of electrographic seizures in rats (ED50 = 34 mg/kg; 12–48 mg/kg, 95% confidence intervals), and only a slightly higher dose was required to control seizures when treatment was delayed to 20 min after seizure onset (ED50 = 48 mg/kg; confidence intervals could not be determined). Pharmacokinetics of EMC and IPC in rats The pharmacokinetics of EMC, IPC, and their individual enantiomers was studied following intraperitoneal administration (50 mg/kg) of each enantiomer as well as the racemate to rats. The dose was chosen for the PK study as the intermediate dose among the various ED50 values. The plasma concentration–time plots of EMC and IPC and their individual enantiomers are presented in Figures 3 and 4. The PK parameters, calculated by noncompartmental analysis, are summarized in Tables S1 and S2.
1949 VPA Carbamate Derivatives Table 1. Anticonvulsant activity and neurotoxicity of EMC (racemate) and its two individual enantiomers (2S)-EMC and (2R)-EMC following intraperitoneal administration to mice and intraperitoneal or oral administration to rats ED50 (95% confidence interval) (mg/kg) Anticonvulsant test
EMC racematea
(2S)-EMC
(2R)-EMC
VPAb
Mice-i.p.
103 (90–124) 120 (108–131) 33 (23–47) 157 (136–185) 16 (11–21) 19 (13–28) 58 (45–70) 64 (39–92) 35 (20–73) >500
85 (67–101) 113 (81–184) 61 (49–74) 203 (172–243) 27 (24–29) 17 (10–24) 77 (56–91) 71 (47–101) 37 (26–48) 107 (65–152)
83 (70–101) 106 (91–121) 61 (56–65) 134 (120–148) 36 (24–50) 29 (18–36) 51 (31–69) 48 (36–59) ND >250
263 (237–282) 220 (177–268) 126 (95–152) 398 (356–445) 140 (110–178)c 195 (157–242)c 275 (239–322)c 484 (324–677) 646 (466–869) 784 (503–1,176)
Rats-i.p.
Rats-p.o.
Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) 6 Hz–32 mA Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50)
ND, not determined. a Data taken from Hen et al.10 b Data taken from Shekh-Ahmad et al.38 c Data taken from L€ oscher and Nolting.39
Table 2. Anticonvulsant activity and neurotoxicity of IPC (racemate) and its two individual enantiomers [(2S)-IPC and (2R)-IPC] following intraperitoneal administration to mice and rats ED50 (95% confidence interval) (mg/kg) Anticonvulsant test Mice-i.p.
Rats-i.p.
Maximal electroshock seizure (MES) 6 Hz–32 mA 6 Hz–44 mA Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50)
IPC racemate
(2S)-IPC
(2R)-IPC
84 (68–103) 66 (53–82) 79 (71–85) 78 (59–109) 133 (109–158) 24 (18–31) 40 (30–49) 54 (36–68)
123 (110–137) ND ND 188 (174–222) 176 (152–217) 31 (24–38) >50 50 (39–63)
77 (63–95) 51 (32–68) 64 (47–83) 144 (117–167) 159 (133–180) 27 (21–35) 31 (25–38) 47 (37–56)
ND, not determined.
Figure 3. Plasma concentration–time plots of the EMC (racemate) and its two individual enantiomers [(2S)-EMC and (2R)-EMC] obtained following intraperitoneal administration of 50 mg/kg (of each compound) to rats. Epilepsia ILAE
Figure 4. Plasma concentration–time plots of the IPC (racemate) and its two individual enantiomers [(2S)-IPC and (2R)-IPC] obtained following intraperitoneal administration of 50 mg/kg (of each compound) to rats. Epilepsia ILAE Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1950 T. Shekh-Ahmad et al. Racemic EMC and IPC and their individual enantiomers had similar clearance and volume of distribution values that ranged between 3.8 and 5.5 L/h/kg and 2.7 and 4.6 L/kg, respectively. Consequently, the half-lives of the EMC and IPC individual enantiomers were short and ranged between 25 and 42 min. Teratogenicity of EMC (racemate) and its two enantiomers The teratogenic potential of racemic EMC and its two enantiomers was assessed for their ability to induce gross morphologic defects in the Swiss Vancouver/Fnn mice, which are highly susceptible to VPA-induced exencephaly. VPA, at a dose of 2.7 mmol/kg, was embryotoxic and teratogenic, causing an almost twofold increase in the resorption rate compared to the control group (11.9% vs. 6.3%, respectively) and neural tube defects (NTDs) in 29.1% of live fetuses. At a lower dose of 1.8 mmol/kg, VPA was still embryotoxic (13.6% of resorptions), but the number of fetuses with exencephaly (2) was not statistically significant. In contrast to VPA, racemic EMC and its two enantiomers did not cause a statistically significant increase of NTDs at doses of 286 or 429 mg/kg or 1.8 or 2.7 mmol/kg (see Table S3). These doses are 3 to 10 times higher than EMC anticonvulsant-ED50 values, when tested at the higher 2.7 mmol/kg dose (Table S3).
Discussion Approximately 30% of patients with epilepsy are not seizure free despite therapy with the existing medications. In addition, AED therapy, particularly with VPA, is associated with severe side effects (e.g., teratogenicity).22–24 There are three major approaches for designing and developing new AEDs: A mechanism-based approach focusing on a new mechanism of action (MOA) that is not currently possessed by any existing AED.25 The second approach is the design of more potent follow-up compounds to existing AEDs, which will circumvent problems associated with the first-generation AEDs.26–28 The third (phenotyping or empirical) approach is based on the screening of drug candidates in anticonvulsant animal (rodent) models of epilepsy that have established their predictability since the discovery of phenytoin in 1938.15,26,29 In this study, we investigated the individual enantiomers of two lead chiral carbamates designed by the phenotyping and the second-generation approaches.10 A reasonable prediction of a compound’s potential as a new AED candidate is based on the characterization of the compound’s anticonvulsant profile in a variety of anticonvulsant animal models. Activity in various anticonvulsant models may indicate a wide antiepileptic spectrum of activity.26,30 Although there are many animal models of anticonvulsant drug efficacy, the MES and scMet seizure models remain the “gold standards” in early stages of discovery of Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
new AEDs. These models have gained an appreciable degree of predictability since the MES was first utilized in the discovery of the anticonvulsant activity of phenytoin (1938).26,27,30 The LTG-resistant amygdala-kindled rat model displays a resistant profile consistent with therapyresistant seizure and is amenable to medium through-put screening.18,31 Carbamazepine, LTG, phenytoin, and topiramate are inactive in this model, while VPA (300 mg/kg) and retigabine (40 mg/kg) effectively blocked the seizures. In the current study, racemic IPC (30 mg/kg) effectively blocked seizures and decreased the ADD in the LTG-resistant amygdala-kindled rat model and was 10 times more potent than VPA. Screening in animal models is essential in an AED-discovery program, particularly if, like most current AEDs, the drug candidate has multiple or unknown MOAs. In addition; animal models provide an insight to PK–PD correlation of a drug candidate. Given the highly heterogeneous nature of seizure disorders in humans, the complexity of the seizure, and the syndrome involved, it is unlikely that a single anticonvulsant animal model will predict the full therapeutic potential of a drug candidate.26 VPA is an example of a major AED with multiple MOAs that consequently has a wide spectrum of antiepileptic activity in patients,32 and is also effective and approved for the treatment of bipolar disorder and migraine prophylaxis. Similarly, the VPA carbamate derivatives EMC and IPC are likely to have multiple MOAs. EMC (racemate) exhibited a wide spectrum of anticonvulsant activity, including the soman-induced SE like valnoctamide (VCD) and sec-propyl-butyl acetamide (SPD) with similar ED50 values.11,33,34 EMC and IPC and their individual enantiomers had clearance values that were 10 and 2 times higher than those of VCD and SPD. Consequently EMC and IPC half-life was shorter than 1 h. Following intraperitoneal administration to rats, racemic IPC had ED50 values similar to those of EMC. Racemic IPC was equipotent to VCD and SPD (Bialer M, Shekh-Ahmad T, Mawasi H, unpublished data) in the LTG-resistant amygdala-kindled rat model. EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to the lack of enantioselective activity in all anticonvulsant models tested. Bialer et al.35 demonstrated a correlation between the anticonvulsant-ED50 values in mice and rats of various AEDs and their dose and therapeutic average steady-state plasma concentrations in patients with epilepsy. The fact that EMC and IPC enantiomers are 3 to 10 times more potent than VPA in various animal models may cause them to be more potent than VPA in patients. Binding of a racemic drug (e.g., EMC, IPC) to the molecular targets that lead to its antiepileptic activity (e.g., ion channel, a receptor or an enzyme) may be stereospecific, and consequently individual stereoisomers that may display distinguished PK behavior that could lead to stereoselective PD (e.g., SPD).33 Therefore, consideration of chirality should be implemented into PK and PD studies.
1951 VPA Carbamate Derivatives The FDA’s 1992 policy “Statement for Development of the New Stereoisomeric Drugs” triggered the development of single individual stereoisomers instead of racemates.36 This policy coupled with marketing incentives of further profitability as a “line extension” has encouraged companies to look for chiral switches of established chiral drugs that were first introduced to the market as racemic mixtures.37 In the current study, no enantioselectivity was observed in the anticonvulsant activity of EMC and IPC in any of the anticonvulsant animal models. In addition, EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to the absence of enantioselectivity in EMC and IPC anticonvulsant activity. If EMC and/or IPC would exert their anticonvulsant activity via a single mechanism of action, it is likely that they would exhibit a stereoselective PD. The fact that there was no significant difference between racemic EMC and racemic IPC and their individual enantiomers, suggests that their anticonvulsant activity is caused by multiple mechanisms of action.
Conclusions In this study, we report the synthesis and comparative evaluation of the anticonvulsant activity and neurotoxicity of individual enantiomers of two CNS-active carbamate derivatives of VPA. EMC and IPC did not exhibit stereoselective PK, a fact that may contribute to their nonstereoselective anticonvulsant activity in all of the anticonvulsant models. EMC and IPC enantiomers offer an optimal anticonvulsant efficacy and safety profile, supporting these compounds as AED candidates for further development. Neither EMC nor IPC demonstrated a stereoselective anticonvulsant activity. The choice of the optimal individual enantiomer will be based on subsequent anticonvulsant testing coupled with toxicologic analysis.
Acknowledgments This work is abstracted from the PhD thesis of Mr. Tawfeeq ShekhAhmad in a partial fulfillment for the requirements of a PhD degree at The Hebrew University of Jerusalem. The authors wish to thank Drs. John H. Kehne, H. Steve White, and Tracy Chen of the NIH NINDS Anticonvulsant Screening Program (ASP) for testing the compounds in the ASP.
Disclosure or Conflict of Interest Dr. Meir Bialer has received in the last 3 years speakers or consultancy fees from Bial, CTS Chemicals, Desitin, Janssen-Cilag, Johnson & Johnson, Medgenics, Rekah, Sepracor, Teva, UCB Pharma, and Upsher-Smith. Dr. Bialer has been involved in the design and development of new antiepileptics and CNS drugs as well as new formulations of existing drugs. None of the other authors has any conflict of interest to disclose. We, the authors, confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Fisher RS, Acevedo C, Arzimanoglou A, et al. ILAE official report: a practical clinical definition of epilepsy. Epilepsia 2014;55:475–482. 2. Tellez-Zenteno JF, Hernandez-Ronquillo L, Buckley S, et al. A validation of the new definition of drug-resistant epilepsy by the International League Against Epilepsy. Epilepsia 2014;55:829–834. 3. Murray WJ, Kier LB. Noncyclic anticonvulsants. In Vida JA (Ed) Medicinal chemistry, Vol. 15, anticonvulsants. New York: Academic Press, 1977:578–619. 4. Ludwing BJ, Powell LS, Berger FM. Carbamate derivatives related to meprobamate. J Med Chem 1969;12:462–472. 5. Kupferberg HJ. Felbamate. In Levy RH, Matteson RH, Meldrum BS. (Eds) Antiepileptic drugs. 4th Ed. New York: Raven Ress, 1995:791– 827. 6. Bialer M. Comparative pharmacokinetics of the newer antiepileptic drugs. Clin Pharmacokinet 1993;24:441–452. 7. Wong IC, Lhatoo SD. Adverse reactions to new anticonvulsant drugs. Drug Saf 2000;23:35–56. 8. Bialer M, Johannessen SI, Levy RH, et al. Progress report on new antiepileptic drugs: a summary of the Ninth Eilat Conference (EILAT IX). Epilepsy Res 2009;83:1–43. 9. Bialer M, Johannessen SI, Levy RH, et al. Progress report on new antiepileptic drugs: a summary of the Tenth Eilat Conference (EILAT X). Epilepsy Res 2010;92:89–124. 10. Hen N, Bialer M, Yagen B. Syntheses and evaluation of anticonvulsant activity of novel branched alkyl carbamates. J Med Chem 2012;55:2835–2845. 11. Shekh-Ahmad T, Hen N, Yagen B, et al. Stereoselective anticonvulsant and pharmacokinetic analysis of valnoctamide, a CNSactive derivative of valproic acid with low teratogenic potential. Epilepsia 2014;55:353–361. 12. Rowland M, Tozer TN. Clinical pharmacokinetics and pharmacodynamics: concepts and applications. Philadelphia, PA: Lippincott William & Wilkins; 2011. 13. Lothman EW. Seizure circuits in the hippocampus and associated structures. Hippocampus 1994;4:286–290. 14. White HS, Johnson M, Wolf HH, et al. The early identification of anticonvulsant activity: role of the maximal electroshock and subcutaneous pentylenetetrazol seizure models. Ital J Neurol Sci 1995;16:73–77. 15. Smith M, Wilcox KS, White HS. Discovery of antiepileptic drugs. Neurotherapeutics 2007;4:12–17. 16. Woodbury LA, Davenport VD. Design and use of a new electroshock seizure apparatus, and analysis of factors altering seizure threshold and pattern. Arch Int Pharmacodyn Ther 1952;92:97–107. 17. Barton ME, Klein BD, Wolf HH, et al. Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy. Epilepsy Res 2001;47:217–227. 18. Srivastava AK, White HS. Carbamazepine, but not valproate, displays pharmacoresistance in lamotrigine-resistant amygdala kindled rats. Epilepsy Res 2013;104:26–34. 19. McDonough JH, Shih TM. Pharmacological modulation of somaninduced seizures. Neurosci Biobehav Rev 1991;17:203–215. 20. Shih TM, McDonough JH. Neurochemical mechanisms in somaninduced seizures. J Appl Toxicol 1991;17:255–264. 21. Finney DJ. Statistical logic in the monitoring of reactions to therapeutic drugs. Methods Inf Med 1971;10:237–245. 22. Tomson T, Battino D, Bonizzoni E, et al. Dose-dependent risk of malformations with antiepileptic drugs: an analysis of data from the EURAP epilepsy and pregnancy registry. Lancet Neurol 2011;10:609– 617. 23. Molgaard-Nielsen D, Hviid A. Newer generation antiepileptic drugs and the risk of major birth defects. JAMA 2011;305:1996–2002. 24. Vajda JE, Graham J, Roten A, et al. Teratogenicity of the newer antiepileptic drugs—the Australian experience. J Clin Neurosci 2012;19:57–59. 25. L€oscher W, Klitgaard H, Twyman RE, et al. New avenues for antiepileptic drug discovery and development. Nat Rev Drug Discov 2013;12:757–776. 26. Bialer M, White HS. Key factors in the discovery and development of new antiepileptic drugs (AEDs). Nat Rev Drug Discov 2010;9:68–83. Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1952 T. Shekh-Ahmad et al. 27. Bialer M. New antiepileptic drugs that are second generation to existing antiepileptic drugs. Expert Opin Investig Drugs 2006;15:637– 647. 28. Bialer M, Yagen B. Valproic acid: second generation. Neurotherapeutics 2007;4:130–137. 29. Perucca E, French J, Bialer M. Development of new antiepileptic drugs: challenges, incentives, and recent advances. Lancet Neurol 2007;6:793–804. 30. Rogawski MA, Loscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004;5:553–564. 31. Srivastava AK, Alex BA, Wilcox KS, et al. Rapid loss of efficacy to the antiseizure drugs lamotrigine and carbamazepine: a novel experimental model of pharmacoresistance epilepsy. Epilepsia 2013;54:1186–1194. 32. L€ oscher W. Valproic acid mechanism of action. In Levy RH, Mattson RH, Meldrum BS, Perucca E (Eds) Antiepileptic drugs. 5th Ed. New York: Lippincott Williams & Wilkins Publishers, 2002:767–779. 33. Hen N, Shekh-Ahmad T, Yagen B, et al. Stereoselective pharmacodynamic and pharmacokinetic analysis of sec-Butylpropylacetamide (SPD), a new CNS-active derivative of valproic acid with unique activity against status epilepticus. J Med Chem 2013;56:6467–6477. 34. White HS, Alex AB, Pollock A, et al. A new derivative of valproic acid amide possesses a broad-spectrum antiseizure profile and unique activity against status epilepticus and organophosphate neuronal damage. Epilepsia 2012;53:134–146. 35. Bialer M, Twyman RE, White HS. Correlation analysis between anticonvulsant ED50 values of antiepileptic drugs in mice and rats and their therapeutic doses and plasma levels. Epilepsy Behav 2004;5:866– 872. 36. Hubbard WK. FDA policy on period of marketing exclusivity for newly approved drug products with enantiomer active ingredients; request for comments. Fed Reg 1997;62:2167–2169.
Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
37. Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy of chiral switches. Nat Rev Drug Discov 2002;1:753–68. 38. Shekh-Ahmad T, Hen N, McDonough JH, et al. Valnoctamide and sec-butyl-propylacetamide (SPD) for acute seizures and status epilepticus. Epilepsia 2013;54 (Suppl. 6):99–102. 39. L€oscher W, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. IV. Proactive indices. Epilepsy Res 1991;9:1–10.
Supporting Information Additional Supporting Information may be found in the online version of this article: Scheme S1. Total enantioselective synthesis of individual enantiomers of EMC and IPC. Table S1. PK parameters of EMC (Racemate) and its two enantiomers: (2S)-EMC and (2R)-EMC calculated following i.p. administration (50 mg/kg) of each to rats. Table S2. PK parameters of IPC (Racemate) and its two enantiomers: (2S)-IPC and (2R)-IPC calculated following i.p. administration (50 mg/kg) of each to rats. Table S3. Teratogenic effect in the SWV mouse model of EMC and its enantiomers. Data S1. Experimental section: Materials and methods.
Enantioselective pharmacodynamic and pharmacokinetic analysis of two chiral CNS-active carbamate derivatives of valproic acid *1Tawfeeq Shekh-Ahmad, *1Hafiz Mawasi, †John H. McDonough, ‡Richard H. Finnell, ‡Bogdan J. Wlodarczyk, *Eylon Yavin, and *§Meir Bialer Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
SUMMARY
Tawfeeq ShekhAhmad is an advanced PhD student at the Hebrew University who performs epilepsy research.
Objective: 2-Ethyl-3-methylbutyl-carbamate (EMC) and 2-isopropylpentyl-carbamate (IPC) are among the most potent anticonvulsant carbamate derivatives of valproic acid. EMC and IPC are chiral compounds. Consequently, the aim of the current study was to comparatively evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticonvulsant activity) profile of EMC and IPC individual enantiomers. Methods: The anticonvulsant activity of EMC and IPC individual enantiomers was evaluated in several anticonvulsant rodent models including maximal electroshock (MES), 6 Hz psychomotor, subcutaneous (pentylenetetrazole) (scMet), and the pilocarpineinduced and soman-induced status epilepticus (SE). The PK–PD relationship of EMC and IPC individual enantiomers was evaluated following intraperitoneal administration (50 mg/kg) to rats. Induction of neural tube defects (NTDs) was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. Results: In mice and rats, (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the MES and scMet tests, whereas in the 6 Hz test, racemic EMC was more potent than its two individual enantiomers. Racemic EMC exhibited a potent activity in the soman-induced SE model when administered 5 and 20 min after seizure onset with median effective dose (ED50) values of 33 and 48 mg/kg, respectively. (2R)IPC and (2S)-IPC exhibited ED50 values similar to those of racemic IPC in the mouse and rat MES and scMet models. (2R)-IPC had similar ED50 values on the 6 Hz tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpine-induced SE model when given 30 min after seizure onset. Racemic EMC and IPC and their enantiomers had similar clearance (3.8–5.5 L/h/kg) and short half-life (20 old and new antiepileptic drugs (AEDs), approximately 30% of the patients with epilepsy are not seizure free despite receiving AED therapy.2 Many carbamate compounds have demonstrated potential therapeutic uses. The discovery in the 1960s that the old dicarbamate anxiolytic drug meprobamate (Fig. 1) also possessed anticonvulsant activity, prompted the design and evaluation of several anticonvulsant carbamates.3 Structure–activity relationship (SAR) studies of carbamates showed that derivatives with two alkyl groups attached at C-2 possess stronger muscle paralyzing activity, whereas the presence of a phenyl group at the two position enhances anticonvulsant activity.4 Felbamate (Fig. 1), 2-phenyl-1,3-propanediol dicarbamate, lacked anxiolytic properties despite its structural similarity to meprobamate, yet it exhibited a broad anticonvulsant activity.5 Felbamate was approved by the U.S. Food and Drug Administration (FDA) in 1993 as a new AED for monotherapy with great expectations.6 However, it is seldom used in clinical practice because of the fatal aplastic anemia and hepatotoxicity associated with its use.7 Another anticonvulsant carbamate that reached phase III clinical trials was carisbamate (Fig. 1); however, its regulatory dossier (application) to the FDA and European Medicines Agency (EMA) was withdrawn because of lack of consistent efficacy across a clinically relevant dose range.8,9 Hen et al.10 designed and comparatively evaluated the anticonvulsant activity of a series of 19 branched alkyl and aryl carbamates, many of which were valproic acid (VPA) derivatives. Despite the close structural features of the investigated carbamates, only a couple of compounds were active in the maximal electroshock (MES) and the pilocarpine-induced status epilepticus (SE) models.10 Among the most active compounds were: 2-ethyl-3-methylbutyl carbamate (EMC; Fig. 2) and 2-isopropylpentyl carbamate (IPC; Fig. 2). EMC and IPC are chiral compounds possessing one stereogenic center in their chemical structure. Consequently, the aims of the current study were to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticon-
vulsant activity) profile as well as the PK–PD relationship of EMC and IPC individual enantiomers in comparison to their respective racemates.
Materials and Methods Chemicals and reagents See Supporting Information Data S1. Biologic testing/anticonvulsant activity Pharmacokinetic studies Analysis of EMC, IPC, and their individual enantiomers in plasma. Plasma concentrations of each compound were quantified by a gas chromatograph-mass spectrometer (GC-MS) assay. The GC-MS analysis was performed on a Hewlett Packard (HP) 5890 Series II GC apparatus (Hewlett Packard, Palo Alto, CA, U.S.A.) equipped with an HP5989A single quadruple mass spectrometer operating in electron impact (EI) mode, an HP7673 autosampler, an HP MS-DOS Chemstation, and an HP-5MS capillary column (0.25 lm 9 15 m 9 0.25 mm). Plasma (200 ll) was added to the test tubes, followed by 25 ll of methanol and 25 ll of internal standard solution a-fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide (aF-TMCD) 250 lg/ml in methanol)11 and the tubes were thoroughly vortexed. Chloroform (2 ml) was used for the extraction of the compounds. The dry residues obtained after evaporation of 1.8 ml chloroform were reconstituted with 60 ll methanol, of which 1 ll was injected into the GC-MS apparatus. The temperature program was as follows: injector temperature, 200°C; initial temperature, 50°C for 6 min; gradient of 20°C/min until 140°C; gradient of 10°C until 300°C; and hold time, 3 min. The MS parameters were set as follows: source temperature, 200°C; transfer line, 280°C; positive ion monitoring, electron ionization-mass spectrometry (EI-MS) (70 eV). The pressure of the carrier gas, helium, was set at 5 psi. For EI analysis, the ionization energy was 70 eV with a source pressure of 10 6 Torr. Retention times
Figure 1. Chemical structure of meprobamate, felbamate, and carisbamate. Meprobamate: [2-(carbamoyloxymethyl)-2-methylpentyl] carbamate; felbamate: (3-carbamoyloxy-2-phenylpropyl) carbamate; and carisbamate: (S)-2-O-carbamoyl-1-ochlorophenyl-ethanol. Epilepsia ILAE
Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1946 T. Shekh-Ahmad et al.
Figure 2. Chemical structure of 2-ethyl-3methylbutyl-carbamate (EMC) and 2isopropylpentyl-carbamate (IPC) (and their individual enantiomers). Epilepsia ILAE
of EMC, IPC, and internal standard were 11.6, 12.6, and 10.6 min, respectively. Calibration curves were constructed for each analytic run and were linear on the concentration range between 0.5 and 50 lg/ml. Calculation of pharmacokinetic parameters. The PK parameters of each compound were calculated by noncompartmental analysis based on statistical moment theory using PK software Phoenix Winnonlin Tripos L.P. (Pharsight co., Mountain View, CA, U.S.A.).12 The terminal halflife was calculated as 0.693/kz, where kz is the linear terminal slope of the log drug plasma concentration (C) versus time (t) curve. The plasma exposure or area under the C versus t curve (AUC) from zero to infinity was calculated by using the trapezoidal rule with extrapolation to infinity. The mean residence time (MRT) was calculated from the quotient AUMC/AUC, where AUMC is the area under the C 9 t product versus t (first moment-time) curve from zero to infinity. The apparent (total) clearance (CL/F) was calculated from the quotient of Dose/AUC, with F being the absolute bioavailability of the drug after intraperitoneal administration. The apparent volume of distribution (V/F) was calculated from the quotient of CL and kz. The peak plasma concentration (Cmax) and the time to reach Cmax (tmax) were determined by visual inspection. Animals and test substances used for seizure testing. Male albino CF1 mice (18–25 g; Charles River, Portage, MI, U.S.A.) and male albino Sprague-Dawley rats13–15 (100–150 g; Charles River, Wilmington, MA, U.S.A.) were Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
used as experimental animals. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC)–accredited temperature and humidity controlled facility and maintained on a standard 12 h:12 h light–dark (lights on at 06:00 a.m.) cycle with free access to standard laboratory chow Prolab RMH 3000 (Charles River, Wilmington, MA, USA) and water ad libitum. All animal experiments were performed in accordance with the guidelines set by National Institutes of Health and the University of Utah Institutional Animal Care and Use Committee (IACUC). All animals were allowed free access to both food and water, except when they were removed from their cages for the experimental procedure. Except for the kindling studies, animals were used once. All animals were euthanized in accordance with the Institute of Laboratory Resources policies on the humane care of laboratory animals. Each of the EMC and IPC enantiomers was administered (i.p. or p.o.) in 0.5% methylcellulose in a volume of 0.04 ml/10 g body weight in rats. Anticonvulsant tests. In vivo anticonvulsant activity was established by both electroconvulsant and chemoconvulsant seizure tests, which have been described previously.14,15 The electrical tests used were the MES and the 6 Hz seizure test. The chemical test was the subcutaneous pentylenetetrazole (scMet). MES test and 6 Hz test. For the MES and 6 Hz tests, a drop of anesthetic/electrolyte solution (0.5% tetracaine
1947 VPA Carbamate Derivatives hydrochloride in 0.9% saline) was applied to the eyes of each animal prior to placement of the corneal electrodes. The electrical stimulus in the MES test was 50 mA, 60 Hz, for mice and 150 mA, 60 Hz, for rats delivered for 0.2 s by an apparatus similar to that originally described by Woodbury and Davenport.16 Abolition of the hind leg tonic extensor component of the seizure was used as the endpoint. The ability of the test substance to prevent seizures induced by 6 Hz corneal stimulation (32 and 44 mA, 3 s duration) in mice was evaluated at a convulsive current that evokes a seizure in 97% of the population tested (CC97). Seizures of 6 Hz are characterized by a minimal clonic phase that is followed by stereotyped, automatistic behaviors described originally as being similar to the aura of human patients with partial seizures.17 Animals not displaying this behavior were considered protected. scMet test. In this test, a pentylenetetrazole convulsive dose (CD97) is injected subcutaneously to mice (85 mg/kg) and to rats (56 mg/kg). The animals are isolated and are observed for the presence and absence of clonic spasms that persist for at least 5 s. When clonic seizures are not observed, the test compound is considered effective in raising the seizure threshold. Lamotrigine-resistant amygdala kindled rat. Prior to the first kindling stimulation, rats were randomized into two groups, that is, control (vehicle) and IPC- treated. Rats in the vehicle-treated control group received (0.5%, W/V) methylcellulose, i.p., daily before the amygdala stimulation until they were fully kindled. Rats in the IPC-treated group received lamotrigine (LTG; 5 mg/kg, i.p.) 1 h before each amygdala stimulation. Amygdala stimulation and drug treatment were discontinued once rats on both groups were fully kindled. Two days following the last kindling, both groups were challenged with a higher dose of LTG (15 mg/kg, i.p.) given to fully kindled rats. The seizure score and afterdischarge duration (ADD) were recorded for each rat. The average and standard error of the mean (SEM) of the seizure scores and ADD are noted, as are the number of animals protected from seizure (defined as a seizure score < 3).18 Soman-induced SE. An established rodent model of nerve agent–induced status epilepticus (SE) was used; a rat HI-6 pretreatment model.19,20 The model utilized a pretreatment and adjunctive drugs that counter the acute immediate lethal effects of the nerve agent without inhibiting the development of SE. In this model the challenge dose of soman is sufficient to elicit SE in all animals within 5–8 min following the soman challenge. Subjects: Male Sprague-Dawley rats (Crl:DCBR VAF/ Plus) from Charles River Labs, weighing 250–300 g upon receipt, served as subjects. The animals were housed individually in temperature-controlled (21 2°C) and humidity-controlled (50 10%) quarters and maintained
on a 12-h light–dark full spectrum lighting cycle with lights on at 06:00 a.m. Laboratory rat chow and tap water were freely available. Each animal was anesthetized with isoflurane (5% induction; 3–1.5% maintenance, with oxygen) and placed in a stereotaxic device. Two stainless steel screws were placed in the skull bilaterally midway between bregma and lambda and approximately 3 mm lateral to the midline. A third screw was placed over the cerebellum. The screws were connected to a miniature connector with wires and the screws; wires and connector were then anchored to the skull with dental cement. The incision was sutured; the animal was removed from the frame, given the analgesic buprenorphine HCl (0.03 mg/kg, s.c.), and placed on a warming pad for at least 30 min before being returned to the animal quarters. Approximately 7 days elapsed between surgery and experimentation. The animals were typically tested in groups of eight and were randomized among treatment cohorts each test day. The animals were weighed, placed in individual recording chambers, and connected to the recording apparatus. EEG signals were recorded using CDE 1902 amplifiers and displayed on a computer running Spike2 software (Cambridge Electronic Design, Ltd., Cambridge, United Kingdom). Baseline EEG was recorded for at least 20 min. The animals were then pretreated with 125 mg/kg, i.p., of the oxime HI6 to prevent the rapid lethal effects of the soman challenge. Thirty minutes after pretreatment the rats were challenged with 180 lg/kg, s.c., soman (1.6 9 Median lethal dose (LD50) and 1 min later treated with 2.0 mg/kg, i.m., atropine methyl nitrate to inhibit peripheral secretions. The rats were then closely monitored both visually and on electroencephalography (EEG) for seizure onset, which was operationally defined as the appearance of >10 s of continuous rhythmic high-amplitude spikes or sharp waves that were at least twice the baseline amplitude accompanied by a rhythmic bilateral flicking of the ears, facial clonus, and possibly forepaw clonus. The rats received standard medical countermeasures (0.1 mg/kg atropine sulfate + 25 mg/kg 2-pyridine aldoxime methyl chloride (2-PAM Cl admixed to deliver 0.5 ml/kg, i.m., and 0.4 mg/kg, i.m., diazepam) at 5 or 20 min after seizure onset, and then were immediately given a dose (18–100 mg/kg, i.p.) of EMC dissolved in multisol (a solution of propylene glycol, alcohol, and water for injection: 5:1:4). These standard medical countermeasures (atropine, 2-PAM, and in the rat model, diazepam), at the doses and times used, are insufficient by themselves to terminate soman-induced seizures in this model. The rats were monitored for at least 5 h postexposure and then returned to the animal housing room. Twenty-four hours after the exposure, surviving animals were weighed and the EEG again recorded for at least 30 min. Evaluation and categorization of the EEG response by an individual animal to treatment was performed by a technician and an investigator, both well-experienced with the appearance of nerve Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1948 T. Shekh-Ahmad et al. agent–induced EEG seizure activity. The overall rating and timing of different events required consensus between both individuals, who were aware of the treatment conditions of an individual animal. To be rated as having the seizure terminated, all spiking and/or rhythmic waves had to stop, and the EEG had to remain normal for at least 60 min. For each animal in which the seizure was terminated, the latency to seizure termination was measured as the time from when the animal received EMC to the last observable epileptiform event in the EEG recording. Minimal behavioral toxicity tests. Minimal toxicity was identified in rats as minimal motor impairment (MMI) as determined by overt evidence of ataxia, abnormal gait, and stance.
Results Chemistry The general synthesis of the two individual stereoisomers of EMC [(2R)-EMC and (2S)-EMC] and IPC [(2R)-IPC and (2S)-IPC] is depicted in Scheme S1 and detailed in the experimental section (Data S1). The first step of the synthesis was to conjugate the starting material (isovaleric acid) to the chiral auxiliary [(1S,2S)- or (1R,2R)- pseudoephedrine] followed by alkylation (ethyl iodide for EMC; propyl iodide for IPC). Furthermore, the pseudoephedrine was removed by acidic hydrolysis. The enantioselective acid was hydrogenated using LiAlH4, and finally, the alcohol was converted to the desired compound (carbamate) by sodium isocyanate. The synthesized products were purified by crystallization. 1H-NMR (nuclear magnetic resonance) spectra of the synthesized compounds were measured in dimethyl sulfoxide (DMSO) using TMS as an internal standard. Elemental analyses were performed for all the synthesized compounds. Time to peak effects (TPEs) of EMC and IPC stereoisomers and determination of their median effective (ED50) or behavioral toxic dose (TD50) All quantitative in vivo anticonvulsant/behavioral toxicity studies were conducted at TPE previously determined in a qualitative analysis. The TPE of EMC and IPC enantiomers was 0.25 h following intraperitoneal and oral administration. Groups of 4–8 mice or rats were tested with various doses until at least two points were established between the limits of 100% protection or minimal toxicity and 0% protection or minimal toxicity. The dose of drug required to produce the desired endpoint in 50% of animals (ED50 or TD50) in each test, the 95% confidence interval, the slope of the regression line, and the standard error of the mean (SEM) of the slope were then calculated by a computer program based on the method described by Finney.21 Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
Anticonvulsant efficacy of EMC and IPC enantiomers in seizure and epilepsy rodent models The anticonvulsant activity of the individual EMC and IPC enantiomers (in comparison to racemate) is depicted in Tables 1 and 2, respectively. In mice (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the scMet and 6 Hz tests, whereas in the 6 Hz test EMC (racemate) was more potent than its two individual enantiomers. In the rat MES and scMet tests, (2S)-EMC exhibited anticonvulsant activity similar to that of the racemic EMC following intraperitoneal and oral administration. However, the rat-i.p.-ED50 values were two to three times more potent than those obtained orally and the neurotoxicity-TD50 values were lower following oral administration. Our experience with central nervous system–active VPA derivatives (e.g., valnoctamide) is that their neurotoxicity-TD50 values are less toxic when they are given orally due to the slower oral absorption compared to intraperitoneal administration.11 (2R)-IPC and (2S)-IPC exhibited anticonvulsant-ED50 values similar to that of racemic IPC in the mice and rat (i.p.) MES and scMet tests. (2R)-IPC had similar ED50 values at the 6 Hz–32 mA and 6 Hz–44 mA tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpineinduced SE model. There was also no significant difference in the neurotoxicity-TD50 values of IPC individual enantiomers and the racemate. IPC anticonvulsant efficacy in the lamotrigine-resistant amygdala kindled rat model When tested at 40 mg/kg, IPC displayed full protection (8/8) and its seizure score was 1.38/5 (5/5 for the control group), and the ADD duration for the IPC was 50% shorter compared to the control group (48 vs. 91 s). Effect of EMC (racemate) on soman-induced SE Racemic EMC exhibited a potent activity in the somaninduced SE model when administered 5 min after the onset of electrographic seizures in rats (ED50 = 34 mg/kg; 12–48 mg/kg, 95% confidence intervals), and only a slightly higher dose was required to control seizures when treatment was delayed to 20 min after seizure onset (ED50 = 48 mg/kg; confidence intervals could not be determined). Pharmacokinetics of EMC and IPC in rats The pharmacokinetics of EMC, IPC, and their individual enantiomers was studied following intraperitoneal administration (50 mg/kg) of each enantiomer as well as the racemate to rats. The dose was chosen for the PK study as the intermediate dose among the various ED50 values. The plasma concentration–time plots of EMC and IPC and their individual enantiomers are presented in Figures 3 and 4. The PK parameters, calculated by noncompartmental analysis, are summarized in Tables S1 and S2.
1949 VPA Carbamate Derivatives Table 1. Anticonvulsant activity and neurotoxicity of EMC (racemate) and its two individual enantiomers (2S)-EMC and (2R)-EMC following intraperitoneal administration to mice and intraperitoneal or oral administration to rats ED50 (95% confidence interval) (mg/kg) Anticonvulsant test
EMC racematea
(2S)-EMC
(2R)-EMC
VPAb
Mice-i.p.
103 (90–124) 120 (108–131) 33 (23–47) 157 (136–185) 16 (11–21) 19 (13–28) 58 (45–70) 64 (39–92) 35 (20–73) >500
85 (67–101) 113 (81–184) 61 (49–74) 203 (172–243) 27 (24–29) 17 (10–24) 77 (56–91) 71 (47–101) 37 (26–48) 107 (65–152)
83 (70–101) 106 (91–121) 61 (56–65) 134 (120–148) 36 (24–50) 29 (18–36) 51 (31–69) 48 (36–59) ND >250
263 (237–282) 220 (177–268) 126 (95–152) 398 (356–445) 140 (110–178)c 195 (157–242)c 275 (239–322)c 484 (324–677) 646 (466–869) 784 (503–1,176)
Rats-i.p.
Rats-p.o.
Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) 6 Hz–32 mA Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50)
ND, not determined. a Data taken from Hen et al.10 b Data taken from Shekh-Ahmad et al.38 c Data taken from L€ oscher and Nolting.39
Table 2. Anticonvulsant activity and neurotoxicity of IPC (racemate) and its two individual enantiomers [(2S)-IPC and (2R)-IPC] following intraperitoneal administration to mice and rats ED50 (95% confidence interval) (mg/kg) Anticonvulsant test Mice-i.p.
Rats-i.p.
Maximal electroshock seizure (MES) 6 Hz–32 mA 6 Hz–44 mA Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50) Maximal electroshock seizure (MES) Pentylenetetrazole-induced seizure (scMet) Neurotoxicity (TD50)
IPC racemate
(2S)-IPC
(2R)-IPC
84 (68–103) 66 (53–82) 79 (71–85) 78 (59–109) 133 (109–158) 24 (18–31) 40 (30–49) 54 (36–68)
123 (110–137) ND ND 188 (174–222) 176 (152–217) 31 (24–38) >50 50 (39–63)
77 (63–95) 51 (32–68) 64 (47–83) 144 (117–167) 159 (133–180) 27 (21–35) 31 (25–38) 47 (37–56)
ND, not determined.
Figure 3. Plasma concentration–time plots of the EMC (racemate) and its two individual enantiomers [(2S)-EMC and (2R)-EMC] obtained following intraperitoneal administration of 50 mg/kg (of each compound) to rats. Epilepsia ILAE
Figure 4. Plasma concentration–time plots of the IPC (racemate) and its two individual enantiomers [(2S)-IPC and (2R)-IPC] obtained following intraperitoneal administration of 50 mg/kg (of each compound) to rats. Epilepsia ILAE Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1950 T. Shekh-Ahmad et al. Racemic EMC and IPC and their individual enantiomers had similar clearance and volume of distribution values that ranged between 3.8 and 5.5 L/h/kg and 2.7 and 4.6 L/kg, respectively. Consequently, the half-lives of the EMC and IPC individual enantiomers were short and ranged between 25 and 42 min. Teratogenicity of EMC (racemate) and its two enantiomers The teratogenic potential of racemic EMC and its two enantiomers was assessed for their ability to induce gross morphologic defects in the Swiss Vancouver/Fnn mice, which are highly susceptible to VPA-induced exencephaly. VPA, at a dose of 2.7 mmol/kg, was embryotoxic and teratogenic, causing an almost twofold increase in the resorption rate compared to the control group (11.9% vs. 6.3%, respectively) and neural tube defects (NTDs) in 29.1% of live fetuses. At a lower dose of 1.8 mmol/kg, VPA was still embryotoxic (13.6% of resorptions), but the number of fetuses with exencephaly (2) was not statistically significant. In contrast to VPA, racemic EMC and its two enantiomers did not cause a statistically significant increase of NTDs at doses of 286 or 429 mg/kg or 1.8 or 2.7 mmol/kg (see Table S3). These doses are 3 to 10 times higher than EMC anticonvulsant-ED50 values, when tested at the higher 2.7 mmol/kg dose (Table S3).
Discussion Approximately 30% of patients with epilepsy are not seizure free despite therapy with the existing medications. In addition, AED therapy, particularly with VPA, is associated with severe side effects (e.g., teratogenicity).22–24 There are three major approaches for designing and developing new AEDs: A mechanism-based approach focusing on a new mechanism of action (MOA) that is not currently possessed by any existing AED.25 The second approach is the design of more potent follow-up compounds to existing AEDs, which will circumvent problems associated with the first-generation AEDs.26–28 The third (phenotyping or empirical) approach is based on the screening of drug candidates in anticonvulsant animal (rodent) models of epilepsy that have established their predictability since the discovery of phenytoin in 1938.15,26,29 In this study, we investigated the individual enantiomers of two lead chiral carbamates designed by the phenotyping and the second-generation approaches.10 A reasonable prediction of a compound’s potential as a new AED candidate is based on the characterization of the compound’s anticonvulsant profile in a variety of anticonvulsant animal models. Activity in various anticonvulsant models may indicate a wide antiepileptic spectrum of activity.26,30 Although there are many animal models of anticonvulsant drug efficacy, the MES and scMet seizure models remain the “gold standards” in early stages of discovery of Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
new AEDs. These models have gained an appreciable degree of predictability since the MES was first utilized in the discovery of the anticonvulsant activity of phenytoin (1938).26,27,30 The LTG-resistant amygdala-kindled rat model displays a resistant profile consistent with therapyresistant seizure and is amenable to medium through-put screening.18,31 Carbamazepine, LTG, phenytoin, and topiramate are inactive in this model, while VPA (300 mg/kg) and retigabine (40 mg/kg) effectively blocked the seizures. In the current study, racemic IPC (30 mg/kg) effectively blocked seizures and decreased the ADD in the LTG-resistant amygdala-kindled rat model and was 10 times more potent than VPA. Screening in animal models is essential in an AED-discovery program, particularly if, like most current AEDs, the drug candidate has multiple or unknown MOAs. In addition; animal models provide an insight to PK–PD correlation of a drug candidate. Given the highly heterogeneous nature of seizure disorders in humans, the complexity of the seizure, and the syndrome involved, it is unlikely that a single anticonvulsant animal model will predict the full therapeutic potential of a drug candidate.26 VPA is an example of a major AED with multiple MOAs that consequently has a wide spectrum of antiepileptic activity in patients,32 and is also effective and approved for the treatment of bipolar disorder and migraine prophylaxis. Similarly, the VPA carbamate derivatives EMC and IPC are likely to have multiple MOAs. EMC (racemate) exhibited a wide spectrum of anticonvulsant activity, including the soman-induced SE like valnoctamide (VCD) and sec-propyl-butyl acetamide (SPD) with similar ED50 values.11,33,34 EMC and IPC and their individual enantiomers had clearance values that were 10 and 2 times higher than those of VCD and SPD. Consequently EMC and IPC half-life was shorter than 1 h. Following intraperitoneal administration to rats, racemic IPC had ED50 values similar to those of EMC. Racemic IPC was equipotent to VCD and SPD (Bialer M, Shekh-Ahmad T, Mawasi H, unpublished data) in the LTG-resistant amygdala-kindled rat model. EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to the lack of enantioselective activity in all anticonvulsant models tested. Bialer et al.35 demonstrated a correlation between the anticonvulsant-ED50 values in mice and rats of various AEDs and their dose and therapeutic average steady-state plasma concentrations in patients with epilepsy. The fact that EMC and IPC enantiomers are 3 to 10 times more potent than VPA in various animal models may cause them to be more potent than VPA in patients. Binding of a racemic drug (e.g., EMC, IPC) to the molecular targets that lead to its antiepileptic activity (e.g., ion channel, a receptor or an enzyme) may be stereospecific, and consequently individual stereoisomers that may display distinguished PK behavior that could lead to stereoselective PD (e.g., SPD).33 Therefore, consideration of chirality should be implemented into PK and PD studies.
1951 VPA Carbamate Derivatives The FDA’s 1992 policy “Statement for Development of the New Stereoisomeric Drugs” triggered the development of single individual stereoisomers instead of racemates.36 This policy coupled with marketing incentives of further profitability as a “line extension” has encouraged companies to look for chiral switches of established chiral drugs that were first introduced to the market as racemic mixtures.37 In the current study, no enantioselectivity was observed in the anticonvulsant activity of EMC and IPC in any of the anticonvulsant animal models. In addition, EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to the absence of enantioselectivity in EMC and IPC anticonvulsant activity. If EMC and/or IPC would exert their anticonvulsant activity via a single mechanism of action, it is likely that they would exhibit a stereoselective PD. The fact that there was no significant difference between racemic EMC and racemic IPC and their individual enantiomers, suggests that their anticonvulsant activity is caused by multiple mechanisms of action.
Conclusions In this study, we report the synthesis and comparative evaluation of the anticonvulsant activity and neurotoxicity of individual enantiomers of two CNS-active carbamate derivatives of VPA. EMC and IPC did not exhibit stereoselective PK, a fact that may contribute to their nonstereoselective anticonvulsant activity in all of the anticonvulsant models. EMC and IPC enantiomers offer an optimal anticonvulsant efficacy and safety profile, supporting these compounds as AED candidates for further development. Neither EMC nor IPC demonstrated a stereoselective anticonvulsant activity. The choice of the optimal individual enantiomer will be based on subsequent anticonvulsant testing coupled with toxicologic analysis.
Acknowledgments This work is abstracted from the PhD thesis of Mr. Tawfeeq ShekhAhmad in a partial fulfillment for the requirements of a PhD degree at The Hebrew University of Jerusalem. The authors wish to thank Drs. John H. Kehne, H. Steve White, and Tracy Chen of the NIH NINDS Anticonvulsant Screening Program (ASP) for testing the compounds in the ASP.
Disclosure or Conflict of Interest Dr. Meir Bialer has received in the last 3 years speakers or consultancy fees from Bial, CTS Chemicals, Desitin, Janssen-Cilag, Johnson & Johnson, Medgenics, Rekah, Sepracor, Teva, UCB Pharma, and Upsher-Smith. Dr. Bialer has been involved in the design and development of new antiepileptics and CNS drugs as well as new formulations of existing drugs. None of the other authors has any conflict of interest to disclose. We, the authors, confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Fisher RS, Acevedo C, Arzimanoglou A, et al. ILAE official report: a practical clinical definition of epilepsy. Epilepsia 2014;55:475–482. 2. Tellez-Zenteno JF, Hernandez-Ronquillo L, Buckley S, et al. A validation of the new definition of drug-resistant epilepsy by the International League Against Epilepsy. Epilepsia 2014;55:829–834. 3. Murray WJ, Kier LB. Noncyclic anticonvulsants. In Vida JA (Ed) Medicinal chemistry, Vol. 15, anticonvulsants. New York: Academic Press, 1977:578–619. 4. Ludwing BJ, Powell LS, Berger FM. Carbamate derivatives related to meprobamate. J Med Chem 1969;12:462–472. 5. Kupferberg HJ. Felbamate. In Levy RH, Matteson RH, Meldrum BS. (Eds) Antiepileptic drugs. 4th Ed. New York: Raven Ress, 1995:791– 827. 6. Bialer M. Comparative pharmacokinetics of the newer antiepileptic drugs. Clin Pharmacokinet 1993;24:441–452. 7. Wong IC, Lhatoo SD. Adverse reactions to new anticonvulsant drugs. Drug Saf 2000;23:35–56. 8. Bialer M, Johannessen SI, Levy RH, et al. Progress report on new antiepileptic drugs: a summary of the Ninth Eilat Conference (EILAT IX). Epilepsy Res 2009;83:1–43. 9. Bialer M, Johannessen SI, Levy RH, et al. Progress report on new antiepileptic drugs: a summary of the Tenth Eilat Conference (EILAT X). Epilepsy Res 2010;92:89–124. 10. Hen N, Bialer M, Yagen B. Syntheses and evaluation of anticonvulsant activity of novel branched alkyl carbamates. J Med Chem 2012;55:2835–2845. 11. Shekh-Ahmad T, Hen N, Yagen B, et al. Stereoselective anticonvulsant and pharmacokinetic analysis of valnoctamide, a CNSactive derivative of valproic acid with low teratogenic potential. Epilepsia 2014;55:353–361. 12. Rowland M, Tozer TN. Clinical pharmacokinetics and pharmacodynamics: concepts and applications. Philadelphia, PA: Lippincott William & Wilkins; 2011. 13. Lothman EW. Seizure circuits in the hippocampus and associated structures. Hippocampus 1994;4:286–290. 14. White HS, Johnson M, Wolf HH, et al. The early identification of anticonvulsant activity: role of the maximal electroshock and subcutaneous pentylenetetrazol seizure models. Ital J Neurol Sci 1995;16:73–77. 15. Smith M, Wilcox KS, White HS. Discovery of antiepileptic drugs. Neurotherapeutics 2007;4:12–17. 16. Woodbury LA, Davenport VD. Design and use of a new electroshock seizure apparatus, and analysis of factors altering seizure threshold and pattern. Arch Int Pharmacodyn Ther 1952;92:97–107. 17. Barton ME, Klein BD, Wolf HH, et al. Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy. Epilepsy Res 2001;47:217–227. 18. Srivastava AK, White HS. Carbamazepine, but not valproate, displays pharmacoresistance in lamotrigine-resistant amygdala kindled rats. Epilepsy Res 2013;104:26–34. 19. McDonough JH, Shih TM. Pharmacological modulation of somaninduced seizures. Neurosci Biobehav Rev 1991;17:203–215. 20. Shih TM, McDonough JH. Neurochemical mechanisms in somaninduced seizures. J Appl Toxicol 1991;17:255–264. 21. Finney DJ. Statistical logic in the monitoring of reactions to therapeutic drugs. Methods Inf Med 1971;10:237–245. 22. Tomson T, Battino D, Bonizzoni E, et al. Dose-dependent risk of malformations with antiepileptic drugs: an analysis of data from the EURAP epilepsy and pregnancy registry. Lancet Neurol 2011;10:609– 617. 23. Molgaard-Nielsen D, Hviid A. Newer generation antiepileptic drugs and the risk of major birth defects. JAMA 2011;305:1996–2002. 24. Vajda JE, Graham J, Roten A, et al. Teratogenicity of the newer antiepileptic drugs—the Australian experience. J Clin Neurosci 2012;19:57–59. 25. L€oscher W, Klitgaard H, Twyman RE, et al. New avenues for antiepileptic drug discovery and development. Nat Rev Drug Discov 2013;12:757–776. 26. Bialer M, White HS. Key factors in the discovery and development of new antiepileptic drugs (AEDs). Nat Rev Drug Discov 2010;9:68–83. Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
1952 T. Shekh-Ahmad et al. 27. Bialer M. New antiepileptic drugs that are second generation to existing antiepileptic drugs. Expert Opin Investig Drugs 2006;15:637– 647. 28. Bialer M, Yagen B. Valproic acid: second generation. Neurotherapeutics 2007;4:130–137. 29. Perucca E, French J, Bialer M. Development of new antiepileptic drugs: challenges, incentives, and recent advances. Lancet Neurol 2007;6:793–804. 30. Rogawski MA, Loscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004;5:553–564. 31. Srivastava AK, Alex BA, Wilcox KS, et al. Rapid loss of efficacy to the antiseizure drugs lamotrigine and carbamazepine: a novel experimental model of pharmacoresistance epilepsy. Epilepsia 2013;54:1186–1194. 32. L€ oscher W. Valproic acid mechanism of action. In Levy RH, Mattson RH, Meldrum BS, Perucca E (Eds) Antiepileptic drugs. 5th Ed. New York: Lippincott Williams & Wilkins Publishers, 2002:767–779. 33. Hen N, Shekh-Ahmad T, Yagen B, et al. Stereoselective pharmacodynamic and pharmacokinetic analysis of sec-Butylpropylacetamide (SPD), a new CNS-active derivative of valproic acid with unique activity against status epilepticus. J Med Chem 2013;56:6467–6477. 34. White HS, Alex AB, Pollock A, et al. A new derivative of valproic acid amide possesses a broad-spectrum antiseizure profile and unique activity against status epilepticus and organophosphate neuronal damage. Epilepsia 2012;53:134–146. 35. Bialer M, Twyman RE, White HS. Correlation analysis between anticonvulsant ED50 values of antiepileptic drugs in mice and rats and their therapeutic doses and plasma levels. Epilepsy Behav 2004;5:866– 872. 36. Hubbard WK. FDA policy on period of marketing exclusivity for newly approved drug products with enantiomer active ingredients; request for comments. Fed Reg 1997;62:2167–2169.
Epilepsia, 55(12):1944–1952, 2014 doi: 10.1111/epi.12857
37. Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy of chiral switches. Nat Rev Drug Discov 2002;1:753–68. 38. Shekh-Ahmad T, Hen N, McDonough JH, et al. Valnoctamide and sec-butyl-propylacetamide (SPD) for acute seizures and status epilepticus. Epilepsia 2013;54 (Suppl. 6):99–102. 39. L€oscher W, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. IV. Proactive indices. Epilepsy Res 1991;9:1–10.
Supporting Information Additional Supporting Information may be found in the online version of this article: Scheme S1. Total enantioselective synthesis of individual enantiomers of EMC and IPC. Table S1. PK parameters of EMC (Racemate) and its two enantiomers: (2S)-EMC and (2R)-EMC calculated following i.p. administration (50 mg/kg) of each to rats. Table S2. PK parameters of IPC (Racemate) and its two enantiomers: (2S)-IPC and (2R)-IPC calculated following i.p. administration (50 mg/kg) of each to rats. Table S3. Teratogenic effect in the SWV mouse model of EMC and its enantiomers. Data S1. Experimental section: Materials and methods.
