Anticonvulsant Profile and Teratogenicity of N-methyl-tetramethylcyclopropyl Carboxamide: A New Antiepileptic Drug

Epilepsia, 49(7):1202–1212, 2008doi: 10.1111/j.1528-1167.2008.01624.x

FULL-LENGTH ORIGINAL RESEARCH

Anticonvulsant profile and teratogenicityof 3,3-dimethylbutanoylurea: A potential

for a second generation drug to valproic acid∗Jakob Avi Shimshoni, †‡Boris Yagen, ∗Neta Pessah, §Bogdan Wlodarczyk,

§Richard H. Finnell, and ∗†Meir Bialer

∗Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem,Jerusalem, Israel; †David R. Bloom Centre for Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel;

‡Department of Natural Products and Medicinal Chemistry, School of Pharmacy, Faculty of Medicine, The HebrewUniversity of Jerusalem, Jerusalem, Israel; and §Center for Environmental and Genetic Medicine, Institute of

Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, U.S.A.

SUMMARYPurpose: The purpose of this study was to evaluatethe anticonvulsant activity and teratogenic poten-tial of branched aliphatic acylureas represented byisovaleroylurea (IVU), pivaloylurea (PVU) and 3,3-dimethylbutanoylurea (DBU), as potential second-generation drugs to valproic acid (VPA).Methods: The anticonvulsant activity of IVU, PVU,and DBU was determined in mice and rats utiliz-ing the maximal electroshock seizure (MES) andthe pentylenetetrazole (scMet) tests. The abilityof DBU to block electrical-, or chemical-inducedseizures was further examined in three acuteseizure models: the psychomotor 6 Hz model, thebicuculline and picrotoxin models and one modelof chronic epilepsy (i.e., the hippocampal kindledrat model). The induction of neural tube defects(NTDs) by IVU, PVU, and DBU was evaluated af-ter i.p. administration at day 8.5 of gestation toa mouse strain highly susceptible to VPA-inducedteratogenicity. The pharmacokinetics of DBU wasstudied following i.v. administration to rats.

Results: DBU emerged as the most potent com-pound having an MES-ED50 of 186 mg/kg (mice)and 64 mg/kg (rats) and an scMet-ED50 of 66 mg/kg(mice) and 26 mg/kg (rats). DBU underwentfurther evaluation in the hippocampal kindledrat (ED50 = 35 mg/kg), the psychomotor 6 Hzmouse model (ED50 = 80 mg/kg at 32 mA andED50 = 133 mg/kg at 44 mA), the bicuculline- andpicrotoxin-induced seizure mouse model (ED50 =205 mg/kg and 167 mg/kg, respectively). In contrastto VPA, DBU, IVU, and PVU did not induce a sig-nificant increase in NTDs as compared to control.DBU was eliminated by metabolism with a half-lifeof 4.5 h.Conclusions: DBU’s broad spectrum and potent an-ticonvulsant activity, along with its high safety mar-gin and favorable pharmacokinetic profile, make itan attractive candidate to become a new, potent,and safe AED.KEY WORDS: Anticonvulsants, Maximal elec-troshock seizure test, Pharmacokinetics, Sub-cutaneous pentylentetrazole seizure test,Teratogenicity.

The development of new antiepileptic drugs (AEDs)continues to be a major goal for both industry and

Accepted March 19, 2008; Online Early publication April 24, 2008.Address correspondence to Professor Meir Bialer, Department

of Pharmaceutics, School of Pharmacy, Faculty of Medicine, TheHebrew University of Jerusalem, Jerusalem 91120, Israel. E-mail:[email protected]

Wiley Periodicals, Inc.C© 2008 International League Against Epilepsy

academia, since approximately 30% of epileptic patientsare not seizure free and, the currently available AEDs lacksatisfactory safety and tolerability profiles (Holmes, 2007;Perucca et al., 2007; Smith et al., 2007).

Since valproic acid (VPA)’s serendipitous discovery asa potent anticonvulsant, many efforts have been madeto develop safer and more potent analogues and deriva-tives of VPA (Nau & Loscher, 1986; Haj-Yehia & Bialer,1989, 1990; Isoherranen et al., 2003a; Bialer et al.,

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Anticonvulsant Profile and Teratogenicity of 3,3-Dimethylbutanoylurea

2007; Bialer & Yagen, 2007). Structure-pharmacokinetic-pharmacodynamic relationship studies in a series of short-chain amide derivatives of VPA’s isomers and analogues,performed in our laboratory starting 20 years ago, clearlydemonstrated the superior anticonvulsant potency of theamide derivatives compared to their corresponding acids(Haj-Yehia & Bialer, 1989; Bialer, 1991; Isoherranen et al.,2003b; Bialer & Yagen, 2007; Shimshoni et al., 2007).In addition, we have shown that the anticonvulsant activ-ities of the amide derivatives of VPA’s isomers and ana-logues are affected by their pharmacokinetics in generaland by the biotransformation of the amide to the respec-tive acid in particular (Haj-Yehia & Bialer, 1989, 1990;Haj-Yehia et al., 1992). An amide that did not undergohydrolytic metabolism in dogs to its acid was more activethan the amide of a VPA isomer or analogue that did bio-transform to its corresponding acid (Haj-Yehia & Bialer,1989, 1990). Furthermore, the amide derivatives of VPA’sconstitutional isomers were found to be nonteratogenic in amouse model for AED-induced teratogenicity (Isoherranenet al., 2003a, 2003b).

In 1939, numerous acylureas of branched aliphatic acidswere synthesized and found to possess hypnotic and seda-tive properties (Stoughton et al., 1939). Nine years laterSpielman et al. evaluated the anticonvulsant activity of aseries of branched aliphatic acylureas (ureides) in two micemodels for epilepsy: the maximal electroshock seizure(MES) and the subcutaneous pentylenetetrazole (scMet)tests (Spielman et al., 1948). The ureides of branchedaliphatic carboxylic acids with six or seven carbons pos-sessing a tertiary carbon atom in their structures showedbroad-spectrum anticonvulsant activity in both seizuretests (Spielman et al., 1948). The neuropharmacologicaland anticonvulsant properties of butyroylurea and 2,2-dimethylpropanoylurea (PVU) (pivaloylurea) produced athigh doses (400 mg/kg) complete protection against tonicconvulsions in the scMet mouse model (Dar & Fakouhi,1974; Dar, 1976). Recently the urea derivatives of consti-tutional isomers of VPA have been investigated and foundto be active as anticonvulsant (Shimshoni et al., 2007).

Teratogenicity is one of the most severe risk factorsassociated with the therapeutic use of the major AEDs:carbamazepine (CBZ), phenobarbital (PB), VPA, andphenytoin (PHT) (Kaneko & Kondo, 1995; Tomson & Bat-tino, 2005, 2007; Harden, 2007). The overall malformationrate is 11.1% in offspring of AED-treated epileptic moth-ers, while it is 5.7% in the offspring of untreated epilepticmothers (Kaneko & Kondo, 1995). Many efforts have beenmade to design nonteratogenic VPA analogues and deriva-tives with enhanced anticonvulsant activity (Nau et al.,1991; Bialer et al., 1994; Isoherranen et al., 2003b; Bialer& Yagen, 2007). These urea derivatives displayed excellentbroad-spectrum anticonvulsant activity; however, at higherdoses (335 mg/kg) they were teratogenic and embryotoxic(Shimshoni et al., 2007).

The purpose of the current study was to evaluate theanticonvulsant activity and teratogenicity of five and sixcarbons atom (in their acyl moieties) homologues of val-proyl urea: isovaleroylurea (IVU), pivaloylurea (PVU), andDBU in order to establish their anticonvulsant profile andteratogenic potential (Fig. 1). DBU, the most potent anti-convulsant compound that emerged from this study in theMES and scMet tests, underwent further evaluation in thehippocampal kindling rat, the psychomotor 6 Hz mousemodel, the bicuculline- and the picrotoxin-induced seizuremodel. The pharmacokinetics of DBU was studied follow-ing intravenous (i.v.) administration to rats.

METHODS

Chemicals

The acylureas, PVU (MW = 144.2), IVU (MW = 144.2)and DBU (MW = 158.2), were synthesized by coupling theappropriate acylchlorides with urea according to the pro-cedure previously reported by us (Sobol et al., 2004). Thesynthesized products were purified by crystallization andtheir structures were identified by 1H-NMR and GC-MS,and their purity was established by elemental analyses.

Figure 1.Structures of VPA, 4-ene-VPA, and the acylureas: val-proylurea (VPU), TMCU, PVU, IVU and DBU. Carbon2 in PVU, carbon 3 in DBU and carbons 2 and 3 inTMCU are quaternary.Epilepsia C© ILAE

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Animals

Male CF No. 1 albino mice (18–25 g) and male Sprague-Dawley albino rats (100–150 g) were used for the eval-uation of anticonvulsant activity and neurotoxicity of alltest compounds. The animals were maintained in animalfacilities of the University of Utah on a 12-h light: 12-h dark cycle and allowed free access to food and water,except during the short period they were removed fromtheir cages for testing. Animals newly received in the lab-oratory were allowed to acclimatize for 24–48 h to com-pensate for the food and water restriction incurred dur-ing the transit. The animals were maintained and han-dled according to the recommendations of the U.S. De-partment of Health, Education and Welfare publication(NIH) No. 8623, Guide and Use of Laboratory Animals.All animal experiments were approved by the InstitutionalAnimal Care and Use Committee of the University ofUtah.

Virgin SWV/Fnn female mice (2–3 months of age, 20–25 g) and SWV/Fnn male mice (3–4 months of age,25–35 g) raised from a breeding colony at the Institute ofBiosciences and Technology, Houston, TX, U.S.A., wereused in the teratogenicity studies. Mice were housed inclear polycarbonate cages, allowed free access to water andfood (Harlan Tekad, Madison, WI, U.S.A.; Rodent Diet#8604) and maintained on a 12:12-h light/dark cycle inthe vivarium at the Institute of Biosciences and Technol-ogy. The teratogenicity studies were approved by the Insti-tutional Animal Care and Use Committee of Texas A&MUniversity.

Pharmaceutical preparations of test compounds for

the anticonvulsant activity determination

For the evaluation of anticonvulsant activity and toxi-city, the test compounds were suspended in 0.5% methyl-cellulose water solution. The test compounds were given ina concentration that permits optimal accuracy of a dosagewithout the volume contributing excessively to total bodyfluid. The injected volume used in mice was 0.01 ml pergram of body weight, and in rats 0.04 ml per 10 g of bodyweight. Methylcellulose as a vehicle has no anticonvulsantactivity (White et al., 2002).

Anticonvulsant activity and toxicity

The evaluation of the anticonvulsant activity and tox-icity of the new compounds was performed in collab-oration with the NIH-Epilepsy Branch as a part of theNIH-Anticonvulsant Screening Project according to thepreviously described protocols (White et al., 2002).The anticonvulsant activity of all test compounds was eval-uated in mice and rat models of electrically and chemi-cally induced seizures: the MES test and the subcutaneouspentylentetrazole test (scMet). Subsequently the most po-tent test compound was quantitatively evaluated in the rathippocampal kindling model, the mouse psychomotor 6 Hzmodel and the mouse bicuculline and picrotoxin models.

For the MES test, a supramaximal current (50 mA, 60 Hz,0.2 s in mice, and 150 mA, 60 Hz, 0.2 s in rats) was deliv-ered through coroneal electrodes to produce tonic hindlimbextension. Animals not displaying tonic hindlimb exten-sion were considered protected. In the scMet test, mice andrats treated with the test substance were challenged with asubcutaneously (s.c.) administered convulsive dose (CD97)of pentylentetrazole (85 mg/kg in mice and 56.4 mg/kgin rats) and then placed in isolation cages and observedfor the next 30 min for the presence or absence of anepisode of clonic spasms persisting for at least 5 s. Animalsnot displaying a minimally clonic seizure were consideredprotected.

In the hippocampal kindling model a bipolar stimulat-ing electrode was stereotactically implanted in the ven-tral hippocampus (AP, −3.6 mm; ML, 4.9; DV, −5.0 fromdura, incisor bar +5) of adult male SD-rats (250–300 g)under ketamine-xylazine anesthesia. One week after im-plantation of electrodes rats were stimulated with an elec-trical stimulus of 50 Hz, 200 μA for 10 s every 30 minfor 6 h on alternate days until the animals were fully kin-dled (stage 5 of behavioral seizure on the Racine scale,see below). Rats were then permitted a 1-week stimulusfree period prior to i.p. dosing with different doses of thetest substances. The effect of a given compound on thebehavioral seizure score (BSS) and afterdischarge dura-tion (ADD) was evaluated at various times after dosing(i.e., 15, 45, 75, 105, and 135 min). The severity of thebehavioral seizures was scored according to the Racinescale (Racine, 1972): stage 1, mouth and facial tonus;stage 2, stage 1 plus head nodding; stage 3, stage 2 plusforelimb clonus; stage 4, stage 3 plus rearing; stage 5,stage 4 plus repeated rearing and falling. Rats not dis-playing stages 4 and 5 of seizures were considered pro-tected. The ADD was determined by a review of the EEGrecordings.

For the 6 Hz-test, mice treated with the test substancewere challenged with a 6 Hz alternating current (32 mA or44 mA, 3 s duration) delivered via the corneal electrodesto elicit a psychomotor seizure which was characterized bya minimal clonic phase followed by automatistic behav-ior that included jaw chomping and whisker movements,etc. Mice not displaying this behavior were considered pro-tected by the test compound.

In the bicuculline and picrotoxin tests, the administeredconvulsive dose of bicuculline (2.7 mg/kg) and picrotoxin(3.15 mg/kg) was injected s.c. at the previously deter-mined time to peak effect (TPE) (see below) for the testedsubstance and induced convulsions in more than 97% ofthe animals. Individual mice were then placed in isolationcages and observed for the presence or absence of clonicseizures.

The toxicity of the compounds was determined usingthe rotorod test in mice. In rats, the positional sense test,muscle tone test, and gait and stance test in rats was taken

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as the end point of minimal behavioral impairment ac-cording to the protocols described in White et al., 2002.Inability of the rat to perform normally in at least twoof these tests indicates that the rat has some neurologicaldeficit.

Determination of the median effective dose (ED50)

and the median neurotoxic dose (TD50)

For the determination of the ED50 by the respectiveanticonvulsant procedures, doses of the titled compoundswere varied until at least four points were establishedbetween the dose level of no protection and 100% pro-tection. These data were then subjected to probit anal-ysis and the ED50 and 95% confidence intervals werecalculated.

The TD50 was determined by varying the dose of thestudied compounds until four points were established be-tween the dose level that induced no signs of minimal mo-tor impairment in any of the animals, and the dose at whichall the animals were considered impaired. The TD50 andthe 95% confidence intervals were then calculated by pro-bit analysis. The protective index (PI) was calculated bydividing the TD50 by ED50.

Determination of TPE

To determine the TPE for anticonvulsant activity, fivegroups of four animals each were administered an appro-priate dose of test drug and subjected to the MES, scMetor 6 Hz tests at 0.25, 0.5, 1, 2, or 4 h. For toxicity determi-nations, a single group of eight animals was injected andtested for minimal motor impairment at the same time in-tervals. The time interval showing the greatest number ofresponded animals was taken as the TPE.

Teratogenicity study

The highly inbred SWV/Fnn mouse strain with a knownsusceptibility to AED-induced NTDs (Finnell et al., 1988)was used in this study according to the previously pub-lished procedure (Finnell et al., 1997). Dams were matedovernight with male mice and examined on the follow-ing morning for the presence of vaginal plugs. The on-set of gestation was set at 22:00 h on the previous night.On gestation day (E) 8.5, pregnant females received asingle i.p. injection (10 μl pre gram body weight) ofVPA or test compound at 3.6, 2.7 or 2.2 mmol/kg doses.All compounds were dissolved in a 25% water solu-tion of Cremophor EL. Control dams were injected i.p.with equivalent volume of Cremophor EL. CremophorEL is a neutral polyoxyethylene-tri-fatty acid-glycerol, be-ing widely accepted as a nonteratogenic vehicle for ter-atogenicity studies (Finnell et al., 1988). On E 18.5 thedams were killed by CO2 asphyxiation, the abdomenopened and the gravid uteri removed. The locations ofall viable, dead, and resorbed fetuses were recorded,and the fetuses were examined for the presence ofexencephaly.

Pharmacokinetic studies

AnimalsMale Sprague-Dawley rats weighing 275 ± 24 g were

used throughout the study. The animals were maintained at22 ◦C with a 12 h light/dark cycle. Prior to each experimentthe animals were allowed to acclimatize for at least 2 daysin communal cages with standard rat chow and tap waterprovided ad libitum. For urine collection animals were keptin metabolic cages. In all the experiments animals werefasted 12 h before and 4 h after the drug administration.Animal use in the PK studies was approved by the Insti-tutional Animal Care and Use Committee of The HebrewUniversity of Jerusalem.

Drug administrationDBU dissolved in 96% ethanol for injection, was admin-

istered intravenously (i.v., 10 mg/kg through the tail vein).The maximal injected volume of ethanol was 200 μl. Nobehavioral changes were observed in rats. Since DBU isinsoluble in aqueous solutions, it was administered in 96%ethanol for injection.

Collection of blood samples and urineThree rats were sacrificed at each time point and blood

samples were withdrawn by cardiac puncture under deepisoflurane anesthesia. Blood samples were collected at0.05, 0.15, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24 h af-ter dosing. The heparinized test tubes with collected bloodwere immediately centrifuged at 3000 g for 10 min andplasma was separated and stored at −20 ◦C until analyzed.Urine was collected from three rats over 24 h periods in50 ml plastic tubes and stored at −20 ◦C until analyzed.

Analysis of DBU in plasma and urine

Plasma and urine levels of DBU were analyzed byGC/MS. The internal standard (valnoctylurea; 50 μl of25 μg/ml solution in methanol) was added to the dry tubesand the organic solvent was evaporated under reduced pres-sure. A total of 500 μl of plasma (or urine) were subse-quently added to the tubes and mixed thoroughly. The ex-traction was performed with 2 ml of chloroform, which wasthen evaporated under reduced pressure. The dry residueswere reconstituted with 50 μl chloroform, of which 1 μlwas injected into the GC/MS apparatus. The oven temper-ature program was set as follows: initial temperature, 60◦Cfor 3 min; gradient of 20◦C/min until 140◦C; gradient of10◦C/min until 190◦C; hold time, 3 min. The injection portwas at 180◦C; the source temperature was at 180◦C; thetemperature of the interface was at 280◦C. The pressure ofthe carrier gas, helium, was set at 5 psi. For EI analysis,the ionization energy was 70 eV with a source pressure of10−6 Torr. DBU and the internal standard were monitoredfor selected ions at m/z: 59, 83, 102. Retention times ofDBU and the internal standard were 7.1 min and 8.5 min,respectively. Calibration curves were constructed for eachanalytical run and were linear on the concentration range of

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Table 1. Quantitative anticonvulsant data in mice dosed intraperitoneally with short-chain acylureasin comparison to VPA

MESa scMetb Neurotoxicity PIc PICompound (ED50 mg/kg) (ED50 mg/kg) (TD50 mg/kg) (MES) (scMet)

VPAd 263 (237–282)e 220 (177–268) 398 (356–445) 1.5 1.8PVU >250 86 (68–107) 223 (139–299) – 2.6IVU >300 >300 >300 – –DBU 186 (172–212) 66 (48–82) 244 (192–306) 1.3 3.7

aMaximal electroshock seizure test.bSubcutaneous pentylenetetrazole test.cProtective index (TD50/ED50).dData taken from reference White et al., 2002.eThe data in parentheses stands for 95% confidence interval.

0.5–100 μg/ml. The developed method was validated ac-cording to the published guidelines (Shah et al., 2000). Thequality control (QC) concentrations used to assess intradayand interday precision and accuracy were within ±15% ofthe expected theoretical values.

Calculation of PK parameters. The PK parameters werecalculated by noncompartmental analysis based on statisti-cal moment theory using PK software WinNonlin version4.1 (Pharsight Co., Mountain View, CA, U.S.A.).

Chemical stability in the bloodFive test tubes containing 80 μg of DBU were spiked

with 2 ml of fresh rat blood. Following 1 min of vigor-ous vortex the tubes were transferred to the shaking bath at37◦C. After 2, 4, 6, 8, and 12 h the test tubes were with-drawn from the bath, centrifuged at 3000 g for 10 min andevaluated for DBU plasma concentration by GC/MS.

Statistical analysis

Statistical analysis was performed using InStat softwareversion 3.01 (GraphPad Software Inc., San Diego, CA,U.S.A.). The significance of reduction in the kindled ratBSS and ADD was evaluated by two-tailed Mann–Whitneyand Kruskal–Wallis tests. Results are presented as eitherthe ED50 or TD50 with 95% confidence intervals. The ter-

Table 2. Quantitative anticonvulsant data in rats dosed orally with short-chain acylureasin comparison to VPA

MESa scMetb Neurotoxicity PIc PICompound (ED50 mg/kg) (ED50 mg/kg) (TD50 mg/kg) (MES) (scMet)

VPAd 485 (324–677)e 646 (466–869) 784 (503–1176) 1.6 1.2PVU 69 (35–150) 30 (17–46) 228 (196–264) 3.3 7.6IVU >250 83 (60–116) 300 < TD50 < 500 – 3.6 < PI < 6DBU 64 (55–74) 26 (23–28) 143 (102–173) 2.2 5.5

aMaximal electroshock seizure test.bSubcutaneous pentylenetetrazole test.cProtective index (TD50/ED50).dData taken from reference White et al., 2002.eThe data in parentheses stands for 95% confidence interval.

atogenicity data were evaluated for significance by ana-lyzing the contingency table with Fisher’s exact test. A p-value < 0.05 was considered significant.

RESULTS

Anticonvulsant activity

In the mice MES and scMet tests, IVU showed no an-ticonvulsant activity and no signs of minimal motor im-pairment up to 300 mg/kg (Table 1). However, in rats,IVU was active in the scMet test (ED50 = 83 mg/kg)30 min after dosing [i.e. at time of peak effect (TPE)]and had a PI (TD50/ED50) of 3.6 but was inactive in theMES test (Table 2). PVU exhibited anticonvulsant activ-ity in the mice scMet test (ED50 = 86 mg/kg, Table 1),but was inactive in the mice MES test at doses of up to250 mg/kg. In rats, PVU demonstrated anticonvulsant ac-tivities in the MES (ED50 = 69 mg/kg, PI = 3.3) and scMettests (ED50 = 30 mg/kg, PI = 7.6), however, at longerTPE of 1 h and 2 h, respectively, as compared to IVU.PVU had the largest safety margin of the tested acylureas(Table 2).

DBU emerged as the most potent compound in mice,being active in both anticonvulsant tests (MES-ED50 =186 mg/kg and scMet-ED50 = 66 mg/kg); while in rats,

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Table 3. Anticonvulsant activity and behavioral impairment of DBU following intraperitoneal and oraladministration to mice and rats in comparison to VPA

ED50 or TD50 ED50 or TD50

Test (mg/kg) of DBU PIa (DBU) (mg/kg) of VPA PI (VPA)

6 Hz (32mA)b 80 (55–104)c 3 126d (95–152) 3.26 Hz (44mA)b 133 (108–172) 1.8 310d (258–335) 1.3Toxicity (TD50) 244 (192–306) – 398d (356–445) –scBice 205 (145–282) 1.2 589f (470–765) 0.7scPicg 167 (147–187) 1.5 270f (186–356) 1.5Hippocampal kindlingh 35 (23–53) 4 119i –Toxicity (TD50) 143 (102–173) – – –

aProtective index (TD50/ED50).bPsychomotor seizure test in mice induced by low-frequency (6 Hz) stimulus at 32 and 44 mA current.c95% confidence interval.dData taken from Isoherranen et al., 2003c.eSubcutaneous bicuculline test in mice.f Data taken from White et al., 2002.gSubcutaneous picrotoxin test in mice.hTest drug administered intraperitoneally to rats.iData taken from Lothman et al., 1988.

DBU demonstrated slightly better anticonvulsant potencythan that of PVU (Tables 1 and 2). The TPE was 2 hfor the MES and 1 h for the scMet tests. Therefore,we further explored DBUs anticonvulsant properties inthree additional acute seizure models of epilepsy (i.e., themouse 6 Hz model, the mouse bicuculline- and picro-toxin models) and one model of chronic epilepsy (i.e.,the hippocampal kindled rat model) (Tables 3 and 4). Thedata presented in Tables 3 and 4 demonstrated DBU’sbroad anticonvulsant spectrum of activity. DBU’s ED50

value in the 6 Hz psychomotor test increased with in-creasing stimulation currents from 80 mg/kg at 32 mA to133 mg/kg at 44 mA (Table 3). In the chemically inducedseizure models, DBU provided protection against boththe bicuculline and picrotoxin-induced seizures (ED50 =205 mg/kg and ED50 = 167 mg/kg, respectively). In allof the four additional animal models, the TPE of DBUwas 30 min, being shorter than the TPE in the MES andscMet tests.

Table 4. Anticonvulsant activity after i.p. administration of DBU on seizure score and afterdischargeduration in hippocampal kindled rats with stimulus intensity of 200 μA

Time of test (min)

0 15 75 105 135Dose = 12.5 mg/kg

ADDa (s) 57 ± 6 65 ± 10 70 ± 11 47 ± 12 36 ± 17Seizure score 5.0 ± 0.0 5.0 ± 0.0 4.8 ± 0.2 3.5 ± 0.8 2.0 ± 0.9b

Dose = 100 mg/kgADDa (s) 64 ± 13 12 ± 3b 27 ± 5b 36 ± 1 43 ± 2Seizure score 4.8 ± 0.3 0.0 ± 0.0b 0.5 ± 0.3b 0.3 ± 0.3b 0.3 ± 0.3b

aAfterdischarge duration.bSignificantly different from control (p < 0.05).

The results obtained in the hippocampal kindled rats byusing supramaximal stimulation of 200 μA are summa-rized in Tables 3 and 4. DBU was highly active in the fullykindled rat against the secondarily generalized seizures(ED50 = 35 mg/kg).

Teratogenicity and embryotoxicity

The results from the evaluation of the ability of the acy-lureas to induce neural tube defects (NTDs) are summa-rized in Table 5. In the high (3.6 mmol/kg) and middle dosegroups (2.7 mmol/kg) VPA was highly teratogenic, causingNTDs in 53% and 29% of live fetuses’ respectively. Theacylureas tested at equimolar doses induced only singlecases of exencephaly in the SWV fetuses (p > 0.05). Therewere only two fetuses identified with exencephaly after i.p.administration of DBU, one in the highest (3.6 mmol/kg)and one in the middle dose group (2.7 mmol/kg). In the2.7 mmol/kg IVU treatment group and the 3.6 mmol/kgPVU treatment group, a single case of exencephaly was

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Table 5. Teratogenic effect of the acylurea derivatives in a mouse modelof VPA-induced teratogenicity

DoseTreatment mg/kg Exencephalygroupa Route (mmol/kg) Mice strain No. of litters No. of live fetuses Embryo/feto lethality % %

Control i.p. 25% CELb SWV 15 188 6 0VPA i.p. 600 (3.6) SWV 13 107 18.3c 53.3c

i.p. 452 (2.7) SWV 13 141 11.9c 29.1c

i.p. 361 (2.2) SWV 12 145 10.5 3.4IVU i.p. 520 (3.6) SWV 12 135 11.8 0

i.p. 392 (2.7) SWV 12 123 7.5 0.8PVU i.p. 520 (3.6) SWV 13 180 4.5 0.6

i.p. 392 (2.7) SWV 13 170 7.6 0DBU i.p. 671 (3.6) SWV 10 98 29c 1

i.p. 502 (2.7) SWV 12 156 8 0.6

aAll dams received the drugs intraperitoneally or subcutaneously on the morning of day 8 of gestation, as indicated for eachtreatment.

b25% water solution of Cremophor EL.cSignificantly different from control, p < 0.05, Fisher exact test.

observed. While these individual fetuses with exencephalyare above the historic strain background frequency, whichis close to 0, they do not represent a significant level ofteratogenicity. Neither IVU nor PVU induced a signifi-cantly higher incidence of embryo lethality (Table 5). How-ever, DBU was embryotoxic in mice treated at the highestdose (3.6 mmol/kg), causing 29% of resorptions (Table 5).At the lower DBU treatment groups (2.7 mmol/kg and1.8 mmol/kg) no embryotoxicity was observed.

Pharmacokinetics

DBU pharmacokinetics was studied followingi.v. administration (10 mg/kg) to rats. DBU plasmaconcentration-time plots are presented in Fig. 2. DBUplasma concentrations were best fitted to the follow-ing two-compartment open body model equation: C =21e−2.1 + 11e−0.15. DBU undergoes relatively fast equi-librium between the central and the peripheral compart-ments (∼30 min postinjection) and only 12% of the

Figure 2.DBU plasma concentrations obtained following i.v. ad-ministration (10 mg/kg) to rats.Epilepsia C© ILAE

drug is eliminated during the distributive phase. Thepharmacokinetic (PK) parameters of DBU, calculated bynoncompartmental analysis, are summarized in Table 6.DBU was well distributed into the extravascular tissueswith a volume of distribution (Vss) of 0.8 L/kg. Asexpected from the rapid distribution phase, there was nosignificant difference between Vβ and Vss (Rowland &Tozer, 1995). DBU was mainly eliminated by metabolismbut did not biotransform to its corresponding carboxylicacid 3,3-dimethybutyric acid (DBA). Only a smallfraction of DBU (fe = 2.4%) was excreted unchangedin the urine (Table 6). The relatively low clearance ofDBU together with its large volume of distribution,resulted in relatively long half-life in rats (t1/2 = 4.5 h).

Table 6. PK parameters of DBU obtained afterits i.v. administration (10 mg/kg) to rats in

comparison to VPA (74 mg/kg)

Parameter DBU VPAa

CL (L/h·kg) 0.12 0.24CLr (L/h·kg) 0.003 0.004Vβ (L/kg) 0.78 1.4Vss (L/kg) 0.81 0.87t1/2 (h) 4.5 4AUC (mg/L·h) 80 301MRT (h) 6.5 3.5fe (%) 2.4 1.8

CL, total clearance; CLr, renal clearance; Vβ , volume ofdistribution based on linear terminal slope; Vss, volume ofdistribution at steady-state; t1/2, half-life; AUC, area underthe plasma concentration-time curve; MRT, mean residencetime; fe, fraction of the systemically available drug excretedunchanged in the urine.

aData taken from Blotnik et al., 1996.

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DBU’s water solubility is 2.3 mg/ml and the compoundwas stable in whole blood at physiological conditionsover a 12-h period.

DISCUSSION

Since about 30% of the epileptic patients are not seizure-free with the existing medications, there is a substantialneed to develop new, better, and safer AEDs. One of theprimary purposes in designing and developing new AEDsis to prevent the teratogenicity associated with VPA andother frontline AED’s therapy (Perucca et al., 2007).

Although branched aliphatic acylureas have been recog-nized since the early 1930s to possess sedative/hypnotic aswell as anticonvulsant properties, only a few studies quan-titatively analyzed their anticonvulsant activities in vari-ous animal models of epilepsy (Stoughton et al., 1939;Dar & Fakouhi, 1974; Dar, 1976). IVU (Fig. 1) and PVU(Fig. 1) demonstrated anticonvulsant properties in a pre-liminary qualitative screening in the mice MES and scMetmodels (Spielman et al., 1948). The sedative/hypnoticproperties of IVU, PVU, and DBU (Fig. 1) were reportedby Stoughton et al. and their CNS-depressant activitieswere further investigated by Mrongovius (Stoughton et al.,1939; Mrongovius, 1975). In the US, capuride or 2-ethyl-3-methylvaleroylurea, a homologue of IVU, PVU, andDBU, underwent clinical trials as a short-acting hypnoticdrug (Johnson et al., 1972). Bromvaleroylurea, an α-bromoderivative of IVU, is widely used in Japan as a CNS drugwith sedative, hypnotic, analgesic, and antipyretic prop-erties (Kudo et al., 2003). The urea derivatives of VPA’sconstitutional isomers, containing eight carbons in theiracyl moiety, emerged as highly potent broad spectrum an-ticonvulsants in the rat MES, scMet, and mice 6 Hz tests(Shimshoni et al., 2007).

Since previous studies indicated the anticonvulsant ac-tivities of branched aliphatic acylureas (Stoughton et al.,1939; Spielman et al., 1948; Dar & Fakouhi, 1974; Mron-govius, 1975; Dar, 1976; Sobol et al., 2006; Shimshoniet al., 2007), we synthesized IVU, a homologue of VPUas well as PVU and DBU, two analogues of VPU (Fig. 1),possessing a quaternary carbon in their structures, andcharacterized their anticonvulsant potency in the mice andrat MES and scMet models. In addition, the teratogenicprofile IVU, PVU, and DBU was evaluated in mice in com-parison to VPA, a widely used major AED with an increaseteratogenic risk compared to other AEDs (Harden, 2007).We then extended the pharmacological evaluation of DBU,the most potent compound in this group, to additional anti-convulsant animal models and determined its PK profile inrats.

Of the three tested acylureas, IVU was the least potentcompound, lacking anticonvulsant activity in mice at dosesabove 300 mg/kg (Table 1). Only in the rat scMet test IVUdisplayed an anticonvulsant activity (ED50 = 83 mg/kg)

and a three times larger protective index than that of VPA(Table 2). Contrary to our findings, Spielman et al., re-ported that IVU only at doses that caused neurotoxic ef-fects provided protection in the mice MES and scMet tests,(Spielman et al., 1948). PVU demonstrated a broader anti-convulsant profile than IVU, being active in the mice scMettest (ED50 = 86 mg/kg) as well as being 7 and 22 timesmore potent than VPA in the rat MES and scMet tests,respectively (Tables 1 and 2). PVU showed an excellentsafety margin in rats as expressed by its PI-index, beingtwo (MES test) and six (scMet test) times larger than thoseof VPA (Table 2). In contrast to Spielman’s report, PVU inthe mice scMet test (Table 1) demonstrated a wide safetymargin between its anticonvulsant potency and the neuro-toxic effects (Spielman et al., 1948). We also found thatPVU is much more potent in the mice scMet test (ED50 =86 mg/kg) than previously reported by Dar (full protectionat 400 mg/kg), (Dar, 1976). The TPE of both urea deriva-tives in rats was in the range of 0.5–2 h, with IVU demon-strating a shorter TPE of 0.5 h.

DBU displaying a wide spectrum of anticonvulsant ac-tivity in both rats and mice (Tables 1–4), and thus emergedas the most potent compound in this group. A compari-son between the anticonvulsant profiles of DBU and PVU(acylureas containing six and five carbons in their acylmoieties, respectively, Fig. 1), with the urea derivatives ofVPA and its constitutional isomers, containing eight car-bons in the acyl moieties (Shimshoni et al., 2007), re-veals a similar potency in the rat scMet test but a slightlylower anticonvulsant activity of DBU and PVU in therat MES test (Table 2). Hence, a reduction in the num-ber of carbon atoms in the acyl moiety form 8 to 6(DBU) or 5 (PVU), while containing a quaternary car-bon atom in their structures, does not affect their anticon-vulsant potency. The pronounced differences in the phar-macological profile of PVU and IVU, two constitutionalisomers containing quaternary carbon atom in their struc-tures (Tables 1 and 2, Fig. 1), demonstrate that very mi-nor structural change such as the location of a singlemethyl group on the aliphatic chain in the acyl moietyhas a strong impact on the anticonvulsant profile of theresulting compounds. These results presented in Tables 1and 2 are in agreement with a previous report on potentanticonvulsant urea derivatives of the constitutional iso-mers of VPA: valnoctylurea, propylisopropylurea, and di-isopropylurea. DBU exhibited a TPE in the range of 1–2 h,similar to PVU, which is in correspondence to the TPEs ofthe urea derivatives of the constitutional isomers of VPA(Shimshoni et al., 2007).

DBU’s anticonvulsant profile was further characterizedin a battery of various anticonvulsant tests that are summa-rized in Tables 3 and 4. In the 6-Hz psychomotor seizuretest, i.e, a model of refractory epilepsy, DBU demonstrateda remarkable anticonvulsant activity at both 32 and 44 mAcurrents (Table 3). As the stimulus intensity in the 6 Hz

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model increased from 22 mA to 44 mA some AEDs (e.g.,PHT, ethosuximide, and lamotrigine) lost their activity, andlevetiracetam’s (LEV) ED50 increased from 19 mg/kg (at32 mA) to 1,089 mg/kg (at 44 mA) (Barton et al., 2001;Smith et al., 2007; Holmes, 2007). DBU at 44 mA was twoand eight times more potent than VPA and LEV, respec-tively (Smith et al., 2007; Table 3). It appears that DBU is apromising candidate for development as a novel AED to beutilized in patients with therapy-resistant epilepsy (Smithet al., 2007).

In both bicuculline and picrotoxin-induced seizure tests,DBU was 3 and 1.6 times more potent than VPA, respec-tively (Table 3), indicating its ability to elevate the seizurethreshold to chemoconvulsants that act by antagonizingGABAA receptors and blocking chloride channels, respec-tively (White et al., 2002).

In the hippocampal kindling model, DBU was 3.4 timesmore potent (ED50 = 35 mg/kg) than VPA (ED50 =119 mg/kg) in the prevention of secondarily generalizedseizures (Table 3). As summarized in Table 4, DBU dis-played a time- and dose-dependent reduction in seizureseverity. A significant reduction in seizure score wasobserved only 135 min after administration of DBU(12.5 mg/kg, i.p.); however, at the highest dose adminis-tered (100 mg/kg, i.p.), DBU reduced significantly boththe seizure score and ADD by 15 min postadministra-tion (Table 4). The decrease in the seizure score follow-ing administration of 100 mg/kg DBU persisted longer than135 min, whereas no reduction in the ADD was observedafter 105 min (Table 4).

All frontline AEDs exhibit teratogenic effects in humansand experimental animals (Tomson and Battino, 2005,2007). NTDs including spina bifida asperta are amongthe most serious, life-threatening congenital malformationcaused by VPA (Lindhout & Schmidt, 1986). One of theprimary purposes in developing a second-generation toVPA drug is to prevent the teratogenicity associated withVPA therapy, which currently threatens tens of thousandsof pregnancies each year worldwide. The doses of thethree acylureas IVU, PVU, and DBU (Fig. 1) adminis-tered to mice in a direct effort to specifically induce NTDswere six and three times higher than their anticonvulsantED50 values in rats and mice, respectively (Tables 1 and2). Even at these extremely high doses, none of the acy-lureas tested herein appeared to share the degree of terato-genicity currently associated with VPA. Although singlecases of NTDs were noted in experimental fetuses ex-posed to these new compounds at high doses, they ap-pear to be significantly safer from a teratogenic perspec-tive than VPA or other teratogenic AEDs (Table 5). Ofspecial interest is the fact that the same equimolar dosesof the urea derivatives of VPA and its constitutional iso-mers were teratogenic and embryotoxic (Shimshoni et al.,2007). Apparently, reducing the length of the acyl-moietyfrom eight carbons (e.g., VPU and its isomers) to five

or six carbons (e.g., IVU, PVU, and DBU), but pos-sessing a quaternary carbon in its structure may be ofcrucial importance in designing nonteratogenic acylureacompounds.

At the highest dose tested (3.6 mmol/kg), DBU re-vealed high embryotoxic potency, causing more em-bryo/feto lethality than did VPA (Table 5). It is essentialto emphasize that the higher incidence of embryotoxicitywas observed only at doses 3.6 times higher than the an-ticonvulsant ED50 value of DBU in mice. Consequently,DBU possesses a larger safety margin as compared toVPA, which showed a marked teratogenicity and embry-olethality at the same dose range as its anticonvulsant ED50

values.Another severe side effect associated with the use

of VPA therapy is fatal hepatotoxicity, caused by VPAmetabolite(s) with a terminal double bond (e.g., 4-ene-VPAand 2,4-diene-VPA) (Gerber et al., 1979; Zimmermann &Ishak, 1982; Rettie et al., 1987; Baillie, 1992). 4-ene-VPAand 2,4-diene-VPA may also be involved in VPA-inducedteratogenicity (Sankar, 2007). It was recently shown that incontrast to previous concepts (Koenig et al., 1994), the riskof VPA-associated liver failure is not limited to patientsyounger than 2 years, receiving polytherapy and therefore,should be considered in all patients (Koenig et al., 2006).Since DBU and PVU possess a quaternary carbon at the β-and α-position to the carbonyl group, respectively, they, inanalogy to 2,2,3,3-tetramethylcyclopropanecarbonylurea(TMCU, Fig. 1) (Sobol et al., 2005), cannot be biotrans-formed to hepatotoxic metabolites with a terminal doublebond, analogous to 4-ene-VPA and 2,4-diene-VPA formedduring VPA therapy, and therefore the risk of inducing hep-atotoxicity is likely to be lower than that associated withVPA.

DBU’s pharmacokinetic profile was evaluated follow-ing i.v. administration (10 mg/kg) to rats. In rats DBUand VPA exhibit a similar half-life (t1/2 = 4 h), in spiteof the fact that DBU has two carbons less than VPAin its acyl moiety. VPA’s total clearance was twice aslarge as that of DBU (Table 6), but since DBU hada lower volume of distribution their half-life were sim-ilar (Table 6). TMCU (Fig. 1), the urea derivative of2,2,3,3-tetramethylcyclopropanecarboxylic acid (a cyclo-propyl analogue of VPA) (Sobol et al., 2004), was shownto possess a broad spectrum anticonvulsant activity in ratsand mice, and in spite of being urea derivative it wasrapidly eliminated from the body (CL = 0.36 L/h/kg) andconsequently, had a short half-life of 1.6 h (Sobol et al.,2005). Thus, DBU resides longer in the body than do eitherTMCU or VPA, suggesting that a more favorable dosageregimen would be possible in its clinical application.

Although DBU was mainly eliminated by metabolism(fe = 2.4%, Table 6), it was not biotransformed to its corre-sponding acid (DBA). Similarly, the corresponding amideof DBA, namely 3,3-dimethylbutanoyl amide (t1/2 =

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3.4 h), exhibited in dogs a similar lack of amide-to-acidbiotransformation. (Haj-Yehia et al., 1992).

In conclusion, the unique structure characterized by thecombination of a quaternary carbon at the β-position to thecarbonyl and a carboxyurea moiety, coupled with its broadspectrum and high potency of anticonvulsant activity aswell as high safety margin and favorable pharmacokineticprofile, make DBU an attractive candidate for developmentas a new, potent, and safe antiepileptic and CNS drug.

ACKNOWLEDGMENTS

The authors thank Mr. James P. Stables Director of the NIH–NINDS-Anticonvulsant Screening Program (ASP) for testing the compounds intheir anticonvulsant program. This work is abstracted from the Ph.D. the-sis of Mr. Jakob A. Shimshoni in a partial fulfillment for the requirementsof a Ph.D. degree at The Hebrew University of Jerusalem.

Conflict of interest: We, the authors, confirm that we have read the Jour-nal’s position on issues involved in ethical publication and affirm that thisreport is consistent with those guidelines.

MB has received speakers or consultancy fees from The AmericanEpilepsy Society (AES), BIAL, Bioline, Desitin, Gerson Lehrman GroupCouncils, Janssen-Cliag, Jazz Pharmaceuticals, Johnson & Johnson, Ova-tion, NeuroAdjuvants, Neurocrine Biosciences, Novonordisk, Shire, Teva,Valeant, and UCB Pharma. In the last 3 years, the author received re-search grants from Jazz Pharmaceuticals, Johnson & Johnson, Teva, andThe Epilepsy Therapy Development Project and has been involved in thedesign and development of new antiepileptics and CNS drugs. None ofthe other authors has any conflict of interest to disclose.

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