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Keywords:

  • Anticonvulsants;
  • Teratogenicity;
  • Neuropathic and persistent pain;
  • Subcutaneous Metrazol seizure test;
  • 6 Hz psychomotor seizure test;
  • Pharmacokinetics

Summary

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

Purpose: α-Fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide (α-F-TMCD) and α-Cl-TMCD, are α-halo derivatives of TMCD, the corresponding amide of a cyclopropane analog of valproic acid (VPA). This study aimed to comparatively evaluate the pharmacodynamics and pharmacokinetics of α-F-TMCD and α-Cl-TMCD in rodent models of epilepsy and for antiepileptic drug (AED)–induced teratogenicity. The potential of α-F-TMCD as an antiallodynic and antinociceptive compound was also evaluated.

Methods: α-F-TMCD and α-Cl-TMCD were synthesized. α-Cl-TMCD anticonvulsant activity was evaluated in comparison to VPA in the mouse maximal-electroshock-seizure (MES), Metrazol (scMet), and 6-Hz psychomotor-seizure tests. Neurotoxicity was assessed by the Rotorod-ataxia test. Induction of neural tube defects (NTDs) by α-Cl-TMCD and α-F-TMCD was evaluated after intraperitoneal administration to a mouse strain highly susceptible to VPA-induced teratogenicity. The ability of α-F-TMCD to reduce pain was evaluated in the rat spinal nerve ligation (SNL) model for neuropathic pain and in the formalin test. α-F-TMCD and α-Cl-TMCD pharmacokinetics was evaluated following intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats.

Results: α-F-TMCD and α-Cl-TMCD had similar potencies in the 6-Hz test and were more potent than VPA in this model and in the scMet test. Neither induced NTDs, and both exhibited wide safety margins. α-F-TMCD was active in the two pain models, and was found to be equipotent to gabapentin in the SNL model (ED50 = 37 and 32 mg/kg, respectively). Comparative pharmacokinetic analysis showed that α-Cl-TMCD is less susceptible to liver first-pass effect than α-F-TMCD because of lower total (metabolic) clearance and liver extraction ratio.

Conclusions: Based on their potent anticonvulsant activity and lack of teratogenicity, α-F-TMCD and α-Cl-TMCD have the potential for development as new antiepileptics and central nervous system (CNS) drugs.

Despite the availability of more than 20 antiepileptic drugs (AEDs), there is still a substantial need to develop more effective and safer AEDs, since about 30% of the patients with epilepsy are not seizure-free with the existing AEDs. Most of the currently utilized AEDs have side effects, which are sometimes severe and in some cases fatal (Bialer & White, 2010). Furthermore, AEDs are clinically used for the treatment of other nonepileptic central nervous system (CNS) disorders, such as migraine, bipolar disorder, and neuropathic pain (Bialer & White, 2010).

Valproic acid (VPA, 1, Fig. 1), one of the established AEDs, is the least potent of the major AEDs in anticonvulsant animal models (White et al., 2002). However, because of its broad spectrum of anticonvulsant activity, VPA is used for the treatment of various types of epilepsy and nonepileptic CNS disorders, and consequently is the most prescribed AED (Perucca, 2002; Nalivaeva et al., 2009). The clinical utilization of VPA is limited by different side effects, with the most serious of those being teratogenicity (Kaneko et al., 1999; Battino & Tomson, 2007) and hepatotoxicity (Chang & Abbott, 2006; Koenig et al., 2006), which restrict its utilization in women of child-bearing age and in children. To overcome the severe side effects associated with use of VPA and to retain its beneficial antiepileptic and CNS activity, numerous VPA analogs and derivatives have been designed and evaluated, and some of these second-generation VPA compounds have been found to possess favorable pharmacodynamic properties (Bialer & Yagen, 2007).

image

Figure 1.   Chemical structures of VPA (1), TMCA (2), 4-ene-VPA (3), TMCD (4), VPD (5), α-fluoro-TMCD (6), α-chloro-TMCD (7), α-F-hydroxymethyl-trimethylcyclopropanecarboxamide (α-F-CH2OH- TriMCD, 8) and its sulfate (α-F-CH2O-SO3H- Tri-MCD, 9), and glucuronide (α-F-CH2O-Glucuronide-TriMCD, 10) conjugates.

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2,2,3,3-Tetramethylcyclopropanecarboxylic acid (TMCA, 2, Fig. 1) is a cyclic analog of VPA that is inactive in the rat anticonvulsant maximal electroshock (MES) model (ED50 > 150 mg/kg) (Winkler et al., 2005). The presence of two quaternary carbons in TMCA chemical structure at the β-position to the carbonyl group prevents its biotransformation to an hepatotoxic metabolite with a terminal double bond analogous to VPA’s hepatotoxic metabolite 4-ene-valpoic acid (4-ene-VPA, 3,Fig. 1) (Tang et al., 1995; Neuman et al., 2001).

The corresponding amide of TMCA, 2,2,3,3-tetramethylcyclopropanecarboxamide (TMCD, 4, Fig. 1) that is a cyclic analog of VPA’s corresponding amide valpromide (VPD, 5, Fig. 1), is three times more potent than VPA (ED50 = 52 mg/kg) in the rat scMet test (Isoherranen et al., 2002, 2004). We have synthesized and evaluated the anticonvulsant activity of several amide derivatives of TMCA that demonstrated superior anticonvulsant potency compared to their corresponding acid (Bialer et al., 1996; Isoherranen et al., 2002, 2004; Sobol et al., 2004; Shimshoni et al., 2008). Our recent study revealed that substitution of the α-hydrogen to the carbonyl group of TMCD by fluorine, chlorine, and bromine atoms yielded halogenated TMCD derivatives with different anticonvulsant potencies. In the anticonvulsant rat-scMet test, α-fluoro-TMCD (α-F-TMCD, 6Fig. 1) was the most potent, followed by α-chlorine-TMCD (α-Cl-TMCD, 7, Fig. 1), whereas α-bromo-TMCD was inactive (Pessah et al., 2009). The rat scMet-ED50 of 6.4 mg/kg for α-F-TMCD shows that it is the most potent anticonvulsant compound among the hundreds of VPA analogs and derivatives that have been tested (Sobol et al., 2004; Shimshoni et al., 2008; Pessah et al., 2009). α-F-TMCD is also potent in the mouse 6 Hz psychomotor seizure test, the hippocampal kindled rat, and the pilocarpine-induced status models (Pessah et al., 2009). α-Cl-TMCD (7, Fig. 1) was potent in the rat scMet test (ED50 = 27 mg/kg) and in contrast to α-F-TMCD it was also active in the rat MES test (ED50 = 97 mg/kg). α-Br-TMCD was inactive in the mice MES and scMet tests and was also neurotoxic; therefore, it was not tested further, and its pharmacokinetics was not evaluated.

In the current study we evaluated the anticonvulsant activity and teratogenicity in mice models of α-Cl-TMCD and α-F-TMCD. We also analyzed the pharmacokinetics of both α-Cl-TMCD and α-F-TMCD following their intraperitoneal and oral administration to rats. Furthermore, we evaluated the activity of α-F-TMCD in two different rodent models for pain, in order to establish if in addition to its potential to become a new anticonvulsant drug it might also become a promising candidate for development as a new antiallodynic and antinociceptive drug.

Experimental Section: Materials and Methods

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

Chemicals and reagents

All the solvents were of analytical grade or high pressure liquid chromatograpy (HPLC) grade and were purchased from J.T. Baker (Deventer, The Netherlands). Sodium valproate, methylcellulose, and Cremophor EL were purchased from Sigma-Aldrich (Milwaukee, WI, U.S.A.).

Tested compounds

α-F-TMCD and α-Cl-TMCD were synthesized according to previously described procedures (Pessah et al., 2009).

Animals

See Supporting Information.

Test solutions

See Supporting Information.

Anticonvulsant activity and neurotoxicity

The anticonvulsant profile of α-Cl-TMCD was evaluated in the MES, scMet, and 6-Hz psychomotor seizure tests in mice. These tests examined the ability to block electrically, chemically, and 6-Hz corneal stimulation–induced seizures.

In addition to the efficacy studies, the neurotoxicity of α-Cl-TMCD was established using the Rotorod ataxia test. All of these tests were performed as part of the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program (ASP) according to previously described protocols (White et al., 2002). The Behavioral Seizure Score (BSS) of α-F-TMCD was established in the kindled rat model. The behavioral seizures were scored according to the following criteria, as originally described by Racine (Racine, 1972) and as detailed in the Supporting Information.

Antiallodynic activity

The induction of allodynia was performed using the spinal-nerve-ligation (SNL) model as described previously (Kim & Chung, 1992; Kaufmann et al., 2009) and as detailed briefly in the Supporting Information.

Analgesic activity

See Supporting Information.

Induction of neural tube defect (NTD)

See Supporting Information (Finnell et al., 1988, 1997).

PK studies of α-F-TMCD and α-Cl-TMCD in rats

Collection of blood samples and urine

Blood samples were withdrawn by cardiac puncture under deep isoflurane anesthesia, and were collected at 0.25, 0.5, 0.45, 1, 1.5, 2, 3, and 4 h after dosing. Three rats were sacrificed at each of these time points in the intraperitoneal experiments and two rats in the oral experiments. The heparinized test tubes with collected blood were immediately centrifuged at 1770 g for 10 min, and plasma was separated and stored at −20°C until analyzed. Urine was collected from two rats for 24 h after dosing in 50 ml plastic tubes and stored at −20°C until analyzed.

Analysis of α-F-TMCD and α-Cl-TMCD in plasma and urine

Plasma and urine levels of α-F-TMCD and α-Cl-TMCD were analyzed by gas chromatograph mass spectrometer (GC/MS). 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 quadrupole mass spectrometer operating in electron impact (EI) mode, an HP7673 autosampler, an HP MS-DOS Chemstation, and an HP-5MS capillary column (0.25 μm × 15 m × 0.25 mm). N-Methyl-TMCD (5 μg/ml) and α-F-TMCD (25 μg/ml) were the internal standards for α-F-TMCD and α-Cl-TMCD, respectively. Plasma or urine (200 μl) was added to the test tubes, followed by 25 μl of methanol and 25 μl of internal standard solution in methanol, and the tubes were vortexed thoroughly. Dichloromethane, 2 ml, was used for the extraction of the compounds. The dry residues obtained after evaporation of 1 ml dichloromethane were reconstituted with 80 μl dichloromethane, of which 1 μl was injected into the GC/MS apparatus. The temperature program was as follows: injector temperature, 180°C; initial temperature, 40°C for 6 min; gradient of 20°C/min until 140°C; gradient of 10°C until 200°C; hold time, 3–10 min. The MS parameters were set as follows: source temperature, 180°C; transfer line, 280°C; positive ion monitoring, 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. α-F-TMCD, α-Cl-TMCD, and MTMCD were monitored for selected ions at m/z: 159, 144 (α-F-TMCD); 175, 160 (α-Cl-TMCD); 155, 140 (MTMCD). Retention times of α-F-TMCD, MTMCD, and α-Cl-TMCD were 8.4, 9.5, and 9.8 min, respectively.

Calibration curves.  See Supporting Information.

Calculation of pharmacokinetics parameters

The pharmacokinetics parameters were calculated by noncompartmental analysis based on statistical moment theory using PK software Winnonlin version 5.2.1 (Pharsight co., Mountain View, CA, U.S.A.) (Rowland & Tozer, 2009). For further details, see Supporting Information.

Identification of α-F-TMCD metabolites in urine

α-F-TMCD was administered (40 mg/kg, i.p.) to three rats, and urine was collected over 24 h. Two milliliters of the urine samples were initially chromatographed on C-18 SPE column (1 g cartridge; Extra-Sep C18; Merck, White House, PA, U.S.A.). The cartridge was initially conditioned with 3 ml of methanol followed by 3 ml of water. Urine (2 ml) was loaded onto the cartridge and eluted under gravity. The cartridge was subsequently eluted with 3 ml of water. Afterwards, the cartridge was washed with methanol, of which fractions of 1 ml were collected. One microliter of each of the methanol fractions was injected to the GC/MS and analyzed for the presence of α-F-TMCD metabolites according to molecular weights and fragmentation.

Twelve milliliters of urine were lyophilized, and the residue was redissolved in 2 ml of H2O. The solution was chromatographed by a 24-cm–long/2-cm–diameter column containing 15 g C-18, reversed phase as stationary phase. The C-18 column was initially conditioned with 90 ml of methanol followed by 90 ml of water. Urine was carefully loaded on the column and eluted with 90 ml of water followed by 90 ml of methanol. The methanol was collected in fractions of 2 ml. One microliter of each fraction was injected to the GC/MS and analyzed for the presence of α-F-TMCD metabolites, according to molecular weights and fragmentation. Methanol fractions suspected to contain the metabolites were further chromatographed on a 6-cm–long/0.5-cm–diameter column containing 3 g silica gel. The column was eluted with 3% methanol in dichloromethane. Fractions of 1 ml were collected, and 1 μl of each fraction was injected to the GC/MS and analyzed using molecular weights and fragmentation for the presence of metabolites. The fractions containing the metabolites were derivatized and further analyzed for identification of the specific metabolites.

Derivatization procedures

Permethylation.  Fifty microliters of the aliquots of methanol fractions after C-18 SPE cartridge or after silica gel column purifications were evaporated to dryness under reduced pressure and redissolved in 50 μl of dry dimethyl sulfoxide (DMSO). Fifty microliters of CH3I and 8–10 mg of powdered NaOH were added, and the reaction mixture was vortexed vigorously at room temperature for 3 min. One milliliter of water was added, and the products were isolated by extraction with 3 ml of chloroform. The organic phase was subsequently washed with 3 ml of water, dried over MgSO4, and evaporated under reduced pressure. The dry residue was redissolved in 50 μl of chloroform, of which 1 μl was injected to GC/MS and analyzed for the presence of permethylated metabolites.

Silylation.  Fifty microliter aliquots of the methanol fractions after C-18 SPE cartridge or after silica gel column purification were evaporated to dryness under reduced pressure, and the residues were redissolved in 50 μl of anhydrous dichloromethane. The derivatization was performed at 60°C by adding 50 μl of dry pyridine and 50 μl of trimethylchlorosilane and stirring for 30 min. The reaction was quenched by adding 1 ml of water, and the products were extracted with 3 ml of chloroform. The organic phase was subsequently washed with 3 ml of water, dried over MgSO4, and evaporated under reduced pressure. The dry residues were redissolved in 50 μl of chloroform, of which 1 μl was injected into GC/MS and analyzed for the presence of the metabolites containing silyl moiety in their structures.

Blood-to-plasma concentration ratio

See Supporting Information.

Water solubility and Clog p-values

See Supporting Information.

Statistical analysis

The embryonic lethality and teratogenicity of α-F-TMCD and α-Cl-TMCD and their comparison to VPA, were performed by Fisher’s exact test. Analgesic effect of α-F-TMCD in the formalin test was performed using analysis of variance (ANOVA).

Results

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

Anticonvulsant activity

The quantitative anticonvulsant activity (ED50) of α-F-TMCD in rats and mice, and the qualitative anticonvulsant activity of α-Cl-TMCD and α-Br-TMCD in rats and mice were reported in our previous publication (Pessah et al., 2009). In the current study, further quantification of α-Cl-TMCD anticonvulsant activity was performed in the mice MES, scMet, and 6-Hz psychomotor seizure tests. α-Cl-TMCD was active in the MES model test and exhibited better potency than VPA in all other anticonvulsant tests conducted. The anticonvulsant potency of α-Cl-TMCD in mice in comparison to VPA is presented in Table 1. In the MES test, α-Cl-TMCD had an ED50 of 133 mg/kg, with little separation from its neurotoxic dose, TD50 = 144 mg/kg. In the scMet test and in the 6-Hz psychomotor seizure test, however, α-Cl-TMCD exhibited ED50 values of 84 and 76 mg/kg, respectively, and had protective index (PI) values of 1.7 and 1.9, respectively.

Table 1.   Anticonvulsant activity and neurotoxicity of α-Cl-TMCD in comparison to VPA following intraperitoneal administration to mice
Testα-Cl-TMCDVPAa
  1. bMaximal electroshock seizure test.

  2. cED50 or TD50 in mg/kg (mmol/kg).

  3. dSubcutaneous Metrazol seizure test.

  4. eAbnormal neurologic status characterized by behavioral impairment.

  5. fProtective Index (PI = TD50/ED50).

MESb-ED50 mg/kg (mmol/kg) 133c (0.76) 263c (1.82)
scMetd-ED50 mg/kg (mmol/kg)84 (0.48)220 (1.52)
6 Hz (32 mA)-ED50 mg/kg (mmol/kg)76 (0.43)126 (0.88)
Neurotoxicitye TD50 mg/kg144398
PIf (MES)1.11.5
PI (scMet)1.71.8
PI (6 Hz–32 mA)1.93.2

α-F-TMCD demonstrated broad spectrum of anticonvulsant activity, with high potency in the scMet test, 6-Hz psychomotor seizure test, and in the pilocarpine-induced status model test. In the kindled rat model, α-F-TMCD exhibited excellent potency with an ED50 value of 30 mg/kg. (Pessah et al., 2009). Further evaluation of the ability of α-F-TMCD to reduce the behavioral seizure score (BSS) in this model is reported here: α-F-TMCD decreased the behavioral seizure score [mean ± standard deviation (SD)] from 4.3 ± 0.7 to 0.8 ± 0.3 (15 min after dosing), and from 5.0 ± 0.7 to 1–1.8 (45–135 min after dosing) when the dose was increased from 11 to 89 mg/kg as depicted in Table 2. At a dose of 89 mg/kg the rats were fully protected against generalized stage 4 or 5 seizures at the time to peak effect (TPE) and also up to 2 hours post TPE (135 min). At a lower dose, 44 mg/kg, >70% of the rats were fully protected at TPE. At doses of 22–89 mg/kg, α-F-TMCD reduced the afterdischarge duration (ADD) at almost all time points, especially at 89 mg/kg (Table 2).

Table 2.   Effect of α-F-TMCD on behavioral seizure score and afterdischarge duration following intraperitoneal administration to hippocampal kindled rats
Dose (mg/kg)Protected/tested at TPE (15 min)Time after drug administration (min)
0154575105135
Average behavioral seizure score ± SEM
 111/65 ± 04.3 ± 0.75 ± 0.75 ± 0.75 ± 0.74.3 ± 0.7
 222/84.8 ± 0.23.6 ± 0.64.4 ± 0.54.9 ± 0.15 ± 04.3 ± 0.6
 445/75 ± 01.6 ± 0.62.6 ± 0.93.3 ± 0.83.6 ± 0.74.1 ± 0.6
 894/45 ± 00.8 ± 0.31.5 ± 0.51.3 ± 0.61 ± 0.41.8 ± 1.1
Average after discharge duration ± SEM (s)
 11 65 ± 1646 ± 362 ± 1266 ± 1470 ± 1864 ± 12
 22 45 ± 340 ± 349 ± 449 ± 647 ± 350 ± 9
 44 67 ± 1253 ± 850 ± 760 ± 1247 ± 859 ± 12
 89 34 ± 522 ± 963 ± 939 ± 1249 ± 1551 ± 16

Antiallodynic activity of α-F-TMCD

α-F-TMCD demonstrated a broad-spectrum anticonvulsant activity similar to VPA. Because AEDs are used for treating several neuropathic pain syndromes, we wanted to explore the antiallodynic effect of α-F-TMCD in the rat SNL model for neuropathic pain (Fig. 2, Table 3). α-F-TMCD reversed tactile allodynia in the tested animals, especially at doses of 40 and 80 mg/kg. Even at a lower dose (20 mg/kg), animals treated with α-F-TMCD showed an increase in antiallodynic effect, compared to control animals treated with the vehicle (methylcellulose) (Fig. 2). The duration of α-F-TMCD effect lasted between 180 and 240 min, and its ED50 was 37 mg/kg (95% confidence interval = 26–54 mg/kg).

image

Figure 2.   Antiallodynic effect of α-F-TMCD following intraperitoneal administration to rats. Each point represents mean pain threshold for all rats tested; Y error bars indicate standard error of the mean (SEM).

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Table 3.   Antiallodynic activity of α-F-TMCD 30–240 min after intraperitoneal administration to ratsa
α-F-TMCD Dose (mg/kg)Time after α-F-TMCD administration (min)
3060120180240
  1. ED50 = 37 mg/kg (95% confidence interval = 26–54 mg/kg).

  2. aNumber of rats protected/number of rats tested.

804/87/85/86/82/8
404/96/94/94/93/9
201/91/92/91/91/9
100/90/90/91/90/9

Antinociceptive activity of α-F-TMCD

α-F-TMCD (40 mg/kg) was tested in the mice formalin test for acute and inflammatory persistent pain induced by sc injection of formalin to the hind paw of the animal (Dubuisson & Dennis, 1977; Wheeler-Aceto et al.,1990). As can be seen from the area of the effect versus time curve (AUEC) (Fig. 3) obtained for the duration of licking, and in Tables S1 and S2, α-F-TMCD significantly decreased the pain in the inflammatory phase (p < 0.01), 10–40 min after formalin injection. In the acute phase, however, which starts immediately after formalin injection and lasts 9 min, no analgesic effect was observed (Fig. 3, Tables S1 and S2).

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Figure 3.   Analgesic effect (assessed by the formalin test) of α-F-TMCD as obtained following its intraperitoneal administration to mice.

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Neural tube defects (NTDs)

The teratogenic potential of α-Cl-TMCD and α-F-TMCD was assessed for their ability to induce neural tube defects (NTDs) in the SWV/Fnn mice that are highly susceptible to VPA-induced exencephaly (Finnell et al., 1988). Treatment of these mice with VPA resulted in incidence of NTDs in a dose-dependent manner (Table S3). At the highest VPA dose (3.6 mmol/kg), the percentage of fetuses with NTDs was >50%. Furthermore this dose was embryolethal (Finnell et al., 1997).

In contrast, none of the administered doses of α-F-TMCD caused NTDs as depicted in Table S3. The higher doses of α-F-TMCD (4.2 and 3.15 mmol/kg), however, resulted in a high percentage of resorptions (78% and 27%, respectively). α-Cl-TMCD did not induce NTDs, and it did not result-in resorptions at doses of 1.8 and 1.1 mmol/kg, but at the highest dose administered (2.7 mmol/kg) it was lethal to the dams (Table S3).

Pharmacokinetics analysis

The mean plasma concentration-versus-time plots of α-F-TMCD and α-Cl-TMCD following intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats are comparatively presented in Figs 4 and 5. These concentration time plots illustrate multiple peaks, probably due to the low water solubility of α-F-TMCD (1.0 mg/ml) and α-Cl-TMCD (0.9 mg/ml) (Table S4). The fraction of the dose excreted unchanged in urine (fe) of both α-F-TMCD and α-Cl-TMCD was 0.18%. Therefore, α-F-TMCD and α-Cl-TMCD are mainly eliminated by metabolism and their total clearance equals their metabolic clearance. The high metabolic clearance resulted in relatively short t1/2 values of 45 min (α-F-TMCD) and 51 min (α-Cl-TMCD). Because of their low water solubility (Table S4), α-Cl-TMCD and α-F-TMCD had partial oral bioavailability (F′) in rats of 44% and 16%, respectively (relative to intraperitoneal dosing).The other pharmacokinetics parameters calculated for α-F-TMCD and α-Cl-TMCD are presented in Table 4 and their blood-to-plasma ratios were 1.0 and 1.6, respectively (Table S4).

image

Figure 4.   Plasma concentration-time plots of α-F-TMCD obtained following its intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats.

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image

Figure 5.   Plasma concentration-time plots of α-Cl-TMCD as obtained following its intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats.

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Table 4.   PK parameters of α-F-TMCD and α-Cl-TMCD as obtained following their intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats
PK Parameterα-F-TMCD (ip)α-Cl-TMCD (ip)α-F-TMCD (po)α-Cl-TMCD (po)
  1. CL/F, ip or oral clearance; CLr, renal clearance; CLm, metabolic clearance; Vz/F, apparent volume of distribution; t1/2, half-life; AUC, area under the plasma concentration-time curve; AUMC, area under the first moment-time curve; MRT, mean residence time of a drug molecule within the body; Cmax, peak plasma concentration; tmax, time to reach Cmax.

  2. aRelative oral bioavailability compared to intraperitoneal administration.

CL/F (L/h/kg)1.50.98.93.0
CLr (ml/h)0.660.4
CLm (L/h/kg)1.50.9
Vz/F (L/kg)1.61.17.713.4
t1/2β (h)0.750.860.60.8
AUC (mg/Lxh)27.346.06.7120.3
AUMC (mg/Lxh2)39.273.59.8540.1
MRT (h)1.41.61.52.0
Cmax (mg/L)18.225.94.69.7
tmax (h)0.50.250.51.5
F′a (%)1644
fe (%)0.180.18
E (%)4225  

Identification of metabolites of α-F-TMCD

Three metabolites of α-F-TMCD were identified in rat urine following its intraperitoneal administration to rats. α-F-hydroxymethyl-trimethylcyclopropanecarboxamide (α-F-CH2OH-TriMCD, 8, Fig. 1) was the primary metabolite, and its sulfate and glucuronide conjugates (9 and 10, respectively, Fig. 1) were the secondary metabolites. α-F-CH2OH-TriMCD was identified by GC/MS and its retention time and relevant m/z ions are depicted in Table 5. Permethylation and silylation was performed on the fractions obtained from the urine after column chromatography. α-F-CH2O-SO3H-TriMCD and α-F-CH2O-glucuronide-TriMCD were detected as their permethylated derivatives along with the derivatives of α-F-CH2OH-TriMCD. The retention time of the derived metabolites and relevant m/z ions are presented in Table 5.

Table 5.   Retention times and electron ionization mass spectral data of α-F-TMCD and its metabolites as obtained using GC/MSThumbnail image of

Discussion

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

In our recent publication we evaluated the anticonvulsant activity of α-halo-TMCD derivatives in the MES and scMet tests. We have revealed that α-F-TMCD was more potent in the rat scMet test than α-Cl-TMCD. α-Br-TMCD was inactive and neurotoxic in mice, and, therefore, was not further evaluated in other anticonvulsant tests (Pessah et al., 2009). In this study we evaluated in rodents the anticonvulsant activity, pharmacokinetics, and teratogenicity of α-Cl-TMCD in comparison to its analog, α-F-TMCD, and to VPA. The design of α-halo-TMCD derivatives was motivated by pharmacokinetic and pharmacodynamic considerations aiming to enhance brain penetration and thus improve their anticonvulsant potency (compared to VPA and TMCD), as well as to avoid major side effects by circumventing the structural requirements needed for VPA-induced teratogenicity and hepatotoxicity (Bialer & Yagen, 2007). α-F-TMCD and α-Cl-TMCD demonstrated a broad-spectrum of anticonvulsant activity in several seizure tests, particularly in rats, and were highly potent. α-F-TMCD possessed large protective indexes compared to VPA, especially in the rat scMet test (PI = 20 compared to 1.2), and also in the mice scMet and 6-Hz psychomotor (44 mA) tests (Pessah et al., 2009). In the 6-Hz psychomotor seizure test (32 mA), α-Cl-TMCD had a similar ED50 value (76 mg/kg) to that of α-F-TMCD, suggesting that these compounds might also be effective in treating patients with refractory epilepsy (Barton et al., 2001; White et al., 2002). α-Cl-TMCD demonstrated improved PI compared to VPA in the rat (MES PI > 2, scMet PI > 8) (Pessah et al., 2009), and PI of the same magnitude as VPA in the mice. Because teratogenicity is a major side affect of old-generation AEDs, and of some of the new-generation AEDs (i.e. lamotrigine) (Morrell, 1996; Palmieri & Canger, 2002), limiting their clinical use in women of child-bearing age, it is essential to develop nonteratogenic new AEDs (Meador, 2008). Little is currently known about the teratogenicity in humans of the newer AEDs; however, some abnormalities have been found in rats (Morrell, 1996), although these may be related to maternal toxicology (Morrell, 1996; Palmieri & Canger, 2002). Both α-halo-TMCD derivatives were found to be nonteratogenic and did not induce neural tube defects at doses 2–4 times (α-Cl-TMCD) and 5–9 times (α-F-TMCD) higher than their mice-anticonvulsant ED50 values (Tables 1 and 6; Pessah et al., 2009). At the higher doses tested, α-F-TMCD (4.2 mmol/kg) was highly embryotoxic and α-Cl-TMCD (2.7 mmol/kg) was lethal to the pregnant dams (Table S3). No abnormalities that may be related to toxicology have been detected in any of the fetuses.

Several AEDs are used nowadays to treat different types of pain, such as neuropathic pain and persistent pain. Currently, there are only a few effective drugs for the treatment of neuropathic pain, and the available drugs used for the treatment of persistent pain produce severe side effects, especially with prolonged use. Therefore, there is a substantial need to develop new drugs for the treatment of neuropathic and persistent pain (Guindon et al., 2007). Because AEDs are targeted to alter abnormal neuron discharges, and both epilepsy and neuropathic pain share the abnormal neuronal discharges by a similar pathophysiology, it is very likely that AEDs that have a broad spectrum of activity will be useful in treating other disorders such as inflammatory or neuropathic pain (Guindon et al., 2007). Gabapentin and carbamazepine are effective in treating various neuropathic pain syndromes and both are also effective in the mouse formalin test for persistent pain (Shannon et al., 2005; Kaufmann et al., 2009). α-F-TMCD was the most potent compound in the rat scMet test of all the VPA analogs and derivatives we have reported in the literature (Bialer & Yagen, 2007; Shimshoni et al., 2008). This compound was also potent in the 6-Hz psychomotor seizure test, the hippocampal kindled rat, and pilocarpine-induced status model in rats (Pessah et al., 2009). Because α-F-TMCD has a broad and potent anticonvulsant activity, we wanted to explore its antiallodynic effect in the rat SNL model for neuropathic pain. α-F-TMCD exhibited high potency in the rat SNL model with an ED50 value (37 mg/kg, Table 3), equipotent (p > 0.05) to gabapentin (ED50 = 32 mg/kg), one of the leading drugs in the treatment of neuropathic pain (Kaufmann et al., 2009). α-F-TMCD was also active in the mice formalin test (Tables 4 and 5). Although it did not reduce the pain in the acute phase (Fig. 3), unlike carbamazepine and gabapentin (Shannon et al., 2005), it significantly reduced the response in the second inflammatory phase (p < 0.01). VPA possesses weak activity in the SNL model for neuropathic pain (ED50 = 269 mg/kg) (Winkler et al., 2005). Furthermore, VPA is inactive in the formalin test in rats (Shannon et al., 2005). In conclusion, we demonstrated that α-F-TMCD is superior to VPA in the SNL and formalin models for pain, its spectrum of activity is broader than that of VPA, and its SNL-ED50 value is similar to that of gabapentin (Winkler et al., 2005).

In the pharmacokinetics studies the concentration-time plots obtained for α-F-TMCD and for α-Cl-TMCD demonstrated a multipeak profile following intraperitoneal and oral administration to rats (Figs 4 and 5). The oral bioavailability of α-F-TMCD relative to intraperitoneal dosing is low (F′ = 16%). The major elimination of α-F-TMCD is by metabolism, and its liver extraction ratio (E) was calculated to be 42%, assuming that the metabolism occurs primarily in the liver (Table 4) (Rowland & Tozer, 2009). These data indicate that α-F-TMCD is susceptible to first-pass effect when administered orally, and its maximal oral availability (FH) is 58% (FH = 1–E; 100% − 42% = 58%). However, the low oral bioavailability (F′ = 16%) in rats of α-F-TMCD shows that in addition to the liver first-pass effect this compound has incomplete absorption from the gastrointestinal tract (Table 4). The low oral bioavailability of α-F-TMCD in the rat does not necessarily imply that α-F-TMCD will have such low bioavailability humans. It has been reported before that drugs can undergo extensive metabolism rats and possess low oral bioavailability in contrast to their extent of absorption in humans: for example, amitriptyline (F = 6.3% in the rat and 46% in humans) (Bae et al., 2009) and ondansetron (4.07% in the rat and 62% in the human) (Yang & Lee, 2008). Therefore, it not always possible to predict the oral bioavailability in humans from the rat model, especially for drugs that are extensively metabolized, and it is most likely that in humans α-F-TMCD will have higher F values. α-F-TMCD did not undergo hydrolytic biotransformation to its corresponding acid α-F-TMCA but was rather metabolized by oxidation of one of the four methyl groups to α-F-CH2OH-TriMCD (8, Fig. 1). This hydroxy derivative of α-F-TMCD is much more water-soluble and is further partly biotransformed to its sulfate and glucuronide conjugates, which are excreted in the urine and have been identified by GC/MS after their derivatizations (Table 5). α-F-TMCD metabolic profile in rats is similar to that of 2,2,3,3-tetramethylcyclopropane carbonyl urea (Sobol et al., 2005) and pyrethroid ester insecticides (Mihara et al., 1981; Kaneko et al., 1987).

An oxidative biotransformation of one of the methyl groups in the tetramethylcyclopropane moiety to hydroxymethyl is a general pattern during phase1 metabolism of TMCA derivatives (Mihara et al., 1981; Kaneko et al., 1987; Sobol et al., 2005). Therefore, we did not investigate the metabolic pathways of α-CL-TMCD, assuming that it will be metabolized to α-Cl-CH2OH-TriMCD in a similar manner to that of α-F-TMCD (Fig. 1).

Despite its low oral bioavailability, α-F-TMCD is extremely potent (scMet ED50 = 6.4 mg/kg) following oral administration to rats, and the duration of its anticonvulsant effect lasts for 4 h after dosing (Pessah et al., 2009), five times longer than its pharmacokinetic t1/2. Therefore, the pharmacodynamic properties of α-F-TMCD override its pharmacokinetic deficiencies (high clearance and low oral bioavailability). α-Cl-TMCD, in comparison to α-F-TMCD, demonstrated improved bioavailability and a lower clearance value. Despite its relatively higher bioavailability (44%) and lower susceptibility to liver first-pass effect or E value (Table 4), α-Cl-TMCD is about four times less potent than α-F-TMCD in the rat scMet (po) test, and its anticonvulsant effect lasts for only 2 h after dosing (Pessah et al., 2009).

In conclusion, α-F-TMCD and α-Cl-TMCD demonstrated opposite pharmacokinetic–pharmacodynamic profiles. α-Cl-TMCD had a better pharmacokinetics profile and was active in the MES test, but in the scMet and 6-Hz anticonvulsant models it was less potent as compared to α-F-TMCD. α-F-TMCD was significantly more potent than VPA in the scMet, 6-Hz, kindling, and pilocarpine induction seizure models (Pessah et al., 2009). The ability of α-F-TMCD to reduce pain in neuropathic and persistent pain models is an enormous add-on value to its extreme anticonvulsant activity. Based on their potent anticonvulsant activity, lack of teratogenicity, and safety margins both α-F-TMCD and α-Cl-TMCD have the potential to become candidates for development as new, potent, and safe antiepileptic and CNS drugs.

Acknowledgment

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

This work is abstracted from the PhD thesis of Ms. Neta Pessah in partial fulfillment of the PhD degree requirements for The Hebrew University of Jerusalem.

The authors wish to thank Mr. James P. Stables Director of the NIH–NINDS-Anticonvulsant Screening Program (ASP) for testing the compounds in the ASP.

Disclosure/Conflict of Interest

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

Dr. Meir Bialer has received in the last 3 years speakers or consultancy fees from Bial, Bioline, CTS Chemicals, Desitin, Janssen-Cilag, Jazz Pharmaceuticals, Johnson & Johnson, Lunbeck, McKinsey & Company, NeuroAdjuvants, Neurocrine Biosciences, Rekah, Sepracor, Shire, Teva, UCB Pharma, and Upsher-Smith. In the last 5 years, the author received research grants from Jazz Pharmaceuticals, Johnson & Johnson, and The Epilepsy Therapy Development Project, and has been involved in the design and development of new antiepileptics and CNS drugs as well as new formulations of existing drugs. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

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  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Experimental Section: Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure/Conflict of Interest
  8. References
  9. Supporting Information

Table S1. Effect of α-F-TMCD (40 mg/kg) compared to control on the duration of paw licking after formalin injection to mice.

Table S2. Area under the effect versus time curve of paw licking after formalin injection to mice.

Table S3. Teratogenicity data of α-Cl-TMCD in comparison to VPA and α-F-TMCD as obtained after a single intraperitoneal injection of the tested compounds to SWV dams on day 8 of gestation.

Table S4. Blood to plasma ratio, water solubility, and ClogP of α-F-TMCD and α-Cl-TMCD.

Data S1. Materials and Methods.

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EPI_2684_sm_TablesS1-S4.doc112KSupporting info item

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