Analgesic and anticonvulsant activity of new derivatives of 2-substituted 4-hydroxybutanamides in mice
Kinga Sa³at1, Katarzyna Kulig2, Robert Sa³at3, Barbara Filipek1, Barbara Malawska2
1Department of Pharmacodynamics,2Chair of Pharmaceutical Chemistry, Department of Physicochemical Drug Analysis, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
3Faculty of Production Engineering, Warsaw University of Life Sciences, Nowoursynowska 164, PL 02-787 Warszawa, Poland
Correspondence: Kinga Sa³at, e-mail: salat.kinga@gmail.com
Abstract:
Earlier in vitro studies of the compounds marked as GT27, GT28, GT29 and BM128 revealed their inhibitory action towards murine g-aminobutyric acid (GABA) transporters (mGAT1–mGAT4). In the present paper, the pharmacological activity of four g-hydroxy- butyric acid (GHB) amide derivatives was investigated. The following procedures were involved: locomotor activity, hot plate and electroconvulsive threshold tests. The compounds’ influence on motor coordination was evaluated in the chimney test, as well.
Intraperitoneal (ip) administration of the GHB derivatives decreased animals’ locomotor activity (ED50values ranged between 23.79 and 26.37 mg/kg). At a dose of 25 mg/kg (ip) the compounds prolonged the nociceptive reaction time latency in the hot plate assay to various degree and GT28 and GT29 were the most potent ones in this respect. Their analgesic efficacy was particularly pro- nounced 30 min after their administration [percent of maximal possible effect (%MPE) = 16.93 and 22.72, respectively]. The investi- gated GHB derivatives, except for GT29 at 100 mg/kg, increased the electroconvulsive threshold by approximately 4–11 mA as compared to the vehicle-treated mice. In the chimney test they impaired the animals’ motor coordination to various degree. We sug- gest further investigations of the compounds to estimate their biological activity.
Key words:
electroconvulsive threshold, GABA uptake inhibitors, hot plate, mice, motor coordination, tiagabine, tonic hind limb extension
Abbreviations: %MPE – percent of maximal possible effect, ECT – electroconvulsive threshold test, GABA – g-amino- butyric acid, GAD – glutamic acid decarboxylase, GAT – GABA transporters, GHB – g-hydroxybutyric acid, ip – intraperitoneal
Introduction
g-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous
system. The dysfunction of the GABA-ergic system is thought to underlie variable pathophysiological states, which are a consequence of diminished GABA inhibi- tory action. These disturbances often result in anxiety, seizures, schizophrenia, neurodegenerative disorders, motion impairment, insomnia, pain or alcoholism [12, 27, 46].
GABA removal from the synaptic cleft involves specific plasma membrane transporters (GABA trans- porters; GAT) displaying an Na+and Cldependence for transport, situated on glia cells and presynaptic neuronal terminals [34, 46]. So far, there have been
102 Pharmacological Reports, 2012, 64, 102112 Pharmacological Reports 2012, 64, 102112 ISSN 1734-1140
Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
four such GABA transporters discovered in the mouse and other species [11–13, 19, 36, 46, 49]. The cloning techniques have revealed a significant discrepancy in the nomenclature of these species-homologue GABA transporters. The nomenclature GAT1 – GAT4 refers to the mouse transporters (mGAT) and the corresponding transporters in rats and humans are named rGAT-1/
hGAT-1, rBGT-1/hBGT-1, rGAT-2/hGAT-2 and rGAT-3/
hGAT-3, respectively. The rat, mouse and human GAT1 are identical and contain 599 amino acids, whereas two other transporters: rGAT-2 and rGAT-3 are less homologous and consist of 602 and 627 amino acids, respectively [34]. Three types of GAT are high affinity transporters: rGAT-1, rGAT-2 and rGAT-3. The fourth one – BGT-1 is a low affinity GABA transporter [12, 47].
Recent research focus on GAT expression in the brain, as well as the pharmacological evaluation of their inhibitors. Several lines of evidence suggest that such compounds can supply novel pharmacological profiles and therapeutic benefits. Transporters local- ized on glia cells are of special interest in this respect.
The anticonvulsant activity of GAT inhibitors other than tiagabine has become well established in epilep- tic animals and seems to be distinct as compared to ti- agabine. Noteworthy, mixed GAT inhibitors are ex- pected to have much broader spectrum of anticonvul- sant activity than pure GAT1 inhibitors [11, 12, 35, 41]. The inhibition of neuronal or glial re-uptake of GABA significantly increases its concentration [10].
For this reason tiagabine, a drug that inhibits GAT1 function, is of special interest not only as an anticon- vulsant agent useful in the treatment of partial sei- zures [5, 9, 10, 12, 13, 31, 33, 35, 44, 45, 52] but also as an analgesic drug in human and anxiolytic or anti- depressant compound in rodents [27, 51]. Currently tiagabine is being investigated for these and other possible indications (e.g., treatment of psychosis, gen- eral anxiety and sleep disorders, drug addiction, acute and chronic pain, posttraumatic stress) [12, 17, 18, 23, 25, 27, 32, 45, 52].
In our earlier search for new anticonvulsants [24, 38, 39] we selected a group of derivatives of 2-substituted 4-hydroxybutanamides. 4-Hydroxybutanoic acid (g- hy- droxybutyric acid, GHB), the core-structure of these compounds, is an endogenous substance that func- tions as an inhibitory neurotransmitter in the mam- malian central nervous system [8, 37]. In the new structures the carboxylic acid function of GHB was transformed into more lipophilic one (i.e., an arylal-
kylamide group). In the position 2 of GHB, an N-di- phenylmethylpiperazine moiety – a part that mimicked the biaryl moiety of already known GAT inhibitors and a fragment corresponding to the anticonvulsant- active arylpiperazine derivatives of GHB were intro- duced. The synthesis of the compounds as well as their preliminary biological evaluation in the in vitro studies were described previously [24]. In Table 1 the compounds’ IC50values obtained in these former studies [24] of [3H]GABA uptake assays are shown.
The chemical structures of the investigated GHB de- rivatives are shown in Figure 1.
In the present study, the evaluation of anticonvul- sant and analgesic activity of four new, non-selective GAT inhibitors is discussed. The influence on loco- motor activity and motor coordination is also de- scribed.
Tab. 1. IC50values obtained for the investigated compounds in the [3H]GABA uptake assay [24]
Compound mGAT1 IC50
value (µM)
mGAT2 IC50
value (µM)
mGAT3 IC50
value (µM)
mGAT4 IC50
value (µM)
GT27 72.44 44.67 28.84 120.23
GT28 58.88 36.31 30.20 63.10
GT29 63.10 67.61 19.05 97.72
BM128 13.18 27.54 8.71 12.30
Tiagabine§ 0.11 > 100 > 100 800
Inhibitory potency of the compounds tested at four murine GAT (1–4).
Study performed as [3H]GABA uptake assay based on stably trans- fected HEK cells (for details see [22, 24]).§Data from [34]
Fig. 1. Chemical structure of the investigated compounds
Materials and Methods
Animals
The behavioral experiments were carried out at the De- partment of Pharmacodynamics, Pharmaceutical Fac- ulty, Jagiellonian University in Kraków. Adult male Albino Swiss (CD-1) mice weighing 18–25 g were used in the experiments. The animals were kept in groups of 15 mice in cages at a room temperature of 22 ± 2°C, under light/dark (12:12 h) cycle and had free access to food and water. The ambient temperature of the room and the humidity were kept constant throughout all the tests.
Animals for the experiments were selected in a ran- dom way. They were killed by cervical dislocation immediately after the assay. Experimental groups consisted of 5–12 animals/dose and all the animals were used only once. The number of animals was kept at minimum to obtain definite results with the test utilized. Prior to the test, the mice were allowed to ac- climate to the holding cages for a minimum of 2 h.
The experiments were performed between 8 a.m. and 3 p.m. All the procedures were approved by the Local Ethics Committee of the Jagiellonian University in Kraków (ZI/329/2006).
Chemicals
The compounds tested (Fig. 1) were synthesized at the Department of Physicochemical Drug Analysis, Pharmaceutical Faculty, Jagiellonian University in Kraków. The synthesis of the tested compounds was described previously [24].
For the pharmacological studies they were sus- pended in a 0.5% methylcellulose solution (Loba Chemie, Germany) and administered by the intraperi- toneal (ip) route. Control mice were given an appro- priate amount of vehicle (methylcellulose solution) with the exception of the hot plate assay in which each animal served as its own control.
Dosage protocol
In the locomotor activity test various doses of the compounds were tested to obtain the ED50values and finally to choose the dose for the hot plate test, which enabled to avoid a misinterpretation of the results from this test. It is a very well-known fact that seda-
tives and myorelaxants give false positive effect in the hot plate assay, as they are able to prolong the time la- tency of nociceptive responses [53]. The ED50values obtained in the locomotor activity test ranged from 23.79 to 26.37 mg/kg, a dose of 25 mg/kg was tested in the hot plate model.
As the preliminary anticonvulsant activity of the in- vestigated compounds (i.e., pentylenetetrazole-induced seizures and maximal electroshock seizures) provided by the protocols designed by the National Institute of Neurological Disorders and Stroke (NIH; Bethesda, USA) involved doses higher than 100 mg/kg, the abil- ity of the compounds to elevate the electroconvulsive threshold (ECT) and their influence on motor coordi- nation were also tested in similar dose ranges.
Administration protocols
Locomotor activity
The effects of various doses of the investigated com- pounds administered by the ip route were recorded in the photoresistor actimeters (30 cm in diameter, illu- minated by two light beams) connected to a counter for the recording of light-beam interruptions. The mice were placed individually in the actimeters and the number of light-beam crossings was counted dur- ing the 30-min session. Each experimental group con- sisted of 8 mice. For the statistical analysis data ob- tained in the 30th minute of the observation period were used.
The hot plate test
The hot plate test was performed according to a method described by Eddy and Leimbach [15] with a modifica- tion according to £uszczki and Czuczwar [29].
The hot plate apparatus (Hot Plate 2A Type, Omega) has an electrically heated surface and is sup- plied with a temperature-controller that maintains the temperature at 55–56°C. The animals (8 mice per each experimental group) were tested as follows: once to get the baselines (control reaction) and then – 30, 60 and 90 min after ip administration of the com- pound. The time latency until the animals placed on the hot plate either licked their fore or hind paws, shook hind paws or jumped off the hot plate was measured by means of a stop-watch. Mice showing control reaction time greater than 12 s were excluded from the subsequent test.
104 Pharmacological Reports, 2012, 64, 102112
In this procedure a cut-off time was chosen (45 s) to avoid tissue damage and mice not responding within 45 s were removed from the apparatus and assigned a score of 45 s. The maximal possible effect (MPE) was defined as a lack of a nociceptive response to the thermal stimulus and the percentage of MPE (%MPE) was calculated according to the formula (1):
%MPE = [(T1–T0)/(T2–T0)] × 100 (1) where T0= predrug latency, T1 = postdrug latency, T2
= cut-off time (45 s).
Electroconvulsive threshold test
The anticonvulsant potencies of the tested compounds were compared at the previously established time of peak drug effect, i.e., 30 min after their ip injection.
Two doses of each compound (100 and 200 mg/kg) were investigated. Electroconvulsions were produced by an alternating current (duration of the stimulus:
0.2 s; 50 Hz) delivered via standard auricular elec- trodes by an electroshock generator (rodent shocker, type GE; COTM, Bia³ystok, Poland). Tonic hind limb extension (i.e., the hind limbs outstretched 180° to the plane of the body axis) was an indication of seizure episodes. For the evaluation of the electroconvulsive threshold (ECT) at least four groups of animals per dose were used (each group consisted of 5–9 animals).
Those mice were challenged with electroshocks of various intensities to yield 10–30%, 30–50%, 50–70%
and 70–90% of animals with seizures. Then, a median current strength value, defined as current intensity re- quired to induce tonic hind limb extension in 50% of the mice challenged (CS50in mA), was estimated by means of log-probit method [26].
Chimney test
The chimney test was performed according to a method described by Boissier et al. [1] and Dudra- Jastrzebska et al. [14]. Briefly, 30 min after the ip ad- ministration of the compounds (i.e., at the previously established time of peak drug effect) the animals (8 mice per group) had to climb backwards up a tube (25 cm length, 3 cm diameter) with a rough inner sur- face. Motor impairment was indicated by the inability of mice to perform the test within 60 s.
Data analysis
The data from the locomotor activity and hot plate as- says are expressed as the mean ± standard error of the mean (SEM). To compare the results obtained for two different groups of animals (the investigated com- pound group vs. the control group) one-way ANOVA followed by Dunnett’s test was used in these two tests. Median current strengths (CS50) values with their 95% confidence limits were calculated and ana- lyzed by computer log-probit analysis according to Litchfield and Wilcoxon [26].
The results from the chimney test are expressed as a percentage of mice unable to get out of the tube within one minute. Qualitative variables from the chimney test were compared by the use of the Fisher’s exact probability test.
Results
Locomotor activity
The investigated compounds decreased the locomotor activity in a dose-dependent manner. The ED50values ranged from 23.79 mg/kg (for GT28) to 26.37 mg/kg (for GT27). The precise data are shown in Table 2.
The hot plate test
Table 3 demonstrates the antinociceptive activity of the investigated compounds in the hot plate assay. The prolongation of nociceptive reaction time latency was particularly pronounced 30 min after the compounds’
injection. GT28 and GT29 at the dose 25 mg/kg (ip) were antinociceptive (statistically significant at p < 0.05 for GT28 and at p < 0.01 for GT29). The %MPE measured 30 and 60 min after administration of these compounds ranged from 16 to 23%. None of the com- pounds was antinociceptive 90 min after the admini- stration.
Electroconvulsive threshold test
The current strength value that induced a tonic hind limb extension in 50% of mice (CS50) in the vehicle- treated group was 7.06 mA. All the compounds, ex- cept for GT29 at 100 mg/kg, elevated the seizure
106 Pharmacological Reports, 2012, 64, 102112 Tab. 2. Influence of the compounds on locomotor activity
Compound Dose
(mg/kg)
Number of light-beam crossings (mean ± SEM) recorded
Locomotor activity decrease (%)
ED50 (mg/kg)
Vehicle (0.5% MC) – 583.7 ± 72.75 – –
BM128 1 567.6 ± 46.63 2.8 25.95 (11.84–56.91)
5 514.4 ± 49.33 11.9
30 253.0 ± 57.59**** 56.7
60 207.2 ± 29.23**** 64.5
Vehicle (0.5% MC) – 553.5 ± 47.48 – –
GT27 5 432.9 ± 55.49 21.8 26.37 (9.21–75.47)
30 250.6 ± 49.93**** 54.7
60 204.0 ± 36.31**** 63.1
Vehicle (0.5% MC) – 562.6 ± 53.22 – –
GT28 5 413.3 ± 46.87 26.5 23.79 (6.71–84.41)
30 273.2 ± 48.07**** 51.4
60 188.3 ± 16.24**** 66.5
Vehicle (0.5% MC) – 565.9 ± 63.58 – –
GT29 5 465.7 ± 42.74 17.7 25.28 (10.48–61.01)
30 289.0 ± 22.7*** 48.9
60 154.0 ± 22.83**** 72.8
Values shown as the mean ± SEM for n = 8. Compounds administered intraperitoneally. MC – methylcellulose. Results shown regard the 30th min of the observation period. Statistical analysis: one way ANOVA, followed by Dunnett’s multiple comparison test; (*) indicates a significant difference compared to the vehicle-treated group: *** p < 0.01, **** p < 0.001
Tab. 2. Influence of the compounds on locomotor activity
Compound Dose (mg/kg) Number of light-beam crossings (mean ± SEM) recorded
Locomotor activity decrease (%) ED50(mg/kg)
Vehicle (0.5% MC) – 583.7 ± 72.75 – –
BM128 1 567.6 ± 46.63 2.8 25.95 (11.84–56.91)
5 514.4 ± 49.33 11.9
30 253.0 ± 57.59**** 56.7
60 207.2 ± 29.23**** 64.5
Vehicle (0.5% MC) – 553.5 ± 47.48 – –
GT27 5 432.9 ± 55.49 21.8 26.37 (9.21–75.47)
30 250.6 ± 49.93**** 54.7
60 204.0 ± 36.31**** 63.1
Vehicle (0.5% MC) – 562.6 ± 53.22 – –
GT28 5 413.3 ± 46.87 26.5 23.79 (6.71–84.41)
30 273.2 ± 48.07**** 51.4
60 188.3 ± 16.24**** 66.5
Vehicle (0.5% MC) – 565.9 ± 63.58 – –
GT29 5 465.7 ± 42.74 17.7 25.28 (10.48–61.01)
30 289.0 ± 22.7*** 48.9
60 154.0 ± 22.83**** 72.8
Values shown as the mean ± SEM for n = 8. Compounds administered intraperitoneally. MC – methylcellulose. Results shown regard the 30th min of the observation period. Statistical analysis: one way ANOVA, followed by Dunnett’s multiple comparison test. (*) indicates a significant difference compared to the vehicle-treated group: *** p < 0.01, **** p < 0.001
Tab. 3. Analgesic activity of the compounds in the hot plate test
Compound Dose
(mg/kg)
Nociceptive reaction latency (mean ± SEM) in (s) measured at various time-points
Baseline time reaction 30 min 60 min 90 min
BM128 25 10.2 ± 0.59 10.44 ± 1.35 11.26 ± 1.23 7.2 ± 0.6***
%MPE – 1.21 5.35 –
GT27 25 8.39 ± 0.62 9.16 ± 0.94 9.92 ± 0.82 9.78 ± 0.6
%MPE – 3.56 7.13 6.43
GT28 25 7.67 ± 0.59 11.45 ± 1.37* 12.01 ± 1.54* 9.14 ± 0.64
%MPE – 16.93 19.44 6.58
GT29 25 7.42 ± 0.77 12.55 ± 1.22*** 11.08 ± 2.23 8.72 ± 1.02
%MPE – 22.72 16.21 5.76
Values shown as the mean ± SEM for n = 8. Compounds administered intraperitoneally. Statistical analysis: one-way ANOVA, followed by Dun- nett’s test; (*) indicates a significant difference compared to the control value of effect latency: * p < 0.05, *** p < 0.01
threshold as compared to the vehicle-treated animals.
GT27 (100 and 200 mg/kg), GT29 (200 mg/kg) and BM128 (100 mg/kg) were the most potent compounds in this respect as they were able to increase the CS50 value to 16.36; 17.82; 15.73 and 16.78 mA, respec- tively. GT29 at 100 mg/kg did not influence the sei- zure threshold in this test (CS50= 7.1 mA). The data from the experiment are demonstrated in Table 4.
Chimney test
In Table 5 the influence of the investigated com- pounds on motor coordination is presented. Thirteen percent of vehicle-treated mice showed impaired mo- tor coordination in the chimney test. The investigated compounds disturbed the motor coordination to vari- ous extent. GT28 (100 and 200 mg/kg) and GT29 (100 mg/kg) prevented 63% and GT29 (200 mg/kg) 75% of tested animals from getting out of the chim- ney during the observation time. Both tested doses of BM128 influenced the animals’ motor coordination to a lesser degree. Only in case of GT29 (200 mg/kg) the results reached statistical significance (p < 0.05).
Tab. 4. Current strength values for tonic hind limb extension in 50% of mice (CS50) in the ECT
Compound Dose (mg/kg) CS50(mA) SE DT (mA) Threshold increase (%)
Vehicle (0.5% MC) – 7.06 (4.69–9.67) 1.27 – –
BM128 100 16.78 (9.93–25.19) **** 3.89 9.72 138
200 11.29 (10.64–11.95) *** 0.33 4.23 60
GT27 100 16.36 (14.14–18.72) **** 1.17 9.30 132
200 17.82 (13.9–22.20) 2.12 10.76 152
GT28 100 11.66 (10.33–13.06) *** 0.70 4.60 65
200 11.24 (10.14–12.38) *** 0.57 4.18 59
GT29 100 7.1 (4.26–10.28) 1.54 0.04 0.6
200 15.73 (12.95–18.76) **** 1.48 8.67 123
Electroconvulsions produced by an alternating current (duration of the stimulus: 0.2 s; 50 Hz) delivered via standard auricular electrodes. Four groups of mice per one dose (n = 5–9 animals) challenged with electroshocks of various intensities. MC = methylcellulose. Data analyzed by log-probit (Litchfield-Wilcoxon) method.Results presented as median current strengths (CS50in mA with 95% confidence limits in parentheses) required to evoke tonic hind limb extension in 50% of mice tested. In order to facilitate statistical analysis, the 95% confidence limits were trans- formed to standard errors (SE). The log-probit method of Litchfield-Wilcoxon [26] and analysis of variance (ANOVA) followed by the post-hoc Dunnett’s multiple comparisons test were used to provide the appropriate statistical evaluation of the data [30]; *** means significantly different at p < 0.01, **** means significantly different at p < 0.001 vs. vehicle-treated mice. The threshold for vehicle-treated animals was set as a base- line value for calculations of percentage in the threshold increase following the investigated compounds administration. DT – threshold in- crease calculated by subtracting the threshold value of control animals from the threshold value of the drug-treated mice
Tab. 5. Influence of the compounds on the motor coordination in the chimney test
Compound Dose (mg/kg) Percent of animals with motor impairment
Vehicle (0.5% MC) – 12.5
GT27 100 25
200 50
GT28 100 62.5
200 62.5
GT29 100 62.5
200 75*
BM128 100 12.5
200 37.5
Values shown as percent of animals with motor impairment (n = 8).
Compounds administered intraperitoneally. MC – methylcellulose.
Statistical analysis: Fisher’s exact probability test; * means significant at p < 0.05
Discussion
GHB is an endogenous substance, which is thought to be a neurotransmitter or a neuromodulator in the mammalian central nervous system [3, 8, 20, 21, 43].
Several reports emphasize that it may be either a me- tabolite of GABA or its precursor [8, 43]. The biologi- cal and toxicological role of GHB has been well established for many years. Its application in the treat- ment of narcolepsy (cataplexy), sleep and wakefulness regulating properties, its limited use in anesthesia and alcohol dependence as well as recreational abuse due to its euphorigenic and amnestic properties are also widely known [8, 21, 43]. Under experimental condi- tions GHB is used for the induction of absence sei- zures in rodents [8].
In our earlier search for new anticonvulsants a group of GHB derivatives was designed [24]. In vitro studies performed by Kulig et al. [24] proved their affinity for mGAT1–mGAT4. Noteworthy, in the study performed as [3H]GABA uptake assay based on stably transfected HEK cells one of such compounds, namely BM128, was found by these authors to inhibit the GABA uptake potently with slight subtype-selectivity towards mGAT1 (the IC50 values ranged between 8.7–27.54 µM for mGAT1–mGAT4) (see Tab. 1).
In the present paper, the pharmacological activity of four new 2-substituted 4-hydroxybutanamides in several behavioral tests (locomotor activity, hot plate, electroconvulsive threshold and chimney tests) is evaluated. The results obtained in these experiments indicate that the biological activity of the investigated compounds can at least partially be attributed to their previously reported [24] affinity towards GAT.
Many compounds, which have different inhibitory potencies towards mGAT1–mGAT4, are either used as anticonvulsant agents (e.g., tiagabine) or tools served for the research on pharmacological properties of different GAT types. Tiagabine, for instance, is highly selective towards mGAT1 (IC50 = 0.8 µM);
SNAP-5114 is modestly selective towards mGAT4 (IC50= 6.6 µM) but has also affinity for mGAT2 (IC50
= 22 µM) and mGAT3 (IC50 = 20 µM), whereas (S)-EF1502 is mGAT2 inhibitor (IC50= 34 µM) [34].
Another compound, NNC-005 2090 is relatively se- lective towards mGAT2 (IC50= 1.4 µM) having some affinity for mGAT3 (IC50 = 41 µM), mGAT1 and mGAT4 (IC50 = 19 µM and IC50 = 15 µM, respec- tively) [7, 33–35, 41, 47, 48, 54]. Similarly to these
agents, the investigated GHB derivatives influenced in a non-selective manner the activity of all the four types of murine GAT (Tab. 1) [22, 24].
Considering the pharmacological action of the tested compounds in behavioral tests it is necessary to take the role of individual GAT types, their localiza- tion and abundance into account. GAT are heteroge- neously expressed in different brain regions. mGAT1 and mGAT4 are primarily situated on neurons and as- troglial cells (mGAT1) or preferentially in glial cells (mGAT4) [34, 47]. GAT1 is the most abundant of the GABA transporters being expressed in all brain re- gions [12, 34, 47]. Therefore, it seems to be the most promising drug target among the plasma membrane GABA transporters [47]. The localization of mGAT2 and mGAT3 is more diverse; the latter is preferen- tially located on non-neuronal elements [10, 34, 47, 49]. However, since in adult mice the expression of mGAT3 almost exclusively regards the brain menin- ges, the role of this transporter in regulating GABA concentration within the synapses is rather improb- able [12]. Therefore, despite the fact that in the previ- ous studies [24] the investigated compounds, espe- cially BM128, demonstrated high affinity for this GAT type, it does not seem to contribute to the com- pounds’ pharmacological efficacy.
Sedation in humans [5, 35], hypolocomotion and impaired motor coordination in rodents reported for tiagabine are attributed to its influence on mGAT1 [4, 5, 17]. The decreased locomotor activity was also ob- served after ip administration of each of the investi- gated GHB derivatives and, as it is suggested, this ef- fect can be attributed to the compounds’ affinity for mGAT1. Their ED50values obtained in the locomotor activity test were similar (approx. 25 mg/kg), so were their previously established [24] IC50 values for mGAT1 (the lowest for BM128) (Tab. 1). On the other hand, the hypolocomotion induced by the tested compounds can also be explained in another way.
There are some reports demonstrating that GHB- induced alterations in the locomotor activity in rodents are due to its affinity for GABAB receptors [20, 42].
The investigated compounds as GHB derivatives may exert such an effect. However, to elucidate the problem further studies evaluating the compounds’ affinity for GABABmetabotropic receptor are necessary.
The analgesic efficacy of the GHB derivatives was evaluated because the GABA-ergic transmission plays a key role in the inhibitory regulation of the no- ciceptive process, especially within the dorsal horn of
108 Pharmacological Reports, 2012, 64, 102112
the spinal cord [50]. Many GABA-ergic drugs, are antinociceptive in the hot plate model [17, 25, 29, 32, 50]. The analgesic effect observed here is found to be a consequence of supraspinal attenuation of ascending nociceptive input [16, 40]. GAT1 inhibition is re- ported to attenuate excitatory amino acids neurotrans- mission, which results in the antinociceptive effect at the spinal cord level [2]. This activity is thought to in- volve GABABreceptors. Several reports indicate that tiagabine is analgesic in acute (hot plate, tail immer- sion, grid-shock) and chronic pain models (formalin test, dynorphin-induced allodynia) in rodents [17, 25, 29, 31, 32, 50]. This effect is suggested to be a conse- quence of enhanced GABA-ergic transmission and subsequent GABABreceptor activation [17, 50]. The involvement of both GABA receptors (A and B) in tiagabine-induced analgesia in the formalin test has also been reported [25]. Despite the compounds’
chemical similarity to GABA and their affinity for mGAT1 their central antinociceptive effect was not observed in the hot plate model. It is a very well- known fact that the results from the hot plate test should be interpreted with caution, especially in case of agents with sedative, psychotomimetic and myore- laxant properties. Such compounds may present falsely positive effects in this assay. Due to the hy- polocomotion observed in the locomotor activity test the dose 25 mg/kg (ip) was tested in the hot plate model as the first one. At this dose, the investigated compounds exerted weak analgesic activity – the
%MPE value measured at three points of time (30, 60 and 90 min after the intraperitoneal injection) ranged from 1.21% (BM128; 30 min after the injection) to 22.72% (GT29; at the same time point).
The elevation of the electroconvulsive threshold was observed after the ip administration of each of the four GHB derivatives (with no effect of GT29 at 100 mg/kg). As shown earlier [24], the compounds are almost equipotent at mGAT types (with maximum a three-fold selectivity). Therefore, their activity in the ECT test is likely to be a consequence of com- bined inhibition of mGAT1, mGAT2 and mGAT4.
Of note, the inhibition of mGAT1 might be the most important since it is the most abundant GAT followed by mGAT4. The anticonvulsant role of tiagabine, a pure mGAT1 inhibitor, is very well established. In preclinical studies it is effective in those seizure mod- els that originate from cortex and limbic system, but not from hindbrain or brainstem. This corresponds with the GAT1 expression in the brain [5, 12, 13, 41].
Thus, tiagabine protects against kindled seizures (ED50 = 36 µmol/kg for inhibiting kindled focal sei- zures), but is only weakly efficacious (ED50 > 73 µmol/kg) in inhibiting tonic maximal electroshock (MES) seizures originated in the brainstem. In the MES test this drug is reported to be active only at doses two to three-fold higher than doses producing motor impairment (40 mg/kg, ip) [6, 9, 31]. Moreo- ver, as other GABA-ergic drugs, it may exacerbate absence-epilepsy in rodents and humans [10, 12, 29, 41]. In contrast to the MES test, tiagabine is effective in the ECT model in mice [6, 29, 31]. This experimen- tal model corresponds to partial tonic-clonic seizures in men and is much more sensitive to anticonvulsant agents than the standard MES [28, 29, 31].
The physiological role of compounds targeting at GAT other than GAT1 has been poorly established.
Several reports suggest that non-GAT1 inhibitors of GABA transport, especially agents acting at mGAT2 and mGAT4, are very interesting as potential pros- pects for the future epilepsy treatment [34]. Such compounds (e.g., (S)-EF1502 or SNAP-5114) have been shown to possess a broad spectrum of anticon- vulsant activity under experimental conditions in vari- ous models of epilepsy in rodents (e.g., audiogenic seizures in DBA/2 mice, pentylenetetrazole in mice and rats) [7, 12, 13, 33, 54]. Noteworthy, EF1502 ex- erts synergistic anticonvulsant activity administered together with tiagabine without enhancing its behavioral toxicity [33, 34, 54]. SNAP-5114, a semi-selective in- hibitor of mGAT4, is effective in several models of epi- lepsy [35] and it was found to produce a synergistic anti- convulsant effect co-administered with EF1502 but not when it was used with tiagabine [33].
In the chimney test the investigated compounds disturbed motor coordination to various extent (12.5–75%). This effect seems to result from their GABA level increasing properties. The inhibition of GABA uptake increases its concentration in the syn- aptic cleft. This effect may be responsible not only for sedation observed after ip administration of the GHB derivatives in the locomotor activity test but it also explains the behavioral changes in the chimney test (motor impairments, inability to climb the cylinder).
Concluding, the application of tiagabine as an add-on drug for treatment of partial epilepsy is sometimes limited because of its adverse effects which are partly due to its GAT1 inhibitory action. Successful devel- opment of drugs targeting at GABA transporters other than GAT1, especially mGAT2, may provide benefit
and be more advantageous, especially in terms of in- tractable epilepsy treatment.
At present, tiagabine is the only one pure GAT1 in- hibitor used as an antiepileptic compound. Non- selective GABA uptake inhibitors possess several in- teresting properties which can be found useful in the treatment of certain central nervous system disorders.
Conceivably, recent data emphasize that the influence on non-GAT1 transporters may be of special value for the future treatment of epileptic patients. For the time being, the discussion of non-selective vs. selective GAT inhibitors is rather difficult and should be taken with great caution. Non-selective compounds could potentially have a wider spectrum of treatment oppor- tunities, however, this also could be at the expense of greater adverse effects. Therefore, the therapeutic po- tential of the tested GHB analogues should be investi- gated in greater detail. As far as the investigated GHB derivatives are concerned, their potential anticonvul- sant, antidepressant and anxiolytic-like effects are possible. We suggest further research of these com- pounds to elucidate their biological properties. The af- finity to putative GHB or GABAB receptors should also be evaluated.
Acknowledgment:
This experimental work has been supported by the Jagiellonian University grant K/ZBW/000603.
References:
1.Boissier JR, Tardy J, Divierres JC: A new simple method to explore the tranquilizing action: the chimney test (French). Med Exp, 1960, 3, 81–84.
2.Bowery NG, Smart TG: GABA and glycine as neuro- transmitters: a brief history. Br J Pharmacol, 2006, 147, S109–S119.
3.Carai MAM, Lobina C, Maccioni P, Cabras C, Colombo G, Gessa GL: g-Aminobutyric acidB(GABAB)-receptor mediation of different in vivo effects of g-butyrolactone.
J Pharmacol Sci, 2008, 106, 199–207.
4.Castelli MP: Multi-faceted aspects of gamma-hydroxybutyric acid: a neurotransmitter, therapeutic agent and drug of abuse. Mini Rev Med Chem, 2008, 8, 1188–1202.
5.Chiu CS, Brickley S, Jensen K, Southwell A, McKinney S, Cull-Candy S, Mody I, Lester HA: GABA transporter deficiency causes tremor, ataxia, nervousness, and in- creased GABA-induced tonic conductance in cerebel- lum. J Neurosci, 2005, 25, 3234–3245.
6.Chroœciñska-Krawczyk M, Ratnaraj N, Patsalos PN, Czuczwar SJ: Effect of caffeine on anticonvulsant effects
of oxcarbazepin, lamotrigine and tiagabine in a mouse model of generalized tonic-clonic seizures. Pharmacol Rep, 2009, 61, 819–826.
7.Clausen RP, Frølund B, Larsson OM, Schousboe A, Krogsgaard-Larsen P, White HS: A novel selective g-aminobutyric acid transport inhibitor demonstrated a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS. Neurochem Int, 2006, 48, 637–642.
8.Crunelli V, Emri Z, Leresche N: Unravelling the brain targets of g-hydroxybutyric acid. Curr Opin Pharmacol, 2006, 6, 44–52.
9.Czapinski P, Blaszczyk B, Czuczwar SJ: Mechanisms of action of antiepileptic drugs. Curr Top Med Chem, 2005, 5, 3–14.
10.Czuczwar SJ, Patsalos PN: The new generation of GABA enhancers. Potential in the treatment of epilepsy.
CNS Drugs, 2001, 15, 339–350.
11.Dalby NO: GABA-level increasing and anticonvulsant effects of three different GABA uptake inhibitors. Neu- ropharmacology, 2000, 39, 2399–2407.
12.Dalby NO: Inhibition of g-aminobutyric acid uptake:
anatomy, physiology and effects against epileptic sei- zures. Eur J Pharmacol, 2003, 479, 127–137.
13.Dalby NO, Thomsen Ch, Fink-Jensen A, Lundbeck J, Sokilde B, Man CM, Sorensen PO, Meldrum B: Anti- convulsant properties of two GABA uptake inhibitors NNC 05-2045 and NNC 05-2090, not acting preferen- tially on GAT-1. Epilepsy Res, 1997, 28, 51–61.
14.Dudra-Jastrzebska M, Andres-Mach MM, Sielski M, Ratnaraj N, Patsalos PN, Czuczwar SJ, £uszczki JJ:
Pharmacodynamic and pharmacokinetic interaction pro- file of levetiracetam in combination with gabapentin, tiagabine and vigabatrin in the mouse pentylenetetra- zole-induced seizure model: an isobolographic analysis.
Eur J Pharmacol, 2009, 605, 87–94.
15.Eddy N, Leimbach D: Synthetic analgesics. II. Dithien- ylbutenyl- and dithienylbutylamines. J Pharmacol Exp Ther, 1953, 107, 385–393.
16.Hough LB, Nalwalk JW, Stadel R, Timmerman H, Leurs R, Paria BC, Wang X, Dey SK: Inhibition of im- progan antinociception by the cannabinoid CB1 antago- nist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-di- chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A): Lack of obligatory role for endocannabi- noids acting at CB1 receptors. J Pharmacol Exp Ther, 2002, 303, 314–322.
17.Ipponi A, Lamberti C, Medica A, Bartolini A, Malmberg-Aiello P: Tiagabine antinociception in rodents depends on GABABreceptor activation:
parallel antinociception testing and medial thalamus GABA microdialysis. Eur J Pharmacol, 1999, 368, 205–211.
18.Iversen L: Neurotransmitter transporters and their impact on the development of psychopharmacology.
Br J Pharmacol, 2006, 147, S82–S88.
19.Jasmin L, Wu MV, Ohara PT: GABA puts a stop to pain.
Curr Drugs Targets CNS Neurol Disord, 2004, 3, 487–505.
20.Kaupmann K, Cryan JF, Wellendorph P, Mombereau C, Sansig G, Klebs K, Schmutz M et al.: Specific g-hy- droxybutyrate-binding sites but loss of pharmacological
110 Pharmacological Reports, 2012, 64, 102112
effects of g-hydroxybutyrate in GABAB(1)- deficient mice. Eur J Neurosci, 2003, 18, 2722–2730.
21.Koek W, Mercer SL, Coop A: Cataleptic effects of g-hydroxybutyrate (GHB), its precursor g-butyrolactone (GBL), and GABABreceptor agonist in mice: differen- tial antagonism by the GABABreceptor antagonist CGP35348. Psychopharmacology, 2007, 192, 407–414.
22.Kragler A, Höfner GC, Wanner KT: Synthesis and bio- logical evaluation of aminomethylphenol derivatives as inhibitors of the murine GABA transporters mGAT1- mGAT4. Eur J Med Chem, 2008, 43, 2404–2411.
23.Kulig K, Szwaczkiewicz M: The role of structure activity relationship studies in the search for new GABA uptake inhibitors. Mini Rev Med Chem, 2008, 8, 1214–1223.
24.Kulig K, Wieckowski K, Wieckowska A, Gajda J, Pochwat B, Höfner GC, Wanner KT, Malawska B:
Synthesis and biological evaluation of the new amides of 2-substituted 4-hydroxybutyric acid derivatives as GABA uptake inhibitors. Eur J Med Chem, 2011, 46, 183–190.
25.Laughlin TM, Tram KV, Wilcox GL, Birnbaum AK:
Comparison of antiepileptic drugs tiagabine, lamotrigine, and gabapentin in mouse models of acute, prolonged and chronic nociception. J Pharmacol Exp Ther, 2002, 302, 1168–1175.
26.Litchfield JT, Wilcoxon E: A simplified method of evalu- ating dose-effect experiments. J Pharmacol Exp Ther, 1949, 96, 99–113.
27.Liu GX, Cai GQ, Cai YQ, Sheng ZJ, Jiang J, Mei Z, Wang ZG et al.: Reduced anxiety and depression-like behaviors in mice lacking GABA transporter subtype I.
Neuropsychopharmacology, 2007, 32, 1531–1539.
28.Loescher W, Fassbender CP, Nolting B: The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs.
II. Maximal electroshock seizure models. Epilepsy Res, 1991, 8, 79–94.
29.Luszczki JJ, Czuczwar SJ: Isobolographic characteriza- tion of interactions between vigabatrin and tiagabine in two experimental models of epilepsy. Prog Neuropsy- chopharmacol Biol Psychiatry, 2007, 31, 529–538.
30.Luszczki JJ, Ratnaraj N, Patsalos P, Czuczwar SJ: Isobo- lographic analysis of interactions between loreclezole and conventional antiepileptic drugs in the mouse maxi- mal electroshock-induced model. Naunyn Schmiede- bergs Arch Pharmacol, 2006, 373, 169–181.
31.Luszczki JJ, Swiader M, Parada-Turska J, Czuczwar SJ:
Tiagabine synergistically interacts with gabapentin in the electroconvulsive threshold test in mice. Neuropsycho- pharmacology, 2003, 28, 1817–1830.
32.£uszczki JJ, Ko³acz A, Wojda E, Czuczwar M, Przesmycki K, Czuczwar SJ: Synergistic interaction of gabapentin with tiagabine in the hot-plate test in mice:
an isobolographic analysis. Pharmacol Rep, 2009, 61, 459–467.
33.Madsen KK, Clausen RP, Larsson OM, Krogsgaard- Larsen P, Schousboe A, White HS: Synaptic and extra- synaptic GABA transporters as targets for anti-epileptic drugs. J Neurochem, 2009, 109, 139–144.
34.Madsen K, White HS, Clausen RP, Frølund B, Larsson OM, Krogsgaard-Larsen P, Schousboe A: Functional and
pharmacological aspects of GABA transporters. Hand- book of Neurochemistry and Molecular Neurobiology.
Neural Membranes and Transport. Springer Science + Business Media, 3rd ed. Plenum, New York, 2007.
35.Madsen KK, White HS, Schousboe A: Neuronal and non-neuronal GABA transporters as targets for antiepi- leptic drugs. Pharmacol Ther, 2010, 125, 394–401.
36.Magnaghi V: GABA and neuroactive steroid interactions in glia: new roles for old players? Curr Neuropharmacol, 2007, 5, 47–64.
37.Maitre M: The gamma-hydroxybutyrate signalling sys- tem in brain: organization and functional implications.
Prog Neurobiol, 1997, 51, 337–361.
38.Malawska B, Kulig K, Gajda J, Szczeblewski D, Musia³ A, Wiêckowski K, Stables JP: Design, synthesis and pharmacological evaluation of a-substituted N-benzyl- amides of g-hydroxybutyric acid with potential GABA- ergic activity. Part 6. Search for new anticonvulsant com- pounds. Acta Pol Pharm Drug Res, 2007, 64, 127–137.
39.Malawska B, Kulig K, Œpiewak A, Stables JP: Investiga- tion into new anticonvulsant derivatives of a-substituted N-benzylamides of g-hydroxy- and g-acetoxybutyric acid. Part 5. Search for new anticonvulsant compounds.
Bioorg Med Chem, 2004, 12, 625–632.
40.Manzanares J, Julian MD, Carrascosa A: Role of the cannabinoid system in pain control and therapeutic implications for the management of acute and chronic pain episodes. Curr Neuropharmacol, 2006, 4, 239–257.
41.Meldrum BS, Rogawski MA: Molecular targets for antiepileptic drug development. Neurotherapeutics, 2007, 4, 18–61.
42.Nissbrandt H, Engberg G: The GABAB-receptor antago- nist, CGP35348, antagonises g-hydroxybutyrate- and baclofen-induced alterations in locomotor activity and forebrain dopamine levels in mice. J Neural Transm, 1996, 103, 1255–1263.
43.Pardi D, Black J: g-Hydroxybutyrate/sodium oxybate.
Neurobiology, and impact on sleep and wakefulness.
CNS Drugs, 2006, 20, 993–1018.
44.Perucca E: An introduction to antiepileptic drugs.
Epilepsia, 2005, 46, 31–37.
45.Rogawski MA: Molecular targets versus models for new antiepileptic drug discovery. Epilepsy Res, 2006, 68, 22–28.
46.Salat K, Kulig K: GABA transporters as targets for new drugs. Fut Med Chem, 2011, 3, 211–222.
47.Sarup A, Larsson OM, Schousboe A: GABA transporters and GABA-transaminase as drug targets. Curr Drug Targets CNS Neurol Disord, 2003, 2, 269–277.
48.Schousboe A, Larsson OM, Sarup A, White HS: Role of the betaine/GABA transporter (BGT-1/GAT2) for the con- trol of epilepsy. Eur J Pharmacol, 2004, 500, 281–287.
49.Schousboe A, Sarup A, Larsson OM, White HS:
GABA transporters as drug targets for modulation of GABAergic activity. Biochem Pharmacol, 2004, 68, 1557–1563.
50.Smith CGS, Bowery NG, Whitehead KJ: GABA trans- porter type 1 (GAT-1) uptake inhibition reduces stimu- lated aspartate and glutamate release in the dorsal spinal cord in vivo via different GABAergic mechanisms.
Neuropharmacology, 2007, 53, 975–981.
51.Thoeringer CK, Erhardt A, Sillaber I, Mueller MB, Ohl F, Holsboer F, Keck ME: Long-term anxiolytic and antidepressant-like behavioural effects of tiagabine, a selective GABA transporter-1 (GAT-1) inhibitor, coin- cide with a decrease in HPA system activity in C57BL/6 mice. J Pharmacol, 2010, 24, 733–743.
52.Thoeringer CK, Ripke S, Unschuld PG, Lucae S, Ising M, Bettecken T, Uhr M et al.: The GABA trans- porter 1 (SLC6A1): a novel candidate gene for anxiety disorders. J Neural Transm, 2009, 116, 649–657.
53.Vogel HG, Vogel WH: Drug discovery and evaluation.
Pharmacological assays, Springer-Verlag Inc., New York, 1997.
54.White HS, Watson WP, Hansen SL, Slough S,
Perregaard J, Sarup A, Bolvig T et al.: First demonstra- tion of a functional role for central nervous system be- taine/g-aminobutyric acid transporter (mGAT2) based on synergistic anticonvulsant action among inhibitors of mGAT1 and mGAT2. J Pharmacol Exp Ther, 2005, 312, 866–874.
Received: April 16, 2011; in the revised form: August 17, 2011;
accepted: September 15, 2011.
112 Pharmacological Reports, 2012, 64, 102112
