Copyright © 2002 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2002, 54, 731–736 ISSN 1230-6002

PRELIMINARY COMMUNICATION

INFLUENCE OF NEW g-HYDROXYBUTYRIC ACID AMIDE ANALOGUES ON THE CENTRAL NERVOUS SYSTEM ACTIVITY IN MICE

Kinga Sa³at, Anna Mendyk, Tadeusz Librowski, Ryszard Czarnecki, Barbara Malawska* Department of Pharmacodynamics, *Department of Pharmaceutical Chemistry, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland

Influence of new g-hydroxybutyric acid amide analogues on the central nervous sys- tem activity in mice. K. SA£AT, A. MENDYK, T. LIBROWSKI, R. CZARNECKI, B. MALAWSKA. Pol. J. Pharmacol., 2002, 54, 731–736. The present study investigates the activity of four g-hydroxybutyric acid amide ana- logues (BM-68, BM-74, BM-75 and BM-76) in two models of chemically induced sei- zures, i.e. picrotoxin- and pentetrazole-induced seizures and in the thiopental-induced sleep test. The results of pharmacological in vivo experiments with the g-hydroxybutyric acid amide analogues presented below show that the compounds possess variable influ- ence on the central nervous system in mice. Key words: Gamma-hydroxybutyric acid, picrotoxin, pentetrazole, thiopental-induced sleep, mice

INTRODUCTION body tissues with the highest concentration in the mammalian brains. Its role as a possible neuro- g-Hydroxybutyric acid (GHB) is a structural transmitter is still being evaluated. It is involved in analogue of g-aminobutyric acid (GABA). It is the regulation of GABA, dopamine, 5-hydroxy- a naturally occuring short-chain fatty acid con- tryptamine and acetylcholine release. Research in- sidered as a metabolite of GABA able to pass the dicates that GHB produces deep reversible depres- blood-brain barrier [10, 16]. GHB is found in all sion of cerebral metabolism, increases dopamine

 correspondence; e-mail: [email protected] K. Sa³at, A. Mendyk, T. Librowski, R. Czarnecki, B. Malawska concentrations and induces hypothermia. The pre- acid (BM-74), N-(4-fluorbenzylamide) of a-(2- cise function and metabolic pathways of GHB in phenylethylamine)-g-hydroxybutyric acid (BM-68), the brain are complex and not yet fully understood N-benzylamide of a-(2-phenylethylamine)-g-hy- [16, 17]. The synthesis of GHB involves GABA or droxybutyric acid (BM-75) and N-(3,4-dimethoxy- 1,4-butanediol as precursor substances. GHB can benzamide) of a-(2-phenylethylamine)-g-hydroxy- also be synthesized in the mammalian liver from butyric acid (BM-76). g-butyrolactone [10, 11, 15]. Preliminary anticonvulsant evaluation of these The primary effect of GHB consists in the CNS compounds, synthesized at the Department of Phar- depression, and for this reason it was earlier used maceutical Chemistry, Jagiellonian University, for anesthesia. GHB causes a trancelike state that Medical College, Kraków, Poland, provided by the mimics physiological sleep. It may also protect the Antiepileptic Drug Development Program of the CNS from injury during hypoxic episodes, hiberna- National Institute of Neurological Disorders and tion and states of increased metabolic demands. Stroke in Bethesda, USA, included maximal elec- There is also some evidence that GHB has epilepti- troshock(MES)-induced seizures. It showed that form activity resembling petit mal epilepsy [6, 16]. these substances, especially BM-74, afforded pro- Besides, the available data suggest the possible role tection against MES. Neurotoxicity of these sub- of GHB in the management of sleep disorders such stances was assessed in the rota-rod test [4, 6]. as narcolepsy [10, 16], night eating syndrome [8], Taking these facts into account, we attempted to as well as in the treatment of alcohol dependence investigate the activity of BM-68, BM-74, BM-75 because it is able to substitute for ethanol during and BM-76 in two models of chemically induced ethanol withdrawal [1, 2, 10, 16]. seizures (picrotoxin, pentetrazole) and in the GHB has the ability to induce absence seizures. thiopental-induced sleep test. All the experiments The precise way by which GHB causes seizures re- were carried out on mice 45 min after oral admini- mains unclear, though GABA-B receptors and/or stration of the compounds or the vehicle. GHB-mediated presynaptic mechanisms within the thalamocortical circuitry may play a role [3]. Some MATERIALS and METHODS data suggest that GHB induces a selective decrease in the basal and depolarization-induced release of Animals GABA in the cerebral cortex, and further, that this The experiments were carried out on male Al- action of GHB may be an important mechanism by bino Swiss mice (18–24 g). The animals were kept which GHB causes absence seizures [3, 13]. So GHB in groups of 15 mice in type III-1290 cages (26.5 × seems to be a useful compound in animal experi- 42.0 × 15.0 cm) at a room temperature of 22 ± 2°C, ments producing characteristic behavioral changes under 12/12 h light/dark cycle (light on from 7 a.m. that respond selectively to antiabsence antiepileptic to 7 p.m.), and had free access to food (standard drugs such as ethosuximide, as well as influencing laboratory pellets; Bacutil, Motycz, Poland) and the EEG recordings [6, 12]. There is also a possi- water before the experiments. Each experimental bility to use GHB/GABA-B receptor antagonists as group consisted of 6–12 animals/dose and all the drugs in the treatment of this type of seizures in the animals were used only once. The experiments future [5]. were performed between 8 a.m. and 3 p.m. Because of the importance of GABA and GHB as endogenous substances playing a significant role Drugs in the regulation of some physiological and patho- BM-68, BM-74, BM-75 and BM-76 (synthesized logical processes in the mammalian CNS, we car- at the Department of Pharmaceutical Chemistry, ried out experiments to establish the possible in- Jagiellonian University, Medical College, Kraków, fluence of some new GHB amide analogues on the Poland) were given to animals in a form of a sus- central nervous system activity in mice. In our pension in 0.5% methylcellulose (Loba-Chemie, search for new chemical moieties which could act Germany). Picrotoxin (Fluka Chemie AG, Germany), as effective anticonvulsants, we carried out investi- pentetrazole (Cefarm, Poland), thiopental (Bioche- gations on four GHB amide analogues, i.e.: N-ben- mie GmbH, Germany) and ethosuximide (Ronton, zylamide of a-(N-benzylamine)-g-hydroxybutyric ICN-Polfa Rzeszów, Poland) were dissolved in

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0.9% sodium chloride (Rhone-Poulenc Rorer, Fran- 45 min before the administration of thiopental. ce). Diazepam (Valium, Roche, France) was used as Thiopental, a short acting anesthetic, was adminis- a 1% water solution. Control animals were given tered ip (60 mg/kg). The animals were placed on appropriate amounts of vehicle. their backs on warmed (33°C) pad and the duration of loss of the righting reflex was measured until Neurotoxicity they regained it. If there was any doubt as to the re- Neurotoxicity of the investigated compounds appearance of the righting reflex, the mouse was was assessed by means of the chimney test. This placed gently on its back again and, if it righted it- test was carried out 45 min after oral administration self within one minute, this time was considered as of the compounds given to mice at three doses: 30, the endpoint. Mean values of duration of anesthesia 100 and 300 mg/kg. The animals were previously were recorded in control and experimental groups. trained and selected. Then they were placed in The time necessary for the animals to fall asleep a 25 cm long and 3 cm in diameter horizontally (sleep latency) was recorded, as well. located tube which was reversed in such a way that Statistical analysis the mice were able to leave it only climbing back- ward up until they reached another end. The ability The data are expressed as means ± SEM. Stu- of the mice to leave the tube within 1 min indicated dent’s t-test was used to determine the significance the lack of neutotoxic properties of the investigated of differences between mean values of control and compounds [9]. treatment groups (this applies to the thiopental- induced sleep). We also used this test to estimate Picrotoxin-induced seizures the period of time after which the first pentetrazole- and picrotoxin-induced convulsions appeared. To Groups of 6–8 mice were treated orally with determine the significance of differences between the investigated compounds prepared in a form of mean values of the number of convulsions in con- a suspension in 0.5% methylcellulose solution. trol and treatment groups we used the U-Mann- A convulsant dose of picrotoxin (3.2 mg/kg) (ED ) 97 Whitney test. Differences were considered signifi- [14] was injected subcutaneously (sc) into mice cant when p < 0.05. 45 min after the tested compound had been admi- nistered orally. The animals were placed individu- ally in cages and observed for the next 90 min. RESULTS and DISCUSSION Time of onset of seizures, the number of attacks and mortality were assessed. Our investigations on the amide analogues of GHB: BM-68, BM-74, BM-75, BM-76 have Pentetrazole-induced convulsions proven that the compounds do affect CNS in mice. The chimney test showed that the compounds were Groups of 8 mice were treated orally with the not neurotoxic to mice, at least in the range of investigated compounds prepared in a form of doses tested. All the animals were able to leave the a suspension in 0.5% methylcellulose solution. tube in the period of time shorter than 1 min. Dia- A convulsant dose of pentetrazole (70 mg/kg) was zepam which was used as a reference in this test, injected intraperitoneally (ip) into mice 45 min injected ip at a dose of 10 mg/kg prevented 50% of after the tested compound had been administered. animals from leaving the tube within 1 min (ED50). The animals were placed individually in cages and As regards the picrotoxin-induced convulsions, observed for the next 30 min. The absence of sei- all the compounds delayed the onset of seizures in zures showed the ability of the compound to ele- comparison with the control group of animals that vate the seizure threshold. Time of onset of sei- received only the vehicle. Only BM-74 at a dose of zures, the number of attacks and mortality were as- 300 mg/kg accelerated the first convulsive attack. sessed. BM-76 was the most active compound in delaying the onset of seizures. Thiopental-induced sleep The data on the number of attacks are more di- Groups of 10 mice were used in this test. The verse. The lowest dose of all the compounds (30 investigated compounds were given orally in a form mg/kg) decreased the number of the attacks. Higher of a suspension in 0.5% methylcellulose solution doses acted variously: either increased it (BM-68

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Table 1. The influence of the investigated compounds on the picrotoxin-induced seizures in mice

Compound Dose Time of onset of seizures [min] Number of seizures Mortality in the group (mg/kg) (mean ± SEM) (mean ± SEM) (%)

Control – 12.8 ± 0.72.0 0 BM 68 30 13.3 ± 1.3 1.8 ± 0.3 0 100 15.0 ± 1.2 2.17± 0.2 16.67 300 14.2 ± 1.1 2.6 ± 0.2* 20

Control – 18.17± 0.9 2.0 ± 0.3 0 BM 74 30 22.0 ± 2.8 1.6 ± 0.2 0 100 20.0 ± 3.1 2.3 ± 0.5 33.33 300 16.2 ± 0.6 2.0 ± 0.3 0

Control – 12.2 ± 1.0 2.3 ± 0.4 50 BM 75 30 12.4 ± 0.7 2.0 ± 0.4 20 100 14.4 ± 1.9 2.8 ± 0.4 0 300 13.2 ± 2.5 2.0 ± 0.3 33.33

Control – 12.4 ± 0.9 2.6 ± 0.4 0 BM 76 30 17.6 ± 1.9* 2.4 ± 0.5 0 100 16.0 ± 3.9 2.2 ± 0.4 20 300 13.8 ± 0.8 1.6 ± 0.2 60

Control – 17.17 ± 1.8 1.3 ± 0.21 0 Diazepam 0.5 48.8 ± 13.02* 0.67± 0.21 0 1.0 70.0 ± 12.65*** 0.33 ± 0.21* 0 1.5 90.0 ± 0.0**** 0.0 ± 0.0*** 0

* p < 0.05. ** p < 0.02. *** p < 0.01. **** p < 0.001 (U-Mann-Whitney test). Results are expressed as mean ± SEM of 6–8 mice per group. Time of observations = 90 min

100, 300 mg/kg, BM-74, BM-75 100 mg/kg) or de- In case of other tested compounds, this influence creased it (BM-75 300 mg/kg, BM-76 100, 300 was generally dose-dependent. Mortality of the ani- mg/kg). The mortality of animals was slightly af- mals was slightly affected by the compounds in this fected by the compounds. Diazepam used ip at test, namely they either increased it or did not influ- 3 doses as a reference delayed the onset of seizures ence it. and decreased the number of attacks in the dose- Ethosuximide used as a reference drug (130 dependent manner. The more detailed data are mg/kg injected ip [14]) delayed the onset of sei- shown in Table 1. zures over 10 times and significantly decreased the In the model of pentetrazole-induced seizures, number of attacks 8 times in comparison with the all the compounds accelerated the first seizure control group of animals. The precise data are attack in comparison with the control group, and shown in Table 2. BM-76 was the most active compound in this re- The ability of the compounds to influence CNS spect. activity was also confirmed in the thiopental- As regards the number of convulsive attacks, induced sleep test. The sleep-latency was shortened only BM-68 at all the doses we used increased it. by BM-68 and BM-76. Conversely, BM-74 and

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Table 2. The influence of the investigated compounds on the pentetrazole-induced seizures in mice

Compound Dose (mg/kg) Time of onset of seizures [s] Number of seizures Mortality in the group (mean ± SEM) (mean ± SEM) (%)

Control – 139.67± 17.1 1.33 ± 0.21 0 BM68 30 109.0 ± 17.0 1.38 ± 0.18 0 100 121.57± 16.1 1.86 ± 0.4 0 300 118.67± 18.7 1.67± 0.33 0

Control – 148.13 ± 14.2 1.5 ± 0.33 0 BM74 30 136.0 ± 18.3 1.8 ± 0.37 0 100 127.43 ± 12.9 1.29 ± 0.18 14.29 300 76.33 ± 3.7*** 2.0 ± 0.26 0

Control – 135.43 ± 15.4 1.71 ± 0.57 0 BM75 30 82.83 ± 6.1** 2.0 ± 0.45 0 100 125.6 ± 21.0 1.6 ± 0.24 20.0 300 124.57± 24.6 1.86 ± 0.34 0

Control – 288.67± 39.6 1.5 ± 0.34 16.67 BM76 30 124.2 ± 24.2*** 1.2 ± 0.2 0 100 183.14 ± 31.1 1.71 ± 0.42 14.29 300 206.4 ± 56.5 1.2 ± 0.2 20.0

Control – 153.28 ± 11.71.0 ± 0.22 0 Ethosuximide 130 mg/kg ip 1612.25 ± 187.8**** 0.125 ± 0.13** 0

* p < 0.05. ** p < 0.02. *** p < 0.01. **** p < 0.001 (U-Mann-Whitney test). Results are expressed as mean ± SEM of 8 mice per group. Time of observations = 30 min

BM-75 prolonged it. The duration of thiopental- and systemic penicillin show similar proconvulsive induced sleep was generally prolonged to some ex- activity and are considered to be model substances tent, except for BM-68 (30 mg/kg), BM-74, BM-76 for absence-seizures [12, 13]. (100 mg/kg) and BM-75 (30, 100 mg/kg). All the On the other hand, BM-68, BM-74, BM-75 and data obtained in this test are shown in Table 3. BM-76 may weakly act via GABA receptors, Summing up, we may state that the amide ana- which is consistent with their protective activity logues of GHB affect CNS activity, when given proven in the picrotoxin-induced seizures and with to mice. Their anticonvulsant activity depended their chemical similarity to GABA. strongly on the proconvulsive substance used in the CNS-stimulating effect of some of these sub- test. This diverse efficacy of the investigated com- stances proven among other properties in the spon- pounds in different models of seizures remains in taneous locomotor activity test (data not shown) agreement with earlier results [7]. and in the thiopental-induced sleep test might be As the compounds intensify in most cases the a logical consequence of their chemical structure: pentetrazole-induced convulsions, it may be de- all of the investigated compounds are amino acid duced their mechanism of proconvulsive activity. It analogues, so it is likely that in some way they may is probable that they act as agonists of GHB/GABA influence the stimulating amino acidergic system hypothetical receptors, since GHB, pentetrazole within CNS. For the moment being, these are only

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Table 3. The influence of the investigated compounds on thio- 2. Addolorato G., Cibin M., Caputo F.: Gamma-hydro- pental-induced sleep xybutyric acid in the treatment of alcoholism: dosage fractioning utility in non-responder alcoholic patients. Compound Dose Sleep-latency [s] Duration of sleep Drug Alcohol Dependence, 1998, 53, 7–10. (mg/kg, po) (mean ± SEM) [s] (mean ± SEM) 3. Hu R.Q., Banerjee P.K., Snead O.C.: Regulation of gamma-aminobutyric acid release in cerebral cortex in Control – 109.44 ± 9.6 528.44 ± 62.5 the gamma-hydroxybutyric acid model of absence sei- BM68 30 69.0 ± 13.0* 375.63 ± 15.6* zures in rat. Neuropharmacology, 2000, 39, 427–439. 100 56.75 ± 9.1*** 1009.0 ± 168.0** 4. Kupferbeger H.: Antiepileptic drug development pro- gram. A cooperative effort of government and indus- 300 88.13 ± 12.3 596.63 ± 123.3 try, Epilepsia, 1989, 30, Suppl. 1, 51–56. 5. Maitre M., Hechler V., Vayer P., Gobaille S., Cash C., Control – 110.13 ± 5.71465.25 ± 182.5 Schmitt M., Bourguignon J.: A specific g-hydroxybu- tyrate receptor ligand possess both antagonistic and BM74 30 116.5 ± 10.9 1800.0 ± 0.0 anticonvulsant properties. J. Pharmacol. Exp. Ther., 100 139.83 ± 5.5*** 463.0 ± 144.6 1990, 255, 657–663. 300 128.13 ± 5.4* 1714.75 ± 60.0*** 6. Malawska B.: Searching of g-amino- and g-hydroxy- butyric acid analogues with expected anticonvulsant activity (Polish). Wiad. Chem., 2001, 55, 377–402. Control – 99.0 ± 8.71532.0 ± 163.4 7. Malawska B., Kulig K., Antkiewicz-Michaluk L., BM75 30 126.38 ± 3.1*** 1498.13 ± 199.9 Cliffe I., Porter R., Misra A.: Anticonvulsant activities and voltage-sensitive calcium channels receptor affi- 100 134.88 ± 5.9*** 1443.13 ± 181.0 nity of substituted N-benzylamides of g-amino- and 300 126.13 ± 5.0** 1572.0 ± 150.1 g-hydroxybutyric acid. Arch. Pharm. Pharm. Med. Chem., 1999, 332, 167–174. 8. Mazzetti di Pietralata M., Florentino M. T.: Night eat- Control – 104.43 ± 9.6 614.71 ± 109.6 ing syndrome. Preliminary results. Eat Weight Disord. BM76 30 94.38 ± 5.4 1371.0 ± 157.7*** 2000, 5, 92–101. 100 96.0 ± 4.7558.86 ± 97.7 9. Przew³ocka B., Lasoñ W.: Epilepsy: mechanisms and pharmacotherapy (Polish). XV Winter School of the 300 86.5 ± 7.7 685.83 ± 77.1 Institute of Pharmacology, Polish Academy of Sci- ences, Mogilany, Kraków, 1998. * p < 0.05. ** p < 0.02. *** p < 0.01. **** p < 0.001 (Student’s 10. Ramek. M.: Intramolecular hydrogen bonds in hy- t-test). Results are expressed as mean ± SEM of 10 mice per droxy acids. J. Mol. Struct. (Theochem), 1994, 310, group. Time of observations = 60 min 269–278. 11. Snead O.C.: g-Hydroxybutyrate. Life Sci., 1977, 20, our hypotheses which need to be checked in the fu- 1935–1943. 12. Snead O.C.: Gamma-hydroxybutyrate model of gen- ture, as the data presented in this paper are prelimi- eralized absence seizures: further characterization and nary. We keep on investigating the pharmacological comparison with other absence models. Epilepsia properties of these substances and hopefully it will 1998, 29, 361–368. enable us to estimate their potential value. 13. Snead O.C, Hechler V., Vergnes M., Maitre M.: In- creased g-hydroxybutyric acid receptors in thalamus of genetic animal model of petit mal epilepsy. Epi- Acknowledgments. The authors would like to thank lepsy Res., 1990, 7, 121–128. Mrs. Teresa Dobrut for her technical assistance. This study 14. Stevens G., Zingel V.: Progress in Drug Research. was supported by Research Project no. W³/199/P/F from Birkhauser Verlag, Basel, Boston, Berlin, 1995. the Jagiellonian University, Medical College, Kraków, Po- 15. Tunnicliff G.: Significance of g-hydroxybutyric acid land. in the brain. Gen. Pharmacol., 1992, 23, 1027–1034. 16. Tunnicliff G.: Sites of action of gamma-hydroxybu- REFERENCES tyrate (GHB) – a neuroactive drug with abuse poten- tial. J. Toxicol. Clin. Toxicol., 1997, 35, 581–590. 1. Addolorato G., Balducci G., Capristo E.: Gamma- 17. Vayer P., Mandel P., Maitre M.: g-Hydroxybutyrate: hydroxybutyric acid in the treatment of alcohol with- a possible neurotransmitter. Life Sci., 1987, 41, drawal syndrome: a randomized comparative study ver- 1547–1557. sus benzodiazepine. Alcohol-Clin. Exp. Res., 1999, 23, 1596–1604. Received: June 26, 2002; in revised form: August 28, 2002.

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