2 mice

Neurophararmacofogy Vol. 26, No. 9, pp. 1425-1429,1987

0028-3908/S? $3.00 + 0.00 Copyright 0 1987 Pergamon Journals Ltd

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A~IC~~ULSANT

EFFECT OF DENZIMOLIN

DBA/2 MICE

G. B. DE SARRO,’ V. LIBRI,’ C. ASCIOTI,’ R. TF_STA*and G. NISTICO’* ‘Institute of Pharmacology, Faculty of Medicine, University of Reggio Calabria, 88100 Catanzaro and 2Recordati S.p.A. Research Division, Pharmacology Department, Milan, Italy (Accepted 26 .January 1987)

Summary-The anticonvulsant activity of N-[~-[4-~-phenylethyl)phenyl]-~-hydroxyethyl]-imidazole hydrochloride, denzimol, was studied following intraperitoneal administration in DBA/2 mice (seizures induced by sound). Protection against sound-induced seizures was observed after intraperitoneal administration of denzimol (3-1.5 mg/kg). The ED, values for the suppression of tonic, clonic and wild

running phases of sound-induced seizures were 1.24, 2.61 and 6.03 mg/kg, respectively. This protective action was si~ifi~ntly reduced by pretreatment with a~nophylline (25mg/kg i.p.), CGS 8216 (I or Smg/kg i.p.) and Ro 15-1788 (2Smg/kg i.p). The present experiments suggest an involvement of purinergic and benzodiazepine mechanisms in the anticonvulsant action of denzimol. Key words: denzimol, epilepsy, anticonvulsant, DBA/Z mice, aminophylline, Ro 15-1788, CGS 8216.

Denzimol, ~-~-[4-(~-phenylethyl)phenyl]-~-hydroxyethyl]-imidazole hydrochloride (Nardi, Tajana, Leonardi, Pennini, Portioli, Magistretti and Subissi, 1981), is an imidazole derivative, which possesses anticonvulsant properties in several experimental models of epilepsy (Graziani, Tirone, Barbadoro and Testa, 1983). The mechanism of the anticonvulsant activity of denzimol is not known, but preliminary data in animal models suggest an activity similar to that of diphenylhydantoin and, in part, to carbamazepine, but clearly different from the benzodiazepines, barbiturates or sodium valproate (Graziani et al., 1983). In addition, in previous pharmacological studies it has been reported that denzimol potentiated the depressant and antipentylenetetrazol activity of diazepam in mice (Ibba, Mennini and Testa, 1985) perhaps through an increase in the number of benzodiazepine receptors (Mennini, Gobbi and Testa, 1984). Denzimol also inhibits the uptake of adenosine (Masturzo, Salmona, Ceci and Mennini, 1987). Experimental findings suggest the existence of strict interactions between benzodiazepines and anticonvulsant drugs (Schmutz, Bernasconi and Baltzer, 1983; Gallager, Mallorga and Tallman, 1980; Czuczwar, Turski and Kleinrok, 1981). Thus diphenylhydantoin, as well as denzimol, potentiated the antimetrazol effects of benzodiazepines while failing to affect their protective efficiency against bicuculline, isoniazide (Czuczwar et al., 1981) and picrotoxin (Mennini et al., 1984). This finding seems to indicate the existence of some benzodiazepine receptors independent of the y-aminobutyric acid (GABA) receptor-ionophore complex. It has been also sug gested that some effects of benzodiazepine are medim *Author for correspondence.

ated by adenosine (Phillis and Wu, 1981; Phillis, Wu and Coffin, 1983). The aim of the present study was to assess whether denzimol was able to attenuate or to suppress soundinduced seizures in DBA/2 mice. This test system has pre~ously permitted the evaluation of agents acting on monoaminer~c (Horton, Anlezark and Meldrum, 1980) and GABAergic transmission (Horton, Collins, Anlezark and Meldrum, 1979) as well as on excitatory amino acids (Croucher, Collins and Meldrum, 1982). In addition, it was planned to ascertain whether the anticonvulsant effects of denzimol were antagonized by aminophyIline, an adenosine receptor antagonist and by 2-phenylpyrazolo-[4,3-c]quinoline-3(5H)one (CGS 8216) or ethyl-&fluoro5,6-dihydro-S-methyl-6-oxo-4&imidazo-[lJa] [1,4]benzodiazepine-3-carboxylate (Ro 15-1788), two “neutral” benzodiazepine receptor antagonists. METHODS

All experiments were performed with DBA/2 mice, an inbred strain, the weanlings of which are genetically susceptible to audiogenic seizures (Collins, 1972). Exposure to a loud sound within a specific frequency range induces a sequential seizure response in these animals, consisting of an early wild running phase (WR), followed by generalised myoclonus and tonic flexion and extension. Later phases in the seizure sequence do not occur in the absence of the earlier phases. Groups of l&12 mice, of mixed sexes, weight 8-12 g and 21-28 days of age, were studied at each dose. For intraperitoneal (i.p.) administration, denzimol and aminophylline were dissolved in sterile saline, while CGS 8216 and Ro 15-1788 were suspended in water to which a drop of Tween-80 had been added and sonicated until immediately before

1425

G. B. DE SARROet al.

1426

injection. All drugs were injected in a volume equivalent to 1% of the body weight. Forty-five min after administration of denzimol or saline and 30min after administration of a dose of aminophylline, CGS 8216 or Ro 15-1788, which per se did not produce convulsions, the rectal temperature was recorded (Elektrolaboratoriet thennometer type TE3) and the mice were placed individually under a hemispheric perspex dome for observation and auditory stimulation. Thirty set were allowed for habituation and assessment of any behavioural changes. Auditory stimulation (electric bell, generating 109 dB at the level of the mouse) was then applied for 60 set or until tonic extension occurred. The percentage incidence and the timing of each phase of the seizure response was recorded. Each response was then scored as previously described (De Sarro, Croucher and Meldrum, 1984): 0 = no response; 1 = wild running; 2 = clonic phase; 3 = tonic phase; 4 = full seizure response followed by respiratory arrest. The maximum score for each animal was recorded and the mean maximal seizure response between control and drug-treated animals was assessed using Fischer’s exact probability test (seizure response) and the two-tailed t-test for independent groups (rectal temperatures). When applicable, ED,, values (with 95% fiducial limits) for suppression of the principal phases of the seizure response for denzimol were carried out by an IBM 4331 computer using the Bliss program according to Finney (1978). The following compounds were tested: denzimol hydrochloride (Recordati Res. Labs, Milan); aminophylline (Sigma, St. Louis, Missouri); CGS 8216 (Ciba Geigy, Sum-

mit, New Jersey) and Ro 15-1788 (Hoffman-La Roche, Base], Switzerland). RESULTS

Treatment

with denzimol

Denzimol (3-l 5 mg/kg i.p.) produced significant protection (P < 0.05) against the tonic and clonic phases of the audiogenic seizure response in DBA/2 mice 45 min after administration (Table 1). Protection against the initial wild running phase was seen only after 7.5-10 and 15 mg/kg of denzimol administered intraperitoneally (Table 1). Mice treated with the largest doses of denzimol (10 and 15 mg/kg) showed mild sedation. No reduction of locomotor activity, ataxia and a fall in rectal temperature, usually observed with other anticonvulsant compounds, were observed. Bliss analysis of the data indicated the following ED,, values (k 95 confidence limits) for denzimol against the principal phases of the audiogenic seizure response: tonus 1.24 (0.67-2.78) mg/kg; clonus 2.61 (1.614.22) mg/kg: wild running 6.03 (4.947.37) mg/kg. Pretreatment

with aminophylline

Aminophylline (25 mg/kg i.p.), given 30 min before testing, did not significantly modify the phase of audiogenic seizure response in DBA/2 mice. As shown in Table 1, the anticonvulsant properties of injection of denzimol (5, 7.5, 10 and 15 mg/kg i.p.), were reduced by the administration of aminophylline (25 mg/kg i.p.), 15 min after denzimol. A significant increase in the incidence of all phases of the seizure

Table I. The effect of denzimol and aminophylline on audiogenic seizures in DBAj2 mice Dose (m&n) Saline 0.1 0.5

Aminophylline Aminophylline + Denzimol Aminophylline + Denzimol Amininophylline + Denzimol Aminophylline + Denzimol

3 5 7.5 IO I5 25 25

WR

Clonus

100 100 100 100 83 75 50. 0” 0.’ loo

100

100 83 67 501 50. 33” o** 0*+ 100

100

loot

IfJOt

% Response RA Tonus 100 83 75 58 50’ o** o** 0” 0.’ 100

Temp (“C) SR

Meanf

SEM

n

67 67 50 33 I7 o** 0” 0” 0’ 90

3.67 3.5 3.08 2.58 2.0 1.25 0.83 0 0 3.9

38.6 kO.12 38.23 + 0.28 38.44kO.18 38.35kO.16 38.39 f 0.19 38.23 +0.26 38.32 50.22 38.28kO.17 38.12+0.21 37.9 f 0.16

I2 6 I2 I2 12 12 12 12 12 10

70

30

3.0

38.29k0.17

10

10

20

IO

2.0

38.52 f 0.28

10

Wt

40

0

0

1.2

38.65 f 0.16

10

w

IO

0

0

0.6

38.21 k 0.19

10

5 25 7.5 25 10 25 15

Denzimol and aminophylline were administered intraperitoneally alone or in combination to groups (n) of mice and auditory stimulation was commenced 45 min later. The table shows the percentage incidence of the principal phases of the audiogenic seizure response in groups of control and drug-treated animals. Significant reductions in incidence of seizure phase relative to control experiments performed on the same day are denoted: *P < 0.05; l*P -z0.01. WR = Wild running; RA = respiratory arrest; SR = mean maximal seizure response (see Methods for grading). Significant differences in the incidence of seizure phases between groups treated with denzimol alone or with denzimol and aminophylline are denoted: fP < 0.05; ttP
Anticonvulsant

action of denzimol

1427

Table 2. ED, values (+95% confidence limits) for denzimol alone or denzimol administered with the other compounds, on the audiogenic phases in DBA/Z mice Treatment

Seizure phase

ED,,, mglkg

Denzimol

WR CL0 TON

6.03 (4.94-7.37) 1.61 (0.794.22) 1.24(0.67-2.28)

1 1

CL0

9.36 (7.07-10.99)

3.59

CL0

9.65 (7.98-l

Denzimol +

Aminophylline Denzimol + CGS-8216(1 mgjkg)

Potency ratio

1

I .66)

3.70

Denzimol +

CGS-8216 (5 me/kg) Dcnzimol + Ro 15-1788

CL0

12.12(10.18-14.43)

4.64

CL0

7.78 (6.1S9.86)

2.98

WR = Wild running; CL0 = Clonus; TON = Tonus. ED, values for suppression of the WR, CL0 and TON phases were evaluated using the Bliss method probit procedure (Finney, 1978).

response was seen in the groups treated with aminophylline (25 mg/kg), compared to the groups receiving denzimol alone. The EDS, for denzimol against the clonic phase increased approximately 3.59 times (Table 2). No fall in rectal temperature or behavioural changes were observed after aminophylline. The mild sedation seen after the largest dose of denzimol (10 and 15 mg/kg i.p.) were also largely prevented by aminophylline. Pretreatment

anticonvulsant effects of denzimol were partially blocked by the subsequent administration of Ro 15-1788 or CGS 8216, indicating that binding at high affinity central benzodiazepine receptors could be responsible for the anticonvulsant activity of denzimol (Braestrup, S&m&hen, Neff, Nielsen and Petersen, 1982; Jensen, Petersen and Braestrup, 1983). It was recently reported that the benzodiazepine receptor antagonists, CGS 8216 and Ro

with CGS 8216 and Ro 15-1788

Neither CGS 8216 (1 and 5 mg/kg i.p.) nor Ro 15-1788 (2.5mg/kg i.p., 30min before testing) significantly changed the phase of the audiogenic seizure response in DBA/2 mice. As shown in Figure 1, the anticonvulsant effects of denzimol (5, 7.5, 10 and 15 mg/kg Lp.), were reduced by the administration of CGS 8216 (1 and 5 mg/kg i.p.) or Ro 15-1788 (2.5 mg/kg i.p.) 15 min after denzimol (Fig. 1). A significant increase in the incidence of all phases of the seizure response was seen in the groups treated with CGS 8216 or Ro 15-1788, compared to the group receiving denzimol alone. The EDSo for denzimol against the clonic phase increased from 2.98 to 4.64 times (Table 2). No behavioural changes or fall in rectal temperature were seen after CGS 8216 or Ro 15-1788. DISCUSSION

The present study shows that denzimol possesses a significant anticonvulsant action in a model of reflex

epilepsy, i.e. sound-induced seizures in DBA/2 mice. The anticonvulsant activity of denzimol in these experiments is consistent with previous reports concerning maximal electroshock seizures in mice, electroshock activity in rabbits, maximal pentylentetrazol activity in rats and 2,4-dimethylpyrimidine-induced convulsions in mice (Graziani et al., 1983). Previous studies on the mechanism of action of denzimol have suggested that its anticonvulsant effects resemble those of diphenylhydantoin and, in part, of carbamazepine, and they were different from those of the benzodiazepines, barbiturates or sodium valproate (Graziani et al., 1983). In the present study the

-

-J I

1

5

7.5 mg/kg

0

Dmrlma1 akaw

q

CGS 6216

-

15

10

q

lSmg)+da-nlmol

i.p. CGS 6216 (lmg)

q RO

+ denzimol

15 1766+

denrimol

Fig. 1. Effects of various doses of denzimol, alone or in association with CGS 8216 (1 and 5 mg/kg) and Ro 15-1788 (2.5 mg/kg), on the percentage incidence of audiogenic seizure phases in DBA/Z mice. Significant differences between groups treated with denzimol alone or denzimol in association with other compounds are denoted: *P < 0.05 and **P < 0.01.

1428

G. B. DE SARROet al.

15-l 788, diminished the anticonvulsant effect of benzodiazepines and it has been proposed that they may serve as a tool for detecting correlations between the effects of antiepileptics and benzodiazepine receptor binding (Schmutz er al., 1983). In addition, CGS 8216, which was found to block adenosine receptors (Czernick, Petrack, Kalinsky, Psychoyis, Cash, Tsai, Rinehart, Granat, Lowell, Brundish and Wade, 19821, may distinguish whether an effect is due to an interaction with a benzodiazepine binding site, adenosine receptors or both. The results of the present experiments could be explained on the basis of previous data showing that denzimol and phenytoin are able to increase the number of “nanomolar” benzodiazepine receptors in viva (Masturzo et al., 1987). However, the finding of the present study that both aminophylline and CGS 8216 efficiently attenuated the anticonvulsant effects of denzimol suggests that adenosine receptors seem to be involved in the mediation of the anticonvulsant effects of denzimol. It seems possible that the anticonvulsant effects of denzimol depend on an enhancement of purinergic transmission through an inhibition of the uptake of adenosine. In fact, it has been demonstrated that denzimol inhibits the uptake of adenosine by synaptosomes in the brain of the rat (Masturzo et ul., 1987). This idea is also supported by experimental evidence suggesting that other anticonvulsant drugs may exert their effects through an enhancement of adenosine-mediated transmission in the brain (Phillis, Siemens and Wu, 1980b). Thus, Phillis and colleagues have speculated that ~~o~~epines may inhibit the uptake of adenosine (Phillis, Bender and Wu, 1980a) and/or enhance the release of adenosine (Phillis et at., 1980b). In addition, it was reported that purines such as inosine, hypoxanthine and caffeine displace benzodiazepines that bind selectively at central benzodiazepine receptors (Skerritt, Chow, Johnston and Davies, 1982) and benzodiazepines potentiate the effects of iontophoretically applied adenosine (Phi&s, 1979). In addition, recently it was suggested that diphenylhydantoin may exert its anticonvulsant effects by inhibiting the uptake of adenosine (Phillis, 1984) and that the anticonvulsant activity of carbamazepine is prevalently mediated by adenosine receptors (Schmutz et al., 1983). Methylxanthines have been reported to reverse the protective activity of a number of widely used antiepileptic drugs including phenytoin, diazepam, phenobarbital, carbamazepine, sodium valproate and flunarizine (Pole, Bonetti, Pieri, Cumin, Angioi, Mohler and Haefely, 1981; Schmutz et al., 1983; Skerritt, Johnston and Crow, 1983; Phillis, 1984; Weir, Padgett, Daly and Anderson, 1984; Czuczwar, Turski, Ikonomidou and Turski, 1985; De Sarro, Nistico and Meldrum, 1986) and this provide additional support to the hypothesis that adenosine receptors may modulate seizure susceptibility. In conclusion, the present findings and previous

pharmacological studies (Ibba et al., 1985; Masturzo et al., 1987) suggest an involvement of benzodiazepine and adenosine receptor mechanisms in the anticonvulsant effects of denzimol. It seems possible that denzimol may interact with an endogenous ligand, perhaps an endogenous xanthine, which is able to modulate the activity of the GABA~nzodiazepine-Cl-ionophore complex or potentiate adenosine mechanisms, possibly through inhibition of uptake of adenosine and/or enhancement of release of adenosine. However, it remains to be established whether the effects of denzimol are mediated by other receptor sites. The potent anticonvulsant effect of denzimol on the audiogenic seizures in DBAj2 mice raises the possibility that denzimol may be useful in the treatment of some epileptic disorders. Acknowledgemenrs-Our thanks to Dr Brian Meldrum for his helpful discussion and to Mrs Adriana Mastroeni for the excellent typing of the manuscript. Partial support from Italian Council for Research (CNR, Roma) and Italian Ministry for Public Education (MPI, Roma) is gratefully acknowledged.

REFERENCES

Braestrup, C., Schmiechen R., Neff G., Nielsen M. and Petersen E. N. (1982) Interaction of convulsive ligands with benzodiazepine receptors. Science 216: 1241-1243. Collins R. L. (1972) Audiogenic seizures. In: Experimental Models of Epilepsy (Purpura P., Penry J. K., Tower D., Woodbury D. M. and Walter R., Eds), pp. 347-372. Raven Press, New York. Croucher M. J., Collins J. F. and Meldrum B. S. (1982) Anticonvulsant of excitatory amino acid antagonists. Science 216: 899-901.

Czernick A. J., Petrack B., Kalinsky M. J., Psychoyis S., Cash W. D.. Tsai C.. Rinehart R. H., Granat F. R., Lowell R. A.1 Brundish D. E. and Wade R. (1982) CGS 8216: receptor binding characteristics of a potent bendoziazepine antagonist. Life Sci. 30: 363-372. Czuczwar S. J., Turski W. A., Ikonomidou C. and Turski L. (1985) Aminophylline and CGS 8216 reverse the protective action of diazepam against el~tr~nvulsions in mice. Epiiepsia 26: 693-696. Czuczwar S. J., Turski L. and Kleinrok 2. (1981) Diphenylhydantoin potentiate effect of diazepam against pentylentetrazol but not against bicuculline and isoniazide induced seizures in mice. Neuropharmacology 20: 615-679.

De Sarro G. B., Croucher M. J. and Meldrum B. S. (1984) Anticonvulsant actions of DS 103-282: pharmacological studies in rodens and the baboon, Papio papio. Neuropharmaco1og.v 23: 525-530.

De Sarro G. B., Nistico G. and Meldrum B. S. (1986) Anticonvulsant properties of flunezizine on reflex and general&d models of epilepsy. Neuropharmacology 2% 695-70 I. Finney D. J. (1978) Statistical Method in Biological Assay. Charles Griffen, London. Gallager D. W., Mallorga P. and Tallman J. F. (1980) Interaction of diphenylidantoin with benzodiazepines in the CNS. Brain Res. 189: 209-220. Graziani G., Tirone F., Barbadoro E. and Testa R. (1983) Denzimof, a new anticonvuisant drug. I. general anticonvulsant profile. Arzneimittel Forsch. Drug Res. 33: 1155-l 160.

Anticonvulsant Horton R. W., Collins J. F., Anlezark G. M. and Meldrum B. S. (1979) Convulsant and anticonvulsant actions in DBA/Z mice of compounds blocking the reuptake of GABA. Eur. J. Pharmac. 59: 75-83. Horton R. W., Anlezak G. M. and Meldrum B. S. (1980) Noradrenergic influences on sound-induced seizures. J. Pharmac. exp. Ther. 214: 437-442.

Ibba M., Mennini T. and Testa R. (1985) Enhancement of diazepam activities induced by denzimol in mice. Pharmat. Res. Commun. 17: 95-103. Jensen L. H., Petersen E. N. and Braestrup C. (1983) Audiogenic seizures in DBA/Z mice discriminate sensitively between low efficacy benzodiazepine receptor agonists and inverse agonists. Lr@ Sci. 33: 393-399. Masturzo P., Salmona M., Ceci A. and Mennini T. (1987) Effect of benzodiazepine and calcium channel blockers on membrane fluidity and adenosine uptake in rat brain synaptosomes. J. bioi. Chem. (In press). Mennini T.. Gobbi M. and Testa R. (1984) Effects of denzimol ‘on benzodiazepine receptors‘in the CNS: relationship between the enhancement of diazepam activity and benzodiazepine binding sites induced by denzimol in the rat. Life Sci. 35: 181 l-1820. Nardi D., Tajana A., Leonardi A., Pennini R., Portioli F., Maeistretti M. J. and Subissi A. (1981) Svnthesis and antiionvulsant activity of N-(Benzoylalkyl)imidazoles and N-(/I-Phenyl$-hydroxyalkyl) imidazoles. J. Med. Chem. 24: 727-731. ‘Phillis J. W. (1979) Diazepam potentiation of purinergic depression of central neurons. Gun. J. Physiol. Pharmac. 57: 432435.

Phillis J. W. (1984) Interactions of the anticonvulsants diphenylhydantoin and carbamazepine with adenosine on cerebral cortical neurons. Epifepsia 25: 765-772. Phillis J. W., Bender A. S. and Wu P. H. (1980a) Benzo-

action of denzimol

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diazepines inhibit adenosine uptake into rat brain synaptosomes. Brain Res. 195: 494-498. Phillis J. W., Siemens R. K. and Wu P. H. (1980b) Effects of diazepam on adenosine and acetylcholine release from rat cerebral cortex: Further evidence for a purinergic mechanism in action of diazepam. Br. J. Pharmac. 70: 341-348.

Phillis J. W. and Wu P. H. (198 1) The role of adenosine and its nucleotides in central synaptic transmission. Prog. Neurobiol. 16: 187-239. Phillis J. W., Wu P. H. and Coffin V. L. (1983) Inhibition of adenosine uptake into rat brain synaptosomes by prostaglandins, benzodiazepines and other centrally active compounds. Gen. Pharmac. 14: 475-479. Pole P., Bonetti E. P., Pieri L., Cumin R., Angioi R. M., Miihler H. and Haefelv W. E. (1981) Caffeine antanonizes several central effects of hiazepam. Life ,!k 28: 2265-2275.

Schmutz M., Bemasconi R. and Baltzer V. (1983) Benzodiazepine antagonists, GABA and the mode of action of antiepileptic drugs. In: Current Problems in Epilepsy I (Boldy-Moulinier M., Ingvar D. J. and Meldrum B. S., Eds), pp. 378-383. Eurotext, London. Skerritt J. H., Chow S. C., Johnston G. A. R. and Davies L. P. (1982) Purines interact with “central” and “oeriferal” ‘benzodiazepine binding sites. Neurosci. Lett: 34: 63-68.

Skerritt J. H., Johnston G. A. R. and Chow S. C. (1983) Interactions of the anticonvulsant carbamazepine with adenosine receptors. 2. Pharmacological studies. Epilepsia 24: 643450.

Weir R. L., Padgett W., Daly J. W. and Anderson S. M. (1984) Interaction of anticonvulsant drugs with adenosine receptors in the central neurons system. Epilepsia U: 492-498.