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Epileptogenic activity of some β-lactam derivatives: Structure-activity relationship
Neuropharmocolo~y Vol. 28, No. 4, pp. 359-365, 1989 Printed in Great Britain. All rights reserved
Copyright
0028-3908/89 $3.00 + 0.00 0 1989 Pergamon Press plc
EPILEPTOGENIC ACTIVITY OF SOME /3-LACTAM DERIVATIVES: STRUCTURE-ACTIVITY RELATIONSHIP A. DE SARRO,* G. B. DE SARRO, C. Ascron and G. NISTI& Institute of Pharmacology, University of Messine, Piazza XX Settembre 4, 98122 Messine and Institute of Pharmacology, University of Reggio Calabria, Policlinico Mater Domini, via T. Campanella, 88100 Catanzaro, Italy (Accepted 6 Ocrober 1988) Summary-The epileptogenic activity of several derivatives of fl-lactam was compared following their intracerebroventricular administration in rats. At a dose of 0.033 pmol/kg cefazolin was the most powerful epileptogenic compound among the drugs tested; dramatic seizure signs (nodding, clonic convulsions and sometimes escape responses) were observed repeatedly. It was approximately three times more potent than ~nzyl~nicillin and other similar compounds, such as ceftriaxone, cefoperazone and cefamandole. No epileptogenic signs were observed with equimolar doses of cefotaxime, cefonicid and ceftizoxime. All these derivatives differ from the substitution at position 3 and at position 7 of 7-aminocephalosporanic acid. The more convulsant compounds (i.e. cefazolin and ceftezole) are both derivatives of tetrazol and show a marked similarity with pentylentetrazol. In addition, aztreonam, a compound having only the /I-lactam ring substituted with a heterocyclic ring at the 4 position, possessed convulsant properties like those of cefoperazone and cefamandole. This suggests that the j%lactam ring is able to produce epileptogenic activity and it seems likely that substitutions at the 7-aminocephalosporanic or &aminopenicillanic acid may increase or reduce the epileptogenic properties of /I-lactam derivatives. Key rtTor&-seizures,
penicillins, cephalosporins, aztreonam, epilepsy.
The administration of /J-lactam antibiotics (i.e. cephalosporins and penicillins) into the cerebral ventricles, subarachnoidal space or directly onto the cerebral cortex of chicks, cats, dogs, monkeys, mice, rats and man has been reported to induce a focal or generalized epileptic state, initially characterized by spike and wave discharges which are followed by clear tonic-clonic or clonic seizures (Gloor and Testa, 1974; Anderson and Rutledge, 19’79; Nistico, Musolino, De Sarro, Gallitto and Di Perri, 1979;
Nistico, De Sarro, Naccari, Musolino, Rotiroti, Galhtto and Di Perri, 1980a; Nisticb, De Sarro, Rotiroti, Silvestri and Marmo, 1980b; De Sarro, Calo, Bagetta, Anfosso, Marmo, Nistico and Guarino, 1983; Rotiroti, De Sarro, Musolino and Nistico, 1983; Van Gelder, Siatitsas, Menini and Gloor, 1983; Gerald, Massey and Spadaro, 1973). The structural similarity of penicillin with the y-aminobutyric acid antagonist, the convulsant drug bicuculline is of interest (Curtis, Game, Johnston, McCulloch and Maclachlan, 1972), since the decrease in inhibition in the hippocampus produced by penicillin contributes greatly to its epileptogenicity (Dingledine and Gjerstad, 1980). The cephalosporins are a family of B-lactam antibiotics that generally contain the 7-aminocephalosporanic acid nucleus. They differ in their basic structure from the penicillins in that the cephalosporins contain a six-membered dihydrothiazini~ ring instead of a five-membered *To whom correspondence should be addressed.
thiazolidinic ring fused to the /I-lactam portion (Quintiliani, Nightingale, Rossi and Ristuccia, 1984). The ~phalospo~ns resemble penicillins both in their actions as antibiotics (Mandell and Sande, 1985) and as convulsant compounds (Rotiroti et al., 1983). Several authors found that the ability of derivatives of penicillin to produce a seizure focus was abolished after incubation with ~nicillinase, an enzyme which splits the /I-lactam ring of penicillins (Gerald et al., 1973; Hartesveldt, Petit and Isaacson, 1975; Gutnick, Van Duijn and Citri, 1976). However, these studies tend to support the fact that the /I-lactam ring is an essential structural feature for the epileptogeni~ activity of penicillins. Using these @-lactam derivatives, it was of interest to compare their epileptogenic activity and to investigate to which type of radical, applied to the basic structure, the epileptogenic properties of these compounds could be related. Thus, the aim of the present study was to assess the behavioural epileptogenic effects of some new cephalosporins and penicillins after acute injection into a lateral ventricle in freely moving rats. METHODS
Adult male Wistar rats (ZO(r250 g), Charles River (Calco, Como, Italy) were stereotaxically implanted with stainless steel guide cannulae under chloral hydrate anaesthesia (400 mg/kg, Lp.). according to the atlas coordinates of Paxinos and Watson (1982), 359
360
A.
DE SARRO et al.
to permit injection of drugs into the left or right lateral cerebral ventricle. After surgery a minimum of 48 hr was allowed for recovery before the experiments were carried out. Freely moving rats were injected (0.2 pl/min) through the injection cannula which extended approximately 1 mm below the tip of the guide cannula. The animals were placed individually in transparent cages (40 x 25 x 25), and allowed 30min to acclimatise to the new environment. Preliminary studies to ascertain the influence of phosphate buffer solution 67mM, in a volume of 2 and 6.6 ~1, showed no significant behavioural changes. The volume of 6.6 ~1 was used only when it was necessary to inject 3.3 pmol of some p-lactam derivatives (i.e. cefonicid, cefuroxime, cefotaxime, ceftizoxime and piperacilline). Each rat was used only once and at least 8 animals were used for each dose level studied. The seizure response after injection of cephalosporins, penicillins and aztreonam was graded according to the following scale: 0 = normal behaviour; 1 = hyperkinesia, wet-dog shakes and sniffing; 2 = head-tremors and nodding; 3 = clonus of limbs; 4 = clonus of four limbs and/or trunk; 5 = clonus of fore limbs, rearing and falling; 6 = transient tonic-clonic seizures and escape response; 7 = generalized seizures followed by postictal period of fatal outcome. The convulsant doses required to induce a seizure response in 50% of rats (CD,,), injected with /I-lactam compounds and the relative confidence limits were determined according to Litchfield and Wilcoxon (1949). At least 32 animals were used to calculate each CD,, values. The sources of the drugs used were: chloral hydrate, sodium cefazolin and sodium ceftizoxime (Farmitalia Carlo Erba, Res. Labs., Milan, Italy); sodium cefotaxime (Hoechst Frankfurt, Germany); sodium ceftazidime (Sigma-Tau, Pomezia, Italy); so-
dium ceftezole (Schering, Berlin, Germany); sodium ceftriaxone (Hoffman-La Roche, Basel, Switzerland); aztreonam and sodium sodium cefoperazone (Menarini, Res. Labs., Florence, Italy); sodium cefamandole (Eli Lilly & Co, Indianapolis, U.S.A.); sodium cefuroxime (Glaxo, Res. Labs., Verona, Italy); sodium cefonicid (ISF, Milan, Italy); sodium benzylpenicillin (Merck, Sharp & Dohme, Munchen, Germany); sodium azlocilline (Bayer Leverkusen, Germany); sodium piperacilline (Cyanamid, Res. Labs., Catania, Italy). All drugs in the powder form were dissolved in buffer phosphate solution and injected at pH 7.3-7.4. RESULTS
The epileptogenic properties of the p-lactam antibiotics varied according to the type of radical groups attached to the basic structure (Tables l-3). The behavioural seizures consisted of an initial phase, characterized by wet-dog shake episodes followed by head tremor, nodding and clonus of the limbs. After the largest doses of b-lactam antibiotics, clonus of four limbs and/or trunk, rearing, jumping, falling down, transient tonic-clonic seizures, escape responses and generalized seizures, followed by a postictal period or fatal outcome were also evident. All these epileptic signs were dose-related and were observed repeatedly only with short interruptions. Generally, the first behavioural signs were observed within 1.5 min after the infusion and the maximum effect was reached after a minimum delay of 9 min after the injection. Dose-response curves of the epileptiform effects induced by intracerebroventricular injection of benzylpenicillin, some cephalosporins and some ureidopenicillins are shown in Figures 1 and 2; and in Figures 3 and 4. As shown in Figures 1, 2, 3 and 4, cefazolin exhibited the greatest activity
* Cefazolin
A Ceftezole 0 BenzyLpenicilh
7
. Aztreonam l Cefamondole
6
0 + 0 A
5 2 0
Azlocilline PlperaciNine Cefuroxime Cefonicid
t
Dose (@mot) Fig. I. Dose-response curves of intensity of seizures after intracerebroventricular injection of derivatives of B-lactam. Each point represents the mean score for 8-10 animals. The seizure response after administration of derivatives of b-lactam was scored as described in Methods.
Epileptogenic
effect
of some /I-lactam
361
compounds
TableI.Convulsant doses (CD,) of several cephalosporins after intracerebroventricular injection. At least 32 animals were used to calculate each CDw value (+ 95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods 7-Aminocefalosporanic
acid
bOOH(Na) Drugs
RI
Rl
CD., ,"(umol) I. 95% confidence limits
N-CHI 0.017 (0.012-0.024)
\ Cefazoline
N-CHz Ceftezole
0.04 (0.018~.09)
N;/) \ N
Cefamandole OH
/_\ i ct OH
\
0.29 (0.16-0.55) CH,S
8.40 (n.d.)* 'CHZS
Cefuroxime 'CH,OCONH,
3.31 (0.99-I I .07)
*The CD, value for cefonicid was extrapolated and so the 95% confidence limits could not be determined. n.d. = not determined.
among the drugs tested, whereas benzylpenicillin was 2-4 times less potent. In addition, ceftezole, astreonam, cefoperazone, ceftriaxone, cefamandole, ceftazidime, cefotaxime, ceftizoxime and cefuroxime were in this order less potent than cefazolin in inducing epileptic behavioural signs. In fact, very large doses as 1.65 and 3.3 pmol of cefotaxime and ceftizoxime were necessary in order to produce clonus of the fore limbs, rearing, falling down or transient tonic-clonus seizures and escape responses. No clear signs of behavioural seizures were noted even after the administration of 1.0 and 3.3 pmol of cefonicid. DISCUSSION
In the present study, all the fi-lactam antibiotics, with the exception of cefonicid, cefuroxime and ceftizoxime, showed a clear epileptogenic activity. By analysing the structure-activity relationships of the
different compounds, it was found that derivatives with a six-membered dihydrothiazine ring, i.e. cephalosporins, displayed a more marked epileptogenic action than compounds having a five-membered thiazolidine ring i.e. penicillins. In fact, cefazolin was 2-4 times more potent than benzylpenicillin. Compounds having heterocyclic rings both at position 3 and position 7 of 7-aminocephalosporanic acid, such as cefazolin, ceftezole, ceftriaxone, cefoperazone, ceftazidime and cefamandole showed potent epileptogenic activity, more or less marked than that of benzylpenicillin. However, the epileptogenic activity of compounds characterized by having one heterocyclic ring at position 7 of 7-aminocephalosporanic acid, such as cefotaxime, cefonicid and ceftizoxime was less evident than that of benzylpenicillin. Aztreonam, which is characterized by having the only /I-lactam ring with a heterocyclic ring at 4 position, possessed convulsant properties similar to
362
A.
DE SARROet al.
Table 2. Convulsant doses (CD,) several cephalosporins after intraeerebroventricular injection. At least 32 animals were used to calculate each CD.,, value (f 95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods
7-Aminoxefalosporanic acid
COOH (Na) CD, @mol) 95% confidence limits
R,
Druns
N-N / Cefoperazone
\CHsS
>N
x
N
0.30 (0.24-0.37)
I CHr
0.43 (0.250.72)
Ceftriaxone
\
OCH,
\
Ceftazidime
CHs-N
6 3 -
’
0.29 (9.12-0.72)
I
C(CH,)rCOOH
NHa
\
CH,OCOCHs
Cefotaxime
‘H
those of cefoperazone and cefamandole. This suggests that the B-lactam ring is,an important structure responsible for the epileptogenic properties of the /I-lactam derivatives. Azlocilline and piperacilline, which belong to the ureidopenicillin group, showed weak epileptic activity in comparison to benzylpenicillin and cefazolin. This might be due to the largest size of the side chain associated with the ureidopenicillins. In fact, it has been reported that the modification of the side chains at the /3-lactam ring of benzylpenicillin is associated with a decrease
0.57 (0.21-1.54)
1.43 (1.20-1.71)
in epileptogenic potency (Gutnick et al., 1976). It seems possible that differences in the threshold concentration actually reflect differences in the abilities of these compounds to produce their effects on some neuronal receptors. In addition, the log-response curves for cefazolin and the other j?-lactam derivatives sometimes differed significantly from parallelism (see Figs 3 and 4) and this is consistent with the concept that the convulsant activity of these agents may involve different mechanisms. It should be emphasized that cefazolin and cefte-
Epileptogenic
effect of some b-lactam
compounds
363
Table 3. Convulsant doses (CD,) of some penicillins and aztreonam after intracerebroventricular injection. At least 32 animals were used to calculate each CD, values (f 95% confidence limits), according to Litchtield and Wilcoxon (19491 as described in Methods
CD, @mol) 95% confidence limits
Drugs &H,CON~~--~~:
Benzylpenicillin
0.052 (0.03-1.10)
COOH 0
Azlocilline
0.51 (0.35-0.65)
CHs
N-CONHCHCONH
Piperacilline
0.45 (0.31-0.65)
CHs COOH
0.12 (0.04-0.39)
Aztreonam
'0
I
C(CH,),COOH
7
q Cefazolin
F
l Catoperamne
6
0 cettrioxonr l Cettaridime l Cstotaxime A Cettizoxime
0.01
a033
0.1
0.33
Dose
(pmol 1
1
3.3
Fig. 2. Dose-response curves of intensity of seizures after intracerebroventricular injection of derivatives of /34actam. Each point represents the mean score for 8-10 animals. The seizure. response after administration of derivatives of B-lactam was scored as described in Methods.
A. DE SARROet al.
364
_ -
l Ceforolin 0 Cefterck v Cefuroxime l Aztreonam l CefamondoLe 0 Cefonicid ABenzyLpeniciUin
I 0.01
I 0.033
QL 0.1
Dose
I 0.33
I
I
1
3.3
(pmol)
Fig. 3. Dose-response curves for clonus induced by @-lactam compounds after intracerebroventricular injection in rats. Each point represents the mean of 8-10 animals. The incidence of clonic. seizures after administration of derivatives of fi-lactam was used to calculate CD, values (195% confidence limits), according to Lit&field and Wilcoxon (1949) as described in Methods.
7-
l Cefozolin
A 0 A 0 .
Dose
(pm01
Ceftriaxone Ceftizoxime Ceftaridime Cefoperozone Cefotaxime
f
Fig. 4. Dose-response curves for clonus induced by @-lactam compounds after intracerebroventricular injection in rats. Each point represents the mean of 8-10 animals. The incidence of clonic seizures after administration of derivatives of @-lactam was used to calculate CD, values (+95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods.
zole are both tetrazol derivatives and show a marked similarity with the pentylentetrazol, a well known convulsant drug. Such a tetrazol group could be responsible for the greater convulsant activity of these two cephalosporins in comparison to the other derivatives. In addition, it is reasonable to presume that substitution of a heterocyclic ring at position 3 in the cephalosporins is more effective in increasing the convulsant potency than the substitution at position 7, as previously suggested by Kame, Sunami and Tasaka (1983). This view is also supported by the fact that the convulsant activity of cefazolin and ceftezole is greater than that of cefuroxime or
ceftizoxime. In contrast cefonicid which possess a heterocyclic ring at position 3, showed the weakest convulsant effect of all the compounds studied. REFERENCES
Anderson T. E. and Rutledge L. T. (1979) Inhibition in penicillin-induced epileptic foci. Electroenceph. din. Neurophysiol. 46: 498-509.
Curtis D. R., Game C. J. A., Johnston G. A. R., McCulloch R, M. and Maclachlan R. M. (1972) Convulsive action of penicihin. BIain Res. 43: 242-245. De Sarro G. B., Cal6 M., Bagetta G., Anfosso R., Marmo E., Nistico G. and Guarino V. (1983) Comparative epileptogenic properties of cefazolin and benzylpenicillin
Epiieptogenic effect of some /I-lactam compounds after intracaudate microinjection in rats. Acta phurmac, sin. 4: 236-238.
D~ngl~ine R. and Gjerstad L. (1980) Reduced inhibition during epiieptiform activity in the in vitro hippocampal slice. J. Physiol. 305: 291-313. Gerald M. C., Massey J. and Spadaro D. C. (1973) Comparative convulsant activity of various penicillins after intracerebral injection in mice. J. Pharm. Pharmac. 25: 104108. Gloor P. and Testa G. (1974) Generalized penicillin epilepsy in the cat: effects of intracarotoid and intravertebral pentylentetrazol and amobarbital injections. EIecrrokce$h.
clin. Neurophysiol. 36: 499-515.
Gutnick M. J., Van Duijn H. and Citri N. (19%) Relative convulsant potencies ofstructuralanalogues of penicillin. Brain Res. 114: 139-143. Hartesveldt C. V., Petit T. L. and Isaacson R. L. (1975) Epilepto~nic effects of several ~nicillins and penicillin-related compounds in rat neocortex. Epilepsiu 16: 449-45s.
Kame C., Sunami A. and Tasaka K. (1983) ~pilepto~nic activity of ~ephalosp~~ in rats and their structureactivity relationship. Epilepsiu 24: 431439. Litchfield J. T. and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. >harmac. exp+ Ther. 96: 99-113. MandelI G. L. and Sande A. M. (1985) Penkiilins, cephalosporins, and other beta-lactam antiobiotics. In: The Pharmacological Basis qf Therapeutics (Gilman A. G., Goodman L. S., Rall T. W. and Murad F., Eds), pp. 1115-1149. MacMillan, New York.
365
Nistico G., Musolino R., De Sarro G. B., Gall&to G. and Di Perri R. (1979) Effetti epilettogeni della cefazolina in varie specie animali. Riv. Ir. E~eftroenc. ~euro~s~of. 2: 519-523. Nisticb G., De Sarro G. B., Naccari F., Musolino R., Rotiroti D., Gallitto G. and Di Perri R. (1980af Cefazolin: a valid model of experimental epilepsy? In: Epilepsy. A C&nicaf and experimental Rest-arch. ~%#onogr. Neural. Sci., Vol. 5, pp. 14-19. Karger, Base].
Nistico G., De Sarro 6: B., Rotiroti D., Silvestri R. and Marmo E. (1980b) Antagonism by classical antiepileptic and sodium valproate of cefazolin induced experimental epilepsy in rats‘bes. Commun. &em. Path., Pharmac. 29: 429-444. Paxinos 6. and Watson C. (1982) The Rat Bruin in Sfereotaxic Coordinales. Academic Press, New York. Quintiliani R., Nightingale C. H., Rossi J. G. and Ristuccia A. M. (1984) Ceph~osporins: an overview. In: Antimt’crobia! Thera&(Ristuccia A. M. and Cunha B. A., Edsf, pp. 289-303. Raven Press, New York. Rotiroti D., De Sarro G. B., Musolino R. and Nistic6 G. (1983) A new model of excremental epilepsy: the cefazolin-induced epilepsy. In: Epilepsy, an Update of Research and Therupy (Nistico G., Di Perri R. and Meinardi H., Eds), pp. 129-144. Alan Liss, New York. Van Gelder N. M., Siatitsas I., Menini C. and GIoor P. (1983) Feline generalized penicillin epilepsy: changes of glutamic acid and taurine parallel the progressive increase in excitability of the cortex. Epiiepsiu 24: 200-213.
Copyright
0028-3908/89 $3.00 + 0.00 0 1989 Pergamon Press plc
EPILEPTOGENIC ACTIVITY OF SOME /3-LACTAM DERIVATIVES: STRUCTURE-ACTIVITY RELATIONSHIP A. DE SARRO,* G. B. DE SARRO, C. Ascron and G. NISTI& Institute of Pharmacology, University of Messine, Piazza XX Settembre 4, 98122 Messine and Institute of Pharmacology, University of Reggio Calabria, Policlinico Mater Domini, via T. Campanella, 88100 Catanzaro, Italy (Accepted 6 Ocrober 1988) Summary-The epileptogenic activity of several derivatives of fl-lactam was compared following their intracerebroventricular administration in rats. At a dose of 0.033 pmol/kg cefazolin was the most powerful epileptogenic compound among the drugs tested; dramatic seizure signs (nodding, clonic convulsions and sometimes escape responses) were observed repeatedly. It was approximately three times more potent than ~nzyl~nicillin and other similar compounds, such as ceftriaxone, cefoperazone and cefamandole. No epileptogenic signs were observed with equimolar doses of cefotaxime, cefonicid and ceftizoxime. All these derivatives differ from the substitution at position 3 and at position 7 of 7-aminocephalosporanic acid. The more convulsant compounds (i.e. cefazolin and ceftezole) are both derivatives of tetrazol and show a marked similarity with pentylentetrazol. In addition, aztreonam, a compound having only the /I-lactam ring substituted with a heterocyclic ring at the 4 position, possessed convulsant properties like those of cefoperazone and cefamandole. This suggests that the j%lactam ring is able to produce epileptogenic activity and it seems likely that substitutions at the 7-aminocephalosporanic or &aminopenicillanic acid may increase or reduce the epileptogenic properties of /I-lactam derivatives. Key rtTor&-seizures,
penicillins, cephalosporins, aztreonam, epilepsy.
The administration of /J-lactam antibiotics (i.e. cephalosporins and penicillins) into the cerebral ventricles, subarachnoidal space or directly onto the cerebral cortex of chicks, cats, dogs, monkeys, mice, rats and man has been reported to induce a focal or generalized epileptic state, initially characterized by spike and wave discharges which are followed by clear tonic-clonic or clonic seizures (Gloor and Testa, 1974; Anderson and Rutledge, 19’79; Nistico, Musolino, De Sarro, Gallitto and Di Perri, 1979;
Nistico, De Sarro, Naccari, Musolino, Rotiroti, Galhtto and Di Perri, 1980a; Nisticb, De Sarro, Rotiroti, Silvestri and Marmo, 1980b; De Sarro, Calo, Bagetta, Anfosso, Marmo, Nistico and Guarino, 1983; Rotiroti, De Sarro, Musolino and Nistico, 1983; Van Gelder, Siatitsas, Menini and Gloor, 1983; Gerald, Massey and Spadaro, 1973). The structural similarity of penicillin with the y-aminobutyric acid antagonist, the convulsant drug bicuculline is of interest (Curtis, Game, Johnston, McCulloch and Maclachlan, 1972), since the decrease in inhibition in the hippocampus produced by penicillin contributes greatly to its epileptogenicity (Dingledine and Gjerstad, 1980). The cephalosporins are a family of B-lactam antibiotics that generally contain the 7-aminocephalosporanic acid nucleus. They differ in their basic structure from the penicillins in that the cephalosporins contain a six-membered dihydrothiazini~ ring instead of a five-membered *To whom correspondence should be addressed.
thiazolidinic ring fused to the /I-lactam portion (Quintiliani, Nightingale, Rossi and Ristuccia, 1984). The ~phalospo~ns resemble penicillins both in their actions as antibiotics (Mandell and Sande, 1985) and as convulsant compounds (Rotiroti et al., 1983). Several authors found that the ability of derivatives of penicillin to produce a seizure focus was abolished after incubation with ~nicillinase, an enzyme which splits the /I-lactam ring of penicillins (Gerald et al., 1973; Hartesveldt, Petit and Isaacson, 1975; Gutnick, Van Duijn and Citri, 1976). However, these studies tend to support the fact that the /I-lactam ring is an essential structural feature for the epileptogeni~ activity of penicillins. Using these @-lactam derivatives, it was of interest to compare their epileptogenic activity and to investigate to which type of radical, applied to the basic structure, the epileptogenic properties of these compounds could be related. Thus, the aim of the present study was to assess the behavioural epileptogenic effects of some new cephalosporins and penicillins after acute injection into a lateral ventricle in freely moving rats. METHODS
Adult male Wistar rats (ZO(r250 g), Charles River (Calco, Como, Italy) were stereotaxically implanted with stainless steel guide cannulae under chloral hydrate anaesthesia (400 mg/kg, Lp.). according to the atlas coordinates of Paxinos and Watson (1982), 359
360
A.
DE SARRO et al.
to permit injection of drugs into the left or right lateral cerebral ventricle. After surgery a minimum of 48 hr was allowed for recovery before the experiments were carried out. Freely moving rats were injected (0.2 pl/min) through the injection cannula which extended approximately 1 mm below the tip of the guide cannula. The animals were placed individually in transparent cages (40 x 25 x 25), and allowed 30min to acclimatise to the new environment. Preliminary studies to ascertain the influence of phosphate buffer solution 67mM, in a volume of 2 and 6.6 ~1, showed no significant behavioural changes. The volume of 6.6 ~1 was used only when it was necessary to inject 3.3 pmol of some p-lactam derivatives (i.e. cefonicid, cefuroxime, cefotaxime, ceftizoxime and piperacilline). Each rat was used only once and at least 8 animals were used for each dose level studied. The seizure response after injection of cephalosporins, penicillins and aztreonam was graded according to the following scale: 0 = normal behaviour; 1 = hyperkinesia, wet-dog shakes and sniffing; 2 = head-tremors and nodding; 3 = clonus of limbs; 4 = clonus of four limbs and/or trunk; 5 = clonus of fore limbs, rearing and falling; 6 = transient tonic-clonic seizures and escape response; 7 = generalized seizures followed by postictal period of fatal outcome. The convulsant doses required to induce a seizure response in 50% of rats (CD,,), injected with /I-lactam compounds and the relative confidence limits were determined according to Litchfield and Wilcoxon (1949). At least 32 animals were used to calculate each CD,, values. The sources of the drugs used were: chloral hydrate, sodium cefazolin and sodium ceftizoxime (Farmitalia Carlo Erba, Res. Labs., Milan, Italy); sodium cefotaxime (Hoechst Frankfurt, Germany); sodium ceftazidime (Sigma-Tau, Pomezia, Italy); so-
dium ceftezole (Schering, Berlin, Germany); sodium ceftriaxone (Hoffman-La Roche, Basel, Switzerland); aztreonam and sodium sodium cefoperazone (Menarini, Res. Labs., Florence, Italy); sodium cefamandole (Eli Lilly & Co, Indianapolis, U.S.A.); sodium cefuroxime (Glaxo, Res. Labs., Verona, Italy); sodium cefonicid (ISF, Milan, Italy); sodium benzylpenicillin (Merck, Sharp & Dohme, Munchen, Germany); sodium azlocilline (Bayer Leverkusen, Germany); sodium piperacilline (Cyanamid, Res. Labs., Catania, Italy). All drugs in the powder form were dissolved in buffer phosphate solution and injected at pH 7.3-7.4. RESULTS
The epileptogenic properties of the p-lactam antibiotics varied according to the type of radical groups attached to the basic structure (Tables l-3). The behavioural seizures consisted of an initial phase, characterized by wet-dog shake episodes followed by head tremor, nodding and clonus of the limbs. After the largest doses of b-lactam antibiotics, clonus of four limbs and/or trunk, rearing, jumping, falling down, transient tonic-clonic seizures, escape responses and generalized seizures, followed by a postictal period or fatal outcome were also evident. All these epileptic signs were dose-related and were observed repeatedly only with short interruptions. Generally, the first behavioural signs were observed within 1.5 min after the infusion and the maximum effect was reached after a minimum delay of 9 min after the injection. Dose-response curves of the epileptiform effects induced by intracerebroventricular injection of benzylpenicillin, some cephalosporins and some ureidopenicillins are shown in Figures 1 and 2; and in Figures 3 and 4. As shown in Figures 1, 2, 3 and 4, cefazolin exhibited the greatest activity
* Cefazolin
A Ceftezole 0 BenzyLpenicilh
7
. Aztreonam l Cefamondole
6
0 + 0 A
5 2 0
Azlocilline PlperaciNine Cefuroxime Cefonicid
t
Dose (@mot) Fig. I. Dose-response curves of intensity of seizures after intracerebroventricular injection of derivatives of B-lactam. Each point represents the mean score for 8-10 animals. The seizure response after administration of derivatives of b-lactam was scored as described in Methods.
Epileptogenic
effect
of some /I-lactam
361
compounds
TableI.Convulsant doses (CD,) of several cephalosporins after intracerebroventricular injection. At least 32 animals were used to calculate each CDw value (+ 95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods 7-Aminocefalosporanic
acid
bOOH(Na) Drugs
RI
Rl
CD., ,"(umol) I. 95% confidence limits
N-CHI 0.017 (0.012-0.024)
\ Cefazoline
N-CHz Ceftezole
0.04 (0.018~.09)
N;/) \ N
Cefamandole OH
/_\ i ct OH
\
0.29 (0.16-0.55) CH,S
8.40 (n.d.)* 'CHZS
Cefuroxime 'CH,OCONH,
3.31 (0.99-I I .07)
*The CD, value for cefonicid was extrapolated and so the 95% confidence limits could not be determined. n.d. = not determined.
among the drugs tested, whereas benzylpenicillin was 2-4 times less potent. In addition, ceftezole, astreonam, cefoperazone, ceftriaxone, cefamandole, ceftazidime, cefotaxime, ceftizoxime and cefuroxime were in this order less potent than cefazolin in inducing epileptic behavioural signs. In fact, very large doses as 1.65 and 3.3 pmol of cefotaxime and ceftizoxime were necessary in order to produce clonus of the fore limbs, rearing, falling down or transient tonic-clonus seizures and escape responses. No clear signs of behavioural seizures were noted even after the administration of 1.0 and 3.3 pmol of cefonicid. DISCUSSION
In the present study, all the fi-lactam antibiotics, with the exception of cefonicid, cefuroxime and ceftizoxime, showed a clear epileptogenic activity. By analysing the structure-activity relationships of the
different compounds, it was found that derivatives with a six-membered dihydrothiazine ring, i.e. cephalosporins, displayed a more marked epileptogenic action than compounds having a five-membered thiazolidine ring i.e. penicillins. In fact, cefazolin was 2-4 times more potent than benzylpenicillin. Compounds having heterocyclic rings both at position 3 and position 7 of 7-aminocephalosporanic acid, such as cefazolin, ceftezole, ceftriaxone, cefoperazone, ceftazidime and cefamandole showed potent epileptogenic activity, more or less marked than that of benzylpenicillin. However, the epileptogenic activity of compounds characterized by having one heterocyclic ring at position 7 of 7-aminocephalosporanic acid, such as cefotaxime, cefonicid and ceftizoxime was less evident than that of benzylpenicillin. Aztreonam, which is characterized by having the only /I-lactam ring with a heterocyclic ring at 4 position, possessed convulsant properties similar to
362
A.
DE SARROet al.
Table 2. Convulsant doses (CD,) several cephalosporins after intraeerebroventricular injection. At least 32 animals were used to calculate each CD.,, value (f 95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods
7-Aminoxefalosporanic acid
COOH (Na) CD, @mol) 95% confidence limits
R,
Druns
N-N / Cefoperazone
\CHsS
>N
x
N
0.30 (0.24-0.37)
I CHr
0.43 (0.250.72)
Ceftriaxone
\
OCH,
\
Ceftazidime
CHs-N
6 3 -
’
0.29 (9.12-0.72)
I
C(CH,)rCOOH
NHa
\
CH,OCOCHs
Cefotaxime
‘H
those of cefoperazone and cefamandole. This suggests that the B-lactam ring is,an important structure responsible for the epileptogenic properties of the /I-lactam derivatives. Azlocilline and piperacilline, which belong to the ureidopenicillin group, showed weak epileptic activity in comparison to benzylpenicillin and cefazolin. This might be due to the largest size of the side chain associated with the ureidopenicillins. In fact, it has been reported that the modification of the side chains at the /3-lactam ring of benzylpenicillin is associated with a decrease
0.57 (0.21-1.54)
1.43 (1.20-1.71)
in epileptogenic potency (Gutnick et al., 1976). It seems possible that differences in the threshold concentration actually reflect differences in the abilities of these compounds to produce their effects on some neuronal receptors. In addition, the log-response curves for cefazolin and the other j?-lactam derivatives sometimes differed significantly from parallelism (see Figs 3 and 4) and this is consistent with the concept that the convulsant activity of these agents may involve different mechanisms. It should be emphasized that cefazolin and cefte-
Epileptogenic
effect of some b-lactam
compounds
363
Table 3. Convulsant doses (CD,) of some penicillins and aztreonam after intracerebroventricular injection. At least 32 animals were used to calculate each CD, values (f 95% confidence limits), according to Litchtield and Wilcoxon (19491 as described in Methods
CD, @mol) 95% confidence limits
Drugs &H,CON~~--~~:
Benzylpenicillin
0.052 (0.03-1.10)
COOH 0
Azlocilline
0.51 (0.35-0.65)
CHs
N-CONHCHCONH
Piperacilline
0.45 (0.31-0.65)
CHs COOH
0.12 (0.04-0.39)
Aztreonam
'0
I
C(CH,),COOH
7
q Cefazolin
F
l Catoperamne
6
0 cettrioxonr l Cettaridime l Cstotaxime A Cettizoxime
0.01
a033
0.1
0.33
Dose
(pmol 1
1
3.3
Fig. 2. Dose-response curves of intensity of seizures after intracerebroventricular injection of derivatives of /34actam. Each point represents the mean score for 8-10 animals. The seizure. response after administration of derivatives of B-lactam was scored as described in Methods.
A. DE SARROet al.
364
_ -
l Ceforolin 0 Cefterck v Cefuroxime l Aztreonam l CefamondoLe 0 Cefonicid ABenzyLpeniciUin
I 0.01
I 0.033
QL 0.1
Dose
I 0.33
I
I
1
3.3
(pmol)
Fig. 3. Dose-response curves for clonus induced by @-lactam compounds after intracerebroventricular injection in rats. Each point represents the mean of 8-10 animals. The incidence of clonic. seizures after administration of derivatives of fi-lactam was used to calculate CD, values (195% confidence limits), according to Lit&field and Wilcoxon (1949) as described in Methods.
7-
l Cefozolin
A 0 A 0 .
Dose
(pm01
Ceftriaxone Ceftizoxime Ceftaridime Cefoperozone Cefotaxime
f
Fig. 4. Dose-response curves for clonus induced by @-lactam compounds after intracerebroventricular injection in rats. Each point represents the mean of 8-10 animals. The incidence of clonic seizures after administration of derivatives of @-lactam was used to calculate CD, values (+95% confidence limits), according to Litchfield and Wilcoxon (1949) as described in Methods.
zole are both tetrazol derivatives and show a marked similarity with the pentylentetrazol, a well known convulsant drug. Such a tetrazol group could be responsible for the greater convulsant activity of these two cephalosporins in comparison to the other derivatives. In addition, it is reasonable to presume that substitution of a heterocyclic ring at position 3 in the cephalosporins is more effective in increasing the convulsant potency than the substitution at position 7, as previously suggested by Kame, Sunami and Tasaka (1983). This view is also supported by the fact that the convulsant activity of cefazolin and ceftezole is greater than that of cefuroxime or
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