An experimental study on dependence liability of zipeprol

Pharmacological Research,

Vol. 21, No. 2, 1989

223

AN EXPERIMENTAL STUDY ON DEPENDENCE LIABILITY OF ZIPEPROL VINCENZO MORETTI, CAROLINA CONCETTONI, PIETRO GIUSTI and LAMBERTO RE Institute of Experimental and Clinical Medicine, University of Ancona, Via Ranieri 6, 60100 Ancona, Italy Received in final form 21 November 1988

SUMMARY Zipeprol, a piperazine ethanol derivative, is a non-essential but widely used (paediatric) antitussive, which is not legally considered as being capable of creating dependence or abuse liability . A first group of experimental results was obtained assaying the displacement of 1 nM [3 H]naloxone by zipeprol vs morphine on the rat brain homogenate fractions . A second group was carried out on the longitudinal muscle of guinea-pig ileum, using the field stimulation technique, either in the absence or in the presence of naloxone 1 x 10 -5 M or in the absence or in the presence of yohimbine 1x10-1m . Further investigations concerning the pharmacological and the biochemical characterization of the mechanisms involved in abuse liability were carried out by means of the in vitro inhibition of ACh response on the guinea-pig ileum preparation . The results indicate zipeprol as a moderate opioid agonist, which also shows a direct anticholinergic effect, independent of presynaptic a 2 interaction . KEY

woRDs : zipeprol, drug abuse, opioid, anticholinergic drug . INTRODUCTION

Zipeprol, a piperazine ethanol derivative, affects the central mechanism of cough . Studies of acute and chronic toxicity exclude severe side-effects on the cardiovascular system, on the intestinal transit and on the central nervous system [1, 2] . However, recent reports have proved one unquestionable causal connection between zipeprol and central excitatory effects such as seizures (in some cases followed by coma) in paediatric and adult patients [3, 4] . Hyperactivity of cortex and direct neurotoxic action, which are mostly evident with EEG tracings and computerized tomographic scanning of some cerebral areas, are ascribed to zipeprol overdose in young adult habitual abusers [5] . Young adult zipeprol abusers are frequently `historical' addicts [6] who claim that its effects resemble those of opioids [3] . This study therefore aims to verify the described non-opioid activity of zipeprol [ 1 ] and to investigate the mechanism responsible for its abuse liability . 0031-6989/89/020223-07/$03 .00/0

® 1989 The Italian Pharmacological Society



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Pharmacological Research, Vol. 21, No. 2, 1989

MATERIALS AND METHODS Non-opioid activity of zipeprol was verified by means of in vitro studies of [ 3 H]naloxone binding displacement on rat brain homogenate fractions and probing reversal twitch inhibitory effects of zipeprol on the longitudinal muscle of guineapig ileum by naloxone . [ 3 H]Naloxone binding assay

Male Sprague-Dawley rats weighing 200 ± 25 g were used for binding studies . After decapitation, the brain was rapidly dissected out, deprived of cerebellum, and homogenized, according to Pert and Snyder [7], in ice-cold 50 mm Tris-HCI buffer (pH 7 .4 at 22°C) with an Ultraturrax TP 18/10 for 30 s . After centrifugation at 49 000 g for 15 min at 4°C, the resulting pellets were resuspended in the same buffer and preincubated for 30 min at 37°C . After further centrifugation as described above, the pellets were suspended in a volume of buffer so that the resulting protein concentration was 1 mg/ml . Protein concentration was measured using the Lowry method [8], with bovine serum albumin as a standard . Triplicate samples were then prepared in plastic polypropylene tubes incubating 0 . 5 ml of brain membrane suspension for 45 min at 25°C with 1 nM [3H]naloxone and scalar doses of morphine • HCI or zipeprol • HCI, in the presence or in the absence of NaCl 50 mm, with continuous shaking during the reaction time. Reaction was stopped by rapid filtration through Whatman GF/B glass-fibre filters, 2 . 5 cm in diameter, with three 5 ml washes of ice-cold 50 mm Tris-HCl buffer . Radioactivity bound to filters was determined by liquid scintillation spectrometry in a ß-counter (Beckman LS 1800), after 18 hours at rest . Specific binding was defined as the difference in binding in the absence and presence of 1 µM naloxone. Preliminary binding studies were carried out by incubating aliquots of freshly prepared homogenate with eight scalar concentrations of [ 3H]naloxone (ranging between 0 . 25 and 10 nM) to determine Bma,, (maximal saturable binding) and Kd values . Guinea-pig ileum (GPI) and strips of longitudinal muscle-myenteric plexus (LM-MP) studies

Albino male guinea-pigs weighing 400 ± 50 g, receiving water and food ad libitum until at least 15 h before experiments, were used . After sacrifice by a blow on the neck and exsanguination, strips of about 4 cm of ileum, at a distance of at least 15 cm from the terminal ileum, were rapidly excised and bathed at 37°C in Tyrode solution pH 7 . 4, and bubbled with a mixture of 95% 0 2 and 5% CO 2. The LM-MP preparation was obtained from 3 cm segments of ileum according to Kosterlitz et al [9] and Kitchen [10], and mounted in 10 ml thermostatic organ bath bubbled as above . The strips were allowed to equilibrate for 2 h at a resting tension of 0 . 5 g and therefore stimulated applying an electrical field between two platinum ring electrodes with an AG 41 Stimulator using repeated pulses of 60 V of 5 ms and at a frequency of 0 .1 Hz .



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Chemicals. The zipeprol • HCl was a generous gift of Sigma-Tau, Pomezia, Italy ; the acetylcholine bromide (ACh) was obtained from Farmitalia, Milan, Italy ; the yohimbine by Simes, Milan, Italy . The morphine-HCl and naloxone • HCl were purchased from the Sigma Chemical Company, St . Louis, USA; the [3H]naloxone from the NEN Research Products, Dreieich, W . Germany ; the bovine serum albumin from the Fluka AG., Buchs, Switzerland .

RESULTS Preliminary [3H]naloxone binding studies performed to determine the Bmax and Kd values by Scatchard plot gave, in our preparation, the following results : 324 fmol/ mg of protein and 2 . 77 nM; 509 fmol/mg of protein and 1 . 8 nM in the absence and in the presence of 50 mm Na* respectively . Zipeprol competition for 1 nM [3 H]naloxone binding on brain opioid receptors was unexpectedly observed even if at doses 300 times greater than morphine ; Na' 50 mm, according to De Montis et al. [ 11 ] and Ishizuka and Oka [ 12], increased the IC50 morphine and zipeprol values six and two times respectively, suggesting an agonistic trend, for zipeprol as well (Table I and Fig . 1). Approximately the same Hill coefficient was obtained for [3H]naloxone displacement by morphine or zipeprol . Table I Inhibitory effect of morphine and zipeprol on binding of 1 nM [3H]naloxone measured in rat whole brain in the presence or in the absence of 50 mm NaCl Agonist

IC50

n

x

t- test

Hill coefficient SD t- test

P'<

i

5 . 3x10 -9 3. 07

0.05

0.60

0 . 15

Morphine+ 4 3 .84x10-8 1-39X 10 -8 5. 51 NaC150 mm

0.02

0. 56

0 . 11 10 .43 0 .01

4 2 . 19x10 -6 5 . 12x10 -' 8. 59

0.01

0 .67

0 . 12 10 . 71 0 . 01

Zipeprol+ 4 4-81x10-1 2-36x10-5 4. 08 NaCI 50 mm

0. 05

0 . 70

0 . 16

Morphine

Zipeprol

6 6 . 65 x10 -9

SD

P<,

9 . 92 0 .001

8 .65 0 .

Each IC 50 value was obtained from the Hill-plot on 12 scalar concentrations of drug . However, in the guinea-pig LM-MP preparation, in conditions of field stimulation, the inhibition elicited by zipeprol was not counteracted by naloxone 1 x 10 -5 M or more . Even pretreatment with naloxone 1 X 10 -5 M can not prevent the zipeprol inhibitory effect (Fig . 2). An a2-presynaptic interaction, or a direct anticholinergic effect, has been postulated to justify the opioid unrelated inhibitory effect on LM-MP preparation .



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`c d

E a) U o â a

o

Log

(M)

Fig . 1 . Competition curves of morphine and zipeprol for the binding of 1 nM [3H]naloxone. The experimental data obtained assaying 12 increasing concentrations of morphine ( •), morphine +NaC1 50 mm (A), zipeprol (0), zipeprol + NaCl 50 mm (*), were fitted according to the following function : y=100/(1 + K X 10x'") where x is the log (M) of drug, y is the % displacement, n is the Hill constant, and K is the affinity constant (K x when n is equal to 1).

(a)

T Naloxone Zipeprol I x 10 -5 M 4 x IO -6 M

(b)

T T T Zipeprol Noloxone -5 M Naloxone -5 M 4xIO -6 M Ix10 2x10

Fig . 2 . Naloxone does not modify the zipeprol induced inhibition of the guinea-pig ileum LM-MP contractile response to field stimulation, either if the strip was preincubated for 3 min with naloxone and then zipeprol added (a), or if naloxone was added after zipeprol pretreatment (b) . The first hypothesis was verified by comparing the effects of zipeprol on LM-MP preparation (at a dose close to the IC 50 ) and clonidine 5 X 10 -8 M, after pretreatment with yohimbine 1 X 10 -5 M . The results indicate that zipeprol induced inhibition is not prevented by yohimbine, excluding an a2 presynaptic interaction .



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Guinea-pig ileum preparations had been employed to investigate the second hypothesis . Increasing concentrations of ACh (ranging between 1 x 10 -8 M and -1 1 x 10 M) promote a dose dependent twitch that is significantly reduced in the presence of zipeprol 2 x 10 - I M or 5 x 10 -6 M, and more evident both for lower doses of ACh and for greater doses of the inhibitory drug (Figs 3 and 4) .

• Control (ACh)

-,

ACh + zipeprol 2 x 10 M 0 ACh+zipeprol 5x10 -6 M

Fig. 3.

Anticholinergic effect of zipeprol at two different doses on GPI preparation . The dose dependent twitch induced by ACh is significantly reduced by zipeprol with atropine-like mechanism .

• Control (ACh) A

ACh + zipeprol 2 x 10-6 M

O ACh + zipeprol 5 x 10 -6 M

-8 + Log (M)

Fig. 4 . Hill plots of anticholinergic effect of zipeprol on GPI preparation, obtained from 10 increasing doses of ACh plotted in Fig . 3 . Function for control ( •) were : y= 0 . 593 -1 . 168x, for ACh+zipeprol 2 x 10 -6 M (A): y=0 . 590-0 . 727x, for ACh+zipeprol 5 X 10 -6 M (0) : y=0 . 866-0 . 841x, (R=0 . 982, R=0 . 955, R=0 . 988 respectively) .



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DISCUSSION The present data show that zipeprol has an anticholinergic direct effect on GPI and LM-MP preparations and has a moderate central opioid affinity . Our results as in the case of opioid affinity agree with those of Chau et al. [13], who noted that the cough suppressant effects of opioids are mediated by receptors (,u and k type) which are less stereoselective and less naloxone-sensitive than the analgesic receptors. Even the binding of the most effective antitussive drug (( -)codeine), is slightly affected by the presence of Na+ . Moreover, our data suggest that zipeprol acts with different mechanisms on cerebral and peripheral areas . Adopting the Schulz and Goldstein [14] model for explanation of neuronal mechanisms involved on contractions of the longitudinal muscle induced by electrical stimulation, and excluding an opioid interaction, other hypotheses can be formulated . Among these, the following had been investigated : 1 . Zipeprol, similarly to clonidine, is an a 2-agonist showing inhibitory effect on the cholinergic neuron innervating smooth muscle . 2. Zipeprol is an anticholinergic drug acting directly on smooth muscle (with an atropine-like mechanism) . A direct anticholinergic action was actually observed on the smooth muscle, but this action could not be directly related to the understanding of the central effects such as abuse liability . Obviously, more than one mechanism can be involved on the antitussive central effects of the drugs . Furthermore the possibility that the antitussive effects of zipeprol could be mediated by the binding on some 'opioid' receptors distinct from the analgesic ones (as for other opioid antitussive drugs) can not be excluded . With higher doses of the drug, as those taken in overuse or abuse, which may be responsible for the psychotropic effects that a drug addict wants, the analgesic naloxone-sensitive receptors could be involved, even if with a lesser degree of affinity, while the seizures observed in some poisoning cases, probably related to a GABA synthesis inhibition [4], could be associated with the piperazine metabolites . Further study is underway to provide a detailed explanation of the abuse liability mechanisms of the drug .

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5 . Moroni C, Cerchiari EL, Gasparini M, Rota E. Overdosage of zipeprol, a non-opioid antitussive agent . Lancet 1984 ; is 45 . 6 . Rossini L, Moretti V. Rilevamento in fase iniziale di stato di dipendenza da farmaci . Nuovo Boll Farmacol Clin 1986 ; 9 : 355-65 . 7 . Pert CB, Snyder SH. Opiate receptor binding of agonists and antagonists affected differentially by sodium. Molec Pharmacol 1974 ; 10 : 868-79 . 8 . Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent . JBiol Chem 1951 ; 193 : 265-75 . 9 . Kosterlitz HW, Lydon RJ, Watt AJ . The effects of adrenaline, noradrenaline and isoprenaline on inhibitory a- and ß-adrenoceptors in the longitudinal muscle of the guinea-pig ileum. BrJ Pharmacol 1970 ; 39 : 398-413 . 10 . Kitchen I. Textbook of in vitro practical pharmacology. 1st ed . London : Blackwell Scientific, 1984: 52 . 11 . De Montis MG, Devoto P, Bucarelli A, Tagliamonte A . Opioid activity of lefetamine . Pharmacol Res Commun 1985 ; 17 : 471-8 . 12 . Ishizuka Y, Oka T . Regulation of opioid antagonist and mu, kappa or delta agonist binding by guanine nucleotide and sodium . Japan J Pharmacol 1984; 36 : 397-405 . 13 . Chau TT, Carter FE, Harris LS . Antitussive effect of the optical isomers of mu, kappa and sigma opiate agonists/antagonists in the cat . J Pharmacol Exp Ther 1983 ; 226 : 108-13 . 14 . Schulz R, Goldstein A . Morphine tolerance and supersentivity to 5-hydroxytryptamine in the myenteric plexus of the guinea-pig . Nature 1973 ; 244 : 168-9 .