Pharmacotoxicological aspects of levosulpiride

p/wmacological

Research, Vol. 31, No. 2,1995

81

PHARMACOTOXICOLOGICAL FRANCESCO

ROSSI*

ASPECTS OF LEVOSULPIRIDE and ANGELO

FORGIONE

Institute of Pharmacology and Toxicology, Faculty of Medicine and Surgery, Second University of Naples. Accepted 28 October 1994 Levosulpiride is the levorotatory enantiomer of sulpiride, a substituted benzamide indicated as an antipsychotic, antidepressant, antiemetic and antidyspeptic drug, as well as for the treatment of somatoform disorders. In vivo sulpiride displays a number of neuroleptic properties which it shares with all typical neuroleptic drugs; however, it has also a number of divergent characteristics that set it apart as the principal compound of the so-called ‘atypical neuroleptic agents’. The main mechanism of action of levosulpiride consists of blocking the DZ dopaminergic receptors, preferentially located on the presynaptic membranes in the dopaminergic pathways of the brain; this means that sulpiride is a selective autoreceptor blocker. The results of series of experimental trials conducted to evaluate the toxicologic characteristics of levosulpiride are presented. Both the acute, subacute, chronic and local toxicity trials, and the studies on reproduction toxicity, mutagenic potential and oncogenic/carcinogenic potential, demonstrate that levosulpiride is well tolerated by the animals tested (rats, mice, rabbits and dogs) at doses higher than those effective in human therapy. Moreover, the findings from the experimental studies on levosulpiride lead to exclude the toxicity from accumulation, tolerance, dependence or withdrawal syndrome. In conclusion, according to the evaluated preclinical studies, levosulpiride shows pharmacotoxicologic properties which make it suitable for the management of diseases for which the drug is indicated. KEYWORDS: sulpiride, levosulpiride,

dopaminergic

INTRODUCTION Levosulpiride is the levorotatory enantiomer of sulpiride, a substituted benzamide of established clinical use both in Europe and worldwide. Levosulpiride is a benzamide derivative selectively inhibiting the dopaminergic D2 receptors both in the nervous central system (CNS) and in the gastrointestinal tract [l-3]. Between the two enantiometers of sulpiride, the levo-enantiomer is the biologically active form, as shown by pharmacological studies in animals and in humans [3-51. The chemical structure of levosulpiride is S-(-)N-[ l-ethyl-2-pirrolidinyl)methyl]-5-sulfamoyl-2-methoxybenzamide (Figure 1). Levosulpiride is indicated as an antipsychotic, antidepressant, antiemetic and antidyspeptic drug, as well as for the treatment of somatoform disorders [ 11. While levosulpiride shares many characteristics of typical neuroleptic drugs, it also has a number of unique properties that make it an atypical neuroleptic agent.

neuronal pathways, pharmacotoxicologic

surveys.

Since sulpiride was developed in France in 1967 [6] a lot of interest eraised in studying this powerful antiemetic drug which very early showed to possess also interesting and marked antipsychotic properties with some characteristics-first of all the low incidence of side effects-different from those of such as phenothiazines, classical neuroleptics, butyrophenones and thioxanthenes. Like the classic neuroleptic drugs, sulpiride antagonizes a number of effects of dopaminergic drugs, including the emetic effect of apomorphine and L-DOPA in the rat, the sedative effect of apomorphine in man, and the hypotensive effect of apomorphine in man [l, 6-101. In addition, sulpiride, like

H&O Correspondence Naples, Italy.

to: Prof. Francesco

1043-6618/95/02008

Rossi, Via Broggia,

3, 80128

Fig. 1. lLl4/$08.00/0

Molecular formula of levosulpiride. 01995

The Italian Pharmacological

Society

82

Pharmacological Research, Vol. 31, No. 2,1995

B B V Fig. 2.

Central distribution of dopaminergic receptors and neuronal related circ ,uits.

phenotiazines and butyrophenones, induces an increase in dopamine turnover in the nigrostriatal, mesolimbic and mesocortical systems, as well as an increase in prolactin secretion both in laboratory animals and human subjects [ 11, 121. Sulpiride binds selectively and reversibly to the dopaminergic receptors as it could be demonstrated by the displacement of [3H]dopamine and [3H]haloperidol from the rat caudate nucleus membranes [ 131.

PHARMACODYNAMICS Central dopaminergic receptors are concentrated on three neuronal circuits, namely the nigro-striatal, the meso-limbic and the meso-cortical in the mammalian brain, though dopaminergic receptors occur along a variety of other central and peripheral structures [l]. The dopamine-containing cell bodies of dopaminergic neurons are localized mainly in the substantia nigra

Dopamine

Coding sequence Chromosome localization Highest brain density Pituitary Affinity for dopamine Characteristic antagonist Adenylcyclase Characteristic agonist Sulpiride-sensitivity

Mesolimbic fibres Mesocortical tibres Hypothalamic fibres

446

AA

5 q31-q34 Neostriatum

ventral compacta (A9 area) and in the adjacent tegmental area (A10 area). Although with a partial overlapping, a distinguishable topography differentiates the links of A9 and Al0 cells to three principal sets of target, i.e. the neo-striatum, which regulates motor activity, the limbic cortex and other limbic areas, which represent the emotional parts of the brain. Indeed, while the nigro-striatal system essentially originates from A9 cells, A10 neurons project rostrally to innervate the nucleus accumbens, olfactory tubercle, prefrontal cortex, antero-medial striatum and septum, among others (Figure 2) [l, 141. The introduction of radioligand-binding techniques to label dopaminergic receptors showed the existence of distinct receptors for dopamine with different biological and pharmacological specificity (Figure 3) [15]. The stimulation of D, receptors increases the activity of adenylcyclase resulting in cyclicAMP accumulation in the cells, while the stimulation of DZ receptors inhibits the enzyme [16]. D1 receptors are

Table I receptor subtypes

4144143 AA

400 AA

387 AA

11 q22-q23 Neostriatum

3 q13.3 Paleostriatum

11P Medulla frontal cortex Yes Submicromolar Clozapine ? ? Yes

No

Yes

No

Micromolar SCH-23390 Stimulates SKF-82526 No

Micromolar Haloperidol Inhibits Bromocriptine Yes

Nanomolar UH 232 ? Quinpirole Yes

477 AA 4 p16

Hippocampus No Submicromolar SCH-23390 Stimulates SKF-82526 No

pharmacological

Research,

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Vol. 31, No. 2, I995

Auroreeeptor on somatic dendrite (modulating impulse): regulates the neuron firing rate

Calmodulin-dependent regulatory protein

* Post-synaptic cell Fig. 3.

Scheme of a dopaminergic synapsis.

located prevailingly at postsynaptic site, while D2 are located either at presynaptic receptors (‘autoreceptors’) or at post-synaptic sites [ 171. Novel subtypes of dopamine receptors have been recently characterized (Table I) [ 18, 191. D3 receptors are found at high concentrations in limbic and cortical regions of the brain; these regions are typically associated with control of motivation, mood, emotion and more-complex behaviour [ 1,201. Some of these functions are disturbed in the positive psychotic symptoms of schizophrenia. Drugs that preferentially block D3 receptors might therefore be beneficial in psychosis. Atypical neuroleptics, such as levosulpiride, though do not seem to have a selective D3 receptor blocking activity, show to possess a more favourable D2/D3 ratio in their dopamine receptor blockade than typical neuroleptics. Sulpiride is a specific inhibitor of the DZ population of dopaminergic receptors. In particular, sulpiride binds specifically and stereoselectively to a subpopulation of D2 receptors which are typically

located on the presynaptic membrane and which have sodium-dependent functions [ 11, 121. This means that sulpiride is a selective autoreceptor blocker. As a consequence, sulpiride increases dopamine turnover in dopaminergic terminal areas, as defined by increased concentrations of homovanillic acid in the brain, without change in the concentrations of dopamine [ 2 1,221. In vivo sulpiride displays a number of neuroleptic properties which it shares with all typical neuroleptic drugs; however, sulpiride has also a number of divergent characteristics that set it apart as the principal compound of the so-called ‘atypical neuroleptic agents’. Its major advantages being the disinhibiting activity and the occurrence of sedative effects only at very high doses of the drug. Either in rodents [23,24] or in humans [25,26] a complete prevention of apomorphine-induced was levosulpiride sedation observed after pretreatment. In man the pretreatment with a single dose of sulpiride completely prevented all the effects

84

(sedation, sleep, hypotension, nausea and vomiting) induced by apomorphine, a dopaminomimetic drug, while after the same dose of o-sulpiride the subjects studied still complained of nausea, sedation and drowsiness. In rodents 2-5-fold-higher doses of Dsulpiride are required to slightly counteract the sedative effects of apomorphine [27]. Sulpiride induces dose-dependent behavioural effects: while it inhibits apomorphine-induced hyperactivity at low doses, the drug blocks apomorphine-induced stereotypes and causes only slight catalepsy at high doses [28]. The dose-related effect of levosulpiride on these different behaviours may be explained as follows: limbic structures are influenced by levosulpiride by doses lower than those influencing striatum, responsible of extrapiramidal side effects, differently from haloperidol, which affects both structures at the same dosage. n-Sulpiride seems to be devoid of any antagonistic activity versus apomorphine-induced stimulatory effects [27, 281. The potent antiemetic activity of L-sulpiride was demonstrated either in comparison with o-sulpiride and racemic sulpiride in humans [27] and dogs [29] in apomorphine-induced emesis, or in comparison with other effective antidopaminergic drugs in squirrel monkeys [30]. In apomorphine-induced emesis Lsulpiride was several times more potent than Dsulpiride and racemic molecule. So, the data generated in the experimental studies clearly show that L-sulpiride is effective at lower doses and concentrations than the racemic mixture. The pharmacologic advantage of agents with a high intrinsic activity consists in the fact that the therapeutic objectives can be met at comparatively lower tissues concentrations. This lowers the risk of interactions with non-specific receptors (particularly in amigdala and nucleus accumbens), for which classical antidepressant drugs also demonstrated high affinity, and, still more important, it decreases the incidence of side effects that occur independently of the principal action of the drug [ 111. As a matter of fact, a 50% or more reduction of the dosage is one of the most important arguments, but there are several considerations to be made. CNS and gastrointestinal effects of D-sulpiride have not been demonstrated to date while the racemate effects conform to those of levosulpiride. Unlike o-sulpiride, levo-sulpiride is highly effective in all peripheral processes mediated by Dz-receptors. In other, non-Dzreceptor-mediated peripheral processes, such as in the reversion of fenoldopam (D,-agonist) triggered suppression of canine chronotropism induced by stimulation of stellate ganglion [31], in the reversion of inhibition induced by fenoldopam on ganglionic transmission of the superior cervical ganglion in the rat [32], in the inhibition of DA-induced forearm vasodilation in man, after a-adrenoceptor blockade with phenoxybenzamine [33], in the inhibition of fenoldopam-induced forearm vasodilation in man

Pharmacological Research, Vol. 31, No. 2.1995

[34, 351, and in the inhibition of ibopamine-induced diuresis in anaesthetized rats [36], it has been demonstrated that o-sulpiride is the active enantiomer, whereas levosulpiride is much less potent. Since there is general consensus that the therapeutic effect of sulpiride is solely attributable to its affinity for Dz receptors, adverse drug reactions of the dextroenantiomer may be avoided by administering only the levoenantiomer.

PHARMACOKINETICS In rats treated with sulpiride 200 mg kg-’ by oral route the pharmacokinetic parameters were the following [37]: C,,, 6.8 pg ml-‘; t,, 3 h; tin 1.4 h; bioavailability 15%. The low bioavailability of sulpiride following oral administration (about 15%) is due not to the metabolism in the liver, but to a reduced absorption by the gastrointestinal tract [37]. Many metabolites were obtained from rats and dogs orally treated with sulpiride. Particularly, in rats metabolites represented 56% of the administered drug (60 mg kg-‘) and 38% of them were conjugates. In the dog the metabolites represented 15% of the administered sulpiride (50 mg kg-‘), of which 4% were conjugates. However, none of the metabolites documented in these species was found in human urine [38]. In the Rhesus monkeys treated with sulpiride at 10 mg kg-’ by oral or intravenous route, in both the urine and the bile were documented the following fractions of the administered dose: 60-80% sulpiride, lo-30% 5-oxopyrrolidine sulpiride and 3-8% unidentified metabolite [39]. After intramuscular administration (50 mg) to healthy volunteers, the bioavailability of t_-sulpiride is practically complete (about 99%), whereas the oral bioavailability of the same dosage is about 30% [39,40]. The half lives are 6.2, 8.3 and 9.7 h respectively, for the intravenous, intramuscular and oral administration (50 mg), with a total clearance of about 260 ml min-‘. Sulpiride is not bound to plasma proteins to any large extent. In dog plasma 16% of the sulpiride is bound over a concentration range of 1 to 50 pg ml-‘. In human plasma 14% is bound over the same concentration range. Thus the protein binding is constant and independent of sulpiride concentrations in dog plasma and human plasma [38,40,41]. The results of a comparative pharmacokinetic study after single oral administration of D-, L- and racemic sulpiride demonstrated that the pharmacokinetic profiles are largely identical (Fig. 4) (unpublished data). Also the urinary excretion is similar for racemic and L-sulpiride and accounts for about 65-70% of the dose after intravenous administration, totally as unchanged drug [40,4 I]. The mean renal clearance is in the range 260-3 10 ml min-’ after intravenous administration of

Pharmacological Research, Vol. 31, No. 2,1995

$

85

Mice, rats and rabbits were used to study acute toxicity while rats, rabbits and dogs were utilized for subacute toxicity studies; rats, rabbits and dogs were used to study chronic toxicity. The dosages used to study acute, subacute and chronic toxicity purposely exceeded those normally adopted to achieve pharmacologic effects.

120.0

2 kJ 90.0 3 $ 8 2 8

60.0

Single dose toxicity 30.0

f 0

8.0

16.0

24.0

32.0

40.0

48.0

Time (h)

Fig. 4. Pharmacokinetics of levo- (L), dextro- (D) and racemic (R) sulpiride in man (n=12) [37]. Cl, L 25 mg; *, R 50 mg; x, L 50 mg; A, R 100 mg; 0, D 50 mg. the two drugs (L- and racemic-sulpiride) [40,41] and suggests a tubular secretion of sulpiride since clearance exceeded glomerular filtration rate. This conclusion is also supported by data on metabolism of sulpiride [38, 391.

TOXICOLOGY The toxicologic study has been carried out keeping in mind ‘the biological features that relate to possible clinical use of the molecule. We report the findings of studies about toxicity of levosulpiride carried out by us and by other researchers. These international reports belong to the literature repertory held by the drug producer (Ravizza, Milan, Italy). We administered levosulpiride by intravenous and oral routes in rats, intravenously in rabbits and by oral and intraperitoneal routes in mice and rats.

Summary Drug

Trials on acute toxicity have been carried out on different animal species (mice, rats and rabbits) and using different administration routes (intravenous, oral and intraperitoneal). These studies demonstrated that the levosulpiride is well tolerated by all the animal species examined and by using different administration routes. In fact, signs of toxicity are encountered only with doses of some magnitude greater than those foreseen in therapy (22503250 mg kg-‘) (Table II). Racemic sulpiride administered by intraperitoneal route is responsible for an LD50 of 210 mg kg-i in mice and 270 mg kg-’ in rats. On considering that LDso is more or less the same for both racemic compound and levosulpiride, it appears evident that when only the active levoisomer is used in human therapy at half the racemic dosage, the toxic component of the compound is halved.

Subacute toxicity Rats, rabbits and dogs were used for subacute toxicity trials. Haematology and blood chemistry analyses have been selected to have an overall picture of the treatment effects on metabolism, on liver and renal functions, and on complete blood count. The necroscopy was carried out on major organs and apparatuses. This was performed on each animal, while each organ was subjected to histologic examination (Table III). The subacute oral administration of levosulpiride

Table II of LDso (mg kg-‘) values and minimal lethal dose (MLD, mg kg-‘) in mice, rats and rabbits using different administration routes Animal species

Sex

L-sulpiride L-supiride L-sulpiride L-sulpiride L-sulpiride L-sulpiride

rat rat rabbit rabbit rabbit rabbit

M F M F M F

L-sulpiride o-sulpiride L-sulpiride o-sulpiride L-sulpiride o-sulpiride L-sulpiride o-sulpiride

mice mice mice rat rat rat rat rat

M/F MF MP M/F M/F MF MP M/F

Administration

LDso

MLD

i.v. i.v. i.v. i.v. p.0. p.0.

53.65 54.83 41.97 42.52

45 45 35 35 >1500 >1500

p.0. p.0. i.p. i.p. p.0. p.0. i.p.

2450 2280 210 225 2600 2200 270 263

i.p.

route

-

2250 2250 175 175 1500 1500 225 225

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in creatinine kinase among males receiving racemic sulpiride after 13 weeks of treatment. This decrease was still apparent at the end of the recovery phase, particularly in males receiving the middle and highest dose levels. Indeed females receiving racemic of both receiving sulpiride and rats sexes levosulpiride were not affected. Several minor differences were noted between control and treated rats in pituitary and adrenal gland vagina/mammary gland hyperplasia and weights, vaginal mucification. It is considered that these changes, which show a marked degree of consistency for both Land racemic sulpiride, reflect a physiological response to treatment. These changes could be originated from an increase in circulating prolactin concentration. It is known that treatment with dopamine D2 receptor antagonist (such as L- or racemic sulpiride) may cause a blockade of the tubero-infundibular pathway, in which dopamine is believed to act physiologically, via Dz-receptors, as an inhibitor of prolactin secretion. The result of this blockade is responsible for an increase in circulating prolactin. It could be considered that an increased circulating level of this hormone could promote the observed vagina/mammary gland hyperplasia and vaginal mucification and may also give rise to the slight changes in pituitary and adrenal gland weights. L- and racemic sulpiride were administered orally (25-200 mg kg-‘), as capsules, to pure-bred beagle dogs for a period of 13 weeks. The study consisted of six groups, each containing three male and three female or five male and five female dogs. An additional group including five male and five female dogs served as control. Mortality did not occur in any experimental group. Initiation of treatment with L- or racemic sulpiride was associated in most dogs with the following clinical signs: decreased activity, tremor, abnormal

in rats at 150-300 mg kg-’ for 12 weeks is welltolerated as shown by the haematology, blood and does not chemistry and urine analysis, significantly differ from controls. There are, however, weight and food a slight reduction in body consumption and some variations in liver function at the highest dosage (300 mg kg-‘) The subacute administration of levosulpiride by subcutaneous route in rats at 12.5-25 mg kg-’ day-’ for 12 weeks is well-tolerated as shown by laboratory analysis. A slight drop in body weight and variations in liver functions were documented with highest dosage (50 mg kg-’ day-‘). Similar results were obtained by studying the effects of the subacute oral (125-250 mg kg-’ day-’ for 12 weeks) or intramuscular (6.25-12.5 mg kg-’ day-‘) administration of levosulpiride in rabbits. In another trial the subacute oral administration of levosulpiride in rats for 13 weeks at 25, 150 and 900 mg kg-’ day-’ and racemic sulpiride at 50, 300 and 1800 mg kg-’ day-‘, no toxic effect was identified for either compound. However, findings obtained for both tested compounds, notably clinical signs, clinical pathology and mammary gland/vaginal changes are considered to be related to the exaggerated pharmacological action of the compounds. The only toxic effect noted was mortality among females receiving the highest treatment level of racemic sulpiride. In particular, two females receiving the highest dose of racemic sulpiride died during the course of the study. These deaths as a result of treatment cannot be excluded. Clinical signs of somnolence were noted among rats of the highest dose groups of either test substance, were predominant among females, but they particularly those treated with racemic sulpiride. Blood chemistry investigations showed a decrease

Repeated-dose

toxicity

(subacute

Table III toxicity) of levosulpiride (mg kg-’ p.o.) in rats of both sexes. The treatment lasted 12 weeks (7 treatment days per week) Treatment (mg kg-‘)

Findings

Body weight Food comsumption Haematology Blood chemistry BUN Urine analysis Gross pathologic findings Histologic findings

12.5

25

50

150

300

600

900

1800

-

-

* D+

-

-

* D+

D -

D I

-

-

(I,

1

-

-

-

-

(1, I+ -

(2)

(2) (3)

“=P
D=decrease. I=increase. +=mild; ++=moderate; +++=severe. (l)=Alkaline phosphatase I+. (2)=Hyperplasia and vaginal mucification. (3)=0ne female killed (lethargy) and one female died (convulsion).

1 -

-

pharmacological Research, Vol. 31, No. 2,1995

gait, stereotyped behaviour, erythema of the mucous membranes of the buccal cavity, and salivation. Treatment with either compound at 200 mg kg-’ was associated in some dogs with collapse, ptosis, deep respiration and/or bradypnoea. Vomiting was also recorded in some dogs treated with either compound; however, in view of the incidence and distribution throughout the groups, no definite association with treatment could be made. In view of the persistence of several of these signs and since animals from all treatment groups were affected, a decision was made to reduce the daily dose of each group by half from day 7. With the exception of salivation, decreased activity and ptosis (L- or racemic sulpiride, 100-200 mg kg-’ only), the clinical signs described above disappear within 1 or 2 weeks of reducing the dose levels. From the beginning of week 3 slight to moderate development is detected mammary in females receiving either L- or racemic sulpiride. The degree of development is similar for both compounds and shows no relationship to dose. There were also documented: a transient reduction in food consumption during week 1 (levosulpiride or racemic sulpiride, 100 or 200 mg kg-‘); a slight, transient loss in body weight during week 1 (levosulpiride 200 mg kg-‘, racemic sulpiride 100-200 mg kg-‘); increases in plasma cholesterol and phospholipid concentrations during weeks 7 and/or 13, without any relationship to dose. No treatmentrelated change was documented in haematology or urine profiles. A clear reduction in prostate weight is apparent in males treated with L- or racemic sulpiride at all dose. The microscopic study documented hyperplasia and active secretion of the mammary glands of treated females, and atrophy of the prostate glands of treated males.

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mild reduction in weight gain with a slight decrease in food consumption. The haematology and blood chemistry analyses have shown that treatment with levosulpiride subcutaneously at lo-20 mg kg-’ day-’ for 190 days is well tolerated in rats. The findings of the pathologic and histopathologic exams in the treated animals were unremarkable (Table IV). In the study carried out on rats treated for 180 days with 50-100 mg kg-’ day-’ of levosulpiride by oral route, the general health of all animals in all groups remained normal. From a behavioural point of view there was no difference between the groups treated with levosulpiride and the control group. No change in urine analyses were noted in spite of a weight gain that levosulpiride trend. neither This shows determines renal damage, nor influences weight gain after chronic use. The white cell count and the complete blood count, as well as the weight, necroptic and histologic examinations of internal organs carried out in treated animals and in controls, excluded any damage caused by levosulpiride. In trials carried out in rabbits there was a mild, not statistically significant decline in growth compared to controls in the group with the highest dosage (15 mg kg-’ day-‘). Haematology, blood chemistry analyses and gross pathologic, histopathologic analyses have shown that treatment with levosulpiride at 5, 10 or 15 mg kg-’ day-’ by intramuscular route for 180 days is well tolerated in rabbits. In the study carried out on dogs treated for 180 days with 50-100 mg kg-’ day-’ of levosulpiride by oral route, none of the treated animals had behavioural modifications worth noting. Even in the group receiving the highest dose provoked no episode of gastrointestinal intolerance, or symptoms linked to neurotoxicity. No change in blood cell count, blood chemistry,

Chronic toxicity Chronic toxicity trials were carried out in rats, rabbits and dogs for 180 days. Analyses regarding haematology and blood chemistry were selected to have an overall picture, within limits of the effects from treatment on metabolism, on liver function, on renal function, and on complete blood count. The necroscopy was carried out on major organs and apparatuses and was performed on each animal and on each organ by microscopy. The doses used were 10, 20 or 30 mg kg-’ day-’ subcutaneously or 50 or 10 or 100 mg kg-’ day-’ orally for rats, 5, 15 mg kg-’ day-’ intramuscularly for rabbits, and 50 or 100 mg kg-’ day-’ orally for dogs. In the study on 120 rats six animals died during the trials. The necroptic revealed that all six animals exam had bronchopulmonary complications affecting both lungs. This was unrelated to the drug administration. Only in the group of rats treated with the dose of 30 mg kg-’ day-’ by subcutaneous route there was a

Table IV Repeated-dose toxicity (chronic toxicity beyond 3 months) of levosulpiride (mg kg-’ p.o.) in rats of both sexes. The treatment lasted 180 days (6 treatment days per week) Treatment

Findings

10

20

-

-

-

Body weight Food consumption Clinical signs Blood chemistry Complete blood count Gross anatomical and microscopic findings ns=not significant. D=decrease. +/-=mild.

(mg kg-‘)

30

100 ns

-

D+/D+/-

ns ns ns

-

-

ns

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urine composition and body weight, which could be linked to levosulpiride use, was documented. The necroptic and histologic evaluations showed no gross anatomical or microscopic pathologies in the major organs.

Reproduction

toxicity

The oral administration of levosulpiride in rats at 75, 150 or 300 mg kg-’ day-’ is without direct effect on fertility, reproduction or organogenesis; more specifically, the administration of levosulpiride before coupling and 13 days after coupling showed that fertility in rats was unchanged, neither time periods nor sequence of gestation modified, newborn vitality was unaffected, no apparent damage to suckling occurred, and either preimplant or post-implant loss remained unchanged. Moreover, in the second generation coming from the zero generation treated with levosulpiride the newborn vitality overlapped with controls. The oral administration of levosulpiride at lo-25 mg kg-’ in rats and 25-50 mg kg-’ in rabbits during organogenesis caused no statistically significant change in weight gain by pregnant animals, in the number of foetal implants, in the number of dead or undeveloped foetuses, and in the weight of foetal offspring from animals treated some compared to controls. In addition, malformations were observed in treated rats and rabbits as well as in controls. Therefore, there is no substantial difference between control groups and treated animals. Teratogenic process in the two tested animal species can be excluded. Levosulpiride administered at dose of 75, 150 or 300 mg kg-’ by oral route in the final period of gestation and during the 21 days of suckling in rats neither changes fertility or gestation indices, nor modify newborn vitality indices. However, all treated groups have suckling indices somewhat lower than controls. The finding does not mean that there is decreased milk production following treatment. On the contrary, there is an increase in production that leads to better nutrition when there are many young of breast disease (congestion, and appearance inflammation) or when the young are few. These findings may be explained by the fact that levosulpiride under the right endocrine conditions and particularly at the end of pregnancy, induces an increase in milk production.

Mutagenic

potential

The mutagenic potential of levosulpiride was studied by using different methods. The point mutation test in Saccharomyces cerevisiae, the bacterial test of B. Ames on Salmonella tiphymurium, the study on the DNA repairing activity, the chromosomal aberration test in human diploid cells, the DNA repair and damage test (evaluated by mitotic crossing-over conversion in and by gene

Saccharomyces cerevisiae), and the gene mutation test in Schizosaccharomyces Pombe Pl, did not document increase in rate induced by mutation any levosulpiride. Furthermore, levosulpiride was tested in vitro for possible induction of structural and numerical chromosome aberration in PHA-stimulated human lymphocytes. The compound was added to the medium at various concentrations 48 h after the culture had been set up. The mean structural aberration rates for the levosulpiride cultures were between 0.5 and 3.0% (for aberrant metaphases including gaps) or 0 and 1.5% (for aberrant metaphases excluding gaps) and were thus found within the range of variation of long-term in-house negative controls. No aberration other than gaps, breaks and isolated isochromatid fragments was observed. There was no substance-related increase in comparison to the concurrent negative controls. Numerically aberrant metaphases were also included in the evaluation for structural aberrations. Under these experimental conditions the induction of structural chromosomal aberrations and numerical aberrations in the form of hypoploid and polyploid metaphases induced by levosulpiride is ruled out. Oncogeniclcarcinogenic

potential

oncogenic/carcinogenic toxicity of The levosulpiride was tested in 600 Wistar rats and NMRI mice by long-term oral administration. 200 further rats and 200 further mice served as controls. Levosulpiride was given over 104 weeks in rats and 100 weeks in mice in daily dosage of 12.5, 25.0 and 50.0 mg (kg B.W.))’ in the food. The control rats and mice remained untreated. The final mortality was significantly higher, in respect to controls, in rats and mice treated with levosulpiride at the dose of 50.0 mg (kg B.W.)-‘day-’ and it was similar or identic to controls in the groups treated with levosulpiride at doses of 12.5 and 25.0 mg (kg B.W.)-’ day-‘. The researches in rats showed that the lowest and medium tested dosage of levosulpiride [12.5 and 25.0 mg (kg B.W.)-’ day-’ in the food] were in the range of the estimated good tolerated doses and did not influence the behaviour, the external appearance, the faeces, the mortality, the food and drinking water consumption, the haematological and urinary profiles, the body weight development, the weight of examinated organs, sight, hearing, dentition and the number of palpable masses. The highest dose of levosulpiride [50.0 mg (kg B.W.)-’ day-’ in the food] did not modify the behaviour, the external appearance, faeces, the drinking water consumption, the haematological and urinary profiles, sight, hearing, dentition or the number of palpable masses, but it induced a significant decrease of food consumption, of body weight development and organ weights, and a significant increase in SGOT and SGPT. Moreover, in

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rats of both sexes treated with levosulpiride at all tested doses a significant increase of plasma prolactin and a moderate increase of corticosterone and were shown. Macroscopically and aldosterone microscopically there were no signs of oncogenic properties with all the doses of levosulpiride tested. At histopathologic examinations benign neoplasms of skin (epithelial and connectival turnours), soft tissues angiomas, leiomiomas), thyroid gland (lipomas, (follicular adenomas), ovaries (thecagranulosa cell tumours), liver (adenomas) and adrenal medulla (phaeochromocytomas) were observed. Microscopic alterations of liver were observed only with the highest dose of levosulpiride (50.0 mg kg-’ day-‘). With levosulpiride at all doses tested microscopic aspect of hyperactivity of mammary and pituitary glands has been observed. Moreover, in control and treated rats of both sexes aspecific alterations of lungs, kidneys, heart, spleen and liver were documented. Similar results dropped out from the trials on mice by using the same doses of levosulpiride (12.5, 25.0 and 50.0 mg (kg B.W.)-’ day-’ in the food).

Effects of sulpiride Animal species

enantiomers

Local toxicity Researches on local toxicity of levosulpiride administered by intravenous or subcutaneous route showed a perfect tolerability to the injected drug at pharmacologically active doses. In fact, 50 and 25 mg kg-’ in rats respectively by subcutaneous and intramuscular routes and 25 and 50 mg kg-’ administered by the same routes in rabbits constitute a range of security: with these dosages, inspection of the injection site at different times for 7 days after treatment and the autopsy revealed no pathological finding. Only higher doses, certainly beyond any human utilization, can induce irritation and inflammation with oedema, mild bleeding and some necroses. These, however, heal readily as seen in early histogenic repair. In addition, 25 mg kg-’ by oral route and 10-25 mg kg-’ by intraperitoneal route of levosulpiride in rats reduce stress- or reserpin-induced gastric ulcers, while dextrosulpiride has no effect at 25 mg kg-’ orally (Table V). It is well known that in rats emotional stress

Table V on the gastrointestinal

Test

tract and on intestinal

Compound (dose)

transit Results

Preventive and curative effects of sulpiride on gastric ulcers

Male SD rats

Wistar rats

Stress ulcers

Reserpine-induced

ulcer

L-sulpiride 5 mg kg-’ p.o. 25 mg kg-’ p.o. 10 mg kg-’ i.p. 25 mg kg-’ i.p.

No effect Reduction in the number of ulcers found

n-sulpiride 25 mg kg-’ p.o.

No effect

L-sulpiride 5 mg kg-’ p.o. 25 mg kg-’ p.o. 10 mg kg-’ i,p. 25 mg kg-’ i.p.

Reduction in the number of ulcers found

n-sulpiride 25 mg kg-’ p.o.

No effect

L-sulpiride 5 mg kg-’ p.o. 5 mg kg-’ i.p.

No effect No effect

L-sulpiride 50 mg kg-’ p.o. 50 mg kg-’ i.p.

Stimulation of intestinal transit

D-sulpiride 50 mg kg-’ p.o. 50 mg kg-’ i.p.

No effect No effect

Effect of sulpiride on intestinal transit

Albino rats

Pharmacological

90

stimulates intestinal motility through the excitation of dopaminergic neurons involving Dr receptors and exogenous activation of central Dr and DZ receptors similarly stimulate intestinal motility by increasing the occurrence of spike bursts [42]. At 50 mg kg-’ by oral or intraperitoneal route levosulpiride and not dextrosulpiride stimulates intestinal peristalsis by the stimulation of Dr receptors

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PHARMACOLOGICAL AND TOXICOLOGICAL EFFECTS ON CARDIOVASCULAR SYSTEM Acute and chronic studies were carried out in rats and dogs and on rabbits hearts in vitro to assess a possible cardiovascular toxicity of L-sulpiride. Repeated oral administration of levosulpiride (12.5, 25 or 50 mg kg-’ day-’ for 30 days) in normal rats did not modify the following cardiovascular parameters: heart rate, arterial blood pressure, the hypertensive agents (L-noradrenaline, responses to several angiotensin II and occlusion of both common carotid arteries), the hypotensive responses to some drugs (L-isoprenaline, dopamine and acetylcholine), the arrhythmias-inducing cardiotoxic effects of barium chloride, kalium substances (aconitine, chloride and calcium chloride), and plasma adrenaline, noradrenaline and arginine-vasopressin levels. In spontaneously hypertensive rats or animals with DOCA-induced hypertension no modification of arterial blood pressure and of heart rate following repeated oral administration of L-sulpiride (12.5, 25 or 50 mg kg-’ day-’ for 30 days) was documented. Rats sensitized by doxorubicine-induced cardiovascular lesions were also studied. The results show that Lsulpiride never modified the ECGraphic and heart histological features induced by doxorubicine. On the contrary a cardiotoxic drug, such as the tryclic antidepressive substance amitryptiline, provokes a worsening of all the above mentioned parameters, in the same experimental conditions. Acute intravenous injection of levosulpiride (10 mg kg-‘) did not induce modifications of the cardiovascular significant functions (arterial blood pressure, heart rate, and hypertensive and hypotensive responses to various stimuli) in freely moving rats. Intravenous perfusion with levosulpiride (2.5-5 mg kg-’ min-‘) until exitus provoked signs of heart toxicity (bradiarrhythmias) just before the death of animals. These toxic effects were potentiated by 3040% by pretreatment with doxorubicine. In the same experimental conditions the tricyclic antidepressive agent imipramine showed cardiotoxic effects 50-70% higher in comparison to levosulpiride. Levosulpiride injected into cerebral ventricle (III ventricle, lateral right ventricle) or posterior hypothalamus or striatum did not significantly modify any cardiovascular function in freely moving normotensive or hypertensive rats.

Time (min) Fig. 5. Guinea-pig, normally-perfused, isolated heart: influence of levosulpiride, continuously-perfused, on coronary flow, force of contraction and heart rate. *P
The presence of specific dopamine receptors in the heart has been clearly documented, both in mammals [ 18,441 and in molluscs [45]. The direct application of dopamine to isolated heart preparations has been shown to induce both negative and positive inotropic effects depending on the dose. Binding studies on the of mammalian heart dopamine characteristics receptors [18] suggested that they are of the D?: subtype, according to the classification of Goldberg and Kohli [19]. Forgione et al. documented on isolated guinea-pig and rat heart that levosulpiride injected into the heart cannula (3, 10, 50 or 100 pug) has no significant effect on the force of contraction, rate or coronary flow [46]. On the other hand, when dissolved in the perfusion medium in the concentration range 5x lO-4-5x lo-’ M, levosulpiride causes an increase in the coronary flow, prevents the decline in the force of contraction and reduces the heart rate. These effects of levosulpiride are statistically significant at the concentration of 5x 10m4M, both in the guinea-pig (Figure 5) and in the rat heart preparations. On the rat hearts in completely deprived O2 conditions, levosulpiride prolongs the latency to asystolia from 696flOl to 1238+104 s, without significantly modifying the latency to arrhythmia in respect to controls; these data were obtained with a final concentration of levosulpiride of 5~10~~ M. On isolated rat hearts, reperfused with normally oxigenated solution after having been brought to complete asytolia by stopping the perfusion, levosulpiride in the reperfusion medium

91

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saline

L-sulpiride

Fig. 6. Infarct size as estimated by the loss of dehydrogenase activity 7 h after coronary ligature. The height of the histograms shows the infarct size as the percentage (meanfs@ of total left ventricle volume in saline (n=14) or levosulpiride-treated (25 mg kg-’ by intravenous route, 5 min before coronary occlusion) (n=8) rats. *P
significantly improves and accelerates the restoration of heart performance and reduces the duration of arrhythmias (Table VI). These results, obtained with spontaneously-beating, whole heart isolated, preparations, have no obvious explanation. The possibility that the autonomic nerve fibers in the isolated heart may be excited-and hence may release appreciable amounts of the transmitter-by the action potentials of the muscle within which they lie, is rather implausible [47]. It follows that the effect of levosulpiride on isolated heart preparations can hardly be ascribed to the blockade of dopamine receptors. The high doses required to obtain a significant effect rather suggest that the mechanism of action may be aspecific, not related to the occupancy of specific binding sites. Tagliavini et al. [48] documented the effects of levosulpiride on ischaemia- and reperfusion-induced heart damage in rats in viva. In particular, treatment with levosulpiride 5 min before the induction of ischaemia by coronary ligation dose-dependently reduced lethality and development and duration of ventricular dysrhythmias. Post-occlusion intravenous treatment (5 min after coronary ligation) with

Table VI Influence of levosulpiride, continuously perfused (5 X lo-’ M), on latency to and duration of arrhythmias in isolated reperfused rat heart after having being brought to complete asystolia by stopping the perfusion (meank~~~) (n=15). *PeO.O5 vs saline group (Student’s t-test). 1461. Treatment

Saline Levosulpiride

Latency to arrhythmias (s)

277*15 272+35

Duration of arrhythmias (s)

622f120 320+59*

levosulpiride (25 mg kg-‘) also reduced the incidence and the duration of ventricular dysrhythmias. Treatment with levosulpiride (12.5-25 mg kg-‘) 5 min before coronary ligation, dose-dependently reduced absent with the dose of lethality-that was 25 mg kg-*-as well as the incidence and the duration of dysrhythmias. The infarct size, estimated by the loss of dehydrogenase activity 7 h after the coronary occlusion, was greatly reduced by the treatment with levosulpiride 25 mg kg-’ by intravenous route and the infarcts never involved the apex (Fig. 6). These findings show that in viva levosulpiride can attenuate the heart damage induced either by permanent coronary occlusion or by post-ischaemic reperfusion, and would imply that endogenous dopamine plays a detrimental role in conditions of ischaemia or postischaemic reperfusion. However, these effects of in vivo, which confirm the data observed levosulpiride in vitro [46], may be due, at least in part, to some other possible properties of this drug that are unrelated to the blockade of dopamine receptors [48]. Sulpiride dose-dependently inhibits locomotor activity of normotensive Wistar-Kyoto rats, whereas spontaneously hypertensive rats show an increase in locomotor activity in responses to low doses of sulpiride [49]. Concentrations of dopamine metabolites DOPAC and HVA, but not of dopamine itself are significantly increased in frontal cortex, striatum and hypothalamus after treatment with 100 mg kg-’ sulpiride, without any significant difference between spontaneously hypertensive and Wistar-Kyoto rats. These data, supported by those ones carried out by treating rats with sulpiride and document that spontaneously amphetamine, hypertensive rats show differential changes in their response to central dopamine Dz-blockade when compared to Wistar-Kyoto rats [49, 501.

OTHER PHARMACOTOXICOLOGICAL ASPECTS While rats treated with acute or chronic sulpiride did not show signs of catalepsy [51], the intracerebroventricular administration of sulpiride induces catalepsy and tolerance (after chronic treatment) [52]. After chronic treatment with sulpiride, dopamine DZ receptors are up-regulated in the striatum [5 l-541. Sulpiride was also studied in the regulation of the of rats. A small dose nociception response (15 mg kg-‘) of L-dopa facilitates pain slightly, whereas larger dose (100-200 mg kg-‘) can produce effect following an initial an antinociceptive hyperalgesia. Moreover, profound hyperalgesia is revealed by either dopamine D, and D2 receptor of SCH23390 or racemic blockade by means sulpiride, respectively, as well as after a reduction of the presynaptic synthesis of catecholamines by a pretreatment of the animals with the tyrosine

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hydroxylase inhibitor alpha-methyl-DL-p-tyrosine. The enhancement of L-dopa’s hyperalgesic effect of SCH23390 treatment is maximal already at the onset of the effects, whereas sulpiride or alpha-methyl-DLp-tyrosine precipitates the hyperalgesia after certain temporal delay during defined phases of the time course of the effects of large t.-dopa doses. The Di receptor agonist (+)-SKF38393 potentiates both the and antinociceptive effects of hyperalgesic 100 mg kg-’ of L-dopa. This suggests that L-dopa’s net effect on pain is modulated through concentrationdependent, opposing effector systems involving both dopamine stimulatory and inhibitory receptor mechanisms. At high dosing, activation of DZ receptors enhancing dopamine functional activity produces an antinociceptive response that can be antagonized by sulpiride [55]. Accordingly to its specific blockade of DZ receptors and to its accumulation in the pituitary gland [8], levosulpiride stimulates prolactin secretion at doses definitely lower than those required to obtain antipsychotic effects. In agreement with the enantiomer’s effect on central dopaminergic neurones, an intravenous bolus of levosulpiride in humans or the intraperitoneal administration of the drug in rats is more effective than o-sulpiride in stimulating prolactin secretion [56], being D-sulpiride 30-50% less active in comparison to L-sulpiride. Testing different single oral doses of racemic sulpiride in normal women, it was shown that the drug provokes a significant plasma prolactin increase at doses as small as 1 mg; after doses of sulpiride between 3 and 50 mg plasma prolactin levels dose-dependently reach peak values about lo-fold higher in comparison to basal levels [56]. Experiments in rats showed that the hyperprolactinemic effect after a single injection of sulpiride by intravenous route (1 mg kg-‘) is always the same, independently of the enantiomer used (Lsulpiride, o-sulpiride or racemic sulpiride) [56,57]. Furthermore when the treatment with L-sulpiride in rats was prolonged for 15 days, the AUC of prolactin secretion was significantly lower at the end of the treatment in comparison to those of the first day [57]. In agreement with these findings are the observations in normal women [56]. These effects are ascribed to the chronic treatmentinduced up-regulation of dopamine receptors in nigrostriatal system and in pituitary, but not in the limbic-cortical areas [51-54,581. Sulpiride (100 mg intramuscularly) and TRH (500 pg intravenously) simultaneously administered to normal women provoke a significant increase of both plasma prolactin and AUC of prolactin secretion. In contrast, this combined administration does not elicit an enhanced response of plasma TSH [59]. This finding shows that the sites of action of TRH and sulpiride might be different from each other, and these agents work additively with no interaction in human lactotrophs.

Besides tonic gonadotropins secretion and the response to their releasing factors are not affected by acute [60] and repeated sulpiride administration, while the cyclic peak of LH seems to be reduced after chronic sulpiride dosing thus determining amenorrhoea in women [61].

CONCLUSIONS The findings about pharmacological and toxicological characteristics of levosulpiride show the effectiveness and the safety of this drug at 25, 50 and 100 mg by oral route or 25-50 mg by intramuscular or intravenous route. There is therefore a pharmacologic basis that justifies the interest in levosulpiride for the management of dyspeptic syndrome due to slow gastric emptying caused by organic and/or functional factors (post-surgical vomiting and nausea, dyspeptic symptoms induced by antineoplastic agents), and of headache (from vasomotor disturbance, from muscletension, from endogenous depressive, psychotic and reactive states). Pharmacokinetic studies in humans indicate that the concentration of levosulpiride, following oral administration of 50 mg, reaches the plasma peak in about 3 h and this peak is on the average 94 ,ug ml-‘. After 50 mg intravenously the levosulpiride tl/2 is 4.3 h. Levosulpiride is mostly eliminated by the kidneys in the urine. The findings from experimental studies on levosulpiride lead to exclude the toxicity from accumulation, tolerance, dependence or withdrawal syndrome. Furthermore, the drug is locally well tolerated. In conclusion, according to the evaluated preclinical studies, levosulpiride shows peculiar pharmacologic properties. The toxicologic aspects monitored in the above described studies show the good safety of the drug for the therapeutic uses. Therefore, levosulpiride shows pharmacological properties which make it suitable for the management of psychosis, depression, emesis and dyspeptic syndromes, as well as for the treatment of somatic disorders.

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