Reserpine-induced central effects:pharmacological evidence for the lack of centraleffects of reserpine methiodide

Srinivas Nammi, Krishna Murthy Boini, Sushruta Koppula, andSatyanarayana Sreemantula

Abstract: Reserpine, an alkaloid from Rauwolfia serpentina, was widely used for its antihypertensive action. However,its use has been reduced because of its sedative and extra pyramidal symptoms. In the present investigation, reserpinemethiodide (RMI), a quaternary analogue of reserpine, was synthesized and pharmacologically evaluated in rats andmice for its central (barbiturate hypnosis, spontaneous motor activity, body temperature, and avoidance of conditionedresponse) and peripheral actions (blood pressure) in comparison with reserpine. The results indicate that reserpine pro-duced a dose-dependent depression of the central nervous system. RMI at doses equal to and double the equimolardoses of reserpine did not produce any behavioural changes compared with control animals. Nevertheless, both reser-pine and RMI were found to produce dose-dependent reduction in the blood pressure of anaesthetized rats, althoughonly at higher doses of RMI, indicating that quaternization of reserpine not only attenuated the entry of RMI into thecentral nervous system, but also reduced its access to the target tissue in the periphery. It is speculated that thehypotensive actions of RMI may also be due to peripheral depletion of catecholamines.

Key words: resperine methiodide (RMI), reserpine, behaviour, blood pressure, mice, rats.

Résumé : La réserpine, un alcaloïde de Rauwolfia serpentina, a été largement utilisée dans le passé pour son actionantihypertensive. Toutefois, son utilisation a diminué au cours des dernières années en raison de ses symptômes de sé-dation et extrapyramidaux. Dans la présente étude, la réserpine méthiodide (RMI), un analogue quaternaire de la réser-pine, a été synthétisée et évaluée sur le plan pharmacologique pour ses actions centrales et périphériques et comparée àla réserpine. Le changement de comportement des souris et des rats après un traitement à la réserpine ou à la RMI, telqu’évalué par leurs effets sur l’hypnose induite par les barbituriques, l’activité motrice spontanée, la température corpo-relle et l’évitement de la réponse conditionnée, a été considéré comme faisant partie des actions centrales, alors queleur influence sur la pression artérielle des rats anesthésiés normotendus a été mesurée comme faisant partie des ac-tions périphériques. Les résultats ont indiqué que la réserpine a causé une dépression dose-dépendante du système ner-veux central, tel que déterminé par une série de tests sur le comportement des souris et des rats. La RMI, aux doseségales aux doses équimolaires de réserpine ou à des doses équivalant au double de ces doses, n’a pas causé de change-ment de comportement comparativement a ce qui a été observé chez les animaux témoins. Néanmoins, tant la réserpineque la RMI, bien qu’aux aux doses plus élevées de RMI uniquement, ont provoqué une diminution dose-dépendante dela pression artérielle des rats anesthésiés, ce qui indique que la quaternisation de la réserpine a non seulement atténuél’entrée de la RMI dans le système nerveux central, mais qu’elle a aussi réduit son entrée dans le tissu cible en péri-phérique. On émet l’hypothèse que les actions hypotensives de la RMI pourraient aussi être causées par la réductionpériphérique de catécholamines.

Mots clés : réserpine méthiodide (RMI), réserpine, comportement, pression artérielle, souris, rats.

[Traduit par la Rédaction] Nammi et al. 515

Introduction

Reserpine has been used for decades in the treatment ofhypertension and schizophrenia (Magarian 1991; Lin et al.1993; Pittrow et al. 1996). It acts centrally and peripherally

by depletion of biogenic amines viz., noradrenaline, seroto-nin, and dopamine. Its peripheral depletion of amines ismostly responsible for its antihypertensive effect, wheras itsantipsychotic action is due to its central depletion of bio-genic amines (Baumeister et al. 2003). However, due to its

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Received 19 August 2004. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 15 June 2005.

S. Nammi,1,2 K. M. Boini,3 S. Koppula, and S. Sreemantula. Pharmacology Division, Department of Pharmaceutical Sciences,Andhra University, Visakhapatnam 530003, Andhra Pradesh, India.

1Corresponding author (e-mail: nammi@rediffmail.com).2Present address: Section of Endocrinology and Metabolism, Department of Internal Medicine, University of Manitoba, Winnipeg,Canada, R3E 3P4.

3Present address: Department of Physiology, University of Tübingen, Gmelinstraβe 5, D 72076, Tübingen, Germany.

central actions, reserpine produces sedation and Parkinson-ism when used for prolonged periods during hypertensionmanagement (Gareri et al. 2002; Dotra et al. 2002). As aresult, its use has been reduced in treatment for chronichypertensive patients and is limited to a selective patientpopulation (Hughes et al. 1955). Therefore, it would be aworthwhile aim to modify the structure of reserpine to makeit more therapeutically acceptable for the treatment of hyper-tension.

Plummer et al. (1954) and Schneider et al. (1955) wereamong the earlier investigators that demonstrated the sedativeeffects of reserpine in monkeys and sham-rage behaviour incats. Desai et al. (1994) reported sedation followed by inhibi-tion of spontaneous motor activity upon reserpine administra-tion. Studies by Shore et al. (1956) and Salmoiraghi et al.(1956) in mice also indicated that reserpine produces sedationand potentiates the depressant effects of hexobarbital. Beim(1956) and other groups (Kastin et al. 1982; Frances and Si-mon 1987) observed hypothermia in mice after reserpine ad-ministration, which they attributed to depression of centralthermoregulatory mechanisms. Later studies also establishedthe tranquilizing actions of reserpine by measurements ofspontaneous motor activity, conditioned avoidance response,and rotarod behaviour after reserpine treatment in rats (Palfaiet al. 1983).

Attempts have been made to synthesize reserpine deriva-tives with higher and (or) modified activities, and with fewerside effects (Protiva et al. 1960). A number of reserpine ana-logues were found to exert stronger influence on the amineconcentration in the periphery than in the brain as comparedwith reserpine (Trcka and Carlsson 1967).

Based on the poor ability of quaternary derivatives to pene-trate the blood–brain barrier (BBB), a great deal of researchhas been devoted to the quaternization of existing drugs toachieve preferential peripheral action (Brewster et al. 1996;Janowsky 2002). Previous reports have demonstrated the syn-thesis of reserpine and isoreserpine quaternary derivatives, buttheir pharmacology was not studied (Schlittler 1965; Gaskelland Joule 1967). Recent investigations in our laboratory wereaimed on the chemical synthesis and pharmacological or bio-chemical evaluation of novel reserpine quaternary analogues(Nammi et al. 2004; Sreemantula et al. 2004; Sreemantula etal. 2005). In the present investigation, a quaternary analogueof reserpine, namely reserpine methiodide (RMI), was synthe-sized and pharmacologically evaluated for change in the be-haviour of mice and rats, and blood pressure of normotensiveanaesthetized rats in comparison with reserpine.

Materials and methods

ChemistryRMI was prepared as previously described (Schlittler 1965;

Gaskell and Joule 1967). Briefly, the compound was pre-pared by adding the solution of reserpine (2 g, 3.3 mmols)in dichloromethane (20 mL) to methyl iodide (11 mL,176 mmols) and the resulting mixture was kept in the darkfor 2 days. The solid was filtered and washed with cold di-chloromethane and dried under vacuum at 70 °C for 2 h toyield RMI.

Chemicals usedReserpine free base and thiopentone sodium were generous

gifts from Novartis India Ltd. (Mumbai, India) and AbbottLaboratories (Mumbai, India), respectively. Dimethylsulphoxide (DMSO) was purchased from Loba Chemie(Mumbai, India).

The solutions of reserpine and RMI were prepared in100% DMSO and the volume of each dose was adjusted to0.1 mL/100 g body mass for behavioural studies, and0.05 mL/100 g body mass for blood pressure experiments, assuggested by Varma et al. (1987). The doses of RMI werecalculated on equimolar basis of reserpine. In all the experi-ments, control groups were administered equivalent volumesof DMSO.

AnimalsNinety-six albino mice and 90 Wistar rats of both sexes,

weighing 20–25 g and 200–250 g, respectively (CharkabortyEnterprise, Kolkata, India) were used in the study. They wererandomly matched and divided into groups (n=6) and pro-vided with standard pellet diet (Ratan Brothers, Hyderabad,India) and water ad libitum. Animals were acclimatized tothe laboratory conditions for at least 10 days prior to the ex-periment with 12 h light : 12 h dark cycle. The care and useof the animals were approved by the Institutional AnimalEthics Committee and by the government regulatory bodyfor animal research (Regd. No. 516/01/A/CPCSEA).

Effect on barbiturate hypnosis in miceThe general procedure of Kuhn and Van Maanen (1961),

as described by Turner (1965), was employed. The effect ofRMI on the duration of thiopentone-induced hypnosis wasevaluated and compared with reserpine.

Groups of mice were administered intraperitoneally witheither reserpine (0.5, 1, and 2 mg/kg body mass) or RMI (2and 4 mg/kg body mass), 1 hour prior to an intraperitonealinjection of thiopentone (40 mg/kg, dissolved in saline solu-tion). A control group simultaneously received DMSO at0.1 mL/100 g. The mice were turned on their backs after ad-ministration of thiopentone for ten seconds and then re-leased. Loss of righting reflex was considered positive whenmice remained on their backs for at least 1 min. The dura-tion of sleep was assessed as the time that elapsed betweenthe loss of and recovery of righting reflex.

Effect on spontaneous motor activity of miceTo determine effect of reserpine or RMI on the spontane-

ous motor activity of mice, the method of Kuhn and vanMaanen (1961) was slightly modified, and the activity wasregistered with photoactometer (INCO, India).

Groups of mice were administered with reserpine (0.25,0.50, and 1 mg/kg, i.p.) or RMI (1 and 2 mg/kg, i.p.). All theanimals were individually placed in the cage and the countfor motor activity was recorded for 5 minutes before and af-ter drug administration. Counts were recorded every 15 minfor up to 2 h, and then again 4 h after drug administration.The count for the control group receiving only vehicle(DMSO, 0.1 mL/100 g) was also recorded.

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Effect on body temperature of miceGroups of mice were administered with reserpine (0.25,

0.5, and 1 mg/kg, i.p.) or RMI (2 mg/kg, i.p.). Temperature

was measured rectally by inserting the probe of a digitalthermometer (CIE 305) about 1 cm into the rectum beforeand after administration of drugs (Turner 1965). The record-ings were made at various intervals up to 24 h and the ex-periment was performed at room temperature (30 ± 1 °C) forall group including the control (DMSO, 0.1 mL/100 g).

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Fig 1. Diagram illustrating the effect of reserpine and reserpine methiodide (RMI) on thiopentone-induced sleeping time of mice. Eachbar indicates the mean ± SE time elapsed between loss and recovery of righting reflex of 6 animals. Thiopentone (40 mg/kg, i.p.) wasinjected 1 h after the intraperitoneal administration of reserpine, RMI, or the control. *, p < 0.001 vs. control.

Fig 2. Influence of reserpine and reserpine methiodide (RMI) onspontaneous motor activity in mice. Values are the mean ± SEcount (n=6) animals during the 5 min observation period. *, p <0.05 vs. control; **, p < 0.01 vs. control; ***, p < 0.001 vs.control.

Fig 3. Effect of reserpine and reserpine methiodide (RMI) onrectal temperature in mice at various time intervals. Values aremean ± SE of 6 animals. *, p < 0.05 vs. control; **, p < 0.01vs. control; ***, p < 0.001 vs. control.

Effect on conditioned avoidance response in ratsTo determine the effect of RMI on conditioned avoidance

response in rats, the methodology of Cook and Weidley(1957) was used. Briefly, the apparatus consists of a stain-less steel grid floor in a wooden box that produces electricalshocks to rats who can escape the noxious stimulus (shocks)by climbing a centrally located wooden pole. The stimulus isapproximately 0.1 mA of 40 V delivered for a period of30 s. The conditioning stimulus is a buzzer attached to thechamber.

Rats were trained to climb the pole by a shock of 30-s du-ration. Jumping on to the pole functionally terminates theshock. They were later conditioned to climb the pole at thesound of the 15-s buzzer. A 60-trial schedule with 1 minutefor each trial was employed. Satisfactorily pretrained ratsthat avoided over 90% of conditioned responses in a 60 trialschedule were selected in for 3 groups (n=6; reserpine, RMI,control). Rats were administered reserpine (0.25, 0.5, and1 mg/kg, i.p.), RMI (2 mg/kg), or DMSO (0.1 mL/100 g),depending on their group. The change in the conditionedavoidance response after administration compared with pre-treatment response was recorded at 1, 2, 4, 6, 8, and 10 h af-ter drug administration.

Effect on blood pressure of normotensive anaesthetizedrats

The procedure described by Noble (1973) was followed toevaluate the effect of RMI on normotensive anaesthetizedrats in comparison with reserpine. Groups of rats (n=6) wereanaesthetized with an initial dose of thiopentone sodium(40 mg/kg, i.p.) dissolved in isotonic saline solution. Theleft femoral vein was cannulated for administration of sup-

plementary doses of anaesthetic (1/3 the initial dose) anddrug solutions.

Hemodynamic setup was used to record the blood pressureof rats. The blood pressure of each animal was recorded fromleft common carotid artery connected to a mercury manome-ter on kymograph paper. The normal blood pressure of ratswas recorded after stabilization for 30 min. The differentdoses of reserpine (0.25, 0.50, 1, 5, 10, and 15 µg/kg), RMI(10, 25, and 50 µg/kg), and DMSO (control; 0.05 mL/100 g)were studied in separate groups (n = 6) to determine thechange in blood pressure.

Statistical analysesData are expressed as mean ± SE. Statistical analysis was

performed using 1-way analysis of variance (ANOVA), withDunnett’s multiple comparisons test for post-hoc compari-sons. In all the cases, p < 0.05 was considered statisticallysignificant.

Results

Effect on mouse barbiturate hypnosisThe results produced by reserpine and RMI on the barbi-

turate sleeping time in mice are shown in Fig 1. Reserpine(0.5, 1, and 2 mg/kg) produced a dose-dependent increase insleeping time, and had significantly increased sleep times(1 and 2 mg/kg) compared with control animals (p < 0.001).The RMI-treated (2 and 4 mg/kg) group did not differ signif-icantly from the control.

Effect on mouse spontaneous motor activityThe effect of reserpine and RMI on the spontaneous loco-

motor activity of mice are shown in Fig 2. A dose-dependentinhibition of spontaneous motor activity compared with con-trol was observed with reserpine (0.25, 0.50, and 1 mg/kg)but not RMI (1 and 2 mg/kg). The reduction in locomotoractivity by reserpine was of longer duration and continuedwith low activity for periods even after the experiment.

Effect on mouse body temperatureThe effect of reserpine and RMI on the normal body tem-

perature are in Fig 3. Reserpine produced hypothermia in adose-dependent manner. No such change in rectal tempera-ture was observed with RMI (2 mg/kg).

Effect on conditioned avoidance response in ratsThe results produced by reserpine and RMI on the avoid-

ance of conditioned response in rats are shown in Fig 4. Adose-dependent reduction in the conditioned avoidance re-sponse was observed with reserpine (0.25, 0.5, and1 mg/kg), but not RMI. The reduced response continued atall doses even after the termination of the experiment.

Effect on blood pressure of normotensive anaesthetizedrats

The effect of reserpine and RMI on the blood pressure ofnormotensive anaesthetized rats is shown in Table 1. Dose-dependent hypotension was observed in reserpine-treated rats,as well as RMI-treated rats. However, vehicle-treated ratsalso exhibited hypotension (i.e., ~15 mmHg below basal

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Fig 4. Influence of reserpine and reserpine methiodide (RMI) onconditioned avoidance response in rats at various time intervals.Each point represents the mean ± SE of 6 animals calculated aspercentage of predrug response. Rats were pretrained to avoidover 90% of conditioned responses in a 60-trial schedule. *, p <0.05 vs. control; **, p < 0.01 vs. control; ***, p < 0.001 vs.control.

level). This fall in blood pressure was subtracted from theblood pressure recorded after the drug or vehicle administra-tion to determine the actual effect of the drug.

Reserpine (0.5, 1, 5, 10, and 15 µg/kg) produced signifi-cant (p < 0.01) reduction in blood pressure, and the recoverytime was significantly higher than the control (p < 0.01)with an increase in doses. RMI (10, 25, and 50 µg/kg) alsoproduced a significant decrease in blood pressure (p < 0.01),and increased the recovery time (p < 0.01) with increase indose compared with control.

Discussion

The localization of drug effects to the peripheral or centralnervous system has been of great interest to achieve drug ac-tivity at the desired site. From the considerable literatureavailable, it is clear that quaternization reduces the diffusionof drugs through the BBB, and thereby confines their effectsto the periphery (Brewster et al. 1996; Janowsky 2002). Cen-tral nervous system depressants and tranquillizers are knownto potentiate the hypnosis produced by barbiturates, reducemotor activity and body temperature, and avoid conditionedresponses through their central actions. The results of thepresent investigation on the central effects of reserpine cor-relate well with the observations of previous investigators(Shore et al. 1955; Schneider 1955).

It has been well-established that the antihypertensive andtranquilizing actions of reserpine are mediated through thedepletion of biogenic amines in the body (Brodie et al. 1960).The peripheral depletion of amines is responsible for resper-pine’s antihypertensive effect (Baumeister et al. 2003) whiletheir central depletion plays a role in sedation and depres-sion of reserpine (Matsuoka et al. 1964). Reserpine exertsits depleting effect by specifically inhibiting the adenosinetriphosphate-Mg2+-dependent incorporation of biogenic aminesinto storage vesicles (Rudnick et al. 1990). Reserpine, beingcapable of central entry, depletes monoamines including 5-HT,and such action is responsible for its central sedation and tran-quilizing activities (Brodie et al. 1960; Bertler 1961).

RMI, even at doses of twice that of reserpine (on equimolarbasis), did not effect barbiturate sleeping time, spontaneousmotor activity, body temperature, or conditioned avoidanceresponses, possibly because of its nonpenetration throughBBB into the central nervous system. The large body of evi-dence on the quaternary metho-derivatives of centrally activedrugs available today also lends support to our observationswith RMI (Brewster et al. 1996; Janowsky 2002).

To evaluate whether the RMI still retains reserpine’s pe-ripheral blood pressure lowering activity, further experimentswere carried out on the blood pressure of anaesthetized rats.Thus far, the results of RMI on the blood pressure responseof anaesthetized rats confirmed that the peripheral actions ofreserpine molecule are not affected by quaternization. Ourobservation that control-treated animals also exhibited minorhypotensive blood pressure supports previous work (Varmaet al. 1987) that also showed hypotension with DMSO. Re-serpine produced a dose-dependent reduction in blood pres-sure as well as increase in recovery time, again in agreementwith previous investigations (Khatri et al. 1982). The intra-venous doses required to produce hypotension were verysmall, and central effects have been reported absent withsuch doses (Beim and Brunner 1966). As indicated by earlierreports (Burn and Rand 1958; Carlsson et al. 1962), the hypo-tensive effect of reserpine observed in rats is likely due to thedepletion of catecholamines from the peripheral stores.

However, RMI created hypotension at higher doses com-pared with reserpine. Recently, we also reported, based onbiochemical evidence, that RMI produces preferential deple-tion of peripheral biogenic amines in rats (Sreemantula et al.2005) and this was supported in the present observations. Itis further indicated that quaternization of reserpine not onlyrestricts the entry of RMI to central nervous system but alsoreduces RMI movement to the target tissue in the periphery.Hence, relatively higher doses were required to produce a re-serpine-like effect. Mechanistically, it is speculated that thehypotensive actions of RMI could also be due to the periph-eral depletion of catecholamines.

In conclusion, the present study indicates that quaterniz-ation of reserpine does not abolish its hypotensive response,

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Mean arterial pressure (mmHg, n = 6)

Drug Dose (µg/kg) Before drug After drugMeanreduction

Reductiondue to drugb

Recoverytime (min)

Recoverydue to druga

Control (vehicle;DMSO)

0.05 mL 126.2±2.8 110.0±4.1 16.2±1.4 — 0.96±0.1 —

Reserpine 0.25 138.3±4.6 119.5±4.2 18.8±1.4 2.6 1.2±0.2 0.20.5 134.1±5.5 91.2±5.0 42.9±0.9* 26.5 3.1±0.4* 2.11 135.2±4.7 80.5±3.8 54.8±1.6* 38.6 4.9±0.4* 3.95 130.5±5.1 69.0±3.5 61.5±2.8* 45.3 7.1±0.3* 6.110 130.0±4.2 58.7±3.9 71.2±1.3* 55 10.2±0.3* 9.215 131.5±5.1 41.1±2.9 90.4±3.1* 74.2 13.0±0.3* 12

Reserpine methiodide 10 125.0±4.1 88.8±3.6 36.3±1.4* 20.1 3.3±0.3* 2.325 126.3±4.9 73.8±2.8 52.3±2.9* 36.1 4.6±0.3* 3.650 130.0±4.1 52.5±1.7 77.5±3.7* 61.3 7.3±0.3* 6.3

Note: *, p < 0.01. vs. control-treated group.aRecovery due to drug was calculated by subtracting the control recovery time from the recovery time of each dose for each drug.bReduction due to drug was calculated by subtracting the mean reduction of the control from the mean reduction of the drug.

Table 1. Effect of reserpine and reserpine methiodide on the mean arterial pressure of anaesthetized rats.

but only at higher doses. Additionally, the nonsedative na-ture of RMI was exhibited through its inability to exert thecentral actions of reserpine.

Acknowledgements

The authors are indebted to Sri G. Ganga Raju and thescientists of Laila Impex Research Centre (Vijayawada, In-dia) for their encouragement and help in the synthesis ofRMI. The financial support by the Council of Scientific andIndustrial Research (CSIR), New Delhi to Srinivas Nammi isgratefully acknowledged.

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