Indian Journal of Experimental Biology Vol. 43, May 2005, pp. 425-429

Acute analgesic effect of loperamide as compared to morphine after intrathecal administration in rat

Subrata Basu Ray, Dilip Verma & Shashi Wadhwa

Department o f Anatomy, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Received 15 October 2004; revised 3 February 2005

Loperamide, a mu opioid receptor agonist, which is commonly used as an antidiarrhoeal agent has been reported to possess analgesic activity after intrathecal administration. However, the exact analgesic profile, i.e., onset, duration and intensity of analgesia in -relation to morphine is not fully kilOwn. In the present study, the acute analgesic effect of loperamide (5 ~lg) was compared with that of morphine (5 ~lg) and morphine + loperamide (5 ~lg of each) using the tail flick method after intrathecal administration . Naloxone (5 mg/kg) reversibility of the analgesic effect was also studied. The analgesic response of loperamide was significantly higher than morphine. Even after 22 hr, maximum possible effect was greater than 49 %. Naloxone partially antagonized the analgesic effect of loperamide. This suggested that loperamide may be acting through blockade of Ca~+ channels besides activating mu opioid receptors. Loperamide may 'prove to be a better substitute for morphine as spinal analgesic.

Keywords: Analgesia, Calcium channel antagonist, Loperamide, Morphine, Mu-opioid receptor

Morphine produces analgesia by predominantly activating ~-opioid receptors I . Loperamide is a synthetic opioid, which binds to ~-opioid receptors with higher affinity than morphine2

. It is a lipophilic, diphenylpiperidine derivative, which is used occasionally for the treatment of non-bacterial diarrhoeas3

.4. However, it produces potent antinociception when administered locally at the site of inflammation2,s. This is due to its effect on peripheral ~-opioid receptors. Interestingly, co­administration of cereport along with loperamide allows the latter to penetrate the blood brain barrier and produce significant analgesic effect6

. It indicates that loperamide can produce an analgesic effect if directly instilled into the central nervous system or into the cerebrospinal fluid surrounding it.

A recent report has noted the analgesic effect of loperamide in 4-6 weeks mice after intrathecal administration in the formalin tese. However, the analgesic effect of loperamide was not analysed with regard to its onset, duration or its magnitude, which is possible with tail flick method. Also, the analgesic effect was not compared with any other standard opioid analgesic drug like morphine. Hence, the

Phone: 91-0 I 10 26593216 (0) E-mail: sbr_aiims@hotmail.com;dilip.verma@gmail.com; shashi wadhwa@hotmail.com

analgesic profile of loperamide was studied in rat after standardization of the dose. Another reason was that earlier observations from our laboratory have shown that co-administration of morphine and nimodipine through the intrathecal route results in a higher analgesic effect than either of the drugs alone in both rats and mice8

.9

. Incidently, nimodipine is a L­type calcium channel blocker. Besides being an opioid, loperamide has also been shown to directly block multiple calcium channels7

. It appeared that loperamide could produce a higher analgesic effect than morphine alone. The present study has been undertaken to note whether loperamide may be a better substitute to morphine as spinal analgesic.

To evaluate the mechanism of analgesic effect of loperamide (opioidergic or non-opioidergic or both), naloxone an opioid antagonist, was systemically administered after 1 hr of administration of morphine and/or loperamide, when the maximum analgesia was achieved as evident by the tail flick response.

Materials and Methods Adult male Wistar rats (n=56) weighing between

200-225 g were used. The rats were kept in cages with food and water ad libitum. The room temperature was maintained at about 25°C. Prior approval of

. Institutional Animal Ethics Committee (lAEC) was obtained.

426 INDIAN J EXP 1310L, MA Y 2005

The animals were divided into following 7 groups: Group I (n=9): received 5 Ilg of loperamide, intrathecally (it) Group II (n=13): received 51lg of morphine, it Group III (n=6): received 5 Ilg of loperamide + 5 Ilg of morphine, it Group IV (n=6) : received 10 III of normal saline (0.9%), (it) Group V (n=6): received 5 Ilg of loperamide (it) followed by naloxone (5 mg/kg) intraperitoneally (ip) after I hr Group VI (n=8): received 25 Ilg of loperamide, (it), followed by naloxone (5 mg/kg), ip after I hr Group VII (n=6) : received 5 Ilg of morphine, (it) , followed by naloxone (5 mg/kg), ip after 1 hr

Loperamide hydrochloride and naloxone was obtained from Sigma, USA. Morphine was obtained as morphine sulphate I. P. in ampules (15 mg/kg) from Government Pharmacy. Loperamide was dissolved in a solution of polyethylene glycol, normal saline and absolute alcohol in a ratio of 2:2: 1. Loperamide and morphine were administered through single injection in Gr HI. The solvent (for loperamide) was also separately injected (it) into a group of albino Wistar rats (n=3) to investigate whether it had any analgesic effecl.

The total volume injected (it) was 10 ~t1 .

Loperamide was given at various doses (I , 5, 10, 25 , 50 and 100 pg) initially. Higher doses (10-100 ~lg)

frequently produced temporary muscular weakness of the hind limbs arter intrathecal administration, which was ahsent at 5 pg. No analgesic elrect was produced with I pg. A consi stent analgesic cl'l'cct wilhoutside c!'kct s \Vas noted with 5 pg morphine. which was sdected for the present study.

Muscular weakness was tested by the stepping and righting reflexes. which arc tests for motor coordination and runction aftcr intrathecal aciministration ili

. In the fonner , the rat is lifted by tlw tail so as to only slightly lift the hindlimbs off the surface when there is a extension of the hind limbs; when the tail is released, there is return of the hindlimbs to the surface. In the later test, the rat is rolled on to the dorsal aspect; the rat immediately returns to sternal recumbency.

For, injections (it) , the lumbar region of the back was shaved by razor blade. Later the skin was di sinfected with povidone-iodine (5 %) and injections (it) were given using sterile tuberculin syringes. This method was modified from an earlier described

technique 1 I . The assessment of sensitivity to noxious thermal stimuli was done by the tail tlick method using the tail flick apparatus (UGO Basile). Normally, ruts flicked or move away their tails from the heat source between 2-4 sec (baseline latency) . An upper cut off time of 10 sec was observed as further exposure to heat may damage the tail. Statistically 10 sec was regarded as equal to 100% response. The latency to tail flick was measured at 15 min, 30 min, I hr and thereafter every hour till 7 hr after (it) administration. For only loperamide (Gr I) and morphine (Gr II) groups, a repeat measurement was ddne at 22 hr. Maximum possible effect (MPE) was calculated as follows: (Post drug latency-baseline latency)/(cut off time-baseline latency) x 100.

Naloxone (5 mg/kg) was given (ip) I hr after 5~lg loperamide administration in Gr V-VI and morphine administration in Gr VII. The total volume injected (ip) was 1 mllkg of body weight. An additional group (Gr VI) with higher dose of loperamide (25 Ilg) was also administered naloxone. The tail flick latency was measured after 5 min and thereafter at every 5 min till 30 min. This is so because naloxone has a half-life of I hr.

Statistical evaluation was done using Kruskal­Wallis test (ANOV A) . Indi vidual groups were compared by Mann-Whitney U tesl. P- value<O.05 was considered signi ficant.

Results Administration of loperamidc (Gr I) produced a tail

nick resonse that exceeded the CLlt oil limit of I () scc (MPE=IOWYr» by 15 min and remained at this k veltill I hI' (Fig I) . Thereafter, the tail nick response slightl y decreased but remained persistently above l) sec (MPE >H I (iC·) till 7 hr. 'fhe ~~nalges ic response was maintained even 22 hI' after the injection (MP[ >49%). Co-administration or morphine + loperamidc produced a tai I fI ick response that exceeded cut oil level by 15 min ancl which persisted at this level till 2 hr. Further on, it rapidly decreased to 6.8 see at 5 hI' where it reached a plateau till about 7 hI' (7 sec-MPE of 58%). The anti nociceptive effect of morphine alone (Gr II) caused the tail flick response to exceed cut oil le vel at 30 min and persist at this level till I hr. Thereafter, it rapidly declined to reach control reading at predrug state at 5 hI' (9.3% of M PE) and persisted at about this level till 7 hr. Administration of normal saline did not produce significant analgesic response and values for tail tlick test remained close to bascline till 3 hr.

RA Y el al.: ACUTE ANALGESIC EFFECT OF LOPERAMIDE 427

Loperamide (Gr I) produced analgesia, which was significantly higher as compared to morphine (Gr II) between 2 - 7 hr. Even at 22 hr, the analgesic response of Gr I was significantly higher than Gr II. Analgesia in Gr I (Ioperamide) was significantly higher than Gr III (morphine+loperamide) at 5 and 6 hr. Morphine + loperamide produced significantly

-4-Morphine + Loperamide ,---Loperamide,

120 .... Morphine, ..... Saline •

100

so

l 60

UJ Il. :l 40

20

0

-20

8 It)

0 0 0 0 0 0

'" ~ 0 0 0 N ;.; "i

Time (hr)

0 0 0

~ .;; to t.:

Fig. I-Maximum possible efFect of loperamide as compared to morphine and morphine + loperamide. 5 /lg of loperamide produced higher analgesic response as compared to other three groups. In fact it persisted at a significantly higher level even 22 hr after administration as compared to morphine (*). (**) represents significantly higher values for morphine + loperamide as compared to morphine alone. (***) indicated significantly higher values for loperamide as compared to morphine together with loperamide.

higher analgesic than morphine between 2-7 hI'. Analgesic response of morphine ' was significantly higher than control (saline) up to 2 hr after administration.

Naloxone reversed the maximum analgesic response of morphine (Gr VII) to a significant extent as evident by the tail flick response (Fig. 2). It remained at this lower level till 30 min. The significance was determined by Student's t test as we were comparing the same group at different time points. However, for loperamide, such a dramatic decrease in response was absent. In fact, there was fluctuation of the tail flick response. Significant fall in analgesic effect of morphine as compared to loperamide (5 ~g) was observed between I: 15 to 1:30 hr. However, with a higher dose of loperamide (25 ~g), in addition to the above mentioned time points (I: 15 to 1 :30 hr), significant decrease of analgesic effect was also noted at 1:05 hr. This indicated that the analgesic effect of loperamide was relatively resistant to naloxone.

Rats injected with solvent for loperamide (it) did not show any analgesic response. No motor deticits were noted except in one rat from Gr J lasting for 5 min. Specifically this rat showed defective stepping and righting reflexes.

Discussion The present study showed that intrathecal

administration of loperamide (Gr I) produced superior

o Morphine (5 microgram) • Loperamide (5 microgram)

(:J Loperamidc (75 microgram)

~ .,-

ri~ rh rL

I

. ~ *

I cr * *

# #

r ~

I

I

i1 ;}

* * # #

i

r r 0:00 0:15 0:30 1:00 1.05 1:10 1:15 1:20 1:25 1:30

Naloxone

Time (hr)

Fig. 2-Effect of naloxone on tail flick response. Time point of administration was at I hI', immediately after recordina the tail flick response (indicated with arrow). The tail fli ck latency was decreased signiticantly in morphine treated group througho"'ut the 30 min ?bser.vation period as estimated by Student's I-test, as thi s was a comparison within the same group at different time points. When the IIldlvl~ual groups were compared among themselves as determined by ANOY A, loperamide (5 /lg) was significantly higher than morphille (5 /lg) at ~: I~ to I :30 hI' (indicated with #) . Loperamide (25 /lg) treated group significantly above morphine at 1:05 beside5 I: 15 to I :30 hI' (*). SIgnIficance level was taken at P < 0.05

428 INDIAN J EXP BIOL, MAY 2005

analgesic response as compared to morphine (Gr II) when administered by the same route. The analgesic response persisted at a higher level till about 6 hr (MPE >81 %) after loperamide administration as compared to I hr after morphine administration. 49.1 % of the maximum possible analgesic effect (MPE) persisted in loperamide treated animals even after 22 hr. This is remarkable as it shows that analgesia due to loperamide is several fold more effective than morphine.

Interestingly, naloxone failed to reverse the analgesic effect of loperamide and morphine in a uniform manner. It appears that only a part of the analgesia produced by loperamide is mediated through its binding to p.-opioid receptors, indicating that other mechan~ sms may be involved. In this regard, loperamide has been shown to prevent calcium ion influx by blocking high- voltage-activated calcium channels in pyramidal neurons of hippocampus 12. This effect was not antagonized by naloxone, which indicates that it was not mediated through opioid receptors . In this regard, a recent study has shown that calcium influx into neurons of dorsal root ganglia was strongly inhibited by loperamide, which presumely was due to simultaneous blockage of L-, P/Q- and N-type calcium channels7

. It appears that blockade of the calcium channels could be the other mechanism responsible for the potent anti nociceptive action of loperamide. Since calcium plays an important role in the release of neurotransmitters from pre-synaptic sites, blockage of calcium influx may decrease the excitability of neurons of the dorsal horn. This would also include neurons transmitting pain signals and hence the analgesic effect. Even the antidiarrhoeal action of loperamide may be partly due its role in blocking calcium channels l3

. Voltage-gated sodium channels is blocked by loperamide to05

. Moreover, it has been shown that loperamide can also bind to yet unidentified opioid receptors or to heterodimers of opioid receptors 14. All these could be responsible for the higher analgesic effect of loperamide.

Another very important observation in the present study was the decrease in analgesic effect of 5 p.g each of loperamide + morphine as compared to 5 p.g of loperamide alone. A possible explanation could be that morphine prevents binding of loperamide to p.-opioid receptors. Morphine being a drug with lower efficacy may not induce as much analgesia as loperamide 1

• Thus, combining loperamide with any

other p. receptor agonist would prove counter­productive.

Regarding side effects of loperamide, temporary paralysis of hind limbs in about 50% of experimental animals lasting for about 20-30 min with 25 p.g of loperamide. However, with 5 p.g of loperamide, only one rat in Gr I (6.7% of the total) and none in any other group which received loperamide showed a temporary weakness lasting for about 5 min . It is possible that this effect could be due to blockade of calcium channels in the motor neurons of the anterior horn of spinal cord.

In conclusion, the present study indicates that loperamide appears to be a very prolnising drug for producing spinal analgesia of long duration . However, future studies particularly neurotoxicity studies are needed to exclude any long-term damage to the spinal cord.

Acknowledgement Thanks are due to Mr R K Ahuja for statistical

analysis and AIIMS, New Delhi for financial assistance.

References I Fang F G, Fields H L & Lee N M, Ac ti on at the mu receptor

is sufficient ot cxplain the suprasp inal analgesic effect of opiates, J Pharmaco/ £xp Ther, 238 (1986) 1039.

2 DeHaven-Hudkins D L, Cortes Burgos L, Cassel J A, Daubert J D, Dehaven R N, Mansson E, Nagasaka H, Yu G & Yaksh T, Loperamide (ADL 2-1294) an opioid antihyperalgesic agent with peripheral selectivity, J Plwrmacol £xp Th er, 289 (1999) 494.

3 Glitstein H 13 & Akil H, Opioid analgesics, in Goodmall & Gill//{/Il's The pharmacological basis oj' therapeutics (Mcgraw-Hill , USA) 2001, 594.

4 Schinkel A H, Wagenaar E, Mol C A. van Deemter L, P­glycoprotein in the blood-brain barrier of mice inl'illcnccs the brain penetration and pharmacological ac ti vity of many drugs, J Clill III vest, 97 (1996) 2517.

5 Nozaki-Taguchi N & Yaksh T L, Clwracterization of th e anti hyperalgesic ac ti on of a novel pcripheral mu-opioid receptor agon ist - loperamide, All aeslh esiology, 90 ( 1999) 225.

6 Emerich D F, Snodgrass P, Pink M, 13100111 F & Bartus R T. Central analgesic act ions of lopcramidc following transient pcrmeation of the blood brain barrier with cereport™ (RMP-7), ilmill Res, 80 I (1998) 259.

7 Hagiwara K, Nakagawasai 0, Murata A, Yamadera F, Miyoshi I, Tan-No K, Tadano T, Yanagisawa T, lijima T & Murakami M, Analgesic action of loperalllide, an opioid agonist, and its blocking action on voltage-dependent Ca2

+

channels, Neul'Osci Res, 46 (2003) 493. 8 Gupta H, Ray S 13 & Wadhwa S, Synergistic illleraction

between morphine and nilllodipine produces acute analgesic effect on illlrathecal ~dlllini stration in rat rsubmitted fo r

RA Yet 01.: ACUTE ANALGESIC EFFECT OF LOPERAMIDE 429

publication /I/diol/ J Med Res (2004) ; application for patent has been filed (1535/De1/2004 dated 18-8-04)].

9 Verma D, Ray S B, Patro I, Wadhwa S, Enhanced analgesic effect of morphine-nimodipine combination as compared to morphine or nimodipine alone after spinal or systemic administration in mice (unpublished) 2004.

10 Malkmus S A & Yaksh T, Intrathecal catheterization and drug delivery in the rat, in Pail/ research, edited by Z David Luo (Humana Press, New Jersey) 2004, 119.

II Mestre C, Pelissier T, Fialip J, Wilcox G & Eschalier A, A method to perform direct transcutaneous intrathecal injection in rats, J Pharmacol Toxieol Methods, 32 (1994) 197.

12 Church J, Fletcher E 1, Abdel-Hamid K & MacDonald J F, Loperamide blocks high-voltage-activated calcium channe ls and N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurons, Mol Pharmacol, 45 (1994) 747.

13 Raynolds I J, Gould R 1 & Snyder S H, Loperamidc: Blockade of calcium channels as a mechanism for antidiarrhoeal effects, J P/wrmaco[ Exp Ther, 231 (1984) 628.

14 Shannon H E & Lutz E A, Comparison of the peripheral and central effects of opioids agonists loperamide and morphine in the formalin test in rats , Neuropharmacology, 42 (2002) 253.