ARTICLE
Auteur(s) : Elham Khajehali1, Hamid
Mirmohammed Sadeghi1, Mohammed
Rabbani2
1Isfahan Pharmaceutical Sciences Research Centre,
School of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, Isfahan, Iran
2Department of Pharmacology, School
of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, Isfahan, Iran
Opioid analgesics such as morphine are widely used in treatment
of acute and chronic pain but their clinical use is greatly limited
by development of tolerance and dependence produced by prolonged
administration [1, 2]. The overall intensity of physical dependence
is gauged by the severity of the withdrawal syndrome.
A detailed understanding of the molecular and cellular mechanism
of morphine tolerance and dependence is considered to be essential
for treating and the prevention of these phenomena [1, 3, 4]. Based
on the interaction between opiate and non-opiate receptor systems
including N-methyl-D-aspartate (NMDA) [5-7], calcium channels [1,
8, 9], gamma-aminobutyric acid (GABA) [10], and α-adrenergic
receptor in the CNS [11], several drugs have been tested for their
effects on the development of tolerance and physical dependence
[11]. None of these drugs, however, have proven to be completely
effective and without drawbacks. Calcium and magnesium have been
shown to play an important role in morphine tolerance and
dependence. Chronic morphine treatment has been shown to
up-regulate calcium channels. The increase in intracellular
Ca+2 and the consequent neuronal hyperactivity is
related to the development of morphine tolerance and withdrawal
syndromes [1]. NMDA receptor activation by chronic morphine
treatment results in increased permeability of these receptors to
Ca+2 ions and therefore, an increase in intracellular
Ca+2 [5-7]. Magnesium as a NMDA receptor antagonist
blocks the Ca+2 influx and attenuates the development of
morphine tolerance and dependence [6, 12, 13]. Many studies have
investigated the effect of calcium or magnesium separately, but the
effect of these two elements in combination had not been studied.
By administrating a combination of these two elements, besides
getting the benefits of both for the body, a better effect on
attenuating morphine tolerance and dependence may also be
achieved.
Calcium magnesium soft gels (CalMag) are a good source of
calcium and magnesium that are available as a nutritional
supplement. In addition to calcium (1 000 mg) and magnesium
(500 mg), each capsule of CalMag also contains vitamin D, zinc,
lecithin, soybean oil, gelatin, glycerin and water. Calcium and
magnesium supplements such as CalMag capsules are generally given
to aid in the development and maintenance of bones [14]. Our
previous study showed that the acute treatment of morphine tolerant
and dependent mice with CalMag can significantly attenuate morphine
tolerance and withdrawal syndromes [15]. The goal of the present
study was to evaluate the effects of chronic administration of
CalMag on the development of morphine tolerance and dependence in
mice. With chronic administration it may be possible to reduce the
effective dose of CalMag, and therefore, it could be used in the
daily diet of morphine dependent and tolerant individuals. We also
assumed that chronic use of CalMag would have a better effect on
the attenuation of morphine tolerance and dependence. To eliminate
the influence of other components of CalMag capsules, we compared
the effect of CalMag with that of its calcium and/or magnesium
content, separately.
Materials and methods
Animals
Male NMRI mice (Pasteur institute, Tehran, Iran) weighing 25-30 g
were housed in cages of six with controlled room temperature
(22-25oC) in a 12 h light-dark cycle. Food and
water were available ad libitum. Tests were performed only after
the mice had acclimated to the above environment for at least 7
days. All experiments were conducted between 09:00 and 13:00 every
day to avoid any temporal factor (e.g. circadian rhythm). Each
animal was used for only one experimental condition. All
experiments were carried out in accordance with the Guide for the
Care and Use of Laboratory Animals at Isfahan University of Medical
Sciences (2002).
Drugs and method of administration
Morphine sulfate (TEMAD, Iran), was dissolved in saline. Naloxone
hydrochloride (TEMAD, Iran) was used to induce morphine withdrawal
syndrome. Among other things, one capsule of CalMag (Bluebonnet
Nutrition Corporation, Sugar Land, Texas, USA) containing
1 000 mg of calcium and 500 mg of magnesium was carefully
suspended in 100 mL saline plus 0.5% Tween 80. This drug
solution was brought up to appropriate concentration while warming
to 40oC with vigorous shaking. Equal concentrations of
calcium chloride (CaCl2.2H2O) and magnesium
chloride (MgCl2.6H2O) were prepared in saline
and used separately or mixed.
Three different doses (calcium/magnesium: 50/25, 25/12.5 and
12.5/6.25 mg/kg) of CalMag were used in our study. All the drugs
were given in a constant volume of 10 mL/kg body weight, and
the control animals received the equivalent volume of vehicle.
Tolerance paradigms
Groups of 6-13 mice were chosen randomly for each dose of drugs.
The treatment schedule consisted of twice daily s.c. injections of
20 mg/kg morphine at 08:00 and 18:00 for 4 days and 20 mg/kg only
at 08:00 on the 5th day. CalMag, calcium and/or
magnesium and vehicle were administered i.p. 30 min before
morning injections of morphine for 4 days. The control mice (drug
naïve) were treated with just saline (s.c. and i.p.).
Morphine antinociception lasts about 2 hours. Tolerance was
assessed 30 min after the final morphine injection based on
loss of the antinociceptive effect of morphine during 5 days, using
the modified Haffner’s method, the tail-pinch test [16].
For the tail-pinch assay, a flattened clip (about 10 mm
wide) was placed approximately 1 inch from the base of a mouse’s
tail, and only the mice that responded to the clip placement by
turning back and biting the clip within 15 s were used in this
test. Drug-naive mice responded to this pressure by immediately
vocalizing and biting the clip. The mice treated by a single
injection of morphine did not respond to the pain during 15 sec.
The clip was never applied to the mouse’s tail longer than this. An
animal that did not respond before 15 s (cut-off time) was
assigned a latency of 15 s. Response time to pain was reduced
after development of morphine tolerance on day 5.
Morphine withdrawal syndrome
Groups of 6-9 mice were chosen randomly for each dose of drugs.
Morphine was injected s.c. twice daily at 08:00 and 18:00 for 5
days as described by Itoh et al. with the following
modifications [17]. Escalating doses, i.e. 1st day (30
and 30 mg/kg at 08:00 and 18:00, respectively), 2nd day
(45 and 45 mg/kg), 3rd day (60 and 60 mg/kg),
4th day (90 and 90 mg/kg) and 5th day (90
mg/kg at 08:00 only) were administered. CalMag, calcium and/or
magnesium and vehicle were injected (i.p.) 30 min prior to
morphine injections in the morning for 4 days.
Withdrawal signs were precipitated by injecting naloxone (5
mg/kg, i.p.) 2 h after the final injection of morphine.
Immediately after a naloxone challenge, mice were individually
placed in an observation box and withdrawal signs were evaluated
during 20 min by counting the number of jumps and standings.
Qualitative signs (hair raising, fast breathing, eye ptosis and
diarrhea) were rated according to the Gellert and Holtzman rating
scale [18]. This scale consists of graded signs and checked signs.
Graded signs were assigned a weighting factor from 1 to 4 which was
based on the frequency of their appearance.
Statistical analyses
Quantitative data were assessed using t-test and one-way analysis
of variance (ANOVA) with post-hoc Newman-Keuls test and expressed
as mean ± S.E.M. Qualitative scores were analyzed with Mann-Whitney
and expressed as median ± interquartile ranges. In all comparisons,
p < 0.05 was considered significant.
Results
Morphine tolerance
The maximum tolerable dose of CalMag in mice was found to be 50/25
mg/kg and doses above this, caused severe overt reaction (e.g.
tremor and hyperventilation). The tail-pinch test was used to
investigate the effects of chronic administration of CalMag and
calcium and/or magnesium on the development of morphine tolerance.
In naïve mice (control group, n = 10) the response time to pain was
1.9 seconds (figure
1). As depicted in figure 1, administration of
a single dose of morphine at 20 mg/kg produced an antinociceptive
effect that was observed by the lack of response to exerted pain
during the cut-off time (15 s). The mean response time to pain
was gradually shortened from 15 s on the first day to
5.8 s on the 5th day in animals receiving salin
(i.p.) and morphine (s.c.) which shows the development of tolerance
(figure 1). With
the exception of magnesium at 6.25 mg/kg, pretreatment with three
doses of the test compounds significantly inhibited the development
of morphine tolerance (p < 0.05) (figure 1). Chronic
administration of magnesium at 50 mg/kg, was not tolerable in mice,
and caused 4 out 6 mice to die. The effects of different doses of
CalMag (50/25, 25/12.5 and 12.5/6.25) were also compared and, the
results showed no significant differences between them.
Morphine withdrawal signs
In mice treated with morphine for 5 days, naloxone injections
precipitated the standard behavioral signs of withdrawal. In the
drug-naive group, however, the injection of naloxone did not
trigger behavioral changes.
As illustrated in figure 2, pretreatment with
CalMag (50/25 and 25/12.5 mg/kg), the same doses of calcium
/magnesium mixture (Ca + Mg), and calcium (50 and 25 mg/kg)
significantly reduced the number of jumps (p < 0.05). In
addition, only the Ca + Mg at 12.5/6.25 had significant effects on
number of jumps (p < 0.05) and unlike other treatments,
magnesium alone at 6.25 mg/kg worsened the state of withdrawal by
increasing the number of jumps, although this effect was not
significant.
The number of standings was significantly reduced only by 50/25
mg/kg CalMag and 25/12.5 mg/kg Ca + Mg (p < 0.05) (figure 3). From comparison
of different doses of CalMag (50/25, 25/12.5 and 12.5/6.25), only
CalMag at 12.5/6.25 mg/kg was ineffective in reducing the number of
jumps and standings.
The effects of CalMag, calcium and/or magnesium at 50 and 25
mg/kg on 4 qualitative signs of morphine withdrawal are shown in
table 1. The results showed that CalMag
50/25 and 25/12.5 significantly reduced most of these signs. None
of the above test compounds at 12.5 mg/kg produced significant
effects on the qualitative withdrawal signs and was therefore
excluded from the table.
Table 1 Effect of chronic administration of two
different doses of CalMag and calcium and/or magnesium on
naloxone-precipitated morphine withdrawal.
|
Treatments
|
Median behavioural scores
|
|
Fast breathing
|
Hair standing
|
Diarrhea
|
Ptosis
|
|
Saline
|
4 (3.5-4)
|
3 (3-4)
|
3.5 (2.5-4)
|
3 (3-4)
|
|
CalMag (50/25)
|
2* (2-2.7)
|
2* (2-3)
|
2* (1.25-2.7)
|
2* (1.25-2.7)
|
|
Ca (50 mg/kg) + Mg (25 mg/kg)
|
2* (2-2)
|
2.5* (2-3)
|
2 (2-3)
|
3 (3-3)
|
|
Ca (50 mg/kg)
|
2* (1-3)
|
3 (3-3)
|
3 (3-3)
|
2* (2-2)
|
|
Mg (25 mg/kg)
|
3* (2-3)
|
2* (2-3)
|
2* (2-2)
|
2.5* (2-3)
|
|
Saline
|
4 (3-4)
|
3 (3-3.7)
|
4 (3.2-4)
|
4 (3-4)
|
|
CalMag (25/12.5)
|
2* (2-2)
|
3 (2-3)
|
2.5* (2-3)
|
2.5* (2-3)
|
|
Ca (25 mg/kg) + Mg (12.5 mg/kg)
|
2.5 (1-3)
|
3 (2-3)
|
2* (2-3)
|
2.5* (2-3)
|
|
Ca (25 mg/kg)
|
3 (2-3)
|
3 (2-3)
|
2* (2-3)
|
2.5* (2-3)
|
|
Mg (12.5 mg/kg)
|
2.5 (2-4)
|
2.5 (2-3)
|
2.5* (2-3)
|
2* (2-3)
|
Discussion
Previous results showed that acute administration of CalMag could
significantly attenuate the development of morphine tolerance and
dependence [15]. In line with these findings, the current study was
designed to investigate the chronic effects of CalMag capsules. By
chronic administration, it is possible to reduce the effective dose
of the drug and therefore reduce any possible toxicity. To
eliminate the influence of other components of CalMag capsules, we
compared the effects of calcium and/or magnesium separately with
that of CalMag on the development of morphine tolerance and
dependence. The results of the tail-pinch assay showed that
pretreatment with all the test compounds in different doses, except
6.25 mg/kg of magnesium, significantly reduced the development of
morphine tolerance. With the exception of magnesium, the other test
compounds significantly attenuated the signs (jumps and standing)
of naloxone precipitated morphine withdrawal. In addition, most of
qualitative signs of morphine withdrawal were also reduced by
chronic administration of CalMag.
The mechanisms underlying the inhibitory effects of CalMag
capsules on the development of morphine tolerance and dependence
are not well known. The results of this study clearly showed that
the effect of CalMag was mainly due to the calcium and magnesium
contents of the capsules, as chronic administration of calcium and
magnesium also prevented morphine tolerance and dependence. Chronic
morphine treatment has been shown to up-regulate calcium channels.
The increase in intracellular Ca+2 and the consequent
neuronal hyperactivity is associated with the development of
morphine tolerance and withdrawal syndrome [1]. Therefore in
theory, calcium should potentiate the development of morphine
tolerance and dependence, a fact that is in conflict with our
findings. One possible explanation may be the probable
down-regulation of calcium channels due to chronic calcium
administration that reverses the effects of calcium channel
up-regulation by chronic morphine treatment. Some experiments have
investigated the effects of co-administration of calcium with
morphine. Smith et al. found that calcium administration into
the icv space of mice blocked opioid-induced antinociception [19].
In a previous study it was shown that acute administration of
calcium did not attenuate morphine tolerance and dependence [15].
Various studies have investigated the effects of calcium channel
antagonists on the development of morphine tolerance and
dependence. The results are controversial. Seyler et al. found
that verapamil increased morphine analgesic effects in mice [20].
Zarauza et al. observed no significant differences in morphine
antinociception by administration of oral nifedipine or i.v.
nimodipine, and, surprisingly, the nifedipine group had a larger
morphine consumption during 24-48 hours than the control group
[12]. It is probable that calcium channel antagonists are more
effective by intrathecal and epidural routes as was demonstrated in
the study of Choe et al. [21]. The other important underlying
mechanism is NMDA receptor activation by chronic morphine treatment
which results in increased permeability of these receptors to
Ca+2 ions and therefore, an increase in intracellular
Ca+2 [5-7]. Magnesium as a NMDA receptor antagonist
blocks the Ca+2 influx and attenuates the development of
morphine tolerance and dependence [6, 12, 13]. Magnesium-deficiency
has been shown to cause hyperalgesia in rats, a phenomenon that was
considered to be due to spinal NMDA receptor activation [22]. The
effect of magnesium alone on morphine tolerance and dependence is
somehow contradictory. The results of a study by Zerauza
et al. showed no changes in postoperative morphine consumption
by co-administration of magnesium sulfate (i.v.) and morphine [12].
Begon et al. found that the co-administration of magnesium
(i.p.) and morphine (i.v.) potentiated the antinociception of
morphine [13]. Kroin et al. found that intrathecal co-infusion
of magnesium and morphine attenuated the development of morphine
tolerance, and potentiated antinociception [23]. Nechifor
et al. found that co-administration of magnesium acetate
(i.p.) and morphine (i.p.) significantly reduced morphine
withdrawal syndrome in rats [24]. McCarthy et al. found that
intrathecal magnesium co-infusion with morphine prevented the
development of morphine tolerance and reduced postoperative
morphine consumption, but had no effect on reducing withdrawal
signs [6], which is in agreement with our results. It is possible
that in the case of chronic administration of magnesium, the
affinity of NMDA receptors combined with Mg2+ has fallen
to a certain degree.
In a previous study, we showed that acute treatment with CalMag
capsules alleviated morphine withdrawal signs at 50/25 mg/kg [15].
In the present study, chronic administration of CalMag and
especially the mixture of calcium and magnesium, at 50/25 mg/kg,
caused overt reactions (weakness, tremor, hyperventilation and
hypothermia), making the dose intolerable to animals. In this
study, the effective dose for chronic treatment with CalMag in
preventing morphine tolerance and dependence was 25 mg/kg.
Therefore, compared to acute doses, the effective dose of CalMag
can be reduced by chronic administration. Also, in contrast to the
results of the present study, acute administration of calcium was
ineffective in reducing morphine tolerance and dependence, and
acute magnesium could reduce the withdrawal signs. This comparison
leads us to explain the adaptation of calcium channels and NMDA
receptors as a possible consequence of long term drug treatment
which affects the outcome [15]. Our results further highlight the
fact that it is the calcium and magnesium content of the capsules
that provide the necessary action for preventing tolerance and
dependence and not the other contents of the capsules. Since these
capsules are used as nutritional supplements, and so far no
undesirable effects have been reported in humans, further studies
could be designed to evaluate their effects in human morphine
addicts.
Acknowledgments
This research was supported by the Research Department of Isfahan
University of Medical Sciences.
References
1 Littleton JM, Little HJ. Adaptation in Neuronal Calcium Channels
as a Common Basis for Physical Dependence on Central Depressant
Drugs. In: Emmett-Oglesby M, Goudie AJ, eds. Tolerance and
sensitisation to psychoactive drugs. New Jersey: Humana press,
1989: 461-518.
2 Schiller PW. Opioid peptid-drived analgesics. AAPS 2005;
7: 560-5.
3 Nestler EJ, Aghajanian GK. Molecular and cellular
basis of addiction. Science 1997; 278: 58-63.
4 Swapnil G, Parmananda K. Cellular and molecular
mechanisms of drug dependence: An overview and update. Psychiatry
2007; 49: 85-90.
5 Hamdy MM, Noda Y, Miyazaki M, Mamiya T,
Nozaki A, Nitta A, Sayed M, Assi AA,
Gomaa A, Nabeshima T. Molecular mechanisms in
dizocilpine-induced attenuation of development of morphine
dependence: an association with cortical
Ca2+/calmodulin-dependent signal cascade. Behav Brain
Res 2004; 152: 263-70.
6 McCarthy RJ, Kroin JS, Tuman KJ, Penn RD,
Ivankovich AD. Antinociceptive potentiation and attenuation of
tolerance by intrathecal co-infusion of magnesium sulfate and
morphine in rats. Anesth Analg 1998; 86: 830-6.
7 Przewlocki R, Parsons KL, Sweeney DD,
Trotter C, Netzeband JG, Siggins GR, Gruol DL.
Opioid enhancement of calcium oscillations and burst events
involving NMDA receptors and L-type calcium channels in cultured
hippocampal neurons. J Neurosci 1999; 19: 9705-15.
8 Diaz A, Florez J, Pazos A, Hurle MA.
Opioid tolerance and supersensitivity induce regional changes in
the autoradiographic density of dihydropyridine-sensitive calcium
channels in the rat central nervous system. Pain 2000; 86:
227-35.
9 Smith FL, Dombrowski DS, Dewey WL. Involvement
of intracellular calcium in morphine tolerance in mice. Pharmacol
Biochem Behav 1999; 62: 381-8.
10 Gilron L, Biederman J, Jhamandas K,
Hong M. Gabapentin blocks and reverses antinociceptive
morphine tolerance in the rat paw-pressure and tail-flick tests.
Anesthesiology 2003; 98: 1288-92.
11 Watson B, Lingford-hughes A. Pharmacological
treatment of addiction. Psychiatry 2007; 6: 309-12.
12 Zarauza R, Saez-Fernandez AN, Iribarren MJ,
Carrascosa F, Adame M, Fidalgo I, Monedero P. A
comparative study with oral nifedipine, intravenous nimodipine, and
magnesium sulfate in postoperative analgesia. Anesth Analg 2000;
91: 938-43.
13 Begon S, Pickering G, Eschalier A,
Dubray C. Magnesium increases morphine analgesic effect in
different experimental models of pain. Anesthesiology 2002; 96:
627-32.
14 Heaney RP. Nutritional factors in osteoporosis. Annu Rev
Nutr 1993; 13: 287-316.
15 Rabbani M, Sadeghi HM, Gudarzi S. Protective
effects of calcium-magnesium soft gels in morphine tolerant and
dependent mice. Magnes Res 2006; 19: 1-7.
16 Takagi H, Inukai T, Nakama M. A modification
of Haffner’s method for testing analgesics. Jpn J Pharmacol 1966;
16: 287-94.
17 Itoh A, Noda Y, Mamiya T, Hasegawa T,
Nabeshima T. A therapeutic strategy to prevent morphine
dependence and tolerance by coadministration of cAMP-related
reagent with morphine. Methods and Findings 1998; 20: 619.
18 Gellert VF, Holtzman SG. Development and
maintenance of morphine tolerance and dependence in the rat by
schedules access to morphine drinking solutions. J Pharmacol Exp
Ther 1978; 205: 536-46.
19 Smith FL, Stevens DL. Calcium modulation of
morphine analgesia: role of calcium channels and intracellular pool
calcium. Pharmacol Exp Ther 1995; 272: 290-9.
20 Seyler DE, Borowitz JL, Maickel RP. Calcium
channel blockade by certain opioids. Fundam Appl Toxicol 1983; 3:
536-42.
21 Choe H, Kim JS, Ko SH. Epidural verapamil
reduces analgesic consumption after lower abdominal surgery. Anesth
Analg 1998; 86: 786-90.
22 Begon S, Pickering G, Eschalier A,
Mazur A, Rayssiguier Y, Dubray C. Role of spinal
NMDA receptors, protein kinase C and nitric oxide synthase in the
hyperalgesia induced by magnesium deficiency in rats. Br J
Pharmacol 2001; 134: 1227-36.
23 Kroin JS, McCarthy RJ, Von Roenn N,
Schwab B, Tuman KJ, Ivankovich AD. Magnesium sulfate
potentiates morphine antinociception at the spinal level. Anesth
Analg 2000; 90: 913-7.
24 Nechifor M, Chelarescu D, Miftode M. Magnesium
influence on morphine--induced pharmacodependence in rats. Magnes
Res 2004; 17: 7-13.
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