Investigation of Concomitant Administration of Morphine and Methadone
Valerie Gonzales
INTRODUCTION:
Pain management strategies employed for chronically ill children admitted into the
Pediatric Intensive Care Unit (PICU) most often include sedation and analgesia.[1] Provision of
such treatments is most effectively carried out by the delivery of opioid agonists, morphine and
fentanyl.[2,3] Although these medications serve as an immediate remedy for chronic pain,
prolonged infusions have been recognized to have adverse effects. It is known that tolerance
results due to opioid-induced analgesia.[3-6] Thus, intravenous opioid concentrations required
for adequate pain reduction increase throughout the duration of the treatment. Opioid abstinence
syndrome (OAS) results from physical dependency and is especially evident when distressful
symptoms occur upon the discontinuation of the administered drug.[3-6] Undesired neurological
side effects due to OAS consist of: irritability, myoclonus, ataxia, vomiting, sweating, fever,
diarrhea, visual and auditory hallucination, seizures and rejection to food/drink.[5]
In the occurrence of OAS in the pediatric population, methadone, also an opioid, is used
to treat opioid abstinence syndrome.[7] The lack of data from pharmacokinetic tests on the
pediatric population however attributes to the uncertain half-life and detailed pharmacokinetics
of methadone in children. Preliminary studies initially conducted for this project suggest that the
half-life of methadone in the pediatric population is significantly less than expected.
Furthermore, the data suggests that the pediatric population may still experience the symptoms of
OAS because the dosing regimens of methadone are not optimized for children. The dosage
protocol for infants involve scaling down adult dosages based on weight and do not fully
consider differences in pharmacokinetic parameters such as the volume of distribution, receptor
concentrations, clearance, and the half life of the drug among the child. In an effort to mediate
this problem, this study will explore whether the administration of morphine and methadone
simultaneously, rather than in turn as is commonly practiced in the PICU, will attenuate OAS.
Administrating the two opiates in this manner may not only decrease OAS, but may also be more
efficient, requiring lower dosages of the two drugs than what is currently used. Morphine is
chosen for the experiment over fentanyl because preliminary studies found morphine to be
prescribed more frequently.
Methadone and morphine are known to bind to the G-protein coupled mu-opioid
receptors found in the central nervous system that inhibit painful stimuli.[8] These receptors
along with N-methyl-D-aspartate (NMDA) receptors are prevalent in the nucleus accumbens, a
region of the brain known to be directly associated with drug tolerance.[9] Morphine’s agonist
interaction with mu-receptors activates G proteins and then after prolonged exposure causes
calcium ion influx.[12] Studies have shown that opioids indirectly interact with the NMDA
complex through a cascade of events.[8-10] The NMDA complex is a ligand-gated ion channel
that transports calcium ions into the cell, activating ion dependent down stream messengers of
the pain signaling pathway.[11] In addition to activating mu-opioid receptors methadone also
acts as an antagonist at NMDA receptors.[13-15] Owing to this characteristic, methadone has
the potential to extremely reduce calcium ion influx which may contribute to its ability to reduce
opioid tolerance and opioid abstinence syndrome.
Calcium influx is significant because it has been shown to indicate cellular
withdrawal.[16] With chronic therapy, opioid tolerance develops resulting in a compensatory
increase in cAMP in part by an influx of the intracellular calcium ions and increased NMDA
activity.[17] The sudden influx of calcium may be attenuated however if the NMDA receptor is
antagonized during mu-receptor stimulation. By coupling methadone’s NMDA antagonist action
with morphine therapy, it is promising that OAS can be reduced significantly.
Morphine
Mu opioid
receptor
Ca2+
Methadone
1
NMDA
receptors
3
Neurotrans mitt
er-pain
Ca 2+ , cAMP
2
Ca 2+ , cAMP
Pain
DRG cell
membrane
2
Figure 1: Co-administration Scheme of Morphine and Methadone
Mu-opioid and NMDA receptors are prevalent in the dorsal root ganglion cells. 1.
Morphine antagonizes the mu-opioid receptors, which in turn decrease internal calcium
and cAMP, and inhibits the pain-triggered pathway. 2. After prolonged exposure to
morphine, low calcium levels are replenished by the activation of NMDA receptors to
influx calcium. 3. Methadone is both a mu-opioid agonist and an NMDA antagonist,
which can be used to inhibit calcium ion influx at the NMDA receptor.
This study assesses the NMDA regulatory effect by administering morphine and
methadone concomitantly in a pediatric animal model. Mice pups are practical models for a
physiological investigation of tolerance development since the mechanism of action of opiates is
exceptionally comparable between mice and humans. Furthermore, long term opioid exposure
has been shown to cause physical dependence in neotonal mice and rats.[19] Symptoms
associated with opioid withdrawal in mice include the following: sporadic activity, wall climbing
behavior, defecation, abdominal stretches, severe tremors, screaming to touch, and spontaneous
vocalization.[19]
To best simulate the metabolism of children in the targeted age range of one month to 36
months, ten-day-old mice pups were used in the study.[18] An osmotic minipump was surgically
implanted to deliver drug to pups. Upon the discontinuation of the experimental drug mixtures
administered, a behavioral assessment was conducted to determine whether withdrawal
symptoms were attenuated when morphine and methadone were delivered concomitantly. In
addition, parent drugs and metabolites were monitored among each pup to determine the actual
drug levels attained at the point of the behavioral observation.
METHODOLOGY:
Drug Delivery: With IACUC approval thirty ten-day-old C57BL/6 strain mice pups from
the same colony were divided into three experimental groups with ten pups in each group. One
group served as the positive control group that received a morphine/saline infusion via an
implanted osmotic minipump. The negative control group received a methadone/saline infusion.
The experimental group received of morphine/methadone/saline mixture. Each solution was
prepared in sterile isotonic saline and cold sterilized. The mouse pups underwent anesthesia via
hypothermia before surgery. Sterile procedures were use to fill the osmotic minipump, implant
the pump dorsally into the pups, and seal the insertion cut with glue. Specifications of the pump
included delivery of solution at 1 μL/hour for 72 hours and suitable for animals at least ten
grams. Minipumps for the morphine group were loaded to deliver drug at 0.7 mg/kg/hr during
the 72 hour exposure period to adequately make the pups tolerant and dependent.[19] The
methadone group was also administered drug for 0.7 mg/kg/hr, while the combined
morphine/methadone group received total drug at 0.7 mg/kg/hr (0.35 mg/kg/hr morphine and
0.35 mg/kg/hr methadone).
Withdrawal Assessments: Following the 72-hour drug exposure, a 30-minute behavioral
assessment was conducted on each pup by an investigator blinded to the infusion conditions.
Animals were assessed for withdrawal at 1, 3, 6, 8 and 12 hours following the 72-hour drug
delivery period, allotting two pups for each sample time in each of the three experimental
groups. The measurement of spontaneous activity was taken by counting the number of times
the pup crossed a line in a cage measuring 50×31 cm with a grid of 30 squares (8×7 cm). Other
behaviors recorded in the 30 minute interval included micturition, defecation, face washing, wall
climbing, tremors/body-shakes, scream on touch, and spontaneous vocalization. These behaviors
were also quantified as the number of animals that exhibit the sign to the total number of animals
observed. Behavioral data was analyzed with analysis of variance (ANOVA) for the overall
group, with individual comparisons to the controls analyzed by Dunnet’s multiple range test. A
P value less than 0.05 was considered statistically significant.
Blood Samples: Samples were collected immediately following the behavioral
assessment. The pups were put under by inhalation of halothane gas, so as to not alter the
internal drug concentrations. Upon the absence of the foot pinch response, the pups were
decapitated and blood droplets were collected in a test tube. After centrifuging the samples, the
plasma layer was pipeted off and frozen for analysis using liquid chromatography-mass
spectrometry (LC-MS) instrumentation.
RESULTS:
Withdrawal Assessment
A single event of a line crossing or the occurrence of a withdrawal symptom was
considered a score. The total score was calculated by tallying the numbers of occurrences for
each assessment category; spontaneous activity, neonatal withdrawal, and adult withdrawal
pertaining to each experimental drug group. Using the analysis of variance (ANOVA), the
behavioral assessments were not statistically significant due to the sample size (N=30) being too
small to perform the statistical test with the desired power (power=0.800).
Although statistical analysis yielded inconclusive results, the behavioral assessment of
the mouse pups, summarized in figures 1-5, show the general trend that was expected. The
group that received concomitantly administered morphine and methadone had less spontaneous
activity than those that received morphine alone or methadone alone, with the exception of the
four hour surveyed group. Similar trends were observed for the number of neonatal withdrawal
symptoms, adult withdrawal symptoms and the overall withdrawal score displayed in figures 2-4.
Neonatal Withdrawal
Spontaneous Activity
1000
Morphine
Methadone
Combo
800
600
160
160
140
140
120
120
100
100
80
80
60
60
40
40
20
20
400
Average numbers of lines crossed per animal
0
0
2
4
6
8
10
12
14
16
Withdrawal Score
200
0
0
0
Time (hrs)
2
4
6
8
10
12
Time (hrs)
Figure 1
Figure 2
0
Morphine
Methadone
Combo
2
Adult Withdrawal
Total Withdrawal
160
1200
140
1000
Morphine
Methadone
Combo
120
800
100
80
600
60
20
0
10
0
12
2
4
6
8
10
12
Withdrawal Score
40
400
200
0
0
Time (hrs)
Morphine
Methadone
Combo
2
4
6
8
10
12
14
16
Time (hrs)
Figure 3
Figure 4
Withdrawal Summary
700
Figures 1-5: Behavioral Assessment Results
Morphine
Methadone
Combo
600
500
400
300
Each bar represents the tally of withdrawal
symptoms (the total score) witnessed after the
1, 3, 6, 8, or 12 hours period prior to drug
discontinuation for each experimental group.
For figures 1-4, each bar corresponds to data
taken from the observation of two pups.
Withdrawal Score
200
100
0
Spontaneous Activity Neonatal
Adult
Total
Behavioral Assessment
Figure 5
Drug Levels
The results for the in vivo concentrations of the parent drugs and their metabolites just
prior to the behavioral assessments are summarized in figures 6-8. Each bar represents a single
animal. For example, morphine (1) and M3G (1) at 1 hour is data pertaining to one mouse pup,
while morphine (1) and M3G (1) at 3 hours pertains to another mouse pup. Figure 6, which
displays the pup groups that received morphine alone, shows that the drug concentrations
remained relatively constant with respect to time. This trend is also seen in figures 7 and 8, and
in some cases there is an escalation in a drug species as the time variable increases. This trend
indicates that there was a flaw in the pump delivery system; the system should have discontinued
administering drug at time zero to give a decreasing trend in parent drugs with respect to time.
Morphine Group
Morphine (1)
Morphine (6)
M3G (1)
M3G (6)
10000
Figures 6-9: In Vivo Drug Concentrations
(A) Mouse pups in groups one and six
received morphine alone. The concentration
of morphine and its metabolite M3G with
respect to time are shown. (B) Mouse pups in
groups three and four received methadone
alone. The concentration of methadone and its
metabolite EDDP with respect to time is
displayed. (C) Mouse pups in groups two and
five received combined morphine and
methadone. The concentration of morphine,
M3G, methadone, EDDP, and EDMP are
displayed with respect to time.
100
10
1
0
2
4
6
8
10
12
14
Time (hrs)
Methadone Group
100
Concentration (ng/mL)
Concentration (ng/mL)
1000
Methadone (3)
Methadone (4)
EDDP (3)
EDDP (4)
10
1
0
2
4
6
8
Time (hrs)
10
12
14
Morphine/Methadone Group
Morphine (2)
Morphine (5)
M3G (2)
M3G (5)
Methadone (2)
Methadone (5)
EDDP (2)
EDDP (5)
EMDP (2)
Concentration (ng/mL)
1000
100
10
1
0
2
4
6
8
10
12
14
Time (hrs)
DISCUSSION:
Concomitant administration of morphine and methadone was expected to result in the
significant decrease of OAS symptoms experienced by the mice pups receiving the
morphine/methadone cocktail compared with those in the control groups receiving morphine
alone and methadone alone. This would imply that the NMDA receptor plays a significant role
in methadone function, specifically in the attenuation of the symptoms associated with prolonged
morphine exposure. Although the experimental study remains incomplete, based off the
literature, the regulation of calcium levels at the NMDA receptor remains a promising strategy to
manage tolerance induced pain treatment for critically ill children.
In this study, the drug delivery minipump system did not function properly in order to
achieve the desired infusion conditions. According to the blood samples, the pump did not shut
off at 72 hours (time 0) as we intended it to. Instead of seeing a stable trend in parent drug
levels, a decrease in the parent drugs and an increase in metabolites upon drug discontinuation
was expected with respect to time. Stable drug levels would mean that the pups were not in a
withdrawal state during the behavioral assessment since they were still receiving drug.
In order to conclude upon the concomitant strategy, the experiment will be repeated. The
future experiment will employ the same animal model however using a different drug delivery
system to ensure pups undergo withdrawal.
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