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Khalil et al. European Journal of Pharmaceutical and Medical Research
152
QUANTITATIVE ASSESSMENT AND TIME KINETICS OF ZOPICLONE (THE MOST
USED HYPNOTIC DRUG) IN THE STORED BLOOD SAMPLES
Ismail Khalil*, Mohammed Jasim and Ali Kadhim
Bilad Al-Rafidain University College.
Article Received on 18/11/2022 Article Revised on 08/12/2022 Article Accepted on 28/12/2022
INTRODUCTION
This review addresses insomnia disorder (ID), by far the
most common sleep disorder, as well as the second most
common neuropsychiatric disorder, only outnumbered by
the Diagnostic and Statistical Manual of Mental
Disorders comprehensive category of all anxiety
disorders.[1] Insomnia is a subjective experience of poor
or unrefreshing sleep that may be apparent from a
delayed onset or decreased duration of sleep. Insomnia is
an underrecognized and undertreated medical condition
that leads to lifestyle impairment, loss of occupational
productivity, and potential physical harm from accidents
as well as exacerbation of other medical conditions. The
rate of diagnosed insomnia in the UK and North America
is estimated at 515 %, with up to 40 % of the population
experiencing symptoms of daytime sleepiness. Some
studies quote that up to a third of elderly North
Americans are prescribed either a Z-drug or
benzodiazepine for sleep disturbance, an alarming
statistic given the risks associated with hypnotics in the
elderly.[2] Commercially available, non-benzodiazepine
drugs in the USA for the treatment of insomnia:
zaleplon, zolpidem, and eszopiclone (the active
enantiomer of zopiclone). The ideal anti-insomnia drug
is a potent sedative during the night without causing the
same residual sedation during the daytime. Suboptimal
clinical and adverse effects of traditional
benzodiazepines have driven the development of
alternative sedativehypnotic drugs. While hypnosis and
sedation are adequately achieved from oral
benzodiazepines, they invariably alter sleep architecture,
reduce deep (stage 3 and 4) sleep, and lead to
dependence, tolerance, and withdrawal. Furthermore,
benzodiazepines carry the risk of residual daytime effects
SJIF Impact Factor 6.222
Research Article
ISSN 2394-3211
EJPMR
EUROPEAN JOURNAL OF PHARMACEUTICAL
AND MEDICAL RESEARCH
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ejpmr, 2023,10(1), 152-158
ABSTRACT
Background: Zopiclone is a hypnotic short-acting agent used in the treatment of primary insomnia. After oral
administration, zopiclone is rapidly absorbed and has a bioavailability of about 80%. Zopiclone is metabolized
extensively in the liver, but the CYP isoforms involved in its metabolism have not yet been identified. Objective:
To estimate the zopiclone levels in the drawn plasma of volunteers at different time points after storage under
refrigeration. To collect data about the insomnia or sleepiness after 24hours of single oral dose of zopiclone.
Methods: There were totally 42 volunteers belonging to the age groups 22-44 years, 45-60 years and 61-76 years
group. Drug administration: The volunteers were given an oral dose of zopiclone (5mg) tablet and blood collected
at various intervals as follows: 1- After administration, blood samples were collected at 0, 1, 2, 3, 4, 6, 12, 18, 24,
48h and plasma separated for zopiclone estimation. 2- After administration, after 2 hours the blood was collected
and plasma separated, which was stored in refrigerator for analysis at various intervals to see the stability of the
zopiclone. 3- Urinary levels of Zopiclone was also measured in all the groups. Results: In our study, we
demonstrated that following the single administration of oral tablet of Zopiclone, the plasma levels diminish very
slowly taking upto 48 hours. Some traces of Zopiclone were identifiable upto 72 hours (not presented here). The
major difference was the relapse of insomnia very fast among the elderly population and middle aged groups in
comparison to the younger group of 22-44 years. In addition, the storage of plasma even under refrigeration
resulted in fast degradation of zopiclone in the samples. This suggests strongly that, the zopiclone should be
estimated as the sample is fresh. In addition the zopiclone values from stored samples need verification with other
assays. Conclusion: Z-drugs have few distinct advantages over their predecessors, the benzodiazepines, and in
many ways they have similar adverse and toxic effects, especially zopiclone. The effects of Z-drugs largely derive
from their GABA ergic action and pharmacokinetic profiles, which decide the extent of efficacy and toxicity.
Adverse Z-drug effects and toxicity are more likely with poly drug use in therapeutics and co-ingested
psychoactive substances in overdose.
KEYWORDS: Insomnia, Zopiclone.
*Corresponding Author: Ismail Khalil
Bilad Al-Rafidain University College.
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Khalil et al. European Journal of Pharmaceutical and Medical Research
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such as impairment of cognitive and psychomotor
function. Like benzodiazepines, the newer Z-drugs are
-aminobutyric acid-type A
(GABAA) receptor. However, they possess shorter
duration of action and half-life, do not disturb overall
sleep architecture, and cause less residual effects during
daytime hours, making them more clinically attractive
than benzodiazepines.[3] Zopiclone is a hypnotic short-
acting agent whose chemical structure corresponds to a
cyclopyrrolone derivative, which, although not
chemically related to existing hypnotics, presents a
pharmacologic profile similar to the Benzodiazepines
group by binding with high affinity to the
pharmacological receptors of them. Zopiclone will often
work well in the short term, but it is not normally
prescribed for more than two to four weeks. This is
because the body gets used to it within a short period of
time and after this it is unlikely to have the same effect.
The system may also become dependent on it when it is
taken for longer periods of time than this. Zopiclone, like
the other Z-drugs, zolpidem and zaleplon, interacts with
the same molecular target as the benzodiazepines on the
GABA -aminobutyric acid) receptor. Zopiclone is
available as the racemic mixture although the S-
enantiomer, eszopiclone, has a 50 times higher affinity
for the receptor than the R-enantiomer. In terms of
clinical efficacy and adverse reactions, the Z-drugs
showed little difference from the short acting hypnotic
benzodiazepines although the fatality toxicity index
(deaths/106 prescriptions) for zopiclone, 2.1, was much
lower than the 9.9 for temazepam.[4] The Hypnotic
mechanisms of Zopiclone GABAA receptors mediate
inhibitory synaptic transmission in the central nervous
system and are the targets of neuroactive drugs used in
the treatment of insomnia.[5] GABAA receptors are
pentametric membrane proteins that operate as GABA
-aminobutyric acid) ligand-gated chloride channels.
Agonists increase the chloride permeability,
hyperpolarize the neurons, and reduce the excitability.
The receptors are made up of seven different classes of
throughout the brain. Most GABAA receptors are
- - -subunits. . ZOP has a high
affinity for the benzodiazepine binding site and acts at
--
comparable anxiolytic effects with less sedation, muscle
relaxation, or addictive potential.[5]
After oral administration of the racemic drug,
ZOPICLONE is rapidly absorbed from the
gastrointestinal tract, with a bioavailability of
approximately 80%. Plasma protein binding of
ZOPICLONE was reported as 45% in one study and 80%
-1-acid glycoprotein
contribute to protein binding but also other plasma
proteins might be involved (e.g. globulins, lipoproteins).
It has been noticed that the protein binding is stereo
selectivity.[6] reported the first large series of zolpidem
poisoning cases in 1994, where the toxicity
predominantly involved sedation with ingestions up to
1.4g. Rarely did zolpidem cause coma, respiratory
depression, cardiovascular toxicity, or death. Since then,
reports of agitation, hallucinations, psychosis, and coma
from Z-drug overdose have been published. Other
unusual reports include hemolytic anemia and
methemoglobinemia from zopiclone. Early clinical trials
failed to show major morbidity or mortality from Z-
drugs either used therapeutically or in overdose. Over the
past 15 years, increasing red flags from forensic cases,
drug-facilitated crimes, and motor vehicle crash statistics
indicate that mortality from Z-drugs may be similar to
benzodiazepines. Bizarre behavior, falls, accidents, and
other injuries may also lead to death. In the study by
Garnier et al.[7] Stability of Zopiclone in the biological
samples for future analysis In Sweden specimens of
venous whole blood are taken by a nurse or physician,
urine samples are collected by the police officers and
post-mortem samples (e.g. femoral blood, urine, vitreous
humor, hair, liver, brain, kidney and lung) are taken by
forensic pathologists. After sampling all specimens are
sent to one central laboratory for toxicological analysis.
During the transport the samples are stored at ambient
temperature for a period of about 2024 h. However, the
blood samples contain 100 mg sodium fluoride and 25
mg potassium oxalate as preservatives and the urine
samples contain 1% sodium fluoride as a preservative.
Before analysis, the samples are stored in a
refrigerator.[8]
MATERIALS AND METHODS
Volunteers
There were totally 42 volunteers belonging to the age
groups 22-44 years, 45-60 years and 61-76 years group.
3.2 Drug administration:
The volunteers were given an oral dose of zopiclone
(5mg) tablet and blood collected at various intervals as
follows: After administration, blood samples were
collected at 0, 1, 2, 3, 4, 6, 12, 18, 24, 48h and plasma
separated for zopiclone estimation.
After administration, after 2 hours the blood was
collected and plasma separated,, which was stored in
refrigerator for analysis at various intervals to see the
stability of the zopiclone. Urinary levels of Zopiclone
was also measured in all the groups.
Insomnia scale
After 24 hours of administration, the volunteers were
asked about their sleep behaviour and how they feel
about sleepiness or insomnia. They were given a score
on the scale of 10, with 10 being more insomnia while 1
being less insomnia.
Spectrophotometric estimation of Zopiclone 100 µl of
plasma was mixed well with 200 ul of butanol. Then this
was centrifuged at 2500 rpm for 10min at 4C. The
supernatant 100µl was taken and diluted to 1ml by
adding 0.9ml of butanol. The optical density of this
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extract was measured at 251 and 301 nm (reference). A
standard curve of pure Zopiclone was used to calculate
the sample readings.
Statistical analysis
The data expressed as mean ± SE were analyzed by one-
compare the control and treatment groups as well as
among the gro
software. Different alphabet letters indicate significance
difference among the respective groups. In some assays,
* indicates significance difference from control
RESULTS
No much difference in Zopiclone concentration among Males and Females.
Fig. 1: Concentration of Zopiclone in the plasma after 1 hour of administration (single oral dose of 0.5mg).
Zopiclone completely disappeared after two days of single injection (plasma).
Fig. 2: Pharmacokinetic assessment of Zopiclone in plasma from 0-48 hours after single administration.
In the middle age group, the plasma levels decreased slowly, indicating the lower detoxification pathways (probable
explanation).
Fig. 3: Pharmacokinetic assessment of Zopiclone in plasma from 0-48 hours after single administration (45-60
years age group.
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No much difference between elderly and middle aged group in processing the zopiclone.
Fig. 4: Pharmacokinetic assessment of Zopiclone in plasma from 0-48 hours after single administration (61-76
years age group).
Middle aged and elderly people complained of sleeplessness (insomnia) the next day after zopiclone single injection.
They wanted another dose for sleep inducing.
Fig. 5: Insomnia score among volunteers after 24 hours.
Urine analysis revealed the presence of traces after 48 hours also.
Fig. 6: Estimation of Zopiclone in urine samples of volunteers after oral administration (22-44 years group).
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Complete absence urine of males in middle aged group.
Fig. 7: Assessment of Zopiclone excretion in urine samples of volunteers after oral administration (45-60 years
group).
Complete absence among females in elderly. However, the fact is generally the urine levels diminish after 48 hours.
Fig. 8: Monitoring of Zopiclone urine levels from volunteers after oral administration (61-76 years group).
Fig. 9: Effect of storage on the Zopiclane stability among plasma samples (22-44 years group) sampled after 2
hours of administration, the plasma stored in refrigeration.
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Fig. 10: Effect of storage on the Zopiclane stability among plasma samples (45-60 years group) sampled after 2
hours of administration, the plasma stored in refrigeration.
Fig. 11: Effect of storage on the Zopiclane stability among plasma samples (61-76 years group) sampled after 2
hours of administration, the plasma stored in refrigeration.
The levels of zopiclone got disappearing as the plasma
was stored, in fridge. The same sample was analysed for
zopiclone levels. This suggests that, zopiclone should be
assessed as soon as the samples are collected.
DISCUSSION
In our study, we demonstrated that following the single
administration of oral tablet of Zopiclone, the plasma
levels diminish very slowly taking upto 48 hours. Some
traces of Zopiclone were identifiable upto 72 hours (not
presented here).
The major difference was the relapse of insomnia very
fast among the elderly population and middle aged
groups in comparison to the younger group of 22-44
years.
In addition, the storage of plasma even under
refrigeration resulted in fast degradation of zopiclone in
the samples. This suggests strongly that, the zopiclone
should be estimated as the sample is fresh. In addition
the zopiclone values from stored samples need
verification with other assays.
Interestingly there was no much difference in the
responses and the zopiclone stability between the
samples from men and women in each age group
participated.
However, zopiclone along with other drugs should be
assessed as part of the forensic investigation.
CONCLUSION
Z-drugs have few distinct advantages over their
predecessors, the benzodiazepines, and in many ways
they have similar adverse and toxic effects, especially
zopiclone. The effects of Z-drugs largely derive from
their GABAergic action and pharmacokinetic profiles,
which decide the extent of efficacy and toxicity. Adverse
Z-drug effects and toxicity are more likely with polydrug
use in therapeutics and co-ingested psychoactive
substances in overdose. Z-drug poisoning is clinically
similar to benzodiazepine overdose with supportive care
sufficient in managing the majority of cases. The
increasing ability to detect Z-drugs in various biological
matrices is promising for future forensic endeavors.
Postmortem redistribution appears to be significant for
zolpidem and likely also for zaleplon. It is recommended
that public health and drug regulatory authorities
maintain a high level of toxicovigilance with regard to Z-
drugs and their adverse outcomes.
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