Background: Reversal of the enduring effect of rocuronium by neostigmine is a common procedure performed in the Libyan
hospitals. The reversal of the continuing effect of rocuronium by neostigmine was also evaluated.
Methods: eighty adult surgical patients were included in the study using neostigmine 2.5mg (0.05-0.07mg/kg) to reversal the
block induced by rocuronium 0.6 mg/kg. Anaesthesia was induced and maintained using i.v. propofol (2.5mg/kg) and fentanyl
(1.5μg/kg). Reversal neuromuscular function was monitored using clinical signs includes patient responsiveness, subjective
measurements of muscle strength (5 second head lift, hand grasp), eye opening, and tongue extrusion.
Results: Reversal of block was sustained in all patients from
the enduring effect of rocuronium by neostigmine. Ninety-six
patients were had a similar time of recovery but eleventh were not.
There were no serious adverse effects from neostigmine and no
significant changes in any measure of safety.
Conclusions: neostigmine is capable of reversing rocuronium-induced blockade in the Libyan patients by monitoring the
muscle strength, eye opening, and tongue extrusion.
Keywords: Acetylcholinesterase; Neostigmine; Propofol; Fentanyl; Anaesthesia; Rocuronium
Introduction
Acetylcholinesterase (AChE) is catalyzing the quick hydrolysis
of acetylcholine (ACh) to acetate and choline. The main biological
function is being annihilation of impulse transmission at
cholinergic synapses. In addition, AChE is believed to have nonclassical
roles in nerve and muscle growth and in hematopoiesis
[1,2]. Additionally, AChE has been concerned in Alzheimer’s disease
[3,4], hypersensitivity to pesticides and Gulf War syndrome [5].
Rocuronium is frequently used non-depolarizing neuromuscular
blocking agents (NMBAs) to facilitate tracheal intubation and
affording muscle relaxation throughout surgery. Patients receiving
these agents are at danger of outstanding curarization, which is a
cause of postoperative pulmonary complications and might augment
postoperative mortality [6,7]. Anticholinesterases drugs act mainly
by inhibiting acetylcholinesterase and butyryl cholinesterase,
prolonging the existence of acetylcholine at the motor end-plate
[8]. Additionally, anticholinesterases may have a direct agonistic
effect by increasing the release of acetylcholine from presynaptic
nerve terminals [9]. For, edrophonium the maximum effective
dose is 1.0-1.5mg/kg and for neostigmine, is 60-80μg/kg [8]. The
use of two antagonists together is avoided as they are not additive
and insufficient reversal can occur. It is not sensible to administer
extra anticholinesterase if maximal doses of edrophonium (1.5mg/
kg), neostigmine (70μg/kg), or pyridostigmine (350μg/kg) fail to
antagonize the residual blockade and might in turn increase the
weakness [9]. They are combined with atropine or glycopyrrolate
in order to counteract the muscarinic side-effects of these drugs.
Neostigmine is the most potent and the preferred drug [9]. Highdose
neostigmine or unnecessary use of neostigmine could interpret
to increased post-operative respiratory morbidity [10,11]. Current
rules identify the use of reversal with neostigmine based on the
train of four (TOF) monitoring with the neuromuscular monitor.
Neostigmine can be given for reversal in patients: who were
receiving drugs which enhance the action of NMBAs (inhalational
agents). The reversal should not be given in case of TOF counts
four in patients receiving anaesthetic drugs which do not boost the
blockade by NMBAs (intravenous anaesthetics) [10,11]. Moreover,
the rules also identify that if the TOF counts less than 2 reversal
should not be delayed. A lower dose of neostigmine (20μg/kg)
must be considered if TOF counts four and no fade is apparent or if
TOF ratio is 0.4:0.9 on qualitative neuromuscular monitoring [12].
Neostigmine is a cholinesterase inhibitors and it is wildly used in
Libya as indicated in the literature as reversal agents for NMBAs
[13,14]. Sugammadex, which is a novel agent for the reversal
of neuromuscular blockade, is adapted gamma-cyclodextrin.
Sugammadex is able to form a complex with rocuronium, eradicates
it from the circulation and terminates neuromuscular blockade
[15]. Sugammadex is a very safe agent with a little risk of serious
side effects [16]. The high cost of sugammadex, which is one of the
costliest drugs in anesthesia practice, prevents it from being used
in Libya as a standard neuromuscular reversal drug. Even though
it has been suggested by many doctors in Libya that the cost of
sugammadex use in anesthesia could be reduced by shortening the
duration of recovery [17,18], further clinical studies on sugammadex
in Libya are needed to introduce it to the governmental and private
hospitals.
It has been reported that the co-administration of neostigmine
with some Nonsteroidal Anti-inflammatory Drugs (NSAIDs, e.g.
aspirin) resulted in a synergistic interaction, which may provide
evidence of supraspinal antinociception modulation by the increased
acetylcholine concentration in the synaptic cleft of cholinergic
interneurons. The interaction obtained between neostigmine and
the NSAIDs could carry important clinical implications [19,20].
Acute toxicity from using cholinesterase inhibitors is related to the
inhibition of acetylcholinesterase activity at the neuromuscular
junction and in the brain, resulting in depression of circulatory
centers in the medulla, weakness of the muscles of respiration,
and pulmonary edema [21]. Because of the toxicity relates with
inhibition of acetylcholinesterase activity rather than butyryl
cholinesterase activity, it would seem reasonable to spotlight on
genetic variants of acetylcholinesterase. Though genetic variants
of human acetylcholinesterase exist, harmful mutations are
uncommon and occur only in the heterozygous state [22]. The
aim of this study is to investigate the effects of neostigmine’s
introduction on the incidence of residual neuromuscular paralysis
and postoperative Libyan patient outcome at Al-Shyfaa Private
Clinic, Tripoli, Libya.
Materials and Methods
Qualitative and Quantitative Determination of
Neostigmine: The Neostigmine methyl sulphate was obtained
from four different companies which are: Neostigmine methyl
sulphate (Rotexmedica, Germany); Plantigmine (Polifarma,
Turkey); Flagstig (Thexopharma, UK) and Neostigmine (Adeka,
Turkey). The reference standard was obtained from Sigma-Aldrich
(cat. no. 2126). The identification test for the neostigmin was done
using absorbance spectra which were measured on a Jenway UVvisible
spectrophotometer, model 6505 (London, UK) using quartz
cells of 1.00 cm path length. The UV-Vis absorbance spectra were
recorded in the 200-500 nm range, and spectral bandwidth of
3.0 nm. It was performed the baseline subtraction of the water
for the final spectrum of each solution analyzed. An equivalent
to 5 mg of neostigmine methylsulfate Injection according to the
labeled amount was taken and completed to 10 mL with distilled
water, and the absorption spectrum of this solution was measured
by Ultraviolet-visible Spectrphotometry which should exhibit a
principle band at 261nm. In addition the identity of neostigmine
was also determined using HPLC technique by determining the
retention time of the major peak in the chromatogram of the four
Neostigmine methylsulphate brands preparations compared to
the retention time of the major peak in the chromatogram of the
standard preparation. The quantitative assay of the neostigmine
(Neostigmine Methylsulfate Injection) was perofromed by Waters
HPLC using the Japanese Pharmacopoeia (Sixteenth Edition), page
1168.
Molecular Docking: The starting geometry of neostigmine
was constructed using chem3D Ultra (version 8.0, Cambridgesoft
Com., USA). The optimized geometry of neostigmine with the
lowest energy was used in the molecular docking. The crystal
structures of human acetylcholinesterase in a complex with a
transition-state analogue were downloaded from the Protein
Data Bank. The molecular dockings of neostigmine with human
acetylcholinesterase was accomplished by AutoDock 4.2software
from the Scripps Research Institute (TSRI). Firstly, the polar
hydrogen atoms were added into human acetylcholinesterase
and neostigmine molecules. Then, the partial atomic charges of
the human acetylcholinesterase and neostigmine molecule were
calculated using Kollman methods [23]. In the process of molecular
docking, the grid maps of dimensions (62Å X 62Å X 62Å) with a
grid-point spacing of 0.376Å and the grid boxes is centered. The
number of genetic algorithm runs, and the number of evaluations
was set to 100. All other parameters were default settings. Cluster
analysis was performed on the results of docking by using a root
mean square (RMS) tolerance of 2.0Å, and this was dependent on
the binding free energy. Lastly, the dominating configuration of the
binding complex of neostigmine and human acetylcholinesterase
with minimum energy of binding can be determined.
Study Design and Patient Selection: This study was
conducted at APC (Al-Shyfaa Private Clinic, Tripoli, Libya) during
July/August 2018. The protocol was accepted by the Independent
Ethics Committee at the clinic and conducted in fulfillment with
the recent amendment of the Declaration of Libyan Guidelines,
present regulatory requirements and Good Clinical Practice. Eighty
patients were included in the study and they were aged 20-60 years
and experiencing surgery in the supine position under general
anaesthesia (Laparoscopy surgery) which are requiring muscle
relaxation. Patients were excluded if they were anticipated to
have a neuromuscular disorder, difficult intubation for anatomical
reasons; family history of malignant hyperthermia, significant
renal dysfunction; or a known allergy to NMBAs, narcotics, or other
medication used throughout general anaesthesia. Patients receiving
anticonvulsants, antibiotics, or magnesium at a time possible to
hinder with neuromuscular block effect were also excluded. Female
patients who were breastfeeding, pregnant, childbearing potential
were also excluded. All patients have presented written consent.
Subject numbers were allocated to patients in chronological order
of their participation into this study. Anaesthesia was induced with
i.v. propofol (2.5mg/kg) and fentanyl (1.5 μg/kg) and maintained
using a continuous infusion of propofol and additional increments
or infusions of analgesic as needed. After the establishment of
neuromuscular scrutinizing, rocuronium 0.6mg/kg was given as an
i.v. bolus over 10 seconds into a quick running i.v. infusion. Tracheal
intubation was performed once the maximum neuromuscular block
effect is achieved and intermittent positive pressure ventilation
started to achieve a standard end-tidal CO2 concentration (4.5-5.5
kPa). Additional doses of rocuronium 0.1-0.2mg/kg up to a highest
of two dosages were administered if required. Once the last dose of
NMBA was administered and reappearance of T2 then neostigmine
0.05-0.07mg/kg (maximum of 2.5mg) was administered within 10
seconds into a quick-running i.v. infusion.
Monitoring: Using clinical signs comprises patient
responsiveness, individual measurements of eye opening, muscle
strength (5 second head lift, hand grasp), and tongue extrusion.
After extubation (removal of the endotracheal tube (ETT) and it is
the final step in liberating a patient from mechanical ventilation),
clinical evaluation of level of consciousness and neuromuscular
recovery (5 s head lift and general muscle weakness on a scale of
1-9) were carried out each 15 min until the first continued head
lift for 5 second was attained. Oxygen saturation using breathing
frequency and pulse oximetry were watched during anesthesia and
in the recovery room for at least one hour after operation. Heart
rate and arterial blood pressure were traced at the screening,
before administration of rocuronium, before and two, five, ten, and
thirty minutes after administration of neostigmine, and during
the post-anaesthetic visit, which was carried out within the first
twenty-four postoperative hours. Diastolic pressure of ≤45 or ≥95
mm Hg, systolic arterial pressure of ≤90 or ≥160 mm Hg, and heart
rates of ≤50 or ≥120 beats per minutes were admitted as clinically
noteworthy. The ECG was monitored constantly in the operating
theater and the recovery ward in a way consistent with classical
anaesthetic practice APC (Al-Shyfaa Private Clinic, Tripoli, Libya).
Physical examination was done prior to the surgery and at the postanaesthetic
visit. Ten milliliters of blood samples were withdrawn
from every patient for haematology and biochemistry evaluations
prior administration of rocuronium, from four to six hours after
administration of neostigmine, and at the post-anaesthetic visit.
Urine samples were gathered for analysis on the day prior surgery
or on the same day of surgery before anaesthesia and at the postanaesthetic
visit.
Anesthesia and Neuromuscular Block: To evaluate the
efficacy of neostigmine, the time from the start of administration
of neostigmine to recovery was determined. In addition, the clinical
signs of recovery after extubation, but before transfer to the
recovery room and before discharge from the recovery room were
also recorded. The time from administration of the intubating dose
of rocuronium to event of highest block was also studied (i.e. onset
time).
Statistics: Results were expressed as mean ± standard error
(SE). Statistical differences between the three parameters (muscle
strength, eye opening, and tongue extrusion) were evaluated by
one-way analysis of variance (ANOVA). All data were analyzed with
SPSS 10.0 software. P < 0.05 was considered statistically significant.
Results and Discussion
Qualitative and Quantitative Determination of
Neostigmine: The qualitative determination of neostigmine was
confirmed by the presence of the principle band at 261 nm of the UVvisible
absorption spectrum of the four Neostigmine methlsulphate
brands which is corresponding to the spectrum of the neostigmine
reference standard (cat. no. 2126, Sigma-Aldrich) and all spectra
exhibit identical absorption spectrum. In addition, retention time
(9 min) of HPLC chromatogram is also confirming the identity of the
neostigmine. Regarding to the quantitative assay; the HPLC results
have shown that the content profiles of the four brands are within
the pharmacopeia limit which is not less than 93% and not more
than 107% of the labeled amount of Neostigmine methylsulphate
C13H22N2O6S:334.39.
Molecular Docking Analysis: The modeling study was
performed in this paper showed great interactions between
neostigmine and human acetylcholinesterase. The binding energies of
neostigmine and acetylcholine human acetylcholinesterase were
-6.52 and -4.83 kcal/mole, respectively. The geometry of docking
obtained with neostigmine with human acetylcholinesterase as
shown in Figure 1. Neostigmine was able to form hydrogen bonds
(HBs) with the residues Try337 of the enzyme, pi-pi stacking with
the residue Trp86 and Pi-alkyl interaction with the residue Trp86.
In addition, the molecular docking results showed that other amino
acids residues are involved in the interactions with the neostigmine.
Figure 1: Three-dimensional representation of neostigmine
interacting with active site of target macromolecule
human acetylcholinesterase.
Clinical Elements: Eighty patients were randomized to
treatment (rocuronium–neostigmine, n=80). All treated patients
had three efficacy parameters that assessment carried out as shown
in Table 1. The time from the start of administration of the reversal
agent (neostigmine) to recovery, 11 patients used neostigmine had
very late recovery time (≥ 9 min). These data were not excluded
from the analysis. Mean systolic and diastolic arterial pressures
and heart rates were not very similar in all patients. Systolic
arterial pressures of ≥150 or ≤90 mm Hg, diastolic pressures of
≥98 or ≤45 mm Hg, and heart rates of ≥121 or ≤55 beats min−1
were observed in six patients used neostigmine. None of these was
believed clinically important. Full recovery from neuromuscular
block postoperative is crucial since residual neuromuscular block
may augment morbidity (hypoxia, dyspnea, airway obstruction,).
The use and monitoring of classical reversal agent is important
to avoid residual neuromuscular blockage [24,25]. The usual
neostigmine therapeutic dose is 0.04-0.07mg/kg whereas a toxic
dose is 0.08mg/kg. Neostigmine uses has restrictions such as
recurarization, muscarinic effect, weakness of respiratory muscle
and ceiling effect [26,27]. Some adverse effects of neostigmine
are dependent on the dose. Recent studies were performed to
estimate the lowest neostigmine dose to obtain recovery from
neuromuscular block (TOF ratio, TOFR 90%, the lower TOFR value,
the deeper the neuromuscular blockade) lacking adverse effects
[28]. The allocation of essential characteristic in this study is
including age, gender, body weight and occupation for all patients.
The duration of surgery in this study was limited to 30–90 minutes
owing to the pharmacokinetics of rocuronium. Rocuronium has
a moderate duration of action between 35-75 minutes [29,30].
However, Debane et al. has reported that the pharmacological
effect of rocuronium is existing until 120 minutes after single dose
administration [31]. All patients do not need to use additional
neuromuscular blocking agents dose during operation in this study.
Table 1 showed that a dose of neostigmine 2.5mg was as efficient
as attuned dose based on the TOFR value for reversal of single dose
rocuronium 0.6mg/kg. Table 1 showed that the total patients who
reached TOFR ≥ 90% at 0-9 minutes after reversal administration
were 69 and who reached TOFR ≥ 90% at 10-20 minutes after
reversal administration were 11 (underline numbers). However,
the differences were not statistically significant (p=0.718, F=0.331,
F critical 3.034). All patients (except one patients had 19 minutes)
had already reached TOFR ≥ 90% in interval of 0-15 minutes after
reversal administration, the data obtained in this study in which
the mean of the time required to reach TOFR ≥ 90% after reversal
was 0-15 minutes were consistent with the data reported by Fuchs-
Buder et. al which showed that the time required was 10 or more
minutes [32]. In addition to that our results are consistent with
other study by Cappellini et. al who reported that the recovery time
of the patient was 10 minutes after neostigmine 0.01-0.03mg/kg
with a shallow degree of neuromuscular block at the end surgery
[33].
Table 1 also showed that the recovery time of the patient number
25 (who is on Aspirin tablets) was 17-20 minutes after neostigmine
administration and this delayed action could be related to the fact
that co-administration of neostigmine with some aspirin or other
NSAIDs like diclofenac resulted in a synergistic interaction, which
may provide evidence of supraspinal antinociception modulation
by the increased acetylcholine availability in the synaptic cleft of
cholinergic interneurons. The interaction obtained in this study
between neostigmine and the NSAIDs have important clinical
implications and it is consistent with the literature as reported by
Hugo et.al [20]. For the present study, patients No. 12, 41, 45, 53,
59, 61, 64, 72, 74, and 77 had delayed action could be related to
the mutational structure perturbation approach to expose longrange
communication and the presence of conformational switches
in the interior of the acetylcholinesterase enzyme. It is known that
mutations are changes to the base sequence. The base sequence
determines the amino acid sequence. A different base sequence
therefore codes for a different amino acid sequence. Amino acids
interact with each other by H-bonds, ionic bonds, and disulphide
bridges. When the amino acids sequence of the acetylcholinesterase
is changing then these interactions and bonds will be affected
and the 3D shape of the acetylcholinesterase changes (tertiary
structure). acetylcholinesterase work by interacting with substrates
through their ‘active site’ as shown in Figure 1. A change in
acetylcholinesterase shape will change the shape of the active site.
The neostigmine cannot bind to the new shape of the active site so
no acetylcholinesterase-neostigmine complexes are formed easily
so the acetylcholinesterase cannot be inhibited or inhibited very
slowly. Hasin et. al. has reported a total of 13 acetylcholinesterase
single nucleotide polymorphisms (SNPs) and they might have
deleterious adverse drug responses to acetylcholinesterase
inhibitors [34]. In addition, Johnson and Moore have reported that
the homo sapiens acetylcholinesterase mutations and they study
the active site residues and they found the mutations could lead to
differences in both steric and electrostatic properties of the active
site [35]. The delayed onset of action of neostigmine as shown in
Table 1 could be attributed to SNPs and mutation in Libyan patients.
Our findings in addition to the published record suggest that the
genetic variation could effect on neostigmine pharmacological
action. Further studies need on Libyan people to be done in the
future in order to confirm these results.
Table 1: Time (min) from start of administration of neostigmine to recovery.
1M.S.A = Muscle Strength Assessment (5-second head lift, hand grasp),
2E.O = eye opening
3T.E = tongue extrusion
Conclusion
This Libyan study was the first comparative study between the
three parameters (measurements of muscle strength, eye opening,
and tongue extrusion) that are used to reversal of the enduring
effect of rocuronium by neostigmine. It is found that rocuroniuminduced
neuromuscular block and can be reversed by neostigmine.
It increased the amount of safety data available about neostigmine,
although this was not the main reason for the study. Clinical signs
of recovery were similar with the three parameters, which is to
be expected. This indicates that the results presented are realistic
in the Libyan patients. The faster time to recovery with most of
patients (n= 69) compared with eleventh patients (n=11) in this
study is consistent with that previously reported. The rapid time to
recovery with neostigmine observed in Libyan patients is similar to
that reported in previous studies, demonstrating a consistency in
observed efficacy.
