J Young Pharm, 2019;11(1):105-107

Short Communication

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Clozapine Disrupts Endothelial Nitric Oxide Signaling andAntioxidant System for its Cardiovascular Complications
Gayathri M Nair, Dona Sheba Skaria, Teenu James, S K Kanthlal*

Department of Pharmacology, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Science Campus, Kochi, Kerala, INDIA.

ABSTRACT
Objective: Many drugs in current practice require additional safety labels
in order to prevent potential risks to the major organ system. Psychotropic
agent clozapine has been reported to produce myocarditis and other cardiac
complications on repeated use. Our study aimed to establish the role ofclozapine in vascular damage associated with nitric oxide metabolism.
Method: Isolated aortic strips incubated with clozapine at different doselevels were estimated for nitrite release and antioxidant systems such as
glutathione and catalase. Vascular integrity assessment was performedby recording the acetylcholine induced relaxation of phenyephrine precontracted aorta. Result: From our study, it was found that clozapine
depilates the nitric oxide level in the endothelium and enhance the oxidativestress. The aorta fails to relax completely after adding acetylcholine indicatesthe deranged eNOS signaling in the endothelium. Conclusion: From theexperimental findings, it was concluded that clozapine could depress theeNOS regulation and thereby perhaps initiates cardiovascular complications
through subsequent vascular events.
Key words: Endothelium, Nitric oxide, GSH, Catalase, Oxidation.
Correspondence
S.K Kanthlal, Department of Pharmacology, Amrita School of Pharmacy, Amrita
Vishwa Vidyapeetham, AIMS, Kochi, Kerala- 682041, INDIA.
Phone: +91 9787419041
Email: skkanthlal@aims.amrita.edu

DOI: 10.5530/jyp.2019.11.22

INTRODUCTION

MATERIALS AND METHODS

Ample array of drugs have been withdrawn from clinical use due to theirbroad cardiac events such as hypertension, ventricular arrhythmia,
angina etc., accompanied with or without cardiac death. Few such drugs
are Rosiglitazone, Valdecoxib, Rofecoxib, Cisapride, Tegaserod etc, Drug
induced cardiac effects are often predictable, dose dependent and hencecan reduce the incidence of mortality.1 Most drug candidates require
additional safety labels to prevent potential risk like angina, myocarditis,
severe hypertension, etc.
Psychotropic agents are also in the figure which was reported withangina, atherosclerosis, cerebrovascular disease, congenital heart defects,
coronary artery disease, heart attack, myocarditis, peripheral vasculardisease and stroke.2 Clozapine, a tricyclic dibenzodiazepine derivative
one among the most preferred drug due to its greater effectiveness andslumped incidence of extra pyramidal symptoms.3 But criticism ofclozapine still exists since it came to clinical practice for its progressionof metabolic syndromes which provoke diabetes and cardiovasculardisorders.4 In a case study, they observed the signs of acute myocardial
infarction and diagnosed with myocarditis after clozapine treatment for
7 days at a dose of 200mg/day.5 It was reported that clozapine escalatesoxidative mitochondrial stress in neutrophils which probably contributes
to the induction of apoptosis.6 However, there is only negligible information regarding the action of clozapine in the blood vessels. Moreovernone explained about the interaction of clozapine with endothelial nitricoxidation metabolism which is a prime element behind the cardiovascular homeostasis.
Here we present our experimental findings of the effect of clozapine on
vascular nitric oxide imbalance and its physiological outcome using
in vitro aortic ring study.

Materials
Clozapine, Acetylcholine and Phenylephrine were purchased from
Sigma-Aldrich (Milan, Lombardy, Italy). N-(2-Hydroxy ethyl)-piperazine
ethane sulfonic acid, N-(1-Naphthyl) ethylenediamine, sulphanilamide,
orthophosphoric acid, L-Glutathione, 5,5′-Dithiobis(2-nitrobenzoicacid) and potassium dichromate were purchased from HiMedia Ltd(Mumbai).

Preparation of aortic rings
Fresh thoracic aorta was isolated from healthy wistar rats after sacrificing
by carbon dioxide asphyxiation. The aorta was immediately transferred
into ice cold Potassium chloride (150mM). It was then cleaned and sliced
into small pieces of about 1cm length which was used for our study.7 All
the proceedings were performed in controlled conditions.

Experimental evaluation
Aortic rings weighing 100-120mg were transferred to test tubes containing ice cold N-(2-Hydroxy ethyl)-piperazine ethane sulfonic acid(HEPES) buffer and added with different concentrations (0.5,1.0, 1.5, 2.0and 3.0µg/mL) of clozapine. Control aorta was added with HEPES buffer
and the volume was made up to 10X of tissue weight using HEPES buffer.
All the samples were incubated at 37°C for 60min in BOD incubator.

Estimation of nitric oxide release
The amount of nitric oxide released from the aorta was indirectly estimatedfrom its metabolite nitrite using Griess reaction.8 10µl of supernatantsolution from each group was added with 20µl Griess reagent A and B.
Then the volume was made up to 1000µl with ultra-pure water. The

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Nair, et al.: Clozapine Induced Nitric Oxide Imbalancemixture was then incubated at 37ºC for 30min before reading the absorbance at 540nm primary filter and 630nm secondary filter using semi
auto biochemistry analyzer. (STAT FAX 3300, Awareness TechnologyInc, USA).

Vascular reactivity assessment
The thoracic aorta was cut into rings of 3–4 mm length and immediatelytransferred into Kreb’s solution. The rings were suspended on tissue bath
(EMKA Bath-2) containing 10mL Kreb’s solution bubbled with carbogen(95% O2 + 5% CO2) maintained at 37°C.9 The changes in isometric force
were recorded using IOX-2 (Emka Technologies S.A.S, Paris, France).
Baseline tension was adjusted to 2g and all the subsequent measurements were generated above the baseline. The aorta was pre-contractedwith phenylephrine (1 µM/L). Vascular integrity was assessed by adding
acetylcholine (10nM – 1mM) cumulatively and the Ach induced relaxation
was expressed as a percentage reduction of phenylephrine inducedcontraction. The vascular reactivity was again recorded after incubating
the aorta with clozapine at three higher concentrations (1.5, 2.0 and3.0µg/mL) for about 30min.10

Estimation of oxidative markersThe oxidative stress in aorta was accessed by quantifying tissue glutathione level and catalase activity after preparing the aortic homogenate.
For GSH, 100µl of homogenate after centrifugation with 5% sulfosalicylic acid was added with 800µl Na2HPO4 and 100µL Ellman’s reagent.11
The absorbance of the mixture was measured in biochemistry analyzer
at 405nm after 5 min. For estimating catalase activity, 100µL homogenate
was mixed with 1000μL of hydrogen peroxide and incubated for 5min.
2000µL of dichromate-acetic acid mixture was added to the above
solution and kept at 100°C for 10min. The absorbance was measured at
570nm against regent blank after centrifugation (3000 Rpm for 15min).12

Figure 1a: Showing the nitrate release from aorta incubated at different
concentrations of clozapine. b. Vascular reactivity assessment by evaluating
the relaxation effect of acetycholine on phenylephrine pre-contracted aorta.
Data are shown as mean ± SEM (n=3). Data are shown as mean ± SEM (n=3).
*p<0.05,** p<0.01 and ***p<0.001 significantly different from control untreatedgroup. (Clz: Clozapine)
Table 1: Effect of clozapine on aortic oxidative stress.
Treatment

GSH

Catalase activity(µg/L)(pM/mg tissue)(U/mg tissue)

ControlClz 0.5
Clz 1.0
Clz 1.5
Clz 2.0
Clz 3.0

4.16±0.17
2.78±0.07*
2.45±0.05*
2.13±0.05*
1.63±0.05*
1.02±0.02*

0.63±0.028
0.39±0.039*
0.37±0.006*
0.33±0.002*
0.31±0.012*
0.11±0.002*

Data are shown as mean ± SEM (n=3). *p<0.05significantly different from control untreated group. (Clz: Clozapine)

Effect of clozapine on vascular oxidation

Results were generated from 3 independent experiments (n = 3) with
3 replicates and the data analysis was performed with Instat-Pro. The
results were illustrated using Graph-Pad Prism 5 and variations between
results were expressed as standard errors mean (SEM). The data were
statistically analyzed by one-way ANOVA using Tukey–Kramer multiple
comparison test.

Endothelial glutathione level and catalase activity of untreated aorta
was found to be 4.16 pM and 0.63 U respectively. The sulfhydryl groupdeclined significantly in all the clozapine treated groups in a dose
dependent manner. The level was dropped by 24.52% after incubatingwith clozapine at 3.0µg/mL. In case of catalase activity, an abatement of
the enzyme activity at in all dose level was observed. The activity was
found to be monstrously diminished to a maximum of 0.11U at highest
dose level of 3.0µg/mL. (Table 1)

RESULTSDISCUSSION

Effect of clozapine on aortic nitrite

In our laboratory we performed an in vitro study to examine the involvement of endothelial damage for cardiac side effects induced by clozapine.
Clozapine is the last-line therapy to treat schizophrenia after multiple
drug failure in schizophrenic patients. Besides the primary insult
agranulocytosis, potential fatal cardiac effects like myocarditis, dilated
cardiomyopathy, venous thromboembolism and pericarditis are themost proclaimed adverse effects associated with its use.13 Acute toxic
symptoms were evidenced after attaining a plasma concentration above
2000µg/L.14 Based on previous reports and statements regarding clozapine,
we have randomly selected the dose at a range of 500 to 3000µg/L tounderstand the physiological changes based on varying dose levels(therapeutic and toxic dose). The incubation time of 1hr was preferred
after a preliminary exploration by incubating aorta with L-NAME (standardeNOS inhibitor) at 15, 30 and 60 min. The nitrite level was declined
about 60% from after 60min incubation. Endothelial dysfunction is
occasionally pervasive throughout the body as patients with known
atherosclerosis.15 This turns out to be the consequence from inadequatelevels of NO and predominantly it is the baseline risk factors for

Statistical analysis

After incubating aorta with clozapine, no significant reduction of nitrite
was found at 0.5µg/mL and there after a dose dependent drop off wasobserved at each dose level (Figure 1a). Clozapine at the dose of 3.0µg/mL
reduces the aortic NO level to about 35% (48.02µM/L) from theuntreated aorta (73.3µM/L) illustrates the impairment of NO metabolism
which may further leads to subsequent destruction in the cardiovascularsystem.

Effect of clozapine on vascular reactivity
In normal aorta, acetylcholine relaxed the phenylephrine pre contracted
aorta to a maximum of 76.34% at log -4M. This depicts the normal tone
and activity of the healthy endothelium. On incubation with clozapine,
the response gradually declines to 56.32%, 29.63% and 11.35% for clozapine
1.5, 2.0 and 3.0µg/mL respectively indicates the signs of endothelial
disruption which obliviously declines the vascular nitric oxide release.
The results were shown in Figure 1b.
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Journal of Young Pharmacists, Vol 11, Issue 1, Jan-Mar, 2019

Nair, et al.: Clozapine Induced Nitric Oxide Imbalancecardiovascular disorders. Patients met with coronary artery bypass grafting(CABG), displayed reduced endothelial function which is principallyrelated to poor NO bioavailability.16 These findings witnessed NO as acontemporary alternative metric to monitor for secondary prevention.
Our finding certainly showcases the declined nitrate release from aorta
after incubating with clozapine. The deficiency in NO was further confirmed
by vascular reactivity assessment using isolated rat aorta (3 toxic doses
selected based on the significance in reducing nitrite level). Parasympathetic stimulation of the endothelium induces NO-dependent
vasodilatation by eNOS translocation from the plasma membrane. Ach
is known to be the classical neurotransmitter in favoring endothelial
mechanotransduction which facilitates flow-mediated dilatation.17 In
our present study, responses to the endothelium-dependent vasodilatationof acetylcholine (-6 to -4 logM) were significantly blunted in aortic rings
incubated with the drug. Such vasodilator dysfunction extending into
the coronary microcirculation contributes to the ischemic manifestationsof coronary artery disease during myocardial ischemia.18 These findings
strongly evidenced that the eNOS signaling is also desired to be disturbed
during clozapine treatment.
Impaired NO synthase, decreased L-arg uptake and increased lipidoxidation shows detectable Increase Reactive Oxygen species (ROS) levelin the endothelium which promotes endothelial dysfunction. This is
attributed to high oxidative stress and inflammation which can beworsen with other conditions (cold, mental stress, anger) etc. A report
said that patients with cardiac and systemic glutathione deficiency areclosely associated with the impaired functional status and structuralabnormalities of the heart.19 Reduction in tissue and plasma GSH levels
is considered to be the risk factors of CVD. Increased oxidative stressalso inactivates the endogenous antioxidant enzyme by releasing hydrogenperoxide. One such enzyme activity named catalase (CAT) has its
property in blocking the oxidative stress along with enhancing the SuperOxide Dismutase (SOD) activity. Drugs targeting antioxidant enzymes inendothelial cells perhaps offer a future perspective for the revolution ofcompetent cardio-protective remedies and in contrast, any disturbancesin such systems will also provoke the development of deadly disorders.
From our experimental findings it is perhaps obvious that clozapine evenat therapeutic dose can initiates endothelial destruction as well as it was
notified that a fair indication of erratic outcome in the vascular endothelium at toxic dose. These changes in ROS directly relate the activationof inflammatory response which further contributes to vascular abnormality on prolonged exposure. Also, neurohormones including catecholamines and angiotensin II all emerge to induce myocardial changes atleast in part via oxidative stress.20 Together it was capable to wrap up
that, the reported cardiovascular effects of clozapine is perhaps due tothe initiation of NO imbalance which switch on further cascade events
constantly on repeated use and leads to deadly sufferings if used without
safety measures.

CONCLUSION
Our study demonstrates that clozapine agitates nitric oxide metabolism
by altering the eNOS signaling and accelerates oxidative stress in theendothelium. NO deficiency alters the vascular tone which further promotes
the peripheral resistance and may leads to cardiac complications. We
have hypothesized that the physiological consequences of clozapine isdue to its effects in vascular eNOS coupling which triggers oxidativestress.

ACKNOWLEDGEMENT
We gratefully acknowledge Ms Rajitha. P for many helpful discussions
and the technical assistance of Ms. Anju Benny.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ABBREVIATIONS
NO: Nitric oxide; HEPES: N-(2-Hydroxy ethyl)-piperazine ethane sulfonicacid; GSH: Glutathione; ROS: Reactive oxygen species.

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Article History: Submission Date : 23-07-2018; Revised Date : 16-08-2018; Acceptance Date : 13-10-2018.
Cite this article: Nair GM, Skaria DS, James T, Kanthlal SK. Clozapine Disrupts Endothelial Nitric Oxide Signaling and Antioxidant System for its CardiovascularComplications. J Young Pharm. 2019;11(1):105-7.

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