1. CYP450 induced Cardiotoxicity 2. Doxorubicin 3. Doxorubicin induced cardiotoxicity 4. NSAIDS 5. NSAID induced cardiotoxicity 6. Opioids induced cardiotoxicity 7. References

1. ASSIGNMENT ON CARDIOTOXICITY INDUCED BY DOXORUBICIN &
OPIOID AND OTHER TYPES OF PAIN KILLERS AND THE
INVOLVEMENT OF CYP ENZYMES
NAME- Priyansha Singh
REG.NO. PC/2021-14/226
SUBJECT CODE- PC-610
M.S. (PHARM) - Pharmacology &Toxicology (2nd SEMESTER)
DEPARTMENT- PHARMACOLOGY AND TOXICOLOGY
SUBMITTED TO- Dr. Roshan Borkar (Asst. Professor- NIPER Guwahati)

2. HOW DOES CYP ENZYME ATTRIBUTE TO CARDIOTOXICITIES
Fig. no. 1- How is CYP 450 responsible for Cardiotoxicity
Fig. No.2- How does imbalance between EET & 20-HETE cause Cardiotoxicity

3. (A) DOXORUBICIN (Dox)
Dox is a cytotoxic agent that belongs to the Anthracyclineantibiotic class. Dox was isolated froma bacterial
culture of Streptomycespeucetius.Doxis used to treat cancers such as Kaposi's sarcoma, acute lymphocytic
leukaemia, breast cancer, lymphoma, and bladder cancer.Dox prevents DNA and RNA synthesis by inhibiting
topoisomerase II. It appears to attach to the nucleic acids of DNA by a specific insertion of Anthracycline nuclei
into the DNA of human cells. Dox has a damaging effecton cancer cells. Its harmful effectson many body organs
are thought to be linked to nucleotide base insertion and cell membrane lipid binding activities of Dox. Dox
insertion inhibits nucleotide replication and consequently the activity of DNA and RNA polymerases. Dox's
interaction with topoisomerase II to create DNA-rupturable complexes appears to be an efficientmechanism
for Dox'scell-killing function. Doxtreatment of cells resulted in a distinct morphological change in apoptosis, or
programmed cell death. Doxorubicin treatment in patients has both therapeutic and harmful effects.
Metabolism: FollowingDox treatment, it was transformed into its active metabolite doxorubicinol by an
enzyme called NADPH-dependent aldoreductases. The active moiety that gives the antineoplastic effectis
doxorubicinol. These reductases can be found in erythrocytes,kidney cells, and liver cells. After 5 minutes of
Dox injection, the metabolite is detectable in more than 20% of the plasma; after 30 minutes, it reaches 70%,
75% after 4 hours, and 90% after 24 hours.
DOX-InducedCardiotoxicity:Molecular Mechanisms DOX builds up in the heart via interacting with cardiolipin
in the inner mitochondrial membrane. DOX clearance fromthe myocardiallags much behind that of the plasma,
whichmay explain why the heart is especially sensitive to DOX.The mechanisms of therapeutic effectsof
doxorubicin on tumor cells are different from those of the mechanisms of its cardiotoxicity. The proposed
mechanisms of its anti-malignancy effectsinclude intercalation into DNA leading to inhibition of synthesis of
macromolecules, generation of reactiveoxygen species, DNA binding and DNA cross-linking; DNA damage by
inhibition of topoisomerase II, and induction of apoptosis by inhibition of topoisomerase II.The proposed
principal mechanisms of doxorubicin cardiotoxicity are increased oxidative stress, as evident from increased
levels of reactiveoxygen species and lipid peroxidation. Decreased levels of antioxidants and sulfhydrylgroups,
inhibition of nucleic acid and protein synthesis, release of vasoactiveamines, altered adrenergic function and
decreased expression of cardiac-specific genes are other proposed mechanisms. It is possible that more than
one mechanism is operative. It should also be emphasized that the importance of many of the proposed
mechanisms remains to be established.
Oxidative Stress- Doxorubicin appears to induce toxic damage to the mitochondria of cardiomyocytes.Several
mitochondrial enzymes such as NADH dehydrogenase, cytochromeP-450reductase and xanthine oxidase are
involvedin generating oxygen free radicals (reactive oxygen species). Doxorubicin also increases superoxide
formation by increasing endothelial nitric oxide synthase, whichpromotes intracellular hydrogen peroxide
formation.
Gene Expression- The down-regulation of α-actin, myosin light and heavy chains, troponin-I, and desmin
proteins by doxorubicin has been suggested as a potential mechanism of its cardiotoxicity.Decreased
expression of the contractile proteins is associated with myofibrillar loss and reduced myocardial contractile
function.Downregulation of sarcoplasmic reticular ATPase may cause abnormal myocardial diastolic function.
It has been suggested that doxorubicin can inactivate extracellular signal-regulated kinase (ERK).
Apoptosis- There is evidence that doxorubicin induces apoptosis of cardiomyocytes.Formation of hydrogen
peroxide and superoxide has been implicated in doxorubicin-induced cardiomyocytetoxicity.These
intracellular oxidants induce p53, and activated p53 promotes apoptosis of cardiomyocytes.
Doxorubicin binds to the eNOS (endothelial nitric oxide synthase) reductase enzyme and induces Dox semiquinone radical
formation; this radical reduces the free oxygen into the superoxide (O2--) free radical. Enzymatic one-electron reduction
type of reaction is involved in this reactive conversion which shows cardiotoxic effects. Dox completes the one-electron
reduction reaction in the presence of flavoenzymes like NADPH-CYP450 reductase and mitochondrial NADH

4. dehydrogenase. When the drug binds to the eNOS reductase enzyme, there is an imbalance in superoxide free radicals and
nitric oxide levels. Nitric oxide level decreases and the levels of superoxide increase which leads to cardiotoxicity
Fig. no. 3 - A summary of all the mechanisms involved in cardiotoxicity induced by Doxorubicin
Fig. no. 4 – Molecular mechanism of cardiotoxicities caused due to Doxorubicin
DOX administration significantly modulates cardiac expression of CYP450 enzymes and its activity leading to
imbalance between CYP450-mediated cardiotoxic and cardioprotective pathways.This imbalance was
accompanied by a significant production of DHETs and 20-HETE where sEH and hydroxylase enzymes
demonstrate pivotal roles in DOX cardiotoxicity,suggesting another mechanism by which DOX causes
progressive cardiotoxicity.However,CYP induction was a result of acute DOX cardiotoxicity that may be
involvedin its progression to end-stage heart failure.
Inhibition of these enzymes conferred protection against DOX toxicity in the cardiac H9c2 cells. Therefore, sEH
and -hydroxylaseenzymes might be considered as novel targets to treat and/or to protectagainst chronic DOX
cardiotoxicity

5. Fig. no. 5 Pathways for cardiotoxicity mediated by DOX via CYP450 pathway
1. CytochromeP450 (CYP)2J2 is the most expressed CYP in the myocardium. CYP 2J2 converts
Arachidonic acid (AA), into four regioisomers of epoxyeicosatrienoic acids (EETs).These EETsare
cardioprotectivebecause they reduce myocardial infarctsize, reduce ischemia-reperfusion injury and
prevents arrhythmia.
2. DOX potentially inhibits Arachidonic acid (AA) metabolism by CYP2J2whichcontributes to
cardiotoxicity.AA and DOX competes for the binding of activesite in CYP2J2.
3. DOX is then converted into 7- deoxydoxorubicin aglycone(7-de-aDOX),by cytochromeP450reductase
(CPR),increases the amount of 5,6 EETand 8,9 EETand decreasing 11,12 EETand 14,15 EET.5, 6 EET
is not be effectivein promoting the survival of human lung microvascular endothelial cells and 8, 9 EET
to be least cardioprotective.This change in homeostasis further exacerbate the toxicity.
4. DOX induced cardiotoxicity is mediated through the induction of CYP1B1and its associated mid-chain
HETEsmetabolite.
5. CYP1B1has a major role in the pathogenesis of cardiovascular diseases such as ischemic heart disease,
myocardial infarction,hypertension, atherosclerosis, heart failure and cardiac hypertrophy.
6. Cardiotoxic roles of the CYP1B1are primarily mediated through the metabolism of AA into mid-chain
HETEssuch as 5-, 8-, 12-, 15-HETE.
7. 5-HETE have vasoconstrictiveand pro-inflammatory action. 12-HETE act as vasoconstrictorin small
renal arteries and induce apoptosis and cardiotoxicity in mice cardiomyocytes
8. Increased formation of 12- and15- HETE metabolites leads to heart failure by the induction of cardiac
fibrosis. Mid chain HETEsinduce cellular hypertrophy in the human ventricular cardiomyocytesRL-14
cells through MAPKs and nuclear factor-kB(NF-kB) dependent mechanisms.

6. Fig. no. 6- Summarized pathway of DOX mediating cardiotoxicity via CYP2J2
To summarize- DOX inhibits AA metabolism by CYP2J2in a competitive manner. This would lowerthe quantity
of EETs available to counteractthe DOX-initiated cardiotoxic attack. CPRthen converts DOX into 7-de-aDOX.
This metabolite contacts CYP2J2in the PUFAbinding pocketto change the conformation of AA's binding. As a
result, the site of metabolism shifts to favourthe creation of EETsthat are less effectiveat combating
cardiotoxicity,resulting in the construction of straw houses rather than brickhouses to counteract
cardiotoxicity.This disruption in homeostasis may increase the toxicity. Non-cardiotoxic analogues of DOX
inhibit CYP2J2but have no effecton regioselectivity. This combination technique of in vitrokinetic experiments
and in silico analysis sheds light on a potentially new mechanism of disease. This combined strategy of in vitro
kinetic experiments and in silico analysis sheds light on a possibly new DOX toxicity mechanism, namely the
inhibition of EETsand regulation of the site of AA metabolism. These findings lay the groundworkfor future in
vivoresearch into DOX cardiotoxicity.Furthermore, these findings can help in the development of new
analogues with lower cardiotoxicity that do not interfere withCYP2J2'sAA metabolism.

7. (B) NSAIDs induced Cardiotoxicity
Non-steroidal anti-inflammatory drugs (NSAIDs) are medicines that are widely used to relieve pain, reduce
inflammation, and bring down a high temperature. They'reoften used to relieve symptoms of headaches,
painful periods, sprains and strains, colds and flu, arthritis, and other causes of long-term pain.
Cardiotoxic risk depends on dose, duration and frequency of NSAID administration. The Cardiotoxicity
associated with use of NSAIDs might be due to inhibition of prostacyclin synthesis, oxidative stress, increase in
blood pressure and impaired endothelial function. As wecan see in the given figure, NSAIDS release free
radicals whichare responsible for cardiovasculardiseases.
Fig. 7- Flow chart depicting how NSAIDS are responsible for cardiac issues
In the cardiovascularsystem the major sources of ROS generation include the mitochondria, NADPH oxidases,
xanthine oxidoreductases, lipoxygenase, cyclooxygenases,nitric oxide synthases, and cytochromeP450based
enzymes (Figure 2). Continuous exposure of cardiovascular cells to oxidative stress associated with elevated
ROS levels would result in altered cellular homeostasis whichcould be an important contributing factorfor
various cardiovascular conditions.
The role of P450 in drug metabolism has been considered to be a key factorin overcoming the adverse and
toxicologicaleffectsof drugs. The P450 system has been shown to play an important role in activation of
oxygen and ROS generation. Normally, the activeoxygen species are formed in situ during the P450 cyclewhen
it reacts witha substrate. However,uncoupling of the P450 system results in excessive ROS generation and the
P450 system being unable to metabolize a substrate, which leads to oxidative stress and subsequently cellular
damage. NSAIDs may lead to increased ROS levels via the upregulation of P450 in cardiovascular related cells.
Varied distribution and functions of COX-1and COX-2have raised the possibility that non-selective NSAIDs and
selective COX-2inhibitors could also have different effects on other body systems, including the cardiovascular

8. haemostasis. COX-2inhibitors appear to alter the balance of vasoactiveeicosanoids such as prostacyclinand
thromboxane. These two prostanoids are produced by the action of cyclooxygenases.Thromboxane A2 (TXA2)
is primarily synthesized in platelets by COX-1,which leads to platelet aggregation, vasoconstriction,and
smooth muscle proliferation. Conversely, prostacyclin(PGI2) synthesis is largely mediated by COX-2in macro-
vascular endothelial cells and this way, on contrary to COX-1, COX-2results in inhibition of platelet aggregation,
vasodilation, and anti-proliferative effects.
For normal haemostasis, the balance between thromboxane and prostacyclinis important and nonselective
NSAIDs maintain this haemostatic balance by blocking both COX isoforms. However,the selective COX-2
inhibitors lower the level of PGI2 and imbalance the equilibrium between TXA2 and PGI2. This situation leads
to the production of the prothrombic state whichfurther leads to the over-activationof TXA2 that in turn is
responsible for the cardiotoxicity
Fig. 8- Pathways by which NSAIDS can induce cardiovascular diseases
NSAIDS relieve pain by inhibiting the cyclooxygenasepathway (COX pathway). Arachidonic acid is
converted into prostaglandins and thromboxane via this mechanism. Thromboxane A2 (TXA2) and
prostacyclin(PGI2)are the most important metabolites generated during Arachidonic acid metabolism for
maintaining vascular homeostasis. TXA2 and PGI2 have opposing effects.TXA2 is primarily involvedin
vasoconstrictionand platelet aggregation, whereas prostacyclinis involvedin vasodilation and platelet
aggregation inhibition.
COX enzyme exists in twoisoforms: COX-1(molecular weight- 70KDa) and COX-2(molecular weight-
70KDa) (molecular weight- 72 KDa). COX-1 is expressed constitutively in the stomach, intestinal mucosa,
kidneys, and other organs. It protects the mucosal lining of the stomach and is involved in vasoconstriction
and platelet aggregation.
During inflammation, inducible COX-2is activated and produces vasodilation.
Coxibs disrupt the balance between TXA2 and PGI2 that leads to atherosclerosis, thrombosis and other
cardiovascular complications.Coxibs (selective COX-2inhibitor) inhibits endothelial cell synthesis of
prostacyclin.Apart from its role in the inhibition of COX pathway, NSAIDS have been shown to cause
programmed cell death (apoptosis) by-
SU signalling pathway

9. wnregulation of BC1 pathway.
-1.
(C) Opioid induced cardiotoxicity
Fig. no. 9 Opioids causing cardiotoxicity
Opioid overdose is a leading cause of unintended mortality; receptor-mediated versus off-targeteffects
may help distinguish cause of death. Both opioid agonism and antagonism/withdrawal are associated with
adverse cardiovascularevents. Synthetic opioids, including forms available over the counter, can be pro-
arrhythmic. Risk mitigation strategies are needed to address ventilatory depression, ventricular
arrhythmia, and the rising incidence of endocarditis.

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