4. Discussion
Cannabinoid receptors may be found all over the cardiovascular system.
The myocardium, human coronary artery, endothelial, and smooth muscle
cells, as well as pre-synaptic sympathetic nerve terminals that
innervate the cardiovascular system, all express the CB1 receptor. In
addition to the myocardium, CB2 receptors have been discovered in human
coronary endothelium and smooth muscle cells. Endocannabinoids are
generated in endothelium and smooth muscle cells, as well as heart
tissue, and their amounts in the blood may be measured. Despite this,
the endocannabinoid system is unlikely to have a significant role in the
control of cardiovascular function under normal circumstances (30).
JWH-018 is a synthetic cannabinoid with full agonist effect on both CB1
and CB2 receptors, It is known that the CB1 receptor affinity of SCs is
100 times higher than ∆9THC. Due to far more potent receptor activity,
it is quite reasonable to expect the cardiovascular effects to be
stronger and more problematic.
The negative effects of SCs on the cardiovascular system have been shown
in previous studies (31-33). According to our analyses of cardiac
results, the HR response of JWH-018 was found as different due to dose
and duration of application. While single high dose administration
resulted with reduced HR, chronic administration resulted with increased
HR irrespective of JWH-018 dose. In the AHD group reduced HR values were
accompanying with reduced mean BP. In long-term, BP drop was continuing
despite increased HR, especially in the rats are given prolonged high
dose JWH-018. In the related literature, the most frequently reported
cardiovascular effect of SCs are a significant decrease in arterial BP,
heart contraction and HR (34-36). Despite the fact that numerous studies
show that SCs related cardiovascular depression effects are mediated by
CB1 receptors, they may also have vascular and cardiac effects that are
independent of CB1 and CB2 receptors. The role of CB1 receptors in the
vasodepressor response has been demonstrated by the relief of
hypotension when a CB1 selective antagonist is used (37). Hypotension
caused by cannabinoid and the complete absence of bradycardia in CB1
receptor-deficient mice is the main evidence showing the effect of CB1
receptors on these cannabinoid-related effects (38). In anaesthetized
hypertensive mice, Batkai and colleagues discovered that CB1 receptor
agonists reduce contractility and normalize BP (39). Recently it was
shown that presynaptic CB1 receptor stimulation inhibits norepinephrine
release both in vitro and in vivo (40,41). Furthermore,
the hypotensive response to HU-210, a synthetic cannabinoid, remained
intact when sympathetic tone was reduced by ganglionic blockade and
vascular tone was restored by vasopressin infusion, even if the
bradycardic effect was gone (42). Above-mentioned data indicate that
cannabinoid-induced bradycardia is caused in the short term by
inhibition of sympathetic tone to the heart, however the hypotensive
response is directly related to vasodilation (43). According to our
long-term results, increased HR response can be explained by reflex
tachycardia for chronic reduced peripheral resistance and hypotension.
Furthermore, cannabinoids have a strong ability to block acetylcholine
release from heart.
In the current study, the effect of JWH-018 on cardiac structure and
function was evaluated by using transthoracic ECHO. Although we did not
recognize functional and structural changes in terms of ejection
fraction or fractional shortening and any of diastolic function
parameters. Also, level of serum proBNP which is a sign of impaired
cardiac functions was found to be increased in long-term high dose
JWH-018 treated group. It can be accepted that this situation is
compatible with the cardio depressant effect of SCs as proven before.
Pacher et al. highlighted that the hypotensive action of a synthetic
cannabinoid, HU-210, is predominantly due to a decrease in ventricular
contractility in pentobarbital-anesthetized mice in vivo ,
employing pressure-volume conductance (44). In accordance with this
finding, in another study Wagner et al. reported decreased cardiac index
and resulting BP by same synthetic cannabinoid using radiolabeled
microsphere technique (42).
It has been reported in many clinical cases, SCs can cause cardiac
arrhythmias and fatalities. In our preclinic study cardiac arrhytmia
frequency found to be increased in all JWH-018 groups. It has been
noticed that there is QT prolongation in rats in the group treated with
SAHD JWH-018, unlike in other groups. Moreover QT analysis, resembling
arrhytmia risk showed prolongation in long-term cannabinoid use, despite
increased HR especially in the high dose group. Al Kury et al.
previously demonstrated that endogenous cannabis can produce arrhythmias
in rat ventricular myocytes by blocking the function of
voltage-dependent Na+ and L-type
Ca2+ channels in the absence of CB1 and CB2 receptor
activation (45). In another study, Li et al. found that anandamide, an
endocannabinoid, reduced L-type Ca2+ current in
ventricular myocytes and delayed the length of action potential in
cardiac tissues via CB1 but not CB2 receptors. Beside this, anandamide
facilitated the inactivation of L-type Ca2+ current
and inhibited its recovery from inactivation (46). Recently, Yun et al.
investigated the effect of JWH-30, a syntetic cannabinoid on duration of
action potantial and QT interval. They observed that inhibiting the
human ether-a-go-go related gene
(hERG) channels in rabbit purkinje fibers shortened the duration of
action potantial, and that intravenous administration of JWH-030 (0.5
mg/kg) at the ECG measurement in rats lengthened the QT interval (47).
There is mounting evidence that using SCs increases the likelihood of a
clinically significant lengthening of the rate-corrected QT interval of
the ECG. Torsades de pointes is the main arrhythmia connected to delayed
ventricular repolarization and, therefore, QT interval lengthening. This
can progress to deadly ventricular fibrillation and is related to
cellular origin of early-after depolarizations and enhanced
repolarization dispersion. Therefore, QT prolongation caused by both
prescription medications and illicit substances has some relevance (48).
In cases where SCs were detected as a result of toxicological studies
conducted in the autopsy series, body fluids and tissue samples
examined, the causes of death were usually due to cardiac problems.
Cardiac problems identified include causes such as myocardial
infarction, dilated cardiomyopathy, cardiomegaly, arrhythmias, etc.
decontamination (49-51). In the present study, during evaluation of QT
interval and arrhythmia, we have also evaluated the ischemic ECG changes
like ST segment depression and T wave negativity in all JWH-018
administered rats. Although we did not detect any ECG changes resembling
ischemia, all of JWH groups has more ischemic ECG findings despite prior
reported coronary vasodilatory effect of cannabinoids (42). This ECG
changes may be result of decreased BP causing reduced coronary perfusion
or increased HR causing supply demand mismatch. However, we did not find
cardiac troponin-I elevations in serum as a myocardial injury biomarker
in contrast our histopathological observations which showed ischemic
circumstance in the cardiac tissue. Histopathologically, we found
morphological changes compatible with the first 4-12th hours of ischemia
in the SALD and SAHD groups. Although these ischemic changes were not
seen on the ECG findings, we thought that they reflected the decreased
BP result. Histopathological findings related to arrhythmia detected on
ECG were not observed in the JWH-018 groups.
One more thing should be emphasized is the metabolic effect of JWH-018.
The ECS regulates hunger and energy balance in the central nervous
system, principally through managing both the homeostatic and hedonic
components of food intake. By activating CB1 receptors in brain areas
implicated in energy control, both endogenous and exogenous cannabis can
promote food absorption, change the release of orexigenic and anorexic
mediators, and boost hedonic valuation (i.e., the hypothalamus and
mesocorticolimbic system) (52). In contrast, agents with specific
antagonistic effects for the CB1 receptor have been shown to suppress
food intake and reduce body weight in laboratory animals (53). Contrary
to this knowledge, we found that all rats lost weight when long-term
JWH-018 were used, regardless of dose. Cooper reported that among the
common side effects of SCs of moderate severity, there may be a decrease
in body weight due to loss of appetite (54). Dalton et al. reported that
weight loss was linked to a dose-dependent down-regulation of CB1
receptors that lasted throughout chronic exposure (55). We also found
that triglyceride levels in blood lipids were decreased, possibly
related to weight loss of rats.
Previously it is well shown that metabolic enzymes are involved in the
biotransformation of SCs. The main ring involved in the molecular
structure of SCs is metabolized, especially by the CYP1A enzyme (56). In
some studies, CYP2C9 and CYP1A2 enzymes have been responsible for the
metabolism of JWH-018 (57). At least nine mono-hydroxylated metabolites
of JWH-018 have been found. It has been shown in studies that these
metabolites bind to the CB1 and CB2 receptors (58,59). It was found that
the heart value was between 0.16-1.63 ng/mg in the rats in the AHD
group. Also, it was estimated that the heart value was between 0.02-0.08
ng/mg in the SALD group. It is detected at lower levels than the rats in
the ALD group. One of the possible explanations of these results is that
it is oxidized by CYP2C9 and CYP1A2 cytochrome P450 isoforms in drug
metabolism and it was thought that the increase in metabolites and the
storage of substance-metabolites in the organs were due to the effect of
UGT2B7 found in hepatic tissue and UGT1A3 main function isoforms found
in extraheptic tissue in the conjugation step (11). The HRs of the rats
increased statistically significantly in the groups in which JWH-018 was
administered subacutely, compared to the groups in which it was
administered acutely. This circumstance may lead to increase renal
perfusion and increased renal excretion of JWH-018 and its metabolites.
JWH-018 tests on animals have indicated a half-life of some 2 hours
(60,61). Considering that it takes approximately 4-5 half-lives for a
drug to be completely removed from the body, the levels of the drug and
its metabolites may have been low in SA JWH-018 administered groups due
to increased renal clearance with increased HR.
Also, it was determined that the heart value was between 0.02-0.08 ng/mg
in the SAHD group. Based on this relationship, in a clinical study, two
volunteers received 100 and 150 mg SCs containing 2.9% JWH-018. JWH-018
serum concentration peaked 5 min after inhalation, reaching 8.1 mcg/L
and 10.2 mcg/L. However, it is reported that the concentration decreases
rapidly after 1 hour and can not be detected in the 24 th hour (62).
Compared to the AHD group, there was a higher blood level and
substance-metabolite accumulation in the tissues. The reason for this
situation was thought to be the decrease in the metabolic rate of the
drug in excess and/or the toxic effect of the drug on the tissues.
JWH-018 and metabolite determinations are mostly measured in tissues
such as serum, blood, oral fluid, urine, brain, kidney, lung, liver and
spleen. Although JWH-018 has been determined in heart tissue before; the
study was in mice and only the level of JWH-018 was assayed (63). On the
other hand, we aimed to examine the JWH-018 level in the heart tissue
along with the other 5 metabolites, and we determined the levels of 4
metabolites that can be separated from each other correctly. In
addition, unlike the other study, the fact that we looked at 4 separable
metabolites that we reached as consumables, rather than a few
metabolites in serum and heart tissue, constitutes another important
uniqueness of our study.