Abbreviations ANP, atrial natriuretic peptide; CAD, coronary artery disease; CVD, cardiovascular disease; DPP-4, dipeptidyl peptidase-4; eNOS, endothelial NOS; GLP-1, glucagon-like pepti
Trang 1David J Grieve, Centre forExperimental Medicine, Queen’sUniversity Belfast, Institute ofClinical Science Block A,Grosvenor Road, BelfastBT12 6BA, UK E-mail:
Glucagon-like peptide-1 (GLP-1) is an incretin hormone whose glucose-dependent insulinotropic actions have been harnessed
as a novel therapy for glycaemic control in type 2 diabetes Although it has been known for some time that the GLP-1receptor is expressed in the CVS where it mediates important physiological actions, it is only recently that specific
cardiovascular effects of GLP-1 in the setting of diabetes have been described GLP-1 confers indirect benefits in cardiovasculardisease (CVD) under both normal and hyperglycaemic conditions via reducing established risk factors, such as hypertension,dyslipidaemia and obesity, which are markedly increased in diabetes Emerging evidence indicates that GLP-1 also exertsdirect effects on specific aspects of diabetic CVD, such as endothelial dysfunction, inflammation, angiogenesis and adversecardiac remodelling However, the majority of studies have employed experimental models of diabetic CVD and information
on the effects of GLP-1 in the clinical setting is limited, although several large-scale trials are ongoing It is clearly important
to gain a detailed knowledge of the cardiovascular actions of GLP-1 in diabetes given the large number of patients currentlyreceiving GLP-1-based therapies This review will therefore discuss current understanding of the effects of GLP-1 on bothcardiovascular risk factors in diabetes and direct actions on the heart and vasculature in this setting and the evidence
implicating specific targeting of GLP-1 as a novel therapy for CVD in diabetes
Abbreviations
ANP, atrial natriuretic peptide; CAD, coronary artery disease; CVD, cardiovascular disease; DPP-4, dipeptidyl
peptidase-4; eNOS, endothelial NOS; GLP-1, glucagon-like peptide-1; hs-CRP, high sensitivity C-reactive protein;
ICAM-1, intercellular adhesion molecule-1; MI, myocardial infarction; PAI-1, plasminogen activator inhibitor-1; STZ,streptozotocin; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; TLR, toll-like receptor; UKPDS, UnitedKingdom Prospective Diabetes Study; VCAM-1, vascular cell adhesion molecule-1
Trang 2The prevalence of type 2 diabetes mellitus (T2DM) is
increas-ing alarmincreas-ingly with the 2013 figure of 382 million estimated
to rise to 592 million by 2035 (International Diabetes
Federation, 2014) A change in lifestyle coupled with an
increase in obesity has led to a global epidemic, with diabetics
typically carrying a fivefold greater mortality risk as a result of
cardiovascular disease (CVD) compared with non-diabetics
(Stamler et al., 1993), and coronary artery disease (CAD)
being the leading underlying cause (Bertoni et al., 2004) It is
well established that hyperglycaemia plays a central role in
development and progression of CVD associated with
diabe-tes (Nathan, 1996) Indeed, two long-term clinical trials, the
Diabetes Control and Complications Trial/Epidemiology of
Diabetes Interventions and Complications study and the
United Kingdom Prospective Diabetes Study (UKPDS), have
demonstrated that intensive glucose-lowering strategies are
effective in markedly reducing the incidence of microvascular
(e.g retinopathy, nephropathy) and macrovascular (e.g
CAD, stroke) complications in both type 1 diabetes mellitus
(T1DM) and T2DM (Holman et al., 2008), although several
similar large-scale trials have reported limited benefits
(Action to Control Cardiovascular Risk in Diabetes Study
Group, 2008; Duckworth et al., 2009; Ginsberg, 2011)
None-theless, there remains a significant incidence of CVD even in
optimally treated diabetic patients, so it is clear that more
effective strategies are required In this regard, the incretin
peptide hormone, glucagon-like peptide-1 (GLP-1), has
received considerable recent attention
The incretin effect is responsible for augmenting insulin
secretion following nutrient ingestion and GLP-1 together
with its sister hormone, gastrointestinal peptide, account for
up to 60% of post-prandial insulin secretion, leading to rapid
blood glucose reduction (Nauck et al., 1986; Drucker et al.,
1987) Furthermore, they possess an inherent ability to
reduce glucagon secretion (Kreymann et al., 1987), delay gastric emptying (Näslund et al., 1998a) and promote satiety (Flint et al., 1998) The metabolic actions of GLP-1 are medi-
ated by GLP-1 receptor activation and stimulation of cAMPand several downstream kinases, including ERK1/2, PI3K andPKA Under physiological conditions, GLP-1 has a short half-life (∼2 min) as it is rapidly degraded by its endogenous
inhibitor, dipeptidyl peptidase-4 (DPP-4) (Deacon et al.,
1995), resulting in cleavage of two amino acids from nativeGLP-1(7-36) to produce GLP-1(9-36), which acts as a weakGLP-1 receptor antagonist lacking insulinotropic activity
(Green et al., 2004) However, emerging evidence suggests
that ‘metabolically inactive’ GLP-1(9-36) may itself be an
important signalling molecule (Ban et al., 2010; Gardiner
et al., 2010) More detailed information on GLP-1 biology and
signalling is provided by recent review articles (Grieve et al., 2009; Donnelly, 2012; Pabreja et al., 2013).
The unique ability of GLP-1 to promote insulin secretion
in a glucose-dependent manner has been harnessed for ment of T2DM, with GLP-1 receptor agonists resistant toDPP-4 (exenatide, Byetta®; liraglutide, Victoza®), and DPP-4inhibitors (e.g sitagliptin, vildagliptin) now widely used foreffective glycaemic control Interestingly, it is well recognizedthat GLP-1 exerts wide-ranging extra-pancreatic actionsoccurring independently of its established metabolic effects.Indeed, GLP-1 signalling is reported to play several important
treat-roles in the CVS in both health and disease (Grieve et al.,
2009), although it appears that the GLP-1 receptor may not
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d,e Alexander et al., 2013a,b,c,d,e).
Trang 3be as widely expressed as previously thought For example,
recent work suggests that cardiac GLP-1 receptor expression
may be localized to atrial tissue, sinoatrial node and
vascula-ture, with some species variation (Kim et al., 2013; Pyke et al.,
2014; Richards et al., 2014), and that earlier reports of more
ubiquitous expression may be questionable because of poor
antibody selectivity and sensitivity (Panjwani et al., 2012).
Nonetheless, it is clear that GLP-1 exerts important
cardio-vascular actions, although it is only recently that its effects in
the setting of diabetes, a condition synonymous with micro/
macrovascular complications, have been explored This is
clearly important because of the large number of patients
receiving GLP-1-based therapies, in which its cardiovascular
actions are largely unknown This review will therefore
discuss the current understanding of the effects of GLP-1 on
both cardiovascular risk factors in diabetes and direct actions
on the heart/vasculature in this setting and the evidence
implicating specific targeting of GLP-1 as a novel therapy for
CVD in diabetes, with a primary focus on the role of GLP-1
receptor agonists More detailed discussion of the pleiotropic
actions of DPP-4 inhibitors in this setting is provided by
recent review articles specifically focused on this important
aspect of cardiovascular GLP-1 signalling (Scheen, 2013;
Aroor et al., 2014).
Influence of GLP-1 on cardiovascular
risk factors in diabetes
BP and hypertension
Increased BP is an established risk factor for CVD in both
normoglycaemia and T2DM (Turner et al., 1998; Vasan et al.,
2001) Notably, therapeutic reductions in BP and circulating
glucose have an additive effect in decreasing cardiovascular
complications in T2DM patients, as highlighted by the
UKPDS (Stratton et al., 2006) Indeed, in an experimental
setting, chronic GLP-1 infusion inhibits development of
hypertension in Dahl salt-sensitive rats, as well as reducing
cardiac fibrosis and hypertrophy, effects which appear to
occur via a natriuretic/diuretic mechanism independently of
blood glucose (Yu et al., 2003), suggesting that GLP-1 may
confer additional benefits which could be harnessed for the
treatment of hypertension associated with T2DM Consistent
with an indirect BP-lowering effect, it was recently reported
that liraglutide-stimulated reduction of angiotensin
II-induced hypertension in mice was blocked by the natriuretic
peptide receptor antagonist, anantin, in a GLP-1
receptor-dependent manner, but unaltered by the NOS inhibitor, NG
-monomethyl-L-arginine, and that liraglutide induced rapid
increases in atrial natriuretic peptide (ANP) secretion both in
vivo and in isolated perfused hearts, suggesting that observed
BP reduction occurred at least partly via direct activation of
cardiac ANP (Kim et al., 2013) Importantly, in the context of
diabetes, the GLP-1 mimetic, exendin-4, inhibited
develop-ment of both spontaneous and high salt-induced
hyperten-sion in obese db/db mice via beneficial actions on renal
sodium handling (Hirata et al., 2009) Furthermore, it was
recently reported that treatment of insulin-resistant Zucker
rats with the DPP-4 inhibitor, linagliptin, for 8 weeks reduced
BP and improved diastolic function (Aroor et al., 2013).
Interestingly, although chronic administration of GLP-1may prevent development of hypertension, it is widelyreported that acute GLP-1 exposure is associated withincreased BP and heart rate, which predisposes to CVD Forexample, acute infusion of GLP-1(7-36) increased systolic/diastolic BP and heart rate in both normal and insulin-deficient streptozotocin (STZ)-induced T1DM rats (Barragán
et al., 1994), with the same group reporting that
exendin-4-induced increases in BP and heart rate were reversed by the
GLP-1 receptor antagonist, exendin(9-39) (Barragán et al.,
1996), suggesting that these effects occurred via an independent mechanism but involving GLP-1 receptor acti-vation Although similar increases in BP and heart rate aftershort-term GLP-1 administration have been reported in
insulin-various experimental models (Grieve et al., 2009), the data
from clinical studies are less clear For example, GLP-1 sion in a small number of T2DM patients with or withoutCAD for 105 min and 48 h, respectively, had no effect on
infu-heart rate or systolic/diastolic BP (Toft-Nielsen et al., 1999; Nystrưm et al., 2004), whereas 48 h GLP-1 infusion in patients with ischaemic heart failure (Halbirk et al., 2010) and
treatment of T2DM patients with an exendin-transferrinfusion protein or exenatide for 7 or 10 days, respectively
(Kothare et al., 2008; Gustavson et al., 2011), resulted in
elevated heart rate and diastolic BP However, the data fromlonger-term GLP-1 clinical trials are more consistent, with themajority reporting decreased BP and minimal effects on heartrate For example, the Liraglutide Effect and Action in Diabe-tes (LEAD)-4 study, investigating 26 week liraglutide treat-ment in combination with metformin in T2DM patients,reported a modest reduction in systolic BP of 6 mmHg com-
pared with placebo (1.1 mmHg) (Zinman et al., 2009)
Simi-larly, the LEAD-2 study, assessing liraglutide combinationtherapy, reported a 2–3 mmHg decrease in systolic BP versus
a small increase (0.4 mmHg) in the glimepiride control group
(Nauck et al., 2009) Furthermore, a 30 week trial comparing
once-weekly versus twice-daily exenatide injection in nạve T2DM patients observed a significant decrease in
drug-systolic/diastolic BP compared with baseline (Drucker et al.,
2008), while another 20 week trial in obese patients reportedreduced systolic/diastolic BP in response to liraglutide, which
persisted for the 2 year follow-up period (Astrup et al., 2012).
Interestingly, meta-analysis of six clinical trials comprising
2171 T2DM patients found that exenatide treatment for 6months produced maximal systolic BP reduction in individu-als with abnormally high baseline levels, whereas no effects
were observed in normotensive subjects (Okerson et al.,
2010) It should be noted that BP reduction is positively
correlated with weight loss (Neter et al., 2003), so it is possible
that the observed changes after chronic GLP-1 treatment mayoccur secondary to its metabolic effects However, althoughbeneficial effects of GLP-1 on body weight are associated withimproved hypertension, it is clear that this cannot solelyaccount for its vascular effects as several studies have reported
a BP reduction prior to weight loss For example, a combinedmeta-analysis of three 26 week liraglutide trials reporteddecreased BP after only 2 weeks, while maximal weight loss
did not occur until 8 weeks (Gallwitz et al., 2010) Indeed,
heart rate, which is not linked to body weight, was increased
by chronic administration of both liraglutide and exenatide
in T2DM patients in parallel with reduced systolic BP (Garber
Trang 4et al., 2009; Gill et al., 2010) Interestingly, it was recently
reported that GLP-1 secretory function increases with age and
is negatively correlated with systolic BP, suggesting that this
may represent an adaptive response (Yoshihara et al., 2013).
Dyslipidaemia
The pathophysiology of diabetes is commonly considered
largely in terms of associated hyperglycaemia However, it is
increasingly apparent that dyslipidaemia is equally important
and represents a significant risk factor for CVD in diabetic
patients (Reiner et al., 2011) It is likely that impaired insulin
sensitivity contributes to dyslipidaemia in T2DM, which is
associated with reduced GLP-1 secretion Indeed, in addition
to their established glycaemic actions, GLP-1 receptor
activa-tion and DPP-4 inhibiactiva-tion are reported to improve lipid
pro-files in both experimental and clinical diabetes For example,
short-term infusion of GLP-1 in normoglycaemic Syrian
golden hamsters decreased lipid absorption and triglyceride
levels, an effect potentiated by oral glucose (Hein et al., 2013),
suggesting that its incretin action may inhibit intestinal
pro-duction of chylomicrons, which are strongly linked to
ath-erosclerosis (Nakano et al., 2008) Similarly, circulating
triglycerides and fat pad mass in rats with diet-induced
obesity were reduced after 4 week treatment with liraglutide
(Madsen et al., 2010), while 40 day administration of
exendin-4 ameliorated systemic and cardiac insulin
resist-ance and dyslipidaemia in both genetic KKAy and
Furthermore, chronic GLP-1 receptor activation with both
the GLP-1 analogue, CNTO3649, and exendin-4 in
apolipo-protein E3-Leiden transgenic mice, which develop severe
hypercholesterolaemia after high-fat feeding, resulted in
reduced very-low-density lipoprotein (VLDL)-triglyceride
and apolipoprotein B synthesis in parallel with decreased
hepatic triglyceride, cholesterol and phospholipids and
lipo-genesis gene expression (Parlevliet et al., 2012) The
long-acting DPP-4 inhibitor, teneligliptin, is also reported to
decrease circulating triglyceride and free fatty acid levels in
insulin-resistant Zucker fatty rats after 2 week treatment
(Fukuda-Tsuru et al., 2012).
Importantly, the majority of clinical studies have also
demonstrated beneficial outcomes of GLP-1 administration
on lipid metabolism in T2DM, such as reduced circulating
triglycerides and low-density lipoprotein (LDL) cholesterol
(Flock et al., 2007; Drucker et al., 2008; Tremblay et al., 2011),
although one study in which patients were treated with
exenatide for 24 weeks reported a similar plasma lipid profile
versus controls (Moretto et al., 2008) For example, decreased
circulating levels of atherogenic triglyceride-rich lipoproteins
were observed following 4 week vildagliptin monotherapy in
drug-nạve T2DM patients, characterized by specific
reduc-tions in total plasma and chylomicron triglycerides, together
with apolipoprotein B-48 and cholesterol in the chylomicron
subfraction (Matikainen et al., 2006) Furthermore, the
LEAD-4 study found that 26 week liraglutide treatment in
combination with metformin and rosiglitazone decreased
cir-culating LDL cholesterol, triglycerides and free fatty acids in
T2DM patients compared with placebo controls, although it
is interesting to note that these changes were greater in
response to low-dose treatment (Zinman et al., 2009) Indeed,
similar results are reported for DPP-4 inhibitors, which
produce much lower circulating levels of GLP-1 For example,twice-daily sitagliptin led to a significant reduction in circu-lating triglycerides and free fatty acids in a large number ofT2DM patients compared with placebo and glipizide controlgroups, despite similar decreases in fasting plasma glucose
and HbA1c levels (Scott et al., 2007) Interestingly, a single
injection of exenatide was shown to attenuate postprandialincreases in triglycerides, apolipoprotein B-48 and CIII/remnant lipoprotein cholesterol for up to 8 h in patients withimpaired glucose tolerance and recent-onset T2DM (Schwartz
et al., 2010), suggesting that such lipid profile benefits may
not be explained solely by chronic changes in body weight,glucose levels and insulin resistance Indeed, it was recentlyreported that exendin-4 completely reverses hepatic steatosis
in mice fed a high-fat diet via a GLP-1 receptor-dependentmechanism resulting in reduced numbers/size of circulatingVLDL-triglyceride and VLDL-apolipoprotein B particles
(Parlevliet et al., 2012), suggesting that GLP-1 may exert
direct effects on dyslipidaemia in diabetes
Obesity
Although it is well known that obesity significantly increases
the risk of T2DM (Willett et al., 1999), and both are ent risk factors for CVD (Hubert et al., 1983), many estab-
independ-lished diabetes therapies, including sulfonylureas andthiazolidinediones, may increase body weight However,GLP-1 reduces body weight because of beneficial effects onglucagon secretion, gastric emptying and satiety (Kreymann
et al., 1987; Flint et al., 1998; Näslund et al., 1998a), so it
seems likely that impaired GLP-1 secretion observed in
non-diabetic obese individuals (Holst et al., 1982; Näslund et al.,
1998b) may at least partly account for their increased bodyweight Indeed, weight loss improves the postprandial GLP-1
response in severely obese patients (Verdich et al., 2001),
sug-gesting that the two are interlinked Furthermore, a 20 weektreatment with liraglutide was reported to cause significantweight loss in obese individuals and to reduce the incidence
of prediabetes (Astrup et al., 2009), confirming an apparent
role for GLP-1 in weight control which may be harnessed fortherapeutic benefit This assertion is supported by the LEADtrials which have consistently reported a reduction in bodyweight in T2DM patients following liraglutide treatment
(Moretto et al., 2008; Buse et al., 2009; Garber et al., 2009; Nauck et al., 2009; 2013; Zinman et al., 2009) For example, in
the LEAD-2 trial, 26 week combination therapy of liraglutidewith metformin in T2DM patients resulted in increased
weight loss compared with metformin alone (Nauck et al.,
2013) Importantly, weight loss associated with both tide and exenatide treatment is reported to be linked toimproved cardiovascular risk factors, such as HbA1c and BP,
liraglu-and to persist for at least 2 years (Klonoff et al., 2007; Astrup
et al., 2009; 2012), highlighting important benefits of GLP-1
which may not be related to its insulinotropic actions Thelong-term effects of GLP-1 on weight loss may be particularlyimportant as conventional weight loss is typically poorlymaintained in T2DM patients Despite GLP-1 receptor ago-nists promoting weight loss in both diabetic and non-diabeticobese subjects, DPP-4 inhibitors appear to be weight-neutral
(Amori et al., 2007), suggesting that GLP-1 receptor agonists
may exert direct gastrointestinal effects in addition to
improving insulin resistance (Rask et al., 2001), although this
Trang 5could simply be due to differences in circulating GLP-1 levels.
However, postprandial GLP-1 levels are reported to be
increased immediately after gastric bypass surgery, despite
patients remaining obese, indicating that GLP-1 may regulate
appetite and food intake directly (Morinigo et al., 2006).
Indeed, in T2DM patients, GLP-1 promotes satiety, thereby
reducing energy consumption (Gutzwiller et al., 1999), while
in healthy individuals i.v administration of GLP-1(7-36)
slows gastric emptying in a dose-dependent manner (Nauck
et al., 1997).
GLP-1 and vascular disease
Vascular function
Impaired endothelial and vascular function are established as
key initiating factors underlying the development of
micro-vascular and macromicro-vascular complications associated with
diabetes Indeed, it has been known for some time that native
GLP-1(7-36) induces ex vivo dose-dependent vasodilatation
in a number of isolated rodent vessels, including aorta
(Golpon et al., 2001; Green et al., 2008), pulmonary artery
(Richter et al., 1993; Golpon et al., 2001), femoral artery
(Nyström et al., 2005) and mesenteric artery (Ban et al., 2008),
although several different mechanisms have been proposed
For example, some studies indicate that the vasorelaxant
actions of GLP-1 are dependent upon endothelium-derived
NO (Golpon et al., 2001; Ban et al., 2008; Gaspari et al., 2011),
whereas others have proposed endothelium-independent
mechanisms involving mediators such as KATP channels,
cAMP andβ2-adrenoceptor activation (Nyström et al., 2005;
Gardiner et al., 2008; Green et al., 2008) Interestingly,
although several studies suggest that the vascular actions of
GLP-1 are dependent upon the GLP-1 receptor (Gaspari et al.,
2011; Chai et al., 2012), it appears that they may also be
mediated, at least partly, by its truncated metabolite,
GLP-1(9-36), which induces dose-dependent relaxation in both
isolated mouse mesenteric artery (Ban et al., 2008) and rat
aorta (Green et al., 2008) It should be noted that although
the synthetic GLP-1 mimetic, exendin-4, exerts similar
actions in rat aorta (Golpon et al., 2001; Green et al., 2008),
they are of reduced magnitude compared with GLP-1(7-36)
and are absent in mouse mesenteric artery (Ban et al., 2008).
Importantly, the vasorelaxant actions of GLP-1 are also
reported in vivo For example, systemic administration of
GLP-1(7-36) by both bolus dose and short-term infusion in
rats induced hindquarters vasodilatation (Gardiner et al.,
2010) Interestingly, however, GLP-1 promoted
vasoconstric-tion in both mesenteric and renal arteries, while exendin-4
exerted similar vasoconstriction in mesenteric artery but
induced vasodilatation in both hindquarters and renal artery
(Gardiner et al., 2008), suggesting differential vascular effects.
Indeed, a similar study demonstrated that GLP-1(7-36)
infu-sion acutely increased muscle microvascular blood volume in
the absence of changes in microvascular blood flow velocity
or femoral blood flow, in association with increased plasma
NO, muscle insulin clearance/uptake, hindlimb glucose
extraction and muscle interstitial oxygen saturation (Chai
et al., 2012) It should be noted that in contrast to the ex vivo
studies, GLP-1(9-36) failed to modulate vascular function in
rats in vivo when given as either a bolus dose or via short-term
infusion, which together with the fact that DPP-4 inhibitorsprolonged the vascular actions of native GLP-1(7-36) in this
setting (Gardiner et al., 2010), suggest that the actions of this
‘inactive’ metabolite may not be significant in vivo.
Importantly, it appears that the vascular effects of GLP-1are also evident in the setting of diabetes, where they arereported to promote beneficial actions Chronic treatment ofSTZ/nicotinamide T1DM rats with either GLP-1(7-36) orexendin-4 was shown to prevent endothelial dysfunction in
parallel with reduction of blood glucose (Özyazgan et al.,
2005), effects which may be mediated via activation of
endothelial NOS (eNOS) (Goyal et al., 2010) Although
similar studies have not been performed in the setting ofovert T2DM, it was recently reported that chronic treatment
of insulin-resistant Zucker rats with the DPP-4 inhibitor, gliptin, resulted in reduced hypertension in parallel withincreased expression of total/phosphorylated eNOS (Aroor
lina-et al., 2013) Furthermore, high-fat fed apolipoprotein
E-deficient mice treated with a different DPP-4 inhibitor, fluoro-sitagliptin, demonstrated attenuation of endothelialdysfunction in parallel with eNOS activation and improved
des-glucose tolerance (Matsubara et al., 2012) Interestingly,
however, endothelial dysfunction in rat femoral arteryinduced by short-term triglyceride exposure was not affected
by exendin-4 (Nathanson et al., 2009), suggesting that the reported in vivo protective actions may occur via indirect
mechanisms In this regard, it is important to note that thevascular actions of GLP-1 in diabetes are likely to occur, atleast partly, secondary to stimulation of insulin, whichinduces vascular relaxation via Ca2+-dependent activation of
eNOS (Han et al., 1995; Kahn et al., 1998).
In addition to the data supporting important vascularactions of GLP-1 in experimental diabetes, several studieshave reported beneficial functional effects in the clinicalsetting For example, in T2DM patients with stable CAD,acute GLP-1 administration improved brachial artery flow-mediated vasodilatation, an effect not observed in healthy
individuals (Nyström et al., 2004) Comparable effects were
observed in insulin-resistant patients with obesity-relatedmetabolic syndrome, where acute treatment with GLP-1(7-36) enhanced insulin-mediated forearm blood flow responses
to both ACh and sodium nitroprusside in the absence ofchanges in forearm glucose extraction/uptake, while GLP-
1(9-36) did not affect vascular function (Tesauro et al., 2013).
Similarly, in patients with T1DM, brachial artery endothelialdysfunction induced by acute blood glucose modulation wascounteracted by simultaneous infusion of GLP-1 (Ceriello
et al., 2013) Interestingly, GLP-1-induced enhancement of
endothelium-dependent peripheral vasodilatation observed
in non-diabetic individuals is differentially modulated bysulphonylureas, with glyburide abolishing GLP-1-inducedACh-mediated responses which are unaltered by glimepiride
(Basu et al., 2007) Furthermore exenatide, which is
com-monly used for hyperglycaemic control in T2DM, alsoincreased postprandial endothelial function assessed byperipheral arterial tonometry in patients with recent-onsetdisease when given as a single dose, largely secondary to a
reduction in circulating triglycerides (Koska et al., 2010),
although chronic treatment for 3 months in obese betic patients had no additional effect when compared with
Trang 6predia-those receiving metformin (Kelly et al., 2012) While it
appears that clinical GLP-1 administration exerts acute
vas-cular effects in T2DM, data on its chronic actions in this
setting are variable T2DM patients who received exenatide
for a period of 4 months as an adjunct to standard metformin
therapy demonstrated improved brachial artery
flow-mediated dilatation, indicated by elevated peak dilatation
and shear rate which are reflective of improved
macrovascu-lar and microvascumacrovascu-lar function respectively (Irace et al.,
2013) Notably, enhanced vasodilatation in exenatide-treated
patients in this study was significantly greater than that
observed in those receiving glimepiride as an add-on to
met-formin therapy Furthermore, liraglutide treatment for 12
weeks in T2DM patients well controlled on metformin
mono-therapy resulted in an improvement in both circulating
markers of vascular function [asymmetric dimethylarginine,
plasminogen activator inhibitor-1 (PAI-1), E-selectin] and
retinal microvascular endothelial function (Forst et al., 2012).
However, a similar study in a small number of severely obese
T2DM patients chronically treated with GLP-1 receptor
ago-nists for 6 months reported that neither exenatide or
liraglu-tide had any effect on brachial artery endothelial-dependent
flow-mediated dilation (Hopkins et al., 2013), indicating
potential for other confounding factors in this setting which
may need to be considered Furthermore, 6 week treatment
with sitagliptin or alogliptin significantly reduced
flow-mediated dilatation in male T2DM patients (Ayaori et al.,
2013), suggesting that chronically increased physiological
levels of GLP-1 may exert unfavourable vascular actions,
although it is possible that this could be a class-specific effect
of DPP-4 inhibitors warranting further investigation
Inflammation and atherosclerosis
It is well known that the incidence and progression of
endothelial dysfunction is exacerbated in T2DM, secondary
to established risk factors, such as insulin resistance,
dyslipi-daemia and hyperglycaemia Endothelial dysfunction in this
setting is characterized by an elevation in circulating
adhe-sion molecules, such as intercellular adheadhe-sion molecule-1
(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1),
and an increased propensity to develop atherosclerosis which
is typified by inflammatory cell infiltration and plaque
for-mation (Van Gaal et al., 2006) Interestingly, several recent
clinical and experimental studies appear to indicate that
GLP-1 exerts both anti-inflammatory and anti-atherogenic
actions For example, GLP-1 treatment in T2DM patients is
associated with beneficial effects on a number of established
CVD biomarkers, including high sensitivity C-reactive
protein (hs-CRP) and PAI-1, which are important in
athero-sclerosis development (Haffner, 2006) Similarly, 14 week
treatment of T2DM patients with liraglutide resulted in
sig-nificantly reduced PAI-1 levels, and a dose-dependent
decrease in plasma hs-CRP levels (Vilsbøll et al., 2007;
Courrèges et al., 2008), an effect that was also observed after
26 week treatment with exenatide (Bergenstal et al., 2010)
and was over and above that seen in patients treated with
insulin glargine (Diamant et al., 2010) Importantly, the
ben-eficial effects of exenatide on circulating hs-CRP appear to
persist at 1 year treatment in T2DM patients receiving
stand-ard metformin therapy, resulting in reduced levels of both
hs-CRP and leptin (Bunck et al., 2010) DPP-4 inhibitors seem
to exert similar effects as T2DM patients receiving sitagliptinfor 6 months also demonstrated significant reductions inplasma hs-CRP, together with VCAM-1 and associated albu-minuria, which may attenuate glucose excursion and inhibit
vascular inflammation (Horváth et al., 2009; Hattori, 2010).
Interestingly, a recent study demonstrated that cessation ofexenatide treatment resulted in the reversal of benefits on
circulating hs-CRP within 6 months (Varanasi et al., 2011) It
should be noted that although these studies support thesuggestion that GLP-1 may protect against inflammation andatherosclerosis in the clinical setting, it is not possible to drawclear conclusions because of the absence of longer-termstudies specifically assessing effects on disease development.Interestingly, a recent study has reported a positive correla-tion between circulating GLP-1 levels and CAD in both dia-betic and non-diabetic patients undergoing angiographybecause of typical or atypical chest pain, highlighting thepossibility that GLP-1 may exert detrimental effects in this
setting (Piotrowski et al., 2013).
Nonetheless, the majority of clinical data are broadly portive of the anti-inflammatory actions of GLP-1, whichpersist for up to 12 weeks in obese T2DM patients after a
sup-single exenatide injection (Chaudhuri et al., 2011) Indeed, in
this study, GLP-1 was associated with a specific reduction ofseveral inflammatory mediators, including TNF-α, toll-likereceptor-2 (TLR-2) and TLR-4, in parallel with suppression ofNF-κB signalling and MMP-9 activity, which are key initiatingfactors of atherosclerosis Furthermore, in obese T2DMpatients, 8 week liraglutide treatment is reported to decreaselevels of the inflammatory macrophage activation molecule,sCD163, and pro-inflammatory cytokines, TNF-α and IL-6,while increasing levels of the anti-inflammatory adipokine,
adiponectin (Hogan et al., 2014) Importantly, these clinical
observations are supported by a number of experimentalstudies which have specifically assessed the effects of GLP-1
on development and progression of atherosclerosis Forexample, continuous infusion of exendin-4 in both wild-typeand apolipoprotein E-deficient normoglycaemic mice wasreported to decrease monocyte adhesion and development ofatherosclerotic lesions in thoracic aorta, effects proposed tooccur via cAMP/PKA-dependent suppression of inflammation
(Arakawa et al., 2010) These findings were confirmed by a
different group who demonstrated reduced aortic rophage recruitment, foam cell formation and atheroscleroticlesion development in apolipoprotein E-deficient mice after
mac-GLP-1 infusion (Nagashima et al., 2011) Furthermore,
chemokine-induced migration of CD4+lymphocytes is ited by both GLP-1(7-36) and exendin-4 in a GLP-1 receptor-
inhib-dependent manner (Marx et al., 2010), while liraglutide
suppresses NF-κB signalling in HUVECs and THP-1 monocyteadhesion in human aortic endothelial cells via downstreamactivation of several proinflammatory and cell adhesion mol-ecules, including TNF-α, VCAM-1 and E-selectin (Shiraki
et al., 2012; Krasner et al., 2014) Indeed, liraglutide inhibits
TNF-α in human vascular endothelial cells and reduceshyperglycaemia-mediated PAI-1, ICAM-1 and VCAM-1 acti-vation, which is associated with endothelial dysfunction and
accelerated atherogenesis in T2DM (Liu et al., 2009)
Further-more, the DPP-4 inhibitor, des-fluoro-sitagliptin, is reported
to exert cAMP-dependent anti-inflammatory actions in tured human macrophages by increasing GLP-1 levels and to
Trang 7cul-reduce atherosclerotic lesion formation in apolipoprotein
E-deficient mice (Matsubara et al., 2012), while alogliptin
inhibits vascular monocyte/macrophage recruitment and
reduces atherosclerotic burden in high-fat diet-fed, LDL
receptor-deficient mice in association with improvement of
metabolic indices (Shah et al., 2011) Taken together, these
experimental data clearly support an important role for
GLP-1 in protecting against vascular inflammation and
atherogenesis, effects which are borne out by the reported
clinical benefits of GLP-1 treatment on circulating
inflamma-tory mediators and CVD biomarkers However, it is evident
that long-term studies specifically investigating effects on
atherosclerotic disease development and progression are
required to ascertain whether the apparent protective effects
of GLP-1 under both normoglycaemic and diabetic
condi-tions may translate to the clinical setting
Angiogenesis
Abnormal angiogenesis is a hallmark of CVD which is
exac-erbated by diabetes, with impaired neovascularization
con-tributing significantly to progression of ischaemic disease
associated with peripheral and coronary arteries
Interest-ingly, it is becoming apparent that GLP-1 may modulate
angiogenesis suggesting that such actions may underlie some
of its reported beneficial cardiovascular effects For example,
exendin-4 stimulates proliferation of human coronary artery
endothelial cells in a GLP-1 receptor-dependent manner via
downstream activation of eNOS, PKA and PI3K/Akt signalling
(Erdogdu et al., 2010) and promotes in vitro HUVEC
migra-tion, ex vivo aortic sprouting angiogenesis and in vivo blood
vessel formation in Matrigel plugs (Kang et al., 2013), while
native GLP-1(7-36) stimulates in vitro angiogenesis in
HUVECs via Akt, Src and PKC-dependent pathways (Aronis
et al., 2013), suggesting that GLP-1 may directly modulate
neovascularization Importantly, these effects appear to
trans-late to the pathological situation, with several recent studies
reporting that GLP-1 can promote the pro-angiogenic actions
of mesenchymal stem cells in different disease settings
Intracoronary artery delivery of GLP-1 eluting encapsulated
human mesenchymal stem cells in a porcine model of
experi-mental myocardial infarction (MI) resulted in improved left
ventricular function and remodelling which was associated
with increased infarct zone angiogenesis (Wright et al., 2012),
while peri-adventitial treatment of porcine vein grafts with
these cells inhibited neointima formation in parallel with
accelerated adventitial angiogenesis (Huang et al., 2013)
Fur-thermore, the addition of GLP-1 to encapsulated human
mes-enchymal cells significantly improved blood flow recovery
and foot salvage in a mouse model of hindlimb ischaemia via
increased capillary and arteriole formation secondary to
par-acrine activation of VEGF-A (Katare et al., 2013) Although
the majority of work investigating the pro-angiogenic actions
of GLP-1 has been performed in normoglycaemic models, a
recent study reported similar beneficial effects in the setting
of diabetes Impaired myocardial angiogenesis in STZ-treated
T1DM rats and associated fibrosis and diastolic dysfunction
were reversed by genetic deletion of DPP-4 or
pharmacologi-cal inhibition with vildagliptin (Shigeta et al., 2012)
Interest-ingly, this study identified DPP-4 as being membrane-bound
and localized to the cardiac capillary endothelium with
increased expression in diabetes, which together with a
report of increased binding affinity of GLP-1 to the coronaryendothelium but not cardiomyocytes in isolated perfused
T1DM rat hearts (Barakat et al., 2011), supports a key
endothelial-specific role of GLP-1 in this setting Althoughthese data provide supportive evidence for pro-angiogenicactions of GLP-1 in diabetes, it is clear that additional mecha-nistic studies are required using different CVD models inorder to define its precise role Furthermore, it is important toassess the effects of GLP-1 therapy in diabetic patients inorder to investigate whether the apparent pro-angiogeniceffects of GLP-1 translate to the clinical setting and are offunctional relevance
GLP-1 and the diabetic myocardium
The heart is one of the major organ targets of GLP-1 and anincreasing number of studies have investigated the actions ofnative GLP-1(7-36), GLP-1 receptor agonists and DPP-4inhibitors in the context of cardioprotection The majority ofexperimental studies have focused on the effects of GLP-1 incardiac ischaemia and its apparent ability to protect againstacute myocardial damage Indeed, it is well established thatGLP-1 pretreatment and chronic DPP-4 inhibition reduceinfarct size after experimental ischaemia in both small andlarge animal models, which is associated with increased sur-
vival and improved cardiac function (Bose et al., 2005; Ban
et al., 2008; 2010; Timmers et al., 2009) Interestingly, a
recent study employing a rabbit model of ischaemia/reperfusion injury reported protective actions of transferrin-stabilized GLP-1, when given both 12 h prior to ischaemiaand immediately upon reperfusion, suggesting that GLP-1may limit infarct size and contractile dysfunction directly,rather than by preconditioning the heart against ischaemia,
as suggested by previous reports (Matsubara et al., 2011) In
addition to its established beneficial actions against acuteischaemic myocardial damage, GLP-1 also confers protectionagainst contractile dysfunction associated with experimentalchronic post-MI remodelling, dilated cardiomyopathy and
hypertensive heart failure (Nikolaidis et al., 2004a; Poornima
et al., 2008; Liu et al., 2010), with similar results reported in
the clinical setting in response to short-term GLP-1 treatment
(Nikolaidis et al., 2004b; Sokos et al., 2006).
Until recently, only limited data were available on thecardiac actions of GLP-1 in diabetes, but it is becomingincreasingly apparent that GLP-1 also plays a key cardiopro-tective role in this setting This is important as it is wellknown that hyperglycaemia is associated with increased sus-ceptibility to cardiac disease and poor outcomes in both
humans and experimental models (Shiomi et al., 2003; Liu
et al., 2005; Greer et al., 2006; Vergès et al., 2007) Chronic
DPP-4 inhibition with linagliptin improves obesity-relateddiastolic dysfunction in insulin-resistant Zucker rats, but has
no effect on cardiomyocyte hypertrophy and fibrosis (Aroor
et al., 2013) Indeed, exendin-4 has been reported to directly
protect isolated rat cardiomyocytes from high induced apoptosis via inhibition of endoplasmic reticulumstress and activation of sarcoplasmic reticulum Ca2 +ATPase 2a
glucose-(Younce et al., 2013) GLP-1 also appears to protect against
diabetic cardiomyopathy, which is defined as cardiac function in the absence of, or disproportionate to, associated
Trang 8dys-hypertension and CAD and is characterized by marked
colla-gen accumulation and impaired diastolic function (Bugger
and Abel, 2014) Both GLP-1 receptor activation and DPP-4
inhibition attenuate development of cardiac dysfunction,
extracellular matrix remodelling, cardiomyocyte
hypertro-phy and apoptosis in experimental models of T1DM and
T2DM, with various mechanisms proposed including
reduc-tion of lipid accumulareduc-tion, oxidative stress and myocardial
inflammation, and modulation of the MMP-2/tissue inhibitor
of MMP-2 axis, endoplasmic reticulum stress and
microvas-cular barrier function (Shigeta et al., 2012; Liu et al., 2013; Monji et al., 2013; Picatoste et al., 2013; Wang et al., 2013).
Furthermore, it appears that GLP-1 also confers reducing actions in diabetes, which is associated withincreased susceptibility to myocardial ischaemia Forexample, mice made diabetic by a combination of STZ
infarct-Figure 1
Summary of the cardiovascular actions of GLP-1 in diabetes GLP-1 exerts indirect cardiovascular benefits in diabetes secondary to its establishedmetabolic actions and subsequent reduction of cardiovascular risk factors In addition, GLP-1 promotes direct cardiovascular benefits which conferprotection against CVD and heart failure, the latter of which may occur via direct myocardial actions or secondary to reduced hypertension andcoronary atherosclerosis
Trang 9injection and high-fat feeding and treated with the GLP-1
receptor agonist, liraglutide, prior to coronary artery ligation,
demonstrated reduced infarct development and improved
survival compared with those treated with the
glucose-lowering drug, metformin, suggesting that the observed
effects occurred via direct actions on the heart and not
sec-ondary to reduced blood glucose (Noyan-Ashraf et al., 2009).
Similar cardioprotective effects have been reported with
DPP-4 inhibition in experimental diet-induced obesity
(Huisamen et al., 2013), while the infarct-limiting effects of
exendin-4 in mice with T2DM were shown to be mediated by
cAMP-induced PKA activation (Ye et al., 2013) Interestingly,
it has recently been suggested that the infarct-reducing
actions of DPP-4 inhibitors may be glucose-dependent, as
both sitagliptin and vildagliptin were found to only decrease
infarct size in isolated rat hearts subjected to
ischaemia-reperfusion injury when they were perfused with elevated
glucose concentrations ≥7 mmol L−1, with similar results
observed in vivo in diabetic, but not normoglycaemic rats
(Hausenloy et al., 2013) This raises the intriguing possibility
that glucose-lowering may counteract the cardioprotectiveactions of GLP-1 and explain why several large-scale clinicaltrials focused on intensive glucose control in T2DM havefailed to demonstrate significant cardiovascular benefits
(Giorgino et al., 2013) Furthermore, it appears that at least
part of the observed beneficial actions of DPP-4 inhibitorsagainst ischaemia-reperfusion injury may be mediated by thechemokine, stromal cell-derived factor 1α in a GLP-1-
independent manner (Bromage et al., 2014).
In addition to the experimental data highlighting a tective role for GLP-1 in the diabetic heart, importantly, asmall number of studies have assessed its cardiac actions inpatients with diabetes It has been known for some time thatshort-term GLP-1 treatment exerts beneficial effects in clini-cal heart failure in both normoglycaemic and diabeticpatients For example, in a small number of heart failurepatients (New York Heart Association class III/IV), 5 weekinfusion with GLP-1 plus standard therapy improved left
pro-Figure 2
Proposed mechanisms underlying the reported cardiovascular actions of GLP-1 Although it is well established that GLP-1 exerts several beneficialeffects on the CVS relevant to diabetes, such as reduction of metabolic CVD risk factors, BP modulation, improved vascular function, decreasedatherosclerosis, promotion of angiogenesis and attenuation of adverse cardiac remodelling, the precise mechanisms are yet to be established,although several pathways have been proposed which are the focus of further investigation EC, endothelial cell; ECM, extracellular matrix; SDF,stromal cell-derived factor 1α; SR, sarcoplasmic reticulum
Trang 10ventricular ejection fraction and myocardial oxygen
con-sumption compared with those receiving standard therapy
alone, effects that were seen in both diabetic and
non-diabetic patients (Sokos et al., 2006) Furthermore, a small
non-randomized trial of 72 h GLP-1 infusion following
primary angioplasty after acute MI led to improved cardiac
function in both non-diabetic and diabetic patients which
was still evident upon 120 day follow-up (Nikolaidis et al.,
2004b) More recently, a larger randomized trial in patients
presenting with ST-segment elevation MI reported that
exenatide infusion for 15 min prior to primary angioplasty
continued until 6 h post-reperfusion resulted in improved
myocardial salvage at 3 months although no functional
ben-efits were observed (Lønborg et al., 2012) Indeed, two current
clinical trials are assessing the potential of using exenatide as
a post-conditioning agent to reduce reperfusion injury
fol-lowing percutaneous coronary intervention (Effect of
Addi-tional Treatment With EXenatide in Patients With an Acute
Myocardial Infarction, the EXAMI trial, NCT01254123;
Phar-macological Postconditioning to Reduce Infarct Size
Follow-ing Primary PCI, POSTCON II, NCT00835848) InterestFollow-ingly,
in patients with left ventricular diastolic dysfunction, DPP-4
activity in the coronary sinus and peripheral circulation is
reported to be negatively correlated with diastolic function
and increased by co-morbid diabetes (Shigeta et al., 2012),
suggesting that reduced GLP-1 levels in diabetes may underlie
the associated cardiac dysfunction Exenatide has also been
found to modulate myocardial glucose transport and uptake
in T2DM patients dependent upon the degree of insulin
resistance (Gejl et al., 2012), although a similar study
reported that GLP-1-induced increases in resting myocardial
glucose uptake in lean individuals were absent in obese
T2DM patients, with parallel studies in pigs suggesting that
this was due to impaired p38-MAPK signalling (Moberly et al.,
2013) Interestingly, a recent experimental study found that
exendin-4 reduced contractile function and was unable to
stimulate glucose utilization in normal rat hearts in the
pres-ence of fatty acids (Nguyen et al., 2013), despite previous
reports of increased myocardial glucose uptake in response to
GLP-1 in experimental myocardial ischaemia and dilated
car-diomyopathy (Nikolaidis et al., 2005; Zhao et al., 2006;
Bhashyam et al., 2010) Such findings highlight the need for
detailed investigation of the effects of GLP-1 on altered
myo-cardial metabolism in diabetic patients both with and
without cardiac complications, in which the effects of GLP-1
may diverge
Although these clinical and experimental data are clearly
supportive of an important role for GLP-1 signalling in the
diabetic heart, they are largely descriptive with limited focus
on underlying mechanisms Previous studies in experimental
models of heart failure have highlighted several pathways
which may mediate the cardioprotective effects of GLP-1,
including cAMP/PKA, PI3K/Akt, p44/p42MAPK, ERK1/2 (Bose
et al., 2005; Timmers et al., 2009; Ravassa et al., 2011),
together with suggestions of GLP-1 receptor-independent
sig-nalling (Nikolaidis et al., 2005; Sonne et al., 2008) However,
the precise mechanisms underlying the apparent protective
actions of GLP-1 in the diabetic heart, in which GLP-1
sig-nalling is likely to be different, remain unknown and clearly
need to be defined in order to fully assess the therapeutic
Trang 11Summary and future perspective
Over recent years, it has become clear that GLP-1 exerts
important actions on the CVS in both health and disease in
addition to its prototypic effects on glycaemic control (Grieve
et al., 2009) and also confers beneficial effects on the
cardio-vascular risk profile in diabetic patients However, emerging
evidence now strongly suggests that GLP-1 exerts specific
cardiovascular actions in diabetes and may attenuate the
development and progression of associated cardiovascular
complications (summarized in Figure 1), although the precise
mechanisms are yet to be established with several candidate
pathways proposed (see Figure 2) Nonetheless, it is
impor-tant to note that the majority of data pointing towards such
beneficial effects are largely experimental and that equivalent
information on the cardiovascular actions of GLP-1 in the
clinical setting of diabetes is somewhat lacking, particularly
in relation to its chronic effects In this regard, the first
large-scale GLP-1 clinical trials to assess cardiovascular
out-comes after long-term treatment (SAVOR-TIMI 53, EXAMINE)
have recently reported that chronic DPP-4 inhibition with
either saxagliptin or apogliptin had no significant effects on
their primary composite end points of cardiovascular death,
or non-fatal MI/stroke (Scirica et al., 2013; White et al., 2013),
although it should be noted that SAVOR-TIMI 53 reported a
27% increase in hospitalization for heart failure However,
until the results of several ongoing clinical trials investigating
the chronic cardiovascular effects of GLP-1 receptor agonists
are known (summarized in Table 1 together with further
DPP-4 inhibitor trials), in which circulating GLP-1 levels will
be much higher than those achieved using DPP-4 inhibitors,
no conclusions can be drawn on either the potential clinical
cardiovascular benefits or safety of GLP-1-based therapies in
diabetes Nonetheless, the apparent complexity of
cardiovas-cular GLP-1 signalling under both normal and diabetic
con-ditions clearly suggests that it is likely that selective targeting
of specific aspects of CVD may be required in order to realize
the optimal benefits of GLP-1 targeting in this setting
Acknowledgements
The authors’ work is supported by the British Heart
Founda-tion, Diabetes UK and the Medical Research Council
Conflict of interest
None
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Trang 171Division of Molecular Biology, GW Pharmaceuticals, Salisbury, Wiltshire, UK,2GW
Pharmaceuticals, Porton Down Science Park, Salisbury, Wiltshire, UK,3
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Napoli, Italy, and4
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK
Correspondence
Dr John M McPartland, 53Washington Street Ext.,Middlebury, VT 05753, USA
Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of
mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis Synthesized data regardingmechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials The evidence indicatesthat CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects
rimonabant These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given
target
Abbreviations
2-AG, sn-2 arachidonoyl glycerol; AEA, anandamide; CBD, cannabidiol; CV, coefficient of variation; DAGL,
diacylglycerol lipase; FAAH, fatty acid amide hydrolase; FsAC, forskolin-stimulated adenylate cyclase; MAFP,
methylarachidonoyl fluorophosphonate; MAGL, monoacylglycerol lipase; PMSF, phenylmethyl sulfonyl fluoride; THCV,
Trang 18Isolating and identifying the ‘primary active ingredient’ in
Cannabis (the plant) and cannabis (the plant product)
stymied chemists for over 150 years Finally, Gaoni
Δ9-tetrahydrocannabinol (THC) THC and biosynthetically
related and structurally similar plant cannabinoids are now
called phytocannabinoids to distinguish them from
structur-ally dissimilar but pharmacologicstructur-ally analogous
(synthocannabinoids)
THC exerts most of its physiological actions via the
endo-cannabinoid system The endoendo-cannabinoid system consists
of (i) GPCRs for THC, known as cannabinoid receptors; (ii)
endogenous cannabinoid receptor ligands; and (iii) ligand
metabolic enzymes The salient homeostatic roles of the
endocannabinoid system have been roughly portrayed as
‘relax, eat, sleep, forget, and protect’ (Di Marzo et al., 1998).
When malfunctioning, the endocannabinoid system can
contribute to pathological states (Russo, 2004; Di Marzo,
2008)
All vertebrate animals express at least two cannabinoid
receptors The CB1 receptor principally functions in the
nervous system but is expressed in many cells throughout the
body CB2 receptors are primarily associated with cells
gov-erning immune function, such as splenocytes, macrophages,
monocytes, microglia, and B- and T-cells Recent evidence
demonstrates the presence of CB2 receptors in other cells,often up-regulated under pathological conditions (reviewed
in Pertwee et al., 2010).
N-arachidonylethanolamine (anandamide, AEA) and2-arachidonoylglycerol (2-AG) One of AEA’s key biosyn-
thetic enzymes is N-acyl-phosphatidylethanolamine
phos-pholipase D The chief biosynthetic enzymes of 2-AG are twoisoforms of diacylglycerol lipase: DAGLα and DAGLβ Theprimary catabolic enzymes of AEA and 2-AG are fatty acidamide hydrolase (FAAH) and monoacylglycerol lipase(MAGL) respectively COX-2 can also catabolize AEA and
2-AG (Kozak et al., 2002).
Synthetic THC (dronabinol) became clinically available inthe 1980s for indications including anorexia and weight loss
in people with AIDS, and for nausea and vomiting associatedwith cancer chemotherapy Off-label uses include migraine,multiple sclerosis, sleep disorders and chronic neuropathicpain However, the therapeutic window of THC is narrowed
by side effects In clinical trials, dronabinol precipitated phoria, depersonalization, anxiety, panic reactions and para-
dys-noia (Cocchetto et al., 1981).
Psychological side effects occur more frequently withTHC than with whole cannabis (Grinspoon and Bakalar,1997) Only six years after Raphael Mechoulam successfullyisolated THC, one of the authors of this article determined
that THC did not act alone in cannabis (Gill et al., 1970).
Other constituents in cannabis work in a paradoxical capacity
Tables of Links
TARGETS
lipase5-HT1Areceptors PLA2
Adenosine A2Areceptors Ion channelsc
DAGLα, diacylglycerol lipase α Glycineα3 receptors
DAGLβ, diacylglycerol lipase β Nuclear hormone
FAAH, fatty acid amide hydrolase PPARγ (NR1C3)
iNOS, inducible NOS
LIGANDS
2-AG, 2-arachidonoylglycerol5-HT
AEA, anandamideAM251
CBD, cannabidiolCP55940LTB4NORimonabant, SR141716ATHC,Δ9-tetrahydrocannabinolTHCV,Δ9-tetrahydrocannabivarinWIN55212-2
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d,e,f Alexander et al., 2013a,b,c,d,e,f).
Trang 19of mitigating the side effects of THC, but improving the
therapeutic activity of THC
Cannabidiol (CBD) and
Δ9-tetrahydrocannabivarin (THCV)
At last count, 108 phytocannabinoids have been
character-ized in various chemovars of the plant (Hanuš, 2008) The
other phytocannabinoids of greatest clinical interest are CBD
and THCV THC and CBD are ‘sister’ molecules,
biosynthe-sized by nearly identical enzymes in Cannabis – expressions of
two alleles at a single gene locus (de Meijer et al., 2003) THC
and CBD are C21 terpenophenols with pentyl alkyl tails,
whereas THCV is a C19propyl-tailed analogue of THC
Can-nabis biosynthesizes these compounds as carboxylic acids,
for example, THC-carboxylic acid (2-COOH-THC) When
heated, dried or exposed to light, the parent compounds are
decarboxylated
Fundamentally, THC mimics AEA and 2-AG by acting as a
partial agonist at CB1and CB2receptors (Mechoulam et al.,
1998) But rather than simply substituting for AEA and 2-AG,
cannabis and its many constituents work, in part, by
‘kick-starting’ the endocannabinoid system (McPartland and Guy,
2004) CBD, in particular, gained attention early in this
regard Several landmark studies published in the previous
century have shown interactions between CBD and THC (see
Box 1)
Meta-analysis
Many narrative reviews have been written about THC, CBDand THCV, and cannabis as a ‘synergistic shotgun’(McPartland and Pruitt, 1999; McPartland and Russo, 2001;
Mechoulam et al., 2007; Zuardi, 2008; Izzo et al., 2009; Russo,
2011) CBD by itself has many therapeutic properties: lytic, antidepressant, antipsychotic, anticonvulsant, anti-nausea, antioxidant, anti-inflammatory, anti-arthritic andantineoplastic
anxio-The ability of CBD to antagonize THC has been the focus
of many narrative reviews, as well as the studies listed inBox 1 This has led to the assumption that CBD exerts a directpharmacodynamic blockade of THC The pharmacologicalcommunity tends to view CBD and THCV as negative modu-lators of CB1 receptor agonists This view may be due to asuperficial interpretation of the available pharmacologicaldata Hence, CBD and THCV would appear to mirror themechanism of first-generation CB1 receptor inverseagonists known as cannABinoid ANTagonists (‘abants’), such
as rimonabant, taranabant, otenabant and ibipinabant.Rimonabant was developed as an anti-obesity agent and mar-keted as an adjuvant to diet and exercise for weight loss inobese individuals It was subsequently withdrawn from themarket due to adverse psychiatric side effects (Bermudez-Silva
et al., 2010).
Box 1 Landmark 20th century studies regarding the effects of CBD upon THC.a
Animal studies
CBD combined with isomeric tetrahydrocannabinols caused ‘synergistic hypnotic activity in the mouse’ – Loewe and Modell, 1941
CBD inhibited THC effects on mouse catatonia, rat ambulation and rat aggression, but potentiated THC effects on mouse analgesia and ratrope climbing – Karniol and Carlini, 1973
CBD decreased THC suppression of behaviour in rats and pigeons – Davis and Borgen, 1974
CBD potentiated THC-induced changes in hepatic enzymes – Poddar et al., 1974.
CBD increased THC potentiation of hexobarbitone in rats – Fernandes et al., 1974.
CBD increased THC reduction of intestinal motility in mice – Anderson et al., 1974.
CBD reduced THC hypothermia and bradycardia – Borgen and Davis, 1974
CBD blocked THC inhibition of pig brain monamine oxidase – Schurr and Livne, 1976
CBD antagonized THC antinociceptive effects in mice – Welburn et al., 1976.
CBD prevented tonic and clonic convulsions induced by THC – Consroe et al., 1977.
CBD antagonized THC suppression of operant behaviour in monkeys – Brady and Balster, 1980
CBD delayed THC discriminative effects – Zuardi et al., 1981.
CBD prolonged THC cue effects in rats – Hiltunen and Järbe, 1986
CBD antagonized THC catalepsy in mice – Formukong et al., 1988a.
CBD increased THC analgesic activity and anti-erythema – Formukong et al., 1988b.
CBD prolonged and reduced the hydroxylation of THC – Bornheim et al., 1995, 1998.
Human clinical trials
CBD decreased anxiety caused by THC – Karniol et al., 1974.
CBD slightly increased time to onset, intensity and duration of THC intoxication – Hollister and Gillespie, 1975
CBD attenuated THC euphoria – Dalton et al., 1976.
CBD reduced anxiety provoked by THC – Zuardi et al., 1982.
CBD improved sleep and decreased epilepsy – Cunha et al., 1980; Carlini and Cunha, 1981.
CBD decreased cortisol secretion and had sedative effects – Zuardi et al., 1993.
CBD provided antipsychotic benefits – Zuardi et al., 1995.
aFull citations appear in previous reviews (McPartland and Pruitt, 1999; McPartland and Russo, 2001; Russo and Guy, 2006)
Trang 20We hypothesize that CBD and THCV may, under certain
conditions, ‘antagonize’ the effects of THC via mechanisms
other than direct CB1receptor blockade The purpose of this
article is to review mechanistic studies (in vitro and ex vivo) of
CBD and THCV Rather than a narrative review, we will
conduct a meta-analysis The results of this synthesis of
mechanistic studies will be applied to an emerging
discus-sion: do CBD and THCV act as natural ‘abants’?
Methods
Meta-analysis uses an objective, transparent approach for
research synthesis, with the aim of minimizing bias A valid
meta-analysis combines data from independent studies,
identifies sources of heterogeneity among the studies and
manages heterogeneity by placing defined limits upon data
selection (Glass et al., 1981) This methodology usually
focuses upon human clinical trials, but it can be applied to
pre-clinical studies We previously conducted a meta-analysis
on CB1 receptor ligand binding affinity (McPartland et al.,
2007) We follow the guidelines proposed by PRISMA, the
Preferred Reporting Items for Systematic Reviews and
Meta-Analyses (Liberati et al., 2009) See Supporting Information
Appendix S1
Search strategy and study selection
We briefly describe the analysis here For details regarding
inclusion criteria, heterogeneity tests, subgroup analysis,
quality assessment and publication bias, see Supporting
Information Appendix S1
PubMed (www.ncbi.nlm.nih.gov/pubmed/) was searched
from 1988 (beginning with Devane et al., 1988) through
December 2013, using the following Boolean search string:
(cannabidiol OR tetrahydrocannabivarin) AND (animal OR
affinity OR efficacy) NOT (behavioral OR behavioural)
Refer-ences identified by the search strategy were scanned for
inclu-sion and excluinclu-sion criteria by three independent reviewers
who resolved disagreements by consensus
Inclusion criteria included studies of CBD or THCV, their
carboxylic acids (CBD-acid, THCV-acid), as well as CBD- or
THCV-enriched plant extracts (‘botanical drug substances,’
CBD-BDS, THCV-BDS) Included studies reported in vitro or ex
vivo mechanistic data (receptor affinity and efficacy assays),
detailed in Supporting Information Appendix S1
The rather broad search strategy retrieved many articles
that were subsequently excluded as irrelevant Excluded
topics included (i) review articles or publications with
dupli-cated data; (ii) animal studies or in vivo studies without
mechanisms or an identified molecular target; (iii) studies of
synthetic analogues, or metabolites of CBD or THCV; (iv)
human clinical trials lacking mechanistic analysis; urinary
metabolites of CBD and THCV and their use in drug testing;
characterizations of cannabinoid drug delivery systems; and
(v) other irrelevant topics (see Supporting Information
Appendix S1 for elaboration)
Articles meeting inclusion and exclusion criteria were
screened for supporting citations, and antecedent sources
were retrieved The search also included unpublished data
communicated at research conferences, upon approval by the
authors of the data Lastly, we contacted world experts andasked them to contribute unpublished data (see Acknowl-edgements section)
Data extraction and synthesis
Extracted data included ligand (CBD or THCV), assay type,animal species, reported means, sample variance, sample sizeand methodological factors Methodological factors (covari-ates) were extracted for use in subgroup analyses to testwhether they exerted heterogeneous effects upon pooledmeans Methodological factors were pre-specified, chosen in
advance by a priori hypotheses based upon recognized odological diversity among studies, and not undertaken after the results of the studies had been compiled (post hoc
meth-analyses)
Data were synthesized qualitatively (e.g categorical data)
or quantitatively [e.g the continuous variables for affinity (K i)
and efficacy (EMAX)] A quantitatively synthesized result isreported as a pooled mean± SEM Optimal quantitative syn-thesis would employ a weighted pooled mean, which adjustseach study’s mean divided by its SEM, because larger studieswith less variance should carry more ‘weight’ in a meta-analysis Unfortunately, many studies omitted variance data
or sample size
To determine whether pooling was statistically ate, the coefficient of variation (CV) was determined foreach pooled mean The CV measures data dispersion of aprobability distribution, defined simply as the ratio of the SD
appropri-to the mean (Reed et al., 2002) We applied the Cochrane
‘skew test’ to the CV (Higgins and Green, 2005), where CV≥
1 (i.e SD ≥ mean) identifies a skewed mean with excessiveheterogeneity Skewed mean was submitted to Grubb’s test(www.graphpad.com/quickcalcs/) Data identified as signifi-
cant outliers (P< 0.05) were reported in Supporting tion Appendices S2 and S3 and withdrawn from synthesis
Informa-The CV-skew test could not be used for sample sizes of n≤ 3
In those cases, we used simple pooled means
Results
The search algorithm identified 431 potentially relevant cles Many of these publications were review articles or con-cerned topics irrelevant to this review In addition to 174articles that met the predefined selection criteria for rel-evance, we included 28 studies obtained from citation track-ing or unpublished studies See Figure 1 for a flowchart.The quality of statistical reporting has steadily improvedbetween 1984 and 2013 For example, early studies that meas-
arti-ured the K iof CBD reported variance as SD, and sometimesomitted variance data or sample sizes (Supporting Informa-tion Appendix S2) Later studies reported variance as SEM
However, K ivalues are not symmetric around a mean, and thebest way to report non-parametric variance is the use of 95%confidence limits/intervals rather than SEM
CBD has many molecular targets; we grouped them inthree categories The first category addresses the effects ofCBD and THCV at CB1receptors – directly and indirectly Thenext category includes what some researchers consider
an ‘expanded’ endocannabinoid system – other GPCRs (CB2,
Trang 21GPR55, GPR18), and transient receptor potential ion
chan-nels (e.g TRPV1, TRPV2, TRPA1, TRPM8) The third category
includes receptors and ligand enzymes beyond the expanded
system, as well as the arachidonic acid (AA) cascade, nitric
oxide signalling, cytokines, redox signalling and mechanisms
involved in apoptosis
CBD at CB1 receptors: direct and
indirect interactions
The pooled mean affinity of CBD at CB1receptors is K i= 3245
± 803 nM The pooled mean was calculated from 1 human, 3
mice and 11 rat studies (Supporting Information
Appen-dix S2) Species differences were not statistically different,
including the human study (K i = 1510 ± 100 nM) Several
studies omitted quality measures such as statistical data
(vari-ance and/or sample size) Subgroup analysis of the five
meth-odological factors produced surprising results Only one
factor – the use of crude brain homogenates – proved to be
statistically relevant (Supporting Information Appendix S2)
None of the other factors gave rise to data heterogeneity –
species differences, class of tritiated ligand (agonist vs
antagonist) or even the use of non-specific probes (e.g
[3H]TMA-THC) One methodological factor could not be
assessed – no studies used PMSF
Eight studies tested CBD efficacy at CB1receptors, assayed
with forskolin-stimulated adenylate cyclase (FsAC) or
[35S]GTPγS (Supporting Information Appendix S2) Six of
these studies reported no measurable response or inconsistent
dose–response curves hovering near zero One study reported
slight agonism, and one study reported slightly inverse
agonism, both at high concentrations (≥10 μM)
Surprisingly, in some mechanistic studies, the effects of
CBD could be reversed by CB1 receptor inverse agonists, or
were absent in CB1receptor knockout mice (n= 10 studies;
Supporting Information Appendix S2) This suggests that CBD
may exert ‘indirect agonism’ at CB1 receptors – either
aug-menting CB1 constitutional activity or augaug-menting
endocan-nabinoid tone Regarding the first mechanism, Howlett et al.
(1989) presented thermodynamic data that suggest that CBD
may alter CB1receptor activity by increasing membrane
fluid-ity Sagredo et al (2011) showed that Sativex®(a mix of THCand CBD; GW Pharmaceuticals, Salisbury, UK) can up-regulate
CB1receptor gene expression in a rat model of Huntington’sdisease The second mechanism – augmenting endocannabi-noid tone – is supported by studies showing that CBD or
CBD-BDS inhibits AEA hydrolysis by FAAH (n = 5 studies;Supporting Information Appendix S2), with a pooled mean
IC50= 19.8 ± 4.77 μM Four rodent studies show that CBDinhibits the putative AEA transporter, with a pooled mean IC50
= 10.2 ± 3.03 μM Two studies reported CBD increasing 2-AGlevels, 33 or 260% (Supporting Information Appendix S2).CBD may affect the pharmacokinetics of THC when the
two are co-administered (n= 17 studies; Supporting tion Appendix S2) One of these pharmacokinetic studiespredates the isolation of pure THC – Loewe and Modell(1941) stated that CBD ‘synergized’ hypnotic action in miceinduced by isomeric tetrahydrocannabinols CBD may impairTHC hydrolysis by CYP450 enzymes The results of CYPstudies vary due to species differences, timing (CBD pre-administration vs co-administration) and specific CYP isoen-zymes Recent human studies show no pharmacokineticinteraction between THC and CBD at clinically relevantdosing (Supporting Information Appendix S2)
Informa-The effect of CBD on CYPs is important because theseenzymes metabolize THC into 11-OH-THC This metabolitemay be up to four times more psychoactive than THC,according to a rat discriminative study (Browne andWeissman, 1981) The affinity and efficacy of 11-OH-THC at
CB1receptors has not been measured, although the affinity of11-OH-Δ8-THC was K i= 25.8 nM, displacing [3H]HU-243 fromrCB1COS cells (Rhee et al., 1997) In the same assay,Δ9-THC
exhibited K i = 80.3 nM (Rhee et al., 1997) or K i = 39.5 nM
(Bayewitch et al., 1996) The significance of CBD modulating
the metabolism of THC into 11-OH-THC is an importantvariable that remains to be explored
CBD may antagonize cannabinoid-induced effects Six in
vitro studies demonstrate that CBD can antagonize
CP55,940-or WIN55212-2-induced efficacy (SuppCP55,940-orting InfCP55,940-ormation
Appendix S2), with a pooled mean KB= 88.5 ± 18.46 nM This
unexpected KBis 37-fold more potent than the K iof CBD inbinding assays This suggests an indirect mechanism, that is,
mediated by (an)other target(s) The KBof CBD is 147-fold
higher (less potent) than the KB of rimonabant, when this
inverse agonist was run in parallel (n= 3 studies; SupportingInformation Appendix S2) Note that this antagonism can bevisualized by juxtaposing two log concentration–responsecurves; for example, the curve of CP55,940 for stimulation of[35S]GTPγS binding to CB1receptors, compared to the curve ofCP55,940 for such stimulation in the presence of CBD Acompetitive antagonist would produce a parallel rightward
shift in the curve of CP55,940 However, Petitet et al (1998)
found CBD to produce a parallel shift in the curve ofCP55,940 that was downward rather than rightward in itsdirection, an effect that a competitive antagonist is notexpected to produce
CBD at targets in the ‘expanded endocannabinoid system’
The ability of CBD to antagonize a cannabinoid agonist in anefficacy assay may be confounded by the impact of CBD on
Figure 1
Flow diagram of article selection for meta-analysis
Trang 22other receptors or enzymes present in the assay In Table 1, we
summarize studies regarding CBD affinity and efficacy at
other metabotropic receptors and ion channels in the
‘expanded endocannabinoid system.’
CBD shows low affinity at CB2 receptors (Table 1) Its
efficacy at CB2 receptors suggests weak inverse agonism at
concentrations that may not be pharmacologically relevant
However, CBD antagonizes CP55,940 signalling at CB2
recep-tors with a KBpotency not commensurate with its K i This
suggests again an indirect mechanism of action At GPR18,
two studies suggest that CBD acts as a partial agonist and
antagonizes THC (Supporting Information Appendix S2) The
direct affinity and efficacy of CBD at GPR55 has not been
measured, but it can antagonize GPR55 agonists (Supporting
Information Appendix S2)
Table 1 highlights CBD signalling at transient receptor
potential (TRP) channels, which have been characterized as
‘ionotropic cannabinoid receptors’ (Di Marzo et al., 2002).
We find a species difference at TRPV1 channels and the
(Table 1), whereas a single study of rat TRPV1 channels
reports an EMAXof 21% (Qin et al., 2008) At TRPA1 channels,
the pooled mean EMAX of CBD (Table 1) is considerably less
than that of CBD-BDS (163.6± 11.9%; De Petrocellis et al.,
2011) This suggests that terpenoids and other plant
sub-stances in the extract may enhance CBD activity at TRPA1
channels, either pharmacokinetically or
pharmacodynami-cally The same study showed that CBD-BDS also showed
slightly greater efficacy than pure CBD at TRPV1 channels
(Supporting Information Appendix S2) CBD acts as an
antagonist at TRPM8, unlike its agonist activity at other TRP
channels (Table 1)
CBD at other molecular targets
CBD is a promiscuous compound with activity at multipletargets Promiscuity is governed by ligand structure, and can-nabinoids exhibit the characteristics of promiscuous ligands:molecular mass >400 g·mol−1, and partition coefficient(CLogP) scores between 2 and 7 (Hopkins, 2008)
CBD inhibits adenosine uptake (pooled IC50 = 122 nM
minus one outlier, n = 3 studies; Supporting InformationAppendix S2) and this mechanism is likely to exert indirectagonism at adenosine receptors Consistent with this, thebeneficial effects of CBD in animal models of inflammationare blocked by adenosine A2A receptor antagonists (n = 8studies) CBD may also regulate 5-HT levels, although sixstudies report disparate results CBD has slight affinity for the5-HT1Areceptor (K i∼ 16 μM; Supporting Information Appen-dix S2), although its efficacy at 5-HT1Areceptors is inconsist-
ent Several beneficial effects of CBD in vivo are blocked
by 5-HT1A receptor antagonists (Supporting InformationAppendix S2)
CBD exerts a positive allosteric modulation ofα3 glycinereceptors (pooled EC50= 11.0 μM, n = 3 studies; Supporting
Information Appendix S2) NMR analysis revealed a directinteraction between CBD and S296 in the third transmem-brane domain of α3 glycine receptors The cannabinoid-induced analgesic effect was absent in mice lacking theα3
glycine receptors (Xiong et al., 2011) CBD can go nuclear,
as it affects PPARγ receptors (IC50 = 5 μM; SupportingInformation Appendix S2), and its beneficial effects areblocked by PPARγ antagonists (n = 5 studies) A dozen studiesindicate that CBD allosterically modulates other receptors:
α1-adrenoceptors, dopamine D2, GABAA, μ- and δ-opioid
CB2receptor A: versus [3H]CP55,940 binding; human CB2transfected cell cultures, or rat
RBL-2H3 leukemia cells, centrifuged membranes
K i = 3612 ± 1382 nM, n = 6
CB2receptor E: [35S]GTPγS binding; human CB2-CHO cells EMAX= −15% below basal at 10 μM
EC50= 503 ± 2080 nM; n = 1
CB2receptor E: antagonism of CP55,940-induced [35S]GTPγS binding; human CB2-CHO cells KB= 65 ± 54.1 nM, n = 1
GPR55 E: antagonism of agonist-induced signalling; human cell cultures or CB2-CHO cells IC50= 433 ± 42.6 nM, n = 3
TRPV1 channels A: versus [3
H]-resiniferatoxin binding; human TRPV1-HEK293 cell membranes K i = 3600 ± 200 (SD) nM, n = 1
TRPV1 channels E: [Ca2+]ielevation in human TRPV1-HEK293 cells EMAX53.4± 5.03%; n = 4
EC50= 1900 ± 802 nMTRPA1 channels E: [Ca2 +]ielevation in rat TRPA1-HEK293 cell membranes EMAX98.6± 10.39%
aA, affinity; E, efficacy Other abbreviations as defined in the manuscript
bNumber of studies that met the CV-Cochrane ‘skew test’ (see Supporting Information Appendix S2)
Trang 23receptors – some positively, some negatively, none with great
efficacy (Supporting Information Appendix S2) CBD
modu-lates intracellular calcium levels, via T-type and L-type
voltage-regulated Ca2 +channels and mitochondrial Na+/Ca2 +
exchange (n= 7 studies)
The effects on the AA cascade are complex Three studies
indicate that CBD mobilizes AA by stimulating PLA2activity
The direction that AA goes from there is uncertain CBD
inhibits the metabolism of AA to LTB4 by 5-lipoxygenase –
pooled IC50= 3.1 ± 0.75 μM, n = 4 studies, although a fifth
study reported no effect up to 80μM (Supporting
Informa-tion Appendix S2) Contradictory studies report CBD
block-ing the metabolism of AA to PGE1 or TxB2 (via COX-1) or
PGE2(via COX-2) Early studies did not identify which COX
isoenzyme they tested Recent studies indicate that CBD
attenuates serum PGE2in animal models of chronic pain, but
this may or may not correlate with COX-2 protein expression
(Supporting Information Appendix S2)
CBD clearly dampens NO production in animal models of
acute and chronic inflammation – as measured by a reduction
in nitrite levels or inducible NOS (iNOS) protein expression (n
= 15 studies) A dozen studies show that CBD inhibits the
expression of inflammatory cytokines and transcription
factors (IL-1β, IL-2, IL-6, IL-8, TNF-α, IFN-γ, CCL3, CCL4,
NF-κB)
CBD is a potent antioxidant; it reduces reactive oxygen
species (ROS) induced by a variety of toxins and tissue insults
(Supporting Information Appendix S2) However, one study
suggests the opposite – that CBD hydroxyquinone, formed
during hepatic microsomal metabolism of CBD, is capable of
generating ROS and inducing cytotoxicity Indeed, one
mechanism by which CBD induces tumour cell apoptosis
appears to be ROS generation (Supporting Information
Appendix S2) In general, it appears that CBD can induce ROS
in cancer cells and reduce ROS in healthy cells stimulated by
agents that induce ROS formation In fact, even in cancer
cells, CBD inhibits ROS formation induced by H2O2(Ligresti
et al., 2006).
THCV at CB1 and CB2
Affinity studies show that THCV binds to CB1receptors with
significant potency There may be species differences – at
human CB1receptors transfected into CHO cells, mean K i=
5.47± 4.02 nM (n = 2 studies); at mouse brain membranes,
mean K i = 61.0 ± 14.40 nM (n = 2); and at rat brain
mem-branes (n = 1), mean K i= 286 ± 43 nM The rat study used
[3H]SR141716A, whereas others used [3H]CP55,940
(Support-ing Information Appendix S3)
Four studies tested the efficacy of THCV at CB1receptors
THCV did not inhibit or stimulate [35S]GTPγS binding to
mouse whole brain membranes (n= 3 studies) or to rat
mem-branes (cortical, cerebellar or piriform cortical memmem-branes) at
concentrations up to 10μM This suggests that THCV targets
the CB1 receptor as a neutral antagonist rather than as an
antagonist/inverse agonist The single study that tested FsAC
in human CB1 receptors in CHO cells produced signs of
inverse agonism at concentrations of 10, 100 and 1000 nM
(Supporting Information Appendix S3)
In the presence of other cannabinoids, THCV binds to
CB1, in vitro, in a manner that gives rise to competitive
antagonism rather than to agonism More specifically, THCV
produces significant parallel rightward shifts in the logconcentration–response curves of established CB1 receptoragonists such as CP55,940 or WIN55212-2, in [35S]GTPγSbinding to mouse whole brain membranes, and in the inhi-bition of electrically evoked contractions of mouse isolatedvasa deferentia Pooling five studies (Supporting Information
Appendix S3) results in a KB= 64.2 ± 14.14 nM Thus, the KB
of THCV is in the same range of concentrations as its K i.The five studies hint at ‘probe dependence’ That is, THCVhas greater potency when antagonizing WIN55212-2 thanwhen antagonizing CP55,940, although the difference fallsshort of statistical significance (Supporting InformationAppendix S3) Two studies of THCV as an antagonist in thevas deferens assay also hint at probe dependence – thepotency of THCV is not the same for all CB1receptor agonists
In rank order: AEA (KB= 1.2 nM), methAEA (KB= 2.6 nM),
WIN55212-2 (KB= 3.15 nM), CP55,940 (KB= 10.3 nM), THC
(KB= 96.7 nM) (Supporting Information Appendix S3) THCValso reverses WIN55212-2-induced decreases of miniatureinhibitory postsynaptic current frequency at mouse cerebellarinterneuron-Purkinje cell synapses, albeit with much lesspotency than the CB1receptor inverse agonist AM251, whichwas tested in parallel (Supporting Information Appendix S3)
At human CB2 receptors transfected into CHO cells,
THCV has significant affinity, with pooled mean Ki= 124.7 ±
64.55 nM (n = 3 studies; Supporting Information dix S3) THCV acts as a partial agonist at these receptors asmeasured by [35S]GTPγS binding or FsAC assays, pooled EMAX=56.7 from basal at 1–10μM, EC50 = 74.2 ± 34.4 nM (n = 3
Appen-studies; Supporting Information Appendix S3)
THCV may exert ‘indirect agonism’ at CB1and CB2tors by augmenting endocannabinoid tone It does this byinhibiting the putative AEA transporter, inhibiting FAAHactivity and inhibiting MAGL activity, albeit at 25–100μM (n
recep-= 3 studies; Supporting Information Appendix S3) Whether
or not THCV exerts ‘indirect agonism’ to an extent that canovercome its surmountable CB1receptor antagonism requiresfurther experiments
THCV at other targets
Unlike CBD, much less evidence exists for non-cannabinoidreceptor-mediated effects of THCV, and this evidence has to
do mostly with the capability of THCV to interact with
‘thermo-TRP’ channels One study demonstrates that THCVacts as an agonist at rat TRPA1, human TRPV1 and ratTRPV2-4 channels and a potent antagonist at rat TRPM8channels (Supporting Information Appendix S3) Dataregarding its effects at GPR55 are controversial, and thepotentiation of 5-HT1A receptors was observed only atthe concentration of 100 nM (Supporting InformationAppendix S3)
Discussion
Our previous meta-analysis of receptor affinity of several
syn-thetic and natural cannabinoid ligands (McPartland et al.,
2007) involved more studies than this current analysis ofCBD and THCV Subgroup analysis suggests that methodo-logical nuances employed in the larger group of studies (such
as PMSF usage) were absent in studies of CBD and THCV
Trang 24The low affinity of CBD at CB1and CB2receptors
damp-ened the signal-to-noise ratio to such an extent that some
nuances were rendered unnecessary – subgroup analysis
showed no differences arising from methodological factors
that proved important in our previous meta-analysis, such as
species differences and the quality of tritiated ligand THCV,
on the contrary, with its moderate to high potency in
dis-placement assays for CB1and CB2receptors, hints at species
differences in its affinity at CB1receptors, as well as ‘probe
dependence’ in its ability to antagonize other cannabinoids
Pre-clinical animal studies demonstrated that CBD
expands the therapeutic window of THC CBD may
accom-plish this by enhancing the efficacy of THC as well as by
mitigating the ‘central’ side effects of THC These effects have
been known for some time (Box 1) In particular, our
discus-sion below indicates that CBD enhances the efficacy of THC
in preclinical models of multiple sclerosis, muscle spasticity,
epilepsy, chronic pain and inflammation, anorexia and
nausea, diabetes and metabolic syndrome CBD mitigates the
side effects of THC in animal models of psychosis, anxiety
and depression-anhedonia
CBD at CB1 receptors: direct and indirect
interactions
Let us return to our original question: does CBD act as a
natural ‘abant’? Data clearly show that CBD does not act
directly at CB1receptors The affinity of CBD at CB1receptors
(K i= 3245 nM) is at least three orders of magnitude less than
that of rimonabant (K d= 1.0 nM in rat, 2.9 nM in human;
McPartland et al., 2007) Several efficacy studies made direct
comparisons of [35S]GTPγS binding: CBD showed no
measur-able activity, whereas rimonabant elicited strong inverse
agonism with a mean IMAX= −35.5% (Supporting Information
Appendix S2)
The inactivity of CBD in vitro is confirmed by in vivo
studies CBD does not elicit the classic CB1-mediated tetrad of
hypolocomotion, analgesia, catalepsy and hypothermia
(Long et al., 2010) In drug discrimination studies, CBD failed
to generalize with THC (Järbe et al., 1977; Zuardi et al., 1981)
and did not alter the discriminative stimulus effects of THC
(Vann et al., 2008) Its lack of cannabimimetic effects is well
known in humans (Hollister, 1973)
CBD exerts CB1receptor agonist-like activity in some in
vitro functional assays at high concentrations (>10 μM),
which can be reversed by CB1receptor inverse agonists, and is
absent in CB1 receptor knockouts This probably occurs by
CBD augmenting endocannabinoid tone Pooled rodent
studies show moderate inhibition of FAAH and the putative
AEA transporter CBD may also augment endocannabinoid
tone by activating PLA2, thus mobilizing AA – the feedstock
for AEA and 2-AG synthesis
On the contrary, CBD inhibits adenosine uptake, thereby
acting as an indirect agonist at A2Areceptors Agonism of these
receptors in post-synaptic cells prevents glutamate mGlu5
receptor-mediated release of AEA and 2-AG through A2A/
mGlu5heteromers (Lerner et al., 2010) These opposing effects
– CBD augmenting endocannabinoid tone and augmenting
adenosine tone – often strike a balance in favour of the
former: Robust rodent studies indicate that CBD augments
endocannabinoid tone (e.g Campos et al., 2013) One clinical
study showed that AEA levels increased in schizophrenic
patients given CBD 800 mg·day−1(Leweke et al., 2012) Future
in vitro studies of CBD affinity and efficacy in the presence or
absence of PMSF or MAFP would be instructive (these areFAAH inhibitors that prevent the synthesis of AEA) Knockingdown tonic adenosine A2Areceptor signalling may also shed
light on this complex situation (Savinainen et al., 2003) Our second paradoxical finding is the in vitro capacity of
CBD to ‘functionally antagonize’ cannabinoid-induced ity at CB1 receptors However, the KB of CBD (88.5 ±18.46 nM) is far higher (implying less potency) than that of
activ-rimonabant (mean KBof 0.6± 0.41 nM when tested in parallel
in the same studies; Supporting Information Appendix S2)
Indeed, CBD in vivo mostly behaved in a different way from
that of rimonabant For example, CBD produced no effect onfood intake, energy expenditure or insulin sensitivity in obese
mice (Cawthorne et al., 2008) Its anxiolytic, antidepressant
and anti-nausea effects are all opposite to those reported forrimonabant (Pertwee, 2008) (Box 2)
Non-CB1 receptor mechanisms of CBD antagonism of THC
Earlier we proposed that the functional antagonism of THC
by CBD may be mediated by a non-CB1receptor mechanism
of action Pharmacodynamic antagonism arises when onedrug diminishes the effect of another drug by targeting dif-ferent receptors or enzymes For example, dry mouth caused
by a sympathomimetic drug is antagonized by a cholinergicdrug
We propose several non-CB1 receptor mechanisms ofaction:
1 CBD augments AEA levels Both AEA and CBD have ity for TRPV1 channels and signal as agonists (Table 1)
affin-Pre-synaptic TRPV1 channel activation enhances glutamate
release in the spinal cord and brain This may counteract
or antagonize the inhibitory action of pre-synaptic CB1
receptors co-localizing on glutamatergic neurons (Camposand Guimaraes, 2009; Di Marzo, 2010)
2 CBD inhibits adenosine uptake and therefore acts as anindirect agonist at adenosine receptors Functionally CBDdoes the opposite of caffeine, an adenosine receptor
antagonist (El Yacoubi et al., 2000) There are four
adeno-sine receptor subtypes; cannabinoid research has focused
on A2Areceptors (Table 1) Pre-synaptic A2Areceptors formheteromers with CB1receptors on glutamatergic neurons,and A2Areceptor agonism inhibits CB1receptor-mediated
effects in rat striatum (Martire et al., 2011) Post-synaptic
A2Areceptor signalling is a different story, as we mentionedearlier On the contrary, some effects of CBD are reversed
by the A1 receptor-selective antagonist DPCPX (Castillo
et al., 2010; Maione et al., 2011) Possible indirect agonism
of A1receptors would place CBD in the company of zodiazepines, which target A1, as well as GABAA, receptorsand, like benzodiazepines, might also strengthen, and notonly oppose, CBD indirect activation of CB1receptors in a
ben-tissue-specific manner (see Maione et al., 2011 for an
Trang 25catalepsy via 5-HT1A receptors (Gomes et al., 2013) It is
tempting to attribute this again to CBD indirect agonism
via AEA – Palazzo et al (2006) demonstrated that sciatic
nerve chronic constriction injury (CCI) elevates AEA in
the dorsal raphe, resulting in 5-HT-mediated hyperactivity
The study also showed that CCI increased extracellular
5-HT levels, a mechanism likely to be shared by CBD
administration, which suppresses enzymatic depletion of
tryptophan, the precursor of 5-HT (Supporting
Informa-tion Appendix S2)
Non-CB1 receptor mechanisms of CBD
potentiation of THC
CBD potentiates some effects of THC in an additive or
syn-ergistic fashion Williamson (2001) has reviewed the
math-ematical definitions of synergy Wilkinson et al (2005)
provided examples of synergy within polypharmaceutical
cannabis extracts CBD may potentiate the behavioural
effects of THC via pharmacokinetic mechanisms, for
example, increasing the area under the curve of THC in blood
and brain (Klein et al., 2011) Pharmacodynamic interactions
arise when CBD and THC act at separate but interrelated
receptor sites Landmark studies demonstrating the
potentia-tion of THC by CBD or by CBD-rich cannabis extracts are
summarized in Box 1 and Box 3
We propose eight non-CB1mechanism of action by which
CBD may potentiate THC:
1 CBD inhibition of FAAH and consequential increase in
AEA may synergize with THC CB1 receptor agonism in
peripheral injury: AEA suppresses pain through a
periph-eral mechanism (Clapper et al., 2010), whereas THC works
through central CB1receptor-mediated mechanisms CBD,like cannabichromene, another cannabinoid capable ofinhibiting the putative AEA transporter, when injectedinto the periaqueductal grey area increased 2-AG levels by2.6-fold and elicited antinociception in the tail-flick test.The antinociceptive effect was blunted by antagonists of
CB1, adenosine A1 receptors and TRPA1, but not TRPV1
channels (Maione et al., 2011).
2 CBD reduces peripheral hyperalgesia via TRPV1 channels
(Costa et al., 2004) This peripheral mechanism may
hypo-thetically potentiate THC central mechanisms via CB1.Sativex provided better antinociception than THC givenalone, and the difference was likely to be mediated by
TRPV1 channels (Comelli et al., 2008).
3 CBD reduces inflammation and inflammatory cytokines(e.g TNF-α, IL-6, IL-1β) through TRPV1-, A2A- and PPARγ-mediated mechanisms (Supporting Information Appen-dix S2) THC reduces inflammation through separate CB1-and CB2receptor-mediated mechanisms
4 CBD acts as a positive allosteric modulator of glycinereceptors, which contributes to cannabis-induced analge-
sia (Xiong et al., 2011).
5 CBD is an antioxidant and ROS scavenger, more potentthan ascorbate or α-tocopherol – thus, it controls free
radical-associated diseases (Hampson et al., 1998) The U.S.
Department of Health patented this discovery (U.S Patent
No 6630507), despite its classification of cannabis as
a Schedule I substance having ‘no currently accepted
Box 2 Landmark 21st century in vivo studies of CBD functional antagonism of THC.
Animal studies
CBD antagonized THC-induced spatial memory – Fadda et al., 2004.
CBD reversed THC-induced conditioned place aversion – Vann et al., 2008.
CBD reversed THC-induced decrease in social interaction – Malone et al., 2009.
CBD increased hippocampal cell survival and neurogenesis, whereas THC has the opposite effect; the CBD response is absent in CB1 −/−
knockout mice – Wolf et al., 2010.
Human clinical trials and epidemiology studies
CBD reduced THC intoxication and impairment in binocular depth perception (a model of psychosis) – Leweke et al., 2000.
Sativexacompared to THC alone reduced adverse effects in patients with multiple sclerosis – Wade et al., 2003; Zijicek et al., 2003.
CBD counteracted THC somnolence and morning-after memory deficits – Nicholson et al., 2004.
High-THC cannabis with higher dose of CBD caused less anxiety than high-THC cannabis with lower dose of CBD – Ilan et al., 2005.
No difference in appetite and quality of life (QOL) scores between cannabis extract and THC alone – Strasser et al., 2006.
Increased CBD-to-THC ratios in chronic cannabis users inversely correlated with expression of psychotic symptoms – Morgan and Curran,2008
CBD reduced anxiety, skin conductance response and amygdala activity – the opposite of THC effects – Fusar-Poli et al., 2009.
CBD reduced ‘psychotic scores’ of THC – Bhattacharyya et al., 2010.
CBD attenuated the appetitive effects of THC – Morgan et al., 2010a.
Increased CBD-to-THC ratios in chronic cannabis users correlated with a reduction of cognitive and memory deficits – Morgan et al., 2010a Increased CBD-to-THC ratios in chronic cannabis users inversely correlated with liking for drug-related stimuli including food – Morgan et al.,
2010b
Increased CBD-to-THC ratio is associated with lower degrees of negative psychiatric symptoms – Schubart et al., 2011.
No difference in side effect profile between cannabis extract and THC alone – Karschner et al., 2011b.
Increased CBD-to-THC ratios in chronic cannabis users inversely correlated with volume loss in the hippocampus – Demirakca et al., 2011 CBD inhibited THC-elicited paranoid symptoms and hippocampal-dependent memory impairment – Englund et al., 2013.
aSativex®contains THC and CBD in a 1:1 ratio, with minor cannabinoids (5–6%), terpenoids (6–7%), sterols (6%), triglycerides, alkanes,squalene, tocopherol, carotenoids and other minor components derived from the plant material (Guy and Stott,2005)
Trang 26medical use.’ Antioxidants limit neurological damage
fol-lowing stroke, ethanol poisoning or trauma, as well as
animal models of multiple sclerosis, Alzheimer’s,
Parkin-son’s and Huntington’s disease (Supporting Information
Appendix S2)
6 CBD suppression of ROS, TNF-α and IL-1β predictably
reduces NF-κB, which is induced by these stimuli
(Support-ing Information Appendix S2) Elevation of NF-κB occurs
in many inflammatory diseases, such as arthritis,
inflam-matory bowel disease, gastritis, asthma, atherosclerosis
and possibly schizophrenia THC may dampen NF-κB
through a CB2-mediated mechanism (Jeon et al., 1996).
THC-mediated mechanisms may diverge from
et al., 2010) CBD suppression of ROS, pro-inflammatory
cytokines and NF-κB also predictably reduces iNOS, and
consequent NO and peroxynitrite formation (Supporting
Information Appendix S2) Inhibiting iNOS helps control
chronic neurological diseases as well as cardiovascular
disease THC probably reduces iNOS through a different
mechanism (Jeon et al., 1996).
7 CBD modulation of cytosolic Ca2+levels via several
mecha-nisms (voltage-gated Ca2 + channels, mitochondrial Na+/
Ca2+ exchange, adenosine A2A and 5-HT1A receptor
agonism) is likely to contribute to its anticonvulsant
ben-efits, particularly for partial or generalized seizures (Jones
et al., 2012).
8 CBD inhibits cancer growth and induces apoptosis CBD
generates ROS and up-regulates caspase proteases
(Sup-porting Information Appendix S2) The capacity of CBD
to selectively generate ROS in cancer cells likely works
through superoxide-generating NADPH oxidases or by
inducing endoplasmic reticulum and mitochondrial stress
THC inhibition of cancer works through CB1- and CB2
receptor-mediated MAPK/ERK pathways and ceramide
accumulation Combining these mechanisms by using
THC and CBD together, produces synergistic inhibition of
cancer cell growth and apoptosis (Marcu et al., 2010).
THCV at CB1 receptors
As discussed previously, meta-analysis indicates that THCV
acts as a neutral CB1and CB2receptor antagonist, at least in
most in vitro studies Indeed, evidence has also emerged that
THCV can block CB1 receptors in vivo, as indicated by its
ability to oppose (i) anti-nociception induced by THC in themouse tail-flick test and by CP55,940 in the rat hot plate test;(ii) hypothermia induced in mice by THC; and (iii) CP55,940-
induced inhibition of rat locomotor activity (Pertwee et al., 2007; García et al., 2011).
Results from other in vivo experiments have shown that
THCV can suppress food consumption and body weight in
non-fasted mice, like AM251 (Riedel et al., 2009), and like
rimonabant, THCV can reduce signs of motor inhibition in
rats caused by 6-hydroxydopamine (García et al., 2011) It
remains to be established whether THCV produced theseeffects through CB1receptor inverse agonism or because it wascompetitively antagonizing CB1receptor-mediated effects ofone or more endogenously released endocannabinoids.However, it is noteworthy that, like the establishedneutral CB1 receptor antagonists, THCV does not share theability (i) of SR141716A or AM251 to produce signs of nausea
in rats (Rock et al., 2013) or (ii) of SR141716A to produce an
anxiogenic-like reaction in the rat light–dark immersionmodel of anxiety-like behaviour, or a suppression of saccha-rin hedonic reactions of rats in the taste reactivity test of
palatability processing (O’Brien et al., 2013) Furthermore, in some in vivo settings where neutral antagonists produce
effects, THCV does not For example, THCV did not reducefood intake and body weight in obese mice, even though itdid improve insulin resistance in these animals (Wargent
et al., 2013) Furthermore, THCV did not reduce food
deprivation-induced food intake in mice (Izzo et al., 2013).
THCV has anti-epileptiform actions in the rat
pentylene-tetrazole seizure model (Hill et al., 2010) This is in contrast to
rimonabant safety data, where seizures were occasionally
observed (Bermudez-Silva et al., 2010) Rimonabant
signifi-cantly increased seizure duration and seizure frequency in the
rat pilocarpine model of epilepsy (Wallace et al., 2003)
Pro-convulsant effects were also observed with AM251 in a model
of generalized seizures (Shafaroodi et al., 2004).
Also, when given in vivo at doses well above those at
which it can block CB1receptors, THCV produces signs of CB1
receptor activation For example, in experiments performedwith mice, THCV has been shown to induce (i) immobility in
Box 3 Landmark 21st century studies of CBD potentiating the effects of THC.
Animal studies
CBD potentiated THC antinociception – Varvel et al., 2006.
CBD enhanced THC tetrad effects – Hayakawa et al., 2008.
CBD turned an ineffective THC dose into an effective one in colitis – Jamontt et al., 2010.
CBD altered THC pharmacokinetics and augments some THC behavioural effects – Klein et al., 2011.
Sativex compared to THC alone enhanced antinociception in a rat model of neuropathic pain – Comelli et al., 2008.
Human clinical trials and epidemiology studies
CBD plus THC imparted synergistic inhibition of human glioblastoma cancer cell growth and apoptosis – Marcu et al., 2010.
Sativexacompared to THC alone provides greater pain relief and improvement in sleep – Notcutt et al., 2004.
Sativexacompared to THC extract reduced cancer-related pain – Johnson et al., 2010.
Sativexacompared to THC alone reduced abnormalities in psychomotor performance associated with schizophrenia – Roser et al., 2009.
aSativex®contains THC and CBD in a 1:1 ratio, with minor cannabinoids (5–6%), terpenoids (6–7%), sterols (6%), triglycerides, alkanes,squalene, tocopherol, carotenoids and other minor components derived from the plant material (Guy and Stott, 2005)
Trang 27the Pertwee ring test, albeit with a potency 4.8 times less than
that of THC, and (ii) anti-nociception in the tail-flick test
(Gill et al., 1970; Pertwee et al., 2007) Importantly, although
SR141716A was found to antagonize the antinociceptive
effect of THCV, it did not give rise to any significant
antago-nism of THCV-induced hypothermia or ring immobility
(Pertwee et al., 2007) THCV also decreased pain behaviour in
the formalin test; this effect appeared to be CB1 and CB2
receptor-mediated (Bolognini et al., 2010) It remains to be
established how THCV produces apparent CB1receptor
acti-vation in vivo but not in vitro at doses above those at which it
can block CB1receptors both in vivo and in vitro One
possi-bility is the apossi-bility of THCV to inhibit endocannabinoid
re-uptake (see previous discussion), with indirect activation
of cannabinoid receptors Models of intestinal transit are
often used as assays of CB1 receptor activation and THCV
inhibits upper intestinal transit in a dose-dependent manner
(Izzo et al., 2013) However, this effect was not antagonized
by a dose of rimonabant inactive per se This study, together
with the hypothermia and immobility assay, indicates that
THCV can have cannabimimetic effects, which appear to be
independent of the CB1receptor
The ‘abants’ were withdrawn from the market because of
their worsening of anxiety and depression in obese patients
In contrast, THCV has very little effect in animal models of
anxiety and depression For example, the dose of THCV
required to impart anxiogenic effects in the elevated plus
maze is 30-fold higher than that of rimonabant (Izzo et al.,
2013)
The observed similarities or differences between THCV and
rimonabant (Box 4) may depend upon several factors These
include (i) the initial physiopathological status of the cell/
tissue/organ, and the degree of expression therein of
func-tional CB1 receptors, which might affect the activity of
different ligands of the same receptor in different manners; (ii)
the presence or absence in the cell/tissue/organ of non-CB1,
non-CB2receptor targets for THCV – in particular, TRP
chan-nels and CB2receptors, particularly when THCV is
adminis-tered at doses higher than those required for CB1 receptor
antagonism, might counteract some of the CB1 receptor
antagonism-mediated effects of THCV, but not others; (iii) the
fact that at higher doses, THCV might start behaving as a CB1
receptor agonist, thus clearly counteracting some of the effects
due to CB1receptor neutral antagonism; (iv) different
pharma-cokinetic properties of the two compounds, particularly, but
not limited to, under pathological conditions that may altertheir tissue distribution and catabolism
Conclusions
Based upon the meta-analysis of in vitro data, and in vivo data
cited in the Discussion section, we conclude that the cology of neither CBD nor THCV has much in common withthat of the ‘abants’ and of rimonabant in particular WhileCBD does counteract some of the actions of THC, particularly
pharma-in the brapharma-in, it potentiates other actions of THC The capacity
of CBD to modulate several signalling systems and to enhanceendocannabinoid levels might produce varying effects on theendocannabinoid system and CB1 receptors This may beorgan/tissue/cell-dependent, and unlikely due to directmolecular interactions with CB1receptors
Although THCV undoubtedly behaves as a CB1(and CB2)receptor orthosteric ligand in binding assays, and as a neutral
CB1 receptor antagonist in functional assays, its
pharmaco-logical profile in vivo overlaps only in part with that of
rimonabant and other CB1receptor inverse agonists, or evenwith that of synthetic CB1receptor neutral antagonists.Thus, CBD and THCV represent the two opposing ends ofactivity at CB1receptors: a low-affinity CB1 receptor ligand,which can affect CB1 receptor activity in vivo in an indirect
manner, and a high-affinity CB1receptor ligand and potent
antagonist in vitro, which instead produces CB1 receptor
antagonism-mediated effects in vivo only in a few instances These paradoxical examples of how in vivo pharmacology is not always predicted from in vitro mechanistic studies under- lie the necessity of testing compounds in vivo before drawing
any conclusion on their functional activity at a given target.This is especially true regarding compounds that havecomplex pharmacology, such as the phytocannabinoids.However, it must be remembered that whether or not
in vitro mechanistic studies predict in vivo pharmacology
depends, in part, upon pharmacokinetic data – particularly
on the ability of CBD and THCV to reach concentrations in
plasma and tissues similar to the concentrations shown in
vitro to be necessary to interact with certain targets In mice,
the plasma Cmaxof THCV, given at a dose of 30 mg·kg−1eitherp.o or i.p., was 0.24 and 0.88 mg·L−1, and thus lower thanthat of a higher dose of CBD (120 mg·kg−1), which was 2.2and 14.3 mg·L−1(Deiana et al., 2012) A similar scenario was
Box 4 Landmark 21st century in vivo studies concerning THCV.
THCV induced anti-nociception in the tail-flick test – Pertwee et al., 2007.
THCV suppressed food consumption and body weight in non-fasted mice – Riedel et al., 2009.
THCV antagonized the antinociceptive effects of THC in the rat acetic acid model of visceral nociception – Booker et al., 2009.
THCV exerted anticonvulsant actions in the PTZ model of seizure – Hill et al., 2010.
THCV reduced pain behaviour in the formalin test – Bolognini et al., 2010.
THCV imparted neuroprotective effects and relieves symptoms in rat models of Parkinson’s disease – García et al., 2011.
THCV did not produce nausea or a suppression of saccharin hedonic reactions of rats in the taste reactivity test of palatability processing – Rock
et al., 2013.
THCV did not reduce food intake and body weight in obese mice – Wargent et al., 2013.
THCV did not produce an anxiogenic-like reaction in the rat light–dark immersion model of anxiety-like behaviour – O’Brien et al., 2013.
Trang 28described by the same authors also for the brain, although in
this case the difference between THCV and CBD was less
striking, with the Cmax of the former being 0.43 and
1.69μm·g−1and that of CBD being 1.3 and 6.9μm·g−1
In rats, two different vehicles were compared for CBD
(120 mg·kg−1), and the plasma Cmax values were 2 and
2.6 mg·L−1after p.o and i.p administration, respectively, for
cremophor, and 3.2 and 2.4 mg·L−1 for solutol Brain Cmax
values were 8.6 and 6.8μm·g−1after p.o and i.p
administra-tion, respectively, for cremophor, and 12.6 and 5.2μm·g−1for
solutol Rat pharmacokinetic values for THCV (30 mg·kg−1)
were Cmax0.21 and 0.4 mg mL−1for p.o and i.p
administra-tion, respectively, in plasma, and 0.3 and 1.62μm·g−1for oral
and i.p administration, respectively, in the brain Thus, there
are only small differences between the two species, for the
two compounds
No human studies have been published regarding THCV
pharmacokinetics Human subjects given Sativex as a buccal
spray (THC 16.2 mg and CBD 15.0 mg) averaged a CBD Cmax
plasma concentration of 6.7μg·L−1, range 2.0–20.5μg·L−1
(Karschner et al., 2011a) The metabolism of THC
adminis-tered as Sativex was slightly aladminis-tered, as they observed lower
11-OH-THC Cmaxafter Sativex in relation to 15 mg p.o THC
(falling short of significance, P= 0.09) Subjects given eightbuccal sprays of Sativex per day (= 20 mg CBD daily) over 9days averaged CBD plasma concentration of 3.2 ng·mL−1
(Stott et al., 2013).
Thus, given these low Cmaxvalues, many in vitro studies
reporting effects in the micromolar range, especially for CBD,might be regarded as irrelevant Yet, in the clinic, both CBDand THCV, like rimonabant, are likely to be administeredchronically and their tissue concentrations are therefore likely
to accumulate Furthermore, it must be emphasized that a per comparison between the pharmacokinetics and bioavail-ability of CBD or THCV with those of rimonabant and other
pro-‘abants’ cannot be made as data for the latter are scant andmostly unpublished possibly because these compounds arestill proprietary and their development has been interrupted
Figure 2
Algorithm for investigating cannabinoids in mechanistic studies Dashed arrow: Note that in some cases compounds that do not enhance the
binding of high affinity ligands to their receptors might still be allosteric modulators (May et al., 2007).
Trang 29At any rate, much of the confusion about CBD as a
‘rimonabant-like drug’ might have been avoided if one had
followed the algorithm shown in Figure 2 to decide whether,
based upon in vitro pharmacological data, a substance can be
described as a CB1receptor orthosteric ligand first and then as
agonist, inverse agonist, neutral antagonist or functionally
inactive (Figure 2) Indeed, based upon this algorithm, one
might even question the necessity of testing compounds in
functional assays of a given receptor in vitro if they do not
exhibit high affinity in radioligand displacement assays for
that receptor An exception to this rule might be allosteric
ligands, which cannot always be identified by the binding
assays normally used to evaluate the affinity of compounds
for GPCRs (May et al., 2007).
Our results may have important effects on the future
clinical development of CBD as an antipsychotic,
anti-inflammatory and anti-epileptic drug, and of THCV for the
treatment of type 2 diabetes Meta-analysis suggests that
these two phytocannabinoids are very unlikely to produce
the central adverse events typical of THC, as well as the
central adverse events of the ‘abants’
Acknowledgements
This work was partially supported by an unrestricted grant
from GW Pharmaceuticals, Salisbury, UK We thank all the
scientists who responded to our queries regarding their
unpublished results
Conflict of interest
M D is an employee of GW Pharmaceuticals, UK J M M., V
D and R P are consultants for, and receive research funds
from, GW Pharmaceuticals, UK
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Supporting information
Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:http://dx.doi.org/10.1111/bph.12944
Appendix S1 PRISMA 2009 Checklist and extendedmethods section for meta-analysis
Appendix S2The CBD data extraction table, followed bysubgroup meta-regression tests
Appendix S3The THCV data extraction table, followed bysubgroup meta-regression tests
Trang 34computer modelling to
biological effects
Shirin Kahremany1, Ariela Livne2, Arie Gruzman1, Hanoch Senderowitz1
and Shlomo Sasson2
1Division of Medicinal Chemistry, Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan
University, Ramat-Gan, Israel, and2Department of Pharmacology, Institute for Drug Research,
School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
Correspondence
Professor Shlomo Sasson,Department of Pharmacology,Institute for Drug Research,School of Pharmacy, Faculty ofMedicine, The Hebrew University
of Jerusalem, Faculty ofMedicine, Jerusalem 91120,Israel E-mail:
shlomo.sasson@mail.huji.ac.il;Professor Hanoch Senderowitz,Division of Medicinal Chemistry,Department of Chemistry,Faculty of Exact Sciences, Bar-IlanUniversity, Ramat-Gan 52900,Israel E-mail:
dimers interact with other co-activator proteins and form active complexes that bind to PPAR response elements and promotetranscription of genes involved in lipid metabolism It appears that various natural fatty acids and their metabolites serve as
ligand binding domain (LBD) of the receptor Here, we introduce an original computational prediction model for ligand
calculating binding probabilities of 82 different natural and synthetic compounds from the literature These compounds were
accuracy of between 70% and 93% (depending on ligand type) This new computational tool could therefore be used in thesearch for natural and synthetic agonists of the receptor
Abbreviations
ALPHA, amplified luminescent proximity homogeneous assay; CARLA, co-activator-dependent receptor ligand assay;DBD, DNA binding domain; LBD, ligand binding domain; LIC, ligand-induced complex formation assay; MUFA,monounsaturated fatty acids; NTD, N-terminal domain; PPRE, PPAR response elements; PUFA, polyunsaturated fattyacids; RMSD, root mean squared deviation; RXR (also known as NR2B receptors), retinoid X receptor; SFA, saturatedfatty acids; SP, scintillation proximity competition assay; TR-FRET, time resolved-fluorescence resonance energy transfer
Trang 35The PPAR family
PPARs are ligand-activated transcription factors of the nuclear
hormone receptor superfamily Peroxisome proliferation in
rat liver treated with the anti-hyperlipidaemic compound
clofibrate was reported over 40 years ago (de Duve, 1969)
Subsequently, several other compounds with similar effects
were identified and were collectively termed peroxisome
pro-liferators Issemann and Green cloned the first member of the
family from rat liver, and in 1990 coined the term
‘peroxi-some proliferator-activated receptor’ (Issemann and Green,
1990) Two other members of the family were identified in
1992 and the group now consists of three major subtypes:
PPARα, PPARδ and PPARγ (Dreyer et al., 1993) These receptors
regulate major metabolic pathways, including carbohydrate
utilization, fatty acid oxidation and lipogenesis (Wagner and
Wagner, 2010) They form heterodimers with members of the
retinoic acid receptor (RXR) family and subsequently interact
in a stereospecific manner with PPAR response elements
(PPRE) in DNA to assemble active transcriptional complexes
(Wahli and Michalik, 2012) PPRE sequences are composed of
double hexameric motifs, separated by a short spacer
sequence and organized in a direct, inverted or everted
manner (Kumar and Thompson, 1999) PPARα is
predomi-nantly expressed in the liver and primarily regulates lipid
metabolism (Pyper et al., 2010) PPARγ is mostly expressed in
adipose tissues and controls adipogenesis and carbohydrate
metabolism (Astapova and Leff, 2012)
Roles of PPARδ in the regulation of glucose
and lipid metabolism
The role of the ubiquitously expressed PPARδ in the
regula-tion of physiological and pathological processes in different
tissues have been intensely investigated (Coll et al., 2009;
Ehrenborg and Krook, 2009; Lee et al., 2009; Wagner and
Wagner, 2010; Wolf, 2010; Ehrenborg and Skogsberg, 2013;
Skerrett et al., 2014) The regulation of lipid and glucose
metabolism is considered a major function of PPARδ (Coll
et al., 2009) For instance, it orchestrates the expression of
genes involved in lipid metabolism in mature adipocytes(Wolf, 2010) Other studies have shown that PPARδ activationimproves the plasma lipid profile in humans and in primates
(Oliver et al., 2001; Wallace et al., 2005; Sprecher et al., 2007; Riserus et al., 2008; Thulin et al., 2008) and significantly
decreases high fat diet-induced obesity in rodents (Tanaka
et al., 2003) In addition, PPARδ-null mice exhibited lower
accumulation of lipids in adipose tissue stores (Peters et al., 2000; Sprecher et al., 2007) The specific PPARδ agonistGW501516 increases blood high-density lipoprotein and
decreases triglyceride levels in rhesus monkeys (Oliver et al., 2001) and preventes obesity in mice (Wang et al., 2003).
Furthermore, PPARδ activation reduces intestinal cholesterol
absorption in mice (van der Veen et al., 2005).
PPARδ also plays an important role in the regulation ofperipheral insulin sensitivity and attenuates symptoms of the
metabolic syndrome (Tanaka et al., 2003; Lee et al., 2006; Riserus et al., 2008) Decreased lipid accumulation in skeletal
muscles following PPARδ activation along with proliferation
of mitochondria and a consistent increase in fatty acids dation in skeletal muscles were also linked to PPARδ-
oxi-dependent amelioration of insulin resistance (Dressel et al.,
2003; Ehrenborg and Skogsberg, 2013) Similar beneficialeffects of PPARδ activation were observed in insulin-resistant
obese rhesus monkeys (Oliver et al., 2001) Others showed
that PPARδ stimulation improved glucose tolerance, loweredpostprandial levels of plasma insulin and glucose, reducedhepatic glucose output by increasing glycolysis and thepentose phosphate shunt and augmented fatty acids synthe-
sis and triglycerides content in the liver (Tanaka et al., 2003; Lee et al., 2006; Chen et al., 2008) Conversely, PPARδ-null
mice were glucose intolerant and exhibited a low metabolic
rate (Lee et al., 2006).
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).
Trang 36Protective role of PPARδ in the development
of atherosclerosis
Active PPARδ may also prevent or delay the development of
atherosclerosis For example, treatment with the PPARδ
agonist L-165041 resulted in reduced monocyte recruitment
to human endothelial cells by reducing the expression of
vascular cell adhesion molecule-1, the secretion of monocyte
chemotactic protein-1 and of the inflammatory cytokines
IL-6 and IL-8 (Rival et al., 2002; Jiang et al., 2009; Liang et al.,
2010) Furthermore, activation of PPARδ by the selective
ago-nists GW0742 and GW501516 augmented the expression of
antioxidant genes, (e.g superoxide dismutase-1, catalase and
thioredoxin), and attenuated the generation of reactive
oxygen species in vascular endothelial cells (Fan et al., 2008).
Others suggested that activated PPARδ augmented cholesterol
efflux from macrophages in atherosclerotic lesions and
thereby decreased transendothelial migration of leucocyte/
monocytes into the arterial wall (For review see Barish et al.,
2008; Piqueras et al., 2009).
PPAR δ protects against pathophysiological
processes in the nervous system
The findings that PPARδ-deficient mice exhibited abnormal
neurophysiological processes, such as decreased myelination,
augmented inflammatory reactions and low score in memory
tests, suggest a critical role for PPARδ in neuronal
develop-ment and function (Peters et al., 2000) Interestingly, CNS
inflammation has been associated with increased level of
inflammatory markers, astrogliosis andτ
hyperphosphoryla-tion (Barroso et al., 2013) Moreover, it was the lack of PPARδ
function in the brain has been linked to increased
vulnerabil-ity to ischaemic insults because of defective antioxidant
responses (Arsenijevic et al., 2006; Pialat et al., 2007)
Addi-tional data, generated from effects of selective PPARδ agonists
in the brain, suggest that PPARδ activation could protect
against neurodegenerative processes (Polak et al., 2005;
Iwashita et al., 2007; Kalinin et al., 2009; Paterniti et al., 2010;
Yin et al., 2010; Martin et al., 2013).
Roles of PPARδ in embryonic, organ and
tissue development
Attempts to generate PPARδ knockout mouse models were
difficult because of high rates of embryo lethality (Michalik
et al., 2001; Barak et al., 2002) Yet, these models revealed
important roles of PPARδ in blastocyst hatching, embryo
implantation, myelination, lipid metabolism and adiposity
and epidermal cell proliferation (Lim et al., 1999; Peters et al.,
2000; Barak et al., 2002; Huang et al., 2007) Furthermore,
PPARδ seemed also involved in morphological adaptive
dif-ferentiation of various tissues, such as skeletal muscles
(Ehrenborg and Krook, 2009), and the development of
oxi-dative type I fibres (Luquet et al., 2003; Wang et al., 2004).
Recent studies suggest that PPARδ regulates cell growth (Lee
et al., 2009): for instance, it increased the number and size of
intestinal polyps and stimulated proliferation of vascular
smooth muscle cells, pre-adipocytes and epithelial cells
(Jehl-Pietri et al., 2000; Hansen et al., 2001; Zhang et al., 2002;
Gupta et al., 2004; Burdick et al., 2006) Importantly, several
PPARδ synthetic agonists exhibited carcinogenic potential
(Ehrenborg and Skogsberg, 2013)
In conclusion, because PPARδ regulates myriad cell andorgan functions, it has become a desirable target for drugdiscovery Development of selective PPARδ agonists for thetreatment and prevention of symptoms of the metabolic syn-drome attracts attention and is highly sought Similarly,PPARδ-selective agonists with enhanced neuroprotective andanti-atherosclerotic properties are of a great interest Yet thecarcinogenic potential of PPARδ agonists should be fullyinvestigated in order to create selective agonists devoid of thisproperty Therefore, PPARδ structural research, detailed analy-sis of ligand binding to the receptor and its activation on thebasis of comprehensive structure activity relationship analy-sis are required to reach these goals
The structure of PPAR δ
Generally, PPARs are organized in four functional domains:the N-terminal domain (NTD), the DNA binding domain(DBD), which includes two zinc fingers, the hinge domain
and the ligand binding domain (LBD) (Schmidt et al., 1992).
The NTD is the most varied domain among the differentPPARs (Helsen and Claessens, 2014) This domain, which isrelatively short, is believed to mediate ligand-independentactivity by promoting protein–protein interactions withco-activators or co-suppressors and by inducing conforma-tional modifications that allow allosteric interactions
(Zieleniak et al., 2008; Helsen and Claessens, 2014)
Interest-ingly, the NTD has not yet been resolved in the full lengthcrystal structure of PPARδ and its precise regulatory interac-tions remain to be resolved The DBD is the most conserveddomain among the various PPARs Two zinc fingers form thefunctional core structure, where theα-helix in the first zincfinger promotes the recognition of specific sequence in thePPRE in the DNA The second zinc finger mediates the het-rodimerization with RXR (Helsen and Claessens, 2014) Otherareas in the DBD further stabilize the DNA-PPAR complex bydirect interactions with the minor groove of the DNA and/oralternatively by stabilizing the interactions with other
partner in the dimer complex (Hsu et al., 1998; Shaffer and Gewirth, 2002; Roemer et al., 2006) The hinge domain,
which separates the DBD from the LBD in all PPARs, contains
a nuclear localization signal and amino acid side chains
ame-nable to post-translational modifications (Anbalagan et al., 2012; Clinckemalie et al., 2012).
The structure of the LBD is similar among the various
PPAR (Bourguet et al., 1995; Renaud et al., 1995): it consists of
a complex of 13 α-helices and four-stranded β-sheets thatform a cavity, which is markedly larger than similar cavities
in other members of the nuclear receptor superfamily TheY-like shaped LBD in PPARs is composed of 25 amino acids(Figure 1): arm-1 forms a ‘tunnel’ that transverses from thesurface of the protein to its internal part, while arm-2 andarm-3 form a cavity Arm-2 is considerably polar, whereasarm-1 and arm-3 are hydrophobic Over 80% of the aminoacid residues in the binding cavity are highly conservedamong the three PPAR isotypes, including four polar residues
in arm-2, which form critical hydrogen bonds with polarmoieties (predominantly carboxylic acids) of ligands Corre-spondingly, PPAR ligands are usually characterized by thepresence of a polar moiety in a predominantly non-polar
molecule (Zoete et al., 2007) Ligand binding selectivity to the
various PPARs is conferred by subtle modifications in the
Trang 37structure of their LBD (Batista et al., 2012; Carrieri et al.,
2013) It has been shown that a single amino acid mutation
in the LBD can dramatically change ligand recognition by the
receptor (Xu et al., 1999) The large volume of the binding
cavity allows several binding options (dynamic binding
equi-librium) of the lipophilic domain of ligands in the LBD, as
was documented for the interaction of eicosanoic acid with
the LBD PPARs (Xu et al., 2001) or for ragaglitazar with
PPARα/γ (Ebdrup et al., 2003) Interestingly, arm-1 forms a
very narrow tunnel in PPARδ LBD, which restricts the entry of
compounds with bulky moieties near the polar head This
structure eliminates the binding of various PPARsα/γ ligands
to the PPARδ LBD (Xu et al., 1999) This structure also
explains the relatively small number of known ligands to
PPARδ in comparison with the two other isotypes For
instance, the bulky acidic head group in thiazolidinediones
permits their binding interaction with PPARγ but not with
PPARδ Yet the relatively small phenoxyacetic acid moiety in
the PPARδ agonist GW501516 fits well the narrow entry
tunnel to the LBD (Oliver et al., 2001).
PPARδ activation
The current hypothesis of PPAR activation claims that the
binding of a ligand to the LBD triggers conformational
changes in the entire receptor Such changes that occur in
helix 12 of the LBD enable interactions of co-activator
mol-ecules (e.g steroid receptor co-activator-1) with the receptor
(Kallenberger et al., 2003) These conformational changes
also increase the strength of the binding interaction between
PPARs and their cognate partner RXR to form active dimers
that interact with PPRE in gene promoters (Okuno et al.,
2001) The fact that PPARs have significant constitutive ity in the total absence of ligands and co-activators indicatesthat the classical ligand-dependent activation of PPARs is not
activ-exclusive (Issemann and Green, 1990; Hallenbeck et al.,
1992)
This review focuses on ligand binding selectivity and sequent activity of PPARδ The human PPARδ is made of 441amino acids that are organized in the classical NTD (aa 1–70),DBD (aa 71–145), which includes two zinc fingers (aa 74–94and 111–133), the hinge domain (aa 146–254) and the LBD
sub-(aa 254–441) (Schmidt et al., 1992) The secondary structure
of the protein consists of 10β-strands and 15 α-helices While
a full crystal structure of PPARγ has been recently reported
(Chandra et al., 2008), most structural information on PPARδ
is restricted to analysis of LBD and hinge domain crystals
(Fyffe et al., 2006).
It has been shown that certain saturated, unsaturated and polyunsaturated fatty acids (SFA, MUFA andPUFA) and eicosanoids and prostaglandins activate PPARδ
mono-(Benetti et al., 2011) Recently, we have reported that
4-hydroxynonenal (4-HNE) and 4-hydroxydodecadienal(4-HDDE), the peroxidation products of PUFA, activate PPARδ
in cultured endothelial cells and pancreatic β-cells (Riahi
et al., 2010; Cohen et al., 2011b; 2013) The mechanism by
which these two 4-hydroxyalkenals activate PPARδ is unclear.These chemically reactive aldehydes avidly form covalentbonds with nucleophilic groups of amino acids (i.e histidine,lysine, arginine or cysteine) Yet it has been reported thatsteric hindrance prevents 4-HNE covalent interaction withhistidine residues in the LBD of PPARδ (Coleman et al., 2007)
1997), scintillation proximity competition assay (SP) (Xu
et al., 1999), time resolved-fluorescence resonance energy
transfer (TR-FRET) (Naruhn et al., 2010) and amplified nescent proximity homogeneous assay (ALPHA) (Jin et al.,
lumi-2011)
CARLA reflects molecular consequences of ligand binding
to the receptor and is based on the principle that binding ofligands to a nuclear receptor increases the binding affinity of
a labelled ‘broad spectrum’ co-activator protein [35S]-SRC1 tothe receptor complex Principally, CARLA detects productivephysical interactions between two proteins in the presence of
a ligand and is not aimed at determining the association of
ligands with the LBD per se LIC is based on the observation
that ligand-activated nuclear receptors bind to response ments in DNA as dimers Thus, a gel shift mobility assay isemployed to detect the capacity of PPAR-RXR complexes withpotential ligands to interact with PPRE-containing DNAsequences The SP competition assay uses the changes in lightemission when PPARδ, immobilized on scintillant micro-scopic beads with radiolabelled ligand (e.g [3H]-GW2433),interacts with test compounds TR-FRET analysis is an inte-grated method that includes two fluorescent techniques: first,FRET interaction between two fluorophores, a donor and an
ele-Figure 1
PPARδ crystal structure with the cavity in the ligand binding site
(bkue surface) PDB code: 3GWX
Trang 38acceptor, which triggers energy transfer from the donor to the
acceptor Second, TRF (time resolved fluorescence) method
takes advantage of the long-lived fluorophores lanthanides,
which enables the elimination of non-specific signals Briefly,
Fluormone™ Pan-PPAR Green (Life Technologies, Carlsbad,
CA, USA) is mixed with test compounds, followed by the
addition of a mixture of the PPARδ-LBD and terbium anti-GST
(glutathione-S-transferase) antibody When the Fluormone
Pan-PPAR Green is bound to the receptor, energy transfer
from the terbium-labelled antibody to the tracer occurs and
resolved as a high TR-FRET ratio Competitive ligand binding
to PPARδ is detected by the test compound’s ability to
dis-place the tracer and a reduced FRET signal The ALPHA
bead-based assay resolves the interactions of a fluorophore donor
with its acceptor The donor bead contains a high
concentra-tion of photosensitizers, which converts ambient oxygen to a
more excited singlet state when excited at 680 nm This bead
is covered with an antibody against PPARδ The acceptor bead
is a chemiluminescer that is covered with the LBD of PPARδ
The binding interaction between the antibody and the LBD
across the two beads results in singlet-induced luminescence
PPARδ ligands interfere with the binding interaction between
the beads and lower the luminescence
Using the CARLA, Krey et al identified eicosatetraynoic
acid, eicosapentanoic acid, linoleic acid, linolenic acid,
8(S)-hydroxyeicosatetraenoic [8(S)HETE] acid and bezafibrate as
high-affinity ligands to the LBD of PPARδ (Krey et al., 1997)
Forman et al (1997) used LIC to compare the binding
inter-actions of ligands with PPARδ LBD with their ability to
trans-activate the PPREX3-TK-LUC vector in transfected cells Of
all compounds tested, the best binding affinities were
reported for carbaprostacyclin (cPGI, a synthetic analogue of
PGI2), followed by iloprost (another PGI2 analogue),
arachi-donic and linoleic acid Interestingly, the transactivation
capacity of these compounds in the cell-based assay was
dif-ferent: arachidonic acid > cPGI > iloprost > linoleic acid
Unlike the results of the above-mentioned CARLA assay,
8(S)-HETE exhibited insignificant binding interaction with
PPARδ LBD in the LIC assay and no activity in the cell-based
assay Also, eicosapentanoic acid, predicted to be an
activa-tor of PPARδ in the CARLA, lacked significant binding
inter-actions with the LBD in the LIC assay, while exhibiting a
marked transactivation capacity in the cell-based assay
Moreover, some compounds that interacted positively in the
activation assay (e.g PGA1, PGA2 or 15d-PGJ2) lacked a
sig-nificant binding interaction with the LBD Xu et al (1999)
also tested some of these putative PPARδ ligands in the SP
assay and ranked them as follows: arachidonic acid>
eicosa-pentanoic acid> linoleic acid > linolenic acid These
find-ings correspond well with some of the results of CARLA and
LIC assays Interestingly, the most potent fatty acid tested in
the SP assay was γ-linoleic acid Other potent ligands were
oleic acid, stearic acid, palmitic acid and palmitoleic acid
Using the TR-FRET assay, Naruhn et al (2010) found that
15-HETE was most potent in inducing the binding of a
co-activator-derived peptide to the PPARδ LBD in vitro,
fol-lowed by arachidonic acid and 15-HpETE Yet 8-HETE,
12-HETE and 12-HpETE had no significant interaction with
the LBD Using the ALPHA screen assay, Jin et al (2011)
confirmed that iloprost was bound to the LBD of PPARδ and
PPARα More studies utilizing other methods report that
some of the above-mentioned compounds and dins could interact with PPARδ (Yu et al., 1995) Coleman
prostaglan-et al (2007) identified several arachidonic and linoleic acid
metabolites (e.g 12/15-HpETE, 15-HETE) as potent tors of PPARδ Riahi et al (2010) and Cohen et al (2011b)introduced 4-hydroxyalkenals as another class of PPARδ acti-vators The peroxidation products of arachidonic acid,4-HDDE and 4-HNE, transactivated PPREX3-TK-LUC intransfected vascular endothelial cells and cultured pancreaticβ-cells
GW0742 and TIPP204 (Sznaidman et al., 2003), and amino
acid side chains in the LBD Hitherto, no crystal structure ofthe whole monomeric PPARδ protein or its heterodimer withRXR has been reported We decided to use the X-ray crystal-lographic data of the above-mentioned complexes with syn-
thetic ligands and create an in silico model that may predict
the degree of possible direct interactions of natural ligands(fatty acids and their metabolites) and other synthetic ligandswith the PPARδ LBD This model also tests the assumptionthat both large (e.g long fatty acid and their metabolites) andsmall (e.g hydroxyalkenals) ligands may enter and interactwithin the bulky cavity in the LBD
Our model was developed based on previously publishedcrystallographic data of 16 PPARδ LBD-ligand complexesand validated on a database of 82 compounds (SFA, MUFA,PUFA and some of their enzymatic and non-enzymaticmetabolites and several synthetic compounds; see Tables 2and 3) These were evaluated for their potential to bind toPPARδ LBD, using pharmacophore modelling and dockingsimulations Because the binding site of the protein con-tains two histidine residues (H323, H449) that are impor-tant for ligand binding, we also examined the effect of theprotonation states of these two residues on the ligandbinding modes The molecular modelling methods used for
the following in silico analysis are given in the Supporting
is in line with a previous study of PPARγ agonists (Iwata et al.,2001) This binding hypothesis was transformed into a phar-macophore model by superposing the 16 crystallographicligands in their complex (i.e bioactive) conformations Theresulting pharmacophore was further refined by adding
Trang 39Table 1
Activity data for the 16 crystallographic ligands of PPARδ bound to the ligand binding domain
Protein Data Bank code Ligand structure EC 50 (nM) Reference
explore.do?structureId=3d5f
Trang 40Table 1
Continued
Protein Data Bank code Ligand structure EC 50 (nM) Reference