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The endocannabinoid system is activated by the binding of natural arachidonic acid derivatives (endogenous cannabinoids or endocannabinoids) as lipophilic messengers to cannabinoid receptors CB1 and CB2. The endocannabinoid system comprises also many hydrolytic enzymes responsible for the endocannabinoids cleavage, such as FAAH and MAGL. These two enzymes are possible therapeutic targets for the development of new drugs as indirect cannabinoid agonists. Recently a new family of endocannabinoid modulators was discovered; the lead of this family is the nonapeptide hemopressin produced from enzymatic cleavage of the α-chain of hemoglobin and acting as negative allosteric modulator of CB1. Hemopressin shows several physiological effects, e.g. antinociception, hypophagy, and hypotension. It is still matter of debate whether this peptide, isolated from the brain of rats is a real neuromodulator of the endocannabinoid system. Recent evidence indicates that hemopressin could be a by-product formed by chemical degradation of a longer peptide RVD-hemopressin during the extraction from the brain homolysate. Indeed, RVD-hemopressin is more active than hemopressin in certain biological tests and may bind to the same subsite as Rimonabant, which is an inverse agonist for the CB1 receptor and a μ-opioid receptor antagonist. These findings have stimulated several studies to verify this hypothesis and to evaluate possible therapeutic applications of hemopressin, its peptidic derivatives and synthetic analogues, opening new perspectives to the development of novel cannabinoid drugs.
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Hemopressin Peptides as Modulators of the Endocannabinoid System and
their Potential Applications as Therapeutic Tools
G. Macedonioa, A. Stefanuccia, C. Maccallinia, S. Mirzaieb, E. Novellinoc and A. Mollicaa,*
aDipartimento di Farmacia, Universita` di Chieti-Pescara ‘‘G. d’Annunzio’’, Chieti, Italy; bDepartment of Biochemis-
try, Islamic Azad University, Sanandaj, Iran; cDipartimento di Farmacia, Universita` di Napoli ‘‘Federico II’’, Naples,
Received: September 5, 2016
Revised: September 29, 2016
Accepted: October 1, 2016
DOI: 10.2174/0929866523666161007
Abstract: The endocannabinoid system (ECS) is activated when natural arachi-
donic acid derivatives (endogenous cannabinoids or endocannabinoids) bind as
lipophilic messengers to cannabinoid receptors CB1 and CB2. The ECS comprises
many hydrolytic enzymes responsible for the endocannabinoids cleavage. These
hydrolases, such as fatty acid amide hydrolase (FAAH) and monoacylglyceride
lipase (MAGL), are possible therapeutic targets for the development of new drugs
as indirect cannabinoid agonists. Recently, a new family of endocannabinoid
modulators was discovered; the lead structu re of this family is the nonapeptide
hemopressin produced from enzymatic cleavage of the α-chain of hemoglobin and
acting as negative allosteric modulator of CB1. Hemopressin shows several
physiological effects, e.g. antinociception, hypophagy, and hypotension. However, it is still a matter
of debate whether this peptide, isolated from the brain of rats, is a real neuromodulator of the ECS.
Recent evidence indicates that hemopressin could be a by-product formed by chemical degradation
of a longer peptide RVD-hemopressin during the extraction from the brain homolysate. Indeed,
RVD-hemopressin is more active than hemopressin in certain biological tests and may bind to the
same subsite as Rimonabant, which is an inverse agonist of CB1 and a µ-opioid receptor antagonist.
These findings have stimulated several studies to verify this hypothesis and to evaluate possible
therapeutic applications of hemopressin, its peptidic derivatives, and synthetic analogues, opening
new perspectives to the development of novel cannabinoid drugs.
Keywords: Opioid, rimonabant, cannabinoid, hemopressin, THC.
The endocannabinoid system (ECS) is an important and
only partially understood neuromodulator of physiologic
pathways controlling diverse roles in signaling and maintain-
ing human health. Furthermore, it is involved in a broad
range of biological processes, such as locomotor activity,
energy balance, and homeostasis at central and peripheral
levels [1]. The ECS controls memory, addiction, appetite,
food intake, metabolic functions [2], and is involved in neu-
roprotection [3,4], modulation of nociception [5], pain sensa-
tion, cognition, and behavioral responses to reward mecha-
nism and stress. It also influences the modulation of inflam-
matory, immune, endocrine responses and intracellular
events that control the proliferation of several types of cancer
cells, thereby producing antitumor effects [6-9]. The endo-
cannabinoid pathways influence also the cardiovascular
*Address correspondence to this author at the Dipartimento di Farmacia,
Universita` di Chieti-Pescara ‘‘G. d’Annunzio’’, Chieti, Italy;
Tel/Fax: +3908713554476; E-mail:
(cardiac rhythm, blood pressure) and respiratory system
(broncospasm). Because of the high complexity of the ECS,
its wide distribution beyond the central nervous system
(CNS), and its evolutionary conservation, the accurate mo-
lecular characterization of its constituents is useful to im-
prove the knowledge of the cannabinoids activity.
The ECS comprises cannabinoid receptors CB1 and CB2
and their endogenous ligands called endocannabinoids. Can-
nabinoid receptors are present throughout the body in cell
membranes. Their stimulation produces a variety of physiol-
ogic processes, which are most likely more numerous than
for other receptor systems [10]. The term “cannabinoid” de-
rives from the plant of Cannabis sativa (Figure 1), because it
contains many compounds mimicking endocannabinoid ef-
fects by binding to the same receptors.
The major psychoactive Cannabis constituent is Δ
tetrahydrocannabinol (Δ9-THC) (Figure 2), which the G-
protein-coupled cannabinoid receptor CB1 and modulates
A. Mollica
2 Protein & Peptide Letters, 2016, Vol. 23, No . 12 Macedo nio et al.
Figure 1. Cannabis sativa plant.
Figure 2. Δ9-THC structure.
cannabinoid receptor CB2. In the last few years, several other
non-cannabinoid plant constituents have been reported to
bind to CB receptors as well [11].
Δ9-tetrahydrocannabinol is the psychoactive component
isolated from Cannabis generally called Marijuana, which
has a long history as medicinal plant. It has been used in
textile fibers and oils, for medicinal purposes and as a
recreational drug due to its potent pharmacological effects
(e.g. sensory distortion, panic, anxiety lowered reaction time,
increased heartbeat, vasodilatation and mouth dryness) [12].
Δ9-THC is the most famous alkaloid among the phyto-
cannabinoids, but also cannabidiol and cannabinol have
gained the interest of researchers, due to a variety of healing
properties. Currently, the term cannabinoid includes all com-
pounds interacting with peripheral and central CB receptors,
which can be divided as:
Phytocannabinoids (psychoactive and not): terpenes
with resorcinol or a benzopyrane,
Endogenous cannabinoids (endocannabinoids), and
Synthetic agonists for therapeutic or scientific research.
The discovery of cannabinoid receptors was casual be-
cause it happened during an accurate study of rat DNA
screening to search the genome for neurokyne receptor [13].
Two different types of cannabinoid receptors have been
identified so far, namely CB1 and CB2, which were cloned
in the early 1990s from mammalian tissues [14-16].
Both CB1 and CB2 receptors belong to the superfamily
of Gi/o proteins coupled receptors (GPCRs) constituted by
seven transmembrane domains. They inhibit voltage-
sensitive calcium channels and adenyl cyclase, while
activating inwardly rectifying potassium channels and MAP
kinase. CB1 and CB2 show different tissue distributions,
activation mechanisms, and release mechanisms of second
messengers. In particular, CB1 has a relatively long extracel-
lular N-terminal domain and lacks a signal sequence. This
receptor exhibits an unusually high sequence identity across
species, whereas CB2 is less conserved. The CB1 receptor is
extensively expressed in forebrain of the CNS, especially in
axons and pre-synaptic terminals, hippocampus, cortex and
cerebellar granule cells, playing a role in memory, mood,
sleep, appetite, and pain sensation [6-9].
CB1 is a pre-synaptic heteroreceptor modulating
neurotransmitters release when activated in dose-dependent
and pertussis toxin-sensitive manners. It activates a Gi
protein, which decreases intracellular cAMP concentration
by inhibiting adenylate cyclase, while increasing the
mitogen-activated protein (MAP) kinase concentration. In
some rare cases, CB1 receptor activation may be coupled to
Gs protein, which stimulates adenylate cyclase. cAMP is a
second messenger coupled to a variety of ion channels,
including the positively influenced inwardly rectifying
potassium channels and calcium channels, via protein
kinases A (PKA) and C (PKC). The highest expression of
the CB2 receptor (sequence similarity between the two
subtypes is 44%) is found in immune cells, cells of
macrophage lineage, B-cells, natural killer cells, monocytes,
polymorphonucleate neutrophils [17], T8 and T4 cells, and
the brain [18,19].
TRPV1 receptors are localized in the ECS and they can
be activated and sensitized by mild acidification, bradykinin,
nerve-growth factor, anandamide, arachidonic acid metabo-
lites (e.g. N-arachidonoyl-dopamine (NADA) and N-
oleoyldopamine), lipoxygenase products, leukotriene B4,
prostaglandins, adenosine, and ATPhttp:// - B29
[20]. Synthetic cannabinoid WIN 55,212-2 inhibits TRPV1
through calcineurin-mediated receptor protein dephosphory-
lation. CB1 and δ-opioid receptors are co-expressed with
TRPV1 on sensory fibers [21], which is particularly impor-
tant for drug development, because the pharmacological ac-
tivity of some compounds could be related to interactions
with different receptors and the phosphorylation state of
Cannabinoid agonists appear to be promising tools for
treating and managing neuronal disorders, loss of body
weight, nausea/vomiting, and pain. In addition, development
of selective CB1 inverse agonists and antagonists have been
of great interest for therapeutic use in addictive disorders,
pain, appetite suppression, and blood pressure reduction.
Eannabinoid receptors are activated mainly by lipophilic
compounds including endocannabinoids, such as anan-
damide (the amide of arachidonic acid and ethanolamine)
and 2-arachidonoylglylcerol (2-AG [22]. After synthesis and
release, these lipophilic messengers act on nearby cannabi-
noid receptors. Their physiological effects are primarily
mediated through the CB1 receptor and then they are rapidly
inactivated by uptake and degradation [23]. A striking
Hemopressin Peptides as Modulators of the Endocannabinoid System Protein & Peptide Letters, 2016, Vol. 2 3, No. 12 3
difference between the endocannabinoids and many classic
neurotransmitters is that endocannabinoids appear to be
'formed on demand' rather than pre-synthesized and stored in
synaptic vesicles [24].
A number of other fatty acid-containing compounds have
also been identified as potential endocannabinoids; these
include amides, virodamine, and noladin ether (Figure 3).
The degradation of endocannabinoids is achieved by two
specific enzymes, the fatty acid amide hydrolase (FAAH)
and the monoacylglyceride lipase (MAGL) enzymes. FAAH
degrades anandamide, whereas the MAGL degrades 2-AG.
FAAH is an integral membrane protein consisting of 597
amino acids with a highly conserved amidase sequence rich
in glycine and serine that hydrolyzes bioactive amides
including anandamide, to free fatty acid and ethanolamine
[25]. FAAH belongs to the serine hydrolase enzyme family
with a single N-terminal transmembrane domain. In 2002, its
structure was determined by X-ray crystallography [26]. It is
more active at alkaline pH (optimum at pH 9). MAGL is a
33-kDa membrane-associated enzyme of the serine
hydrolase superfamily [27]. It is worth noting that 2-AG and
anandamide can be degraded by COX-2 and LIPOX (Figure
4) [25].
Hemopressin (PVNFKFLSH) is a bioactive nonapeptide
derived from the α-chain of hemoglobin. It was originally
isolated from rat brain homogenate as a substrate for en-
dopeptidase 24.15 (thimet oligopeptidase), endopeptidase
24.16 (neurolysin), and ACE [28]. Hemopressin elicits a
weak dose-dependent hypotensive effect in mice, rats, and
rabbits, and is endowed with significant CB1 receptor-
selective antagonist activity [28,29]. Subsequently, in vivo
studies revealed that administration of hemopressin causes
significant non-opioid anti-nociceptive effects in rats
[30,31]. The cellular target of hemopressin is the CB1 recep-
tor and in vitro assays confirmed that hemopressin acts a
selective inverse agonist on CB1 [31]. The peptide showed a
similar binding profile and affinity (EC50 = 0.35 nM) to CB1
as rimonabant [31]. Indeed, a docking study suggested that
hemopressin and rimonabant bind to the same CB1 receptor
binding pocket. Circular dichroism and NMR spectroscopy
indicated a regular turn structure in the central portion [32]
of hemopressin and its truncated biologically active fragment
Hp(1-6), which is critical for an effective interaction with the
receptor [33]. Rimonabant is a selective CB1 receptor an-
tagonist that has not been approved by the U.S. Food and
Drug Administration (FDA) due to the high risk of psychiat-
ric side effects; it was withdrawn from the European market
in 2009 [34].
Effect on Blood Pressure
Hemopressin causes hypotension in anesthetized rats. Al-
though the mechanism mediating this response are still un-
clear, it could involve ion channels activation or blockade,
release of nitric oxide (NO) and vasodilator peptides (e.g.
atrial natriuretic factor) or inhibition of endogenous pepti-
dase activity leading to increased circulating levels of hy-
potensive peptides. Enalapril scarcely influenced the pres-
sure responses to hemopressin in comparison to the effect
seen in bradykinin (BK) induced hypotension. Hemopressin
improves the hypotensive response to BK without interfering
with hypertension provoked by angiotensin II, although it is
still unclear if this response is selective for BK or not.
Treatment of normotensive rats with CB1 antagonists alone
does not influence blood pressure; in fact the baseline blood
pressure is similar in CB1 knockout mice and their wild-type
littermates [35,36].
Hypotensive effects of anandamide were observed in anes-
thetized normotensive rats and the lack of hypotension after
anandamide transport blocking [37] indicating the absence of
the endocannabinergic ‘tone’ in the maintenance of normal
blood pressure. In contrast, anandamide and Δ9-THC pro-
voke longer lasting hypotension in spontaneously hyperten-
4 Protein & Peptide Letters, 2016, Vol. 23, No . 12 Macedo nio et al.
sive rats (SHR) than in normotensive rats, which is inde-
pendent from the absence or presence of anesthesia [38, 39].
Antinociceptive Action
The role of hemopressin in nociception was demonstrated in
several experimental models of pain. The rat paw pressure
uses pressure as a mechanical stimulus for directly activating
nociceptors of C and Aδ fibers, resulting in a motor response
that leads to paw withdrawal. This model is widely used to
study analgesic drugs with peripheral activity [33]. Hemo-
pressin reverts hyperalgesia induced by carrageenan and
bradykinin injected concomitantly or 2.5 h after injection of
the phlogistic agents [40]. These effects are not blocked by
naloxone, suggesting that opioid receptors are not involved
and that the effect of hemopressin on pain sensitivity is me-
diated by chemical neurotransmitters released during in-
flammatory hyperalgesia [41]. Data for C-terminally trun-
cated hemopressin fragments indicate that the full sequence
is not essential for the expression of anti-nociceptive activity,
while hemopressin and its two fragments Hβ(1-6)
(PVNFKF) and Hβ(1-7) (PVNFKFL) were similarly effec-
tive in exerting the anti-hyperalgesic action. However,
shorter fragments (PVNFK and PVNF) were inactive [33].
It is worth noting that the order of activity of these fragments
on blood pressure is the exact opposite of that seen for anal-
gesia. Although the mechanisms involved in the anti-
hyperalgesic effect of hemopressin remain to be character-
ized, the results obtained so far suggest a non-opioid path-
way in regulating inflammatory pain [42], which could be
explored further to develop therapeutic drugs based on the
hemopressin sequence [43].
Reduction of Appetite
Hemopressin decreases dose-dependently nighttime food
intake in normal male rats and mice as well as in obese male
mice when administered centrally or systemically without
adverse side effects [44]. N-terminally extended peptides of
hemopressin, i.e. RVD- and VD-hemopressin, are CB1 ago-
nists [45] indicating that a difference of only two or three
residues determines the antagonistic or agonistic activity.
The signaling pathways for the N-terminally extended pep-
tide agonists are distinct from the classic G-protein-mediated
pathway of lipid-based and synthetic agonists, resulting in a
robust and sustained increase in Ca2+ release. It remains un-
clear whether hemopressin is the endogenous peptide or in-
stead it is only the cleavage product of the longer RVD-
hemopressin peptide; the Asp-Pro bond is one of the most
labile peptide bond under acidic conditions applied during
the extraction of rat brains [46]. Mass spectrometry study of
mouse brain extracts prepared by different conditions did not
identify hemopressin but RVD-hemopressin [46].
Mediation of Neuronal Plasticity
Endocannabinoids serve as mediators of short- and long-term
neuronal plasticity [47]. The behaviors involved are diverse
and include movement, sensory learning, analgesia, anxiety,
Hemopressin Peptides as Modulators of the Endocannabinoid System Protein & Peptide Letters, 2016, Vol. 2 3, No. 12 5
and appetitive, to name a few. Actually, the most relevant for
therapeutic inventions appear to be obesity (involving both
central and peripheral mechanisms - alternatively
homeostatic and hedonistic mechanisms) and craving-based
disorders, such as alcohol and tobacco dependency [48].
Many of the mechanisms underlying are CB1-mediated and
their integration with other pathways including motivation,
reward, and satiety remain unclear [47].
Potential-driven endocannabinoids release induces a
long-lasting membrane potential hyperpolarization in hippo-
campus cells, which depends on the activation of neuronal
CB2 receptors modulating sodium-bicarbonate co-transporter
activity, instead of CB1 receptors. The CB2 activation hap-
pens in self-regulatory manner altering hippocampus cells
function and modulating gamma oscillations in vivo, thus
providing evidence for the neuronal expression of CB2 recep-
tors [49]. Although excitement remains over the finding of
peptide modulators of the cannabinoid system, additional
research is necessary to better understand the generation and
regulation of peptide ligands, hemopressin’s inconsistent
activity, and hemopressin-related peptides modulation of the
cannabinoid receptors [48]. It is possible that RVD-
hemopressin and hemopressin, which differ in three amino
acids, elicit completely opposite responses at the CB1 recep-
tor namely agonist and inverse agonist effects, but to validate
this concept systematic structure-activity studies are needed.
Being involved in many diseases, numerous selective
agonists and antagonists for CB receptors have been devel-
oped. The therapeutic applications of cannabinoid receptor
antagonists depend on ligand selectivity [50]. In particular,
selective CB1 receptor antagonists have been studied for
their possible therapeutic use in the treatment of obesity
[45,51], drug abuse, and heroin addiction [52]. Many com-
pounds have been synthesized and rimonabant (also known
as SR141716; trade names Acomplia, Zimulti) has been
extensively studied for its application as anoressant drug [53]
(Figure 5).
Although studies demonstrated its efficacy in treating
obesity and addictive disorders, severe adverse effects have
been reported on the CNS, which prevented its approval in
the United States and suspended its use elsewhere. In fact,
the US Food and Drug Administration rejected rimonabant
because the clinical trials suggested a higher incidence of
anxiety, depression and suicide behaviors, following pro-
longed administration [54]. Selective cannabinoid agonists
and antagonists lacking adverse side effects, but maintaining
therapeutic benefits, are highly desired and represent a strong
need in medicinal chemistry. URB597 (KDS-4103) is an
irreversible inhibitor of FAAH leading to an accumulation of
anandamide in the CNS and in periphery, where it can
activate cannabinoid receptors [55]. Thereby, alterations
caused by the direct stimulation of the cannabinoid system
by an exogenous compound like Δ9-THC might be avoided
[55]. Recently a new FAAH inhibitor (Bia 10-2474) was
tested as a new anti-dolorific drug for neuropathic pain: it
increases levels of the anandamide in the CNS and peripheral
tissues, but it was withdrawn from a clinical trial due to
potentially serious side effects [56].
The discovery and identification of the endocannabinoid
system are important for the development of new drugs, al-
though its physiological role are not yet completely revealed.
Certainly, it is involved in the regulation of many physio-
logical functions including cellular communication, locomo-
tor activity, and the mediation of nociceptive, endocrine,
immune, and inflammatory responses, but it is also impli-
cated in many pathological processes. Endocannabinoids
have anti-proliferative, anti-nociceptive, and neuroprotective
BIA 10-2474
URB 597
6 Protein & Peptide Letters, 2016, Vol. 23, No . 12 Macedo nio et al.
effects. They influence the cardiovascular and respiratory
systems including blood pressure, bronchospasm, and car-
diac rhythm. Hemopressin increases the levels of endocan-
nabinoids, while the treatment with URB597 enforced he-
mopressin-induced anti-nociceptive effects. This suggests
that it might increase endocannabinoid levels and in turn
activate the descending inhibitory pain pathway inducing
analgesia [57].
Extended and truncated derivatives of hemopressin are
orally active anti-nociceptive compounds. They are partially
resistant to proteolysis and can cross the blood-brain barrier
[58]. Furthermore, truncated hemopressin-7 is as potent as
hemopressin-9, as they may be able to bind to the same re-
ceptor allosteric site. However, further studies are necessary
for the delineation of Hp(1-7) binding site and its pharma-
cological significance in mammalian species. We can con-
clude that this new class of peptides derived from hemo-
pressin may represent an important target for the develop-
ment of peptide tools to investigate the ECS.
The authors confirm that this article content has no
conflict of interest.
Declared none.
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... CB 2 R is mainly expressed in the immune system and in hematopoietic cells, but it is also detected in some other peripheral tissues (Galiègue et al., 1995). At the protein level, CB 1 R and CB 2 R share 44% amino acid homology (Howlett and Abood, 2017;Macedonio et al., 2016). The crystal structures of CB 1 R (Hua et al., 2016;Shao et al., 2016) (PDB ID: 5TGZ) and CB 2 R (Li et al., 2019) (PDB ID: 5ZTY) were determined using AM6538 and AM10257 as CB 1 R and CB 2 R stabilizing antagonists, respectively. ...
... The similar result was also present in an immunoaffinity mass spectrometry-based study in which more N-terminally extended peptides named pepcans (peptide endocannabinoids) were identified in the brain and plasma and Hp remained unidentified (Bauer et al., 2012). So, Hp was postulated to be generated from the cleavage of longer peptides under acidic conditions (Macedonio et al., 2016;Gomes et al., 2009;Bomar and Galande, 2014). RVD-Hpα, the Nterminally extended peptides of Hp, which was found in the studies (Gomes et al., 2009) (Bauer et al., 2012) mentioned above, was considered the endogenous peptide (Gomes et al., 2009;Bauer et al., 2012). ...
Cannabinoid receptors (CBRs) are part of the endocannabinoid system, which is involved in various physiological processes such as nociception, inflammation, appetite, stress, and emotion regulation. Many studies have linked the endocannabinoid system to neuroinflammatory and neurodegenerative disorders such as Parkinson's disease, Huntington's chorea, Alzheimer's disease, and multiple sclerosis. Hemopressin [Hp; a fragment of the hemoglobin α1 chain (95-103 amino acids)] and related peptides [VD-Hpα and RVD-Hpα] are peptides that bind to CBRs. Hp acts as an inverse agonist to CB1 receptor (CB1R), VD-Hpα acts as an agonist to CB1R, and RVD-Hpα acts as a negative allosteric modulator of CB1R and a positive allosteric modulator of CB2R. Because of the critical roles of CBRs in numerous physiological processes, it is appealing to use Hp and related peptides for therapeutic purposes. This review discusses their discovery, structure, metabolism, brain exposure, self-assembly characteristics, pharmacological characterization, and pharmacological activities.
... Barriers such as the rapid development of tolerance to ∆ 9 -THC, lack of highly specific inhibitors, and difficulty separating centrally and peripherally mediated effects lessen the ability to safely target this complex signalling system for BP management. Despite this, a new hope comes from the discovery of small molecules with different mechanisms of modulating the ECS, e.g., pepcans, which are a large family of eCB peptides capable of CBR antagonism or allosteric modulation of CBRs depending on their amino acid sequence length [83][84][85]. ...
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In the past, cannabis was commonly associated with mysticism and illegality. Fortunately, in recent years perspectives and discourses have changed. More prominence has been given to the rigorous scientific effort that led to the discovery of cannabis’ many physiological actions and endogenous signalling mechanisms. The endocannabinoid system is a complex and heterogeneous pro-homeostatic network comprising different receptors with several endogenous ligands, numerous metabolic enzymes and regulatory proteins. Therefore, it is not surprising that alterations and dysfunctions of the endocannabinoid system are observed in almost every category of disease. Such high degree of pathophysiological involvement suggests the endocannabinoid system is a promising therapeutic target and prompted the translation of resurgent scientific findings into clinical therapies. Shifting attitudes toward cannabis also raised other matters such as increased patient awareness, prescription requests, self-medication, recreational use, recognition of new knowledge gaps, renewed scientific activity, and seemingly exponential growth of the cannabis industry. This review, following a general overview of cannabis and the endocannabinoid system, assiduously describes its role within the context of cardiovascular diseases, paying particular attention to the Janus influence that endocannabinoid system modulators can have on the cardiovascular system.
... They are variously distributed in the central nervous system (CNS), specifically in axons and pre-synaptic terminals, hippocampus, cortex, where they are implicated in the control of memory, sedation, hypothermia, hypotension, and pain sensation [8][9][10] (Fig. 2). The endocannabinoid system is also known to modulate anxiety and feeding behavior interacting with different ligands, as in the case of Hemopressin and RVD-hemopressin [11][12][13]. ...
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Nowadays cardiovascular diseases (CVDs) are the major causes for the reduction of the quality of life. The endocannabinoid system is an attractive therapeutic target for the treatment of cardiovascular disorders due to its involvement in vasomotor control, cardiac contractility, blood pressure and vascular inflammation. Alteration in cannabinoid signalling can be often related to cardiotoxicity, circulatory shock, hypertension, and atherosclerosis. Plants have been the major sources of medicines until modern eras in which researchers are experiencing a rediscovery of natural compounds as novel therapeutics. One of the most versatile plant is Cannabis sativa L., containing phytocannabinoids that may play a role in the treatment of CVDs. The aim of this review is to collect and investigate several less studied plants rich in cannabinoid-like active compounds able to interact with cannabinoid system; these plants may play a pivotal role in the treatment of disorders related to the cardiovascular system.
... There is growing evidence indicating that peptides that control appetite (e.g., kisspeptin, orexins, spexin, adropin, apelin, phoenixin, ghrelin, amylin, and pancreatic peptides) also modulate the endocrine activity of endocrine glands as well as lipid and glucose metabolism [1][2][3][4][5][6]. Moreover, some peptides are involved in regulating the endocannabinoid system and, through it, food intake, e.g., hemopressin, a small peptide derived from the α-chain of hemoglobin, reduces appetite through increased levels of endocannabinoids [7,8]. On the other hand, endogenous cannabinoids can also increase the secretion of feeding-regulated hypothalamic neuropeptides [9]. ...
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Neuropeptide B (NPB) is a peptide hormone that was initially described in 2002. In humans, the biological effects of NPB depend on the activation of two G protein-coupled receptors, NPBWR1 (GPR7) and NPBWR2 (GPR8), and, in rodents, NPBWR1. NPB and its receptors are expressed in the central nervous system (CNS) and in peripheral tissues. NPB is also present in the circulation. In the CNS, NPB modulates appetite, reproduction, pain, anxiety, and emotions. In the peripheral tissues, NPB controls secretion of adrenal hormones, pancreatic beta cells, and various functions of adipose tissue. Experimental downregulation of either NPB or NPBWR1 leads to adiposity. Here, we review the literature with regard to NPB-dependent control of metabolism and energy homeostasis.
... Hemopressin reduces food intake without causing any obvious adverse side effects [2,111,112]. Nevertheless, further studies are needed in order to confirm that these effects are due to the direct action of hemopressin on CB1R [113], whereas pregnenolone binding to CB1R does not modify the binding of agonists, but reduces body weight gain in diet-induced obese mice and it does not induce anxiety [30,109]. ...
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The endocannabinoid system (ECS) employs a huge network of molecules (receptors, ligands, and enzymatic machinery molecules) whose interactions with other cellular networks have still not been fully elucidated. Endogenous cannabinoids are molecules with the primary function of control of multiple metabolic pathways. Maintenance of tissue and cellular homeostasis by functional fine-tuning of essential metabolic pathways is one of the key characteristics of the ECS. It is implicated in a variety of physiological and pathological states and an attractive pharmacological target yet to reach its full potential. This review will focus on the involvement of ECS in glucose and lipid metabolism, food intake regulation, immune homeostasis, respiratory health, inflammation, cancer and other physiological and pathological states will be substantiated using freely available data from open-access databases, experimental data and literature review. Future directions should envision capturing its diversity and exploiting pharmacological options beyond the classical ECS suspects (exogenous cannabinoids and cannabinoid receptor monomers) as signaling through cannabinoid receptor heteromers offers new possibilities for different biochemical outcomes in the cell.
... 4,5 As the endogenous ligands of cannabinoid receptors,anandamide (AEA) and 2arachidonoylglycerol (2-AG) are synthesized and degraded by specific enzymes. 4,[6][7][8][9] Then, the regulation of cellular 2-AG level is crucial for the ECS modulation. 2-AG is mainly hydrolyzed by monoacylglycerol lipase (MAGL), which is a serine hydrolase with 303 amino acid residues. ...
Monoacylglycerol lipase (MAGL) is the major enzyme that catalyzes the hydrolysis of monoacylglycerols (MAGs). MAGL is responsible for degrading 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and specific tissues. The inhibition of MAGL could attenuate the inflammatory response. Here, we report a series of reversible non-covalent MAGL inhibitors via virtual screening combined with biochemical analysis. The hit, DC630-8 showed low-micromolar activity against MAGL in vitro, and exhibited significant anti-inflammatory effects.
... Thus, the receptor can be modulated under hyperactivaton by agonists instead of being blocked. Endogenous NAMs have been described, such as hemopressin (106), pregnenolone, and the family of cannabinoid peptides named pepcans (107). Pepcans might be useful for downregulating CB 1 receptor activity. ...
There is an urgent need for developing effective drugs to combat the obesity and Type 2 diabetes mellitus epidemics. The endocannabinoid system plays a major role in energy homeostasis. It comprises the cannabinoid receptors 1 and 2 (CB1 and CB2), endogenous ligands called endocannabinoids and their metabolizing enzymes. Because the CB1 receptor is overactivated in metabolic alterations, pharmacological blockade of the CB1 receptor arose as a promising candidate to treat obesity. However, because of the wide distribution of CB1 receptors in the central nervous system, their negative central effects halted further therapeutic use. Although the CB2 receptor is mostly peripherally expressed, its role in metabolic homeostasis remains unclear. This review discusses the potential of CB1 and CB2 receptors at the peripheral level to be therapeutic targets in metabolic diseases. We focus on the impact of pharmacological intervention and/or silencing on peripheral cannabinoid receptors in organs/tissues relevant for energy homeostasis. Moreover, we provide a perspective on novel therapeutic strategies modulating these receptors. Targeting CB1 with peripherally restricted antagonists, neutral antagonists, inverse agonists, or monoclonal antibodies could represent successful strategies. CB2 agonism has shown promising results at preclinical level. Beyond classic antagonism and agonism targeting orthosteric sites, the recently described crystal structures of CB1 and CB2 open new possibilities for therapeutic interventions with negative and positive allosteric modulators. The challenge of simultaneously targeting CB1 and CB2 might be possible by developing dual-steric ligands. The future will tell whether these promising strategies result in a renaissance of the cannabinoid receptors as therapeutic targets in metabolic diseases.
... While the major molecular targets of hemopressin peptides are the cannabinoid receptors, the mechanisms mediating the hypotensive effect are still unclear. However, different explanations have been suggested, such as vasodilation, release of nitric oxide (NO), ion channels activation or blockade, or even inhibition of endogenous peptidase activity leading to increased circulating levels of hypotensive peptides (Blais et al., 2005;Lippton et al., 2006;Macedonio et al., 2016). Recently, the effects of hemopressin extended peptide VD-Hp on blood pressure at the spinal level were analyzed. ...
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Classically, the endocannabinoid system (ECS) consists of endogenous lipids, of which the best known are anandamide (AEA) and 2 arachidonoylglycerol (2-AG), their enzyme machinery for synthesis and degradation and their specific receptors, cannabinoid receptor one (CB1) and cannabinoid receptor two (CB2). However, endocannabinoids also bind to other groups of receptors. Furthermore, another group of lipids are considered to be endocannabinoids, such as the fatty acid ethanolamides, the fatty acid primary amides and the monoacylglycerol related molecules. Recently, it has been shown that the hemopressin peptide family, derived from α and β chains of hemoglobins, is a new family of cannabinoids. Some studies indicate that hemopressin peptides are expressed in the central nervous system and peripheral tissues and act as ligands of these receptors, thus suggesting that they play a physiological role. In this review, we examine new evidence on lipid endocannabinoids, cannabinoid receptors and the modulation of their signaling pathways. We focus our discussion on the current knowledge of the pharmacological effects, the biosynthesis of the peptide cannabinoids and the new insights on the activation and modulation of cannabinoid receptors by these peptides. The novel peptide compounds derived from hemoglobin chains and their non-classical activation of cannabinoid receptors are only starting to be uncovered. It will be exciting to follow the ensuing discoveries, not only in reference to what is already known of the classical lipid endocannabinoids revealing more complex aspects of endocannabinoid system, but also as to its possibilities as a future therapeutic tool.
Monoacylglycerol lipase (MAGL) is the enzyme degrading the endocannabinoid 2-arachidonoylglycerol and it is involved in several physiological and pathological processes. The therapeutic potential of MAGL is linked to several diseases, including cancer. The development of MAGL inhibitors has been greatly limited by the side effects associated with the prolonged MAGL inactivation. Importantly, it could be preferable to use reversible MAGL inhibitors in vivo, but nowadays only few reversible compounds have been developed. In the present study, structural optimization of a previously developed class of MAGL inhibitors led to the identification of compound 23, which proved to be a very potent reversible MAGL inhibitor (IC50 = 80 nM), selective for MAGL over the other main components of the endocannabinoid system, endowed of a promising antiproliferative activity in a series of cancer cell lines and able to block MAGL both in cell-based as well as in vivo assays.
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Monoacylglycerol lipase (MAGL) is an attractive therapeutic target for many pathologies, including neurodegenerative diseases, cancer as well as chronic pain and inflammatory pathologies. The identification of reversible MAGL inhibitors, devoid of the side effects associated to prolonged MAGL inactivation, is a hot topic in medicinal chemistry. In this study, a novel phenyl(piperazin-1-yl)methanone inhibitor of MAGL was identified through a virtual screening protocol based on a fingerprint-driven consensus docking (CD) approach. Molecular modeling and preliminary structure-based hit optimization studies allowed the discovery of derivative 4, which showed an efficient reversible MAGL inhibition (IC50 = 6.1 µM) and a promising antiproliferative activity on breast and ovarian cancer cell lines (IC50 of 31–72 µM), thus representing a lead for the development of new and more potent reversible MAGL inhibitors. Moreover, the obtained results confirmed the reliability of the fingerprint-driven CD approach herein developed.
Full-text available
Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.
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Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted.
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Direct-acting cannabinoid receptor ligands are well known to reduce hyperalgesic responses after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Hemopressin (Hp) is a nonapeptide that selectively binds CB1 cannabinoid receptors (CB1 receptors) and exerts antinociceptive action in inflammatory pain models. We investigated the effect of Hp on neuropathic pain in rats subjected to chronic constriction injury (CCI) of the sciatic nerve, and explored the mechanisms involved. Oral administration of Hp inhibits mechanical hyperalgesia of CCI-rats up to 6hours. Hp treatment also decreases Egr-1 immunoreactivity (Egr-1Ir) in the superficial layer of the dorsal horn of the spinal cord of CCI rats. The antinociceptive effect of Hp seems to be independent of inhibitory descending pain pathway since methisergide (5HT1A receptor antagonist) and yohimbine (α-2 adrenergic receptor antagonist) were unable to prevent Hp antinociceptive effect. Hp decreased calcium flux on DRG neurons from CCI rats, similarly to that observed for AM251, a CB1 receptor antagonist. We also investigated the effect of Hp on potassium channels of CCI rats using UCL 1684 (a blocker of Ca(2+) -activated K(+) channels) which reversed Hp-induced antinociception. Furthermore, concomitant administration of URB-584 (FAAH inhibitor) but not JZL-184 (MAGL inhibitor) potentiates antinociceptive effect of Hp in CCI rats indicating an involvement of anadamide on HP-induced antinociception. Together, these data demonstrate that Hp displays antinociception in pain from neuropathic etiology through local effects. The release of anandamide and the opening of peripheral K(+) channels are involved in the antinociceptive effect.
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Rationale: Introduction of sedation protocols has been associated with improved patient outcomes. It is not known if an update to an existing high-quality sedation protocol, featuring increased patient assessment and reduced benzodiazepine exposure, is associated with improved patient process and outcome quality metrics. Methods: This was an observational before (n = 703) and after (n = 780) cohort study of mechanically ventilated patients in a 24-bed trauma-surgical intensive care unit (ICU) from 2009 to 2011. The three main protocol updates were: (1) requirement to document Richmond Agitation Sedation Scale (RASS) scores every 4 hours, (2) requirement to document Confusion Assessment Method-ICU (CAM ICU) twice daily, and (3) systematic, protocolized deescalation of excess sedation. Multivariable linear regression was used for the primary analysis. The primary outcome was the duration of mechanical ventilation. Prespecified secondary endpoints included days of delirium; the frequency of patient assessment with the RASS and CAM-ICU instruments; benzodiazepine dosing; durations of mechanical ventilation, ICU stay, and hospitalization; and hospital mortality and ventilator associated pneumonia rate. Results: Patients in the updated protocol cohort had 1.22 more RASS assessments per day (5.38 vs. 4.16; 95% confidence interval [CI], 1.05-1.39; P < 0.01) and 1.15 more CAM-ICU assessments per day (1.49 vs. 0.35; 95% CI, 1.08-1.21; P < 0.01) than the baseline cohort. The mean hourly benzodiazepine dose decreased by 34.8% (0.08 mg lorazepam equivalents/h; 0.15 vs. 0.23; P < 0.01). In the multivariable model, the median duration of mechanical ventilation decreased by 17.6% (95% CI, 0.6-31.7%; P = 0.04). The overall odds ratio of delirium was 0.67 (95% CI, 0.49-0.91; P = 0.01) comparing updated versus baseline cohort. A 12.4% reduction in median duration of ICU stay (95% CI, 0.5-22.8%; P = 0.04) and a 14.0% reduction in median duration of hospitalization (95% CI, 2.0-24.5%; P = 0.02) were also seen. No significant association with mortality (odds ratio, 1.18; 95% CI, 0.80-1.76; P = 0.40) was seen. Conclusions: Implementation of an updated ICU analgesia, sedation, and delirium protocol was associated with an increase in RASS and CAM-ICU assessment and documentation; reduced hourly benzodiazepine dose; and decreased delirium and median durations of mechanical ventilation, ICU stay, and hospitalization.
Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission. The neuronal expression of CB2Rs has been a matter of long-standing debate. Stempel et al. demonstrate that CB2Rs are expressed in hippocampal principal cells and modulate neuronal function both in vitro and in vivo.
Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both μ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation.
The orally active, α-hemoglobin derived hemopressin (PVNFKFLSH, Hp(1–9)) and its truncated (PVNFKFL, Hp(1–7) and PVNFKF, Hp(1–6)) and extended ((R)VDPVNFKFLSH, VD-Hp(1–9) and RVD-Hp(1–9)) derivatives have been postulated to be the endogenous peptide ligands of the cannabinoid receptor type 1 (CB1). In an attempt to create a versatile peptidic research tool for the direct study of the CB1 receptor–peptide ligand interactions, Hp(1–7) was radiolabelled and in vitro characterized in rat and CB1 knockout mouse brain membrane homogenates. In saturation and competition radioligand binding studies, [3H]Hp(1–7) labelled membrane receptors with high densities and displayed specific binding to a receptor protein, but seemingly not to the cannabinoid type 1, in comparison the results with the prototypic JWH-018, AM251, rimonabant, Hp(1–9) and RVD-Hp(1–9) (pepcan 12) ligands in both rat brain and CB1 knockout mouse brain homogenates. Furthermore, functional [35S]GTPγS binding studies revealed that Hp(1–7) and Hp(1–9) only weakly activated G-proteins in both brain membrane homogenates. Based on our findings and the latest literature data, we assume that the Hp(1–7) peptide fragment may be an allosteric ligand or indirect regulator of the endocannabinoid system rather than an endogenous ligand of the CB1 receptor.
Endocannabinoids (eCBs) are endogenous lipid mediators involved in a variety of physiological, pharmacological, and pathological processes. While activation of the eCB system primarily induces inhibitory effects on both GABAergic and glutamatergic synaptic transmission and plasticity through acting on presynaptically expressed CB1 receptors in the brain, accumulated information suggests that eCB signaling is also capable of facilitating or potentiating excitatory synaptic transmission in the hippocampus. Recent studies show that a long-lasting potentiation of excitatory synaptic transmission at Schaffer collateral (SC)-CA1 synapses is induced by spatiotemporally primed inputs, accompanying with a long-term depression of inhibitory synaptic transmission (I-LTD) in hippocampal CA1 pyramidal neurons. This input timing-dependent long-lasting synaptic potentiation at SC-CA1 synapses is mediated by 2-arachidonoylglycerol (2-AG) signaling triggered by activation of postsynaptic N-methyl-d-aspartate receptors, group I metabotropic glutamate receptors (mGluRs), and a concurrent rise in intracellular Ca(2+). Emerging evidence now also indicates that 2-AG is an important signaling mediator keeping brain homeostasis by exerting its anti-inflammatory and neuroprotective effects in response to harmful insults through CB1/2 receptor-dependent and/or -independent mechanisms. Activation of the nuclear receptor protein peroxisome proliferator-activated receptor-γ apparently is one of the important mechanisms in resolving neuroinflammation and protecting neurons produced by 2-AG signaling. Thus, the information summarized in this review suggests that the role of eCB signaling in maintaining integrity of brain function is greater than what we thought previously.