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Protein & Pep tide Letters, 2016, 23, 1-7 1
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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,
A R T I C L E H I S T O R Y
Received: September 5, 2016
Revised: September 29, 2016
Accepted: October 1, 2016
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.
INTRODUCTION TO THE ENDOCANNABINOID
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 . The ECS controls memory, addiction, appetite,
food intake, metabolic functions , and is involved in neu-
roprotection [3,4], modulation of nociception , 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: firstname.lastname@example.org
(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.
CANNABINOIDS AND CANNABINOID RECEPTORS
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 . 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
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 .
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) .
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 .
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 , 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://
www.nature.com/nrd/journal/v6/n5/full/nrd2280.html - B29
. 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 , 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.
THE ENDOCANNABIN OID
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 . 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 . 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 .
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
. 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 . It is
more active at alkaline pH (optimum at pH 9). MAGL is a
33-kDa membrane-associated enzyme of the serine
hydrolase superfamily . It is worth noting that 2-AG and
anandamide can be degraded by COX-2 and LIPOX (Figure
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 . 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 . The peptide showed a
similar binding profile and affinity (EC50 = 0.35 nM) to CB1
as rimonabant . 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 
of hemopressin and its truncated biologically active fragment
Hp(1-6), which is critical for an effective interaction with the
receptor . 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 .
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
Hypotensive effects of anandamide were observed in anes-
thetized normotensive rats and the lack of hypotension after
anandamide transport blocking  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-
Figure 3. Fatty acid-containing compounds identified as endocannabinoids.
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].
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 . Hemo-
pressin reverts hyperalgesia induced by carrageenan and
bradykinin injected concomitantly or 2.5 h after injection of
the phlogistic agents . 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 . 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 .
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 , which could be
explored further to develop therapeutic drugs based on the
hemopressin sequence .
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 . N-terminally extended peptides of
hemopressin, i.e. RVD- and VD-hemopressin, are CB1 ago-
nists  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 . Mass spectrometry study of
mouse brain extracts prepared by different conditions did not
identify hemopressin but RVD-hemopressin .
Mediation of Neuronal Plasticity
Endocannabinoids serve as mediators of short- and long-term
neuronal plasticity . The behaviors involved are diverse
and include movement, sensory learning, analgesia, anxiety,
Figure 4. Degradation of endocannabinoids by FAAH and MAGL.
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 .
Many of the mechanisms underlying are CB1-mediated and
their integration with other pathways including motivation,
reward, and satiety remain unclear .
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 . 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 . 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.
SYNTHETIC MODULATORS OF ENDOCANNABI-
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 . 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 . 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 
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 . 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 . Thereby, alterations
caused by the direct stimulation of the cannabinoid system
by an exogenous compound like Δ9-THC might be avoided
. 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 .
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
Figure 5. Chemical structures of rimonabant and synthetic derivatives URB597 and BIA 10-2474.
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
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
. 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.
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
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