Vascular pharmacology of a novel cannabinoid-like compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16) in the rat

Article abstract of DOI:10.1038/sj.bjp.0707470

Background and purpose: A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water-soluble agonist, a

Full text of DOI:10.1038/sj.bjp.0707470

British Journal of Pharmacology (2007) 152, 751–764
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RESEARCH PAPER
Vascular pharmacology of a novel cannabinoid-like
compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-
N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16) in
the rat
PM Hoi1,5, C Visintin2,5, M Okuyama2, SM Gardiner3, SS Kaup1, T Bennett3, D Baker4,
DL Selwood2 and CR Hiley1
1Department of Pharmacology, University of Cambridge, Cambridge, UK; 2The Wolfson Institute for Biomedical Research, University
College London, London, UK; 3School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, UK; 4Institute of
Cell and Molecular Science, Barts and The London, Queen Mary’s School of Medicine and Dentistry, London, UK
Background and purpose: A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-
cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel
water-soluble agonist, as a vasorelaxant potentially acting at non-CB1, non-CB2 cannabinoid receptors in the vasculature.
Experimental approach: VSN16 and some analogues were synthesized and assayed for vasodilator activity in the rat third
generation mesenteric artery using wire myography. Also carried out with VSN16 were haemodynamic studies in conscious
rats and binding studies to CB1 receptors of rat cerebellum.
Key results: VSN16 relaxed mesenteric arteries in an endothelium-dependent manner. The vasorelaxation was antagonized by
high concentrations of the classical cannabinoid antagonists, rimonabant and AM 251, as well as by O-1918, an antagonist at
the abnormal-cannabidiol receptor but not at CB1 or CB2 receptors. It did not affect [3H]CP55,940 binding to CB1 receptors in
rat cerebellum. The vasorelaxation was not pertussis toxin-sensitive but was reduced by inhibition of nitric oxide synthesis,
Ca2 þ -sensitive Kþ channels (KCa) and TRPV1 receptors. In conscious rats VSN16 transiently increased blood pressure and
caused a longer-lasting increase in mesenteric vascular conductance. Structure-activity studies on vasorelaxation showed a
stringent interaction with the target receptor.
Conclusions and implications: VSN16 is an agonist at a novel cannabinoid receptor of the vasculature. It acts on the
endothelium to release nitric oxide and activate KCa and TRPV1. As it is water-soluble it might be useful in bringing about
peripheral cannabinoid-like effects without accompanying central or severe cardiovascular responses.
British Journal of Pharmacology (2007) 152, 751–764; doi:10.1038/sj.bjp.0707470; published online 24 September 2007
Keywords: VSN16; cannabinoid receptors; endothelium; rat mesenteric artery; haemodynamics; O-1918; vasodilator;
rimonabant; AM 251; GPR55
Abbreviations: DCM, dichloromethane; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide;
K
Ca, Ca2 þ -sensitive Kþ channels; L-NAME, NG-nitro-L-arginine methyl ester; nH, Hill slope; O-1918, (À)-1,3-
dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol; TRPV1, transient receptor potential vanilloid 1;
VSN16, (7)-3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide; VSN16-R, (R)-3-
(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide; VSN16-S, (S)-3-(5-dimethylcar-
bamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide
Introduction
There is growing evidence for a novel cannabinoid receptor
in the vasculature. White and Hiley (1997a) showed that, in
the isolated mesenteric arterial bed of the rat, anandamide,
an endocannabinoid, causes vasorelaxation that is sensitive
to the cannabinoid receptor antagonist rimonabant (for-
merly designated SR141716A). However, although the major
active constituent of cannabis, D9-tetrahydrocannabinol
causes relaxation, this is insensitive to pertussis toxin and
the antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-di-
chorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251)
Correspondence: Dr CR Hiley, Department of Pharmacology, University of
Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
E-mail: crh1@cam.ac.uk
5These authors contributed equally to this work.
Received 31 May 2007; revised 28 August 2007; accepted 28 August 2007;
published online 24 September 2007

VSN16 pharmacology in rat mesenteric artery
752
PM Hoi et al
(O’Sullivan et al., 2005), which suggests that there is no CB1
receptor-mediated vasorelaxation in this vascular bed as the
CB1 receptor is sensitive to both the antagonist and the
acetonitrile/water (1:1, 20 ml) before being added to the
resin suspension and shaken for 20 min. The resin was
filtered off, washed with acetonitrile/water (3:1) and the
solvent removed from the filtrate under vacuum. The residue
was purified by short flash column chromatography on silica
gel (dichloromethane/MeOH/AcOH, 1–8% methanol gradi-
ent, with 1% AcOH) to yield the desired compound. As an
alternative (method A0), the treatment of the crude material
with DOWEX50 WX80 was performed in the presence of
methanol instead of acetonitrile/water (3:1).
toxin. Also rimonabant is
a high-potency antagonist
(Ki ¼ 21 nM) at CB1 receptors (Rinaldi-Carmona et al., 1995),
but high concentrations of 1 mM or more are required to
antagonize the vascular actions of anandamide (White and
´
Hiley, 1997a; Chaytor et al., 1999; Jarai et al., 1999).
Mesenteric arterial vasodilatation in rat can also be induced
by abnormal cannabidiol, a behaviourally inactive agent;
this relaxation is also sensitive to rimonabant (Ho and Hiley,
2003b) but occurs in mice lacking both the CB1 and CB2
Method B: general procedure for amide couplings. Triethyla-
mine (2 mmol) was added to a solution of the alkynoic acid
(1 mmol) in dry tetrahydrofuran (6 ml) under an N2 atmo-
sphere, and then cooled at À101C. To the reaction mixture,
ethyl chloroformate (1 mmol) was added and then stirred for
further 15 min at À101C. In the meantime, a solution of
amine hydrochloride (3 mmol), water (0.88 ml), triethyla-
mine (6 mmol) and tetrahydrofuran (1.76 ml) was prepared
and added dropwise to the reaction mixture. The reaction
was left warming up to 51C in 1.5 h and then stirred at room
temperature for a further 30 min. The mixture was poured
into a 1:1 mixture of saturated brine and saturated NaHCO3
(50 ml) and then extracted with dichloromethane (DCM;
5 Â 50 ml). The organic layer was evaporated under vacuum,
the residue was purified by short column chromatography
on silica gel (DCM/MeOH, 1–10% methanol gradient) to give
the desired compound.
´
receptors (Jarai et al., 1999). The vascular responses to
abnormal cannabidiol can be antagonized in a competitive
fashion by its analogue (À)-1,3-dimethoxy-2-(3-3,4-trans-p-
´
menthadien-(1,8)-yl)-orcinol (O-1918) (Offertaler et al.,
2003), which also antagonizes the vascular actions of
´
anandamide (Offertaler et al., 2003) as well as vasorelaxation
to other putative endocannabinoids such as virodhamine
(Ho and Hiley, 2004) and oleamide (Hoi and Hiley, 2006).
The behavioural effects of most cannabinoids, such as
sedation and the risk of the development of dependence,
together with their long pharmacokinetic half-lives, limit
their utility as therapeutic agents (Williamson and Evans,
2000; Pacher et al., 2006). Nevertheless, an extract of the
cannabis plant, with the trade name Sativex, has gained a
licence for use in pain relief in multiple sclerosis in Canada
although it is argued that additional benefit in this
preparation might be conferred by the presence at a carefully
controlled ratio not only of D9-tetrahydrocannabinol but
also of the behaviourally inactive cannabidiol (Smith, 2004).
The desirability of separating the behavioural effects of
cannabinoids from their therapeutic potential in diseases
such as multiple sclerosis has led to a search for novel
compounds. Here we report the vascular activity of one such
compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-
1-methyl-ethyl)benzamide (VSN16). This agent is water
soluble and we show that it causes vasorelaxation of a rat
resistance artery in a manner that is sensitive to both
O-1918 and rimonabant although it does not bind to rat
CB1 cannabinoid receptors; VSN16 is therefore a candidate
agonist at a novel vascular receptor for cannabinoids.
Method C: general method for Lindlar hydrogenation. Quino-
line (25ml, 0.21 mmol), palladium on barium sulphate
reduced (5%) (360 mg) and the alkyne (1 mmol) were
combined in methanol (15 ml) and stirred under atmo-
spheric pressure of hydrogen until the 1H NMR of the crude
showed that the reduction was complete. The catalyst was
removed by filtration through
a pad of celite, which
was washed several times with methanol. The filtrate was
evaporated under vacuum and the product was purified by
preparative HPLC.
Chemical synthesis of VSN16
N-(2-hydroxy-1-methyl-ethyl)-3-iodobenzamide 1: At room
Methods
temperature,
imide (EDCI: 40.3 mmol) was added to
1-ethyl-3-(30-dimethylaminopropyl)carbodi-
a
solution of
Chemistry: general synthetic procedures
3-iodobenzoic acid (40.3 mmol), in dry CH2Cl2 (180ml) under
an N2 atmosphere, followed by triethylamine (60.45 mmol).
The mixture was stirred at room temperature for a further
5 min before adding DL-alaninol (40.3 mmol) and subse-
quently stirring the mixture for 16 h. The reaction mixture
was washed with a mixture of saturated brine and saturated
NaHCO3 (1:1; 2 Â 150 ml) followed by saturated brine
(100 ml). The organics were separated and dried over MgSO4
and the solvent evaporated under vacuum. The residue was
purified by flash column chromatography on silica gel
(DCM/MeOH, 1–8% methanol gradient) to afford compound
1 (13.6 mmol, 34% yield; Figure 1) as an off-white solid.
d(1H)(CDCl3); 1.41 (3H, d, J 6.8 Hz), 3.70 (1H, dd, J1 5.5, J2
10.9 Hz), 3.80 (1H, dd, J1 2.9, J2 10.9 Hz), 4.38 (1H, m), 6.46
Method A: general procedure for Sonogashira coupling reac-
tion. Tetrakis(triphenyl-phosphine)palladium(0) (2 mol%)
and copper(I) iodide (7 mol%) were added to pyrrolidine
(15 ml) and stirred at room temperature under a nitrogen
atmosphere, for 5 min. N-(2-hydroxy-1-methyl-ethyl)-3-io-
dobenzamide 1 (1 mmol) was added and stirred for an
additional 15 min at room temperature. The alkyne (1 mmol)
was added and the reaction mixture was stirred at 601C for
3 h. The reaction mixture was concentrated under vacuum
and the residue treated with DOWEX50 WX80 (10 Â weight
of the starting material), which had been washed with
acetonitrile (3 Â 20 ml) and then suspended in a mixture of
acetonitrile/water (3:1, v vÀ1). The residue was dissolved in
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
753
Figure 1 Scheme showing the synthetic pathway of VSN16 and some analogues from 3-iodobenzoic acid via N-(2-hydroxy-1-methyl-ethyl)-
3-iodobenzamide (1) using the palladium-catalysed Sonogashira coupling reaction. Alkenes are shown in the Z configurations as these are the
forms isolated in the synthesis. Reagents: (a) EDCI, DCM, Et3N, room temperature; (b) (i) tetrakis(triphenylphosphine)palladium(0), Cu(I)I,
pyrrolidine, 601C; (ii) DOWEX50 WX80 acetonitrile; (c) (i) EtOCOCl, Et3N, À101C; (ii) Et3N, H2O, tetrahydrofuran, substituted amine HCL, 01C
to 51C; (d) quinoline, palladium on barium sulphate (5%), MeOH, H2 atmospheric pressure ; (e) borohydride polymer-supported,
(CH3COO)2Ni Á 4H2O, MeOH, H2 atmospheric pressure; (f) bis(triphenylphosphine)palladium(II) chloride, Cu(I)I, Et3N, DMF, 601C; (g)
DOWEX50 WX80 MeOH; (h) concentrated NH3, room temperature.
(1H, m), 7.27 (2H, t, J 7.8 Hz), 7.93 (1H, d, J 7.88 Hz), 8.21
(1H, s). d(13C) (CDCl3); 17.49 (CH3), 48.53 (C2), 67.19 (CH2),
94.59 (C), 126.79 (CH), 129.58 (CH), 130.62 (CH), 136.37
d(1H)(CDCl3); 1.29 (3H, d, J 6.8 Hz), 1.81–1.94 (2 H, m),
2.37–2.47 (4H, m), 2.91 (3H, s), 3.00 (3H, s), 3.38–3.64 (2H,
m) 4.19–4.43 (1H, m) 6.78 (1H, d, J 7.2 Hz), 7.29 (1H, t,
J 7.7 Hz), 7.42 (1 H, d, J 7.7 Hz), 7.68 (1H, d, J 7.8 Hz), 7.75
(1H, s). d (13C) (CDCl3); 17.42(CH3), 19.36 (CH2), 24.45
(CH2), 32.30 (CH2), 35.83(CH3), 37.67 (CH3), 48.51 (CH),
66.90 (CH2), 80.91 (C), 90.92 (C), 124.60 (C), 126.85 (CH),
128.85 (CH), 130.39 (CH), 134.58 (CH), 135.13 (C), 167.63
(C), 172.87(C). MS (ES) m/z 317 (M þ H).
.
(CH), 136.83 (C), 166.71(C). Calculated C10H11NO2I 1/2H2O:
C 38.23%, H 3.85%, N 4.46%; found: C 38.95%, H 3.80%,
N 4.08%.
6-[3-(2-Hydroxy-1-methyl-ethylcarbamoyl)phenyl]-hex-5-
ynoic acid 3: The iodobenzamide (1; Figure 1) (6.5mmol) was
coupled with 5-hexynoic acid using method A (Kadota et al.,
1999) giving product 3 (6.42 mmol, 99% yield; Figure 1).
d(1H)(CDCl3); 1.49 (3H, d, J 6.8 Hz), 2.14 (2H, t, J 7.2 Hz),
2.67-2.76 (4H, m), 3.83–3.90 (2H, m) 4.39–4.45 (1H, m) 7.64
(1H, t, J 7.7 Hz), 7.76 (1H, d, J 7.7 Hz), 7.99 (1H, d, J 7.8 Hz),
8.10 (1H, s). d(13C) (CD3OD); 17.47 (CH2), 19.99 (CH3), 36.25
(CH2), 66.54 (CH2), 81.82 (C), 91.53 (C), 126.03 (C), 128.05
(CH), 129.99 (CH), 131.75 (CH), 135.69 (CH), 136.58 (C),
168.538 (C). MS (CI) m/z 290 (M þ H).
3-(5-Dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-
ethyl)benzamide: By Lindlar-catalysed reduction using
method C, from alkyne 5 (0.10 g, 0.3 mmol) a mixture of
VSN16 and the fully reduced compound 3-(5-dimethylcar-
bamoyl-pentyl)-N-(2-hydroxy-1-methyl-ethyl)-benzamide
were obtained, which was separated by reverse-phase HPLC
chromatography (20% acetonitrile/80% water 20 min iso-
cratic program) (VSN16, 34 mg, yield 35%).
d(1H)(CDCl3); 1.31 (3H, t, J 6.8 Hz), 1.81–1.91 (2H, m),
2.26–2.39 (4H, m), 2.90 (3H, s); 2.98 (3H, s); 3.65 (2H, dd, J1
5.5, J2 11.2 Hz), 3.83 (2H, dd, J1 3.2, J2 11.2 Hz), 4.27–4.30
(1H, m), 5.68–5.77 (1H, m), 6.46 (1H, d, J 11.6 Hz), 7.24–7.33
3-(5-Dimethylcarbamoyl-pent-1-ynyl)-N-(2-hydroxy-1-methyl-
ethyl)benzamide 5: 6-[3-(2-hydroxy-1-methyl-ethylcarba
moyl)phenyl]-hex-5-ynoic acid 3 (Figure 1) (0.377 mmol) was
reacted with dimethylamine hydrochloride using method B
to obtain 5 (0.115 g, 0.363 mmol; 96% yield).
(1H, m), 7.38 (1H, d,
J 7.6 Hz), 7.74–7.79 (2H, m).
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
754
PM Hoi et al
d(13C)(CDCl3); 16.93 (CH3), 24.80 (CH2), 28.22 (CH2), 32.51
(CH2), 35.73 (CH), 37.45 (CH), 48.32 (CH2), 66.73 (CH2),
126.20 (CH), 126.35 (CH), 128.58 (CH), 129.12 (CH), 131.88
(CH), 132.63 (CH), 134.70 (C), 137.5 (C), 168.00(C),
173.11(C). Theoretical mass: (M þ H) 318.19433. Measured
mass: (M þ H) 318.19507 MS (ES) m/z 319 (M þ H).
based on those showing specific inhibition in previous
experiments (White and Hiley, 1997a, 1998a, b; Ho and
Hiley, 2003b). None of these agents, except O-1918, had any
effect on the tone induced by methoxamine; the mean
tension in the test for endothelial function was 15.770.5 mN
as compared with 15.670.6 mN (n ¼ 64) after preincubation
with the antagonists or inhibitors. For O-1918 (30 mM), the
mean tension developed by methoxamine in the endothelial
integrity test was 14.670.7 mN and was 17.470.7 mN after
application of the antagonist (n ¼ 15). A vehicle control with
dimethyl sulfoxide (DMSO) was obtained for all compounds
(except VSN16, which was dissolved in water) by adding an
appropriate volume of vehicle alone to methoxamine-
precontracted vessels. Since considerable variations were
observed in the relaxant responses to the novel compounds,
all the experiments were done in paired fashion with control
and test experiments conducted in vessels from the same
branch of artery from the same animal.
Myograph studies
Male Wistar rats (300–400 g; Charles River UK Ltd, Kent, UK)
were killed with an overdose of sodium pentobarbital
(120 mg kgÀ1, i.p.; Sagatal, Rhone Merieux, Harlow, Essex,
UK); all animal care and use was in accordance with the UK
Animal (Scientific Procedures) Act 1986. After rapid removal
of the mesenteric arterial bed, it was placed into cold Krebs–
Henseleit buffer solution of the following composition (mM):
NaCl, 118; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25;
CaCl2, 2.5; D-glucose, 5.5. The Krebs–Henseleit solution also
contained 10 mM indomethacin and was bubbled with 95%
O2/5% CO2 to give a pH of 7.4.
ˆ
´
Third-generation (small) mesenteric arteries (internal
diameter, 29473 mm; basal tone after normalization,
3.970.1 mN, 240 vessels) were then dissected free and
cleaned of adherent tissue. Two-millimetre-long segments
were mounted in a wire myograph (Danish Myo Technology,
Aarhus, Denmark) maintained at 371C in Krebs–Henseleit
solution, gassed with 95% O2/5% CO2, and normalized as
described previously (White and Hiley, 1997a). Tension was
measured and recorded on a PowerLab recording system (AD
Instruments, Hastings, Sussex, UK) connected to an Apple
Macintosh computer. The presence of a functional endothe-
lium was tested by precontracting the vessels with methox-
amine (10 mM), and then adding carbachol (10 mM) to cause
relaxation; an intact endothelium was shown by relaxations
490% of the methoxamine-induced precontraction. When
the endothelium was not required, vessels were denuded by
Data and statistical analysis for myograph experiments
Responses are expressed as the percentage relaxation of the
precontraction induced by 10 mM methoxamine. Data are
given as the mean7s.e.m. and n indicates the number of
rats. When a defined maximum relaxation response was
observed, the data were fitted to a logistic equation of the
following form:
R ¼ ðRmax Á An Þ=ðEC50 Á An þ An
Þ
H
H
H
where R is the reduction in tone, A the concentration of the
agonist, Rmax the maximal reduction of established tone,
nH the slope function and EC50 the agonist concentration
giving half the maximal relaxation. The curve-fitting was
carried out using KaleidaGraph (Synergy Software, Reading,
PA, USA).
When concentration–relaxation curves did not reach a
clearly defined maximal relaxation, thus rendering curve-
fitting inappropriate, the mean EC50% was determined
whenever possible. The EC50% was determined from the
individual experimental concentration–response curves as
the concentration of agonist required to produce 50%
relaxation of the tone induced by methoxamine.
rubbing the intimal surface with
a human hair, and
successful endothelial removal was confirmed by absence
of a vasorelaxant response (o10% of the precontraction) to
carbachol.
Experimental protocols in the myograph
Statistical comparisons of concentration–response curves
were made by two-way analysis of variance of the whole data
set, followed by the Bonferroni post-hoc test for determining
significant differences between treatment groups (StatView
4.5 for the Macintosh; Abacus Concepts Inc., Berkeley, CA,
USA). P-values less than 0.05 were considered to be
statistically significant.
After determination of the presence or absence of endothe-
lium, vessels were left for 15–30 min and then precontracted
submaximally with 10 mM methoxamine (yielding approxi-
mately 80% of the maximal response). When a stable level
of tone was obtained (mean, 15.170.3 mN; n ¼ 170), concen-
tration–response curves were generated by cumulative addi-
tion of the compound under investigation and each
concentration was left for 10 min before addition of the
next one.
Where the effect of VSN16 was investigated in the
presence of antagonists (rimonabant, AM 251, O-1918 or
capsazepine), apamin, charybdotoxin, NG-nitro-L-arginine
methyl ester (L-NAME) or capsaicin, these agents were added
to the organ bath 30 min before, and then were present
during, the construction of the concentration–response
curve. Note that in the case of pertussis toxin, preincubation
was for 2 h. The concentrations of these inhibitors were
Haemodynamic studies
Male Sprague–Dawley rats (n ¼ 8, 380–450 g, Charles River
UK) were anaesthetized with fentanyl and medetomidine
(300 mg kgÀ1 of each i.p.) and had miniaturized pulsed
Doppler flow probes sutured around the left renal and
superior mesenteric arteries, and around the distal abdom-
inal aorta (to monitor hindquarters flow). After surgery,
anaesthetic reversal and the provision of analgesia was
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
755
achieved using atipamezole and buprenorphine (1 mg kgÀ1
and 0.02 mg kgÀ1, respectively, s.c.). At least 10 days after
probe implantation, animals were re-anaesthetized (fentanyl
and medetomidine, as above), and catheters were implanted
in the distal abdominal aorta (via the ventral caudal artery)
for monitoring mean arterial blood pressure and heart rate,
and in the right jugular vein for the administration of
substances. Anaesthesia was reversed and analgesia provided
using atipamezole and buprenorphine as above. The proce-
dures were approved by the University of Nottingham
Ethical Review Committee and performed under a Home
Office Project Licence; the fitness of the animals between
surgical stages was certified by the Named Veterinary
Surgeon.
concentration was determined by a BioRad (Hemel Hemp-
stead, Hertfordshire, UK) protein assay.
Binding assays were carried out in triplicate in a final
volume of 1 ml buffer (50 mM Tris–HCl, 1 mM EDTA, 3 mM
MgCl2, 3 mg mlÀ1 bovine serum albumin, pH 7.4). Cerebellar
membrane (50 mg protein) was incubated with 62 pM [3H]CP
55940 and non-specific binding (B30–50% of the total) was
determined as the residual binding in the presence of
rimonabant or unlabelled (À)-cis-3-[2-hydroxy-4-(1,1di
methylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol
(CP 55940) (1 mM for both). Incubation was for 60 min at
301C and the reaction was terminated by the addition of ice-
cold wash buffer (10 mM Tris buffer, pH 7.4) and vacuum
filtration using a 24-well Brandel cell harvester (SEMAT
Technical (UK) Ltd, St Albans, Hertfordshire, UK) and GF/B
filters (Whatman, Maidstone, Kent, UK) that had been
presoaked in wash buffer at 41C for 2 min. After drying the
filters for 10 min, they were placed in 4 ml of Emulsifier-Safe
(Perkin-Elmer, Waltham, MA, USA) for more than 3 h before
measuring the radioactivity bound by liquid scintillation
spectrometry.
Experimental protocol for haemodynamics
Experiments were run 48 h following catheterization when
the animals were fully conscious and moving freely, with
access to food and water ad libitum. After a period of baseline
recording of at least 60 min, (R)-3-(5-dimethylcarbamoyl-pent-
1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16-R)
was administered as i.v. bolus doses (0.1 ml) of 1, 3 and
15 mg kgÀ1. The order of dose administration was rando-
mized with at least 3 h between doses.
Data analysis of radioligand binding
Results are expressed as the percentage inhibition of specific
binding of the cannabinoid agonist, [3H]CP 55940. Data are
given as the mean7s.e.m. and n indicates the number of
membrane preparations from different groups of rats. When
possible data were fitted to a logistic equation:
Data acquisition and analysis in haemodynamic studies
Continuous recordings of cardiovascular variables were
made using a customized, computer-based system (Haemo-
dynamics Data Acquisition System; University of Limburg,
Maastricht) connected to a transducer amplifier (Gould
model 13-4615-50) and a Doppler flowmeter (Crystal Biotech
VF-1 mainframe (pulse repetition frequency 125 kHz) fitted
with high-velocity (HVPD-20) modules). Raw data were
sampled by the haemodynamics data acquisition system
every 2 ms, averaged every cardiac cycle, and stored to disc at
5-s intervals. Data were analysed offline using software
(Datview, University of Limburg, Maastricht), which inter-
faced with the haemodynamics data acquisition system.
Values were then exported into custom-designed software
(Biomed, University of Nottingham) for statistical analysis
using Friedman’s test.
nH
nH
nH
I ¼ ðImax Á ½Lꢀ Þ=ðIC50 þ ½Lꢀ
Þ
where I is the percentage inhibition of [3H]CP 55940 binding
to the rat membrane preparation, [L] the concentration of
the competing ligand, Imax the maximal inhibition of
radioligand binding, nH the Hill slope and IC50 the
concentration of the competing ligand that reduced the
specific binding by 50%. Curve-fitting was carried out as
before and Ki values were determined from IC50 values using
a Kd of 0.36 nM for [3H]CP 55940 determined by competition
against unlabelled CP 55940 (see Results). Statistical compar-
ison of concentration–inhibition curves was as for myo-
graph-obtained relaxation curves.
Drugs
Binding experiments with [3H]CP 55940
Methoxamine hydrochloride, carbachol, charybdotoxin,
L-NAME (Sigma Chemical Company, Poole, Dorset, UK) and
apamin (Calbiochem, Nottingham, UK) were dissolved in
deionized water as was the VSN16 synthesized as described
Male Wistar rats (250–350 g; Charles River UK) were killed
by CO2 asphyxiation (under Schedule 1 of the UK Animal
(Scientific Procedures) Act 1986) and decapitated. Brains
were removed quickly and the cerebella were dissected for
preparation of a membrane homogenate from fresh or
thawed tissue which had been frozen at À801C until use.
The cerebella were suspended in ice-cold buffer (50 mM Tris–
HCl, 1 mM EDTA, 3 mM MgCl2, pH 7.4) and homogenized
in a glass mortar using 15 strokes of a Teflon pestle. The
homogenate was centrifuged at 1000 g at 41C for 5 min, the
pellet was discarded and the supernatant was centrifuged
at 13 000 g for 30 min at 41C. The resulting pellet was
resuspended in 5 ml homogenization buffer and the protein
above. Indomethacin (Sigma) was dissolved in 5% (w vÀ1
)
NaHCO3 solution. Capsaicin (Sigma), rimonabant (a gener-
´
ous gift from Sanofi-Synthelabo, Montpellier, France), AM
251 (Tocris Cookson, Bristol, UK), CP 55940 (Tocris Cook-
son) and O-1918 (a generous gift from Dr George Kunos,
National Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, Bethesda, MD, USA) were
dissolved in 100% ethanol. For haemodynamic studies,
fentanyl citrate was from Janssen-Cilag (High Wycombe,
Buckinghamshire, UK); medetomidine hydrochloride
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VSN16 pharmacology in rat mesenteric artery
756
PM Hoi et al
(Domitor) and atipamezole hydrochloride (Antisedan) were
from Pfizer (Sandwich, Kent, UK); buprenorphine (Veter-
gesic) was from Alstoe Animal Health (York). [side chain-
several different analogues by reacting alkyl 11 with different
amines as shown in Figure 2.
2,3,4(N)-3 H]CP 55940 (specific activity 6.66 TBq mmolÀ1
)
was purchased from Perkin-Elmer.
Vasorelaxant effects of VSN16 and its enantiomers
(R)-3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-
methyl-ethyl)benzamide (VSN16-R), (S)-3-(5-dimethylcarba-
moyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide
(VSN16-S) and VSN16 (the latter being used to designate the
racemic mixture of the enantiomers) had similar potency at
relaxing methoxamine-induced tone in the endothelium-
Results
Chemistry
VSN16 is a water-soluble (420 mg mlÀ1) benzamide deriva-
tive that was designed to be a cannabinoid-like compound
that would not show central actions. The synthetic route for
the synthesis of benzamide analogues utilizes simple,
versatile chemistry, the key step is the palladium-catalysed
Sonogashira coupling reaction, which is used to insert a
variety of alkyl side chains into the N-(1-hydroxypropan-2-
yl)-3-iodobenzamide 1 (Figure 1). 3-Iodobenzoic acid was
reacted with DL-alaninol in the presence of a diimide (EDCI)
to give amide 1 in good yield. Palladium-catalysed coupling
of the amide with the alkyne acid in the presence of Cu(I)I
and pyrrolidine proceeded smoothly to give alkynes 2 and 3.
The acids were quantitatively transformed into 4, 5, 6 using
ethylchloroformate and the appropriate substituted amine.
Lindlar-catalysed reduction (Hopper et al., 1998) of alkyne 5
or 6 yielded the alkene 7, VSN16 or 8. Sonogashira coupling
of hex-5-yne nitrile with 1 provided 9 after Lindlar reduc-
tion. When acid 2 was treated with DOWEX50 WX80 in
methanol, the corresponding methyl ester was obtained and
the unsubstituted amide 10 by aminolysis. We obtained
intact small mesenteric artery of the rat (VSN16-R: EC50
110769 nM, Rmax ¼ 91.679.6%; VSN16-S: EC50 ¼ 140717 nM,
VSN16: Rmax
¼
Rmax ¼ 71.071.8%;
EC50 ¼ 8873 nM,
¼
86.776.4%; n ¼ 6 for all; Figure 3). Statistical analysis shows
that VSN16-R is a little more potent than VSN16-S (Po0.05),
although there was no significant difference in potency
between VSN16-R and the racemic mixture.
Effects of cannabinoid receptor antagonists
Figure 4a shows that O-1918 (30 mM), an antagonist for the
novel cannabinoid receptor in the vasculature, markedly
affected the vasorelaxation to VSN16. There was a 20-fold
shift to the right of the concentration–response curve and
the maximal relaxation was also reduced to about half the
control value (control: EC50 ¼ 1773 nM, Rmax ¼ 80.972.6%;
O-1918: EC50 ¼ 341791 nM, Rmax ¼ 36.272.5%; n ¼ 5;
Po0.01). The cannabinoid receptor antagonist rimonabant
(3 mM) also produced a prominent reduction of the relaxation
Figure 2 Scheme showing the synthetic pathway to obtain the enantiomers of VSN16 (*the chiral centre) and some analogues from the ester
of 3-iodobenzoic acid via the alkyl 11 using the appropriate amines for the coupling reaction. Alkenes are shown in the Z configurations as
these are the forms isolated in the synthesis. Reagents: (a) (i) tetrakis(triphenylphosphine)palladium(0), Cu(I)I, pyrrolidine, 601C; (ii) DOWEX50
WX80 acetonitrile; (b) (i) EtOCOCl, Et3N, À101C; (ii) Et3N, H2O, tetrahydrofuran, substituted amine HCL, 0–51C; (c) NaOH 1 M water; (d)
EDCI, DCM, Et3N, room temperature; (e) quinoline, palladium on barium sulphate (5%), MeOH, H2 atmospheric pressure; (f) borohydride
polymer-supported (CH3COO)2Ni Á 4H2O, MeOH, H2 atmospheric pressure.
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VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
757
control, 60.875%; apamin with charybdotoxin and
L-NAME, 7.373%; n ¼ 4; Po0.01).
-20
0
VSN16
VSN16-R
VSN16-S
Involvement of TRPV1
Both functional desensitization of the vanilloid system by
capsaicin (10 mM pretreatment for 30 min) and preincubation
with capsazepine (an antagonist at the vanilloid TRPV1
receptor; 3 mM) significantly inhibited the relaxation induced
by VSN16 (control: EC50 ¼ 1472 nM, Rmax ¼ 76.872.2%;
capsaicin: EC50 ¼ 643745 nM, Rmax ¼ 66.578.0%; capsaz-
epine: EC50 ¼ 55712 nM; Rmax ¼ 54.372.2%; both n ¼ 4;
Po0.05; Figure 4e). In addition, while the capsaicin
pretreatment or incubation with O-1918 alone did not
completely abolish the relaxant response induced by
VSN16, combination of the two had an additive effect,
20
40
60
80
100
further reducing the relaxation and leaving
a residual
relaxation at 3 mM VSN16 of 14.772.9% (n ¼ 3; Figure 4e).
10-10
10-9
10-8
10-7
10-6
10-5
Concentration of VSN16 (M)
Effects of pertussis toxin on VSN16-induced relaxation
The relaxation produced by VSN16 was not inhibited by
preincubation with pertussis toxin at a concentration of
400 ng mlÀ1 for 2 h (relaxation by VSN16 at 10 mM: control,
58.174.0%; pertussis toxin, 53.174.0%; n ¼ 4; P40.05). This
same treatment and batch of toxin did inhibit relaxation to
oleamide in another study carried out during this work (Hoi
and Hiley, 2006).
Figure 3 Concentration–response curves for relaxation of methox-
amine-induced tone by VSN16 and its enantiomers in the isolated
small mesenteric artery of the rat. Relaxation was determined in the
presence of a functional endothelium (n ¼ 6 for all). Values are
shown as means and vertical lines represent the s.e.m. from paired
experiments. The curves drawn are those obtained from the curve-
fitting procedure and values for Rmax and EC50 are given in the text.
leaving a residual relaxation of only 24.675.2% (n ¼ 4;
Po0.01; Figure 4b). Similarly, the selective CB1 receptor
antagonist, AM 251 (1 mM), blocked the VSN16-induced
response with a residual relaxation of 25.274.8% at the
highest concentration of VSN16 available (n ¼ 4; Po0.01;
Figure 4b). A lower concentration of AM 251 (0.1 mM) had
no significant effect on relaxations to VSN16 (control:
EC50 ¼ 35717 nM; AM 251: EC50 ¼ 52726 nM; n ¼ 5).
Haemodynamic studies with VSN16
Resting cardiovascular variables before administration of
VSN16-R at 1, 3 and 15 mg kgÀ1 were: heart rate, 36978,
364710 and 380713 beats minÀ1
; mean arterial blood
pressure, 10873, 10872 and 10871 mmHg; renal vascular
conductance, 10079, 9579 and 97710 (kHz mmHgÀ1) 103;
mesenteric vascular conductance, 6175, 5674 and 6374
(kHz mmHgÀ1
)
103; hindquarters vascular conductance,
4973, 5175 and 4974 (kHz mmHgÀ1) 103.
Role of the endothelium and endothelial factors
At doses of 1 and 3 mg kgÀ1, there were no significant
cardiovascular effects of VSN16-R (Figure 5). At a dose of
15 mg kgÀ1, there was a prompt and short-lived rise in blood
pressure (Po0.05 between 1 and 4 min) and fall in renal
vascular conductance (Po0.05 between 1 and 3 min), and a
slower onset, longer-lasting rise in mesenteric vascular
conductance (Po0.05 between 10 and 25 min); there were
no changes in hindquarters vascular conductance or heart
rate (Figure 5).
VSN16-induced vasorelaxation was abolished by the removal
of endothelium (n ¼ 4; Figure 4c) but the presence or absence
of indomethacin (10 mM) did not have any significant effect
(relaxation by VSN16 at 10 mM: control, 59.674%; indo-
methacin, 57.374%; n ¼ 6; P40.05). In contrast, Figure 4d
shows that inhibition of nitric oxide synthase by L-NAME
caused a rightwards shift of the concentration–response
curve (Po0.01); although solubility limitations prevented
definition of a true maximum response, the shift at the
level of 50% relaxation of tone was 3.1-fold (control:
EC50% ¼ 0.3570.05 mM; L-NAME: EC50% ¼ 1.170.1 mM; n ¼ 3).
Inhibition of the Ca2 þ -sensitive Kþ channels (KCa) that
are associated with the activity of endothelium-dependent
hyperpolarizing factor, using a combination of apamin and
charybdotoxin (both at 50 nM), significantly inhibited the
relaxation induced by VSN16 (relaxation by 10 mM VSN16:
control, 60.875%; apamin and charybdotoxin, 13.773%;
n ¼ 8; Po0.01). Addition of L-NAME (300 mM) to the combin-
ation of apamin plus charybdotoxin further reduced the
relaxant response by VSN16 (relaxation by 10 mM VSN16:
Relaxant effects of analogues of VSN16
A structure–activity study was undertaken with a series of
analogues of VSN16. As shown in Figure 6, only compound 9
(with code number VSN15) showed a vasorelaxant effect
that, at a concentration of 30 mM, was significantly greater
than that evoked by the DMSO vehicle. Some vasorelaxation
was seen with compound 7, but this did not reach
significance relative to the DMSO control. For compound
9, the vasorelaxant effect was concentration-dependent but,
as a maximal response was not found, an EC50 could not be
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
758
PM Hoi et al
-20
0
-20
0
20
40
60
80
100
20
40
60
80
100
Control
+ AM 251 (1µM)
+ Rimonabant (3µM)
Control
+ O-1918 (30µM)
-10
-9
10
-8
10
-7
10
-6
10
-5
-10
-9
10
-8
10
-7
10
-6
10
-5
10
10
10
10
Concentration of VSN16 (M)
Concentration of VSN16 (M)
-20
0
-20
0
20
40
60
80
100
20
40
60
80
100
Endothelium intact
Endothelium denuded
Control
+ L-NAME (300µM)
-10
10
-9
10
-8
10
-7
10
-6
10
-5
-10
10
-9
10
-8
10
-7
10
-6
10
-5
10
10
Concentration of VSN16 (M)
Concentration of VSN16 (M)
-20
0
20
40
60
Control
+ Capsaicin (10µM)
80
+ Capsazepine (3µM)
+ Capsaicin (10µM) and O-1918 (30µM)
100
-10
-9
10
-8
10
-7
10
-6
10
-5
10
10
Concentration of VSN16 (M)
Figure 4 Effects of endothelial removal, inhibitors and cannabinoid antagonists on the concentration–response curves for VSN16-induced
relaxation of methoxamine-induced tone in the isolated small mesenteric artery of the rat. All vessels had an intact endothelium except where
stated. (a) Antagonism by O-1918 (30 mM, n ¼ 5). (b) Antagonism by AM 251 (1 mM) or rimonabant (3 mM) (both n ¼ 4). (c) Removal of
endothelium abolished the relaxant response to VSN16 (n ¼ 4). (d) Effect of L-NAME (300 mM; n ¼ 3). (e) Effects of pretreatment with capsaicin
for 30 min (10 mM), or of the presence of capsazepine (3 mM) or combination of capsaicin pretreatment and O-1918 (30 mM) (n ¼ 3–4). All values
are shown as means and vertical lines represent the s.e.m. from paired experiments; n shows the number of animals in each data set. The
curves drawn are obtained from the curve-fitting procedure and values for Rmax and EC50 are given in the text.
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
759
0
20
40
*
60
Compound 7
Compound 12
Compound 13
Compound 20
DMSO vehicle
Compound 8
Compound 9
Compound 10
80
100
10-9
10-8
10-7
10-6
10-5
10-4
Concentration (M)
Figure 6 Concentration–response curves for relaxation of methox-
amine-induced tone by analogues of VSN16. Relaxation was
determined in the presence of a functional endothelium (n ¼ 4–6).
The effects of the appropriate amounts of vehicle (dimethyl
sulfoxide; DMSO) are also shown; the highest amount of vehicle
added gave 0.6% v vÀ1 in the bath. Values are shown as means and
vertical lines represent the s.e.m. from paired experiments.
*Significantly different from the relaxation obtained with the DMSO
vehicle alone (Po0.05).
Figure 5 Haemodynamic effects of VSN16-R, administered i.v.,
in conscious rats. Values are shown as means and vertical lines
represent the s.e.m. (n ¼ 8). *Significantly different from the control
value (Po0.05).
relaxant activity after VSN16, significantly inhibited the
specific binding of [3H]CP 55940 by 2575% at a concentra-
tion of 100 mM (n ¼ 4) but compound 7, which showed
some vasorelaxant activity, did not (inhibition at
100 mM ¼ À4715%; n ¼ 4).
determined. However, it is B80-fold less potent than the
parent VSN16 as its EC50% of approximately 28 mM compares
to 0.35 mM for VSN16 (cf Figure 4).
Discussion
VSN16 was designed to be a cannabinoid-like compound
which would not show central actions and which might be
beneficial in multiple sclerosis. It is water-soluble, unlike the
analogues also tested here, and, in addition to neuronal
effects to be reported elsewhere, we have now found it to
induce vasorelaxation in the isolated third-generation
mesenteric artery of the rat. Its actions show only slight
stereoselectivity, are strictly endothelium-dependent, appar-
ently involve the vanilloid receptor system and are sensitive
to cannabinoid antagonists including one (O-1918) active at
a currently unidentified cannabinoid receptor in the vascu-
Effects on [3H]CP 55940 binding
Unlabelled CP 55940 inhibited the binding of [3H]CP 55940
according to
a
simple one-site competition model
(Ki ¼ 0.3670.05 nM, nH ¼ 0.7670.05, n ¼ 3) and the maximal
inhibition was 5575% of the total bound [3H]CP 55940. The
cannabinoid receptor antagonist rimonabant inhibited the
specific binding of [3H]CP 55940 to rat cerebellar membranes
in
a
concentration-dependent manner (Ki ¼ 9.071.3 nM,
nH ¼ 0.8170.07, n ¼ 5). The maximum inhibition by rimo-
nabant at 1 mM was 5674% of the total bound; since this is
comparable to the effect of CP 55940, rimonabant (1 mM) was
used to determine non-specific binding in further experi-
ments. The putative antagonist at the vascular cannabinoid
receptor, O-1918, showed limited inhibition of radioligand
binding (1571% of the specific binding at 30 mM, the highest
concentration that could be used). VSN16 (300 mM) did not
inhibit [3H]CP 55940 binding to rat cerebellar membranes
(inhibition ¼ À1575%; n ¼ 4). Interestingly, compound 9,
the compound in the series showing the greatest vaso-
´
lature (Offertaler et al., 2003).
The role of cannabinoid receptors
The endothelium-dependent vasorelaxation induced by
VSN16 was antagonized by two CB1 receptor antagonists,
rimonabant (Showalter et al., 1996) and AM 251 (Lan et al.,
1999). A similar reduction in the relaxation at the highest
agonist concentration available brought about by both
antagonists could indicate involvement of the cannabinoid
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VSN16 pharmacology in rat mesenteric artery
760
PM Hoi et al
CB1 receptor. However, this is unlikely because, firstly, a
lower concentration of AM 251 (100 nM), which might be
expected to give a shift of over 100-fold in the concentra-
tion–response curve based on its Kdo1 nM (Gatley et al.,
1997), did not antagonize the responses to VSN16. Secondly,
CB1 receptors, even if they are present in some parts of the
vasculature (Darker et al., 1998; Ho and Hiley, 2004), have
not been reported to contribute to any vasodilator action
in the rat mesenteric vasculature (White and Hiley, 1998a;
Wagner et al., 1999; O’Sullivan et al., 2005).
like compounds in the production of vasorelaxation by
VSN16. However, the nature of these receptors cannot be
clearly identified, as the relaxant effect of VSN16 is not
consistent with action at CB1 receptors. Nor does its
pharmacology agree totally with the profile of the putative
endothelial abnormal cannabidiol receptor in the mesentery
as the latter has been reported to be insensitive to AM 251
(Ho and Hiley, 2003b). Clearly, further detailed investigation
is necessary for characterizing the cannabinoid receptor,
or receptors, involved in these responses. Thus far no other
receptor has been definitively identified as a cannabinoid
receptor although the orphan receptor GPR55 has been put
forward as a candidate (Baker et al., 2006). It has been
contended that GPR55 is unlikely to be a cannabinoid
receptor on the basis of the ‘functional fingerprint’ of amino
acids involved in ligand binding at CB1 and CB2 receptors
(Petitet et al., 2006), but the value of this particular analysis is
questionable as cannabinoid ligands are known to bind to
GPR55 (Brown and Wise, 2001; Drmota et al., 2004). It would
be interesting to determine the actions of VSN16 at this
receptor, especially as its mRNA has been identified in whole
rat mesenteric artery (Hoi, 2007). Indeed, we have some
preliminary data that show that, at a concentration of 10 mM,
it increases intracellular Ca2 þ in HEK293 cells stably
expressing GPR55 in a manner that is sensitive to rimona-
bant (3 mM) but not pretreatment with pertussis toxin.
(Values as fold increase over baseline7s.e.m. Control:
1.7070.06, n ¼ 30; with rimonabant: 1.0670.02, n ¼ 30,
Po0.001, Student’s t-test. Control: 1.7670.10, n ¼ 38; after
pretreatment for 20 h with toxin: 1.8570.02, n ¼ 41, not
significant.)
The interpretation of the effects of the antagonists is not
simple as they did not produce a parallel shift in the
concentration–response curve but rather depressed the
maximum response. However, the lack of documented CB1
receptor-mediated responses in the third-generation mesen-
teric artery of the rat as well as the observation that the
responses to VSN16 are insensitive to pertussis toxin (while
CB1 receptor-mediated responses are sensitive to the toxin;
Matsuda et al., 1990) indicate that its actions are not likely to
be due to CB1 receptor activation.
The absence of involvement of the CB1 receptor in the
responses to VSN16 is further substantiated by the results
with [3H]CP 55940 binding to rat cerebellar membranes. Rat
cerebellum has been shown to be a tissue rich in CB1
receptors whereas the CB2 receptor is very sparse (Griffin
et al., 1999) and the affinity observed for rimonabant in rat
cerebellum in this study is similar to those reported in the
literature for its binding to CB1 receptors. Here, the Ki value
for rimonabant was 9.0 nM, which compares with those
reported at stably expressed human CB1 receptors (5.6–
12.3 nM; see Howlett et al., 2002 for review), though a
slightly higher affinity at rat brain CB1 receptors
(Ki ¼ 1.98 nM) was reported by Rinaldi-Carmona et al.
(1994). The absence of any inhibitory effect of VSN16 on
specific [3H]CP 55940 binding shows that it does not bind to
the CB1 receptor in rat tissues.
Processes involved in vasorelaxation to VSN16
The relaxant response to VSN16 was sensitive to the
inhibition of nitric oxide synthase by L-NAME, though there
also was a role for KCa as the responses were sensitive to the
combination of apamin with charybdotoxin; this combin-
ation has been extensively used to show a role for endo-
Interestingly, the vasorelaxation induced by VSN16 was
also inhibited by O-1918, which has been proposed by
´
Offertaler et al. (2003) to be a selective antagonist for the
´
putative endothelial ‘abnormal-cannabidiol receptor’ (Jarai
thelium-dependent hyperpolarization in a vasorelaxant
et al., 1999) or ‘anandamide receptor’ (Wagner et al., 1999);
effect (Chataigneau et al., 1998). As the responses were
abolished by removal of the endothelium, VSN16 is the first
cannabinoid-like agonist that displays an entirely endothe-
lium-dependent vasorelaxation. Other cannabinoids that
have been investigated until now induce relaxation that is
wholly or partly endothelium-independent (for example, CP
55940, HU 210, palmitoylethanolamide and WIN 55212-2;
White and Hiley, 1997a, b, 1998a: anandamide; Ho and
O-1918 blocks the action of both abnormal cannabidiol and
´
anandamide in rat blood vessels (Offertaler et al., 2003) and,
as shown here, has a very low binding potency at rat
cerebellar CB1 receptors. Furthermore, the concentrations of
rimonabant and AM 251 used in this study to show
antagonism are much higher than those needed to block
CB1 receptors, and sensitivity to high concentrations of
rimonabant is a feature of responses mediated by the
postulated vascular cannabinoid receptor (Ho and Hiley,
2003b).
´
Hiley, 2003a: abnormal cannabidiol; Offertaler et al., 2003;
Ho and Hiley, 2003b: virodhamine, Ho and Hiley, 2004).
These cannabinoids and endocannabinoids therefore show a
wide range of mechanisms to bring about their vascular
actions that are sometimes very markedly different. For
example, unlike the vasorelaxation caused by VSN16,
responses induced by abnormal cannabidiol are not affected
by L-NAME but are similar in that they are sensitive to
The relaxant response to VSN16 was entirely dependent on
a functional endothelium. It therefore resembles those to
anandamide and abnormal cannabidiol in that the vaso-
relaxation they induce shows some endothelium depen-
´
dence and sensitivity to rimonabant (Offertaler et al., 2003).
´
Therefore, VSN16 could be an agonist for a novel endothelial
cannabinoid receptor, or receptors, activated by anandamide
or abnormal cannabidiol. Taken together, there is strong
evidence for the involvement of receptors for cannabinoid-
apamin plus charybdotoxin (Offertaler et al., 2003; Ho and
Hiley, 2003b). In the absence of a certain identification of
the receptor, or receptors, mediating the endothelium-
dependent responses to the cannabinoids, it is difficult to
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VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
761
untangle these diverse mechanisms to see how they are
activated by the receptors concerned. The fact that VSN16 is
entirely endothelium-dependent in its actions might make it
a more useful agent in resolving the pathways involved than
those compounds, which show both endothelium-mediated
and -independent mechanisms.
endocannabinoid is due to activation of TRPV1 leading to
the release of the vasodilator calcitonin gene-related peptide
from sensory nerves (Zygmunt et al., 1999). In this respect,
functional studies on sensory neurogenic vasorelaxation to
electrical field stimulation in the rat-isolated mesenteric
arterial bed have provided evidence that non-CB1, non-CB2
cannabinoid receptors might exist on capsaicin-sensitive
sensory nerves in the rat mesenteric bed (Ralevic and
Kendall, 2001; Duncan et al., 2004). However, since the
responses to VSN16 were completely abolished by removal of
the endothelium, it seems unlikely that the cannabinoid
receptors of the sensory nerves are involved in the vasodi-
lator response that it induces in the rat mesenteric artery.
The situation concerning the inhibition of VSN16 func-
tion with other cannabinoid and vanilloid receptor-related
antagonists currently appears complex and will remain so
until the identification of the target and its signalling
cascade. While CB1- and TRPV1-binding agents modified
VSN16 function, currently VSN16 cannot be shown to bind
to either of these receptors (see above for results on the CB1
receptor). This suggests either that VSN16 may bind to
allosteric sites or there is a complex interaction of the
different receptor systems within a cascade that controls
vasodilatation or that the current reagents have off-target
specificities. It is already known that rimonabant has actions
at other than CB1 receptors (White and Hiley, 1998b) and
can inhibit the actions of abnormal cannabidiol at the
As with some endocannabinoids, relaxation to VSN16 was
sensitive to capsazepine, a competitive antagonist of TRPV1,
and to functional desensitization of the vanilloid receptor by
capsaicin pretreatment; both drugs significantly reduced the
maximum response and, moreover, to similar levels. VSN16
therefore shares with anandamide (Zygmunt et al., 1999; Ho
and Hiley, 2003a) and its non-hydrolysable derivative
methanandamide (Ralevic et al., 2000), the property of
acting through the TRPV1 receptor system. This ability to
act through vanilloid receptors is shared by a few com-
pounds structurally related to each other by having an amide
bond and an aliphatic side chain. These include olvanil (N-
vanillyloleamide: Szallasi and Blumberg, 1999) and the
putative endocannabinoid N-arachidonoyl dopamine
(Huang et al., 2002; O’Sullivan et al., 2004) and they are
clearly quite different structurally from VSN16. In contrast,
other endocannabinoids including 2-arachidonoyl glycerol
(Zygmunt et al., 1999) and virodhamine (Ho and Hiley,
2004), as well as abnormal cannabidiol and synthetic
cannabinoids, including HU 210, WIN 55212-2 and
CP 55940, do not behave in this way (Zygmunt et al., 1999;
´
´
Offertaler et al., 2003; Ho and Hiley, 2003a, b). It is clear
‘abnormal-cannabidiol receptor’ (Jarai et al., 1999; Ho and
therefore that quite different structure–activity properties are
associated with vasorelaxation mediated by TRPV1 and the
as-yet-undefined cannabinoid receptor(s) of the rat mesen-
teric artery but it must be borne in mind that it is not
possible to determine at this stage if VSN16 directly or
indirectly stimulates the vanilloid receptor although pre-
liminary data suggest that it is not a direct stimulant (V Di
Marzo, personal communication).
Hiley, 2003b), whereas we have shown that the abnormal
cannabidiol receptor is relatively insensitive to the actions of
AM 251 (Ho and Hiley, 2003b). Likewise, O-1918 inhibits the
abnormal-cannabidiol responses at its vascular receptor. The
finding that VSN16 is sensitive to inhibition by O-1918, and
AM 251, but is pertussis toxin-insensitive suggests that the
vascular target for VSN16 may be distinct from the receptor
for abnormal cannabidiol.
In spite of this, the relaxation of the mesenteric artery in
response to VSN16 is unlikely to occur via sensory nerves,
since the effect is entirely endothelium-dependent. Thus,
VSN16 might activate a TRPV1 receptor in the endothelium.
Poblete et al. (2005) reported that they could detect the
mRNA for the rat TRPV1 receptor in the endothelial cell layer
in the mesenteric arterial bed, in addition to its expression in
sensory nerves. They also showed that anandamide (at a sub-
vasorelaxant concentration) and capsaicin elicited nitric
oxide release, and that this was reduced by the TRPV1
receptor antagonists 50-iodoresiniferatoxin, SB 366791 and
capsazepine, as well as by removal of the endothelium or
nitric oxide synthase inhibition.
Recently it has been reported that abnormal cannabidiol
and O-1918, AM 251 and rimonabant bind to the orphan
receptor GPR55 and signal via pertussis toxin-insensitive
¨
mechanisms (Sjogren et al., 2005; Johns et al., 2007).
However, although GPR55 does not appear to mediate the
vasodilator effects of abnormal cannabidiol (Johns et al.,
2007), the receptor has been implicated in control of blood
¨
pressure in a pathological system (Sjogren et al., 2005). These
points underline further that work is needed to clarify the
interactions of VSN16 with GPR55.
Systemic cardiovascular effects
Moreover, the sensitivity of vasorelaxation to capsaicin
treatment does not always imply the involvement of TRPV1
receptors; for example, D9-tetrahydrocannabinol stimulates
calcitonin gene-related peptide release from perivascular
sensory nerves in TRPV1 receptor-knockout mice, and it
was speculated that a novel receptor or ion channel on the
sensory nerves might be involved (Zygmunt et al., 2002).
Indeed, the relaxant effects of anandamide in blood vessels
are not only sensitive to capsaicin pretreatment, but also to
the calcitonin gene-related peptide antagonist, CGRP(8–37),
which suggests that some of the vasorelaxant action of the
In view of the actions on the isolated third-generation artery,
which is considered to make some contribution to mesen-
teric vascular resistance, a haemodynamic study was per-
formed in conscious rats. At doses of 1 and 3 mg kgÀ1, VSN16
caused no significant changes in heart rate, blood pressure or
regional vascular conductances. However, at the highest
dose used, 15 mg kgÀ1, there was a transient pressor response
that was accompanied by a decrease in renal vascular
conductance. Interestingly, at this dose, there was
a
significant increase in mesenteric conductance, which would
be consistent with the mesenteric vasodilatation observed in
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
762
PM Hoi et al
isolated vessels. These results may reflect a sparse distribu-
tion of receptors for VSN16 in the vasculature since a dose of
5 mg kgÀ1 VSN16-R gave a peak concentration of 7 mg mlÀ1
(22 mM) at 5 min after i.v. injection (data not shown); this
concentration is B20 times that of its EC50 in the mesenteric
artery. The relatively small in vivo effect on blood pressure of
VSN16 could suggest that in vitro vasorelaxation of third-
order mesenteric arteries has poor predictive value in
generalized blood pressure effects. Alternatively, there is a
possibility that there might be a strain difference in the
distribution of receptors for VSN16 as the myograph studies
used Wistar rats while the systemic haemodynamics were
carried out in Sprague–Dawley rats.
therapeutic activity in models of multiple sclerosis (D Baker,
unpublished results).
A limited number of analogues is presented here to
illustrate the structure–activity relationships; in general,
very few structural alterations are tolerated. Thus changes
to the ethanolamide group, for example, to 2-fluoroethyla-
mide, as in compound 13, or to cyclopropylamide (com-
pound 12) caused loss of activity, though these groups are
tolerated in simple anandamide analogues (Ryan et al.,
1997). The interactions of the terminal carboxamide group
with the receptor seem to be similarly precise with
apparently conservative changes to the monomethyl amide
or the unsubstituted amide not tolerated. In contrast, a
cyano group was allowed in this position (compound 9,
which is also termed VSN15). These data indicate that
H-bond donor groups at this position are unfavourable
whereas H-bond acceptor groups are apparently allowed.
Reducing the length of the alkyl side chain as in compound
7 again reduced the activity markedly.
The modest haemodynamic effects of VSN16-R contrast
with the complex picture presented by anandamide, which
some of us have previously found to evoke both a pressor
response, at all doses studied between 0.075 and 3 mg kgÀ1
,
as well as vasoconstriction not only in the mesenteric but
also in the renal and hindquarters vascular beds (Gardiner
et al., 2002). At higher doses (2.5 and 3 mg kgÀ1), ananda-
mide both slowed the heart and decreased the blood pressure
while the hindquarters vasoconstriction was followed by an
increase in vascular conductance. None of these effects was
sensitive to AM 251. This pattern shows limited similarity
to those responses observed with VSN16-R: both induce
systemic pressor responses and increase renal vascular
resistance. However, anandamide had no effect on mesen-
teric vascular resistance while VSN16-R did not affect
hindquarters conductance which was increased by higher
doses of anandamide. This suggests that the two compounds
do not share sites of action that can be readily identified at
the systemic level even though the mesenteric artery effects
of both anandamide and VSN16-R observed in the myograph
share the property of sensitivity to the novel antagonist
O-1918.
Compound 7, which showed some vasorelaxant activity,
is like VSN16 in that it did not inhibit [3H]CP 55940 binding
at CB1 receptors in rat cerebellar membranes. Thus, it might
be suggested that chemicals of this series do not have any
ability to bind to classical cannabinoid receptors. However,
compound 9, which was the most effective vasodilator after
VSN16, brought about modest inhibition of the binding of
[3H]CP 55940 to the CB1 receptor; thus although the
interaction is weak, at least one of the series has the ability
to bind to the CB1 receptor even if, for the reasons discussed
above, this receptor is not responsible for the vasorelaxant
activity of the series.
Conclusions
Nevertheless, although VSN16-R had a modest effect in
normotensive rats, it remains to be determined if it may
bring about hypotension under pathological conditions.
However, the lack of cardiovascular effect would be bene-
ficial in a drug developed for use in non-cardiovascular
conditions such as multiple sclerosis. Indeed, other work
with VSN16 have shown that it has therapeutic potential
in this condition as it reduces spasticity in vivo in the
autoimmune encephalomyelitis mouse model of the disease
(Okuyama et al., 2005; D Baker, unpublished results).
VSN16 represents a novel water-soluble vasorelaxant that
produces
a
strictly endothelium-dependent response
through several mechanisms, possibly involving novel
cannabinoid receptor-dependent and -independent path-
ways. Thus, results reported here support the proposal that
an additional cannabinoid receptor (or receptors) different
from either the CB1 or the CB2 receptor is responsible for the
actions of VSN16. The sensitivity to O-1918 suggests that the
proposed endothelial cannabinoid receptor, the ‘abnormal-
cannabidiol’ or ‘anandamide’ receptor might contribute to
some of its effects, although it is possible that another
unknown but similar entity also exists. One candidate target
is GPR55. Structure–activity studies of analogues in the VSN
series suggest a very stringent interaction between the
compound and the binding pocket of the potential receptor
in the vasculature.
Chirality and structure–activity relationships
VSN16 showed little stereospecificity in its activity as a
vasodilator. This probably indicates either that the part of
the molecule that includes the chiral centre does not interact
with the receptor or that the element of the binding surface
that interacts with this region does not have a structure
which is sensitive to the orientation of the side chain. Until
the target and the binding moieties of VSN16 are better
defined, the role of the chiral elements in relation to
function will remain unclear. Based on the in vitro experi-
ments presented here, VSN16-R is slightly more active than
VSN16-S and this is seen also in relation to the in vivo
Acknowledgements
This work was supported by a Wellcome Trust University
Translation Award (076001/Z/04/Z), the Brain Research Trust
and the Bloomsbury Bioseed Fund.
British Journal of Pharmacology (2007) 152 751–764

VSN16 pharmacology in rat mesenteric artery
PM Hoi et al
763
Conflict of interest
Johns DG, Behm DJ, Walker D, Ao Z, Shapland EM, Daniels DA et al.
(2007). The novel endocannabinoid receptor GPR55 binds atypical
cannabinoids but does not mediate their vasodilator effects. Br J
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Kadota I, Shibuya L, Lutete LM, Yamamtoto Y (1999). Palladium/
benzoic acid catalyzed hydroamination of alkynes. J Org Chem 64:
4570–4571.
VSN16 is subject to patent WO2005/080316 granted to
M Okuyama, D Selwood, C Visintin, D Baker, G Pryce and
University College, London.
Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D et al. (1999).
Structure–activity relationships of pyrazole derivatives as canna-
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Structure of a cannabinoid receptor and functional expression of
the cloned cDNA. Nature 346: 561–564.
Okuyama M, Selwood D, Visintin C, Baker D, Pryce G (2005).
University College London. Modulators of cannabinoid receptors.
Patent WO2005/080316.
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