Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog

Article abstract of DOI:10.1016/j.biopha.2021.111861

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8–13) analog. The Tyr¹¹ residue of NT(8–13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys⁸ and Trp¹¹ to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (K_i 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t_1/2 > 24 h) compared with NT (t_1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED₅₀ = 4.6 μg/kg) and tonic (ED₅₀ = 7.1 μg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.

Full text of DOI:10.1016/j.biopha.2021.111861

Biomedicine & Pharmacotherapy 141 (2021) 111861
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor
type 2 (NTS2) analgesic macrocyclic analog
,
1
a,
Magali Chartiera , Michael Desgagne 1, Marc Sousbiea, Charles Rumsbya, Lucie Chevillardb,
´
a
a
a
´
a
´
´
`
´ ˆ
,*,2
ˆ ´ a
Lea Theroux , Lounes Haroune , Jerome Cote , Jean-Michel Longpre , Pierre-Luc Boudreault ,
Eric Marsault 2, Philippe Sarreta
a,
´
a
´
Institut de Pharmacologie de Sherbrooke, Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke,
QC, Canada
b
´
Inserm, UMR-S 1144, Universite de Paris, Paris, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neu-
rotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by
activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-
independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic proper-
ties of the first constrained NTS2 macrocyclic NT(8–13) analog. The Tyr11 residue of NT(8–13) was replaced with
a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization
reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit
its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for
NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability
profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted
dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain
models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model.
Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and
potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause
hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are
physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic
potential of NTS2-selective analogs for the management of pain.
Macrocycle
Metabolic stability
Analgesia
Pain
Opioid-sparing effects
1. Introduction
reveal that over 75% of patients with chronic pain taking prescribed
analgesic medications are not satisfied with their current available
Chronic pain management represents a daily public health challenge,
as 20% of the global population lives with different types of disabilities,
including physical impairment and psychological distress [1]. Despite a
better understanding of the underlying mechanisms of pain and the
identification of novel pain targets [2], epidemiological studies still
treatment, mainly due to the development of debilitating side effects
[3]. In light of the current opioid crisis, alternative options to conven-
tional pain medications that exhibit high analgesic effectiveness, a low
risk of adverse effects and no abuse liability are needed to adequately
relieve chronic pain [2,4].
´
* Correspondence to: Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, 3001, 12e Avenue Nord,
´
Sherbrooke, Quebec, Canada J1H 5N4.
´
E-mail addresses: Magali.Chartier@USherbrooke.ca (M. Chartier), Michael.Desgagne@USherbrooke.ca (M. Desgagne), Marc.Sousbie@USherbrooke.ca
´
(M. Sousbie), Charles.Rumsby@USherbrooke.ca (C. Rumsby), luciechevillard@gmail.com (L. Chevillard), Lea.Theroux@USherbrooke.ca (L. Theroux), Lounes.
ˆ ´
´
Haroune@USherbrooke.ca (L. Haroune), Jerome.Cote@USherbrooke.ca (J. Cote), Jean-Michel.Longpre@USherbrooke.ca (J.-M. Longpre), Pierre-Luc.Boudreault@
´
USherbrooke.ca (P.-L. Boudreault), Philippe.Sarret@USherbrooke.ca (E. Marsault), philippe.sarret@usherbrooke.ca (P. Sarret).
1
Authors contributed equally to this work.
2
Authors contributed equally to directing this study.
https://doi.org/10.1016/j.biopha.2021.111861
Received 22 March 2021; Received in revised form 22 June 2021; Accepted 28 June 2021
Available online 3 July 2021
0753-3322/© 2021 The Authors.
Published by Elsevier Masson SAS. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
Neurotensin (NT) is an endogenous tridecapeptide (pGlu-Leu-Tyr-
residue of NT(8–13), which was previously reported in linear peptides to
play a critical role in receptor subtype selectivity [23,45,46,55–61], was
replaced with a Trp11 followed by side-chain to side-chain macro-
cyclization between Lys8 and Trp11. The pharmacodynamic and phar-
macokinetic properties of the resulting constrained NT(8–13)
macrocyclic compound, designated CR-01-64, were then assessed, with
a particular emphasis on antinociceptive activity, interactions with the
opioid system and potential NT-related adverse effects.
Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) that functions as a neu-
romodulator/neurotransmitter in the central nervous system (CNS),
modulating dopamine transmission, stimulating hypothalamic-
pituitary-adrenal activity and exerting potent hypothermic and anal-
gesic effects [5–11]. In the periphery, NT also behaves as a paracrine and
endocrine hormone of the cardiovascular system and the digestive tract
[12,13]. Regarding its analgesic action, NT was found to produce a
potent opioid-independent antinociceptive response, since the admin-
istration of the opioid antagonists, naloxone or naltrexone is not effec-
tive in reversing NT-induced analgesia [14–22]. Importantly,
compelling evidence also indicates that the analgesic potencies of NT
and its analogs are superior to morphine at equimolar doses in various
pain models [23–26].
2. Materials and methods
2.1. Peptide synthesis
All peptides were synthesized on a 50 μmol scale using 2-chlorotrityl
NT exerts its antinociceptive action through the activation of two
distinct G protein-coupled receptors (GPCRs), namely, NTS1 and NTS2
[8,9,27,28]. Both receptors and the NT peptide are highly abundant in
the ascending and descending pain control pathways, particularly in the
dorsal root ganglia, spinal cord dorsal horn, periaqueductal gray, raphe
magnus and pallidus, and rostroventral medulla [28–37]. However, in
contrast to NTS2, which is predominantly expressed in brain structures
associated with pain modulation, NT binding to NTS1 also regulates
blood pressure, myocardial contractility, core body temperature and
gastrointestinal motility [12,13,38–40]. Consequently, the NTS2 re-
ceptor is attracting interest in drug discovery for the development of
new chemical entities producing analgesia with minimal adverse effects.
Structure-activity relationship (SAR) studies have shown that the C-
terminal NT(8–13) fragment retains the full biological activity of NT,
thereby defining it as the essential amino acid sequence for the devel-
opment of NT(8–13)-based analgesics [41]. Since then, several NT
(8–13) analogs harboring reduced amine bonds and site-specifically
modified unnatural amino acids have been developed to improve re-
ceptor binding affinity, subtype selectivity, efficacy/potency and pep-
tide stability. Among these compounds, NTS1-selective agonists
(PD149163 and NT72) were reported to induce antinociceptive effects
following central administration [31,32,42–44]. Likewise, centrally
injected NTS2-selective analogs (JMV431 and NT79) have been shown
to successfully reverse nociceptive behaviors in both acute and chronic
pain models [23,26,33,45–47].
chloride resin from Matrix Innovation (maximum loading 0,89 mmol/
g). Commercially available Fmoc-protected amino acids were purchased
from Chem-Impex, Matrix Innovation and Sigma-Aldrich at >95% pu-
rity. Amino acid coupling was performed on an orbital shaker at 150
RPM using 6 mL of solid-phase synthesis reactors equipped with a 20 µm
frit from Applied Separations.
2.1.1. Standard resin washing procedures
The resin was washed using a standard sequence of DMF, DCM,
iPrOH, DCM, and DMF, with ~3 mL of solvent per reactor in each wash.
When a dried resin was required, two additional washes with DCM were
performed.
2.1.2. Resin loading
Standard 2-chlorotrityl chloride resin was allowed to react with 4 mL
of DCM containing 0.5 mmol/g of the first amino acid and 1,5 eq. of
DIPEA for at least 2 h. The resin was then washed using the standard
procedure described above. Unreacted chlorotrityl sites were capped
using a 7/2/1 solution of DCM/methanol/DIPEA for at least 20 min.
When loading was uncertain, 10 mg of the loaded resin were isolated
and deprotected using 2 mL of 20% piperidine in DMF for 10 min. A
spectrophotometric analysis of the supernatant was performed to esti-
mate the dibenzofulvene concentration, which reflects amino acid
loading.
In recent years, the drug development of peptide therapeutics has
received renewed interest from the pharmaceutical industry. To date,
over 50 peptide drugs targeting GPCRs have been approved by the FDA
for clinical use, most of them for the treatment of type 2 diabetes mel-
litus and other metabolic disorders, as well as oncology indications [48].
Among the chemical strategies used to optimize the properties of NT
(8–13), peptide cyclization has shown great success in improving the
physicochemical properties of active NT analogs targeting NTS1 [49,
50]. Indeed, macrocyclization often increases proteolytic stability
through restricted accessibility to the cleavage sites [51–53]. Further-
more, cyclization allows control of the peptide conformation and thus
mimics secondary structures and improves ligand-receptor binding.
Macrocyclic compounds thus combine the ability to present remote
pharmacophores while maintaining a semirigid structure [51]. In
addition, macrocycles that possess better absorption, distribution,
metabolism, and excretion/pharmacokinetics (ADME/PK) properties
may display an increased ability to cross barriers, as evidenced by the
cyclic NT(8–13) nonselective analog (JMV2012) exhibiting central
bioavailability [54]. Ring size is also an important factor when consid-
ering an appropriate synthesis strategy. Therefore, we recently reported
the optimal macrocyclization site for NT(8–13) and a potent new NT
(8–13) macrocyclic scaffold [49,50]. This macrocyclic compound dis-
played an excellent affinity for NTS1, as well as potent analgesia. As
expected, however, it was associated with hypothermia and
hypotension.
2.1.3. Fmoc deprotection
Fmoc-protected peptides were deprotected in the solid phase using
piperidine 20% v/v in DMF for 5 min, and this step was repeated once.
Standard wash procedures were subsequently performed.
2.1.4. Coupling of amino acids
When coupling commercially available and inexpensive amino acids,
5 eq. were reacted with 5 eq. of HATU and 6 eq. of DIPEA in 4 mL of
DMF. The reaction mixture was stirred on an orbital shaker for at least
20 min. Standard wash procedures were then applied.
2.1.5. Coupling of expensive amino acids
Expensive amino acids were coupled using 2 eq. amino acid in the
presence of 2 eq. of HATU and 3 eq. of DIPEA in 4 mL of DMF. The re-
action mixture was stirred on an orbital shaker for at least 2 h. Standard
wash procedures were then applied.
2.1.6. Synthesis of unnatural Fmoc-Trp(N-Allyl)-OH
2.1.6.1. Boc-Trp-OMe. Triethylamine (5,75 mL, 41,2 mmol) was added
to flame-dried RBF with L-Trp methyl ester HCl (10 g, 39.3 mmol) in
THF (100 mL) under Ar and cooled on ice. Di-tert-butyl dicarbonate (9 g,
41,2 mmol) in THF (40 mL) was subsequently added slowly. The reac-
tion mixture was stirred at room temperature overnight. The product
was then evaporated under vacuum and partitioned in 50:50 AcOEt:
H2O. The organic phase was washed with water once and brine twice,
Here, we describe the synthesis and in vitro/in vivo characterization
of the first constrained NTS2 macrocyclic NT(8–13) analog. The Tyr11
2

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
dried with MgSO4 and evaporated under a vacuum. Purity was
confirmed using TLC. Flash chromatography was not performed (100%
conversion via TLC).
2.2. Peptide purification
The dried crude mixture obtained in the previous step was solubi-
lized in a 1:1 ACN/H2O mixture (1–1.8 mL) and purified on a Waters
preparative HPLC-MS column (XSELECTTM CSHTM Prep C18 (19 ×
100 mm) packed with 5 µm particles, UV detector 2998, MS SQ Detector
2, Sample manager 2767 and a binary gradient module). The eluents
used were acetonitrile to which 0,03% aqueous ammonia was added, as
well as water with 20 mM NH4CO3.
2.1.6.2. Boc-Trp(N-Allyl)-OMe. Cesium carbonate (12.4 g, 37.7 mmol)
was added to a solution of Boc-Trp methyl ester (3.33 g, 10.4 mmol) in
DMF (~22 mL) in a pressurized vessel and stirred for 2–3 min. Allyl
bromide (2.71 mL, 31.3 mmol) was then added slowly, and the reaction
mixture was stirred at 90 ◦C for 4 h until the disappearance of the
starting material was detected using TLC. After cooling to room tem-
perature, the solvent was evaporated overnight under airflow. The crude
product was solubilized in DCM with a small amount of AcOEt and
purified using flash chromatography on silica gel 20% AcOEt:hexanes,
yielding 2.98 g of Na-Boc-Nind-AllylTrp-OMe as a slightly yellow oil.
First, a 10-min run was performed with the eluents listed above and a
gradient of 5% ACN to 95% ACN (0–10 min) to estimate the %ACN
where the product was eluted. A second run was performed using the
following eluent program: [(%ACN) - 10 - (%ACN) + 5] (0–15 min).
Fractions collected were analyzed on a Waters UPLC-MS system (column
Acquity UPLC® CSHTM C18 (2.1 × 50 mm) packed with 1.7 µm parti-
cles) using ACN and water + 0,1% formic acid. The separation method
used in this step was 0–0.2 min: 5% ACN; 0.2–1.5 min: 5–95% ACN;
1.5–1.8 min: 95% ACN; 1.8–2.0 min: 95–5% ACN; 2.0–2.5 min: 5% ACN.
Pure fractions were grouped and lyophilized. HRMS spectra of the
peptides were also recorded using a maXis ESI-Q-TOF in ESI+ mode.
2.1.6.3. Boc-Trp(N-Allyl)-OH. Lithium hydroxide (1.19 g, 49.9 mmol)
in H2O (~35 mL) was added to a solution of Na-Boc-Nind-AllylTrp-OMe
(2.98 g, 8.31 mmol) in THF (20 mL). The reaction mixture was stirred at
room temperature for 30 min until TLC indicated complete conversion
(3:7 AcOEt:hexanes with 1% acetic acid). THF was then evaporated
under reduced pressure. The solution was acidified using 1 M HCl,
AcOEt was added, and the organic phase was washed with water and
brine. The organic layer was dried over Na2SO4 and concentrated to
yield 2.98 g of Na-Boc-Nind-AllylTrp-OH as a colorless oil. The product
was not purified.
2.3. Competitive radioligand binding assay using hNTS1 and hNTS2
receptors
CHO-K1 cells stably expressing hNTS1 (ES-690-C from PerkinElmer,
´
Montreal, Canada) or 1321N1 cells stably expressing hNTS2 (ES-691-C
´
from PerkinElmer, Montreal, Canada) were cultured in DMEM/F12 at
2.1.6.4. Trp(N-Allyl)-OH. A TFA/TIPS/H2O mixture with a 95/2.5/2.5
vol ratio (~30 mL) was added to Na-Boc-Nind-AllylTrp-OH (2.98 g, 8.65
mmol) and stirred for 30 min to completion, as assessed using TLC. The
solution was evaporated and diluted with DCM for 3 subsequent co-
evaporations to remove all TFAs. The product was not purified.
37 ◦C in a humidified chamber with a 5% CO2 atmosphere. Culture
media were supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/
mL streptomycin, 20 mM HEPES, and 0.4 mg/mL G418. Cells expressing
hNTS1 were frozen when they reached 80% confluence. They were
scraped off the dish in 10 mM Tris buffer and 1 mM EDTA, pH 7.5, and
◦
centrifuged at 15,000 g for 5 min at 4 C. The pellet was then resus-
2.1.6.5. Fmoc-Trp(N-Allyl)-OH. Nind-AllylTrp-OH (2.11 g, 8.64 mmol)
was dissolved in H2O and THF (~50 mL of both) at a pH adjusted to 7–8
using sodium bicarbonate. Fmoc chloride (2.31 g, 8.84 mmol) was dis-
solved in THF (~15 mL), and both solutions were combined, with the pH
observed and adjusted during the reaction accordingly to maintain it at
approximately 7–8. After 3 h of stirring, THF was evaporated, and the
solution was extracted with Et2O three times. Then, the pH of the
aqueous solution was adjusted to 2 using 1 N HCl before washing with
EtOAc until extraction was complete, as assessed using TLC. EtOAc
fractions were combined, washed with brine, dried with Na2SO4 and
evaporated. Biotage chromatography of 3.109 g of the crude product
yielded 1.120 g of Na-Fmoc-Nind-AllylTrp-OH.
pended in 1 mL of binding buffer. Cells expressing hNTS2 were also
frozen when they reached 80% confluence. They were scraped off the
dish in PBS and 0.5 mM EDTA, pH 7.5, and sonicated for 5 min (pulse 30
s/5 s off, amplitude 40%). They were ultracentrifuged at 100,000 g for
60 min at 4 ◦C. The pellet was resuspended in 1 mL of freezing buffer
(PBS, glycerol, and 0.5 mM EDTA), sonicated for 1 min (pulse 30 s/5 s
off, amplitude 50%) and further diluted in binding buffer. Competitive
radioligand binding experiments were performed by incubating 15 μg of
cell membranes expressing the hNTS1 receptor with 45 pM 125I-[Tyr3]-
NT (2200 Ci/mmol) or 50 μg of cell membranes expressing the hNTS2
receptor with 300 pM 125I-[Tyr3]-NT in binding buffer (50 mM Tris-HCl,
pH 7.5, 0.2% BSA). The samples were incubated with increasing con-
centrations of analogs ranging from 10ꢀ 11 to 10ꢀ 4 M for 60 min at 25 ◦C,
and then, the binding reaction mixture was transferred to PEI-coated 96-
well filter plates (glass fiber filters GF/B, Millipore, Billerica, MA). The
reaction was terminated by filtration, and the plates were washed three
2.1.7. Ring-closing metathesis
The dried resin (200 mg) and 0.2 eq of Grubbs-Hoveyda 2nd gen-
eration (6.3 mg, 0.01 mmol) were added to a flame-dried, argon-filled
microwave tube containing a stir bar. The resin and catalyst were
degassed for 10 min under an argon flow and were then suspended in 3
mL of anhydrous dichloroethane. The reaction mixture was then heated
in a CEM Discover microwave for 1 h at 70 ◦C. Standard wash proced-
ures were applied.
times with 200 μL of ice-cold binding buffer. Glass filters were then
counted using a γ-counter (2470 Wizard2, PerkinElmer, Mississauga,
Ontario, Canada). Nonspecific binding was measured in the presence of
10ꢀ 5 M unlabeled NT(8–13) and represented less than 5% of total
binding. Data for NT(8–13) were normalized to the control to exclude
unwanted sources of variation. IC50 values were determined from
competition curves as the unlabeled ligand concentration inhibiting
50% of 125I-[Tyr3]-NT-specific binding. Ki values were determined using
the Cheng-Prusoff equation from IC50 values [62]. Competitive radio-
ligand binding data were plotted using the nonlinear regression
One-site-Fit log(IC50) and presented the means ± SEM of three inde-
pendent experiments, each performed in triplicate. The binding assay
was performed in triplicate to ensure the reliability of single values.
2.1.8. Simultaneous cleavage of the resin and Boc-protected groups
A solution of 95/2,5/2,5% TFA/TIPS/H2O (~2.5 mL) was added to
the dried resin described above and allowed to react for 2 h on an orbital
shaker. The acidic solution was filtered, and the filtrate was poured into
~10 mL of tert-butyl methyl ether. The solution was centrifuged at 3000
RPM at 4 ◦C for 10 min, and the supernatant was analyzed using UPLC-
MS to confirm the absence of the peptide and then discarded. If the
peptide was still present, the supernatant was either evaporated and
copurified with the precipitate or extracted with 3:1 water/ACN,
lyophilized and then purified with the crude material. The precipitate
was subsequently evaporated in vacuo.
2.4. Plasma and cerebrospinal fluid (CSF) stability assays
Rats were anesthetized with 2.5% isoflurane for blood collection and
3

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
with ketamine/xylazine (87 mg/kg: 13 mg/kg, i.m.) for CSF collection.
Rat plasma was obtained by centrifuging blood at 13,000 rpm for 5 min
at 4 ◦C. Rat CSF was collected from the cisterna magna. Six microliters of
calculated for each dose in the tail-flick test. Then, ED50 values were
determined using the dose-response stimulation log(agonist) vs response
(four parameters).
a 1 mM aqueous solution of the peptide was incubated with 27 μL of rat
plasma or rat CSF at 37 ◦C for 24 h (or for 1, 2, 3 and 5 min for NT(8–13)
in plasma and for 8, 16 and 24 h for NT(8–13) in CSF). Proteolytic
2.5.4. Formalin test
The analgesic effects of compounds were assessed using the formalin
test as a model of persistent pain. Before testing, animals were accli-
matized to manipulations and the experimental apparatus for three
consecutive days (30 min/day). On the test day, 5 min after intrathecal
degradation was quenched by adding 70 μL of a 10% trichloroacetic acid
and 0.5% nicotinamide solution followed by immediate vortexing.
Samples were centrifuged (13,000 rpm, 5 min, 4 ◦C), and the superna-
tant was filtered through a 4 mm nylon 0.2 µm syringe filter and then
analyzed using UPLC-MS (Waters 2695 with ACE C18 column 2.0 × 100
mm, 2.7 µm spherical particle size and Electrospray micromass ZQ-2000
from Waters). Data were analyzed with GraphPad Prism 7 software
using a one-phase decay equation and presented as the half-life from
three independent experiments.
injection of the compound at increasing doses ranging from 3 to 60
μg/
kg, rats received a subcutaneous injection of 50
μL of diluted formal-
dehyde (1.85%; i.e., 5% formalin; Bioshop, Burlington, Ontario) into the
plantar surface of the right hind paw. Rats were then placed in clear
Plexiglas chambers (30 × 30 × 30 cm) positioned over a mirror angled at
45◦ to allow an unobstructed view of the rats’ paws. Their behavior was
then observed for the next 60 min. The intraplantar injection of formalin
produced a biphasic nociceptive response typical of this tonic pain
model [65]. The two distinct phases of spontaneous pain behaviors that
occur in rodents are proposed to reflect first the direct effect of formalin
on sensory receptors (acute phase, 0–9 min) and longer-lasting pain due
to inflammation and central sensitization (inflammatory phase, 21–60
min). Following the injection of formalin, mean nociceptive scores were
determined for each 3 min block of the 60 min period by measuring the
amount of time spent in each of four behavioral categories, as previously
described [66,67]: 0, the injected paw is comparable to the contralateral
paw; 1, the injected paw has little or no weight placed on it but still
touches the ground; 2, the injected paw is elevated and is not in contact
with any surface; 3, the injected paw is licked, bitten, or shaken. Be-
haviors believed to represent higher levels of pain intensity were given
higher weighted scores. The weighted average pain intensity score
ranging from 0 to 3 was then calculated by multiplying the time spent in
each category by the category weight, summing these products, and
dividing by the total time in a particular time interval. The pain score
was thus calculated using the following formula (1T1 + 2T2 +
3T3)/180, where T1, T2, and T3 are the duration (in seconds) spent in
behavioral categories 1, 2, or 3, respectively, during each 180 s block.
The area under the curve (AUC) was calculated for all durations of the
test (0–60 min). Data are presented as the means ± SEM of 5 rats treated
with each dose.
2.5. In vivo analgesic assay
2.5.1. Animals, housing and habituation
Experiments were performed with adult male Sprague-Dawley rats
weighing 225–300 g (Charles River laboratories, St. Constant, Canada).
Rats were housed in groups of two per cage on Aspen shavings in a quiet
room, maintained on a 12 h light/dark cycle and allowed ad libitum
access to food and water. All experimental procedures reported in this
´
study were approved by the Animal Care Committee of Universite de
Sherbrooke in accordance with policies and directives of the Canadian
Council on Animal Care. Furthermore, they were performed in agree-
ment with the ARRIVE (Animals in Research: Reporting In Vivo Exper-
iments) guidelines and the United States NIH [63].
2.5.2. Intrathecal administration
Rats were lightly anesthetized with a flow of 2% isoflurane (Baxter
Corporation, Mississauga, ON, Canada) and oxygen (2 L/min) and
injected intrathecally using a 27G needle at the L5-L6 intervertebral
space with saline or the test compound diluted in 0.9% saline at con-
centrations ranging from 3 to 60 μg/kg.
2.5.3. Tail-flick test
Acute pain was assessed using the tail-flick test (Tail-Flick Analgesia
meter V2.00, Columbus Instruments, Columbus, Ohio, USA), which
measures heat-induced pain in animals and is reflective of injected
compound analgesic efficacy [64]. The test measures sensitivity to a
high-intensity light beam focused on the rat’s tail. The tail-flick appa-
ratus was set at a light intensity of 6, and a cutoff of 10 s was used.
Testing involved measurements of the latency for the rat to withdraw its
tail from the path of the light beam, which corresponds to the measure of
pain sensitivity. Before testing, animals were individually acclimatized
to manipulations and the behavioral apparatus 5 min/day for three
consecutive days. On the test day, a latency baseline was recorded before
the drug injection. The compound was diluted in 0.9% saline and
2.5.5. Complete Freund’s adjuvant-induced chronic inflammatory pain
The von Frey test was used to measure mechanical allodynia in a
complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain
model. Chronic inflammatory pain was induced with an intraplantar
injection of 100 μL of CFA into the plantar surface of the rat’s left hind
paw. The injected volume (injected as a 1:1 emulsion of oil and saline
0.9%) contained the equivalent of 200 g of lyophilized bacterial
μ
membranes (Mycobacterium butyricum) for a final concentration of 8 mg/
mL. An intraplantar injection of saline 0.9% was administered to sham
animals. CFA injections were performed under isoflurane/oxygen
anesthesia. Rats were acclimatized to the Plexiglas enclosures, the mesh
floor and the filament for 3 days prior to testing. On test days, rats were
allowed 10 min to habituate to the enclosures. The von Frey test was
performed before (day 0) and 3, 7 and 14 days after the CFA injection.
Allodynia was determined using calibrated von Frey hairs. The blunt
filaments were applied against the mid-plantar surface of the animal’s
hind paw. Filaments with increasing resistance were used, ranging from
2 to 15 g of force. The paw withdrawal threshold (PWT) was then
determined, as previously described [68]. Fifty grams were considered
the upper cutoff. Five values were recorded alternately for both ipsi-
lateral and contralateral hind paws at intervals of 7 s. The effect of a 60
µg/kg dose of the compound or saline on allodynia was assessed at 15,
30, 60 and 120 min following i.t. administration on days 7 and 14. The
% antiallodynic effect was calculated using the following formula: %
injected at increasing doses ranging from 3 to 60 μg/kg. Morphine was
injected intrathecally at an equimolar dose to CR-01-64 (30 µg/kg = 35
nmol/kg) or intravenously at 1 mg/kg, alone or in combination with
CR-01-64 at 30 µg/kg. The NTS2 antagonist NTRC-844 was diluted in
DMSO and was injected intrathecally at a 10x equimolar dose to
CR-01-64 (350 nmol/kg), alone or in combination with CR-01-64 at 30
µg/kg. Naloxone was injected intraperitoneally at 10 mg/kg 30 min
before the administration of CR-01-64. The effects of the compound or
saline on thermal nociception were assessed every 10 min for up to 60
min following i.t. administration. Tail-flick latency was then converted
into the percent maximal possible effect (% MPE) at the time of maximal
peak of analgesia. % MPE was calculated according to the following
formula: %MPE = [(Test latency) – (Saline latency)]/[(Cutoff) – (Saline
latency)] × 100. Data are presented as the means ± SEM of 6 animals for
each treatment dose. The half-maximal effective dose (ED50) of the
compound was determined based on the % MPE at 20 min, which was
antiallodynic effect = 100 × (PWTcompound - PWTsaline)/(PWTsham
-
PWTsaline). Data are presented as the means ± SEM of 6 rats per
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M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
condition.
silastic tubing, with external and internal diameters of 0.96 and 0.58
mm, respectively (Becton Dickinson, Franklin Lake, NJ, USA). The
catheter was subcutaneously tunneled and fixed at the back of the neck.
Heparinized saline (100 UI/mL) was injected into the catheter to avoid
thrombosis and catheter obstruction, and the wound was closed. Rats
were then returned to their individual cages for a recovery period of at
least 24 h, allowing complete anesthesia washout. On the day of
experimentation, rats were placed in horizontal Plexiglas cylinders (6.5
cm internal diameter, up to 20 cm adjustable length) (Harvard Appa-
ratus, Inc., Holliston, MA, USA). Following an i.v. injection of 0.2 mg/kg
2.6. Body temperature measurement
Body temperature was measured using a thermistor probe inserted
into the rat’s rectum. Animals were individually acclimatized to ma-
nipulations and thermistor probes for three consecutive days. On the test
day, temperatures were measured before (baseline) and every 10 min for
up to 60 min following intrathecal drug administration. The compound
was dissolved in 0.9% saline and injected at the highest dose of 60 μg/
CR-01-64, 300
μL of blood were sampled from catheterized rats at 5, 10,
kg. Changes in body temperature (Δ body temperature) were deter-
mined from the baseline for each time point and each animal. Data are
presented as the means ± SEM of 6 rats per condition.
15, 30, 60, and 120 min postinjection. Blood samples were collected in
Eppendorf tubes containing 30
μL of sodium heparin (Sandoz, Bou-
cherville, Canada). After centrifugation (5000 rpm, 10 min), plasma was
isolated and stored at ꢀ 80 ◦C until analysis.
2.7. Blood pressure measurement
2.9.2. Cerebrospinal fluid PK
Rats were anesthetized with ketamine/xylazine (87 mg/kg: 13 mg/
kg, i.m.) and placed in a supine position on a heating pad. Mean, systolic
and diastolic arterial blood pressure and heart rate were measured
through a catheter (PE 50 filled with heparinized saline) inserted into
the right carotid artery and connected to a Micro-Med transducer (model
TDX-300, USA) linked to a blood pressure Micro-Med analyzer (model
BPA-100c). Another catheter (PE 10 filled with heparinized saline) was
inserted in the left jugular vein to administer bolus injections of the
compound at 0.01 and 0.1 mg/kg (volume 1 mL/kg, over 5–10 s) or
0.9% saline. Blood pressure was recorded each second for up to 1000 s
following the intravenous injection. Changes in mean arterial blood
pressure (ΔMABP) were determined from the basal pressure of rats. Data
are presented as the means ± SEM of 5 rats per condition.
Rats received an i.t. injection of 60 µg/kg CR-01-64 diluted in 0,9%
NaCl. Shortly before sampling, rats were deeply anesthetized with ke-
tamine/xylazine (87 mg/kg: 13 mg/kg, i.m.) and fixed on a stereotaxic
frame using ear bars. The head was maintained downward at approxi-
mately 45◦ to place the cisterna magna in the proper position [69]. A
27G winged perfusion needle connected to a syringe was inserted into
the cisterna magna. Approximately 150 μL of cerebrospinal fluid (CSF)
were then collected at 10, 30, 60, 90, 120, 180, 240, 360 or 480 min
after the CR-01-64 injection. Only one CSF sample was collected from
each rat, and three replicates were performed for each sampling time.
Rats were then euthanized, and CSF samples were stored at ꢀ 80 ◦C until
analysis.
2.9.3. Measurements of CR-01-64 concentrations in plasma and CSF
The CR-01-64 concentration was analyzed using a Sciex Qtrap
6500+ mass spectrometer (Ab Sciex LLC, Framingham, MA, USA)
coupled to a microflow M3 liquid chromatography apparatus equipped
with an HSST3 column (100 mm × 1 mm, 1.8 µm equipped with a 0.2
2.8. Ex vivo ileum relaxation assay
Smooth muscle relaxation assays of the distal rat ileum were per-
formed in isolated organ baths. Male Sprague-Dawley rats were anes-
thetized with an intramuscular injection of ketamine/xylazine (87 mg/
kg: 13 mg/kg, i.m.) and euthanized by surgical rupture of the abdominal
aorta. Tissues were rapidly removed, cut into 1 cm-long strips, cleaned
of fat and feces, and mounted vertically in 15 mL baths. The baths were
filled with oxygenated (95% O2 and 5% CO2) and thermoregulated
(37 ◦C) Krebs solution (NaCl 118.1 mmol/L, KCl 4.7 mmol/L, CaCl2 2.5
mmol/L, KH2PO4 1.2 mmol/L, MgSO4 1.2 mmol/L, NaHCO3 25 mmol/L,
and D-glucose 5.5 mmol/L; pH 7.4). Strips were stretched with a resting
tension of 1.0 g followed by an equilibration period of at least 60 min
with replacement of the Krebs solution every 15 min. At the beginning of
each experiment, ileum preparations were challenged with 100 nM
carbachol until a constant contractile response was achieved, with
multiple washes between stimulations. Tissues were then precontracted
with the same dose of carbachol and presented analog concentrations
ranging from 10ꢀ 11 to 10ꢀ 5 M. Noncumulative concentration-response
curves were established; thus, analogs were added to the baths as a
single addition following washout of the previous dose. Muscular con-
tractions were measured isometrically with force transducers (Radnoti
Glass Technology Inc.) and recorded on multichannel chart recorders
(PowerLab 8/30, ADInstruments) linked to LabChart Pro software
(version 8.1.8). The percentage of ileum relaxation was calculated for
each individual contraction. EC50 values were determined by calculating
the dose response – stimulation Log(agonist) vs. response (four param-
eters) - using GraphPad Prism 7 and presented as the means ± SEM of
three different experiments.
mm fritted prefilter). The solvent flow rate was set to 50 μL/min, and the
column temperature was maintained at 35 ◦C. The sample volume
injected was 2 L. The mobile phase was 0.10% formic acid/water (A)
μ
and 0.10% formic acid/methanol (B). The elution gradient started with
5% eluent B for 1 min, increased to 10% in 2.5 min, and then increased
to 20% over 3.5 min. Solvent B was then pumped up to 95% over 1 min
and held at that concentration for 2 min. It was then increased to 100%
over 1 min and held at that concentration for 3 min. The initial solvent
mixture was then re-established over 1 min and held for 2 min. The
equilibration time was 3 min, and the total run time was 16 min. Mass
spectrometry analysis was performed using a positive electrospray
ionization (ESI+) source in multiple reaction monitoring mode (MRM).
The optimized parameters were obtained by directly infusing the
analytical standard solution at 10 ng/mL as follows: CUR at 30; Caz 1 at
20; capillary voltage of 5.5 kV; source temperature of 150 ◦C and des-
olvation temperature of 350 ◦C. Two product ions (transitions) were
used; the most abundant transition was used for quantification, whereas
the second most abundant transition was used for qualification. MS/MS
acquisition and data processing were performed with Analyste 1.6.3,
and quantification was performed with multiquad software from Sciex
LLC.
2.9.4. Plasma and CSF PK analyses
Plasma and CSF PK were analyzed using Phoenix WinNonlin (Cer-
tara, Princeton, NJ, USA). Plasma CR-01-64 PK parameters were
assessed after the i.v. injection using a two-compartmental model with a
first-order elimination including the initial plasma concentration (C0),
central volume of distribution (V1), elimination rate constant (k10),
transfer rate constants between central and peripheral compartments
(k12 and k21), volume of distribution at steady state (VSS), total clearance
2.9. In vivo pharmacokinetics (PK)
2.9.1. Blood PK
On the day before the experiment, animals were anesthetized with
ketamine/xylazine (87 mg/kg: 13 mg/kg, i.m.) and placed in a supine
position on a heating pad. The jugular vein was catheterized using 20 cm
(Cl), distribution and elimination rate constants (α and β), distribution
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M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
and elimination half-life (t1/2α and t1/2β) mean residence time (MRT)
two main classes of available drugs: biologics and small molecules.
Among the strategies used to improve peptide druggability, macro-
cyclization has been shown to be effective at providing suitable con-
formations for proper ligand-receptor interactions [51]. Peptide
macrocyclization indeed increases the affinity and selectivity for a
specific target, as well as the metabolic stability, permeability and
pharmacokinetic properties [71,72]. The current pipeline of macro-
cycles includes over 80 approved drugs, with almost half of them
belonging to the cyclic peptide class [72–74]. Among the well-known
examples of cyclic peptide drugs on the market, eight currently target
GPCRs. These drugs include somatostatin analogs targeting different
SSTR subtypes (SSTR1, 2, 3 and 5), such as octreotide, lanreotide and
pasireotide, which are used clinically as oncological drugs or to treat
Cushing’s disease and acromegaly; the synthetic analog of the vaso-
pressin antidiuretic hormone desmopressin, which is administered
orally or as an intranasal spray to treat diabetes insipidus, polydipsia
and polyuria through its binding to V2 receptors; the synthetic oxytocin
pitocin, which is used to stimulate uterine smooth muscle contractility
during labor by binding to oxytocin receptors (OXTR); the cyclic peptide
and area under the concentration-time curve (AUCinf). CSF CR-01-64 PK
parameters were assessed after the i.t. injection using
a non-
compartmental analysis including the peak plasma concentration
(Cmax), time to achieve Cmax (Tmax), terminal elimination rate constant
(λz), elimination half-life (t1/2 λz), volume of distribution (VZ/F), total
clearance (ClZ/F), mean residence time (MRT) and area under the
concentration-time curve (AUClast).
2.10. Data and statistical analysis
All experimental protocols, data, and statistical analyses described in
this study comply with the recommendations on experimental design
and analysis in pharmacology [70]. Animals were randomized to
treatment groups, and binding experiments were performed by in-
vestigators who were blinded to the compound. Reference compounds
were tested by several experimenters to avoid potential bias in the
assessment of pain, with consistent results.
For the behavioral tests, baseline latency in the tail-flick test, AUC of
the pain scores in the formalin test and baseline paw withdrawal
threshold on days 7 and 14 in the von Frey test followed a normal (or
Gaussian) distribution, as determined using the Shapiro-Wilk normality
test. Parametric tests were then performed to increase statistical power.
For ANOVA, post hoc tests were conducted only if F was significant and
no variance in homogeneity was detected. Data are presented as the
means ± SEM (or means ± SD for PK studies). All graphs and statistical
analyses were performed using GraphPad Prism 9 (GraphPad software,
La Jolla, CA, USA). Two-way ANOVA followed by Dunnett’s multiple
comparison test was used to determine differences in the tail-flick la-
tency, paw withdrawal threshold and body temperature between the
saline-treated group and groups treated with different doses of CR-01-64
or different compounds at equimolar doses (*), between groups coad-
ministered CR-01-64+ morphine and morphine ($) or CR-01-64 (!), or
between groups administered CR-01-64 and morphine (?), or to deter-
mine differences in paw withdrawal threshold between sham and saline
groups (#). The %MPE and AUC for the formalin test and % anti-
allodynic effect were analyzed using one-way ANOVA followed by
Dunnett’s multiple comparison test to compare the effect of saline
treatment and different doses of CR-01-64. The %MPE in the acute pain
model or the AUC in the tonic pain model were calculated for each dose
of the compound to determine the half-maximal effective dose (ED50) of
CR-01-64. Then, ED50 values were determined using the dose-response
stimulation log(agonist) vs response (three parameters). Nonlinear
regression analyses using three parameters were also performed to
determine the half-maximal inhibitory concentration (IC50) of neuro-
tensin in the ileum relaxation assay. For binding affinities, Ki values ±
SEM were calculated from the IC50, which were derived from the
resulting dose-response curves. A difference in response between the
compound and saline was considered significant with p-values of *p <
0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. A difference in the
response between sham animals and saline-treated animals was
considered significant with a p-value ####p < 0.0001. A difference in
the response between the CR-01-64 and morphine treatments was
considered significant with the following p-values: ?p < 0.05, ??p <
0.01, and ???p < 0.001. A difference in the response between groups
coadministered CR-01-64+ morphine and animals treated with
morphine ($) or CR-01-64 (!) alone was considered significant with the
following p-values: $$p < 0.01, $$$$p < 0.0001, !p < 0.05, !!!p < 0.001,
and !!!!p < 0.0001.
analog of
α-MSH, bremelanotide, which primarily functions as an
agonist of the melanocortin receptors MC3 and MC4 and is approved for
sexual arousal disorder; and finally the calcitonin derivatives miacalcin
and elcatonin that act on the amylin (AMY1) and calcitonin (CT) re-
ceptors, respectively, and are used to treat Paget disease, hypercalcemia
and pain caused by osteoporosis. Importantly, several cyclic peptide
drug candidates targeting other GPCRs are also in active clinical
development. Among those candidates being investigated in ongoing
phase III trials, balixafortide, a potent and highly selective bicyclic
peptide antagonist of the CXCR4 chemokine receptor, is used to manage
tumor growth and metastatic breast cancer. Motixafortide, which was
developed to treat human cancers, specifically pancreatic cancer, is a
cyclic synthetic peptide that also acts as a selective blocker of CXCR4.
Livoletide, a first-in-class analog of ghrelin, is used to treat hyperphagia
and obesity in patients with Prader-Willi syndrome, a rare genetic dis-
ease. Finally, setmelanotide, an MC4 cyclic agonist octapeptide, was
developed as an anti-obesity medication.
Of particular importance, peptide cyclization has shown great suc-
cess in generating active macrocyclic NT(8–13) analogs targeting NTS1
[54,75–78]. Accordingly, we have recently delineated the optimal ring
size and macrocyclization sites for NT(8–13), leading to the develop-
ment of high-affinity NTS1 cyclic agonists exhibiting better resistance to
proteases [49,50]. In the present study, we focused on the design of
NTS2-selective macrocyclic NT(8–13) peptides showing effective anal-
gesia and few of the adverse effects of NT known to be related to NTS1
activation (i.e., hypotension, hypothermia and ileum relaxation). In this
process, the Tyr11 residue of NT(8–13) was replaced with Trp11 to
improve selectivity towards NTS2. Side-chain to side-chain macro-
cyclization was then performed using ring-closing metathesis (RCM)
between Lys8 and Trp11 to constrain the peptide conformation and
improve its biological and metabolic properties. The resulting NT(8–13)
macrocyclic peptide, designed CR-01-64, was synthetized using
Fmoc-based solid phase peptide synthesis (SPPS) on 2-chlorotrityl
chloride resin with standard procedures (Fig. 1) [49,50].
3.2. Pharmacodynamic profile of CR-01-64
3.2.1. CR-01-64 displays potent affinity and selectivity for NTS2
Dose-concentration displacement curves of [125I]-neurotensin by CR-
01-64 were generated using membranes prepared from cells stably
expressing either hNTS1 or hNTS2 (Fig. 2). CR-01-64 displayed high
affinity for NTS2 (Ki of 7.0 ± 2.9 nM), while the affinity for NTS1 was
significantly decreased (871 ± 190 nM), thus yielding a macrocyclic
compound showing 125-fold selectivity in binding affinity for NTS2. As
NTS2-selective linear peptides were previously reported to induce
analgesic responses in various animal pain models [23,26,33,44–47], we
then decided to assess the analgesic effects of CR-01-64 on the responses
3. Results and discussion
3.1. Design and synthesis of a neurotensin receptor type 2 (NTS2)
macrocyclic analog
Peptide-based therapeutics occupy a structural niche between the
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M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
Fig. 1. Scheme of the synthesis of the NTS2 macrocyclic analog CR-01-64.
to thermal, chemical and mechanical pain stimuli in animal models of
different experimental pain paradigms.
saline-treated rats (Fig. 3A). The calculated percentage of the maximum
possible effect (% MPE) determined at 20 min postinjection revealed
that CR-01-64 inhibited the nociceptive tail-flick reflex in a dose-
dependent manner, reaching 95.4% inhibition at the highest dose
3.2.2. In vivo analgesic activity
tested (Fig. 3B). The resulting median effective analgesic dose (ED50
)
was estimated to be 11.1 µg/kg (i.e., 13 nmol/kg; Fig. 3C). Importantly,
coadministration with the selective NTS2 antagonist NTRC-844
completely abolished the analgesic effect of CR-01-64, indicating the
sole role of the NTS2 receptor in CR-01-64-mediated analgesia (Fig. 3D)
[79]. In this thermal acute nociceptive test, CR-01-64 provided pain
relief similar to that provided by the previously described NTS2 analog
JMV431 (Boc-Arg-Arg-Pro-Tyr-psi(CH2NH)Ile-Leu-OH) [33]. However,
3.2.2.1. CR-01-64 produces potent nonopioid antinociceptive effects and
potentiates morphine-induced analgesia. The potential antinociceptive
effect of CR-01-64 was first assessed using the radiant heat tail-flick test,
which consists of measuring the withdrawal latency (rapid flick) to
painful thermal stimuli applied to the tail. Intrathecal (i.t.) injections of
CR-01-64 at doses ranging from 10 to 60 µg/kg significantly increased
the tail-flick latency up to 40–50 min after administration compared to
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Biomedicine & Pharmacotherapy 141 (2021) 111861
Fig. 2. Curves showing the displacement of [125I]-neurotensin by NT(8–13) and CR-01-64 on hNTS1 (A) and hNTS2 receptors (B). Data are presented as the
means ± SEM of at least three separate experiments.
Fig. 3. Analgesic effect of CR-01-64 on tail-flick latencies in Sprague-Dawley rats. (A) Time course of the analgesic effect of an acute intrathecal injection of CR-01-64
at doses ranging from 3 to 60 µg/kg. (B) The percentage of maximal possible effect (%MPE) was calculated at 20 min postinjection, where the antinociceptive
response was maximal for each dose. (C) Determination of the analgesic effective dose (ED50) of CR-01-64. (D-E) Antagonism of the effect of the antinociceptive
action of CR-01-64 by either NTRC-844 (NTS2 antagonist) or naloxone (µ-opioid antagonist). NTRC-844 was administered intrathecally at a 10× equimolar dose of
CR-01-64, and naloxone was administered intraperitoneally at 10 mg/kg 30 min before the administration of CR-01-64. Comparison of the effects of CR-01-64,
morphine, and their combination on acute pain. Morphine was administered intrathecally at an equimolar dose to CR-01-64 (30 µg/kg = 35 nmol/kg; D) or
intravenously at 1 mg/kg (E). (A-D) Comparisons of the time courses for the effects of four doses of CR-01-64 and saline (*), between morphine and saline (*), and
between CR-01-64 and morphine (?). (E) Comparisons of the time courses for the analgesic effects of CR-01-64+ morphine and morphine ($) or CR-01-64 (!). The
results are presented as the means ± SEM (N = 6/group). For time courses (A, D, and E), comparisons were performed using two-way ANOVA with repeated
measures followed by multiple comparisons using Dunnett’s correction. For %MPE (B), comparisons were performed using one-way ANOVA with repeated measures
followed by multiple comparisons using Dunnett’s correction between groups treated with saline and CR-01-64 at different doses. Nonlinear regression analyses using
three parameters were used to determine the dose-effect relationship. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, $$p < 0.01, $$$$p < 0.0001, !p < 0.05,
!!!p < 0.001, !!!!p < 0.0001, ?p < 0.05, ??p < 0.01, and ???p < 0.001.
CR-01-64 appeared to be approximately three times more potent than
JMV431, which exhibits an ED50 of 33 nmol/kg. This increased potency
might be due to the inherent properties of the macrocycle, which im-
poses a conformational constraint that potentially minimizes the bind-
ing entropy to achieve the most favorable conformation in the binding
pocket of NTS2 [80]. We previously reported the synthesis and char-
acterization of a series of nonselective NT(8–13) macrocyclic analogs
formed via RCM between an ortho allylated tyrosine residue at position
11 and the side chain of an alkene-functionalized amino acid at position
8 of NT(8–13) [50]. The best compound of this series ([Orn
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Biomedicine & Pharmacotherapy 141 (2021) 111861
(All)-Lys-Pro-Tyr(OAll)]-Ile-Leu13), with an ED50 of 4.6 µg/kg, was less
effective than CR-01-64, achieving 50% pain relief at the highest dose
compared to 95.4% MPE for CR-01-64. Importantly, this macrocyclic
analog also displayed a high binding affinity for NTS1 (Ki 15 nM) and
therefore induced significant hypotensive and hypothermic effects,
which limits its clinical use for pain management [50].
NT release within the periaqueductal gray (PAG) [84,85]. Therefore,
certain levels of interaction between opioid and NT systems and some
uncertainty as to the NT receptor subtype involved in this
cross-regulation exist.
In the tail-flick assay, despite its long-acting action, i.t. administra-
tion of an equimolar dose of morphine (35 nmol/kg), which is the gold
standard opioid analgesic, was not as effective as CR-01-64 in increasing
the withdrawal response latency to thermal nociceptive stimuli
(Fig. 3D). This result is consistent with previous findings showing that
NT-related compounds exhibit superior analgesic efficacy to equimolar
doses of morphine in models of acute, tonic and neuropathic pain
[23–26]. Of obvious importance is whether CR-01-64-induced analgesia
was independent of the endogenous opioid system. Rats were pretreated
with naloxone (10 mg/kg; i.p.) 30 min before the administration of
We next assessed the potential functional crosstalk between opioi-
dergic and neurotensinergic systems. Indeed, compelling evidence has
shown complex interactions between NT and opioid neurotransmission
[4,9,81]. For instance, antisense peptide nucleic acids targeting the mu
opioid receptor (MOR) do not affect the analgesic response to NT [82].
In contrast, we previously reported a significant decrease in morphine
analgesia in NTS1-null mice [83]. In addition, a microinjection of MOR
agonists in discrete brain regions produces antinociception by inducing
Fig. 4. Analgesic effect of an acute intrathecal
injection of CR-01-64 on formalin-evoked pain-
related behaviors in Sprague-Dawley rats. (A)
Evaluation of the doses (from 3 to 60 µg/kg)
inducing the antinociceptive effects of CR-01-
64. (B) The area under the curve (AUC) was
calculated for all durations of the test. (C)
Determination of the analgesic effective dose
(ED50). The results are presented as the mean-
s ± SEM (N = 5/group). Comparisons between
the AUCs of groups treated with saline and CR-
01-64 at different doses were performed using
one-way ANOVA with repeated measures fol-
lowed by multiple comparisons using Dunnett’s
correction. Nonlinear regression analyses using
three parameters were used to determine the
dose-effect relationship. ***p < 0.001 and
****p < 0.0001.
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Biomedicine & Pharmacotherapy 141 (2021) 111861
CR-01-64 to investigate the effects of opioid antagonists. Interestingly,
naloxone did not block or reduce the analgesic action of CR-01-64 in our
study (Fig. 3E). To our knowledge, these results are the first to show the
nonopioid action of NT analogs acting at the NTS2 receptor.
median effective analgesic dose of CR-01-64 was estimated to be 7.1 µg/
kg, similar to that obtained in the tail-flick acute pain paradigm
(Fig. 4C). When focusing on the inflammatory phase, CR-01-64 was
effective at reducing both spinal (lifting) and supraspinal (licking/
biting/shaking) pain behaviors, while other NTS2-selective compounds,
such as JMV-431 and levocabastine, only affected the spine-related
lifting behaviors [47].
The use of multimodal analgesia, which is defined as the adminis-
tration of at least two analgesic drugs with different mechanisms of
action, represents a successful strategy for the management of a wide
range of pain conditions by minimizing the adverse effects associated
with opioid treatment while improving pain control through the syn-
ergistic or additive actions of drug combinations [86,87]. Here, we
evaluated whether a multimodal pharmacological approach combining
NTS2 and opioid medications was potentially useful as a treatment op-
tion for the management of some pain conditions. For this purpose,
morphine was administered intravenously at 1 mg/kg, alone or in
combination with CR-01-64 (30 µg/kg; i.t.). As shown in Fig. 3E,
morphine combined with CR-01-64 significantly increased the tail-flick
latency compared to CR-01-64 or morphine alone, thus indicating that
NT analogs are able to potentiate the analgesic effects of morphine.
Accordingly, the combination of opioids with NT derivatives was found
to increase analgesia through synergistic or additive effects [88–90].
3.2.2.3. CR-01-64 alleviates mechanical hypersensitivity in a rat model of
chronic inflammatory pain. We next investigated the analgesic effec-
tiveness of CR-01-64 in a complete Freund’s adjuvant (CFA)-induced
chronic inflammatory pain model. Following a unilateral injection of
CFA into the plantar surface of the rat hind paw, rats exhibited pro-
gressive swelling of the ipsilateral hind paw, accompanied by persistent
hypersensitivity to nonpainful mechanical stimuli caused by a series of
von Frey filaments. The paw withdrawal threshold was significantly
reduced as early as day 3 post-CFA injection and was maintained at least
until day 14 compared to sham animals (Fig. 5A). On days 7 and 14 post-
CFA, the intrathecal injection of CR-01-64 at 60 µg/kg induced an in-
crease in the paw withdrawal threshold, which lasted up to 60 min
(Fig. 5B and C). Indeed, CR-01-64 exerted potent antiallodynic effects,
inducing more than 80% pain relief, compared to saline-treated rats. To
our knowledge, CR-01-64 is the first NT analog with selectivity for NTS2
reported to date that shows analgesic efficacy in the CFA model of
chronic inflammatory joint pain.
3.2.2.2. CR-01-64 reduces formalin-evoked nociceptive behaviors. The
antinociceptive effect of the macrocyclic compound CR-01-64 was then
measured in the persistent inflammatory pain model induced by
formalin by monitoring the paw licking, biting, shaking and lifting
nociceptive behaviors induced by an intraplantar injection of diluted
formalin (5%) into the right hind paw. Formalin-induced pain produced
a typical biphasic behavioral response characterized by an early acute
phase (0–9 min) and a sustained inflammatory phase (21–60 min)
separated by a transient inactive interphase. Intrathecal injection of CR-
01-64 dose-dependently reduced formalin-induced nociceptive behav-
iors in both the acute and inflammatory phases (Fig. 4A). Compared to
saline-treated rats, the reduction in pain intensity score defined as the
area under the curve (AUC) was effective even at the very low dose of
3.2.3. Other NT-mediated physiological effects
NT is involved in the regulation of a number of other physiological
functions. Among them, central or systemic administration of NT ana-
logs has been shown to produce mild hypothermia and to exert neuro-
protective effects on various neurological conditions, such as ischemic
stroke and traumatic brain injury [40,91–94]. Peripherally, NT also
regulates the gastrointestinal and cardiovascular systems, inducing
changes in gastric and colonic motility and modulating blood pressure
and myocardial contractility [12,13,38,39,95]. Importantly, preclinical
3 μg/kg, producing complete pain relief at 30 μg/kg (Fig. 4B). The
Fig. 5. Anti-allodynic effect of CR-01-64 on a
chronic inflammatory pain model. (A) Mechan-
icalanti-allodyniceffectsofCR-01-64(60 µg/kg,
i.t.) were determined at days 7 and 14 after the
induction of inflammatory pain by complete
Freund’s adjuvant (CFA). The percent of anti-
allodynic effect was determined at the maximal
analgesic effect, 10 min post-injection, on day 7
(B) or 14 (C). The results are presented as the
means ± SEM (N = 6/group). For the analysis of
the time course of the effects on days 3, 7 and 14,
comparisons between sham animals and CFA
rats treated with saline (#) or between CFA rats
receiving either saline or CR-01-64 (*) were
performed using two-way analysis of variance
for repeated measures followed by multiple
comparisons using Dunnett’s correction. For the
% of anti-allodynic effect determined on days 7
and 14, comparisons between sham animals and
CFA rats treated with saline (#) or CFA rats
receiving either saline or CR-01-64 (*) were
performed using one-way analysis of variance
for repeated measures followed by multiple
comparisons using Dunnett’s correction.
****p < 0.0001 and ####p < 0.0001.
10

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
studies performed on NTS1-deficient mice or with the NTS1-selective
antagonist SR48692 revealed that all of these physiological effects are
exclusively associated with NTS1 activation [45,96–99]. We therefore
decided to characterize the effects of CR-01-64 on the regulation of body
temperature, blood pressure and ileum relaxation.
bodies and fibers in various nuclei of the hypothalamus known to con-
trol thermoregulation, such as the arcuate nucleus and the preoptic area
[37].
3.2.3.2. CR-01-64 exerts moderate hypotensive effects at high doses. In
addition to its hypothermic effects, intravenous (i.v.) or intra-
cerebroventricular (i.c.v.) injection of NT analogs results in a significant
decrease in blood pressure [38,100–102]. We thus evaluated the hypo-
tensive action of CR-01-64 and NT(8–13) by monitoring the variation in
arterial blood pressure (ΔMABP) using continuous intracarotid mea-
surements. As observed in a previous study [103], an i.v. injection of NT
(8–13) (0.1 mg/kg) induced a severe and sustained decrease in blood
pressure characterized by a triphasic response (Fig. 6B). A short decrease
of ꢀ 25 mmHg occurred rapidly (first phase) before a swift return to
basal levels (second phase) followed by a prolonged decrease to
ꢀ 50 mmHg (third phase). At a 10-fold lower dose (0.01 mg/kg; i.v.), NT
(8–13) did not elicit the last sustained hypotensive phase. In compari-
son, an i.v. injection of CR-01-64 at the lowest dose of 0.01 mg/kg
produced slight and transient hypotension lasting approximately two
minutes, while a dose of 0.1 mg/kg induced moderate triphasic hypo-
tension that was less than a similar dose of NT(8–13) and lasted up to
15 min (Fig. 6B). In the same experimental paradigm, nonselective
3.2.3.1. An analgesic dose of CR-01-64 does not induce hypothermia. The
ability of CR-01-64 to modulate body temperature was then monitored
at 10 min intervals for 60 min following i.t. administration and reported
as the variation in temperature (Δ body temperature) from baseline
before the injection. As shown in Fig. 6A, CR-01-64 did not produce
hypothermia at the maximal analgesic dose tested (i.e., 60 µg/kg). In
contrast, an injection of NT(8–13) at the same dose induced short-term
hypothermia, while the NTS1-selective agonist PD149163 (h-LysΨ
[CH2NH]Lys-Pro-Trp-Tle-Leu-OEt), an NT(8–13) analog that contains a
reduced amine bond to improve its resistance to proteases, was
responsible for a sustained decrease in body temperature, even at a
lower dose of 30 µg/kg (Fig. 6A). Accordingly, nonselective macrocyclic
analogs of NT(8–13) also induced a dose-dependent decrease in body
temperature for over 1 h [50], while NTS2-selective ligands did not
induce signs of hypothermia when injected centrally [45]. These results
are also consistent with the presence of NTS1-immunopositive cell
Fig. 6. Effects of CR-01-64 on body tempera-
ture, blood pressure, and smooth muscle
contraction in Sprague-Dawley rats. (A) Varia-
tion in body temperature measured after the
intrathecal injection of CR-01-64 and NT(8–13)
at 60 µg/kg, PD149163 (NTS1-selective
agonist) at 30 µg/kg or saline. (B) The change
in mean arterial blood pressure (ΔMABP)
recorded after the intravenous injection of NT
(8–13) or CR-01-64 at 0.01 or 0.1 mg/kg to
Sprague-Dawley rats. (C) Percent ileum relaxa-
tion monitored following an incubation with
increasing concentrations of CR-01-64 or NT
(8ꢀ 13) ranging from 10ꢀ 11 to 10ꢀ 5 M. The re-
sults are presented as the means ± SEM (N = 6/
group for body temperature measurements and
N = 5/group for ΔMABP measurements). For
body temperature, comparisons between
groups treated with saline and PD149163 or
CR-01-64 were performed using two-way anal-
ysis of variance for repeated measures followed
by multiple comparisons using Dunnett’s
correction. Nonlinear regression analyses using
three parameters were performed for % ileum
relaxation. The NT(8–13) EC50 value was ob-
tained from the resulting dose-response curve.
*p < 0.05, **p < 0.01, ***p < 0.001, and
****p < 0.0001.
11

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
macrocyclic NT(8ꢀ 13) analogs produced strong and persistent hypo-
tension, similar to NT(8–13) [50]. The remaining hypotensive action of
CR-01-64 is probably related to its ability to bind to NTS1, since the
3.3.2. Pharmacokinetic profile of CR-01-64
To our knowledge, the plasma and cerebrospinal fluid (CSF) phar-
macokinetics (PK) of NTS2 compounds have never been evaluated and
are therefore of interest in the development of new analgesics. Opti-
mizing the ADME properties of a molecule is indeed a challenging part of
the whole drug discovery process since the PK profile has a major effect
on the likelihood of a drug’s success and will also help determine the
appropriate route of administration in a clinical setting. Here, we sub-
sequently determined the PK profiles of CR-01-64 by collecting plasma
and CSF samples at various time points following i.v. and i.t. adminis-
tration, respectively.
NTS2-selective compound NT79 ((NαMe)Arg-Arg-Pro-D-3,1-Nal-tLeu--
Leu) does not lower blood pressure when injected intraperitoneally at a
high dose of 5 mg/kg in freely moving rats [46]. Despite this moderate
decrease in blood pressure, CR-01-64 offers a wider therapeutic window
than nonselective NT analogs.
3.2.3.3. Ileum relaxation is not induced by CR-01-64. NT is a gastroin-
testinal peptide known to induce relaxation of colonic smooth muscles
[39,95,104,105]. We thus investigated whether CR-01-64 was able to
modulate the smooth muscle tone of the isolated rat ileum prestimulated
with carbachol (Fig. 6C). Compared to NT(8–13), which produced
strong relaxation of the carbachol-contracted strips in the organ bath
with an EC50 of 3.9 nM, CR-01-64 was not effective at relaxing isolated
segments of smooth muscles at concentrations of up to 1 µM. These re-
sults support the predominant role of NTS1 in mediating the relaxation
of the ileum. Moreover, NT agonists fail to induce contractile responses
of isolated ileal strips in mice deficient in NTS1 [96].
The time course of plasma concentrations following an i.v. injection
of CR-01-64 at 0.2 mg/kg, as well as in CSF after an i.t. injection of
60 µg/kg are shown in Fig. 8A and B. The associated PK parameters are
shown in Tables 1 and 2. The plasma PK of CR-01-64, which were
analyzed using a two-compartmental model, displayed rapid elimina-
tion with a short elimination half-life and high elimination rate constant
and clearance. CR-01-64 also exhibited high distribution volumes.
Exposure to CR-01-64 and the mean residence time were low. Indeed,
plasma PK revealed that CR-01-64 was rapidly removed from plasma
with first-order elimination kinetics (t1/2β = 44.7 min and Cl = 0.38 L/
min/kg), and was CR-01-64 broadly distributed (V1 = 2.63 L/kg and VSS
= 9.44 L/kg), indicating tissue diffusion. We hypothesize that renal
excretion probably contributes to its rapid elimination from plasma,
given the peptidic nature of CR-01-64.
3.3. Pharmacokinetics of CR-01-64
3.3.1. Stability in plasma and cerebrospinal fluid exceeds 24 h
As a peptide, NT exhibits low oral bioavailability and low resistance
to proteolytic degradation, which limits its clinical therapeutic use
[106]. Its short half-life of approximately 2 min is due to rapid cleavage
by three metalloendopeptidases (24.11, 24.15, and 24.16) at the peptide
bonds Arg8-Arg9, Pro10-Tyr11 and Tyr11-Ile12 [107–110]. The metabolic
stabilities of CR-01-64 and NT(8–13) were thus assessed in rat plasma
and cerebrospinal fluid (CSF) at 37 ◦C for up to 24 h, and their degra-
dation profiles were analyzed using UPLC-MS. As expected, rapid
degradation of NT(8–13) was observed in plasma with a half-life of
2 min (Fig. 7A). Unlike blood, the stability of NT(8–13) in CSF was
higher (half-life = 20 h), probably due to a lower proteolytic enzyme
activity profile (Fig. 7B).
Likewise, the PK of CR-01-64 in CSF, which were analyzed using
noncompartmental analysis, exhibited rapid elimination with a short
elimination half-life and high elimination rate constant and clearance.
The macrocycle was broadly distributed with a large volume of distri-
bution. Its exposure and mean residence time were low. The PK analysis
in CSF indicated a delayed Cmax at 90 min, as CR-01-64 was injected
intrathecally between the L5-L6 intervertebral space and collected
above in the cisterna magna. It also exhibited rapid clearance from the
CSF and a high distribution (t1/2 λz = 55.49 min and VZ/F = 0.26 L/kg),
indicating tissue diffusion. Interestingly, CSF concentrations of CR-01-
64 represented less than 1% of the administered dose (60 µg/kg),
consistent with its tissue diffusion. CR-01-64 therefore appears to
exhibit a favorable PK profile in CSF, which may govern its long-lasting
analgesic effects. Together, these results indicate that the macro-
cyclization of NT analogs is a successful strategy to develop stable
compounds and to improve the ADME properties.
In both plasma and CSF, CR-01-64 exhibited excellent stability with a
half-life of 24 h, indicating that macrocyclization limits peptide bond
hydrolysis. Indeed, macrocyclization protects well-recognized degra-
dation sites, notably Ile12-Leu13, Pro10-Tyr11 and dibasic Arg8-Arg9 site
amide bonds, which are readily hydrolyzed by enzymes, such as the
angiotensin-converting enzyme and enkephalinases that are expressed
at high levels in rat synaptic brain membranes [107–109]. In addition,
the conformational restriction of the peptide backbone creates unfa-
vorable folding, with presumably fewer beta-strand-like properties, thus
preventing recognition by endopeptidases [111]. Therefore, CR-01-64
shows lower polarity and improved stability in plasma and CSF
compared to NT(8–13).
4. Conclusions
There is a very high need for improving the management of acute
and persistent pain since its pharmacotherapy remains often limited. To
support the development of analgesics, more predictive assessments of
target engagement and response to drug treatment are needed. For
instance, the incorporation of translational tools, such as validated
pharmacodynamic (PD) biomarkers can provide important information
Fig. 7. Metabolic stability of CR-01-64 in rat biological fluids. In vitro stability of NT(8–13) and CR-01-64 in rat plasma (A) and cerebrospinal fluid (CSF) (B). The
half-life (t1/2) was calculated as the disappearance of half of the compound (% remaining) determined using UPLC-MS. Error bars represent the means ± SEM of at
least three separate experiments using each compound.
12

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
Fig. 8. Pharmacokinetic profile of CR-01-64 in rat biological fluids. (A) Plasma CR-01-64 concentration-time profiles following an intravenous injection of 0.2 mg/kg
CR-01-64 in SD rats show the observed (blue circle) and predicted (black dashed line) concentrations using a two-compartment model (N = 6). (B) CSF CR-01-64
concentration-time profiles following an intrathecal injection of 60 µg/kg CR-01-64 in SD rats show the observed (blue circle) and mean (black dashed line) con-
centrations (N = 27 rats). For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
about the pharmacological effects of a drug on its target, prove its effi-
Table 1
cacy throughout clinical phases and facilitate regulatory approval [112].
Plasma pharmacokinetic parameters following an intravenous injection of
Unfortunately, unlike the field of oncology, which has been transformed
0.2 mg/kg CR-01-64 in Sprague-Dawley rats. Parameters were determined using
by the use of biomarkers, to our knowledge, such biomarkers do not yet
a two-compartmental model (N = 6).
exist in the field of chronic pain [113,114]. Monitoring of blood or CSF
Parameters
Units
Plasma
PD biomarkers that rise or fall in response to drug treatment therefore
has the potential to facilitate the assessment of the efficacy and safety
issues of non-addictive pain therapeutics as well as to reduce attrition
and high-failure rate in the development of analgesics [115].
Estimation
CV (%)
C0
V1
k10
k12
k21
VSS
Cl
ng/mL
L/kg
75.97
2.63
49.81
49.86
32.32
29.67
32.10
45.18
19.89
28.3
minꢀ 1
minꢀ 1
minꢀ 1
L/kg
0.14
The need for safe and effective alternatives to opioids has never been
greater than during the current opioid epidemic. While opioids are
recognized as the most effective pain-relieving drugs, the use of non-
opioid analgesics and opioid-sparing multimodal therapy has the po-
tential to limit the adverse effects of opioids by reducing the dose used.
Importantly, this alternative strategy also provides an approach to
mitigate the recognized dangers associated with opioid misuse and
abuse. Regarding its analgesic action, CR-01-64 produces potent and
effective antinociceptive responses to different sensory modalities (e.g.,
thermal, chemical and mechanical stimuli) in various models of acute,
persistent, and chronic pain. Importantly, the analgesic effect of CR-01-
64 does not appear to depend on the endogenous opioid system, since
the administration of the opioid antagonist naloxone is not effective at
reversing NT-induced analgesia. By activating a different mechanism of
action, CR-01-64 combined with morphine further improves pain relief
at lower doses of morphine than morphine alone. Overall, these results
highlight the potential use of NTS2 as nonopioid analgesics or in a
multimodal regimen with morphine to achieve more effective analgesia
with reduced adverse effects.
0.06
0.02
9.44
L/kg/min
minꢀ 1
minꢀ 1
min
0.38
α
0.21
β
0.016
3.23
29.5
t1/2
α
28.2
t
1/2β
min
44.7
29.5
MRT
min
25.12
532.17
30.27
19.87
AUCinf
µg min/L
C0, initial plasma concentration; V1, central volume of distribution; k10, elim-
ination rate constant; k12 and k21, transfer rate constants between central and
peripheral compartments; VSS, volume of distribution at the steady state; Cl,
total clearance; α, distribution rate constant, β, elimination rate constant, t1/2α,
distribution half-life, t1/2β, elimination half-life; MRT, mean residence time;
AUCinf, area under the concentration-time curve. Values are presented as the
means ± SD.
Table 2
CSF pharmacokinetic parameters following an intrathecal injection of 60 µg/kg
CR-01-64 in Sprague-Dawley rats. Parameters were determined using a non-
compartmental analysis (N = 27 rats).
Funding
Parameters
Units
CSF
This work was supported by funding from the Canadian Institutes of
Health Research (FDN-148413 to P.S.), the Natural Sciences and Engi-
neering Research Council of Canada and the Canadian Foundation for
Innovation. P.S. is the holder of Canada Research Chair Tier 1 in the
Neurophysiopharmacology of Chronic Pain and a member of the FRQS-
Estimation
Tmax
Cmax
λz
min
90
ng/mL
minꢀ 1
L/kg
236,310
0.01
VZ/F
ClZ/F
0.26
´
funded Quebec Pain Research Network. E.M. is a member of the FRQNT-
L/kg/min
min
0.003
55.49
121.62
18,363
´
funded Proteo and the FRQS-funded Quebec Network for Drug Research
t
1/2 λz
MRT
min
networks. M.C.’s Ph.D. is funded by the FRQS. M.D. is a recipient of a
AUClast
µg min/L
fellowship from the Centre de Recherche du CHUS.
Cmax, peak plasma concentration; Tmax, time to achieve Cmax; λz, elimination
rate constant; VZ/F, volume of distribution; ClZ/F, total clearance; t1/2 λz, ter-
minal elimination half-life; MRT, mean residence time; AUClast, area under the
concentration-time curve. Values are presented as the means ± SD.
Dedication
In memory of our close colleague, best friend and research director,
´
Professor Eric Marsault, who passed away far too early.
13

M. Chartier et al.
Biomedicine & Pharmacotherapy 141 (2021) 111861
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Magali Chartier: Investigation, Formal analysis, Writing - original
´
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´
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Validation, Writing - original draft, Visualization. Marc Sousbie:
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´
´
`
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´ ˆ
ˆ ´
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´
Michel Longpre: Formal analysis, Resources, Writing - review & edit-
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& editing, Funding acquisition.
´
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Declaration of Competing Interest
[28] P. Sarret, F. Cavelier, Neurotensin and Its Receptors, Reference Module in
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