Synthesis and Preclinical Evaluation of the First Carbon-11 Labeled PET Tracers Targeting Substance P1-7

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00518

Two potent SP_1-7 peptidomimetics have been successfully radiolabeled via [¹¹C]CO₂-fixation with excellent yields, purity, and molar activity. l-[¹¹C]SP_1-7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[¹¹C]SP_1-7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[¹¹C]SP_1-7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP_1-7 in spinal cord for l-[¹¹C]SP_1-7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[¹¹C]tracers did not reduce the tracers' brain and spinal cord exposure. In summary, PET scanning of l- and d-[¹¹C]SP_1-7-peptidomimetics confirms rapid blood-brain barrier and blood-spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP_1-7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.

Full text of DOI:10.1021/acs.molpharmaceut.8b00518

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Article
Synthesis and Preclinical Evaluation of the First
Carbon-11 Labeled PET Tracers Targeting Substance P
1-7
Aleksandra Pekosak, Janez Ž. Bulc, Špela Korat, Robert C. Schuit, Esther Kooijman, Ricardo Vos, Marissa
Rongen, Mariska Verlaan, Kevin Takkenkamp, Wissam Beaino, Alex J. Poot, and Albert D. Windhorst
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00518 • Publication Date (Web): 18 Oct 2018
Downloaded from http://pubs.acs.org on October 19, 2018
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Molecular Pharmaceutics
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Synthesis and Preclinical Evaluation of the First
Carbon-11 Labeled PET Tracers Targeting Substance
P1-7
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Aleksandra Pekošak, Janez Ž. Bulc§, Špela Korat§, Robert C. Schuit, Esther Kooijman, Ricardo
Vos, Marissa Rongen, Mariska Verlaan, Kevin Takkenkamp, Wissam Beaino, Alex J. Poot, Albert
D. Windhorst
Department of Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, the
Netherlands
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Abstract:
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Two potent SP1-7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation in
excellent yields, purity and molar activity. L-[11C]SP1-7-peptidomimetic exhibited promising ex
vivo biodistribution profile. Metabolite analysis showed that L-[11C]SP1-7-peptidomimetic is stable
in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic
stability can be significantly improved by substituting L-Phe for D-Phe, preserving 70% more of
intact tracer and resulting in better brain and spinal cord tracer retention. PET scanning confirmed
moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for L-
and D-[11C]SP1-7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment
with natural peptide SP1-7 in spinal cord for L-[11C]SP1-7-peptidomimetic. On the contrary,
blocking using cold analogues of L- and D-[11C]tracers did not reduce the tracers’ brain and spinal
cord exposure. In summary, PET scanning of L- and D-[11C]SP1-7-peptidomimetics confirms rapid
blood-brain-barrier and blood-spinal-cord-barrier penetration. Therefore, further validation of
these two tracers targeting SP1-7 is needed in order to define a new PET imaging target and select
its most appropriate radiopharmaceutical.
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Table of Contents
O
BEMP
DMSO
O
H2N
NH2
[
11C]CO2
*
O
NH
11C
O
NH2
RT
Ki = 5.3 nM
L-[11C]1
H-Phe-NH2 or
H-D-Phe-NH2
Substitution to D-Phe
Rapid Brain and Spinal Cord
Uptake
Stable in Brain and Spinal Cord
Favourable Biodistribution
D-[11C]2
Improved Metabolic
Stability in Plasma
Keywords: Substance P1-7, CO2 fixation, Carbon-11, Metabolism, Biodistribution, PET Imaging
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Introduction
Substance
P
(SP) (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) is
a
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neurotransmitter and neuromodulator involved in a variety of neurological functions. Its role in
pain transmission has attracted particular interest, since SP binds preferably to the neurokinin 1
receptor (NK-1R), however the SP N-terminal major bioactive fragment SP1-7 (H-Arg-Pro-Lys-
Pro-Gln-Gln-Phe-OH) acts through binding to a specific site apart from any known neurokinin or
opioid receptor. The absence of binding of SP1-7 to the NK-1R is attributed to the lack of the
amidated amino acid sequence Gly9-Leu10-Met11-NH2 on the C-terminus.1–7
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SP1-7 has been shown to be present in various central nervous system (CNS) areas, such as
hypothalamus, nucleus accumbens, ventral tragemental area, and in the dorsal and ventral part of
spinal cord, confirming that the heptapeptide is related to pain and reward processing when
produced in the CNS by endopeptidases. 8–10 Even though the binding sites in mouse and rat are
known, the receptor for SP1-7 has not yet been cloned or characterized, making its mechanism of
action remaining unclear.8,11 Recently, in vitro and in vivo studies showed improved binding
affinity of amidated C-terminus SP1-7-NH2 compared to the endogenous fragment SP1-7, as well as
increased potency effect to mediate a reduced expression of opioid withdrawal through the SP1-7
binding site.12,13 In addition, the SP1-7 analog D-SP1-7 (D-Pro2-D-Phe7-SP1-7) was able to block SP1-7
induced antinociception and showed binding affinity to SP1-7 binding site equipotent to natural SP1-
1,14,15
.
Interestingly, the SP1-7-NH2 showed higher anti-allodynic activity after intraperitoneal
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administration compared to SP1-7 despite the poor in vivo stability of both peptides.7
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Aiming to develop a peptidomimetic with a long duration of action and be able to cross the
blood-brain barrier (BBB), Fransson et al. performed extensive structure-activity relationship
(SAR) studies resulting in a lead dipeptide H-Phe-Phe-NH2 (Ki of 1.5 nM; rat spinal cord
membrane) as small molecule to target the SP1-7 binding site.5,12 Further fine-tuning, by replacing
the N-terminal Phe with a benzylcarbamate group, resulted in a peptidomimetic (S)-benzyl (1-
amino-1-oxo-3-phenylpropan-2-yl)carbamate L-1 with a Ki of 5.2 nM, determined by using rat
spinal cord membranes preparation (Figure 1).1,11,16 Its reasonable binding affinity, excellent
membrane permeability, low efflux, moderate in vitro clearance and ability to enter the CNS
according to the in vivo infusion study, make SP1-7 an interesting target for PET tracer
development.
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H2N
NH
NH
NH2
O
N
NH2
O
H2N
HN
O
OH
O
HN
O
H
N
O
N
O
N
H
O
Ki = 1.6 nM
NH2
O
SP1-7
H2N
NH
NH
O
O
H
*
O
NH
N
NH2
O
11C
O
NH2
H2N
NH2
O
O
N
NH2
H2N
Ki = 5.3 nM
L-[11C]1
HN
Ki = 1.5 nM
H-Phe-Phe-NH2
NH2
O
HN
O
N
H
N
O
O
N
O
and
H
O
Ki = 0.3 nM
D-[11C]2
NH2
O
SP1-7-NH2
Figure 1: Structure of SP1-7 and SP1-7-NH2 as leads for the development of small molecules, H-
Phe-Phe-NH2 and the peptidomimetics L-1 and D-2. Red 11C indicates radiolabeling position using
the [11C]CO2-fixation method.
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Neuropathic pain is clinically characterized by spontaneous and evoked type of pain
significantly impairing the quality of patients’ life whereas its effect in treating represents a major
unsolved problem.17 Developing PET-tracers able to image and evaluate neuropathic pain would
provide a valuable tool for better estimation of the incidence and prevalence of neuropathic pain,
and treatment development.18 SP1-7 is a relatively large molecule and current radiolabeling
techniques require a strong modification of the native peptide structure hampering the BBB
penetration. Nonetheless, the dipeptide peptidomimetics are more attractive alternative and could
be viable neuro PET tracers able to cross the BBB. PET imaging is a powerful technique that
provides direct quantitative information on the levels of brain and spinal cord exposure, target
engagement and pharmacological activity.19,20 In our study, we used dipeptide peptidomimetics
(Figure 1) for radiolabeling with carbon-11 in order to investigate the SP1-7 binding site and provide
insights in the potential neuropathic pain PET imaging.
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The merits of carbon-11 (t1/2=20.4 min, 99% β+, 0.96 MeV) as a choice radionuclide for PET
tracer synthesis have been well recognized. The ubiquity of carbon atoms in all pharmacologically
active compounds makes it an attractive isotope for radiolabeling, preserving the favorable
physicochemical properties.21–28 With the recent developments in carbon-11 chemistry, routinely
employed complex processes and appropriate special equipment, radiochemists are able to label
various compounds. Direct fixation of [11C]CO2, published by Hooker et al.29 and Wilson et al.30,
addressing the carbon-11 labeled carbamate functional group, enables us to label the SP1-7
peptidomimetic (S)-benzyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate L-1 or (R)-benzyl (1-
amino-1-oxo-3-phenylpropan-2-yl)carbamate D-2 (Figure 1) to serve as a research tool to
investigate the SP1-7 binding site. To achieve this goal, we firstly investigated their physiochemical
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properties using novel tool CNS PET multi parameter optimization (MPO) algorithm31,32, as well
as commercial platform for in silico prediction of physicochemical, absorption, distribution,
metabolism, and excretion (ADME), and toxicity properties, to provide valuable information if
compounds of interest meet a distinctive set of criteria for CNS PET ligand. As prediction models
provided enviable starting point for PET ligand discovery effort, we investigated the feasibility of
carbon-11 radiolabeling of compounds 1 and 2 without further modifications. Finally, we
evaluated their biodistribution, in vivo metabolic stability and assessed their in vivo behavior in
brain and spinal cord using PET imaging.
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Experimental Section
Materials and Methods
Chemicals were obtained commercially from Sigma-Aldrich (Zwijndrecht, the Netherlands) and
Bachem (Bubendorf, Switzerland), and used without further purification, unless stated otherwise.
Solvents were purchased from Biosolve (Valkenswaard, the Netherlands) and Acros (Landsmeer,
the Netherlands), and used as received, unless stated otherwise. All deuterated solvents were
purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands). Reactions were performed at
ambient temperature unless stated otherwise and monitored by thin layer chromatography on pre-
coated silica 60 F254 aluminum plates (Merck, Darmstadt, Germany). Spots were visualized by UV
light (254 nm) and ninhydrine or bromocresol green staining. Solvents were evaporated under
reduced pressure using a rotary evaporator (Rotavapor® R II, Flawil, Switzerland). Flash column
chromatography was performed on Büchi (Flawil, Switzerland) Sepacore system (comprising a C-
620 control unit, a C-660 fraction collector, two C-601 gradient pumps and a C-640 UV detector)
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equipped with Büchi Sepacore pre-packed flash columns. Yields refer to purified and
spectroscopically pure compounds.
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NMR spectroscopy was performed on a Bruker Avance 250, 400 or 500 MHz spectrometer
(Billerica, MA, USA, exact magnetic field is reported in the chemical syntheses section) with
chemical shifts (δ) reported in parts per million (ppm) relative to the solvent (CDCl3, 1H 7.26 ppm,
13C 77.16 ppm; D2O, 1H 4.79 ppm; (CD3)2SO, 13C 39.52 ppm). Splitting patterns are indicated as
s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet and br: broad peak, and J, coupling constant
in Hz. Electrospray ionization-high resolution mass spectrometry (ESI-HRMS) was carried out
using Bruker microTOF-Q instrument in a positive ion mode (capillary potential of 4500 V) and
interpreted with Bruker Compass Data Analysis 4.0 software.
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Analytical HPLC was performed on a Shimadzu SPD-20A system (Shimadzu Corporation,
Japan) and Jasco PU-2089 Plus station (Easton, MD, USA) with a Alltima C18 5 μm (250 × 4.6
mm) column (Grace, Breda, the Netherlands) (A) with Jasco UV-2075 Plus UV detector (254 nm)
and NaI radioactivity detector (Raytest, Straubenhardt, Germany) at ambient temperature with a
flow rate 1 mL·min-1. Acetonitrile (C), acetonitrile+0.1% TFA (D), water (E), water+0.1% TFA
(F) and buffer 1 (4 mM sodium formate and 4% dimethylformamide) (G) were used as mobile
phases. Chromatograms were acquired with LabSolutions 5.85 software (Shimadzu Corporation,
Japan) and Raytest GINA Star software (version 5.8; Straubenhardt, Germany).
*Method A: solvents D/F (v/v), linear gradient, 0 - 1 min: 35% D; 1 - 19 min: 35-65% D; 19 -
20 min: 65 - 35% D; 20 - 21 min: 65% D.
Semi-preparative isocratic HPLC was performed on Jasco UV-2087 Plus station with a Alltima
C18 5 μm (250 × 10 mm) column (Grace, Breda, the Netherlands) (J) using C/G (40/60, v/v) as a
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eluent, flow rate 5 mL·min-1 (Method B), a Jasco UV-2075 Plus UV detector (254 nm), a
homemade radioactivity detector and Jasco ChromNAV CFR software (version 1.14.01).
HPLC conditions for metabolite analysis and LC-MS/MS analysis are for convenience reported
under appropriate section.
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Prediction Models:
 ACD Labs - Percepta
Full array of physiochemical, ADME and toxicity properties, next to BBB transport parameters
were predicted using the commercial package of ACD/Percepta (version: 14.2.0 (Build 2972)).
 CNS PET Multi Parameter Optimization (MPO)
CNS PET MPO value was calculated using a set of six physiochemical parameters:
-
-
-
-
-
-
molecular weight (MW),
lipophilicity, calculated partition coefficient (ClogP),
calculated distribution coefficient at pH=7.4 (ClogD),
topological polar surface area (tPSA),
ionization constant of the most basic center (pKa) and
number of hydrogen bond donors (HBD).
following the publication of Zhang et al.31 and standard commercial packages described in the
paper of Wager et al.33:
List of standard commercial packages: Biobyte (Bio-Loom; version 5) for ClogP, ACD/Percepta
for ClogD at pH=7.4, tPSA, pKa, ChemDraw (professional; version: 16.0.1.4 (77)) for Mw and
HBD.
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Calculation of the Radiochemical Yield by Analytical HPLC
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Radiochemical yield, determined by analytical HPLC of the crude reaction mixture, defined as
radiochemical conversion (RCC), was determined by HPLC as the percentage of converted
[11C]CO2 to desired product [11C]1 or [11C]2 in the crude reaction mixture using an analytical
HPLC method described in the radiochemistry section under each procedure and is based on the
area under the curve (AUC) of the radioactivity profile of HPLC analysis.
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Calculation of the Radiochemical Yield
Radiochemical yield (RCY) was calculated as the quotient of measured activity of the isolated
product at the end of the synthesis (EOS) and the measured activity at the end of the cyclotron
bombardment (EOB) and expressed as a percentage. Radiochemical yield has been corrected for
decay from the EOB. No corrections have been made for material losses (potentially volatile
radioactive species or residual activity in the vial, tubes and syringes).
Calculation of the Molar Activity
Molar activity (AM) of the final tracer, expressed as GBq·µmol-1, was determined by
measurement of the UV absorbance (AUC) of a known amount of radioactivity under identical
analytical HPLC conditions used to generate a mass calibration curve for the corresponding non-
radioactive standard.
The Mass Calibration Curve
The mass calibration curve was performed using samples of the reference compound 1 due to
strong UV absorption and is based on the AUC of HPLC analysis.
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Chemical Syntheses:
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(S)-benzyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (1)
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Starting compound (S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanoic acid (80.0 mg, 267
µmol, 1 eq) and di-tert-butyl dicarbonate (125 mg, 573 µmol, 2 eq) were dissolved in 3 mL of 1,4-
dioxane and placed on a magnetic mixer. Pyridine (18.0 µL, 267 µmol, 1 eq) and ammonium
hydrogen carbonate (58.0 mg, 746 µmol, 2.8 eq) were added to the solution. The reaction mixture
was flushed with nitrogen and left stirring overnight (17 h). Solvents were evaporated in vacuo.
The remaining material in the vessel was dissolved in 7 mL of ethyl acetate (EtOAc), washed three
times with 5 mL of 5 mM H2SO4 and three times with 5 mL of brine. The organic phases were
combined, dried over MgSO4, filtered and evaporated in vacuo. Compound 1 was obtained in 80%
yield as a white crystalline powder after precipitation in ice-cold n-hexane. 1H NMR (250.13 MHz,
DMSO-d6): δ (ppm) = 7.50 (br s, 1H, -NH-), 7.31 – 7.19 (m, 10H, 10x Ar-H), 7.07 (s, 2H, -NH2),
4.91 (s, 2H, -CH2-O), 4.19 – 4.10 (m, 1H, -CH-CH2-), 3.00 (dd, J1=22 Hz, J2=8 Hz, 1H, CH2-
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CH-), 2.75 – 2.66 (m, 1H, CH2-CH-). C NMR (125.81 MHz, DMSO-d6): δ (ppm) = 174.05 (-
CONH2), 160.09 (-COONH-), 138.61 (Ar), 129.61 (Ar), 128.76 (Ar), 128.53 (Ar), 127.85 (Ar),
126.74 (Ar), 74.94 (-CH2O-), 65.64 (-CH-), 37.91 (-CH2CH-). HRMS: Calculated for C17H18N2O3
298.1317, measured 299.1225 (M+H+) and 321.1225 (M+Na+). HPLC purity: ˃99% (determined
by HPLC: column A, method A, Rt = 11.8 min).
Synthesis of (R)-benzyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (2)
Starting compound (R)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanoic acid (100 mg, 334
µmol, 1 eq) and di-tert-butyl dicarbonate (183 mg, 668 µmol, 2 eq) were dissolved in 3 mL of 1,4-
dioxane and placed on a magnetic mixer. Pyridine (27.0 µL, 334 µmol, 1 eq) and ammonium
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hydrogen carbonate (75.0 mg, 868 µmol, 2.6 eq) were added to the solution. Nitrogen athmosphere
was introduced before the solution was left stirring overnight (18 hrs). Solvents were evaporated
in vacuo. The remaining material in the vessel was dissolved in 7 mL of EtOAc, washed three
times with 5 mL of 5 mM H2SO4 and three times with 5 mL of brine. The organic phases were
combined, dried over MgSO4, filtered and evaporated in vacuo. Compound 2 was obtained in 55%
yield as a white crystalline powder after precipitation in ice-cold n-hexane. 1H NMR (400.13 MHz,
DMSO-d6): δ (ppm) = 7.47 (br s, 1H, -NH-), 7.41 – 7.21 (m, 10H, 10x Ar-H), 7.07 (s, 2H, -NH2),
4.94 (s, 2H, -CH2-O), 4.19 – 4.16 (m, 1H, -CH-CH2-), 3.00 (dd, J1=16 Hz, J2=4 Hz, 1H, CH2-
CH-), 2.74 (t, J=12 Hz, 1H, CH2-CH-). 13C NMR (125.81MHz, DMSO-d6): δ (ppm) = 173.88 (-
CONH2), 153.30 (-COONH-), 138.77 (Ar), 137.52 (Ar), 128.93 (Ar), 128.73 (Ar), 128.49 (Ar),
127.88 (Ar), 126.66 (Ar), 65.58 (-CH2O-), 65.54 (-CH-), 37.96 (-CH2CH-). HRMS: Calculated for
C17H18N2O3 298.1317, measured 299.1378 (M+H)+ and 321.1199 (M+Na)+. HPLC purity: ˃95%
(determined by HPLC: column A, method A, Rt = 11.8 min).
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Radiosynthesis:
Radionuclide production procedure for carbon-11
An IBA Cyclone 18/9 (IBA, Louvain-la Neuve, Belgium) cyclotron was used for [11C]CO2
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production by the N(p,α)11C nuclear reaction performed in a 0.5% O2/N2 gas mixture. After
trapping of [11C]CO2 from the target in a stainless trap dispersed in liquid N2, [11C]CO2 was
transferred to the desired reaction vessel using a 10 mL·min-1helium flow.
Procedure of the manual synthesis of (S)-[11C]benzyl (1-amino-1-oxo-3-phenylpropan-2-
yl)carbamate
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H-L-Phe-NH2 was placed in a pre-dried reaction vessel in a research hot-cell and dissolved in
DMSO containing BEMP. [11C]CO2 (4 - 6 GBq) was transferred through a sicapent (phosphorus
pentoxide) column to the mixture using helium flow of 10 mL·min-1 from a CO2 trap. When the
activity in the vessel reached its maximum, DMSO containing benzyl chloride was manually added
to the reaction vessel with a syringe, heated to 75 °C and left to stir with helium flow (10 mL·min-1)
for 2 - 10 min. Approximately 50 µL of the reaction mixture was then quenched with 300 µL of
solution of acetonitrile and water (1/1; v/v) and analyzed on HPLC (Column A; Method A) to
determine the radiochemical yield expressed as radiochemical conversion of the reaction.
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Procedure for the automated synthesis of (S)-[11C]benzyl (1-amino-1-oxo-3-phenylpropan-
2-yl)carbamate ([11C]1) and of (R)-[11C]benzyl (1-amino-1-oxo-3-phenylpropan-2-
yl)carbamate ([11C]2)
L-Phe-NH2 or D-Phe-NH2 (1.0 mg, 3.3 µmol) was placed in a pre-dried reaction vessel dissolved
in DMSO (0.15 mL) containing BEMP (8.3 µL) and fixed in the home-build synthesis module
located in the hot-cell.34 [11C]CO2 (45 GBq) was transferred through a sicapent column to the
mixture using helium flow (10 mL·min-1). When the activity in the vessel reached its maximum,
benzyl chloride (4.0 µL) in DMSO (0.15 mL) was automatically added to the reaction vessel,
which was then heated to 75 °C and left to stir with helium (10 mL·min-1) for 5 min. The mixture
was then cooled to 25 °C, quenched with a solution of acetonitrile and water (1/1; v/v; 1.5 mL) and
injected on a preparative HPLC column (Column J, Method B). The product, eluting between 12
and 14 min, was collected (Supporting information: Figure 2), diluted in 7 mL of water and loaded
on a pre-conditioned Sep-Pak C18 column. The Sep-Pak C18 column was washed with 5 mL of
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water, before the product was eluted with 1 mL of ethanol (EtOH). Saline solution (9 mL) was
added to the product, forming the final injectable radioactive tracer.
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[11C]1 was obtained in a decay-corrected radiochemical yield of 27.9 ± 10.9% (n=10) with an
isolated non-decay-corrected yield of 5.1 ± 2.2 GBq (n=10) of the product with a radiochemical
purity >99%, an average AM of 93.2 ± 46.9 GBq·µmol-1 (n=9) within 24 - 26 min (n>10). The
identity of the product was confirmed with analytical HPLC (Column A; Method A) by co-
injection of the product and non-labeled 1 (Supporting information: Figure 3).
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[11C]2 was obtained in a decay-corrected radiochemical yield of 25.5 ± 9.7% (n=5) with an
isolated non-corrected yield of 3.9 ± 1.9 GBq (n=5) of the product with a radiochemical purity
>99%, an average AM of 69.4 ± 11.4 GBq·µmol-1 (n=4) within 23 - 29 min (n=5). The identity of
the product was confirmed with analytical HPLC (Column A; Method A) by co-injection of the
product and non-radiolabeled 2.
Animals
Adult healthy male Wistar rats (200 – 360 g; 7 – 10 weeks; Charles River International Inc,
Sulzfeld, Germany) were housed in groups of maximal 6 in a conventional cage and kept in a
ambient with a 12-hour light/dark cycle, and constant ambient temperature (19 - 21 °C) and
humidity level (52 - 70%). Rats were provided with water and food pellets (Teklad Global Diet
2016, Envigo; Horst, the Netherlands) ad libitum. The rats were allowed to adapt to the laboratory
environment for one week before the experiment. Animal experiments were performed in
accordance with the European Community Council Directive (2010/63/EU) for laboratory animal
care and the Dutch Law on animal experimentation. The experimental protocol was validated and
approved by the local committee on animal experimentation of the VU University Medical Center.
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Ex vivo Biodistribution
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Healthy male Wistar rats (200 - 250 g) were injected with the radiotracer (25.8 ± 7.6 MBq; 0.74
± 0.21 nmol; n=16) via the tail vein under isoflurane anesthesia (2 - 2.5% in 1 L·min-1). Animals
were sacrificed and dissected at 5, 15, 30 and 60 min (n=4 per time point) post-injection. Blood,
heart, lungs, liver, kidney, duodenum, urine, tail, bone, spleen, large brain, cerebellum, brain stem,
spinal cord top and spinal cord mid were collected, weighed and counted for radioactivity in a
Wallac Computagama 1210 or a Wallac Universal Gamma Counter 1282. Biodistribution data are
expressed as the percentage of injected dose per gram (%ID/g). Errors are standard error of the
mean (SEM).
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Metabolites Analysis (Plasma and Brain)
Healthy male Wistar rats (200 - 330 g) were injected with the tracer [11C]1 (27.3 ± 7.3 MBq;
0.50 ± 0.22 nmol; n=12) or [11C]2 (32.4 ± 3.4 MBq; 1.80 ± 0.19 nmol; n=9) via the tail vein under
isoflurane anesthesia (2 – 2.5% in 1 L·min-1). The rodents were sacrificed at 5, 15 and 45 min post
injection for the tracer [11C]1 (n=4 per time point) and at 5, 15 and 45 min post injection for the
tracer [11C]2 (n=3 per time point). Blood (2.0 - 5.0 mL) was collected via heart puncture into
heparin tubes (DB Vacutainer, LH, Becton Dickinson, Franklin Lakes, NY, USA) and centrifuged
at 4000 r.p.m for 5 min at 4 °C (Hettich Universal 32, Andreas Hettich GmbH & Co. KG,
Tuttlingen, Germany). The brain was dissected and one hemisphere was put in a falcon tube
containing 50% acetonitrile in saline (4 mL), and homogenized using a dispenser (IKA T18 B
Ultra-Turrax, IKA-Werke GmbH & Co KG, Staufen, Germany) before centrifugation at 4000
r.p.m. for 5 min at 4 °C. The other hemisphere was counted for radioactivity for recovery
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calculations. Plasma and brain supernatant were separated from blood cells and brain precipitate,
respectively.
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About 1 mL of plasma was diluted with 2 mL of 0.15 M hydrochloric acid and loaded onto a
tC2 Sep-Pak cartridge, which was pre-activated by elution with 3 mL of methanol and 6 mL of
water, respectively. The cartridge was washed with 3 mL of water to collect the rest of the polar
radioactive metabolites. The polar radioactive metabolite fraction was defined as the two combined
fractions. Next, the tC2 Sep-Pak cartridge was eluted with 1.5 mL of methanol and 2 mL of water
to collect the mixture of non-polar radioactive metabolites. About 2 mL of the brain supernatant
was ultra-centrifuged (Eppendorf 5417c, Eppendorf, Hamburg, Germany) and 1 mL was injected
onto an HPLC system. All non-polar metabolites and polar brain metabolites fractions were
immediately analyzed using HPLC to determine the percentage of the intact [11C]1 or [11C]2.
HPLC was performed on the Dionex Ultimate 3000 System station, equipped with 1 mL loop, with
Phenomenex Gemini C18, 250 × 10 mm, 5 µm, column using gradient elution of acetonitrile+0.1%
TFA (D) and water+0.1% TFA (F) in water. The HPLC gradient ran for 14 min decreasing the
concentration of eluent F from 90% to 20% in 11 min, 2 min with 20% of F and back to original
setting (90% F) at flow 5 mL·min-1. All separated fractions of the brain supernatant were counted
for radioactivity in a Wizard Gamma Counter 1470 or 2480 (Wallac, PerkinElmer, Waltham, MA,
USA). The percentage of the intact tracer in the non-polar plasma fractions was determined by the
online radioactivity detection by HPLC analysis for [11C]1 or [11C]2 (Rt = 11.7 min). AUC,
providing the percentage of the intact tracer and the metabolites, has been corrected for decay.
Results are expressed as percentage of the intact tracer, polar metabolites, non-polar metabolites
± standard deviation.
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Metabolites Analysis (Spinal Cord)
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Healthy male Wistar rats (330 - 360 g) were injected with the tracer [11C]1 (32.8 ± 8.6 MBq;
0.63 ± 0.17 nmol; n=4) via the tail vein under isoflurane anesthesia (2 – 2.5% in 1 L·min-1). The
rodents were sacrificed at 15 min post injection for the tracer [11C]1 (n=4 per time point). Whole
spinal cord was collected via cutting the muscles and breaking the spine, and was put in a falcon
tube containing 50% acetonitrile in saline (2 mL), and homogenized with a dispenser (IKA T18 B
Ultra-Turrax, IKA-Werke GmbH & Co KG, Staufen, Germany) before centrifugation at 4000
r.p.m. for 2 min at ambient temperature (Eppendorf 5417c, Eppendorf, Hamburg, Germany).
Spinal cord supernatant and precipitate were separated and about 1.5 mL of was immediately
analyzed using HPLC to determine the percentage of the intact [11C]1. HPLC was consistent of
the Dionex Ultimate 3000 System station, equipped with 1 mL loop, with Phenomenex Gemini
C18, 250 × 10 mm, 5 µm, column using gradient elution of acetonitrile+0.1% TFA (D) and
water+0.1% TFA (F). The HPLC gradient ran for 14 min decreasing the concentration of eluent F
from 90% to 20% in 11 min, 2 min with 20% of F and back to original setting (90% F) at flow 5
mL·min-1. The percentage of the intact tracer in the non-polar plasma fractions was determined by
the online radioactivity detection by HPLC analysis for [11C]1 (Rt = 11.6 min). Area under the
curve, providing the percentage of the intact tracer ot the metabolites, has been corrected for decay.
Results are expressed as percentage of the intact tracer, metabolites ± standard deviation. All
separated fractions (one fraction of 30 s) of the spinal cord supernatant were counted for
radioactivity in a Wizard Gamma Counter 1470 or 2480 (Wallac, PerkinElmer, Waltham, MA,
USA).
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LC-MS/MS Method
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LC–MS/MS analysis was performed on a Jasco X-LC HPLC system (Easton, PA, USA) with an
AB Sciex QTRAP 5500 mass spectrometer (Concorde, Ontario, Canada). The Jasco system
consisted of two pumps (X-LC 3180PU), a degasser (X-LC 3080DG), a mixer (X-LC 3080MX),
a column oven (X-LC 3080CO) and an auto sampler (X-LC3159AS). Data were collected with
two data boxes (LV 2080-03 and LC-Net II/ACD).
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A Kinetex Biphenyl C18 column (2.6 μ, 100 A, 100 × 2.10 mm; Phenomenex, Torrance, CA,
USA) at 25 °C was used for chromatographic separation. A gradient elution was used at a flow
rate of 0.5 mL·min-1. The mobile phase consisted of a mixture of acetonitrile (C) and 0.1% formic
acid in water according to the following scheme: 0 min 20% C, 90% C in 3.5 min, 90% C for 3.6
min, 20% C in 5 min at 25 ºC. MS parameters: TurboIon Spray voltage was set at 5.5 kV, source
temperature at 750 °C. Based of full-scan MS and MS/MS spectra of each analyte, the most
abundant fragment ions were selected and the mass spectrometer was set to monitor the transitions
of the precursors to the product ions as followed: m/z 299.1  255.1 for the intact tracer, m/z
315.1 271.1 for the hydroxylated metabolite and m/z 475.1  299.1 for the glucoronated
metabolite.
PET Imaging
Dynamic PET imaging was performed on a healthy male Wistar rats (200 – 320 g; n=10) using
Nanoscan small animal PET/CT and PET/MR scanners (Mediso Ltd., Budapest, Hungary) with
identical PET components. The rats were anaesthetized with 3% and 1 – 2.5% isoflurane in oxygen
(1 L·min-1) for induction and maintenance, respectively. Rats were positioned on the scanner bed
and respiratory rate was monitored during the entire time of scanning. A computed tomography
(CT) or magnetic resonance imaging (MRI) scan was performed for 5 or 15 min, respectively,
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followed by intravenous (i.v.) administration of [11C]1 (13.1 ± 5.8 MBq; 0.14 ± 0.06 nmol; n=4)
or of [11C]2 (14.7 ± 1.5 MBq; 0.37 ± 0.24 nmol; n=3) via tail vein cannula at the start of a dynamic
PET scan of 60 min. PET data were normalized, and corrected for scatter, randomization,
attenuation, decay and dead time. List-mode PET data were re-binned in 18 successive frames: 4
× 5, 4 × 10, 2 × 30, 3 × 60, 2 × 300, 1 × 600, 1 × 900 and 1 × 1200 s, which were reconstructed
using a fully 3-dimensional reconstruction algorithm (Tera-TomoTM) using 4 iterations and 6
subsets, resulting in an isotropic 0.4 mm voxel dimension. Static reconstruction was also
performed on the total duration of the scan (60 min). Images were analyzed using the freely
available AMIDE software (A Medical Image Data Examiner; version 1.0.4), whereas images in
Figure 6 are made using VivoQuant (version 3.0, InVicro, Boston, MA, USA). Regions of interest
(ROI) were drawn manually around whole brain (ellipsoid) and spinal cord (elliptic cylinder).
Results are expressed as standardized uptake values (SUVs) and error bars indicate standard
deviation using Graph Pad Prism (Version 5.02, Graph Pad Software Inc., La Jolla, CA, USA).
For blocking experiments, rats were injected with the natural heptapeptide SP1-7 or 1 or 2,
depending on the investigated PET tracer. The SP1-7 was diluted in DMSO and then further diluted
with saline to the final concentration (5% DMSO). Peptide was injected intraperitoneally (i.p.)
(185 nmol·kg-1), according to previous experiments by Jonsson1, 15 min prior to tracer [11C]1
administration (tracer administration per rat: 22.3 ± 0.5 MBq; 0.30 ± 0.01 nmol; n=2). Compound
1 was dissolved in EtOH and further diluted with saline to the final concentration (20% EtOH). 1
was injected slowly i.v. in the tail vein (3 µmol·kg-1), according to previous experiments by
Fransson35, 3 and 20 min prior to tracer [11C]1 administration (tracer administration per rat: 34.0
± 10.8 MBq; 0.55 ± 0.14 nmol; n=3 for 3 min and 16.4 ± 4.3 MBq; 0.22 ± 0.04 nmol; n=2 for 20
min). Same dose and formulation conditions applied for compound 2, with the exception that it
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was injected only 3 min prior to tracer [11C]2 administration (tracer administration per rat: 16.8 ±
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10.1 MBq; 0.25 ± 0.15 nmol; n=2).
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Results
Prediction Models
The characteristics of the SP1-7 peptidomimetics for brain penetration were anticipated prior to
synthesis and radiolabeling. ACD/Labs Percepta’s Drug profiler and BBB module report
confirmed optimal physiochemical and ADME profile, as well as brain penetration sufficient for
CNS activity (Supporting Information). CNS PET MPO algorithm was calculated following the
publication of Zhang et al.31 and displayed value of 3.6, which is in the desirable range for BBB
penetration (Supporting Information).
Chemistry
Reference compounds ((S)-benzyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate 1 and ((R)-benzyl
(1-amino-1-oxo-3-phenylpropan-2-yl)carbamate 2) were synthesized according to patented
procedure36 in a one-step amidation reaction (Supporting Information; Scheme 1) from a
commercially available 2-(((benzyloxy)carbonyl)amino)-3-phenylpropanoic acids. Reference
compounds were further characterized according to Fransson et al.35 Overnight amidation at
ambient temperature followed by extraction and pre-crystallization in ice-cold n-hexane yielded
L-stereoisomer 1 in 80% yield and D-stereoisomer 2 in 55% yield.
Radiochemistry
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O
O
O
BEMP
DMSO
-O
O
NH
H2N
N
[11C]CO2
NH2
11C
O
11C
O
NH2
NH2
DMSO
75 ºC
RT
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L-[11C]1 or
D-[11C]2
H-Phe-NH2 or
H-D-Phe-NH2
Scheme 1: Radiosynthesis of SP1-7 carbamate analogues [11C]1 and [11C]2.
The general synthesis towards the SP1-7 carbamate analogues [11C]1 and [11C]2 is outlined in
Scheme 1. The synthesis involved 2 steps: direct [11C]CO2 trapping at ambient temperature and
11C-carboxybenzylation at elevated temperature. The preparation of SP1-7 carbamate analogue
[11C]1/[11C]2 was achieved without the isolation of the synthetic intermediate in a one-pot reaction
to establish an efficient procedure for the desired product (Supporting Information; Table 1). In
order to optimize this procedure, the following parameters have been thoroughly investigated:
amount of reagents and solvent, reaction time and helium flow. These parameters were changed
systematically one by one per experiment.
Trapping of cyclotron produced [11C]CO2 was >90%, when [11C]CO2 was bubbled thought the
precursor solution in dry DMSO with the aid of 3.5 - 5.0 eq of 2-tert-butylimino-2-diethylamino-
1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP), which served as trapping reagent and a
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catalyst for the carboxylation reaction. The C-carboxybenzylation to the desired product was
achieved in high radiochemical yield (>85%; Supporting Information, Table 1), determined by
analytical HPLC, using 4 - 5 eq of benzyl chloride, dissolved in DMSO, at 75 °C under helium
stirring (10 mL·min-1). Excellent conversion rates were attained for both reaction times of 5 and
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10 min, whereas time of 5 min was selected to decrease the total synthesis time. The volume of
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DMSO was increased to 300 µL for a smoother automation of the synthesis procedure.
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Importantly, N-benzylation or other significant side products were not observed during the C-
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carboxybenzylation reaction (Supporting Information; Figure 1).
The next step, purification of the PET-tracer was achieved using a reverse-phase C18 semi-
preparative HPLC (Supporting Information; Figure 2 and 3). Subsequent reformulation towards
an injectable solution provided [11C]1 (5.1 ± 2.2 GBq) in a radiochemical purity of >99% and AM
of 93.2 ± 46.9 GBq·µmol-1 in 23 - 29 min with an overall RCY of 27.9 ± 10.9% (corrected for
decay; n=10) formulated in an i.v. injectable solution containing 10% EtOH in saline, ready for in
vivo experiments.
In the same manner, we performed the reaction with H-D-Phe-NH2 as precursor. As expected,
the D-isomer displayed the same properties and the radiosynthesis of [11C]2 provided an isolated
non-corrected yield of 3.9 ± 1.9 GBq (n=5) in a radiochemical purity of >99% and AM of 69.4 ±
11.4 GBq·µmol-1 (n=4) in 26 - 29 min (n=5) with an overall RCY of 25.5 ± 9.7% (corrected for
decay; n=5) formulated in an i.v. injectable solution (10% EtOH in saline).
Ex Vivo Biodistribution
In order to assess the brain and spinal cord penetration potential of [11C]1 an ex vivo
biodistribution study was performed. Healthy male Wistar rats were injected with [11C]1 (25.8 ±
7.6 MBq; 0.74 ± 0.21 nmol; n=16) and were analyzed for biodistribution at 5, 15, 30 and 60 min
post injection (p.i.) (Figure 2). [11C]1 was cleared via kidneys and liver, and showed moderate
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uptake in well-perfused organs, like lungs and heart. Highest uptake was observed after 5 min in
the brain regions (large brain, cerebellum, brain stem) and spinal cord, showing rapid penetration
and BBB crossing of the SP1-7 peptidomimetic. At later time points we observed a better retention
of the [11C]1 tracer in the spinal cord compared to the brain and the perfused organs.
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11C]1, 5 min
11C]1, 15 min
5
[
[
11C]1, 30 min
11C]1, 60 min
2.0
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Figure 2: Biodistribution of [11C]1 at 5, 15, 30 and 60 min post-injection (p.i.) in healthy male
Wistar rats (n=4 per time point) following i.v. administration of 25.8 ± 7.6 MBq (0.74 ± 0.21 nmol;
n=16) of [11C]1 via the tail vein under isoflurane anesthesia (2 - 2.5% in 1 L·min-1). Values are
expressed in %ID/g ± SEM.
From the data obtained, organ of interest-to-blood ratios were also calculated (Figureꢀ3A). The
brain-to-blood ratios was 1.24 - 1.39 and spinal cord-to-blood ratios was 0.82 - 0.88 for [11C]1.
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[11C]1, 15 min
[11C]1, 30 min
[11C]1, 60 min
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Figure 3: A: Organs-to-blood ratio of [11C]1 at 5, 15, 30 and 60 min (n=4 per time point).Columns
show the region-of-interest to blood ratio ± SEM.
Metabolite Analysis (L-[11C]1)
In vivo stability of [11C]1 was determined by metabolite analysis in healthy male Wistar rats
(n=4 per time point). Rats were injected i.v. with the radiotracer [11C]1 (27.3 ± 7.3 MBq; 0.50 ±
0.22 nmol; n=12) and sacrificed at 5, 15 and 45 min p.i. followed by blood sample collection and
brain dissection. Analysis of the plasma revealed mediocre stability of the tracer with 52, 15 and
11% of intact tracer after 5, 15 and 45 min, respectively, suggesting extensive metabolic
degradation (Table 1). HPLC analysis of the plasma showed the presence of one major metabolites
of [11C]1 after 45 min. This major metabolite had a retention time of 12.7 min and constituted more
than 67% of the non-polar metabolites (Figure 4). Radioactivity recovery of the blood samples was
99.1 ± 0.3, 70.0 ± 2.2 and 65.5 ± 3.7% at 5, 15 and 45 min, respectively. The Sep-Pak cartridge
stickiness at later time points was inevitable, despite additional employment of MeOH.
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In brain supernatant, the percentage of intact tracer in the non-polar fraction was >99.9% at 5
min and 97% after 15 or 45 min (Figure 4D). Radioactivity extraction of the brain supernatant
samples was 77.1 ± 5.2, 71.5 ± 15.7 and 63.0 ± 5.9 % at 5, 15 and 45 min, respectively. These data
confirms rapid penetration and stability of the [11C]1 tracer in brain.
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Table 1: Metabolite analysis of [11C]1 in plasma, brain and spinal cord supernatant.
Spinal
Cord
Plasma
Brain
5 min
[%; SD]
52.3 ±
11.3
15 min
[%; SD]
45 min
[%; SD]
5 min
15 min
45 min
15 min
[%]
[%; SD] [%; SD] [%; SD]
97.3 ±
0.6
96.7 ±
4.5
Intact tracer
15.2 ± 7.9 10.9 ± 3.0
>99.9
97.6
Non-polar
metabolite 1
Non-polar
metabolite 2
Non-polar
metabolite 3
Polar
7.8 ± 5.4
/
7.9 ± 2.4
/
7.7 ± 2.9
1.1 ± 1.1
/
/
/
/
0.9 ± 0.6 1.6 ± 3.2
/
/
/
/
34.3 ± 5.0 40.8 ± 4.4 40.0 ± 1.7
5.2 ± 1.1 6.1 ± 0.4 5.7 ± 0.3
1.8 ± 0.2 1.6 ± 3.2
2.4
/
/
/
metabolites
Additionally, in vivo stability of [11C]1 in spinal cord was preliminary assessed by metabolite
analysis in healthy male Wistar rats (n=4 per time point). Rats were injected i.v. with the
radiotracer [11C]1 (32.8 ± 8.6 MBq; 0.63 ± 0.17 nmol; n=4) and sacrificed at 15 min p.i. followed
by spinal cord dissection. Unfortunately, the percentage of the intact tracer in the spinal cord
supernatant was possible to be assessed only for one rat (Supporting information), where results
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indicate that [11C]1 is stable in spinal cord after 15 min, with a trace unidentified metabolite 2
5
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(Supporting Information; Figure 4).
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Brain
[11C]1
Plasma
[11C]1
[11C]1
[11C]1
A
E
B
C
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D
[11C]2
F
[11C]2
[11C]2
[11C]2
G
H
Figure 4: Metabolites radiochromatograms of the plasma non-polar fraction after: A: 5 min, B: 15
min and C: 45 min after L-[11C]1 injection; E: 5ꢀmin, F: 15 min and G: 45 min after D-[11C]2
injection. Intact L-[11C]1 and D-[11C]2 have a retention time of 11.7ꢀmin. Radiochromatograms of
the total fraction of brain supernatant after 5ꢀmin showing presence of intact tracer: D: L-[11C]1
(Rt of 11.5 min); H: D-[11C]2 (Rt of 11.1 min). HPLC data has been corrected for decay.
Metabolite Analysis (D-[11C]2)
In order to improve the bioavailability of the tracer we investigated the effect of substitution of
L-phenylalanine to D-configuration. We synthesized the D-variant [11C]2and examined the in vivo
stability by metabolite analysis in healthy male Wistar rats (n=3 per time point). The rodents were
injected i.v. with [11C]2 tracer (32.4 ± 3.4 MBq; 1.80 ± 0.19 nmol; n=9) and rats were sacrificed at
5, 15 and 45 min p.i. followed by blood collection and brain dissection. Plasma analysis revealed
improved stability of the tracer with 80, 49 and 37% of intact tracer after 5, 15 and 45 min,
respectively (Table 3). Radioactivity recovery of the blood samples was 95 - 99% at all time points.
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HPLC analysis of non-polar fraction revealed three unidentified metabolites (Figure 4E-G, and
Table 3).
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Table 2: Metabolite analysis of D-isomer [11C]2 in plasma and brain supernatant.
Plasma
15 min
[%; SD]
Brain
15 min
[%; SD]
5 min
[%; SD]
45 min
[%; SD]
5 min
[%; SD]
45 min
[%; SD]
>99.9
Intact tracer 79.6 ± 2.3 49.2 ± 2.0 36.6 ± 1.7 96.4 ± 6.3 96.4 ± 5.2
Non-polar
2.6 ± 0.2
1.8 ± 0.6
9.8 ± 0.8 10.5 ± 4.8 3.6 ± 6.3
3.6 ± 5.2
/
/
/
/
metabolite 1
Non-polar
metabolite 2
Non-polar
metabolite 3
Polar
9.5 ± 1.3
7.2 ± 3.0
/
/
/
/
/
/
11.3 ± 1.1 20.9 ± 1.5 33.6 ± 0.2
3.5 ± 0.5 5.6 ± 0.9 7.3 ± 2.0
metabolites
In brain supernatant, the percentage of intact D-[11C]2 tracer in the non-polar fraction was >96%
after 5, 15 and 45 min (Figure 4B, H). Radioactivity extraction of the brain supernatant samples
was 58.8 ± 13.7, 55.1 ± 22.0 and 63 ± 13.3% at 5, 15 and 45 min, respectively.
LC-MS/MS Method
A product ion scan was performed for 1 to determine high intensity mass fragments in a multiple
reaction monitoring (MRM) method by LC-MS/MS experiments. The transition of 299.1 m/z to
255.1 was chosen where the fragments corresponded to the intact parent ion and a loss of
carboxylic fragment, and the transition of 315.1 m/z to 271.1 was chosen where the fragments
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corresponded to the hydroxylated parent ion and a loss of carboxylic fragment. Non-polar plasma
fractions, after 5 and 15 min p.i, were analyzed demonstrating a trace amount of 2 (Supporting
Information, Figure 5; blue trace) and two mass peaks of 315.1 (Supporting Information, Figure
5; red trace) corresponding to the hydroxylated parent ion.
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Additionally, the transition of 475.1 m/z to 299.1 was chosen to detect the glucoronated parent
ion and the intact parent ion. Rat was pretreated with cold non-radiolabeled 1, injected 5 min before
PET scan as a blocker, where non-polar plasma sample indeed confirmed the glucorinated
metabolite after 60 min [11C]1 p.i. (Supporting Information, Figure 5, green trace). Furthermore,
using this transition the non-polar plasma samples from in vivo stability study of [11C]2 were re-
analyzed, confirming the postulated metabolites (hydroxylation, oxidative deamination and
subsequent glucoronidation) (Supporting Information; Scheme 2).
PET Imaging
Dynamic PET imaging was carried out in healthy rats with [11C]1 and [11C]2 tracers (Figure 5).
The time-activity-curves (TACs) of the PET imaging showing SUVs over time are presented in
Figure 6. Under baseline condition [11C]1 scan revealed good uptake in the brain, reaching a
maximum of 1.5 ± 0.05 SUV (n=4) at 3 min p.i., followed by a rapid clearance within 60 min.
[11C]1 showed consistent uptake in the brain with no variation between different rats (Figure 6A).
In the spinal cord (Figure 6F) the maximum uptake was at 10 min p.i and reached 0.95 ± 0.03 SUV
(n=4).
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C: Spinal Cord: [11C]1
2.7
C
A
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Baseline
Blocking (SP1-7
)
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Blocking (1)
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0.4
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B
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60
Time [min]
Figure 5: Static reconstruction of the PET-image scans in healthy male Wistar rat (Rat 2) with
tracer [11C]1 (20 MBq i.v. injection via tail vein; 3% and 1 – 2.5% isoflurane in oxygen (1 L·min-1)
for induction and maintenance). A: PET/CT between 0 - 60 min coronal image. B: PET/CT
between 0 - 60 min sagittal image. C: Time-activity-curves (TACs) of [11C]1 expressed as SUV ±
SD. TACs of non-blocking (black line) and blocking conditions (colored lines): natural SP1-7
administered 15 min before [11C]1 (red line; n=2) and compound 1 20 min prior to tracer injection
(blue line; n=2).
In order to verify the specific uptake in brain, blocking experiments were performed.
Pretreatment with natural SP1-7 (15 min, i.p., 185 nmol·kg-1) or cold compound 1 (20 min prior to
tracer injection, i.v., 3 µmol·kg-1) did not block brain uptake. Although, when 1 was injected 3 min
prior to tracer injection (i.v., 3 µmol·kg-1), we noticed an increase in the SUV up to 2.0 at 3 min.
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Despite our efforts to block brain uptake [11C]1, pretreatment did not measurably decrease
radiotracer uptake resulting in similar uptake pattern for all scans (Figure 6B and E).
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In the spinal cord pretreatment with the natural SP1-7 showed moderate blocking (0.95 SUV
baseline condition vs 0.87 SUV block) (Figure 5C, black vs. red line). However, self-blocking
with 1 at 3 or 20 min prior tracer injection did not show any blocking (Figure 5C, blue line).
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