Novel kappa opioid receptor agonist as improved PET radiotracer

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Novel kappa opioid receptor agonist as improved PET radiotracer: Development and in vivo evaluation Songye Li, MingQiang Zheng, Mika Naganawa, Hong Gao, Richard Pracitto, Anupama Shirali, Shu-fei Lin, Jo-Ku Teng, Jim Ropchan, and Yiyun Huang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01209 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 10, 2019

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Molecular Pharmaceutics

Novel kappa opioid receptor agonist as improved PET radiotracer: Development and in vivo evaluation

Songye Li*, Ming-Qiang Zheng, Mika Naganawa, Hong, Gao, Richard Pracitto, Anupama Shirali, Shu-fei Lin, Jo-Ku Teng, Jim Ropchan and Yiyun Huang

1PET

Center, Department of Radiology and Biomedical Imaging, Yale University School of

Medicine, New Haven, CT 06520, USA

*Corresponding authors: Songye Li, Yale PET Center, 801 Howard Ave, PO Box 208048, New Haven, CT 06520-8048, USA, Tel: 203-785-3605, Fax: 203-785-2994, e-mail: [email protected].

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Abstract The kappa opioid receptor (KOR) is involved in depression, alcoholism and drug abuse. The current agonist radiotracer 11C-GR103545 is not ideal for imaging KOR due to its slow tissue kinetics in human. The aim of our project was to develop novel KOR agonist radiotracers with improved imaging properties. A novel compound FEKAP ((R)-4-(2-(3,4dichlorophenyl)acetyl)-3-((ethyl(2-fluoroethyl)amino)methyl) piperazine-1-carboxylate) was designed, synthesized and assayed for in vitro binding affinities. It was then radiolabeled and evaluated in rhesus monkeys. Baseline and blocking scans were conducted on a Focus-220 scanner to assess binding specificity and selectivity. Metabolitecorrected arterial activities over time were measured and used as input functions to analyze the brain regional time-activity curves and derive kinetic and binding parameters with kinetic modeling. FEKAP displayed high KOR binding affinity (Ki = 0.43 nM) and selectivity (17 folds over mu opioid receptor and 323 folds over delta opioid receptor) in vitro. 11C-FEKAP was prepared in high molar activity (mean of 718 GBq/μmol, n = 19) and > 99% radiochemical purity. In monkeys 11C-FEKAP metabolized fairly fast, with ~31% of parent fraction at 30 min post-injection. In the brain it exhibited fast and reversible kinetics with good uptake. Pre-treatment with the non-selective opioid receptor antagonist naloxone (1 mg/kg) decreased uptake in high binding regions to the level in the cerebellum, and the selective KOR antagonist LY2456302 (0.02 & 0.1 mg/kg) reduced

11C-FEKAP

specific binding in a dose-dependent manner. As a measure of specific binding signals, the mean binding potential (BPND) values of 11C-FEKAP derived from the multilinear analysis1 (MA1) method were great than 0.5 for all regions except for the thalamus. The novel


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KOR agonist tracer 11C-FEKAP demonstrated binding specificity and selectivity in vivo and exhibited attractive properties of fast tissue kinetics and high specific binding.

Keywords 11C-FEKAP,

kappa opioid receptor, agonist, PET radiotracer, non-human primates



Introduction Opioid receptors (OR) are classified into three major subtypes: Kappa (KOR), mu (MOR) and delta (DOR)1, which belong to the superfamily of G-protein coupled receptors with a common seven transmembrane helical architecture, and are coupled predominantly to the heterotrimeric Gi/Go proteins2. The ORs can be activated by both endogenous and exogenous ligands3 and have been linked to a number of neuropsychiatric disorders4. Early studies on the ORs revealed that the agonists at the MOR and DOR sites are rewarding and reinforcing, while agonists at the KOR sites are aversive5. The KOR distributes abundantly in non-human primate6 and human brains7 with highest density in the cortical regions and has been implicated in substance abuse8, 9, mood-related disorders10 including depression11, 12

and anxiety13, pain14 and neurological diseases such as Alzheimer’s disease15 and

epilepsy16,

17.

As such KOR is an attractive target with therapeutic potentials18.

Visualization, characterization, and quantification of KOR with in vivo imaging techniques such as positron emission tomography (PET) would greatly facilitate the understanding of KOR and its involvement in disease pathology and treatment. According to the two-state theory of G-protein-coupled receptor activation19, agonists bind only with high affinity to, and interact with the active state of the receptor, while antagonist binds with equal affinity to both the active and inactive states. For example, it has been suggested that several neuropsychiatric disorders may be characterized by hypersensitivity for the neurotransmitter dopamine, and an increase in dopamine D2/3 receptors in their active state were associated with this hypersensitivity20. The development of an agonist-antagonist pair of imaging agents will make it possible to


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probe not only the total expression, but also the affinity state of KOR under normal and disease conditions. In fact, recent crystal structure of nanobody-stabilized KOR active state revealed remarkable conformation and binding pocket change between the active and inactive states21. Hence, a suitable pair of KOR PET radiotracers will enable the assessment of the receptor ratio configured in active vs. inactive state. We have successfully developed several KOR antagonist PET radiotracers with 11C-

and 18F-labeling and advanced them to imaging studies in humans22-26. A few KOR

agonists have also been evaluated as PET radiotracers over the past decades, such as ligands from the U-5048827 family and Salvinorin A28, however none of them was validated for use in humans. Up to date

11C-GR103545

is the only KOR agonist PET

radiotracer tested in both rhesus monkeys and baboons29, 30, and subsequently advanced to evaluation in humans. However, when translated to humans, it exhibited slow kinetics in brain, resulting in large test-retest variability of quantitative measures31. Despite its slow tissue kinetics in humans,

11C-GR103545

possesses several

desirable characteristics as PET imaging agent, such as high brain uptake, in vivo KOR binding specificity and selectivity, and high specific binding signals. Therefore we designed a library of novel KOR agonists based on the GR103545 structure backbone to look for PET radiotracers with faster tissue kinetic profiles and/or suitable for labeling with the longer half-life 18F-radionuclide (t1/2 of 109.8 min vs. 20.4 min for C-11), which led to the

discovery

of

methyl

11C-(R)-4-(2-(3,4-dichlorophenyl)acetyl)-3-((ethyl(2-

fluoroethyl)amino)methyl) piperazine-1-carboxylate (named

11C-FEKAP,

Figure 1). In

this report we present the synthesis, in vitro binding assay and in vivo evaluation of this



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novel KOR agonist radiotracer.

Materials and Methods General Information Chemistry FEKAP (Compound 5), its fumarate salt (Compound 6) and optically pure precursor, (S)-4, as well as the reference standard and precursor of the other enantiomer were prepared from racemic piperazine carbaldehyde according to the previously published procedure32. The step-wise synthesis and conditions are shown in Scheme 1, and the detailed synthetic procedures and compound characterizations are shown in supporting information.

Radioligand competition binding assays in vitro The two enantiomers (compound 6 and 8) were submitted to the NIMH Psychoactive Drug Screening Program (PDSP) for assays of their binding affinities to MOR,

KOR

and

DOR

following

the

described

procedures

(https://pdspdb.unc.edu/pdspWeb/?site=assays). Each compound was assayed in triplicate.

Radiochemistry 11C-FEKAP

(11C-5) and its optically opposite enantiomer,

11C-7,

were prepared

from precursor (S)-4 and (R)-4, respectively, and the reaction conditions are shown in Scheme 2.


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General. The semi-preparative HPLC system used in radiochemistry included a Shimadzu™ LC-20A pump, a Knauer K200 UV detector, and a Bioscan γ-flow detector. The analytical HPLC system included a Shimadzu™ LC-20A pump, a Shimadzu™ SPDM20A PDA or SPD-20A UV detector, and a Bioscan γ-flow detector. [11C]Carbon dioxide was produced via the

14N(p,α)11C

nuclear reaction with the PETtrace cyclotron (GE

Medical Systems) using 16.5 MeV proton irradiation of nitrogen gas containing 0.5% oxygen. Waters Classic C18 Sep-Pak cartridges were purchased from Waters Associates. Reagents and solvents were purchased from commercial sources and used without further purification. Pre-treatment of the precursor: To the solution of compound (S)-4 (1 mg) in anhydrous DMF (0.3 mL) were added tetrabutylammonium triflate (TBAOTf, 2.0 mg) and cesium carbonate (Cs2CO3, 2.0 mg). The reaction mixture was sonicated for 1 min and bubbled with CO2 at 25 mL/min for 5 min. Radiosynthesis:

11C-CH

3OTf

was produced as previously described

33, 34

and

bubbled into the CO2-treated solution of the precursor in DMF until activity peaked in the reaction vial. The reaction mixture was heated at 45 °C for 5 min, cooled to room temperature, diluted with 1.5 mL of 0.1 M ammonium formate solution and injected onto the semi-preparative HPLC column (Gemini C18, 10 µm, 10 × 250 mm). The column was eluted with a mobile phase of 52% MeCN and 48% 0.1 M ammonium formate at a flow rate of 5 mL/min. The radioactivity fraction eluting between 14-16 min was collected, diluted with a solution of 400 mg of ascorbic acid (United States Pharmacopeia grade, USP) in 50 mL of water, and then loaded onto a Waters Classic C18 SepPak cartridge. The



SepPak was rinsed with 10 mL of 1 mM HCl and dried. The product was then eluted off the SepPak with 1 mL of USP absolute ethanol (Pharmco) followed by 3 mL of USP saline containing 3 mg of USP ascorbic acid. The resulting solution was passed through a sterile 0.22 µm membrane filter (13 mm, MILLEX GV, Millipore) for terminal sterilization into a vented dose vial containing 7 mg of USP ascorbic acid and 200 μL of 4.2% USP sodium bicarbonate (Abraxis) in 7 mL USP saline (American Regent). The other enantiomer 11C7 was synthesized with the same procedures.

PET Imaging Experiments in Rhesus Monkeys PET Scan Procedures. PET imaging experiments were performed in rhesus monkeys (Macaca mulatta) according to a protocol approved by the Yale University Institutional Animal Care and Use Committee. Three monkeys were used in a total of ten scans. Among them, eight scans with 11C-FEKAP were performed, including five baseline scans to assess kinetic and binding profiles, one blocking scan with the OR antagonist naloxone at 1 mg/kg dose to evaluate binding specificity, two blocking scans with the KOR antagonist LY2456302 (0.02 & 0.1 mg/kg, respectively) to verify binding selectivity. In the blocking scans, the blocking agents were given as a 3-min slow bolus injection at 20 min before radiotracer administration. One baseline scan with the inactive enantiomer 11C7 was carried out to compare the chirality of the radiotracer, and one baseline scan with 11C-GR103545

was conducted for comparison purpose.

The monkey was prepared and monitored in each scan as previously described22. Dynamic PET scans were performed on the Focus 220 scanner (Siemens Medical


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Solutions, Knoxville, TN, USA). Before radiotracer injection, a 9-min transmission scan was obtained for attenuation correction. The radiotracer was administered by an infusion pump over 3 min. Emission data were collected in list mode for 120 min and reformatted into 33 successive frames of increasing durations (6 × 30 s, 3 × 1 min, 2 × 2 min, and 22 × 5 min). Plasma Metabolite Analysis and Input Function Measurement. Arterial blood samples were collected at preselected time points and assayed for radioactivity in whole blood and plasma with calibrated well type gamma counters (Wizard 1480/2480, Perkin Elmer, Waltham, MA, USA). Six samples, drawn at 0, 5, 15, 30, 60 and 90 min, were processed and analyzed to measure the radiotracer metabolite profile by HPLC using the column-switching method

35.

Whole blood samples in EDTA tubes were centrifuged at

2,930 g at 4 °C for 5 min to separate the plasma. Supernatant plasma was collected and activity in 0.2 mL aliquots was counted on a gamma counter. Plasma samples were then mixed with urea (8 M) to denature plasma proteins, filtered through a 1.0 µm Whatman 13 mm CD/X syringe filter and loaded onto an automatic column-switching HPLC system connecting a capture column (4.6 × 19 mm) self-packed with Phenomenex Strata-X polymeric SPE sorbent and eluting with 1% MeCN in water at 2 mL/min for 4 min. The trapped activity in the capture column was then back flushed and eluted through a Phenomenex Luna C18 phenyl hexyl column (4.6 × 250 mm, 5 μm) with 60% MeCN in 0.1 M ammonium formate (pH = 6.4, v/v) at a flow rate of 1.5 mL/min. The eluent fractions were collected with an automated fraction collector (Spectrum Chromatography CF-1). Activity in the whole blood, plasma, filtered plasma-urea mix, filter, and HPLC eluent



fractions was all counted with the automatic γ counters. The un-metabolized parent fraction was determined as the ratio of the sum of radioactivity in fractions containing the parent compound to the total amount of radioactivity collected, fitted with inverted gamma function and corrected for filtration efficiency. The arterial plasma input function (AIF) was then calculated as the product of the total counts in the plasma and the interpolated parent fraction at each time point. Measurement of Radiotracer Free Fraction (fp) in Plasma. Ultrafiltration method was used for measuring the unbound portion (free fraction) of 11C-FEKAP in plasma as previously described22. The fP was determined as the ratio of the radioactivity concentration in the filtrate to the total activity in plasma. Measurements of fP were performed in triplicate for each scan. Measurement of Lipophilicity. Lipophilicity (log P) was determined as previously described22. log P was calculated as the ratio of decay-corrected radioactivity concentrations in 1-octanol and in phosphate buffered saline (PBS, Dulbecco). Six consecutive equilibration procedures were performed until a constant value of log P was obtained. Imaging Analysis and Kinetic Modeling. High-resolution magnetic resonance (MR) images were acquired with a Siemens 3T Trio scanner to assist with image co-registration and anatomical localization of regions of interest (ROIs). The MR image was registered to an atlas and to the PET images, as previously described36. PET emission data were attenuation-corrected using the transmission scan, and dynamic images were reconstructed using a Fourier rebinning and filtered back projection


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algorithm. For each PET scan, radiotracer regional concentrations over time, i.e. timeactivity curves (TACs) were generated for the ROIs. Regional TACs were fitted and analyzed with the one-tissue and two-tissue compartment (1TC, 2TC) models37, as well as the multilinear analysis method 1 (MA1) with a starting time (t*) of 30 min38. Regional distribution volume (VT, mL/cm3) was calculated from kinetic analysis of regional TACs using the metabolite-corrected AIF39. Akaike information criterion (AIC)40 and visual assessment fitting curves were used to evaluate the goodness-of-fits using the MA1, 1TC and 2TC models. Non-displaceable binding potential (BPND) was calculated from regional VT values using cerebellum as the reference region, i.e., BPND = (VT, ROI–VT, Cerebellum)/VT, Cerebellum. Additionally, the simplified reference tissue model (SRTM) was also tested in calculating BPND to evaluate the possible generation of binding parameters without arterial blood samples41. KOR occupancy by the blocking drugs was obtained from occupancy plot using the regional VT from the baseline scan and VT difference between baseline and blocking scans42.

Results Chemistry The synthesis of FEKAP (5) and the precursor for 11C-FEKAP ((S)-4) followed the synthesis route of GR10354529 as depicted in Scheme 1. The racemic aldehyde 1 underwent reductive amination with 2-(ethylamino)ethan-1-ol, followed by conversion of hydroxy group to fluoride using DAST reagent. Deprotection of compound 3 afforded the



racemic compound 4. The two enantiomers were separated by chiral HPLC. The desired (S)-enantiomer ((S)-4) with retention time of 6.60 min was obtained in greater than 99% chemical purity and greater than 99% enantiomeric excess (ee). This compound was used as the precursor for 11C-FEKAP, and also converted to the FEKAP reference standard (5). The free base form of FEKAP was an oil and thus converted to its solid fumarate salt (6) for use in the binding assays because of its ease in storage and handling. The undesired or inactive (R)-enantiomer ((R)-4) was also collected and used in the radiolabeling, and further conversion to the fumarate salt (8) for in vitro binding assays.

In vitro binding assays The inhibition constant (Ki) values of FEKAP (6) were measured at 0.43 nM for KOR, 7.1 nM for MOR and 139 nM for DOR. The other enantiomer (8) displayed much lower binding affinities for the ORs (51.0 nM for KOR, and >10,000 nM for both MOR and DOR). In the same set of binding assays, GR103545 was found to have Ki of 0.23 nM for KOR, 3.1 nM for MOR and 67.0 nM for DOR.

Radiochemistry 11C-FEKAP

was prepared in 8 ± 4% radiochemical yield (decay-uncorrected), > 99%

radiochemical purity, and mean molar activity of 718 GBq/mol at the end of synthesis (EOS, n = 19). Total synthesis time was about 45 min including purification and formulation.


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The inactive enantiomer 11C-7 was prepared from the corresponding precursor ((R)4) in 11% radiochemical yield (decay-uncorrected), > 99% radiochemical purity, and molar activity of 550 GBq/mol (EOS, n = 1).

PET Imaging Experiments in Rhesus Monkeys Injection Parameters. A total of eight PET scans with 11C-FEKAP were performed in three different monkeys. Injected activity was 164.6 ± 26.7 MBq, with injected mass of 0.56 ± 0.64 µg. Plasma Analysis. Results from plasma analysis are shown in Figure 2. Metabolism of 11C-FEKAP was fairly quick, with 31 ± 6% of intact parent compound at 30 min after radiotracer injection, which further decreased to 18 ± 10% and 12 ± 6%, respectively, at 60 and 90 min (n = 8) (Figure 2A). After a bolus injection of 11C-FEKAP, parent radioactivity level in plasma displayed a quick rise to peak and a sharp decline phase, then a slow decrease from 10 min onward (Figure 2B). On the reverse phase HPLC chromatograms, the two major metabolites of 11C-FEKAP appeared with retention times of 1.0 and 7.0 min, respectively, compared with 10.5 min for the parent compound (Figure 2C). The measured log P value of 11C-FEKAP was 2.98 ± 0.17 (n = 5), higher than that of 11C-GR103545 (1.82 ± 0.02, n = 12). The free fraction of 11C-FEKAP in plasma was 13 ± 4% (n = 8), lower than that of 11C-GR103545 (42 ± 6%, n = 2). Brain Analysis. The regional TACs from baseline scans of both 11C-FEKAP and 11C-7

are shown in Figure 3. As expected, a baseline scan with the inactive enantiomer,



11C-7

Page 14 of 38

(Figure 3B), displayed homogeneous regional uptake, indicating a lack of specific

binding and chirality of radiotracer binding. Shown in Figure 4 are representative PET images summed from 20-40 min postinjection of 11C-FEKAP in a baseline and a blocking scan with naloxone. In the monkey brain 11C-FEKAP exhibited heterogeneous distribution (Figure 4A, middle) and blocking with naloxone significantly reduced its binding (Figure 4A, right). Regional TACs from the same baseline and blocking scans are shown in Figure 5. Brain uptake of 11C-FEKAP was high, with peak SUV around 5 in the cingulate cortex (Figure 5A). After entering the monkey brain, the radiotracer localized to KOR-rich regions. The highest concentrations were observed in the cortical areas, while the lowest uptake was seen in the cerebellum. Tissue kinetics of

11C-FEKAP

was rapid and reversible. Regional concentrations of the

radiotracer reached peak levels within 20 min after injection, followed by a moderate rate of clearance over time. Pretreatment with naloxone and LY2456302 brought regional uptake levels in high binding regions to that in the lowest binding region of cerebellum (Figure 5B & 5C), demonstrating the binding specificity and selectivity of

11C-FEKAP.

Further blocking with LY2456302 at two different doses (0.02 and 0.1 mg/kg) reduced the regional concentrations of

11C-FEKAP

in a dose-dependent fashion (Figure 5C & 5D),

indicating the saturability of 11C-FEKAP binding. Regional TACs were processed with the 1TC, 2TC models and MA1 method to generate binding parameters (Figure 6A). The 2TC model showed apparently better fits than the 1TC model (AIC[2TC] < AIC[1TC]). Regional distribution volumes (VT) estimated with the MA1 method correlated well with those from 2TC (VT, MA1 = 1.0 VT, 2TC


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+ 0.10, r2 = 0.99). Listed in Table 1 are regional VT values derived from MA1 analysis (t* = 30 min). The inactive enantiomer exhibited VT values largely indistinguishable among the brain regions, reflecting non-specific binding. Regional BPND values were calculated from MA1 VT values using cerebellum as the reference region, along with those calculated from SRTM (Table 2). The rank order of BPND is as follows: cingulate cortex > globus pallidus > insula > caudate ≈ frontal cortex > temporal cortex > thalamus, which is consistent with the reported KOR distribution in monkey brains43, 44. SRTM BPND values correlated well with those from MA1 (BPND, SRTM = 0.94 BPND, MA1 – 0.01, r2 = 0.99), as shown in Figure 6B. Pre-treatment with blocking agents significantly reduced VT across the monkey brain regions. Using the MA1-derived VT values, receptor occupancy was calculated to be 77% with 1 mg/kg of naloxone. The KOR selective antagonist LY2456302 induced 84% and 93% receptor occupancy at the 0.02 mg/kg and 0.1 mg/kg doses, respectively. Comparison of 11C-FEKAP with 11C-GR103545. Regional TACs from the baseline scans of 11C-FEKAP and 11C-GR103545 are shown in Figure 3. Levels of regional brain uptake and distribution pattern are similar for these two radiotracers. The tissue kinetics of 11C-GR103545

(Figure 3C) is evidently much slower. Binding parameters for these two

radiotracers are very similar (Table 3) with slightly higher regional VT and BPND values for 11C-GR103545.



Page 16 of 38

DISCUSSION In this study we describe the development and in vivo evaluation of a novel KOR agonist PET radiotracer

11C-FEKAP

in rhesus monkeys, and its comparison with

11C-

GR103545. Currently, 11C-GR103545 is the only agonist PET radiotracer available for clinical research studies and the most superior among all the KOR radiotracers that have been tested in terms of brain uptake and in vivo binding characteristics. The only disadvantage is its slow tissue kinetics, which led to unreliable derivation of binding parameters and high test-retest variability. The impetus for this study was to look for a KOR agonist radiotracer with faster kinetics than

11C-GR103545,

therefore we took GR103545 as the lead

compound and developed a library of KOR agonists through modifications of the GR103545 structure. The design of FEKAP focused on two tactics: 1) open the pyrrolidine ring to allow more freedom of movement for the molecule, which tends to lower the binding affinity, i.e., fast dissociation rate from the receptor binding sites and thus fast tissue kinetics; 2) incorporate functional groups feasible for radiolabeling with the longer half-life 18F-radionuclide (radioactive t1/2 of 109.8 min vs. 20.4 min for C-11), which would allow longer scanning time to capture the wash-out phase of the radiotracer even if the tissue kinetics remains slow. FEKAP and its 11C-radiolabeling precursors were prepared in good yield, and the racemic precursor was resolved by chiral HPLC into pure enantiomers with > 99% ee. As expected, in vitro binding assays showed that FEKAP indeed displayed lower affinity for KOR (Ki of 0.43 nM vs. 0.23 nM for GR103545). The novel radiotracer 11C-FEKAP and


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its inactive enantiomer 11C-7 were then produced from the enantiomeric pure precursors and 11C-MeOTf with good radiochemical yield, purity and molar activity. In rhesus monkeys 11C-FEKAP was metabolized at a fairly rapid rate, with parent fraction of 31 ± 15% at 30 min post injection. Two major radioactive metabolites were detected in the blood, which appear to be much more polar than the parent radiotracer, and thus are unlikely to enter the brain and complicate the quantitative analysis of PET imaging data. The free fraction of this tracer in plasma is relatively high and can be reliably measured. Measured log P of 11C-FEKAP fits in the range of PET radiotracers that is predicted to have good permeability through the blood-brain barrier45. Indeed, 11C-FEKAP readily entered the monkey brain and accumulated in regions known to have high KOR densities, such as the cortex and striatum. Regional TACs demonstrated fast and reversible kinetics. Highest tissue uptake levels were found in the globus pallidus and cingulate cortex. Peak uptake was reached within 20 min in all brain regions. A baseline scan of the inactive enantiomer,

11C-7,

displayed only non-specific binding, consistent with its low binding

affinity to cloned human KOR in vitro (51 nM vs. 0.43 nM for FEKAP). In the blocking study, pre-treatment of the monkey with the OR antagonist naloxone induced great reductions in brain uptake, demonstrating the binding specificity of

11C-FEKAP.

The

blocking scans with 0.02 and 0.1 mg/kg of the KOR-selective antagonist LY2456302 were carried out in two different monkeys. Note that the lower uptake in the scan with a lower dose of LY2456302 (0.02 mg/kg) resulted from the lower brain uptake of 11C-FEKAP in that individual monkey (Figure 5C and 5D). Nonetheless, dose-dependent blockade of 11C-



Page 18 of 38

FEKAP binding was observed (see Table 1), thus confirming the binding selectivity and saturability of the radiotracer. Taken together, experiments in rhesus monkeys indicated that binding of 11C-FEKAP in the brain is saturable, specific, and selective for KOR. In comparison of kinetic models for PET data analysis, the 2TC model provided good fits to regional TACs and was considered to be an appropriate model for estimation of binding parameters. Further comparison between the 2TC and MA1 methods revealed that MA1 produced regional VT estimates well correlated with those from 2TC. However, the 2TC model sometimes produced implausible VT values, therefore the MA1 method was selected to generate binding parameters. Regional VT values in the ROIs showed high binding in the cortical and striatal regions and low binding in cerebellum, consistent with the KOR autoradiography results in nonhuman primates 6. Previous studies with KOR radiotracers indicated that cerebellum can be used as a reference region in non-human primates to calculate BPND25. This was also true for 11CFEKAP, as cerebellum VT remained largely unchanged in the blocking scans. Regional BPND values estimated by SRTM using cerebellum as the reference region exhibited excellent correlation with those derived from MA1 VT values. Compared with 11C-GR103545, the novel radiotracer 11C-FEKAP presented similar brain uptake and distribution pattern. Furthermore,

11C-FEKAP

displayed a faster

metabolism rate and much more rapid tissue kinetics: peak uptake in less than 20 min for 11C-FEKAP

and gradual clearance from the monkey brain vs. peak uptake not observed

after 120 min for 11C-GR103545 in most brain regions (Figure 3A and 3C). Therefore, we also expect greatly improved kinetic profile of

11C-FEKAP


in the human brain in

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comparison with

11C-GR103545,

despite the slower kinetics in higher species as

demonstrated in previous studies30,

31.

The difference in specific binding signals, as

measured by regional BPND values, is small between 11C-FEKAP and 11C-GR103545. Both radiotracers provide BPND values of > 0.5 in most brain regions, levels of specific binding signals that can be reliably and accurately estimated by quantitative kinetic modeling analysis, and one of the important characteristics for a suitable and effective neuroimaging radiotracer46. Although limited number of scans (two scans with 11C-FEKAP and one scan with 11C-GR103545) were conducted in the same monkey for within-subject comparison, both radiotracers have been used in other studies and tested in multiple monkeys (a total of 6 scans with 11C-FEKAP in 3 monkeys and 13 scans with 11C-GR103545 in 4 monkeys). In these between-subject comparison, the kinetic and binding profiles of the two radiotracers matched those from the within-subject comparison. In addition, 11C-FEKAP bears a fluorine atom in its structure, which creates the potential for future radiolabeling with the longer half-life 18F-radionuclide, and thus the potential for wide availability and central production and distribution.

CONCLUSIONS We have successfully developed a novel KOR agonist PET radiotracer, 11C-FEKAP, and carried out a detailed evaluation in non-human primates. This novel radiotracer exhibits favorable metabolic, pharmacokinetic and in vivo binding profiles. A side-by-side comparison between

11C-FEKAP

and

11C-GR103545

indicates similarly high specific



binding signals, but a faster tissue kinetics with

11C-FEKAP.

Page 20 of 38

Given the desirable

characteristics of this novel radiotracer, it has been advanced to evaluation in humans.

AUTHOR INFORMATION Corresponding authors*: Songye Li, PO Box 208048, PET Center, Yale University, School of Medicine, New Haven, CT 06520, USA, Tel: 203-785-3605, Fax: 203-785-2994, e-mail: [email protected]. ORCID: Songye Li: 0000-0002-6096-8756 Note: The authors declare no conflict of interest.

ACKNOWLEDGEMENT This work was supported by the NIH/NIMH grant R21MH092664 and R33MH092664. The authors thank the staff at the Yale PET Center for their expert assistance in this work, especially the non-human primate team.

Supporting Information.

Detailed Synthetic procedures and compound characterization of FEKAP, its fumarate salt and 11C-labeling precursor as well as the reference standard and precursor of the other enantiomer.


Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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29. Nabulsi, N. B., Zheng, M. Q., Ropchan, J., Labaree, D., Ding, Y. S., Blumberg, L., and Huang, Y. (2011) [11C]GR103545: novel one-pot radiosynthesis with high specific activity, Nucl Med Biol 38, 215-221. 30. Talbot, P. S., Narendran, R., Butelman, E. R., Huang, Y., Ngo, K., Slifstein, M., Martinez, D., Laruelle, M., and Hwang, D. R. (2005)

11C-GR103545,

a radiotracer for

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 28 of 38

TABLE 1. MA1 derived regional distribution volume (VT, mL/cm3) of 11C-FEKAP in the monkey brain. Brain regions Cingulate cortex

Globus pallidus

Insula

Caudate nucleus

Frontal cortex

Temporal cortex

Thalamus

Cerebellum

Baseline (n = 5)

16.5±4.8

16.1±5.1

14.5±3.9

9.8±2.5

6.4±1.9

5.2±1.9

Inactive enantiomer

5.2

5.2

4.8

5.5

5.1

5.7

4.4

4.7

4.8

Naloxone (1 mg/kg)

9.0

7.4

7.3

6.8

8.1

6.6

6.5

6.6

6.2

LY2456302 (0.1 mg/kg)

11.8

10.4

9.8

10.2

9.9

9.6

8.6

7.7

10.4

LY2456302 (0.02 mg/kg)

6.8

7.6

6.1

5.1

5.6

5.0

5.0

4.7

4.6

Putamen

10.7±3.7 11.3±1.9 10.6±3.2


Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


TABLE 2. MA1 and SRTM derived regional binding potential (BPND) of 11C-FEKAP in the monkey brain. Brain regions BPND

MA1

SRTM

Cingulate cortex

Globus pallidus

Insula

Caudate nucleus

Frontal cortex

Putamen

Temporal cortex

Thalamus

Baseline (n = 5)

2.22±0.31

2.11±0.46

1.83±0.29

1.05±0.22 1.28±0.41 1.08±0.43

0.92±0.21

0.25±0.14

Naloxone (1 mg/kg)

0.44

0.18

0.17

0.10

0.29

0.06

0.04

0.05

LY2456302 (0.1 mg/kg)

0.13

0.00

-0.06

-0.03

-0.05

-0.08

-0.18

-0.27

Baseline (n = 4)

2.10±0.25

1.78±0.42

1.68±0.28

0.86±0.20

0.28±0.16

Naloxone (1 mg/kg)

0.45

0.18

0.22

0.17

0.42

0.01

0.07

0.08

LY2456302 (0.1 mg/kg)

0.28

0.16

0.11

0.10

0.01

-0.02

-0.05

-0.03

1.04±0.23 1.15±0.44 1.07±0.53



Page 30 of 38

TABLE 3. Comparison of binding parameters derived from MA1 between baseline scans of 11C-FEKAP and 11C-GR103545 in the same monkey. Brain Regions Globus pallidus

Cingulate cortex

Insula

Caudate

Frontal cortex

Putamen

Temporal cortex

Thalamus Cerebellum

VT (mL•cm-3) 11C-FEKAP

(n = 2) 11C-GR103545

(n = 1)

20.0±4.5

20.3±5.6

17.5±4.7

13.6±3.9

12.6±2.6

12.2±3.6

11.8±3.0

7.8±2.4

6.9±2.3

39.3

53.7

39.5

34.4

29.1

28.5

27.4

16.3

14.0

BPND 11C-FEKAP

(n = 2) 11C-GR103545

(n = 1)

1.96±0.33

1.97±0.17

1.57±0.18

0.99±0.09

0.86±0.23

0.78±0.06

0.74±0.15

0.14±0.02

-

1.80

2.83

1.82

1.45

1.08

1.04

0.96

0.16

-


Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


O a

H

N O

N

N

OH

O

Cl

1

N

N

b

O

Cl

2

F

c Cl

N

Cl

N

Cl

N

Cl

3 O

O N

HN

F

O N

F Cl

N O 4

N

N

O

Cl

N

HN

e

O

Cl

O HO

N

HN

e Cl

N O (R)-4

Cl

O

Cl O

Cl

O

Cl

5

F

F

N OH 6

O

O

Cl

N

N

O

f Cl

N

(S)-4

d

F

N

N

F

f Cl

N O

Cl

N

N

O O HO

F Cl

N OH

O

O

7

Cl

8

Reagents and conditions: a. 2-(Ethylamino)ethan-1-ol, NaBH(OAc)3, (CH2Cl)2, 0 oC then r.t., 16 h; b. Diethylaminosulfur trifluoride (DAST), CH2Cl2, 0 16 h; c. Pd/C (5 %), HCl, H2, THF/H2O, r.t., 3 h; d. Chiral HPLC separation; e. Methyl chlorofomate, TEA, CH2Cl2, r.t., 16 h; f. Fumaric acid, MeOH/Et2O, 0 oC, 16 h.

oC,

SCHEME 1. Synthesis of FEKAP, its 11C-labeling precursor and the inactive enantiomer



F HN

TBAOTf, Cs2CO3, CO2 Cl

N O

F

O

N

Cl

DMF, r.t., 5 min

O

Page 32 of 38

N

11

Cl

N O

F

O

N

C-MeOTf

H311C

O

N N

45 oC, 5 min 11

SCHEME 2. Radiosynthesis of 11C-FEKAP (11C-5).


Cl O

Cl

(S)-4

N

C-FEKAP

Cl

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Figure images O

11

O

O C O

O H311C

N

O

N

O O O 11

C-Salvinorin A

11

O

C-GR103545

N

N

Cl O

O

F

O H311C

N

Cl

N O

Cl 11

Cl

C-FEKAP

FIGURE 1. Chemical structures of KOR agonist radioligands.



B

A 11

1.0

C-FEKAP C-GR103545

11

0.5

0.0 0

20

40

60

Time (min)

80

100

Plasma activity (SUV)

Parent Fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 34 of 38

C 4

11

C-FEKAP C-GR103545

Metabolites

11

3

11

C-FEKAP Parent

2

60 min 30 min 15 min 5 min

1 0 0

20

40

60

80

100

Time (min)

0

2

4

6

8

FIGURE 2. Parent fraction in plasma (A) and metabolite-corrected plasma activity over time (B) for GR103545, and HPLC chromatograms from metabolite analysis of 11C-FEKAP (C).


10

12

14

Time (min)

11C-FEKAP

and

11C-

Page 35 of 38

B

C 5

4

4

3

3

SUV

5

2 1 0 0

Cingulate cortex Insula Frontal cortex Temporal cortex Caudate nucleus Putamen Thalamus Cerebellum

2

40

60

80

Time (min)

100

120

0 0

4 3 2 1

1 20

5

SUV

A

SUV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


20

40

60

80

100

Time (min)

120

0 0

20

40

60

80

100

120

Time (min)

FIGURE 3. Brain regional TACs from baseline scans of 11C-FEKAP (A) and its inactive enantiomer (B), in comparison with 11C-GR103545

(C).



FIGURE 4. MR (left) and summed PET SUV images from 20-40 min after 11C-FEKAP injection from a baseline scan (middle), and a blocking scan (right) with naloxone (1 mg/kg).


Page 36 of 38

Page 37 of 38

B 5

5

4

4

3

3

SUV

SUV

A

2 1 0 0

20

40

60

80

100

2

0 0

120

Time (min)

20

D

4

4

3

3

SUV

5

2 1

40

60

80

100

120

100

120

Time (min)

5

0 0

Cingulate cortex Insula Frontal cortex Temporal cortex Caudate nucleus Putamen Thalamus Cerebellum

1

C

SUV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


2 1

20

40

60

80

100

120

Time (min)

0 0

20

40

60

80

Time (min)

FIGURE 5. Regional TACs of 11C-FEKAP from a baseline scan (A) and blocking scans with 1 mg/kg naloxone (B), 0.02 mg/kg LY2456302 (C) and 0.1 mg/kg LY2456302 (D).



B Caudate nucleus Cingulate cortex Cerebellum 1T fit 2T fit MA1 fit (t*=30 min)

120,000 90,000 60,000 30,000 0 0

20

40

60

80

100

120

2.5

BPND (SRTM)

A Concentration (Bq/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 38 of 38

2.0

Y = 0.9401X - 0.0128

1.5 1.0 0.5 0.0 0.0

0.5

1.0

1.5

2.0

2.5

BPND (MA1)

Time (min)

FIGURE 6. Comparison of fitting with 1TC, 2TC models and MA1 methods in selected brain regions (A) and correlation of SRTM and MA1 BPND values (B).


Novel kappa opioid receptor agonist as improved PET radiotracer

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