Radiosynthesis and Evaluation of a C-11 Radiotracer for Transient Receptor Potential Canonical 5 in the Brain

Abstract

Purpose:

TRPC5 belongs to the mammalian superfamily of transient receptor potential (TRP) Ca²⁺- permeable cationic channels and has been implicated in various CNS disorders. As part of our ongoing interest in the development of a PET radiotracer for imaging TRPC5, herein we explored the radiosynthesis, in vitro and in vivo evaluation of a new C-11 radiotracer [¹¹C]HC070 in rodents and nonhuman primates.

Procedures:

[¹¹C]HC070 was radiolabeled utilizing the corresponding precursor and [¹¹C]CH₃I via N-methylation protocol. Ex vivo biodistribution study of [¹¹C]HC070 was performed in Sprague-Dawley rats. In vitro autoradiography study was conducted for the rat brain sections to characterize the radiotracer distribution in the brain regionals. Micro PET brain imaging studies of [¹¹C]HC070 were done for 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice for 0–60 min dynamic data acquisition after intravenous administration of the radiotracer. Dynamic PET scans (0–120 min) for the brain of cynomolgus male macaques were performed after the radiotracer injection.

Results:

[¹¹C]HC070 was efficiently prepared with good radiochemical yield (45 ± 5%, n = 15), high chemical and radiochemical purity (>99%), and high molar activity (320.6 ± 7.4 GBq/μmol, 8.6 ± 0.2 Ci/μmol) at the end of bombardment (EOB). Radiotracer [¹¹C]HC070 has good solubility. The ex vivo biodistribution study showed that [¹¹C]HC070 had a quick rat brain clearance. Autoradiography demonstrated that [¹¹C]HC070 specifically binds to lTRPC5-enriched regions in rat brain. MicroPET study showed the peak brain uptake (SUV value) was 0.63 in 129S1/SvImJ TRPC5 knockout mice compared to 1.13 in 129S1/SvImJ wild-type mice. PET study showed that [¹¹C]HC070 has good brain uptake with maximum SUV of ~2.2 in the macaque brain, followed by rapid clearance.

Conclusions:

Our data showed that [¹¹C]HC070 is a TRPC5 specific radiotracer with high brain uptake and good brain washout pharmacokinetics in both rodents and nonhuman primates. The radiotracer is worth further investigating of its suitability to be a PET radiotracer for imaging TRPC5 in animals and human subjects in vivo.

Introduction

Transient receptor potential canonical 5 (TRPC5) belongs to the family of seven mammalian TRPC moieties (TRPC1–7) which are Ca²⁺-permeable cationic channels. The brain widely expresses TRPC5 and this has been implicated in a wide range of physiological and pathological mechanisms of neurological and psychiatric diseases [1–4]. These findings have driven several TRPC5 inhibitors like BI 135889 (Boehringer Ingelheim) and GFB-887 (Goldfinch Bio) into phase 1/2 clinical trials for treating kidney disease and CNS disorders [5–6]. To develop a TRPC5 radiotracer, we previously reported [¹¹C]HC608 (IC₅₀ = 6.2 nM) (Figure 1) as a TRPC5 PET radiotracer with relatively low brain standard uptake (SUV). Meanwhile, low solubility of [¹¹C]HC608 was observed for this dose formulation and a potent non-ionic solubilizer was required [7–8]. These unfavorable properties prevented further investigation of the clinical suitability of [¹¹C]HC608.

Fig. 1.

The structures of two C-11 TRPC5 radiotracers [¹¹C]HC070 and [¹¹C]HC608.

The xanthine-based analogue, HC070, a structure closely related to HC608, was reported by Hydra Biosciences/Boehringer Ingelheim [9]. In vitro calcium flux assay and whole-cell manual patched clamp assay showed compound HC070 is a potent inhibitor towards human TRPC5 with an IC₅₀ of 9.3 nM and 0.52 nM respectively [10]. In vitro patch clamp assay also showed HC070 was at least 400-fold selective over a wide range of molecular targets including ion channels such as transient receptor potential cation channel subfamily vanilloid (TRPV) protein channels, and transient receptor potential cation channel subfamily melastatin (TRPM) protein channels; receptors such as dopamine D₁ and D₂ receptors, NMDA receptors, and TNF-α receptors; and enzymes such as acetylcholinesterase and tyrosine hydroxylase [10]. Recent Cryo-electron microscopy (cryo-EM) structural studies also explored the molecular mechanisms of potency and selectivity of HC070 towards human TRPC5 [11]. Furthermore, in vivo studies indicated that HC070 is orally bioavailable and carries anxiolytic and antidepressant function [10].

The molecular structure of HC070 with a chloride replacing the trifluoromethyl group in HC608, may improve its hydrophilicity and solubility in aqueous solution [12–13]. Furthermore, the slight structural difference of HC070 from HC608 may also improve the radiopharmaceutical profile of the C-11 labeled radiotracer. Therefore, we decided to radiosynthesize [¹¹C]HC070 and perform initial in vitro, ex vivo and in vivo evaluation of [¹¹C]HC070 and compare it with [¹¹C]HC608 [7]. PET brain studies in nonhuman primate were also carried out to check its brain uptake and brain washout pharmacokinetics. Our data suggested that [¹¹C]HC070 exhibits improved solubility in ethanol/saline medium with lower percentage of residue retaining inside surface of a glass viral or a syringe. Our autoradiography data also showed that [¹¹C]HC070 has higher accumulation in TRPC5-enrich regions of the rat brain tissue and [¹¹C]HC070 has higher brain uptake in 129S1/SvImJ wide type mice than the 129S1/SvImJ TRPC5 knockout mice, indicating that [¹¹C]HC070 accumulation in the brain has TRPC5 specificity. More importantly, [¹¹C]HC070 displays higher brain uptake and good brain washout pharmacokinetics in both rodents and nonhuman primate compared to radiotracer [¹¹C]HC608. Together, [¹¹C]HC070 could be a useful preclinical tool for in vivo investigating TRPC5 function in living animals.

Materials and Methods

General

All reagents and solvents (ACS or HPLC grade) employed in the syntheses were purchased from Sigma-Aldrich or other commercial vendors without further purification.

Thin-layer chromatography (TLC) was conducted with 0.25 mm silica gel plates 60 F₂₅₄ (EMD Chemicals Inc, Billerica, MA) and visualized by exposure to UV light (254 nm) or stained with potassium permanganate. Flash column chromatography was performed using 230–400 mesh silica gel (SiliCycle Inc, Quebec, Canada) particle. Nuclear magnetic resonance (NMR) spectra were obtained either on a Varian spectrometer 400 MHz with CDCl₃ as solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm, and coupling constants are reported in hertz. The following abbreviations are used to describe multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br. (broad). High resolution mass spectrum (HRMS, m/z) reports were acquired by a Bruker MaXis 4G Q-TOF mass spectrometer with electrospray ionization source.

Male Sprague Dawley rats (Charles River, Wilmington, MA), male 129S1/SvImJ wild-type (The Jackson Laboratory, Bar Harbor, ME) and 129S1/SvImJ TRPC5 knockout mice that were kindly provided by Dr. Vadim Bolshakov at Harvard Medical School for our in-house breeding colony were used in the studies.

Chemistry and radiochemistry

Reference compounds HC608, HC070 and intermediates 1–5 in Scheme 1 were synthesized and fully characterized as reported previously [7].

Scheme 1.

Synthesis of the precursor 7 and radiosynthesis of [¹¹C]HC070.

Reagents and conditions: (a) Br₂, H₂O, 90%; (b) 1-(bromomethyl)-4-chlorobenzene, K₂CO₃, DMF, 85%; (c) (2-(chloromethoxy) ethyl)trimethylsilane, DBU, DMF, 0 ^oC to 60 ^oC, 65%; (d) 2-(3-bromopropoxy)tetrahydro-2H-pyran, K₂CO₃, DMF, 60 ^oC, 72%; (e) 3-chlorophenol, K₂CO₃, DMF, 80 ^oC; (f) HCl (aq), EtOH, reflux, 68%; (g) [¹¹C]CH₃I, K₂CO₃, DMF, 90 ^oC, 5 min.

7-(4-Chlorobenzyl)-8-(3-chlorophenoxy)-1-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-3-((2-(trimethyl-silyl)ethoxy) methyl)-3,7-dihydro-1H-purine-2,6-dione (6)

A mixture of 5 (626 mg, 1.0 mmol), 3-chlorophenol (154 mg, 1.2 mmol), K₂CO₃ (166 mg, 1.2 mmol) in DMF (10 mL) was heated at 80 °C for 12 h. After cooling to room temperature (RT), the resultant mixture was poured into brine and extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, and evaporated under reduced pressure to yield 6 as colorless oil. The crude material could be used for next step directly without further purification.

7-(4-Chlorobenzyl)-8-(3-chlorophenoxy)-1-(3-hydroxy-propyl)-3,7-dihydro-1H-purine-2,6-dione (7)

Concentrated hydrochloric acid (1.5 mL, 12 M) was added dropwise to a solution of 6 above in EtOH (20 mL), then the reaction mixture was refluxed for about 12 h until the starting material was disappeared using TLC monitoring the reaction. The reaction mixture was cooled and concentrated in vacuo, and then quenched by 10 mL NaHCO₃ (sat.), diluted by water (30 mL) and ethyl acetate (50 mL). The organic layer was washed with saturated aqueous NaCl (30 mL), dried over Na₂SO₄, and concentrated in vacuum. After purification by silica column chromatography (hexane/ethyl acetate, 1:2 to 1:3, v/v)), compound 7 was obtained as a white solid (267 mg, 58% for 2 steps). Mp:180–181 ^oC. ¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 7.48–7.39 (m, 6H), 7.34–7.27 (m, 2H), 6.36 (s, 2H), 4.41 (t, J = 5.0 Hz, 1H), 3.84 (t, J = 7.0 Hz, 2H), 3.39 (dd, J = 11.2 Hz, 5.9 Hz, 2H), 1.68–1.61 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 154.66, 153.56, 153.06, 150.56, 144.67, 135.36, 133.63, 132.59, 131.28, 129.58, 128.76, 126.05, 120.56, 119.05, 102.50, 58.82, 45.69, 37.49, 31.09. HRMS (m/z) [M + H]⁺ calculated for C₂₁H₁₉Cl₂N₄O₄ 461.0783, found:461.0781.

For radiosynthesis, [¹¹C]CO₂ was produced through the ¹⁴N (p, α)¹¹C nuclear reaction using JSW BC-16/8 cyclotron of Washington University Medical School. The [¹¹C]methyl iodide ([¹¹C]CH₃I) was generated by reduction of [¹¹C]CO₂ using a nickel catalyst under atmosphere of H₂ at 360 ^oC, followed by iodination at 690 ^oC. The [¹¹C]MeI was immediately transferred under N₂ gas flow into a solution of 7 (0.4 mg, 0.87 μmol) and K₂CO₃ (2.0 mg) in DMF (300 μL). When the radioactivity reached a plateau (measured by the proximal radiation detector), the sealed reaction vessel was heated at 90 °C for 5 min during which the vial was shaken occasionally using a long clamp. Then the oil bath was removed and the mixture was cooled and quenched with a mixture solution composed of 0.5 mL DMSO and 0.7 mL milli-Q water. The diluted reaction mixture was loaded onto a reverse phase semi-preparative C-18 Agilent column (Zorbax SB-C18, 9.2 mm ID × 250 mm, 5 μm). Using 62% MeCN and 38% ammonium formate (0.1 M, pH = 4.5) as mobile phase and, a flow rate of 4 mL/min, UV detector wavelength of 254 nm, the desired radioactive product was collected from 14 to 15 min and diluted with 50 mL of Milli-Q water, and then passed through a plus short C-18 Sep-Pak cartridge (Part No. WAT020515, Waters cooperation, Milford, MA). After using 20 mL of Milli-Q water to rinse the C-18 cartridge, the radioactive product was eluted into a dose vial using 1.0 mL absolute ethanol and then 9.0 mL saline (v/v) to formulate a dose solution for intravenous (IV) injection for animal studies.

LogD_7.4 Measurement

The LogD_7.4 value was measured similar to the lipophilicity measurement of “Shake Flask Method” with minor modification. Briefly, varying volumes of tracer of [¹¹C]HC070 (1 μL, 2.5 μL, 5 μL) solution were added into 3 centrifuge tubes containing n-octanol/phosphate-buffered saline (PBS, 0.1 M, pH 7.4, 500 μL/500 μL). Both n-octanol and PBS were presaturated with each other prior to use. Each resulting mixture was vortexed for 1 min, followed by centrifugation at 3500 × g for 3 min at room temperature. An aliquot of 10 μL of PBS and 10 μL of n-octanol was taken out separately, and the radioactivity in each aliquot was counted with a gamma counter. The LogD_7.4 value was determined according to the following equation:

LogD_7.4 = log{[cpm/g (n-octanol)]/[cpm/g (PBS)]}. The measurements were performed 3 times in total.

Ex vivo Biodistribution Study

A solution of [¹¹C]HC070 (~3.7 MBq/100 μL) was injected into Sprague Dawley (SD) rats (male; 7 weeks old; 200–220 g) via tail vein. Rats (n = 4 for each time point) were euthanized under isoflurane and oxygen inhalation anesthesia at 5-, 30-, and 60-min post tracer injection. Tissues of interests, including blood, lung, liver, spleen, kidney, muscle, fat, heart, brain, pancreas, thymus, small intestine, tail were collected, weighed, and counted using an automated Beckman Gamma counter. All radioactivity measurements were background and decay-corrected. The uptake of each organ was calculated as a percentage of the injected dose per gram of wet tissue (% ID/g).

In vitro autoradiography study of the rat brain

To check the distribution of the radiotracer within the brain, in vitro autoradiography was performed on brain section slides from adult Sprague Dawley (SD) rats. Animals (male; 7 weeks old; 200–220 g) were euthanized under anesthesia, brains rapidly removed and frozen then 20 micron frozen sections were prepared. Slides were incubated with [¹¹C]HC070 (1.11 MBq/slide) radiotracer in 500 μL of binding buffer (1 × Ringer buffer, pH 7.4) at room temperature for 30 min. The cold HC608 (1 μM) was used for a blocking study. After incubation, brain slides were washed (2 min each) in the following buffers sequentially: 1 × PBS; 10% EtOH in 1 × PBS; 30% EtOH in 1 × PBS; 1 × PBS. Then the rat brain sections were exposed on the Storage Phosphor Screen in an imaging cassette in −20 ^oC for 3.0 hours at dark. The uptake of [¹¹C]HC070 was visualized with a Fuji Bio-Imaging Analyzer FLA-7000 (Fuji Photo Film, Tokyo, Japan).

MicroPET imaging study of the mouse brain

MicroPET Scans studies were carried out by using an Inveon PET/CT scan system (Siemens Inc., Knoxville, TN, USA) to check the distribution of the radiotracer in the brain. 129S1/SvImJ wild-type and 129S1/SvImJ TRPC5 knockout mice (n = 3 for each group, 20 ~ 22 g) were anesthetized with oxygen mixed with 2% (v/v) isoflurane delivered via a nose cone before 26-gauge catheters were placed in the lateral tail vein for bolus injection of radiotracer. PET scans image data were acquired from 0–60 min post injection of [¹¹C]HC070 (~10 MBq, injected mass ~0.12 μg) via the tail vein catheter. Dynamic PET images were reconstructed and analyzed using the Inveon Research Workstation with built in software IRW 4.2 program (Siemens Inc., Knoxville, TN). All radioactivity data were decay-corrected to the injection time and the standard uptake value (SUV) was calculated by the following equation: SUV = nCi/cm³ × weight (g) ∕ dose injection (nCi). Then time-activity curves of [¹¹C]HC070 in the brain were generated.

MicroPET brain study of the nonhuman Primate

To check the pharmacokinetics of [¹¹C]HC070 in nonhuman primate brain, brain imaging studies were done in three adult male cynomolgus macaques (9–10 kg) using a microPET Focus 220 scanner (Concorde/CTI/Siemens Microsystems, Knoxville, TN, USA). Animal A underwent one PET with [¹¹C]HC608 scan [7]. Animal B underwent one PET with [¹¹C]HC608 scan and two PET with [¹¹C]HC070 scans. Animal C underwent two PET with [¹¹C]HC070 scan for imaging data acquisition. Prior to PET study, the animal was fasted for 12 h and then initially anesthetized with injection of ketamine (10 – 20 mg kg⁻¹) and glycopyrrolate (0.13 mg/kg) intramuscularly. Upon arrival at the scan room, the animal was intubated, and anesthetized with 0.8 – 2.0 % isoflurane/oxygen throughout whole PET imaging data acquisition. After a percutaneous venous catheter was placed for radiotracer injection and the head of the animal was positioned supine in the adjustable head holder with the brain in the center of the field of view, a 10 min transmission scan was performed to check positioning prior to a 45 min transmission scan for attenuation correction. Subsequently, a 0–120 min dynamic PET scan was performed (3 × 1-min, 4 × 2-min, 3 × 3-min, and 20 × 5 min frames) after venous injection of ∼0.35 GBq of [¹¹C]HC070 (injected mass ~4.2 μg).

Results

Chemistry and radiochemistry

The synthesis of the precursor and compound HC070 followed our reported procedure with necessary modification [7]. Briefly, the precursor 7 of preparing [¹¹C]HC070 was achieved through four steps, starting with commercially available xanthine (1H-Purine-2,6-dione) reacted with 3-chlorophenol shown in Scheme 1. The intermediate compounds 2, 3, 4, and 5 were generated as reported in literature [7]. Compound 6 was obtained via a nucleophilic substitution reaction on the bromide of 5 with K₂CO₃ as base. The desired precursor 7 for radiolabeling was carried out in one step via simple hydrolysis of the two protecting groups of compound 6.

The radiosynthesis of [¹¹C]HC070 was accomplished starting with precursor 7 via N-methylation by employing the strategy of making [¹¹C]HC608 as depicted in Scheme 1. After reverse-phase HPLC purification of the radioactive product and formulation with 10% absolute ethanol in saline, the chemical and radiochemical purities of [¹¹C]HC070 (t_R = 5.3 min) were measured using an analytical HPLC system (Agilent Zorbax SB-C18, 4.6 mm ID × 250, 5 μm), UV wavelength of 254 nm, mobile phase of CH₃CN/0.1 M HCOONH₄ (v/v, 82/18, pH = 4.5), flow rate = 1.0 mL/min). The identification of [¹¹C]HC070 was authenticated by co-injection with unlabeled HC070.

The total radiosynthesis took ~50 min from end of bombardment (EOB) to the dose formulation with radiochemical yield of 45 ± 5% (n = 15) decay-corrected to EOB, >99% radiochemical purity, and the molar activity was 320.6 ± 7.4 GBq/μmol (8.6 ± 0.2 Ci/μmol, EOB).

Measurement of the Distribution Coefficient (LogD_7.4 value)

An assessment of the candidate compound’s lipophilicity property is one of the most important and fundamental measurements in central nervous system drug development. By using liquid−liquid partition between n-octanol and PBS buffer (pH 7.4), the so-called “shake flask method” [14], the logD_7.4 values for [¹¹C]HC070 and [¹¹C]HC608 were determined to be 1.08 ± 0.04 and 1.20 ± 0.03 respectively (n = 3). These results indicated that both two radiotracers possess favorable lipophilicity for blood brain barrier penetration, and [¹¹C]HC070 is more hydrophilic than [¹¹C]HC608.

Biodistribution Studies in Rats

The tissue distribution data of [¹¹C]HC070 in male adult Sprague Dawley (SD) rats was presented in Table 1. Radiotracer uptake at 5, 30 and 60 min was expressed as the percentage of the injected dose per gram (%ID/g) of tissue. For peripheral tissues, high uptake of [¹¹C]HC070 (>1% ID/g) was observed in several organs including liver, kidney, heart, pancreas and small intestine at 5 min. After the initial high uptake of [¹¹C]HC070, the radioactivity in the majority tissues decreased quickly, similar to [¹¹C]HC608 [7].

Table 1.

Biodistribution of [¹¹C]HC070 at 5, 30, and 60 min post injection in SD rats (n = 4)

Tissues	Tracer uptake (%I.D./gram)
	5 min	30 min	60 min
Blood	0.13±0.02	0.12±0.02	0.12±0.01
Heart	0.94±0.12	0.33±0.03	0.20±0.03
Lung	0.72±0.07	0.41±0.07	0.23±0.07
Muscle	0.12±0.01	0.17±0.02	0.13±0.02
Fat	0.07±0.02	0.31±0.09	0.35±0.12
Pancreas	1.33±0.30	0.47±0.01	0.33±0.06
Spleen	0.73±0.17	0.20±0.02	0.13±0.01
Kidney	1.28±0.28	0.46±0.03	0.33±0.05
Thymus	0.47±0.08	0.24±0.02	0.13±0.03
Liver	2.65±0.40	1.70±0.18	1.88±0.13
Small intestine	1.16±0.11	3.01±0.23	3.01±1.71
Brain	0.47±0.10	0.18±0.01	0.09±0.02

Upon intravenous radiotracer administration, [¹¹C]HC070 had a good brain uptake as 0.45 at 5 min, then decreased to 0.17 at 30 min, and 0.10 at 60 min. For [¹¹C]HC608, the brain uptake was 0.51, 0.37 and 0.25 at 5 min, 30 min and 60 min, respectively [7]. The brain uptake ratio for [¹¹C]HC070 was 4.5:1 from 5 min to 60 min while the ratio was 2.0:1 from 5 min to 60 min for [¹¹C]HC608.

In vitro autoradiography study

Encouraged by the good brain uptake of [¹¹C]HC070 in SD rats, we further studied the brain regional distribution of [¹¹C]HC070 and binding specificity in the brain through in vitro autoradiography on SD rat brain slices (Fig. 2). After incubation, brain sections were washed and radioactivity accumulation was visualized using a phosphor imaging system. The autoradiograms showed that [¹¹C]HC070 had the highest accumulation in the TRPC5-enriched colliculus, striatum, cortex, and thalamus regions; these results are similar to those reported in the literature [15–18]. More importantly, the addition of the highly potent and selective TRPC5 inhibitor HC608 at 1 μM significantly reduced binding. Thus, autoradiography indicated that [¹¹C]HC070 specifically binds TRPC5 sites in rat brain sections.

Fig. 2.

Autoradiography study of [¹¹C]HC070 in rat brain sections. The most left panel is the horizontal, coronal, and sagittal section anatomic structure of H&E staining from same sections that are used for autoradiography studies; the middle panel (red color) is the autoradiography signal of [¹¹C]HC070 in brain regions with the dose of 1.11 MBq /slide; the most right panel (light green) is autoradiography signal of the radiotracer [¹¹C]HC070 incubated with HC608 (1 μM) as a TRPC5 blocking agent. Autoradiography study showed that [¹¹C]HC070 had the highest accumulation in the TRPC5-enriched colliculus, striatum, cortex, and thalamus regions. The TRPC5 compound HC608 (1 μM) is able to reduce the radioactivity signal in the tissue slides very well. * hipp-Hippocampus, cereb-Cerebellum, colli-colliculus, striat-striatum, thala-thalamus.

MicroPET imaging of [¹¹C]HC070 in the mouse brain

To determine the in vivo binding specificity of [¹¹C]HC070 for TRPC5, dynamic PET brain imaging was conducted in 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice, respectively. The brain uptake of [¹¹C]HC070 in 129S1/SvImJ wide type mice peaked at ~ 1.0 min with maximum SUV of ~1.13, then washed out rapidly over 60 min (Fig. 3). As a comparison, the brain uptake of [¹¹C]HC070 in 129S1/SvImJ TRPC5 knockout mice reached with a maximum value of 0.63 within 2 min, and then washout from the brain. Notably, the uptake of [¹¹C]HC070 in 129S1/SvImJ TRPC5 knockout mice brain was significant lower from 0 to 60 min.

Fig. 3.

PET brain studies of [¹¹C]HC070 in 129S1/Svlmj control mice and TRPC5 knockout mice. Dynamic PET scans from 0 to 60 min were performed. The brain uptake of [¹¹C]HC070 was significant lower in TRPC5 knockout mice, compared to 129S1/SvImJ normal control mice.

MicroPET brain study of [¹¹C]HC070 in a nonhuman primate

To determine the suitability of [¹¹C]HC070 to be a PET radiotracer for imaging TRPC5 in NHP brain, microPET brain imaging [¹¹C]HC070 in the male cynomolgus macaques was performed. The representative summed images from 0–120 min were co-registered with MRI images to accurately identify the regions of interest (Fig. 4). The time-activity curve (TAC) revealed high initial brain uptake of [¹¹C]HC070, at 3–5 min post-injection, the brain uptake (standardized uptake value, SUV) reached a maximum (~2.2), followed by a fast washout pharmacokinetics from the macaque brain. Compared with [¹¹C]HC608 (maximum SUV ~ 0.9 at 5 min), [¹¹C]HC070 had a on average 2.4 fold higher brain uptake (2.20 at 5 min) at initial time, and also higher uptake after 5 min until 120 min. The tissue time activity curves for different brain regions of [¹¹C]HC070 in nonhuman primate revealed rapid brain uptake and regional heterogeneity, with the highest radioactivity observed in the prefrontal cortex (SUV =2.88).

Fig. 4.

PET study of [¹¹C]HC070 and [¹¹C]HC608 in nonhuman primate. A) Representative images of [¹¹C]HC070 in NHP brain (images showed the sum uptake of a 120 min scan). B) The total brain uptakes (SUV) of [¹¹C]HC070 (n = 4) and [¹¹C]HC608 (n = 2) in male adult cynomolgus macaque. [¹¹C]HC070 had an average of 2.4-fold higher brain uptake at 2.20 at 5 min compared to [¹¹C]HC608 (Data represents mean ± SEM); C) Brain regional uptake of [¹¹C]HC070 in the brain of a nonhuman primate.

Discussion

The radiosynthesis of [¹¹C]HC070 was performed in a similar fashion as for [¹¹C]HC608. Using an HPLC mobile phase to quench and dilute the reaction mixture of [¹¹C]HC070 after methylation led to white precipitate that caused a challenge for loading the reaction mixture onto an HPLC column for subsequent purification. Nevertheless, decreasing the amount of the precursor and employing DMSO/water system to quench the reaction resolved this problem smoothly. In addition, previously, 5% solubilizer and emulsifying agent named Kolliphor HS 15 was required to formulate the final dose of [¹¹C]HC608. While for [¹¹C]HC070, little percentage of retaining was detected inside the surface of a glass vial or a syringe without Kolliphor HS15 addition in dose solution.

The lipophilicity of a potential radiotracer can predict blood−brain barrier (BBB) permeability. For a neuroimaging PET radiotracer, the desirable LogD_7.4 value, an index of molecular lipophilicity ranges from 1.0 to 3.0 [19]. Based on the lipophilicity measurement results, HC070 has lower logD_7.4 value than HC608. The increased hydrophilicity of HC070 correlates well with the molecular structure disparity since chloride is more lipophobic than trifluoromethyl group in the structure of HC608 [20–21]. This may explain the improved solubility of [¹¹C]HC070 compared to [¹¹C]HC608. More importantly in the aqueous dose solution. The higher hydrophilicity of [¹¹C]HC070 enables its better BBB permeability to have higher brain uptake and good brain washout pharmacokinetics.

Based on the ex vivo distribution study in SD rats, [¹¹C]HC070 has the tissue distribution pattern similar to [¹¹C]HC608 [7]. While [¹¹C]HC070 exhibited faster brain washout pharmacokinetics from rats with 5 min/60 min brain uptake ratio of 4.5 for [¹¹C]HC070 (2.0 for [¹¹C]HC608). Moreover, both [¹¹C]HC070 and [¹¹C]HC608 cleared from plasma primarily through hepatobiliary metabolism as the decrease of radioactivity in small intestines parallels increased uptake in liver.

The subsequent in vitro autoradiography binding assays in rat brain tissue further confirmed [¹¹C]HC070 was a favorable radiotracer for imaging TRPC5 in the brain. Similar to [¹¹C]HC608, [¹¹C]HC070 strongly binds to TRPC5-enriched brain regions in rats. The brain tissue uptake of [¹¹C]HC070 was significantly blocked by the addition of HC608 at 1 μM. Consistently, we have previously showed that HC070 could also greatly reduce the rat brain uptake of [¹¹C]HC608 [7]. This result confirmed that both HC608 and HC070 are highly potent and selective for TRPC5 and each radiotracer has binding specificity for TRPC5 in the brain tissues.

Compared with 129S1/SvImJ wild-type (WT) mice, the 129S1/SvImJ TRPC5 knockout mice have a unique phenotype including a reduction in epileptogenesis and excitotoxicity [22], an enhancement in joint inflammation and hyperalgesia [23], etc. We demonstrated the ability of [¹¹C]HC070 to provide TRPC5-specific binding property in 129S1/SvImJ wild-type and TRPC5 knockout mice. Peak brain uptake (SUV, mean ± SD) was 0.66 ± 0.06 in TRPC5 knockout mice, whereas the maximum SUV of 1.13 ± 0.08 was observed in 129S1/SvImJ wild-type mice, indicating [¹¹C]HC070 brain uptake in mice has TRPC5 specificity.

The superiority of [¹¹C]HC070 compared to [¹¹C]HC608 was also confirmed by microPET brain studies in nonhuman primate. [¹¹C]HC070 had remarkably higher initial brain uptake in the macaque. In addition, [¹¹C]HC070 has excellent BBB penetration with a maximum SUV of 2.2 at 3–5 min in the brain, then washed out gradually from the brain.

Taken together, our evaluation efforts suggest that [¹¹C]HC070 is a good PET radiotracer for studying the TRPC5 functions in the brain of a living animal. The optimized radiolabeling approach circumvented precipitation that hampered purification. Radiotracer [¹¹C]HC070 displayed improved solubility in formulated dosing solution reducing the percentage of the residual retaining inside surface of a glass vial or syringe after transfer or injection. Additionally, microPET brain studies in rodents and nonhuman primates confirmed its high brain uptake, as well as accumulating in the brain correlated well the with TRPC5 expression. Future PET studies of [¹¹C]HC070 using animal models of diseases to detect the changes of TRPC5 expression level in binding sites compared to the healthy control animals, and biological quantification of the TRPC5 expression in postmortem brain tissues of patients with TRPC5 related diseases and healthy normal controls will be performed for determining if [¹¹C]HC070 is worth transferring into clinical investigation for human use.

Conclusions

Our preliminary ex vivo biodistribution, in vitro autoradiography and microPET study in 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice suggested that [¹¹C]HC070 has good brain uptake and high binding in TRPC5-enriched brain regions. PET brain imaging study in a nonhuman primate brain demonstrates a significantly higher brain uptake of [¹¹C]HC070 compared to [¹¹C]HC608, as well as a faster washout pharmacokinetics, indicating [¹¹C]HC070 is more favorable to be a TRPC5 radiotracer than [¹¹C]HC608. The minor change of structure between [¹¹C]HC070 and [¹¹C]HC608 significantly improved the solubility and radiopharmaceutical profiles, suggesting further optimization of xanthine analogues has high potential to identify a clinically suitable PET radiotracer for imaging TRPC5 in vivo.