Development of New Positron Emission Tomography Radiotracer for

Nov 2, 2016 - Translational imaging methods such as positron emission tomography (PET) are uniquely suited to visualize targets in inaccessible tissue...
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Development of New Positron Emission Tomography Radiotracer for BET Imaging Changning Wang,* Frederick A. Schroeder, and Jacob M. Hooker Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, United States ABSTRACT: The bromodomain and extraterminal domain (BET) inhibitors have been extensively studied for tumor treatment in the past few years. Recently, BETcontaining proteins have been reported to play a key role in brain functions, such as learning and memory. BET proteins have also been shown to be a potential therapeutic target for substance abuse disorders. Development of a molecular probe for noninvasive imaging will elucidate the distribution and functional roles of BET in the living subject and accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and pilot imaging of a novel BET imaging agent, [11C]MS417. Our imaging results demonstrate that this probe has moderate brain uptake, good specificity, good selectivity, and appropriate kinetics and distribution. [11C]MS417 is an ideal lead compound for further optimization of clinical BET PET radiotracer tools and MS417 could be used as a blood-brain-barrier-penetrant compound for preclinical research. KEYWORDS: Bromodomain, PET, radiotracer, [11C]MS417

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in neuronal function and mediates the transcriptional regulation underlying learning and memory.21 Bioluminescent probes have been demonstrated to measure epigenetic enzyme activity, including for bromodomain-containing chromatin modifying enzymes;22 however, there are no tools to investigate BET expression and impact in the brain noninvasively. Translational imaging methods such as positron emission tomography (PET) are uniquely suited to visualize targets in inaccessible tissues. With an in vivo BET neuroimaging tool, it will be possible to evaluate BET across multiple regions of the brain and other organs in the living subjects or to understand the impact of BET expression on functional interactions. Herein, we report a novel PET radiotracer ([11C]MS417), a carbon-11-labeled version of the highly potent BET inhibitor (MS41723,24), and demonstrate its pharmacokinetic properties in pilot imaging studies. Future studies will focus on optimization of the PET imaging probe and characterization of BET expression in rodents.

pigenetics refers to functional modifications to the genome that do not involve a change in the DNA sequence. The epigenetic enzymes responsible for the addition of histone posttranslational modifications are collectively known as “writers”, of which the most studied are the histone acetyltransferases (HATs), histone methyltransferases (HMTs), and kinases. Enzymes that remove these marks are called “erasers” and include histone deacetylases (HDACs), demethylases, and phosphatases. By binding to acetylated lysine residues on histone tails, bromodomain containing proteins (BCPs) function as epigenetic “readers” and play a key role in epigenetic regulation of gene transcription.1,2 Bromodomains are highly conserved protein modules, comprised of approximately 110 amino acids.3,4 There are at least 56 human bromodomains in 42 BCPs identified to date.5−7 A subset of BCPs, the bromodomain and extraterminal domain (BET) proteins, contain two bromodomains each (Nterminal bromodomain (BD1) and C-terminal bromodomain (BD2)): thus BETs can each engage with two acetylated histones. Four BET members are found in humans: BRD4, which acts as a mitotic bookmark and transcription facilitator; BRD3, which binds to acetylated GATA1; BRD2, a regulator of body energy balance; and BRDT, which is expressed selectively in the testis. Among them, BRD4 is the best-studied member of the BET family.2 There are several BET inhibitors that have recently been reported, such as JQ-1,8 I-BET-762,9−11 I-BET-15112−14 and OTX015.15,16 They provide powerful tools to study the roles of BET proteins in different cellular processes and the therapeutic potential of BET inhibition, particularly in tumors.17−20 Recently, more research has been focused on the role of BET in brain disorders. BRD4 has been reported to have a key role © XXXX American Chemical Society

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RESULTS AND DISCUSSION

Physiochemical Properties of MS417. MS417 is a thienodiazepine-based compounds that was first reported for their binding activity for BRD4 by the Mitsubishi Tanabe Pharma Corporation.24 MS417, which has a methyl ester instead of a tert-butyl ester moiety, is structurally almost identical to the prototypical BET inhibitor, (+)-JQ1 (Figure 1). Received: September 2, 2016 Accepted: November 2, 2016 Published: November 2, 2016 A

DOI: 10.1021/acschemneuro.6b00288 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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min scan when administered by intravenous bolus injection (0.9−1.1 mCi per animal), as shown in Figure 2.

We recognized the methyl ester was recognized as ideal radiolabeling position for introducing carbon-11 isotope.

Figure 1. (+)-JQ1 behaves as a highly selective inhibitor of BRD4 (IC50 = 77 and 33 nM for BRD4(1/2), respectively). Its analogue, MS417, has high bind affinity toward BRD (Kd = 25.4−36.1 nM).

Figure 2. Rodent in vivo PET imaging with [11C]MS417 reveals blockable uptake in brain. [11C]MS417 shows blockade after pretreatment (5 min, i.v.) with MS417 at 1.0 mg/kg.

The reported crystal structure of the BRD4-BD1·MS417 reveals that MS417 is embedded in the acetyl-lysine binding pocket, forming a hydrogen bond between the triazole ring and the conserved Asn-140 in BRD4-BD1.23 Similar to (+)-JQ1, MS417 is not in contact with Asp-144, which is unique in BRD4-BD1. Therefore, MS417 binds BRD4-BD1 and BRD4BD2 with similar affinity of Kd = 36.1 ± 7.8 and 25.4 ± 3.4 nM, respectively. 23 MS417 is highly specific for the BET bromodomains, with weaker binding to bromodomains within other BCPs including CBP, BRD7, or BPTF and no binding at all to other BCPs including BAZ1B, BAZ2B, ATAD2, ASH1L, SMARCA4, PCAF, PHIP-1, TAF1L, and TAF1.23 Synthesis of the Precursor of MS417 and the PET Imaging Agent, [11C]MS417. The preparation of the precursor of MS417 was straightforward (Scheme 1). The tert-butyl ester in MS417 was hydrolyzed in the presence of sodium hydroxide to form the precursor. [11C]MS417 was labeled with [11C]CH3I (Scheme 1). Cyclotron-produced [11C]CH3I was trapped into a sealed vial with precursor in DMSO solution in the presence of potassium carbonate followed by heating at 80 °C for 3 min. The reaction was then quenched with water and purified by semipreparative HPLC. Including formulation, [11C]MS417 was prepared in 35−40 min after end of bombardment with adequate radiochemical yields (4−15%, uncorrected for decay and based on trapped [11C]CH3I) and high radiochemical purity (>95%). In Vivo PET-CT Imaging with [11C]MS417 in Rodents. To test [11C]MS417 as a radiotracer in vivo, we conducted PET imaging focused on the rat brain. Using PET-CT, we determined that [11C]MS417 exhibited moderate blood-brain barrier (BBB) penetration and slow signal decrease over the 60

To investigate the specificity of [11C]MS417, we performed PET imaging studies in rats with 5 min i.v. pretreatment of unlabeled MS417 at 1.0 mg/kg. We found that administration of unlabeled MS417 blocked [11C]MS417 binding in brain by approximately 20%, measured as the percent change in wholebrain radioactivity between peak uptake at 2 min and the lowest uptake at 60 min (Figure 2). Application of [11C]MS417 as a PET probe can be used to quantify BET density in cancer and brain disorders, such as neurodegenerative diseases and mood disorders and, via blocked binding signal in the brain, for demonstrating target engagement of BRD4 inhibitor therapeutics. In Vivo PET-MR Imaging with [11C]MS417 in Baboon. In baboons, we found moderate brain uptake of [11C]MS417 based on PET-MR focused on the head. Similar to the blocking results we obtained in rats, injection of unlabeled MS417 (0.5 mg/kg, i.v.) changed tracer kinetics in specific brain regions including cerebellum, cortex and thalamus (Figure 3). Physiological effects (i.e., respiration rate, blood pressure, heart rate and end-tidal CO2) were unchanged at the administered dose of MS417 or [11C]MS417.

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CONCLUSION In summary, [11C]MS417 binds to BET with selectivity and specificity in rats and baboons and provides a preclinical tool for quantitative imaging of BET density in vivo. [11C]MS417 can be used as a PET radioligand for studying BET expression in disorders, and would also valuable for evaluation of potential drugs in vivo. The further optimization to improve the brain uptake is ongoing.

Scheme 1. Precursor for Radiolabeling Was Prepared from (+)-JQ1a

Radiolabeling condition: precursor (1 mg), [11C]CH3, K2CO3 (5 mg), in 300 μL DMSO, 3 min, 80 °C. Radiochemical yield (RCY): 4-15% (nondecay corrected to trapped [11C]CH3I).

a

B

DOI: 10.1021/acschemneuro.6b00288 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 3. Preliminary data. NHP in vivo PET imaging with [11C]MS417 reveals blockable uptake in brain. (A) Summed PET-MR images (0−30 min) following injection with [11C]MS417 at baseline. (B) [11C]MS417 shows blockade in whole brain (WB) and brain regions such as cortex (CTX), thalamus (THA), and cerebellum (CB) after pretreatment with MS417 at 0.5 mg/kg (5 min, i.v.).

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mixture was stirred at 60 °C for 8 h, cooled to room temperature. Acidification with 1 M HCl to pH 7−8 (pH paper). The product was extracted with ethyl acetate (10 mL x 3), the organic layer was the combined and washed with brine (20 mL), dried (Na2SO4), filtered, and the solvent was removed under vacuum to obtain precursor (0.014 g, 81%) as white solid. LC-MS calculated for C19H17ClN4O2S expected [M]: 400.0; Found [M-H]+: 400.9. Radiosynthesis of [11C]MS417. [11C]CH3I was trapped in a TRACERlab FX-M synthesizer reactor (General Electric) preloaded with a solution of precursor (1.0 mg) in dry DMSO (300 μL). The solution was stirred at 80 °C for 3 min and water (1.2 mL) was added. The reaction mixture was purified by reverse phase semipreparative HPLC (Phenomenex Luna 5u C8(2), 250 mm × 10 mm, 5 μm, 5.0 mL/min, 35% H2O + 0.1% TFA/65% CH3CN + 0.1% TFA, 254 nm) and the desired fraction was collected. The final product was reformulated by loading onto a solid-phase exchange (SPE) C-18 cartridge, rinsing with water (5 mL), eluting with EtOH (1 mL), and diluting with saline (0.9%, 9 mL). The chemical and radiochemical purity of the final product was tested by analytical HPLC (Agilent Eclipse XDB-C18, 150 mm × 4.6 mm). The identity of the product was confirmed by analytical HPLC with additional coinjection of MS417 reference standard. The average time required for the synthesis from end of cyclotron bombardment to end of synthesis was 35−40 min. The average radiochemical yield was 4−15% (nondecay corrected to trapped [11C]CH3I). Chemical and radiochemical purities were ≥95%. Specific activity of [11C]MS417 (at time of injection): 0.7−1.3 mCi/nmol. Rodent PET-CT Acquisition and Post Processing. Male Sprague−Dawley rats were utilized in pairs, anesthetized with inhalational isoflurane (Forane) at 3% in a carrier of 2 L/min medical oxygen, and maintained at 2% isoflurane for the duration of the scan. The rats were arranged head-to-head in a Triumph Trimodality PET/ CT/SPECT scanner (Gamma Medica, Northridge, CA). Rats were injected standard references or vehicle via a lateral tail vein catheterization at the start of PET acquisition. Dynamic PET acquisition lasted for 60 min and was followed by computed tomography (CT) for anatomic coregistration. PET data were reconstructed using a 3D-MLEM method resulting in a full width at half-maximum resolution of 1 mm. Reconstructed images were exported from the scanner in DICOM format along with an anatomic CT for rodent studies. These files were imported to PMOD (PMOD

METHODS

All reagents and solvents were of ACS-grade purity or higher and used without further purification. Analytical separation was conducted on an Agilent 1100 series HPLC fitted with a diode-array detector, quaternary pump, vacuum degasser, and autosampler. Mass spectrometry data were recorded on an Agilent 6310 ion trap mass spectrometer (ESI source) connected to an Agilent 1200 series HPLC with quaternary pump, vacuum degasser, diode-array detector, and autosampler. [11C]CO2 (1.2 Ci) was obtained via the 14N (p, α) 11 C reaction on nitrogen with 2.5% oxygen, with 11 MeV protons (Siemens Eclipse cyclotron), and trapped on molecular sieves in a TRACERlab FX-MeI synthesizer (General Electric). [11C]CH4 was obtained by the reduction of [11C]CO2 in the presence of Ni/ hydrogen at 350 °C and recirculated through an oven containing I2 to produce 11CH3I via a radical reaction. All animal studies were carried out at Massachusetts General Hospital (PHS Assurance of Compliance No. A3596-01). The Subcommittee on Research Animal Care (SRAC) serves as the Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital (MGH). SRAC reviewed and approved all procedures detailed in this paper. PET-CT imaging was performed in anesthetized (isoflurane) rats (Sprague−Dawley) to minimize discomfort. Highly trained animal technicians monitored animal safety throughout all procedures, and veterinary staff were responsible for daily care. All rats were socially housed in cages appropriate for the physical and behavioral health of the individual animal and were given unlimited access to food and water, with additional nutritional supplements provided as prescribed by the attending veterinary staff. PET-MR imaging was performed in anesthetized (ketamine, isoflurane) baboon (Papio anubis) to minimize discomfort. Highly trained animal technicians monitored animal safety throughout all procedures, and veterinary staff were responsible for daily care. All animals were socially housed in cages appropriate for the physical and behavioral health of the individual animal and were fed thrice per diem, with additional nutritional supplements provided as prescribed by the attending veterinarian. Audio, video, and tactile enrichment was provided on a daily basis to promote psychological well-being. No nonhuman primates were euthanized to accomplish the research presented. Chemical Synthesis. To a solution of (+)-JQ1 (0.02 g, 0.044 mmol) in MeOH (2 mL) was added 1 M NaOH (10 mL). The C

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ACS Chemical Neuroscience Technologies, Ltd.) and manually coregistered using six degrees of freedom. Rodent PET-CT Image Analysis. Volumes of interest (VOIs) were drawn manually as spheres in brain regions guided by high resolution CT structural images and summed PET data, with a radius no less than 1 mm to minimize partial volume effects. Time−activity curves (TACs) were exported in terms of decay corrected activity per unit volume at specified time points with gradually increasing intervals. The TACs were expressed as percent injected dose per unit volume for analysis. Baboon PET-MR Acquisition. Papio Anubis baboons, deprived of food for 12 h prior to the study, were included in the PET-MR scans. Atropine 0.05 mg/kg was used intramuscularly to prevent excessive secretion (15 or 30 min of ketamine and xylazine). Anesthesia was induced with intramuscular xylazene (0.5−2.0 mg/kg) and ketamine (10 mg/kg). After endotracheal intubation and insertion of V-line and A-line, anesthesia was maintained using isoflurane (1−1.5%, 100%O2 or 50/50 O2/N2O, l L/min). During anesthesia, we monitored heart rate, RR, O2sat, BP, and ETCO2. The baboon was catheterized antecubitally for radiotracer injection, and a radial arterial line was placed for metabolite analysis. PET-MR images were acquired in a Biograph mMR scanner (Siemens, Munich, Germany), and PET compatible 8-channel coil arrays for nonhuman primate brain imaging with a PET resolution of 5 mm and field of view of 59.4 and 25.8 cm (transaxial and axial, respectively). Dynamic PET image acquisition was initiated followed by administration of the radiotracer in a homogeneous solution of 10% ethanol and 90% isotonic saline. An MEMPRAGE sequence began after 30 min of the baseline scan for anatomic coregistration. To characterize the specific binding of [11C]MS417, a second imaging experiment was carried out in which unlabeled MS417 was administered intravenously 5 min prior the acquisition. Both scans were carried out in the same animal on the same day, separated by 2.5 h. In both scans, 4−5 mCi of [11C]MS417 was administered to the baboon. Dynamic data from the PET scans were recorded in list mode and corrected for attenuation. Baboon data were reconstructed using a 3D-OSEM method resulting in a full width at half-maximum resolution of 4 mm. Baboon PET-MR Image Analysis. The analysis was carried out in PMOD (PMOD3.3, PMOD Technologies Ltd., Zurich, Switzerland). VOIs were drawn manually as spheres in brain regions guided by highresolution MPRAGE MRI image. TACs were exported from the cerebellum, cortex, and thalamus VOIs for analysis.

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by the NIH Shared Instrumentation Grant Program and/or High-End Instrumentation Grant Program; specifically, Grant Numbers S10RR017208, S10RR026666, S10RR022976, S10RR019933, and S10RR029495. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Martinos Center radiopharmacy and imaging staff (Helen Deng, Judit Sore, Kari Phan, Garima Gautam, Samantha To, Grae Arabasz, and Shirley Hsu) for help with nonhuman primate experiments. The authors are grateful to Dr. Aijun Zhu for assisting the rodent PET-CT study.

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ABBREVIATIONS BRD4, bromodomain-containing 4; BET, bromodomain and extraterminal domain; BCPs, bromodomain containing proteins; MRI, magnetic resonance imaging; PET, positron emission tomography; CT, computed tomography; EOS, end of synthesis; RCY, radiochemical yield; HAT, histone acetyltransferase; HMT, histone methyltransferase; HDAC, histone deacetylase; PET, positron emission tomography; BCP, bromodomain containing protein

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, CNY 149, Room 5.022A, 149 13th Street, Charlestown, MA 02179. Phone: 617724-9390. E-mail: [email protected]. Author Contributions

C.W. discovered, synthesized, and purified [11C]MS417. C.W. and J.M.H. planned experiments and performed data analysis. C.W. and F.A.S. performed the experiments. The manuscript was written through contributions of all the authors. All authors have given approval to the final version of the manuscript. Funding

This research was supported by the Harvard/MGH Nuclear Medicine Training Program from the Department of Energy under Grant DE-SC0008430 (to C.W.). This research was carried out at the Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided by the Center for Functional Neuroimaging Technologies, P41EB015896, a P41 Regional Resource supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health. This work also involved the use of instrumentation supported D

DOI: 10.1021/acschemneuro.6b00288 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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