Comparative Analysis of Folate Derived PET Imaging Agents with

(19-28) Overexpressed in epithelial-derived cancers, FR-α is found in high levels in lung, ovary, kidney, breast, myelogenous, and brain malignancies...
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Comparative Analysis of Folate Derived PET Imaging Agents with [18F]-2-Fluoro-2-deoxy‑D‑glucose Using a Rodent Inflammatory Paw Model Sumith A. Kularatne,† Marie-José Bélanger,‡ Xiangjun Meng,‡ Brett M. Connolly,‡ Amy Vanko,‡ Donna L. Suresch,‡ Ilonka Guenther,‡ Shubing Wang,§ Philip S. Low,† Paul McQuade,‡ and Dinko González Trotter*,‡ †

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States Imaging Department, Merck Research Laboratories, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States § Biometrics Research Department, Merck Research Laboratories, Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States ‡

S Supporting Information *

ABSTRACT: Activated macrophages play a significant role in initiation and progression of inflammatory diseases and may serve as the basis for the development of targeted diagnostic methods for imaging sites of inflammation. Folate receptor beta (FR-β) is differentially expressed on activated macrophages associated with inflammatory disease states yet is absent in either quiescent or resting macrophages. Because folate binds with high affinity to FR-β, development of folate directed imaging agents has proceeded rapidly in the past decade. However, reports of PET based imaging agents for use in inflammatory conditions remain limited. To investigate whether FR-β expressing macrophages could be exploited for PET based inflammatory imaging, two separate folate-targeted PET imaging agents were developed, 4-[18F]-fluorophenylfolate and [68Ga]-DOTA-folate, and their ability to target activated macrophages were examined in a rodent inflammatory paw model. We further compared inflamed tissue uptake with 2-[18F]fluoro-2-deoxy-D-glucose ([18F]-FDG). microPET analysis demonstrated that both folate-targeted PET tracers had higher uptake in the inflamed paw compared to the control paw. When these radiotracers were compared to [18F]-FDG, both folate PET tracers had a higher signal-to-noise ratio (SNR) than [18F]-FDG, suggesting that folate tracers may be superior to [18F]-FDG in detecting diseases with an inflammatory component. Moreover, both folate-PET imaging agents also bind to FR-α which is overexpressed on multiple human cancers. Therefore, these folate derived PET tracers may also find use for localizing and staging FR+ cancers, monitoring response to therapy, and for selecting patients for tandem folate-targeted therapies. KEYWORDS: PET imaging, [18F]-FDG, folate-PET, activated macrophages, inflammatory diseases

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INTRODUCTION While activated macrophages mainly serve to fight against foreign pathogens,1 they can also play a vital role in inflammatory diseases such as rheumatoid arthritis (RA),2 atherosclerosis,3 systemic lupus erythematosus (SLE),4 irritable bowel disease,5 psoriasis,6 and diabetes.7 Both genetic and pharmacological studies demonstrate that the release of cytokines (IL-1, IL-6, TNF-α), chemokines (MCP-1), metalloproteinase, and reactive oxygen species (ROS) by the activated macrophages constitutes the main process through which the pro-inflammatory phenotype is maintained.8 Moreover, release of chemokines by activated macrophages can cause improper recruitment of other immune cells, such as activated T cells, that can further worsen the disease state.9 While the © 2013 American Chemical Society

initial cause of the majority of inflammatory diseases remains unknown, they affect over 10 million people in the US every year. Therefore, early diagnosis and treatment of the disease is critical in minimizing chronic, progressive, and debilitating systemic effects. Early diagnosis of inflammatory diseases requires a method with enough sensitivity to detect and monitor inflamed tissues. Although conventional radiography is considered the accepted diagnostic modality for diseases such as RA, it may detect later Received: Revised: Accepted: Published: 3103

March 19, 2013 June 26, 2013 July 2, 2013 July 2, 2013 dx.doi.org/10.1021/mp4001684 | Mol. Pharmaceutics 2013, 10, 3103−3111

Molecular Pharmaceutics

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mL) followed by dimethyl formamide (DMF, 3 mL). A solution of 20% piperidine in DMF (3 × 3 mL) was added to the resin, and argon was bubbled for 5 min. The resin was washed with DMF (3 × 3 mL) and isopropyl alcohol (i-PrOH, 3 × 3 mL). After swelling the resin in DMF, a solution of p-FPh-COOH (1.5 equiv), HATU (1.5 equiv), and DIPEA (2.0 equiv) in DMF was added. Argon was bubbled for 2 h, and resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin in DCM, a solution of 1 M HOBt in DCM/trifluoroethane (TFE) (1:1) (2 × 3 mL) was added. Argon was bubbled for 1 h, the solvent was removed, and resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin in DMF, a solution of fluorenylmethoxycarbonyl (Fmoc)-PEG6-OH (1.5 equiv), HATU (1.5 equiv), and DIPEA (2.0 equiv) in DMF was added. Argon was bubbled for 2 h, and resin was washed with DMF (3 × 3 mL) and iPrOH (3 × 3 mL). The above sequence was repeated for two more coupling steps for conjugation of Fmoc-Glu-(OtBu)-OH and N10-TFA-Ptc-OH. The final product was cleaved from the resin using a trifluoroacetic acid (TFA):H2O:triisopropylsilane cocktail (95:2.5:2.5) and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum, then purified using preparative RP-HPLC [solvent gradient: 1% B to 50% B in 25 min; A = 10 mM NH4OAc, pH = 7; B = CH3CN]. Acetonitrile removed under vacuum, then freeze-dried to yield 4 as a yellow solid (32.3 mg, 61%). RP-HPLC: Rt = 8.5 min (A = 10 mM NH4OAc, pH = 7.0; B = CH3CN, solvent gradient: 1% B to 50% B in 10 min, 80% B wash 15 min run); 1H NMR (DMSO-d6/D2O) δ 1.88 (m, 1H); 2.03 (m, 1H); 2.15 (t, J = 7.4, 2H); 2.28 (t, J = 6.4, 2H); 3.05 (m, 4H); 3.20−3.60 (m, x); 4.23 (m, 1H); 4.48 (s, 2H); 6.64 (d, J = 8.8 Hz, 2H); 7.28 (t, J = 8.9 Hz, 2H); 7.64 (d, J = 8.8 Hz, 2H); 7.90 (t, J = 8.9 Hz, 2H); 8.63 (s,1H); LC/MS (ESI) (m/z): (M + H)+ calcd. for C43H58FN10O13, 941.37, found, 941.40; UV/vis: λmax = 280 nm. Folate-PEG6-EDA (5). Folate linker was synthesized similar to compound 4 using solid phase peptide synthesis using 1,2diaminoethane trityl resin. Yellow solid (82.6 mg, 86%). Analytical RP-HPLC: Rt = 8.5 min (A = 10 mM NH4OAc, pH = 7.0; B = CH3CN, solvent gradient: 1% B to 50% B in 10 min, 80% B wash 15 min run); 1H NMR (DMSO-d6/D2O) δ 1.88 (m, 1H); 2.03 (m, 1H); 2.15 (t, J = 7.4, 2H); 2.28 (t, J = 6.4, 2H); 3.05 (m, 4H); 3.20−3.60 (m, x); 4.23 (m, 1H); 4.48 (s, 2H); 6.64 (d, J = 8.8 Hz, 2H); 7.64 (d, J = 8.8 Hz, 2H); 8.63 (s, 1H); LC/MS (ESI) (m/z): (M + H)+ calcd. for C36H55N10O12, 819.37, found, 819.18; UV/vis: λmax = 280 nm. DOTA-Folate (6). To a solution of DOTA-NHS (3.3 mg, 6.7 μmol) and folate-PEG-EDA (5) linker (5 mg, 6.1 μmol) in DMSO, DIPEA was added. The reaction was stirred at room temperature for overnight and purified using RP-HPLC. Yellow solid (6.8 mg, 93%). Analytical RP-HPLC: Rt = 7.7 min (A = 0.1% TFA, pH = 2.0; B = CH3CN, solvent gradient: 1% B to 50% B in 10 min); 1H NMR (DMSO-d6/D2O) δ 1.88 (m, 1H); 2.03 (m, 1H); 2.15 (t, J = 7.4, 2H); 2.28 (t, J = 6.4, 2H); 3.05 (m, 4H); 3.20−3.60 (m, x); 4.23 (m, 1H); 4.48 (s, 2H); 6.64 (d, J = 8.8 Hz, 2H); 7.64 (d, J = 8.8 Hz, 2H); 8.63 (s, 1H); HRMS (ESI) (m/z): (M + H)+ calcd. for C53H82N13O19, 1205.37, found, 1205.40; UV/vis: λmax = 280 nm. Radiochemical Synthesis of 4-[18F]-Fluorophenylfolate (7). N-Succinimidyl 4-[18F]-fluorobenzoate was synthesized as reported in the literature.37 To a solution of N-succinimidyl 4-[18F]-fluorobenzoate in CH3CN (200 μL) 0.1 M borate buffer (1.5 mL) containing folate-PEG6-EDA (0.4 mg, 0.9

stages of the disease, such as joint space narrowing and bone erosions, whereas it may not detect synovitis and bone marrow edema.10−13 While magnetic resonance imaging (MRI) offers an assessment of the rheumatoid synovium with improved sensitivity, it may also be expensive for routine screening, limited to one joint region at each session, and time-consuming. In contrast, ultrasonography technology is readily accessible in the clinic, but it cannot play a role in the evaluation of pathology below the bone surface.10−13 Motivated by the above limitations, much effort has been devoted to develop morphological, functional, and molecular imaging procedures such as single photon emission computed tomography (SPECT), positron emission tomography (PET), PET-CT, and PET-MRI.14−17 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]FDG) is currently in use in the clinic for PET imaging to diagnose inflamed sites.17 However, [18F]-FDG is not a specific marker for inflammatory diseases, and it only detects increases in glucose metabolism, which could be elevated due to a number of other reasons.17 Therefore, development of an imaging agent that would be able to specifically detect inflammation in its earliest stages would be beneficial. Based on the limited distribution of FR in normal tissues tied with higher receptor expression levels on various disease cells, folic acid (FA) remains an attractive and high affinity ligand for the selective delivery of therapeutic and imaging agents to FR+ cancer cells and activated macrophages.18 To date, four isoforms of FR have been identified (FR-α, FR-β, FR-γ, and FR-δ); however, only FR-α and FR-β are expressed in adequate numbers for use in diagnostic and therapeutic applications.19−28 Overexpressed in epithelial-derived cancers, FR-α is found in high levels in lung, ovary, kidney, breast, myelogenous, and brain malignancies.19−23 In contrast, FR-β is expressed on activated macrophages associated with inflammatory disease states, but not on quiescent or resting macrophages.19,24−26 Importantly, most cells accumulate their required FA (vitamin B9) via a reduced folate carrier or proton coupled folate transporter, which is unable to transport folate conjugates.19,29,30 Owing to this selectivity to cells overexpressing FR receptor, folate-targeted 99mTc and 111In imaging agents have been developed as potential SPECT imaging agents for the detection of FR+ cancers and inflammatory conditions in the clinic.31−34 Although SPECT isotopes 99mTc and 111In radioisotopes could be accessed easily when compared to PET isotopes such as 18F and 64Cu, PET imaging can offer several advantages over SPECT scans including higher sensitivity and resolution in a clinical setting.35 Therefore, a folate-targeted PET imaging agent may be ideal for detection of inflammatory diseases via selective uptake by activated macrophages. Herein, we report the design and synthesis of folate-targeted [18F] and [68Ga] PET imaging agents and their uptake in a rodent hind paw inflammation model in comparison to [18F]-FDG.

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EXPERIMENTAL SECTION Synthesis and Characterizations. N10-TFA-Pteroic Acid (Ptc, 3). N10-TFA-Pteroic acid was synthesized according to a literature protocol.36 Pale yellow solid (12.79 g, 98%). RPHPLC: Rt = 12.8 min (A = 10 mM NH4OAc, pH = 7.0; B = CH3CN, solvent gradient: 1% B to 50% B in 30 min); 1H NMR (DMSO-d6/D2O) δ 4.48 (s, 2H); 6.64 (d, J = 8.8 Hz, 2H); 7.64 (d, J = 8.8 Hz, 2H); 8.63 (s, 1H); EI-HRMS (m/z): (M + H)+ calcd. for C16H12F3N6O4, 409.29; found, 409.28. 4-Fluorophenylfolate (4). Universal NovaTag resin (100 mg, 0.53 mM) was swollen with dichloromethane (DCM, 3 3104

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× 256 × 95 voxels, 0.90 × 0.90 × 0.80 mm3, no attenuation and no scatter correction). For each hind paw, a region of interest (ROI) was drawn on the summed [18F]-FDG image using PMOD 2.95 (PMOD Technologies, Adliswil, Switzerland) and standardized uptake value (SUV) calculated. [18F]-FDG uptake was corrected for folate contamination that was determined by fitting a monoexponential decay to the last 2 pts of the nondecay corrected folate tracers and extrapolating this fit to the [18F]FDG scan times. To measure the ability of a tracer to detect changes, a sensitivity measurement is defined as the difference of the SUV between inflamed (IP) and normal paws for each rat divided by the within-tracer standard deviation of SUVIPSUPNP; i.e.,

μmol) was added. The resulting solution was stirred at room temperature for 30 min. 4-[18F]-Fluorophenylfolate was purified using a Gemini RP HPLC column (flow rate = 5 mL/min, 30%B − 80%B in 15 min, A = 0.5% ammonium formate and B = CH3CN). After purification, 4-[18F]fluorophenylfolate was loaded onto a C18 Sep-pak cartridge and eluted with ethanol (1 mL), diluted to the desired volume with sterile water. Radiochemical purity was determined by injection on an analytical HPLC (Waters 2795) system equipped with a Waters 996 UV detector and β-RAM model 4 Radio-HPLC detector (IN/US Systems, Brandon FL) at a flow rate of 1 mL/min using a Gemini RP HPLC column (150 × 4.6 mm I.D.; flow rate = 1 mL/min, 30%B − 80%B in 15 min, A = 0.1% formic acid and B = CH3CN). Yellow solid (0.5 mg, 68%). Radiochemical Synthesis of [68Ga]-DOTA-Folate (8). Radiolabeling and purification was performed on an automated Modular-Lab system (Eckhart & Ziegler Eurotope GmbH) configured for 68Ga peptide labeling. 68Ga was obtained in the form of 68GaCl3 from a 68Ge/68Ga generator (Eckhart & Ziegler Eurotope GmbH) by elution with 0.1 M HCl (2 mL/ min), with 68GaCl3 being trapped on a Ag 1-X8 anion exchange resin (BioRad, Hercules, CA). The resin was washed with a 1 mL solution containing 1.58% HCl (30%, ultrapure), 80.0% acetone, and 18.42% milli-Q H2O to remove any metal impurities. 68Ga was removed from the resin via a 400 μL solution containing 0.53% HCl (30%, ultrapure), 97.56% acetone, and 1.91% milli-Q H2O and transferred to a reaction vessel containing 75 μg of DOTA-folate and 5 mL of H2O. The reaction mixture was heated to 80 °C for 5 min. [68Ga]-DOTAfolate was purified via a C18 light Sep-Pak (Waters Corp., Milford, MA) that had been preconditioned with EtOH (3 mL) followed by Milli-Q H2O (3 mL). After loading onto the SepPak, unreacted [68Ga]GaCl3 was removed by washing with Milli-Q H2O (5 mL), followed by elution of the [68Ga]-DOTAfolate via a 1 mL 70% EtOH solution. This was then diluted with 5 mL of PBS. Radiochemical purity was determined by injection on an analytical Waters 2795 HPLC system equipped with a Waters 996 UV detector and β-RAM model 4 RadioHPLC detector (IN/US Systems, Brandon FL) at a flow rate of 1 mL/min using a Gemini RP HPLC column (150 × 4.6 mm I.D.; flow rate = 1 mL/min, 30%B − 80%B in 15 min, A = 0.1% formic acid and B = CH3CN). Induction of Inflammatory Model. All of the procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Merck, West Point. Sprague− Dawley (SD) rats were purchased from Taconic (NY, USA), maintained on rodent chow, and housed in a standard 12 h light−dark cycle for the duration of the study. SD rats were injected subcutaneously with 0.1 mL of Complete Freund’s Adjuvant (CFA, 1 mg/mL) in one hind paw and monitored for 72 h. microPET Imaging. At 72 h after administering CFA, animals were anesthetized with 4% isoflurane and maintained on 1−3% isoflurane throughout the PET imaging session. The rats were administered either 4-[18F]-fluorophenylfolate (∼9.9 ± 0.1 MBq, n = 3) or [68Ga]-DOTA-folate (∼10.9 ± 2.0 MBq, n = 4) via tail vein and imaged for 60 min using a small-animal PET system (Focus220, Siemens Medical Solution, Hoffman Estate, IL). After 60 min the rats were then injected with [18F]FDG (∼28.6 ± 1.6 MBq, n = 7) via tail vein and imaged for an additional 90 min. All microPET scans were reconstructed using maximum a posteriori reconstruction (MAP, β = 0.2, 256

sensitivity = (SUVIP − SUVNP)/σIP ‐ NP

The within-tracer signal-to-noise ratio (SNR) is the mean of the sensitivities. A paired t test was applied to compare the mean differences in sensitivities between folate and [18F]-FDG, to determine which tracer showed statistically greater inflammatory signal compared to its inflammatory signal variance. Autoradiography and Immunostaining. At 72 h after CFA had been administered, a single SD rat was euthanized by carbon dioxide inhalation 30 min after receiving [18F]-FDG and soft tissue from both inflamed and normal paw footpads was collected and immediately frozen on dry ice. Ten μm thick sections were mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh PA). The slides were exposed to a phosphor image plate for 18 h and scanned on a Fuji BAS-5000 phosphor imager. Ex vivo autoradiographic images were generated using MCID Analysis software. After radioactive decay, the scanned slides were fixed in icecold acetone/ethanol (3:1 v/v) and washed in PBS-Tween 20 (PBST). Endogenous peroxidase activity was blocked followed by blocking with 2.0% goat serum/0.1% Triton-X 100/0.5% Tween 20/0.1% cold fish skin gelatin/1.0% BSA/0.05% sodium azide/0.01 M PBS, pH 7.2. The slides were incubated with mouse anti-rat CD68 (MCA341; Serotec, Raleigh NC) for 1 h at room temperature in a humid chamber and washed in PBST. After incubation with HRP-conjugated goat anti-mouse IgG polymer (Envision+; Dako, Carpenteria CA), slides were washed in PBST, and bound antibody was visualized with 3′,3-diaminobenzidiene, yielding a brown reaction product. Finally, the slides were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene, and coverslipped. Low-magnification digital images (1×) were captured using an Aperio Scanscope (Aperio; Vista CA), and high-magnification images were captured with a Nikon E-1000 microscope equipped with a Nikon DXM1200 digital camera.

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RESULTS Given the overexpression of FR-β in activated macrophages, which play a pivotal role in inflammatory conditions, we designed and synthesized folate-targeted [18F] and [68Ga] PET imaging agents to monitor adjuvant induced inflammation in a SD rat model. To minimize possible steric effects that could impede binding of the agent to FR, a polar and uncharged PEG linker was incorporated between folate and prosthetic group. Synthesis of Folate-Targeted PET Imaging Agents. Chemistry. To synthesize a regioselective derivative of the FA, synthesis of N10-protected pteroic acid (3) is required (Scheme 3105

dx.doi.org/10.1021/mp4001684 | Mol. Pharmaceutics 2013, 10, 3103−3111

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Scheme 1. Synthesis of N10-TFA Protected Pteroic Acida

a

Reagent and conditions: (a) ZnCl2, CPG/tris base, 30 °C; (b) (i) TFAA, (ii) 3% TFA.

Scheme 2. Synthesis of 4-Fluorophenylfolatea

a

Reagent and conditions: (a) (i) 20% piperidine/DMF, rt, 10 min; (ii) p-F-C6H4-COOH, HATU, DIPEA/DMF, 2 h; (b) (i) 1M HOBt in DCM/ TFE (1:1), 1 h; (ii) Fmoc-PEG6-COOH, HATU, DIPEA/DMF, 2 h; (c) (i) 20% piperidine/DMF, rt, 10 min; (ii) Fmoc-Glu-OH, HATU, DIPEA/ DMF, 2 h; (d) (i) 20% piperidine/DMF, rt, 10 min; (ii) N10-TFA-Ptc, HATU, DIPEA/DMF, 2 h; (e) (i) 2% NH2NH2/DMF, (ii) TFA:TIPS:water (95:2.5:2.5).

indicating that 4 has a high affinity for the FR. This further confirms the hypothesis that conjugation of a prosthetic group to folate via PEG linker will not dramatically compromise the affinity of folate for its cognate receptor. microPET Imaging. As seen in Figure 2, both folate PET tracers accumulated predominantly in the inflamed paw with no substantial radioactivity in other organs except the kidneys (Figure 3). Significant uptake in kidneys was anticipated, since the apical membrane of the proximal tubule of the kidney has been known to express high levels of FR.38 Moreover, it may also be possible that the probes are excreted through kidneys due to low molecular weights and half-life (most of the folate conjugates has