Frederick T. Chin

Persistent Dopamine Functions of Neurons Derived from Embryonic Stem Cells in a Rodent Model of Parkinson Disease

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long‐term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D2 receptors was normalized in the host striatum. These data suggest that ES cell‐derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.

MicroPET of tumor integrin αvβ3 expression using 18F-labeled PEGylated tetrameric RGD peptide ( 18F-FPRGD4)

In vivo imaging of αvβ3 expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin αvβ3–binding affinity due to the polyvalency effect. Here we report an example of 18F-labeled tetrameric RGD peptide for PET of αvβ3 expression in both xenograft and spontaneous tumor models. Methods: The tetrameric RGD peptide E{E[c(RGDyK)]2}2 was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate α-amino group. NH2-mini-PEG-E{E[c(RGDyK)]2}2 (PRGD4) was labeled with 18F via the N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer 18F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies. Results: The decay-corrected radiochemical yield for 18F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from 18F-F−. The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. 18F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of 18F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK). Conclusion: The tetrameric RGD peptide tracer 18F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin αvβ3 expression in cancer and other angiogenesis related diseases.

Pilot Pharmacokinetic and Dosimetric Studies of 18F-FPPRGD2: A PET Radiopharmaceutical Agent for Imaging αvβ3 Integrin Levels

PURPOSE To assess the safety, biodistribution, and dosimetric properties of the positron emission tomography (PET) radiopharmaceutical agent fluorine 18 ((18)F) FPPRGD2 (2-fluoropropionyl labeled PEGylated dimeric RGD peptide [PEG3-E{c(RGDyk)}2]), which is based on the dimeric arginine-glycine-aspartic acid (RGD) peptide sequence and targets α(v)β(3) integrin, in the first volunteers imaged with this tracer. MATERIALS AND METHODS The protocol was approved by the institutional review board, and written informed consent was obtained from all participants. Five healthy volunteers underwent whole-body combined PET-computed tomography 0.5, 1.0, 2.0, and 3.0 hours after tracer injection (mean dose, 9.5 mCi ± 3.4 [standard deviation] [351.5 MBq ± 125.8]; mean specific radioactivity, 1200 mCi/mmol ± 714 [44.4 GBq/mmol ± 26.4]). During this time, standard vital signs, electrocardiographic (ECG) readings, and blood sample values (for chemistry, hematologic, and liver function tests) were checked at regular intervals and 1 and 7 days after the injection. These data were used to evaluate tracer biodistribution and dosimetric properties, time-activity curves, and the stability of laboratory values. Significant changes in vital signs and laboratory values were evaluated by using a combination of population-averaged generalized estimating equation regression and exact paired Wilcoxon tests. RESULTS The administration of (18)F-FPPRGD2 was well tolerated, with no marked effects on vital signs, ECG readings, or laboratory values. The tracer showed the same pattern of biodistribution in all volunteers: primary clearance through the kidneys (0.360 rem/mCi ± 0.185 [0.098 mSv/MBq ± 0.050]) and bladder (0.862 rem/mCi ± 0.436 [0.233 mSv/MBq ± 0.118], voiding model) and uptake in the spleen (0.250 rem/mCi ± 0.168 [0.068 mSv/MBq ± 0.046]) and large intestine (0.529 rem/mCi ± 0.236 [0.143 mSv/MBq ± 0.064]). The mean effective dose of (18)F-FPPRGD2 was 0.1462 rem/mCi ± 0.0669 (0.0396 mSv/MBq ± 0.0181). With an injected dose of 10 mCi (370 MBq) and a 1-hour voiding interval, a patient would be exposed to an effective radiation dose of 1.5 rem (15 mSv). Above the diaphragm, there was minimal uptake in the brain ventricles, salivary glands, and thyroid gland. Time-activity curves showed rapid clearance from the vasculature, with a mean 26% ± 17 of the tracer remaining in the circulation at 30 minutes and most of the activity occurring in the plasma relative to cells (mean whole blood-plasma ratio, 0.799 ± 0.096). CONCLUSION (18)F-FPPRGD2 has desirable pharmacokinetic and biodistribution properties. The primary application is likely to be PET evaluation of oncologic patients-especially those with brain, breast, or lung cancer. Specific indications may include tumor staging, identifying patients who would benefit from antiangiogenesis therapy, and separating treatment responders from nonresponders early.

Pilot Comparison of ⁶⁸Ga-RM2 PET and ⁶⁸Ga-PSMA-11 PET in Patients with Biochemically Recurrent Prostate Cancer.

Glu-NH-CO-NH-Lys-(Ahx)-[68Ga(HBED-CC)] (68Ga-PSMA-11) is a PET tracer that can detect prostate cancer relapses and metastases by binding to the extracellular domain of PSMA. 68Ga-labeled DOTA-4-amino-1-carboxymethyl-piperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (68Ga-RM2) is a synthetic bombesin receptor antagonist that targets gastrin-releasing peptide receptors. We present pilot data on the biodistribution of these PET tracers in a small cohort of patients with biochemically recurrent prostate cancer. Methods: Seven men (mean age ± SD, 74.3 ± 5.9 y) with biochemically recurrent prostate cancer underwent both 68Ga-PSMA-11 PET/CT and 68Ga-RM2 PET/MRI scans. SUVmax and SUVmean were recorded for normal tissues and areas of uptake outside the expected physiologic biodistribution. Results: All patients had a rising level of prostate-specific antigen (mean ± SD, 13.5 ± 11.5) and noncontributory results on conventional imaging. 68Ga-PSMA-11 had the highest physiologic uptake in the salivary glands and small bowel, with hepatobiliary and renal clearance noted, whereas 68Ga-RM2 had the highest physiologic uptake in the pancreas, with renal clearance noted. Uptake outside the expected physiologic biodistribution did not significantly differ between 68Ga-PSMA-11 and 68Ga-RM2; however, 68Ga-PSMA-11 localized in a lymph node and seminal vesicle in a patient with no abnormal 68Ga-RM2 uptake. Abdominal periaortic lymph nodes were more easily visualized by 68Ga-RM2 in two patients because of lack of interference by radioactivity in the small intestine. Conclusion: 68Ga-PSMA-11 and 68Ga-RM2 had distinct biodistributions in this small cohort of patients with biochemically recurrent prostate cancer. Additional work is needed to understand the expression of PSMA and gastrin-releasing peptide receptors in different types of prostate cancer.

Positron Emission Tomography Imaging of Drug‐Induced Tumor Apoptosis with a Caspase‐Triggered Nanoaggregation Probe

Drug Design: An (18)F-labeled caspase-3-sensitive nanoaggregation positron emission tomography tracer was prepared and evaluated for imaging the caspase-3 activity in doxorubicin-treated tumor xenografts. Enhanced retention of the (18)F activity in apoptotic tumors is achieved through intramolecular macrocyclization and in situ aggregation upon caspase-3 activation (see picture).

PET of Malignant Melanoma Using 18F-Labeled Metallopeptides

Melanocortin type 1 receptor (MC1R), also known as α-melanocyte–stimulating hormone (α-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An 18F-labeled linear α-MSH peptide (18F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH2 [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized α-MSH peptide, ReO[Cys3,4,10,d-Phe7,Arg11]α-MSH3–13 (ReCCMSH(Arg11)), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg11) could be a good platform for the further development of an 18F-labeled probe for PET of MC1R-positive malignant melanoma. Methods: In this study, the metallopeptide Ac-d-Lys-ReCCMSH(Arg11) was synthesized using conventional solid-phase peptide synthesis chemistry and a rhenium cyclization reaction. The resulting peptides were then labeled with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The 18F-labeled metallopeptides were further tested for their in vitro receptor binding affinities, in vivo biodistribution, and PET imaging properties. Results: Both isomers of Ac-d-Lys-ReCCMSH(Arg11), named as RMSH-1 and RMSH-2, were purified and identified by high-performance liquid chromatography. The binding affinities of RMSH-1 and RMSH-2 and their respective 19F-SFB–conjugated peptides (19F-FB-RMSH-1 and 19F-FB-RMSH-2) were all determined to be within nanomolar range. Both 18F-labeled metallopeptides showed good tumor uptake in the B16F10 murine model, with high MC1R expression, but much lower uptake in the A375M human melanoma xenografted in mice, indicating low MC1R expression. 18F-FB-RMSH-1, when compared with 18F-FB-RMSH-2, displayed more favorable in vivo performance in terms of slightly higher tumor uptakes and much lower accumulations in the kidney and liver at 2 h after injection. Small-animal PET of 18F-FB-RMSH-1 and -2 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptakes and poorer tumor–to–normal organ contrasts were observed for the A375M model than for the B16F10 model. 18F-FB-RMSH-1 and -2 showed higher tumor uptake and better tumor retention than did 18F-FB-NAPamide. Conclusion: Specific in vivo targeting of 18F-FB-RMSH-1 to malignant melanoma was successfully achieved in preclinical models with high MC1R expression. Thus, the radiofluorinated metallopeptide 18F-FB-RMSH-1 is a promising molecular probe for PET of MC1R-positive tumors.

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-ZEGFR:1907 and 18F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al18F to produce Al18F-NOTA-ZEGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole (18F-CBT) was conjugated to Cys-ZEGFR:1907 to produce 18F-CBT-ZEGFR:1907. Binding affinity and specificity of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al18F-NOTA-ZEGFR:1907 or 18F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al18F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for 18F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al18F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.

New positron emission tomography (PET) radioligand for imaging σ-1 receptors in living subjects.

σ-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([(18)F]FTC-146, [(18)F]13). [(18)F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/μmol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [(18)F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [(18)F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

Integrin-targeted molecular imaging of experimental abdominal aortic aneurysms by (18)F-labeled Arg-Gly-Asp positron-emission tomography.

Background—Both inflammation and neoangiogenesis contribute to abdominal aortic aneurysm (AAA) disease. Arg-Gly-Asp–based molecular imaging has been shown to detect the integrin &agr;v&bgr;3. We studied a clinical dimeric 18F-labeled Arg-Gly-Asp positron-emission tomography (PET) agent (18F-FPPRGD2) for molecular imaging of experimental AAAs. Methods and Results—Murine AAAs were induced in Apo-E–deficient mice by angiotensin II infusion, with monitoring of aortic diameter on ultrasound. AAA (n=10) and saline-infused control mice (n=7) were injected intravenously with 18F-FPPRGD2, as well as an intravascular computed tomography contrast agent, then scanned using a small-animal PET/computed tomography scanner. Aortic uptake of 18F-FPPRGD2 was quantified by percentage-injected dose per gram and target-to-background ratio. Focal increased PET signal was found in AAA lesions, but not in normal control aortae, confirmed by quantitative analysis (median percentage-injected dose per gram [interquartile range], 2.05 [1.05–2.85] versus 0.63 [0.43–0.83], P=0.003; median target-to-background ratio [interquartile range], 2.72 [2.31–3.49] versus 1.44 [1.10–1.52], P=0.0008). Ex vivo autoradiography demonstrated high uptake of 18F-FPPRGD2 into the AAA wall, with immunohistochemistry showing substantial cluster of differentiation (CD)-11b+ macrophages and CD-31+ neovessels. Target-to-background ratio of AAAs on PET did not correlate with AAA diameter (r=−0.29, P=0.41) but did strongly correlate with both mural macrophage density (r=0.79, P=0.007) and neovessel counts (r=0.87, P=0.001) on immunohistochemistry. Conclusions—PET imaging of experimental AAAs using 18F-FPPRGD2 detects biologically active disease, correlating to the degree of vascular inflammation and neoangiogenesis. This may provide a clinically translatable molecular imaging approach to characterize AAA biology to predict risk beyond size alone.

18F-FPPRGD2 PET/CT: Pilot Phase Evaluation of Breast Cancer Patients

PURPOSE To present data from the first prospective pilot phase trial of breast cancer participants imaged with fluorine 18 ((18)F)-2-fluoropropionyl-labeled PEGylated dimeric arginine-glycine-aspartic acid ( RGD arginine-glycine-aspartic acid ) peptide (PEG3-E[c{ RGD arginine-glycine-aspartic acid yk}]2) ( FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) ), a radiopharmaceutical agent used in positron emission tomographic (PET) imaging. MATERIALS AND METHODS The local institutional review board approved the HIPAA-compliant protocol. Written informed consent was obtained from each patient. Eight women (age range, 44-67 years; mean age, 54.3 years ± 8.8 [standard deviation]) with newly diagnosed or recurrent breast cancer were recruited between November 2010 and February 2011. (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/computed tomographic (CT) and (18)F-fluorodeoxyglucose ( FDG fluorine 18 fluorodeoxyglucose ) PET/CT examinations were performed within 3 weeks of each other. Dynamic (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET and two whole-body static (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/CT scans were obtained. During this time, vital signs and electrocardiograms were recorded at regular intervals. Blood samples were obtained before the injection of (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) and at 24 hours and 1 week after injection to evaluate for toxicity. A nonparametric version of multivariate analysis of variance was used to assess the safety outcome measures simultaneously across time points. A paired two-sample t test was performed to compare the maximum standardized uptake values ( SUVmax maximum standardized uptake value ). RESULTS (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) was well tolerated, without noticeable changes in vital signs, on electrocardiograms, or in laboratory values. A total of 30 lesions were evaluated at (18)F- FDG fluorine 18 fluorodeoxyglucose PET/CT and (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/CT. The primary breast lesions had (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) uptake with SUVmax maximum standardized uptake value of 2.4-9.4 (mean, 5.6 ± 2.8) 60 minutes after injection, compared with (18)F- FDG fluorine 18 fluorodeoxyglucose uptake with SUVmax maximum standardized uptake value of 2.8-18.6 (mean, 10.4 ± 7.2). Metastatic lesions also showed (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) uptake, with SUVmax maximum standardized uptake value of 2.4-9.7 (mean, 5.0 ± 2.3) at 60 minutes, compared with (18)F- FDG fluorine 18 fluorodeoxyglucose uptake with SUVmax maximum standardized uptake value of 2.2-14.6 (mean, 6.6 ± 4.2). CONCLUSION Data from this pilot phase study suggest that (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) is a safe PET radiopharmaceutical agent. Evaluation of (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) in participants with breast cancer demonstrated significant uptake in the primary lesion and in the metastases. Larger cohorts are required to confirm these preliminary findings.