Koon Pak

Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine) coordination complexes as cell death imaging agents

The aim of this study was the development of (⁹⁹m)Tc labeled bis(zinc(II)-dipicolylamine) (Zn²⁺-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 was labeled with (⁹⁹m)Tc via the (⁹⁹m)Tc-tricarbonyl core ([(⁹⁹m)Tc(CO)₃-1]³⁺) or via HYNIC ((⁹⁹m)Tc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for [(⁹⁹m)Tc(CO)₃-1]³⁺. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding to apoptotic cells. (⁹⁹m)Tc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-reperfusion injury was used to further explore the ability of the (⁹⁹m)Tc-labeled Zn²⁺-DPA coordination complexes to target cell death. Selective accumulation could be detected for both tracers in the area at risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were 3.82 for [(⁹⁹m)Tc(CO)₃-1]³⁺ and 5.45 for (⁹⁹m)Tc-HYNIC-1.

Deep-red fluorescent imaging probe for bacteria.

A versatile deep-red fluorescent imaging probe is described that is comprised of a bis(zinc(II)-dipicolylamine) targeting unit covalently attached to a pentamethine carbocyanine fluorophore with Cy5-like spectroscopic properties. A titration assay based on fluorescence resonance energy transfer is used to prove that the probe selectively associates with anionic vesicle membranes whose composition mimics bacterial cell membranes. Whole-body optical imaging experiments show that the probe associates with the surfaces of both Gram-positive and Gram-negative bacteria cells, and it can target the site of bacterial infection in a living mouse. In vivo accumulation at the infection site and subsequent clearance occurs more quickly than a structurally related near-infrared bis(zinc(II)-dipicolylamine) probe. The fact that the same deep-red probe molecule can be used for spectroscopic assays, cell microscopy, and in vivo imaging studies, is an important and attractive technical feature.

Radiolabeled Zn-DPA as a potential infection imaging agent

INTRODUCTION A zinc-dipicolylamine analog (Zn-DPA) conjugated with a fluorophore (PSVue®794) has been shown to image bacterial infections in mice. However, radiolabeled Zn-DPA has not previously been considered for nuclear imaging of infection. METHODS Both 111In-labeled DOTA-biotin and Zn-DPA-biotin were combined using streptavidin (SA) as a noncovalent linker. Mice injected intramuscularly with Streptococcus pyogenes (infection model) or with lipopolysaccharide (LPS) (inflammation model) were coinjected intravenously with 6 μg of DPA as PSVue794 and as 111In-DOTA-biotin/SA/biotin-Zn-DPA. Periodic fluorescent and SPECT (single photon emission computed tomography)/CT (computed tomography) images were acquired, and biodistributions were obtained at 22 h. RESULTS Histological examination confirmed the validity of both the infection and inflammation animal models. Both the whole-body optical and nuclear images showed obvious accumulations in the target thigh in both models at all time points. At 22 h, the average target thigh accumulation of 111In was 1.66%ID/g (S.D. 0.15) in the infection mice compared to 0.58%ID/g (S.D. 0.07) in the inflammation mice (P<.01), and the 111In target/normal thigh ratio was 2.8 fold higher in the infection animals compared to the inflammation animals. CONCLUSIONS These preliminary results show that Zn-DPA within streptavidin targets S. pyogenes-infected mice similarly to its free fluorescent analogue. The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.

Early prediction of tumor response to treatment: pre-clinical validation of 99mTc-duramycin

Noninvasive imaging of cell death can provide an early indication of the efficacy of tumor treatment, aiding clinicians in distinguishing responding patients from nonresponding patients early on. 99mTc-duramycin is a SPECT tracer for cell death imaging. In this study, our aim was to validate the use of 99mTc-duramycin for imaging the early response of tumors to treatment. Methods: An in vitro binding assay was performed on COLO205 cells treated with 5-fluorouracil (3.1, 31, or 310 μM) and oxaliplatin (0.7 or 7 μM) or radiation (2 or 4.5 Gy). 99mTc-duramycin cell binding and the levels of cell death were evaluated after treatment. In vivo imaging was performed on 4 groups of CD1-deficient mice bearing COLO205 human colorectal cancer tumors. Each group included 6 tumors. The first group was given irinotecan (100 mg/kg), the second oxaliplatin (5 mg/kg), the third irinotecan (80 mg/kg) plus oxaliplatin (5 mg/kg), and the fourth vehicle (0.9% NaCl and 5% glucose). For radiotherapy studies, COLO205 tumors received 4.5 Gy, 2 fractions of 4.5 Gy in a 24-h interval, pretreatment with an 80 mg/kg dose of irinotecan combined with 2 fractions of 4.5 Gy in a 24-h interval, or no treatment (n = 5–6/group). Therapy response was evaluated by 99mTc-duramycin SPECT 24 h after the last dose of therapy. Blocking was used to confirm tracer specificity. Radiotracer uptake in the tumors was validated ex vivo using γ-counting, cleaved caspase-3, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) histology. Results: Chemotherapy and radiotherapy increased 99mTc-duramycin binding to COLO205 cells in a concentration/dose- and time-dependent manner, which correlated well with cell death levels (P < 0.05) as analyzed by annexin V and caspase 3/7 activity. In vivo, 99mTc-duramycin uptake in COLO205 xenografts was increased 2.3- and 2.8-fold (P < 0.001) in mice treated with irinotecan and combination therapy, respectively. Blocking with unlabeled duramycin demonstrated specific binding of the radiotracer. After tumor irradiation with 4.5 Gy, 99mTc-duramycin uptake in tumors increased significantly (1.24 ± 0.07 vs. 0.57 ± 0.08 percentage injected dose per gram in the unirradiated tumors; P < 0.001). γ-counting of radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, P < 0.001) and TUNEL (r = 0.81, P < 0.001) staining. Conclusion: We demonstrated that 99mTc-duramycin can be used to image induction of cell death early after chemotherapy and radiotherapy. It holds potential to be translated into clinical use for early assessment of treatment response.

Non-invasive in vivo imaging of arthritis in a collagen-induced murine model with phosphatidylserine-binding near-infrared (NIR) dye.

IntroductionDevelopment of non-invasive molecular imaging techniques that are based on cellular changes in inflammation has been of active interest for arthritis diagnosis. This technology will allow real-time detection of tissue damage and facilitate earlier treatment of the disease, thus representing an improvement over X-rays, which detect bone damage at the advanced stage. Tracing apoptosis, an event occurring in inflammation, has been a strategy used. PSVue 794 is a low-molecular-weight, near-infrared (NIR)-emitting complex of bis(zinc2+-dipicolylamine) (Zn-DPA) that binds to phosphatidylserine (PS), a plasma membrane anionic phospholipid that becomes flipped externally upon cell death by apoptosis. In this study, we evaluated the capacity of PSVue 794 to act as an in vivo probe for non-invasive molecular imaging assessment of rheumatoid arthritis (RA) via metabolic function in murine collagen-induced arthritis, a widely adopted animal model for RA.MethodsMale DBA/1 strain mice were treated twice with chicken collagen type II in Freund’s adjuvant. Their arthritis development was determined by measuring footpad thickness and confirmed with X-ray analysis and histology. In vivo imaging was performed with the NIR dye and the LI-COR Odyssey Image System. The level of emission was compared among mice with different disease severity, non-arthritic mice and arthritic mice injected with a control dye without the Zn-DPA targeting moiety.ResultsFluorescent emission correlated reliably with the degree of footpad swelling and the manifestation of arthritis. Ex vivo examination showed emission was from the joint. Specificity of binding was confirmed by the lack of emission when arthritic mice were given the control dye. Furthermore, the PS-binding protein annexin V displaced the NIR dye from binding, and the difference in emission was numerically measurable on a scale.ConclusionsThis report introduces an economical alternative method for assessing arthritis non-invasively in murine models. Inflammation in feet and ankles can be measured longitudinally using the PSVue 794 probe for cell death and with a commonly available multipurpose imager. This technique provides metabolic and functional information that anatomical measurement of footpad swelling or visual determination of arthritic index cannot. It also may decrease the number of animals required per experiment because tissue damage will not necessarily require evaluation by harvesting joints for histology.

99mTc-Duramycin SPECT imaging of early tumor response to targeted therapy: a comparison with 18F-FDG PET.

Molecular imaging of cell death may provide a detailed readout of the cellular response to novel therapies and prognostic information on tumor treatment efficacy, assisting in the design of individualized therapy. We compared the predictive power of cell death imaging using 99mTc-duramycin with the current gold standard 18F-FDG for treatment response evaluation after targeted therapy. Methods: Early therapy response evaluation was assessed by 99mTc-duramycin SPECT and 18F-FDG PET imaging in treatment-sensitive COLO205 and treatment-resistant HT29 human colorectal cancer xenografts 24 h after a single dose of conatumumab or IgG1 control. The specificity of 99mTc-duramycin for apoptosis was assessed using 99mTc-linear duramycin control radiotracer. Radiotracer uptake was validated ex vivo by γ-counting and autoradiography and compared with cleaved caspase-3 (CC3) activation and DNA fragmentation (TdT-mediated dUTP nick-end labeling [TUNEL]). Data were analyzed with the Student t test and Pearson correlation. All statistical tests were 2-sided. Results: COLO205 tumor uptake of 99mTc-duramycin was increased 7-fold from baseline in conatumumab- versus IgG1-treated control mice (P < 0.001), in good correlation with histologic analysis of apoptosis (CC3, r = 0.842, and TUNEL, r = 0.894; P < 0.001). No response was detected in HT29 tumors. No change in 99mTc-linear duramycin uptake could be detected in COLO205 tumors after treatment, indicating specificity of the 99mTc-duramycin tumor signal. 18F-FDG uptake was not significantly increased from baseline in conatumumab- versus IgG1-treated COLO205 and HT29 tumor–bearing mice (P = 0.104 and 0.779, respectively) and did not correlate with immunohistochemical evidence of apoptosis. Conclusion: We have demonstrated that 99mTc-duramycin specifically accumulates in apoptotic tumors in which 18F-FDG was not able to differentiate responding from nonresponding tumors early after treatment. 99mTc-duramycin holds promise as a noninvasive imaging radiotracer for early treatment evaluation in the clinic.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Detection of atherosclerotic plaques in ApoE-deficient mice using 99mTc-duramycin

UNLABELLED Apoptosis of macrophages and smooth muscle cells is linked to atherosclerotic plaque destabilization. The apoptotic cascade leads to exposure of phosphatidylethanolamine (PE) on the outer leaflet of the cell membrane, thereby making apoptosis detectable using probes targeting PE. The objective of this study was to exploit capabilities of a PE-specific imaging probe, (99m)Tc-duramycin, in localizing atherosclerotic plaque and assessing plaque evolution in apolipoprotein-E knockout (ApoE(-/-)) mice. METHODS Atherosclerosis was induced in ApoE(-/-) mice by feeding an atherogenic diet. (99m)Tc-duramycin images were acquired using a small-animal SPECT imager. Six ApoE(-/-) mice at 20weeks of age (Group I) were imaged and then sacrificed for ex vivo analyses. Six additional ApoE(-/-) mice (Group II) were imaged at 20 and 40weeks of age before sacrifice. Six ApoE wild-type (ApoE(+/+)) mice (Group III) were imaged at 40weeks as controls. Five additional ApoE(-/-) mice (40weeks of age) (Group IV) were imaged with a (99m)Tc-labeled inactive peptide, (99m)Tc-LinDUR, to assess (99m)Tc-duramycin targeting specificity. RESULTS Focal (99m)Tc-duramycin uptake in the ascending aorta and aortic arch was detected at 20 and 40weeks in the ApoE(-/-) mice but not in ApoE(+/+) mice. (99m)Tc-duramycin uptake in the aortic lesions increased 2.2-fold on quantitative imaging in the ApoE(-/-) mice between 20 and 40weeks. Autoradiographic and histological data indicated significantly increased (99m)Tc-duramycin uptake in the ascending aorta and aortic arch associated with advanced plaques. Quantitative autoradiography showed that the ratio of activity in the aortic arch to descending thoracic aorta, which had no plaques or radioactive uptake, was 2.1 times higher at 40weeks than at 20weeks (6.62±0.89 vs. 3.18±0.29, P<0.01). There was barely detectable focal uptake of (99m)Tc-duramycin in the aortic arch of ApoE(+/+) mice. No detectable (99m)Tc-LinDUR uptake was observed in the aortas of ApoE(-/-) mice. CONCLUSIONS PE-targeting properties of (99m)Tc-duramycin in the atherosclerotic mouse aortas were noninvasively characterized. (99m)Tc-duramycin is promising in localizing advanced atherosclerotic plaques.

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA

INTRODUCTION Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.

Bis(Zinc-Dipicolylamine), Zn-DPA, a New Marker for Apoptosis

PURPOSE To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA). METHODS Adult Wistar rats were given unilateral intravitreal injections of 3 μL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 μL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III β-tubulin and vimentin were performed. RESULTS Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480-positive cells in the RGC layer were labeled by FG and III β-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480-positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480-positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL. CONCLUSIONS Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.