Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with 89Zirconium oxine for PET imaging.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products. METHODS: To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89Zirconium-oxine (89Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells. RESULTS: We observed that radiolabeling of CAR-T cells with 89Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells. CONCLUSIONS: Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89Zr-oxine retain critical product attributes and suggest 89Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Cysteine-specific 89Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas.

Introduction: Positron emission tomography (PET) using radiolabeled Abs as imaging tracer is called immuno-PET. Immuno-PET can verify therapeutic Ab delivery and can noninvasively quantify global levels of target expression in tumors of living subjects. The interleukin-2 receptor α chain (IL-2Rα; CD25) is a promising target for immune therapy and radioimmunotherapy of lymphomas. Immuno-PET could facilitate this approach by visualizing CD25 expression in vivo. Methods: We prepared 89Zr-anti-CD25 IgG specifically labeled to sulfhydryl moieties by maleimide-deferoxamine conjugation. Results and Discussion: CD25(+) SUDHL1 human T-cell lymphoma cells showed high anti-human 89Zr-CD25 IgG binding that reached 32-fold of that of CD25(-) human lymphoma cells and was completely blocked by excess unlabeled Ab. In SUDHL1 tumor-bearing nude mice, pharmacokinetic studies demonstrated exponential reductions of whole blood and plasma activity following intravenous 89Zr-anti-CD25 IgG injection, with half-lives of 26.0 and 23.3 h, respectively. SUDHL1 tumor uptake of 89Zr-CD25 IgG was lower per weight in larger tumors, but blood activity did not correlate with tumor size or blood level of human CD25, indicating minimal influence by circulating soluble CD25 protein secreted from the lymphoma cells. 89Zr-CD25 IgG PET allowed high-contrast SUDHL1 lymphoma visualization at five days. Biodistribution studies confirmed high tumor 89Zr-CD25 IgG uptake (8.7 ± 0.9%ID/g) that was greater than blood (5.2 ± 1.6%ID/g) and organ uptakes (0.7 to 3.5%ID/g). Tumor CD25-specific targeting was confirmed by suppression of tumor uptake to 4.3 ± 0.2%ID by excess unlabeled CD25 IgG, as well as by low tumor uptake of 89Zr-labeled IgG2a isotype control Ab (3.6 ± 0.9%ID). Unlike CD25(+) lymphocytes from mouse thymus that showed specific uptake of anti-mouse 89Zr-CD25 IgG, EL4 mouse lymphoma cells had low CD25 expression and showed low uptake. In immunocompetent mice bearing EL4 tumors, anti-mouse 89Zr-CD25 IgG displayed low uptakes in normal organs as well as in the tumor. Furthermore, the biodistribution was not influenced by Ab blocking, indicating that specific uptake in nontumor tissues was minimal. 89Zr-CD25 IgG immuno-PET may thus be useful for imaging of T-cell lymphomas and noninvasive assessment of CD25 expression on target cells in vivo.

Preparation of 177Lu-PSMA-617 in Hospital Radiopharmacy: Convenient Formulation of a Clinical Dose Using a Single-Vial Freeze-Dried PSMA-617 Kit Developed In-House.

OBJECTIVE: In the recent time, endoradionuclide therapy for metastatic castration-resistant prostate carcinoma employing 177Lu-PSMA-617 has yielded encouraging results and several clinical trials with the agent are currently ongoing. Routine preparation of 177Lu-PSMA-617 patient doses can be made simpler and convenient, if the ingredients essential for radiolabeling are made available in a ready-to-use lyophilized form. METHODS: PSMA-617 freeze-dried kit was formulated and used for the preparation of 177Lu-PSMA-617 clinical dose with high radiochemical purity using low/medium specific activity 177Lu. Detailed radiochemical studies were performed to determine the maximum activity and volume of 177LuCl3, which can be added in the kit for the formulation of 177Lu-PSMA-617. Studies were also performed to determine the shelf life of the kit to ensure its long-term usage. Studies were performed in buffer as well as human serum medium to determine the stability of the 177Lu-PSMA-617 complex after storing in respective media up to 7 days postpreparation. About ten patient doses of 177Lu-PSMA-617 were administered, and posttherapy scans were acquired. RESULTS: The formulated freeze-dried kit of PSMA-617 could be radiolabeled with an average percentage radiochemical purity > 98.53 ± 0.38. The freeze-dried kit was found suitable for tolerating up to 0.5 mL of 177LuCl3 (in 0.01 N HCl) and specific activity of 555 MBq/µg (15 mCi/µg) for the preparation of the patient dose of 177Lu-PSMA-617. The 177Lu-PSMA-617 complex prepared using the freeze-dried kit of PSMA-617 was observed to maintain % radiochemical purity (RCP) of 96.74 ± 0.87 and 94.81 ± 2.66, respectively, even after storing up to 7 days in buffer and human serum, respectively. 177Lu-PSMA-617 prepared using the in-house formulated freeze-dried kit of PSMA-617 exhibited accumulation in metastatic lesions picked up in a pretherapy PET scan. Reduction in number as well as size of lesions was observed in posttherapy scans acquired after two months of administering the first therapeutic dose of 177Lu-PSMA-617. CONCLUSIONS: The freeze-dried kit of PSMA-617 could be used for the preparation of 177Lu-PSMA-617 with high radiochemical purity (>98%) in a reproducible manner. 177Lu-PSMA-617 prepared using the developed kit was successfully evaluated in patients suffering from metastatic prostate cancer.

Targeted design of a recombinant tracer for SPECT renal imaging.

Rationale: A robust radiopharmaceutical has high uptake in the target and low retention in non-target tissues. However, traditional tracers for renal imaging that chemically chelate 99mTc are excreted through the renal route with transient resident time in the kidney. Following a rational design approach, we constructed a protein-based radiotracer, designated PBT-Fc, to sequentially bind tubular neonatal Fc-receptor and subsequently proximal tubular basement membrane for its targeted sequestration in kidney parenchyma. In this process, the tracer participates in physiologic glomerular filtration and tubular reabsorption while escaping lysosomal catabolism and urinary clearance. Methods: To specifically target renal receptors in navigating the urinary passage in the kidney, we produced a recombinant fusion protein with two separate functional parts: a polybasic PBT segment derived from human Vascular Endothelial Growth Factor and Fc segment of IgG1. The chimeric fusion of PBT-Fc was labeled with radionuclide 99mTc and tested in rodent models of kidney diseases. Planar scintigraphy and single-photon emission computerized tomography (SPECT) were performed to evaluate renal-specificity of the tracer. Results: When injected in mouse and rat, following a brief 10 - 15 min dynamic redistribution phase in circulation, ~ 95% of the [99mTc]-PBT-Fc signal was concentrated in the kidney and lasted for hours without urinary loss or surrounding tissue activities. Long-lasting tracer signals in the kidney cortex in conjunction with SPECT greatly augmented the image quality in detecting pathological lesions in a variety of disease models, including ischemic acute kidney injury, drug-induced renal toxicity, and chronic kidney disease from renin-angiotensin system (RAS) overactivation. Conclusion: Exclusive renal retention of the recombinant radiotracer greatly facilitated static-phase signal acquisition by SPECT and achieved submillimeter spatial resolution of kidney alternations in glomerular and tubular disease models.

Scientific research and product development in the United States to address injuries from a radiation public health emergency.

The USA has experienced one large-scale nuclear incident in its history. Lessons learned during the Three-Mile Island nuclear accident provided government planners with insight into property damage resulting from a low-level release of radiation, and an awareness concerning how to prepare for future occurrences. However, if there is an incident resulting from detonation of an improvised nuclear device or state-sponsored device/weapon, resulting casualties and the need for medical treatment could overwhelm the nation's public health system. After the Cold War ended, government investments in radiation preparedness declined; however, the attacks on 9/11 led to re-establishment of research programs to plan for the possibility of a nuclear incident. Funding began in earnest in 2004, to address unmet research needs for radiation biomarkers, devices and products to triage and treat potentially large numbers of injured civilians. There are many biodosimetry approaches and medical countermeasures (MCMs) under study and in advanced development, including those to address radiation-induced injuries to organ systems including bone marrow, the gastrointestinal (GI) tract, lungs, skin, vasculature and kidneys. Biomarkers of interest in determining level of radiation exposure and susceptibility of injury include cytogenetic changes, 'omics' technologies and other approaches. Four drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of acute radiation syndrome (ARS), with other licensures being sought; however, there are still no cleared devices to identify radiation-exposed individuals in need of treatment. Although many breakthroughs have been made in the efforts to expand availability of medical products, there is still work to be done.

Assessment of absorbed dose for Zr-89, Sm-153 and Lu-177 medical radioisotopes: IDAC-Dose2.1 and OLINDA experience.

OBJECTIVE: In this article, IDAC-Dose2.1 and OLINDA computer codes are compared as they are the most widely used software tools for internal dosimetry assessment at the present time. OLINDA/EXM personal computer code was created as a replacement for the widely used MIRDOSE3.1 code. IDAC-Dose2.1 was developed based on the ICRP specific absorbed fractions and computational framework of internal dose assessment given for reference adults in ICRP Publication 133. IDAC uses cumulated activities per administered activity in hours and calculates the absorbed dose and the effective dose. The program calculates the dose in the Eckerman stylized family phantoms. It is useful in standardizing and automating internal dose calculations, assessing doses in clinical trials with radiopharmaceuticals, making theoretic calculations for existing pharmaceuticals, teaching, and other purposes. METHODS: To produce such a comparison, the results of this work were compared with available published data in the literature on radiopharmaceuticals. Radiopharmaceuticals with 89Zr, 153Sm, 177Lu radionuclides are used as the basis for the comparison. 89Zr, 153Sm, 177Lu radionuclides are regarded as the future of radiopharmaceutical treatment. For 89Zr, two different labelled carriers, Zr-89_cMAb U36 and Zr-89 Panitumumab, were used on patients. RESULTS: The results show a clear difference in terms of absorbed dose of the Zr-89 radiopharmaceuticals for red bone marrow when calculated by IDAC-Dose2.1 (0.76 mGy/MBq), while the estimated absorbed dose in literature results is 0.07 mGy/MBq and 0.14 mGy/MBq when the calculation is done by OLINDA program. In the case of 177Lu-EDTMP, the absorbed dose in red bone marrow is in reasonable agreement (0.63 mGy/MBq and 0.8 mGy/MBq for IDAC-Dose2.1 and OLINDA, respectively). A significant difference was found for the absorbed dose in the bone surface, which was almost twice as high for OLINDA (2.1 mGy/MBq for IDAC-Dose2.1 and 5.4 mGy/MBq for OLINDA). In some direct cases, the calculated absorbed dose in the urinary bladder wall with OLINDA is ten times higher compared to WinAct (which was utilized to calculate the total activity in the organs and tissues) and IDAC 2.1. These results are considered key to greater accuracy in internal dose calculation.

Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9-13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 as compared to [177Lu]Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

Dose Coefficient Calculation for Use in Dosimetry Assessment of a Fission-Based Weapon.

In the event of a fission-based weapon or improvised nuclear device (IND) detonation, dose coefficients can be harnessed to provide dose assessments for defense, emergency preparedness, and consequence management, as well as to prospectively inform the assessment of radiation biomarkers and development of medical prophylaxis countermeasures for defense and homeland security stakeholders and decision-makers. Although dose coefficients have previously been calculated for this group, they would apply specifically to the studied population, the 1945 Japanese cohort, after which their anthropomorphic computational phantoms were modeled. For this reason, applications to other populations may be limited, and instead, an assessment of a more standardized population is desired. We employed a series of computational human phantoms representing international reference individuals: UF/NCI voxel phantom series containing newborn, 1-, 5-, 10-, 15-, and 35-year-old males and females. Irradiation of the phantoms was simulated using the Monte Carlo N-Particle transport code to determine organ dose coefficients under four idealized irradiation geometries at three distances from the detonation hypocenter at Hiroshima and Nagasaki using DS02 free-in-air prompt neutron and photon fluence spectra. Through these simulations, age-specific dose coefficients were determined for individual organs. Various articulated PIMAL stylized phantoms were simulated as well to estimate the effect of body posture on dose coefficients and determine the effect of posture on dosimetric estimation and reconstruction. Results additionally demonstrate that 137Cs and the Watt fission spectra are not ideal general surrogate sources for fission weapons, which may be considered for experimental testing of medical countermeasures. Supplementary data provided tabulates the compilation of organ dose-rate coefficients in this study.

Calcium Carbonate Core-Shell Particles for Incorporation of 225Ac and Their Application in Local α-Radionuclide Therapy.

Actinium-225 (225Ac) radiolabeled submicrometric core-shell particles (SPs) made of calcium carbonate (CaCO3) coated with biocompatible polymers [tannic acid-human serum albumin (TA/HSA)] have been developed to improve the efficiency of local α-radionuclide therapy in melanoma models (B16-F10 tumor-bearing mice). The developed 225Ac-SPs possess radiochemical stability and demonstrate effective retention of 225Ac and its daughter isotopes. The SPs have been additionally labeled with zirconium-89 (89Zr) to perform the biodistribution studies using positron emission tomography-computerized tomography (PET/CT) imaging for 14 days after intratumoral injection. According to the PET/CT analysis, a significant accumulation of 89Zr-SPs in the tumor area is revealed for the whole investigation period, which correlates with the direct radiometry analysis after intratumoral administration of 225Ac-SPs. The histological analysis has revealed no abnormal changes in healthy tissue organs after treatment with 225Ac-SPs (e.g., no acute pathologic findings are detected in the liver and kidneys). At the same time, the inhibition of tumor growth has been observed as compared with control samples [nonradiolabeled SPs and phosphate-buffered saline (PBS)]. The treatment of mice with 225Ac-SPs has resulted in prolonged survival compared to the control samples. Thus, our study validates the application of 225Ac-doped core-shell submicron CaCO3 particles for local α-radionuclide therapy.

CD38-Targeted Theranostics of Lymphoma with 89Zr/177Lu-Labeled Daratumumab.

Lymphoma is a heterogeneous disease with varying clinical manifestations and outcomes. Many subtypes of lymphoma, such as Burkitt's lymphoma and diffuse large B cell lymphoma, are highly aggressive with dismal prognosis even after conventional chemotherapy and radiotherapy. As such, exploring specific biomarkers for lymphoma is of high clinical significance. Herein, a potential marker, CD38, is investigated for differentiating lymphoma. A CD38-targeting monoclonal antibody (mAb, daratumumab) is then radiolabeled with Zr-89 and Lu-177 for theranostic applications. As the diagnostic component, the Zr-89-labeled mAb is highly specific in delineating CD38-positive lymphoma via positron emission tomography (PET) imaging, while the Lu-177-labeled mAb serves well as the therapeutic component to suppress tumor growth after a one-time administration. These results strongly suggest that CD38 is a lymphoma-specific marker and prove that 89Zr/177Lu-labeled daratumumab facilitates immunoPET imaging and radioimmunotherapy of lymphoma in preclinical models. Further clinical evaluation and translation of this CD38-targeted theranostics may be of significant help in lymphoma patient stratification and management.

Biokinetic Method on Simultaneous Intake of Radionuclides from Multiple Intake Scenarios for Application in Internal Exposures.

Biokinetics underlies the basis for assessment of internal exposures. This paper develops a biokinetic method on simultaneous intake of radionuclides from multiple intake scenarios in internal exposures. With numerical techniques that transform the whole biokinetics between the coupled and decoupled representations of the same problem, this method applies to coupled biokinetics with complex structures and has no restrictions of practical importance on the number of intake scenarios, the number of intake parent radionuclides and decay products, and the complexity of decay relationships between parent and progeny nuclides. For illustration, this method is applied to an assumed case of mixed inhalation and ingestion of weapon-grade plutonium material for reference workers that is focused on Pu and Am. Due to coupled biokinetics between the direct intake and ingrowth parts in different intake pathways, the multiple intake results (the contents of lungs, daily excretions, and cumulative contents) display richer behaviors as compared to single intake cases. This method benefits both the prospective and retrospective assessment of internal exposures for complex intake cases in actual applications.

Power Function Retention of Radionuclides in a Wound.

NCRP Report 156 describes soluble radionuclide retention kinetics in a wound, segregated into four retention categories: weak (W), moderate (M), strong (S), and avid (A). An alternate single-parameter model, the negative power function, t, is presented in this paper to describe the time behavior of radionuclide retention. With this mathematical description, γ is a single parameter that can be used to assign the wound retention category rapidly. Using the power function description of wound retention, the various wound categories present as straight lines on log scales with different slopes corresponding to the various retention categories. Regression analysis of average retention values in NCRP 156 shows γ = 0.735 ± 0.132, 0.514 ± 0.015, 0.242 ± 0.016, and 0.053 ± 0.023 for the weak, moderate, strong, and avid categories, respectively. A case study is presented (REAC/TS Registry case 1284) where a power function is shown to fit retention data in a Pu/Am hand wound up to 2,000 d (5.4 y) post-accident.

Cross-species/cross-modality physiologically based pharmacokinetics for biologics: 89Zr-labelled albumin-binding domain antibody GSK3128349 in humans.

Two-pore physiologically-based pharmacokinetics (PBPK) for biologics describes the tissue distribution and elimination kinetics of soluble proteins as a function of their hydrodynamic radius and the physiological properties of the organs. Whilst many studies have been performed in rodents to parameterize the PBPK framework in terms of organ-specific lymph flow rates, similar validation in humans has been limited. This is mainly due to the paucity of the tissue distribution time course data for biologics that is not distorted by target-related binding. Here, we demonstrate that a PBPK model based on rodent data provided good to satisfactory extrapolation to the tissue distribution time course of 89Zr-labeled albumin-binding domain antibody (AlbudAb™) GSK3128349 in healthy human volunteers, including correct prediction of albumin-like plasma half-life, volume of distribution, and extravasation half-life. The AlbudAb™ used only binds albumin, and hence it also provides information about the tissue distribution kinetics and turnover of that ubiquitous and multifunctional plasma protein.

Internal doses in experimental mice and rats following exposure to neutron-activated 56MnO2 powder: results of an international, multicenter study.

The experiment was performed in support of a Japanese initiative to investigate the biological effects of irradiation from residual neutron-activated radioactivity that resulted from the A-bombing. Radionuclide 56Mn (T1/2 = 2.58 h) is one of the main neutron-activated emitters during the first hours after neutron activation of soil dust particles. In our previous studies (2016-2017) related to irradiation of male Wistar rats after dispersion of 56MnO2 powder, the internal doses in rats were found to be very inhomogeneous: distribution of doses among different organs ranged from 1.3 Gy in small intestine to less than 0.0015 Gy in some of the other organs. Internal doses in the lungs ranged from 0.03 to 0.1 Gy. The essential pathological changes were found in lung tissue of rats despite a low level of irradiation. In the present study, the dosimetry investigations were extended: internal doses in experimental mice and rats were estimated for various activity levels of dispersed neutron-activated 56MnO2 powder. The following findings were noted: (a) internal radiation doses in mice were several times higher in comparison with rats under similar conditions of exposure to 56MnO2 powder. (b) When 2.74 × 108 Bq of 56MnO2 powder was dispersed over mice, doses of internal irradiation ranged from 0.81 to 4.5 Gy in the gastrointestinal tract (small intestine, stomach, large intestine), from 0.096 to 0.14 Gy in lungs, and doses in skin and eyes ranged from 0.29 to 0.42 Gy and from 0.12 to 0.16 Gy, respectively. Internal radiation doses in other organs of mice were much lower. (c) Internal radiation doses were significantly lower in organs of rats with the same activity of exposure to 56MnO2 powder (2.74 × 108 Bq): 0.09, 0.17, 0.29, and 0.025 Gy in stomach, small intestine, large intestine, and lungs, respectively. (d) Doses of internal irradiation in organs of rats and mice were two to four times higher when they were exposed to 8.0 × 108 Bq of 56MnO2 (in comparison with exposure to 2.74 × 108 Bq of 56MnO2). (e) Internal radiation doses in organs of mice were 7-14 times lower with the lowest 56MnO2 amount (8.0 × 107 Bq) in comparison with the highest amount, 8.0 × 108 Bq, of dispersed 56MnO2 powder. The data obtained will be used for interpretation of biological effects in experimental mice and rats that result from dispersion of various levels of neutron-activated 56MnO2 powder, which is the subject of separate studies.

Internal dosimetry studies of 170Tm-EDTMP complex, as a bone pain palliation agent, in human tissues based on animal data.

Radiopharmaceuticals with therapeutic applications are designed to deliver high doses of radiation to target organs with minimizing unwanted radiation to healthy tissues. Owing to the potential of targeted radiotherapy to treat a wide range of malignancies, 170Tm -EDTMP was developed for possible therapeutic applications. This study describes absorbed dose prediction of 170Tm-EDTMP in human organs after animal injection which is determined via medical internal radiation dose (MIRD) and MCNP-4C code methods. It was estimated that a 1-MBq administration of 170Tm-EDTMP into the human body would result in an absorbed dose of 37.9 mGy (MIRD method) and 38.02 mGy (MCNP-4C code) in the bone surface after 60 days post injection. Highest and lowest difference between MIRD and MCNP results are for lung and bone surface respectively. Finally, the results show that there is a good agreement between MIRD method and MCNP-4C simulation code for absorbed dose estimation.

Molecular Imaging of Inflammation in a Mouse Model of Atherosclerosis Using a Zirconium-89-Labeled Probe.

BACKGROUND: Beyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions. MATERIALS AND METHODS: Mal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe-/- mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls. RESULTS: PET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe-/- mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe-/- mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe-/- mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe-/- mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001). CONCLUSION: 89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe-/- mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.

Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with 177Lu-Acridine.

OBJECTIVE: A DNA intercalating agent reversibly stacks between the adjacent base pairs of DNA and thus is expected to exhibit preferential localization in the tumorous lesions as tumors are associated with enhanced DNA replication. Therefore, radiolabeled DNA intercalators are supposed to have potential to be used in targeted tumor therapy. Working in this direction, an attempt was made to radiolabel 9-aminoacridine, a DNA intercalator, with 177Lu, one of the most useful therapeutic radionuclides, and study the potential of 177Lu-acridine in targeted tumor therapy. Experiments. 9-Aminoacridine was coupled with p-NCS-benzyl-DOTA to facilitate radiolabeling, and the conjugate was radiolabeled with 177Lu. Different reaction parameters were optimized in order to obtain 177Lu-acridine complex with maximum radiochemical purity. In vitro stability of the radiolabeled complex was studied in normal saline and human blood serum. Biological behavior of the radiolabeled agent was studied both in vitro and in vivo using the Raji cell line and fibrosarcoma tumor-bearing Swiss mice, respectively. RESULTS: 177Lu-acridine complex was obtained with ~100% radiochemical purity under the optimized reaction conditions involving incubation of 1.5 mg/mL of ligand with 177Lu (1 mCi, 37 MBq) at 100°C at pH ~5 for 45 minutes. The complex maintained a radiochemical purity of >85% in saline at 6 d and >70% in human serum at 2 d postpreparation. In vitro cellular study showed uptake of the radiotracer (5.3 ± 0.13%) in the Raji cells along with significant cytotoxicity (78.06 ± 2.31% after 6 d). Biodistribution study revealed considerable accumulation of the radiotracer in tumor 9.98 ± 0.13 %ID/g within 1 h postadministration and retention therein till 6 d postadministration 4.00 ± 0.16 %ID/g with encouraging tumor to nontarget organ uptake ratios. CONCLUSIONS: The present study, although preliminary, indicates the potential of 177Lu-acridine and thus radiolabeled DNA intercalators in targeted tumor therapy. However, further detailed evaluation is required to explore the actual potential of such agents in targeted tumor therapy.

Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. EXPERIMENTAL DESIGN: We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. RESULTS: Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. CONCLUSIONS: Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

A New Pharmacokinetic Model Describing the Biodistribution of Intravenously and Intratumorally Administered Superparamagnetic Iron Oxide Nanoparticles (SPIONs) in a GL261 Xenograft Glioblastoma Model.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPIONs) have displayed multifunctional applications in cancer theranostics following systemic delivery. In an effort to increase the therapeutic potential of local therapies (including focal hyperthermia), nanoparticles can also be administered intratumorally. Therefore, the development of a reliable pharmacokinetic model for the prediction of nanoparticle distribution for both clinically relevant routes of delivery is of high importance. MATERIALS AND METHODS: The biodistribution of SPIONs (of two different sizes - 130 nm and 60 nm) radiolabeled with zirconium-89 or technetium-99m following intratumoral or intravenous injection was investigated in C57/Bl6 mice bearing subcutaneous GL261 glioblastomas. Based on PET/CT biodistribution data, a novel pharmacokinetic model was established for a better understanding of the pharmacokinetics of the SPIONs after both administration routes. RESULTS: The PET image analysis of the nanoparticles (confirmed by histology) demonstrated the presence of radiolabeled nanoparticles within the glioma site (with low amounts in the liver and spleen) at all investigated time points following intratumoral injection. The mathematical model confirmed the dynamic nanoparticle redistribution in the organism over a period of 72 h with an equilibrium reached after 100 h. Intravenous injection of nanoparticles demonstrated a different distribution pattern with a rapid particle retention in all organs (particularly in liver and spleen) and a subsequent slow release rate. CONCLUSION: The mathematical model demonstrated good agreement with experimental data derived from tumor mouse models suggesting the value of this tool to predict the real-time pharmacokinetic features of SPIONs in vivo. In the future, it is planned to adapt our model to other nanoparticle formulations to more precisely describe their biodistribution in in vivo model systems.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine ß93 is the sole attachment moiety to the αß-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.