Slow Pharmacokinetics Research Articles

Bioorthogonal Diels-Alder Click Chemistry-Based Pretargeted PET Imaging Strategy for Monitoring Programmed Death-Ligand 1 Expression.

The development of antibody tracers for positron emission tomography (PET) imaging enables real-time monitoring of tumor expression of programmed cell death ligand 1 (PD-L1) in vivo, aiming to facilitate the selection of immunotherapy responders. However, the slow pharmacokinetics of the antibodies in vivo limits their applications in PET imaging with commonly used radionuclides with short half-lives. In this study, we developed a pretargeted PET imaging strategy based on Diels-Alder (IEDDA) click chemistry to overcome these limitations. Atezolizumab and durvalumab, the most commonly used PD-L1 antibodies in cancer immunotherapy, were selected and compared in the development of the pretargeted PET imaging strategy. Fluorine-18-labeled derivatives of methyl tetrazine ([18F]Tz, [18F]PEG6-Tz, and [18F]PEG12-Tz) were tested in biodistribution and PET imaging of A549-PDL1 xenografts (PD-L1 positive) pretargeted with the trans-cyclooctene (TCO)-functionalized atezolizumab/durvalumab. The biodistribution and imaging results indicated that atezolizumab-TCO/[18F]PEG12-Tz was more suitable for pretargeted PET imaging strategy, and the optimal interval time was 48 h after atezolizumab-TCO administration, where the atezolizumab-TCO/[18F]PEG12-Tz pretargeted approach clearly delineated the A549-PDL1 tumor with a tumor-to-muscle ratio of 5.33, while the ratios are 3.39 and 2.39 for durvalumab/[18F]PEG12-Tz and mock-pretargeting controls, respectively. In conclusion, a pretargeted 18F-immuno-PET imaging technology was successfully established on atezolizumab. The high-contrast PET images of the A549-PDL1 tumor models demonstrate that the pretargeting strategy incorporating short-lived fluorine-18 is viable in identifying tumors with high PD-L1 expression, marking this strategy as a potential candidate for further clinical translation.

Targeting Breast Cancer Using 177 Lu-Labeled Trastuzumab and Trastuzumab Fragment : First-in-Human Clinical Experience.

A monoclonal antibody, trastuzumab, is used for immunotherapy for HER2-expressing breast cancers. Large-sized antibodies demonstrate hepatobiliary clearance and slower pharmacokinetics. A trastuzumab fragment (Fab; 45 kDa) has been generated for theranostic use. Fab was generated by papain digestion. Trastuzumab and Fab have been radiolabelled with 177 Lu after being conjugated with a bifunctional chelating. The affinity and target specificity were studied in vitro. The first-in-human study was performed. The bifunctional chelating agent conjugation of 1-2 molecules with trastuzumab and Fab was detected at the molar ratio 1:10 in bicarbonate buffer (0.5 M, pH 8) at 37°-40°C. However, 2-3 molecules of bifunctional chelating agent were conjugated when DMSO in PBS (0.1 M, pH 7) was used as a conjugation buffer at a molar ratio of 1:10. The radiolabelling yield of DOTA-conjugated Fab and trastuzumab at pH 5, 45°C to 50°C, with incubation time 2.5-3 hours was 80% and 41.67%, respectively. However, with DOTAGA-conjugated trastuzumab and Fab, the maximum radiolabelling yield at pH 5.5, 37°C, and at 2.5-3 hours was 80.83% and 83%, respectively. The calculated K d of DOTAGA Fab and trastuzumab with HER2-positive SKBR3 cells was 6.85 ± 0.24 × 10 -8 M and 1.71 ± 0.10 × 10 -8 M, respectively. DOTAGA-Fab and trastuzumab showed better radiolabelling yield at mild reaction conditions.177 Lu-DOTAGA-Fab demonstrated higher lesion uptake and lower liver retention as compared with 177 Lu-DOTAGA-trastuzumab. However, 177 Lu-DOTAGA-Fab as compared with 177 Lu-DOTAGA-trastuzumab showed a relatively early washout (5 days) from the lesion. 177 Lu-DOTAGA-Fab and trastuzumab are suitable for targeting the HER2 receptors.

Abstract 6033: Preclinical evaluation of an actinium-225 labeled PSMA-targeting small molecule (225Ac-PSMA-Trillium (BAY 3563254)) for the treatment of metastatic castration resistant prostate cancer (mCRPC)

Abstract PSMA is a clinically validated target for the treatment of mCRPC. Since the linear energy transfer and degree of induced DNA damage of alpha particles far exceeds that of beta particles, several investigational PSMA-targeted small molecules labeled with the alpha-emitter actinium-225 (225Ac) have shown encouraging clinical signs of efficacy. However, xerostomia is often found as a dose-limiting side effect, presumably due to low levels of PSMA expression and efficient uptake of small molecules in salivary glands. To reduce these unwanted side effects a PSMA-targeted radioligand comprising a triad of important features was investigated. It consists of a high-affinity PSMA binder for specific tumor-targeting; a customized albumin-binding moiety designed to prolong plasma residence time and increase the therapeutic index by improving tumor uptake while reducing salivary gland uptake; and a chelator for efficient complexation of 225Ac. From a series of compounds containing the same high-affinity PSMA-targeting moiety, but different chain lengths and albumin-binding domains, compound RPS-072 containing a PEG-3 and PEG-8 chain and an iodobenzyl moiety as albumin-binder was selected for further evaluation (J Nucl Med 2019; 60:656-663). This compound was the basis for the PSMA-Trillium with a DOTA chelator for imaging applications in humans (NCT05773703) or a macropa chelator for 225Ac labeling resulting in 225Ac-PSMA-Trillium. In vitro preclinical characterization of 225Ac-PSMA-Trillium was performed to test affinities for PSMA and albumin. In a SPR assay, Kd was measured as 4.88x1011 M, Kon as 1.4x107 1/Ms and Koff as 6.6x10-4 1/Ms. 225Ac-PSMA-Trillium demonstrated good stability up to 48 h and induced potent in vitro cytotoxicity in the LNCaP subclone C4-2 (0.114 kBq/ml). In vivo biodistribution of 225Ac-PSMA-Trillium showed consistent with the albumin binding property a relatively slow pharmacokinetics profile with 5 % ID/g still in the blood after 24 h and tumor accumulation peaking at ~ 20 % ID/g after 5-7 days. Tumor penetration was investigated using autoradiography which demonstrated fast, homogeneous, and even distribution across the tumor within 15 mins after injection. This property was also reflected by strong therapeutic efficacy in several PrCa models. In the LNCaP model a tumor growth inhibition was observed at a single dose dependent efficacy at 150 kBq/kg and 300 kBq/kg and increased the time to reach 400 mm3 by 35 days for the 300 kBq/kg group. In the androgen independent PDX KuCaP-1 model, a single dose of 250 kBq/kg 225Ac-PSMA-Trillium induced strong tumor growth inhibition over the course of 35 days. A first-in-human trial with 225Ac-PSMA-Trillium is anticipated to start in the first half of 2024. Citation Format: Sabine Zitzmann-Kolbe, Alex Papple, Thorsten Poethko, Ingrid Moen, Christoph Schatz, Shawn Hillier, John W. Babich, Alan Cuthbertson, Urs B. Hagemann. Preclinical evaluation of an actinium-225 labeled PSMA-targeting small molecule (225Ac-PSMA-Trillium (BAY 3563254)) for the treatment of metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6033.

Preclinical characterisation of gallium-68 labeled ferrichrome siderophore stereoisomers for PET imaging applications

BackgroundSiderophores are small iron-binding molecules produced by microorganisms to facilitate iron acquisition from the environment. Radiolabelled siderophores offer a promising solution for infection imaging, as they can specifically target the pathophysiological mechanisms of pathogens. Gallium-68 can replace the iron in siderophores, enabling molecular imaging with positron emission tomography (PET). Stereospecific interactions play a crucial role in the recognition of receptors, transporters, and iron utilisation. Furthermore, these interactions have an impact on the host environment, affecting pharmacokinetics and biodistribution. This study examines the influence of siderophore stereoisomerism on imaging properties, with a focus on ferrirubin (FR) and ferrirhodin (FRH), two cis–trans isomeric siderophores of the ferrichrome type.ResultsTested siderophores were labelled with gallium-68 with high radiochemical purity. The resulting complexes differed in their in vitro characteristics. [68Ga]Ga-FRH showed less hydrophilic properties and higher protein binding values than [68Ga]Ga-FR. The stability studies confirmed the high radiochemical stability of both [68Ga]Ga-siderophores in all examined media. Both siderophores were found to be taken up by S. aureus, K. pneumoniae and P. aeruginosa with similar efficacy. The biodistribution tested in normal mice showed rapid renal clearance with low blood pool retention and fast clearance from examined organs for [68Ga]Ga-FR, whereas [68Ga]Ga-FRH showed moderate retention in blood, resulting in slower pharmacokinetics. PET/CT imaging of mice injected with [68Ga]Ga-FR and [68Ga]Ga-FRH confirmed findings from ex vivo biodistribution studies. In a mouse model of S. aureus myositis, both radiolabeled siderophores showed radiotracer accumulation at the site of infection.ConclusionsThe 68Ga-complexes of stereoisomers ferrirubin and ferrirhodin revealed different pharmacokinetic profiles. In vitro uptake was not affected by isomerism. Both compounds had uptake with the same bacterial culture with similar efficacy. PET/CT imaging showed that the [68Ga]Ga-complexes accumulate at the site of S. aureus infection, highlighting the potential of [68Ga]Ga-FR as a promising tool for infection imaging. In contrast, retention of the radioactivity in the blood was observed for [68Ga]Ga-FRH. In conclusion, the stereoisomerism of potential radiotracers should be considered, as even minor structural differences can influence their pharmacokinetics and, consequently, the results of PET imaging.

Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer

Abstract Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy. Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.

Deep Eutectic Solvents for Subcutaneous Delivery of Protein Therapeutics.

Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic-based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold-standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic-based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.

Antibody-based Radiopharmaceuticals as Theranostic Agents: An Overview.

Since the inception of antibodies as magic bullets for targeting antigens with high specificity for various in vitro and in-vivo detection and therapy applications, the field has evolved, and remarkable success has been achieved not only in the methods of development of these targeting agents but also in their applications. The utilization of these moieties for the development of antibody-based radiopharmaceuticals for diagnostic and therapy (theranostic) purposes has resulted in the availability of various cancer-targeting agents suitable for clinical applications. The high affinity and specificity of antibodies towards the target antigens overexpressed on tumors render them an excellent carrier molecules for radionuclide delivery. Although intact antibodies have high potential as imaging and therapeutic agents, a major drawback of intact antibody-based radionuclide targeting is their slow pharmacokinetics and poor penetration into solid tumors. In contrast to large intact antibodies, engineered antibody fragments, such as minibodies, diabodies, single-chain variable region fragments (scFvs), nanobodies, and non-antibody protein scaffolds-based moieties, retain the specificities and affinities of intact antibodies in addition to improved pharmacokinetics for imaging and therapy of solid tumors. These engineered carrier molecules are not only amenable for simple and robust radiolabeling procedures but also provide high contrast images with minimal radiotoxicity to vital organs. However, in various instances, rapid clearance with sub-optimal tumor accumulation, limiting renal dose, and cross-reactivity of these radiolabeled engineered smaller molecules have also been observed. Herein, we review current knowledge of the recent methods for the development of antibody-based targeting moieties, the suitability of various engineered formats for targeting tumors, and radiolabeling strategies for the development of radioformulations. We discuss promising antibody-based and non-antibody- based affibody radiopharmaceuticals reported for clinical applications. Finally, we highlight how emerging technologies in antibody engineering and drug development can be amalgamated for designing novel strategies for cancer imaging and therapy.

28P Development 68Ga trastuzumab Fab and bioevaluation by PET imaging in HER2/neu-expressing breast cancer patients

A target-specific mAb for the HER2 receptor is trastuzumab, which is used for treatment of breast cancer. This mAb coupled with radionuclides β or α particle emitters are used for radioimmunotherapy. However, radioimmunotherapy with the whole mAb is still limited due to slow pharmacokinetics. This leads to the slow elimination of trastuzumab from the blood and low tumor to blood and tumor to background ratios. To limit this, a small molecule Fab was introduced but the role in HER2 detection by immuno-PET imaging is unknown.

Synthesis and ex vivo biodistribution of two 68Ga-labeled tetrazine tracers: Comparison of pharmacokinetics

Pretargeted PET imaging allows the use of radiotracers labeled with short-living PET radionuclides for tracing drugs with slow pharmacokinetics. Recently, especially methods based on bioorthogonal chemistry have been under intensive investigation for pretargeted PET imaging. The pharmacokinetics of the radiotracer is one of the factors that determine the success of the pretargeted strategy. Here, we report synthesis and biological evaluation of two 68Ga-labeled tetrazine (Tz)-based radiotracers, [68Ga]Ga-HBED-CC-PEG4-Tz ([68Ga]4) and [68Ga]Ga-DOTA-PEG4-Tz ([68Ga]6), aiming for development of new tracer candidates for pretargeted PET imaging based on the inverse electron demand Diels-Alder (IEDDA) ligation between a tetrazine and a strained alkene, such as trans-cyclooctene (TCO). Excellent radiochemical yield (RCY) was obtained for [68Ga]4 (RCY > 96%) and slightly lower for [68Ga]6 (RCY > 88%). Radiolabeling of HBED-CC-Tz proved to be faster and more efficient under milder conditions compared to the DOTA analogue. The two tracers exhibited excellent radiolabel stability both in vitro and in vivo. Moreover, [68Ga]4 was successfully used for radiolabeling two different TCO-functionalized nanoparticles in vitro: Hepatitis E virus nanoparticles (HEVNPs) and porous silicon nanoparticles (PSiNPs).

Development 68Ga trastuzumab Fab and bioevaluation by PET imaging in HER2/neu expressing breast cancer patients.

Receptors on breast cancer cells play a crucial role in the management of patients. Trastuzumab is a widely used drug for the treatment of HER2/neu expressing tumors. ImmunoPET with trastuzumab is not feasible due to slow pharmacokinetics. Fragment of antigen-binding (Fab) radiolabeled with positron emitters can be used for immunoPET. Fab has been generated by papain digestion and conjugated with the bifunctional chelating agent NOTA. The SDS-PAGE and MALDI-TOF were used to see the integrity of Fab and conjugated Fab. In-vitro stability and target specificity for HER2/neu receptors were performed in plasma and receptor binding with bio-layer interferometry (BLI) techniques. Radiolabeling was standardized with 68GaCl3 and PET imaging was performed in seven patients showing 18F fluorodeoxyglucose (18F-FDG) uptake and correlated with HER2/neu expression by immunohistochemistry. Fab production was optimized at molar ratio 23:1 of trastuzumab and papain at 37 °C with a constant stirrer at 850 rpm for 22-24 h, at pH 8. Conjugation with NOTA was standardized at molar ratio 1:25 of trastuzumab Fab and NOTA. Molecular mass of trastuzumab Fab-NOTA was found approximately 46.3 kDa (~1/3 of intact antibody). Trastuzumab Fab-NOTA showed radiolabelling efficiency of 48-70% with incubation time 15 min at 37-40 °C and pH 4.5-5.0. BLI demonstrated the affinity of trastuzumab, trastuzumab Fab and trastuzumab Fab-NOTA towards HER2/neu receptor with KD of <1pM, ~0.5nM and ~20nM, respectively. All immunohistochemistry proven patients showed uptake in primary breast lesion and lymph nodes. Trastuzumab Fab-NOTA is suitable for radiolabelling with 68Ga and ImmunoPET imaging of HER2/neu receptor.

The Race for Hydroxamate-Based Zirconium-89 Chelators.

Metallic radionuclides conjugated to biological vectors via an appropriate chelator are employed in nuclear medicine for the diagnosis (imaging) and radiotherapy of diseases. For the application of radiolabeled antibodies using positron emission tomography (immunoPET), zirconium-89 has gained increasing interest over the last decades as its physical properties (t1/2 = 78.4 h, 22.6% β+ decay) match well with the slow pharmacokinetics of antibodies (tbiol. = days to weeks) allowing for late time point imaging. The most commonly used chelator for 89Zr in this context is desferrioxamine (DFO). However, it has been shown in preclinical studies that the hexadentate DFO ligand does not provide 89Zr-complexes of sufficient stability in vivo and unspecific uptake of the osteophilic radiometal in bones is observed. For clinical applications, this might be of concern not only because of an unnecessary dose to the patient but also an increased background signal. As a consequence, next generation chelators based on hydroxamate scaffolds for more stable coordination of 89Zr have been developed by different research groups. In this review, we describe the progress in this research field until end of 2020, including promising examples of new candidates of chelators currently in advanced stages for clinical translation that outrun the performance of the current gold standard DFO.

PSMA-Targeted Therapeutics: A Tale About Law and Economics.

1482 Introduction: Thorium-227 (227Th) offers a longer half-life, 18.7 days, than other Targeted Alpha Therapy (TAT) agents currently being evaluated clinically such as actinium-225 (t1/2= 9.9 days) and astatine-211 (t1/2= 7.2 hours). This physical characteristic favors its use with biomolecules that have slower pharmacokinetics like monoclonal antibodies. Utilizing 227Th is also advantageous due to the 5 alpha emissions occurring during decay. Hydroxypyridinonate (HOPO) derivatives have been shown to be effective decorporation agents for actinides owing to their hard oxygen donor atoms and strong complexation. Therefore, utilizing these molecules as chelators for potential nuclear medicine agents should allow for radiometals to be effectively sequestered by the ligand. The biological chemistry of thorium is similar to calcium in that the unbound radiometal tends to localize in bone. Consequently, it is pivotal to establish a thorium complex that possesses high stability and thwarts bone uptake. Methods: 227Th received from Oak Ridge National Laboratory was purified from its daughters on a MP1 BioRad resin. The purified thorium was then complexed to the 3,4,3-(LI-1,2-HOPO) ligand by adding the radiometal to a 0.1 M acetate solution containing the ligand (0.08 mM) for 1 hour at 50 °C and was purified using Chelex resin column. The biological distribution of the 227Th-HOPO complex was compared to the “bare” 227Th (227Th-nitrate in 0.1 M HCl/ 5 M acetate solution) in male CD1 mice (n=3) injected with 1 μCi of either construct. At the prescribed time points (15 m, 1 h, 3 h, 6 h, 24 h, 3 d, 7 d, 14 d, 21 d, and 28 d) the organs of interest were harvested and counted using a gamma counter calibrated for 227Th. The biodistribution data was used to calculate the residence time in each organ and was then entered into the OLINDA software to produce the effective dose in rem/mCi. Terminal SPECT/CT imaging of 227Th-nitrate and 227Th-HOPO was obtained in representative mice at 20 min post injection of 15 µCi. Results: The biodistribution results for the 227Th-nitrate species shows clearance through the kidneys (%ID/g = 26.3 ±5.3, 1-day p.i.). Also, substantial localization in the bone as expected is observed. At 15 minutes p.i. the %ID/g in the bone is 7.1 ± 3.9 and gradually increases through the 7-day timepoint, %ID/g- 28.8 ± 12.8, ending the study 28 days p.i with an accumulation of 33.6 ± 17.5 %ID/g. Conversely the 227Th-HOPO species appears to clear through the hepatobiliary tract with accumulation in the gallbladder (54.2 ± 27.7 %ID/g, 1-hour p.i.) and the intestines (29.5 ± 2.2 %ID/g, 1-hour p.i.). Most significantly the bone localization is relatively low and remains constant, approximately 1.5 %ID/g, through all timepoints. The low bone uptake is also reflected in the dosimetry where the effective dose in the bone for 227Th-nitrate and 227Th-HOPO is 1680 rem/mCi and 192 rem/mCi respectively. The effective dose of the 227Th-HOPO species is at least 1 order of magnitude lower than the nitrate species in all organs. Conclusion: We have shown that the 227Th-HOPO complex is stable in vivo and facilitates rapid elimination from the body which results in lower dose to organs, specifically the bone. The stability of the 227Th-HOPO complex suggests that it has potential for use in TAT. Acknowledgments: This work has been funded by BNL LDRD 21-00 and Seed grant under B&R code YN0100000. We would also like to thank all those who assisted with the project.

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.

Evaluation of 18F‐JNJ‐067 as a tau tracer

AbstractBackgroundCurrent tau PET tracers have obvious shortcomings. 18F‐JNJ‐64326067 (18F‐JNJ‐067) is a new tau tracer that binds to aggregated tau in Alzheimer’s Disease (AD) brain tissue with Ki=2.4nM. Our objective was to evaluate 18F‐JNJ‐067 in individuals with AD (N=3), Mild Cognitive Impairment (MCI, N=3), Progressive Supranuclear Palsy (PSP, N=2) and healthy controls (HC, N=4).MethodFollowing a 50‐70min 11C‐PIB PET scan (Siemens Biograph PET/CT), 12 subjects underwent 90 min dynamic PET scan after 18F‐JNJ‐067 injection. MPRAGEs were acquired on a Siemens 3T and segmented using FreeSurfer. Time activity and SUVR curves were viewed for commentary on steady state. Logan DVR quantification was calculated from 35‐90min (k2ref=0.025 min‐1 derived from SRTM2 analysis) using inferior cerebellar gray as a reference region. Data quantification was done in native space; images were warped to template space for visualization.ResultSubject information is shown in figure 1; all AD and MCI subjects were PIB positive with MMSE scores from 18‐30, all PSP and HCs were PIB negative. Steady state was not reached in ROIs with high levels of tracer retention within 90 minutes (SUVR curves shown in figure 2). Figure 1 shows native space DVR quantification for ROIs. ADs had 18F‐JNJ‐067 binding in typical AD regions, one MCI had elevated binding, the others showed low binding (DVR images in figure 3). HCs showed no binding in entorhinal cortex, and had elevated signal in basal ganglia, midbrain, superior cerebellar gray, and white matter but no binding in the choroid plexus or meninges (figure 4). MMSE was correlated with Braak I/II (r2=0.80), Braak III/IV (r2=0.73) and Braak V/VI (r2=0.50) ROIs.Conclusion18F‐JNJ‐067 shows relatively slow pharmacokinetics in brain regions with high target density, has low levels of probable off‐target binding in the basal ganglia and white matter but not elsewhere, and low entorhinal signal in HCs and MCIs. Binding in AD patients reflects a typical pattern of tau distribution seen with other tracers, and MCI patients are intermediate. Braak ROIs are significantly correlated to MMSE. These data suggest that 18F‐JNJ‐067 is suitable for investigation of the AD continuum.

Radiolabeled proteins for the development of biopharmaceuticals

Positron emission tomography (PET) is a molecular imaging technique that visualizes pathophysiology in the body using radiotracers at tracer doses (~μg). PET would provide the information regarding not only pharmacokinetics of radiolabeled compounds, but also target engagements, patient selection, and biomarkers. Previously small molecules are widely used as radiotracers, but recently biopharmaceuticals are launched, providing a novel type radiotracer. In general, antibodies are radiolabeled by long physiological half-lives radionuclides such as Cu-64 and Zr-89 because of their slow pharmacokinetics. However, shorter half-lives radiolabeled tracers (C-11 and F-18) might be suitable on the point view of radiation. Now small protein ligands such as affibodies (~7 kDa) are developed as a radiotracer. We are trying to develop a novel approach to label proteins for PET imaging, which are based on radiolabeled amino acids and cell-free protein synthesis system. In this review, we introduced the topics of protein-based PET tracers.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

A Pretargeted Imaging Strategy for Immune Checkpoint Ligand PD-L1 Expression in Tumor Based on Bioorthogonal Diels-Alder Click Chemistry.

The use of antibodies as tracers requires labeling with isotopes with long half-lives due to their slow pharmacokinetics, which creates prohibitively high radiation dose to non-target organs. Pretargeted methodology could avoid the high radiation exposure due to the slow pharmacokinetics of antibodies. In this investigation, we reported the development of a novel pretargeted single photon emission computed tomography (SPECT) imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry. The radioligand [99mTc]HYNIC-PEG11-Tz was achieved by the synthesis of a 6-hydrazinonicotinc acid (HYNIC) modified 1,2,4,5-tetrazine (Tz) and subsequently radiolabeled with technetium-99m (Tc-99m). The stability of [99mTc]HYNIC-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Atezolizumab was modified with trans-cyclooctene (TCO). The [99mTc]HYNIC-PEG11-Tz and atezolizumab-TCO interaction was tested in vitro. Pretargeted H1975 cell immunoreactivity binding and saturation binding assays were evaluated. Pretargeted biodistribution and SPECT imaging experiments were performed in H1975 and A549 tumor-bearing modal mice to evaluate the PD-L1 expression level. [99mTc]HYNIC-PEG11-Tz was successfully radiosynthesized with a specific activity of 9.25MBq/μg and a radiochemical purity above 95% as confirmed by reversed-phase HPLC (RP-HPLC). [99mTc]HYNIC-PEG11-Tz showed favorable stability in NS, PBS, and FBS and rapid blood clearance in mice. The atezolizumab was modified with TCO-NHS ester to produce a conjugate with an average 6.4 TCO moieties as confirmed by liquid chromatograph-mass spectrometer (LC-MS). Size exclusion HPLC revealed almost complete reaction between atezolizumab-TCO and [99mTc]HYNIC-PEG11-Tz in vitro, with the 1:1 Tz-to-mAb reaction providing a conversion yield of 88.65 ± 1.22%. Pretargeted cell immunoreactivity binding and saturation binding assays showed high affinity to H1975 cells. After allowing 48h for accumulation of atezolizumab-TCO in H1975 tumor, pretargeted in vivo biodistribution revealed high uptake of the radiotracer in the tumor with a tumor-to-muscle ratio of 27.51 and tumor-to-blood ratio of 1.91. Pretargeted SPECT imaging delineated the H1975 tumor clearly. Pretargeted biodistribution and SPECT imaging in control groups demonstrated a significantly reduced tracer accumulation in the A549 tumor. We have developed a HYNIC-modified Tz derivative, and the HYNIC-PEG11-Tz was labeled with Tc-99m with a high specific activity and radiochemical purity. [99mTc]HYNIC-PEG11-Tz reacted rapidly and almost completely towards atezolizumab-TCO in vitro with the 1:1 Tz-to-mAb reaction. SPECT imaging using the pretargeted strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) demonstrated high-contrast images for high PD-L1 expression H1975 tumor and a low background accumulation of the probe. The pretargeted imaging strategy is a powerful tool for evaluating PD-L1 expression in xenograft mice tumor models and a potential candidate for translational clinical application.

A method for the minimally invasive drug monitoring of mitotane by means of volumetric absorptive microsampling for a home-based therapeutic drug monitoring.

Mitotane is the only currently approved treatment for adrenocortical carcinoma (ACC), a rare endocrine malignancy. Plasma levels within the range of 14 to 20mgL-1 are correlated with higher clinical efficacy and manageable toxicity. Because of this narrow therapeutic index and slow pharmacokinetics, therapeutic drug monitoring is an essential element of mitotane therapy. A small step towards the therapeutic drug monitoring (TDM) by volumetric absorptive microsampling (VAMS) was made with this work. A simple method enabling the patient to collect capillary blood at home for the control of mitotane blood concentration was developed and characterized using MITRA™ VAMS 20μL microsampler. Dried blood samples were extracted prior to HPLC-UV analysis. Mitotane and the internal standard dicofol (DIC) were detected at 230nm by ultra-violet detection after separation on a C8 reversed phase column. The assay was validated in the range of 1 to 50mgL-1. Dried samples were stable at room temperature and at 2-8°C for 1week. At 37°C, a substantial amount of the analyte was lost probably due to evaporation. Hematocrit bias, a common problem of conventional dried blood techniques, was acceptable in the tested range. However, a significant difference in recovery from spiked and authentic patient blood was detected. Comparison of mitotane concentration in dried blood samples (CDBS) by VAMS with venous plasma in patients on mitotane therapy demonstrated poor correlation of CDBS with the concentration in plasma (CP). In conclusion, application of VAMS in clinical routine for mitotane TDM appears to be of limited value in the absence of a method-specific target range.

C-547, a 6-methyluracil derivative with long-lasting binding and rebinding on acetylcholinesterase: Pharmacokinetic and pharmacodynamic studies

C-547, a potent slow-binding inhibitor of acetylcholinesterase (AChE) was intravenously administered to rat (0.05 mg/kg). Pharmacokinetic profiles were determined in blood and different organs: extensor digitorum longus muscle, heart, liver, lungs and kidneys as a function of time. Pharmacokinetics (PK) was studied using non-compartmental and compartmental analyses. A 3-compartment model describes PK in blood. Most of injected C-547 binds to albumin in the bloodstream. The steady-state volume of distribution (3800 ml/kg) is 15 times larger than the distribution volume, indicating a good tissue distribution. C-547 is slowly eliminated (kel = 0.17 h−1; T1/2 = 4 h) from the bloodstream.Effect of C-547 on animal model of myasthenia gravis persists for more than 72 h, even though the drug is not analytically detectable in the blood. A PK/PD model was built to account for such a pharmacodynamical (PD) effect. Long-lasting effect results from micro-PD mechanisms: the slow-binding nature of inhibition, high affinity for AChE and long residence time on target at neuromuscular junction (NMJ). In addition, NMJ spatial constraints i.e. high concentration of AChE in a small volume, and slow diffusion rate of free C-547 out of NMJ, make possible effective rebinding of ligand. Thus, compared to other cholinesterase inhibitors used for palliative treatment of myasthenia gravis, C-547 is the most selective drug, displays a slow pharmacokinetics, and has the longest duration of action. This makes C-547 a promising drug leader for treatment of myasthenia gravis, and a template for development of other drugs against neurological diseases and for neuroprotection.

Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression.

Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.