ABSTRACT
Chemotherapy is the first-line treatment for cancer, but its systemic toxicity can be severe. Tumor-selective prodrug activation offers promising opportunities to reduce systemic toxicity. Here, we present a strategy for activating prodrugs using radiopharmaceuticals. This strategy enables the targeted release of chemotherapeutic agents due to the high tumor-targeting capability of radiopharmaceuticals. [18F]FDG (2-[18F]-fluoro-2-deoxy-D-glucose), one of the most widely used radiopharmaceuticals in clinics, can trigger Pt(IV) complex for controlled release of axial ligands in tumors, it might be mediated by hydrated electrons generated by water radiolysis resulting from the decay of radionuclide 18F. Its application offers the controlled release of fluorogenic probes and prodrugs in living cells and tumor-bearing mice. Of note, an OxaliPt(IV) linker is designed to construct an [18F]FDG-activated antibody-drug conjugate (Pt-ADC). Sequential injection of Pt-ADC and [18F]FDG efficiently releases the toxin in the tumor and remarkably suppresses the tumor growth. Radiotherapy is booming as a perturbing tool for prodrug activation, and we find that [18F]FDG is capable of deprotecting various radiotherapy-removable protecting groups (RPGs). Our results suggest that tumor-selective radiopharmaceutical may function as a trigger, for developing innovative prodrug activation strategies with enhanced tumor selectivity.
ABSTRACT
Antimony-119 (119Sb) holds promise for radiopharmaceutical therapy (RPT), emitting short-range Auger and conversion electrons that can deliver cytotoxic radiation on a cellular level. While it has high promise theoretically, experimental validation is necessary for 119Sb in vivo applications. Current 119Sb production and separation methods face robustness and compatibility challenges in radiopharmaceutical synthesis. Limited progress in chelator development hampers targeted experiments with 119Sb. This review compiles literature on the toxicological, biodistribution and redox properties of Sb, along with existing Sb complexes, evaluating their suitability for radiopharmaceuticals. Sb(III) is suggested as the preferred oxidation state for radiopharmaceutical elaboration due to its stability in vivo and lack of skeletal uptake. While Sb complexes with both hard and soft donor atoms can be achieved, Sb thiol complexes offer enhanced stability and compatibility with the desired Sb(III) oxidation state. For 119Sb to find application in RPT, scientists need to make discoveries and advancements in the areas of isotope production, and radiometal chelation. This review aims to guide future research towards harnessing the therapeutic potential of 119Sb in RPT.
ABSTRACT
Alzheimer disease is a neurodegenerative disorder with limited treatment options. It is characterized by the presence of several biomarkers, including amyloid-ß aggregates, which lead to oxidative stress and neuronal decay. Targeted α-therapy (TAT) has been shown to be efficacious against metastatic cancer. TAT takes advantage of tumor-localized α-particle emission to break disease-associated covalent bonds while minimizing radiation dose to healthy tissues due to the short, micrometer-level, distances traveled. We hypothesized that TAT could be used to break covalent bonds within amyloid-ß aggregates and facilitate natural plaque clearance mechanisms. Methods: We synthesized a 213Bi-chelate-linked benzofuran pyridyl derivative (BiBPy) and generated [213Bi]BiBPy, with a specific activity of 120.6 GBq/µg, dissociation constant of 11 ± 1.5 nM, and logP of 0.14 ± 0.03. Results: As the first step toward the validation of [213Bi]BiBPy as a TAT agent for the reduction of Alzheimer disease-associated amyloid-ß, we showed that brain homogenates from APP/PS1 double-transgenic male mice (6-9 mo old) incubated with [213Bi]BiBPy exhibited a marked reduction in amyloid-ß plaque concentration as measured using both enzyme-linked immunosorbent and Western blotting assays, with a half-maximal effective concentration of 3.72 kBq/pg. Conclusion: This [213Bi]BiBPy-concentration-dependent activity shows that TAT can reduce amyloid plaque concentration in vitro and supports the development of targeting systems for in vivo validations.
ABSTRACT
Immunotherapy has transformed cancer treatment. Nevertheless, given the heterogeneity of clinical efficacy, the multiplicity of treatment options available and the possibility of serious adverse effects, selecting the most effective treatment has become the greatest challenge. Molecular imaging offers an attractive way for this purpose. ImmunoPET provides specific imaging with positron emission tomography (PET) using monoclonal antibodies (mAb) or its fragments as vector. By combining the high targeting specificity of mAb and the sensitivity of PET technique, immunoPET could noninvasively and dynamically reveal tumor antigens expression and provide theranostic tools of several types of malignancies. Because of their slow kinetics, mAbs require radioelements defined by a consistent half-life. Zirconium 89 (89Zr) and Copper 64 (64Cu) are radiometals with half-lives suitable for mAb labeling. Radiolabeling with a radiometal requires the prior use of a bifunctional chelate agent (BFCA) to functionalize mAb for radiometal chelation, in a second step. There are a number of BFCA available and much research is focused on antibody functionalization techniques or on developing the optimum chelating agent depending the selected radiometal. In this manuscript, we present a critical account of radiochemical techniques with radionuclides 89Zr and 64Cu and their applications in preclinical and clinical immuno-PET imaging.
ABSTRACT
INTRODUCTION: Microtubule (MT) stability is crucial for proper neuronal function. Understanding MT dysregulation is critical for connecting amyloid beta (Aß) and tau-based degenerative events and early changes in presymptomatic Alzheimer's disease (AD). Herein we present positron emission tomography (PET) imaging properties of our MT-PET radiotracer, [11C]MPC-6827, in multiple established AD mouse models. METHODS: Longitudinal PET, biodistribution, autoradiography, immunohistochemistry, and behavioral studies were conducted at multiple time points in APPswe/PSEN1dE9 (APP/PS1), P301S-PS19 (P301S), 5xFAD, and age-matched control mice. RESULTS: Longitudinal [11C]MPC-6827 brain imaging showed significant increases in APP/PS1, P301S, and 5xFAD mice compared to controls. Longitudinal MT-PET correlated positively with biodistribution, autoradiography, and immunohistochemistry results and negatively with behavior data. DISCUSSION: Our study demonstrated significant longitudinal [11C]MPC-6827 PET increases in multiple AD mouse models for the first time. Strong correlations between PET and biomarker data underscored the interplay of MT destabilization, amyloid, and tau pathology in AD. These results suggest [11C]MPC-6827 PET as a promising tool for monitoring MT dysregulation early in AD progression. HIGHLIGHTS: Longitudinal positron emission tomography (PET) imaging studies using [11C]MPC-6827 in multiple established Alzheimer's disease (AD) mouse models revealed an early onset of microtubule dysregulation, with significant changes in brain radiotracer uptake evident from 2 to 4 months of age. Intra-group analysis showed a progressive increase in microtubule dysregulation with increasing AD burden, supported by significant correlations between PET imaging data and biodistribution, autoradiography, and molecular pathological markers. [11C]MPC-6827 PET imaging demonstrated its efficacy in detecting early microtubule alterations preceding observable behavioral changes in AD mouse models, suggesting its potential for early AD imaging. The inclusion of the 5xFAD mouse model further elucidated the impact of amyloid beta (Aß) toxicity on inducing tau hyperphosphorylation-mediated microtubule dysregulation, highlighting the versatility of [11C]MPC-6827 in delineating various aspects of AD pathology. Our study provides immediate clarity on high uptake of the microtubule-based radiotracer in AD brains in a longitudinal setting, which directly informs clinical utility in Aß/tau-based studies.
ABSTRACT
Bismuth-213 is a radionuclide of interest for targeted alpha therapy and is supplied via a radiochemical generator system through the decay of 225Ac. Radionuclide generators employ longer lived "parent" radionuclides to routinely supply shorter-lived "daughter" radionuclides. The traditional 225Ac/213Bi radiochemical generator relies on an organic cation exchange resin where 225Ac binds to the resin and 213Bi is routinely eluted. These resins degrade when they absorb large doses of ionizing radiation (>1 × 106 Gy/mg), which has been observed when the loading activity of 225Ac exceeds 2.59*109 Bq (70 mCi). Herein we report the development of an electrochemical generator for the supply of 213Bi that has the potential to overcome this limitation. Bismuth-213 spontaneously electrodeposits onto nickel foils in 0.1 M hydrochloric acid at 70 °C. Using this method, we were able to plate an average of 73 ± 4 % of the 213Bi in solution and obtain a final 213Bi recovery of 65 ± 8 % in 0.1 M citrate pH 4.5 via reverse electrolysis using titanium as the cathode. The recovered 213Bi had an average radiochemical purity of >99.8 % and was successfully used to radiolabel DOTATATE with an average radiochemical yield of 85.1 % (not optimized).
ABSTRACT
Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.
Subject(s)
Click Chemistry , Copper , Cycloaddition Reaction , Fluorine Radioisotopes , Peptides , Click Chemistry/methods , Fluorine Radioisotopes/chemistry , Peptides/chemistry , Copper/chemistry , Isotope Labeling/methods , Automation , Catalysis , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/chemical synthesisABSTRACT
The establishment of a compliant radiopharmacy facility within a university setting is crucial for supporting fundamental and preclinical studies, as well as for the production of high-quality radiopharmaceuticals for clinical testing in human protocols as part of Investigational New Drug (IND) applications that are reviewed and approved by the U.S. Food and Drug Administration (FDA). This manuscript details the design and construction of a 550 ft2 facility, which included a radiopharmacy and a radiochemistry laboratory, to support radiopharmaceutical development research and facilitate translational research projects. The facility was designed to meet FDA guidelines for the production of aseptic radiopharmaceuticals in accordance with current good manufacturing practice (cGMP). A modular hard-panel cleanroom was constructed to meet manufacturing classifications set by the International Organization of Standardization (ISO), complete with a gowning room and an anteroom. Two lead-shielded hot cells and two dual-mini hot cells, connected via underground trenches containing shielded conduits, were installed to optimize radioactive material transfer while minimizing personnel radiation exposure. Concrete blocks and lead bricks provided sufficient and cost-effective radiation shielding for the trenches. Air quality was controlled using pre-filters and high-efficiency particulate air (HEPA) filters to meet cleanroom ISO7 (Class 10,000) standards. A laminar-flow biosafety cabinet was installed in the cleanroom for preparation of sterile dose vials. Noteworthy was a laminar-flow insert in the hot cell that provided a shielded laminar-flow sterile environment meeting ISO5 (class 100) standards. The design included the constant control and monitoring of differential air pressures across the cleanroom, anteroom, gowning room, and controlled research space, as well as maintenance of temperature and humidity. The facility was equipped with state-of-the-art equipment for quality control and release testing of radiopharmaceuticals. Administrative controls and standard operating procedures (SOPs) were established to ensure compliance with manufacturing standards and regulatory requirements. Overall, the design and construction of this radiopharmacy facility exemplified a commitment to advancing fundamental, translational, and clinical applications of radiopharmaceutical research within an academic environment.
ABSTRACT
A set of 211At-astatoarenes were synthesized from corresponding trimethylgermyl arenes with radiochemical conversions (RCCs) of up to 90%. Both electron rich and electron poor substrates were successfully radiolabeled at room temperature (RT) using relatively low precursor amounts (0.15 µmol/ 0.02 mL solvent (7.5 mM)). Ready access to ortho-, para- and meta- astatinated arenes was achievable. Optimized reaction conditions were successfully applied to label a poly (ADP-ribose) polymerase (PARP) inhibitor with a RCC of approx. 50%. We believe that trimethylgermanyl derivatives are a viable addition to the astatination precursor toolbox and facilitate astatination of arenes. The developed labeling method should easily be applicable for productions under good manufacturing practice (GMP).
ABSTRACT
A new automated radiosynthesis of [11C]2-(2,6-difluoro-4-((2-(N-methylphenylsulfonamido)ethyl)thio)phenoxy)acetamide ([11C]K2), a radiopharmaceutical for the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, is reported. Although manual syntheses have been described, these are unsuitable for routine production of larger batches of [11C]K2 for (pre)clinical PET imaging applications. To meet demands for the imaging agent from our functional neuroimaging collaborators, herein, we report a current good manufacturing practice (cGMP)-compliant synthesis of [11C]K2 using a commercial synthesis module. The new synthesis is fully automated and has been validated for clinical use. The total synthesis time is 33 min from end of bombardment, and the production method provides 2.66 ± 0.3 GBq (71.9 ± 8.6 mCi) of [11C]K2 in 97.7 ± 0.5% radiochemical purity and 754.1 ± 231.5 TBq/mmol (20,382.7 ± 6256.1 Ci/mmol) molar activity (n = 3). Batches passed all requisite quality control testing confirming suitability for clinical use.
Subject(s)
Acetamides , Carbon Radioisotopes , Positron-Emission Tomography , Radiopharmaceuticals , Receptors, AMPA , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/chemistry , Carbon Radioisotopes/chemistry , Acetamides/chemical synthesis , Acetamides/chemistry , Receptors, AMPA/metabolism , Radiochemistry/methods , Automation , Chemistry Techniques, Synthetic , Sulfonamides/chemical synthesis , Sulfonamides/chemistryABSTRACT
Positron emission tomography imaging of misfolded proteins with high-affinity and selective radioligands has played a vital role in expanding our knowledge of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The pathogenesis of Huntington's disease, a CAG trinucleotide repeat disorder, is similarly linked to the presence of protein fibrils formed from mutant huntingtin (mHTT) protein. Development of mHTT fibril-specific radioligands has been limited by the lack of structural knowledge around mHTT and a dearth of available hit compounds for medicinal chemistry refinement. Over the past decade, the CHDI Foundation, a non-for-profit scientific management organisation has orchestrated a large-scale screen of small molecules to identify high affinity ligands of mHTT, with lead compounds now reaching clinical maturity. Here we describe the mHTT radioligands developed to date and opportunities for further improvement of this radiotracer class.
Subject(s)
Huntingtin Protein , Positron-Emission Tomography , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntingtin Protein/chemistry , Ligands , Humans , Protein Aggregates/drug effects , Mutation , Huntington Disease/diagnostic imaging , Huntington Disease/metabolism , Huntington Disease/genetics , Radiopharmaceuticals/chemistryABSTRACT
After the first atomic bomb test in Alamogordo in July 1945, followed by the Hiroshima and Nagasaki bombs in August 1945, radioecology became recognized as a branch of ecology in response to the radioactive fallout associated with the subsequent proliferation of atmospheric nuclear weapons testing which continued throughout the Cold War. In parallel, environmental radiochemistry emerged in the 70s to understand the chemical behavior of possible nuclear contaminants of the environment. In this discussion we stress the need to crosslink radioecology and chemical speciation, where radiochemistry and radioecology should meet to go beyond the present state of the art. Accordingly, we are seeking a methodology that calls for several angles of investigation: speciation (chemistry), toxicology (physiology and biology), accumulation data (environmental studies), distribution (geochemistry).
ABSTRACT
BACKGROUND: Parkinson's disease is a neurodegenerative disorder that is characterized by a degeneration of the dopaminergic system. Dopamine transporter (DAT) positron emission tomography (PET) imaging has emerged as a powerful and non-invasive method to quantify dopaminergic function in the living brain. The PET radioligand, [18F]FE-PE2I, a cocaine chemical derivative, has shown promising properties for in vivo PET imaging of DAT, including high affinity and selectivity for DAT, excellent brain permeability, and favorable metabolism. The aim of the current study was to scale up the production of [18F]FE-PE2I to fulfil the increasing clinical demand for this tracer. RESULTS: Thus, a fully automated and GMP-compliant production procedure has been developed using a commercially available radiosynthesis module GE TRACERLab FX2 N. [18F]FE-PE2I was produced with a radiochemical yield of 39 ± 8% (n = 4, relative [18F]F- delivered to the module). The synthesis time was 70 min, and the molar activity was 925.3 ± 763 GBq/µmol (250 ± 20 Ci/µmol). The produced [18F]FE-PE2I was stable over 6 h at room temperature. CONCLUSION: The protocol reliably provides a sterile and pyrogen-free GMP-compliant product.
ABSTRACT
BACKGROUND: The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biannual highlight commentary to update the readership on trends in the field of radiopharmaceutical development. MAIN BODY: This selection of highlights provides commentary on 24 different topics selected by each coauthoring Editorial Board member addressing a variety of aspects ranging from novel radiochemistry to first-in-human application of novel radiopharmaceuticals. CONCLUSION: Trends in radiochemistry and radiopharmacy are highlighted. Hot topics cover the entire scope of EJNMMI Radiopharmacy and Chemistry, demonstrating the progress in the research field in many aspects.
ABSTRACT
Herein, we disclose that pyridinium salts derived from abundant (hetero)anilines represent a novel precursor class for nucleophilic aromatic substitution reactions with [18F]fluoride. The value of this new 18F-fluorodeamination is demonstrated with the synthesis of over 30 structurally diverse and complex heteroaryl 18F-fluorides, several derived from scaffolds that were yet to be labelled with fluorine-18. The protocol tolerates heteroarenes and functionalities commonly found in drug discovery libraries, and is amenable to scale-up and automation on a commercial radiosynthesiser.
Subject(s)
Aniline Compounds , Fluorine Radioisotopes , Pyridinium Compounds , Salts , Aniline Compounds/chemistry , Pyridinium Compounds/chemistry , Pyridinium Compounds/chemical synthesis , Salts/chemistry , Fluorine Radioisotopes/chemistry , Halogenation , Molecular StructureABSTRACT
Positron emission tomography (PET) is becoming increasingly important in nuclear medicine and drug discovery. To date, the development of many potential PET tracers is hampered by the lack of suitable synthetic pathways for their preparation. This is particularly true for the highly desired radiolabeling of compounds bearing [18F]CF3-groups. For instance, S(O)nCF3-groups (n=0, 1, 2) serve as structural motif in a range of biologically active compounds, but their radiosynthesis remains largely unprecedented (for n=1, 2). Herein, we describe general methods for the radiosynthesis of 18F-labeled aryl trifluoromethyl sulfones, -sulfoxides, and -sulfides. All three methods are operationally straightforward, start from widely available precursors, i.e., sulfonyl fluorides and thiophenols, and make use of the recently established [18F]Ruppert-Prakash reagent. Further, the syntheses display good functional group tolerance as demonstrated by the 18F-labeling of more than 40â compounds. The applicability of the new method is demonstrated by the radiolabeling of three bioactive molecules, optionally to be used as PET tracers. In a broader context, this work presents a substantial expansion of the chemical space of radiofluorinated structural motifs to be used for the development of new PET tracers.
ABSTRACT
The difluoromethyl functionality has proven useful in drug discovery, as it can modulate the properties of bioactive molecules. For PET imaging, this structural motif has been largely underexploited in (pre)clinical radiotracers due to a lack of user-friendly radiosynthetic routes. This Minireview provides an overview of the challenges facing radiochemists and summarises the efforts made to date to access 18F-difluoromethyl-containing radiotracers. Two distinct approaches have prevailed, the first of which relies on 18F-fluorination. A second approach consists of a 18F-difluoromethylation process, which uses 18F-labelled reagents capable of releasing key reactive intermediates such as the [18F]CF2H radical or [18F]difluorocarbene. Finally, we provide an outlook for future directions in the radiosynthesis of [18F]CF2H compounds and their application in tracer radiosynthesis.
ABSTRACT
BACKGROUND: (S)-[18F]FETrp is a promising PET radiotracer for imaging IDO1 activity, one of the main enzymes involved in the tryptophan metabolism that plays a key role in several diseases including cancers. To date, the radiosynthesis of this tryptophan analogue remains highly challenging due to partial racemization occurring during the nucleophilic radiofluorination step. This work aims to develop a short, epimerization-free and efficient automated procedure of (S)-[18F]FETrp from a corresponding enantiopure tosylate precursor. RESULTS: Enantiomerically pure (S)- and (R)-FETrp references as well as tosylate precursors (S)- and (R)-3 were obtained from corresponding Na-Boc-(L and D)-tryptophan in 2 and 4 steps, respectively. Manual optimisation of the radiolabelling conditions resulted in > 90% radiochemical conversion with more than 99% enantiomeric purity. Based on these results, the (S)-[18F]FETrp radiosynthesis was fully automated on a SynChrom R&D EVOI module to produce the radiotracer in 55.2 ± 7.5% radiochemical yield, 99.9% radiochemical purity, 99.1 ± 0.5% enantiomeric excess, and molar activity of 53.2 ± 9.3 GBq/µmol (n = 3). CONCLUSIONS: To avoid racemisation and complicated purification processes, currently encountered for the radiosynthesis of (S)-[18F]FETrp, we report herein significant improvements, including a versatile synthesis of enantiomerically pure tosylate precursor and reference compound and a convenient one-pot two-step automated procedure for the radiosynthesis of (S)-[18F]FETrp. This optimised and robust production method could facilitate further investigations of this relevant PET radiotracer for imaging IDO1 activity.
ABSTRACT
Uranium is a natural radioelement (also a model for heavier actinides), but may be released through anthropogenic activities. In order to assess its environmental impact in a given ecosystem, such as the marine system, it is essential to understand its distribution and speciation, and also to quantify its bioaccumulation. Our objective was to improve our understanding of the transfer and accumulation of uranium in marine biota with mussels taken here as sentinel species because of their sedentary nature and ability to filter seawater. We report here on the investigation of uranium accumulation, speciation, and localization in Mytilus galloprovincialis using a combination of several analytical (Inductively Coupled Plasma Mass Spectrometry, ICP-MS), spectroscopic (X ray Absorption Spectroscopy, XAS, Time Resolved Laser Induced Fluorescence Spectroscopy, TRLIFS), and imaging (Transmission Electron Microscopy, TEM, µ-XAS, Secondary Ion Mass Spectrometry, SIMS) techniques. Two cohorts of mussels from the Toulon Naval Base and the Villefranche-sur-Mer location were studied. The measurement of uranium Concentration Factor (CF) values show a clear trend in the organs of M. galloprovincialis: hepatopancreas â« gill > body ≥ mantle > foot. Although CF values for the entire mussel are comparable for TNB and VFM, hepatopancreas values show a significant increase in those from Toulon versus Villefranche-sur-Mer. Two organs of interest were selected for further spectroscopic investigations: the byssus and the hepatopancreas. In both cases, U(VI) (uranyl) is accumulated in a diffuse pattern, most probably linked to protein complexing functions, with the absence of a condensed phase. While such speciation studies on marine organisms can be challenging, they are an essential step for deciphering the impact of metallic radionuclides on the marine biota in the case of accidental release. Following our assumptions on uranyl speciation in both byssus and hepatopancreas, further steps will include the inventory and identification of the proteins or metabolites involved.
Subject(s)
Mytilus , Uranium , Water Pollutants, Radioactive , Mytilus/chemistry , Mytilus/metabolism , Animals , Uranium/analysis , Water Pollutants, Radioactive/analysis , Mass SpectrometryABSTRACT
Because carbon-11 (11C) radiotracers cannot be shipped over long distances, their use in routine positron emission tomography (PET) studies is dependent on the production capabilities of individual radiochemistry laboratories. Since 2003, 11C-labeled Pittsburgh compound B ([11C]PiB) has been the gold standard PET radiotracer for in vivo imaging of amyloid ß (Aß) plaques. For more than two decades, researchers have been working to develop faster, higher-yielding, more robust, and optimized production methods with higher radiochemical yields for various imaging applications. This review evaluates progress in [11C]PiB radiochemistry. An introductory overview assesses how it has been applied in clinical neurologic imaging research. We examine the varying approaches reported for radiolabeling, purification, extraction, and formulation. Further considerations for QC methods, regulatory considerations, and optimizations were also discussed.