Formulation and radiochemical evaluation of a freeze-dried mixed peptide kit for the preparation of 68Ga-labeled peptides for PET imaging of somatostatin receptor positive neuroendocrine cancers

⁶⁸Ga-labeled DOTA-coupled somatostatin analogue peptides are regularly being used for the PET imaging of patients suspected to be suffering from various types of neuroendocrine cancers over-expressing somatostatin receptors. The article describes the formulation of a freeze-dried mixed peptide kit containing equal amounts of DOTA-TATE and DOTA-NOC and the radiochemical evaluation of the kit for the easy and convenient preparation of ⁶⁸Ga-labeled mixed radiopeptides. The simultaneous use of two different peptides in the kit is expected to perceive more cancerous lesions and may have a wider applicability in the diagnosis of neuroendocrine cancers.     

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting

The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of ⁶⁸Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [⁶⁸Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.     

Determination of HEPES in <Superscript>68</Superscript>Ga-labeled peptide solutions

A practical and reliable HPLC method was used for the determination of 2-[4-N-(2-hydroxyethyl)-1-piperazinyl]-N′-ethanesulfonic acid (HEPES) content in the ⁶⁸Ga-labeled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-octreotide (DOTANOC). Linearity of this method was observed in a concentration range of 0.01–10 mg mL⁻¹ and the quantitative limit (signal to noise = 11) was determined as 10 μg mL⁻¹. The HEPES concentration in the final products of ⁶⁸Ga-DOTANOC was typically lower than the detection limit. Pure water and HEPES buffer as reaction medium were investigated using various activities of gallium-68. It was demonstrated that the presence of HEPES buffer consistently furnished very high radiochemical purity of ⁶⁸Ga-DOTANOC, which remained stable for several hours post-labeling. Evidence is provided that in addition to its role as a buffer, HEPES also functions as a radioprotectant agent.     

Research on extracted <Superscript>90</Superscript>Y with P204 in lipiodol for liver cancer

Research on extracted ⁹⁰Y with di(2-ethylhexyl) orthophosphoric acid (P204) in lipiodol for liver cancer was made to evaluate the stability of extracted ⁹⁰Y with P204 in lipiodol (⁹⁰Y-P204-lipiodol) in serum of newly-born cattle and human’s blood. At first, P204 (extractant) was dissolved in lipiodol (organic phase). Secondly, ⁹⁰Y was extracted to organic phase after adding ⁹⁰Y solution into test tube with P204 and lipiodol in it. The extracting efficiency with 0.01 mol/l P204 could reach 99.4%. The stability of ⁹⁰Y-P204-lipiodol has been experimented in physiological saline solution as preparation for further stability experiment. The result indicated that the extracted ⁹⁰Y lost 0.02%–0.36% in physiological saline solution. The results of further stability experiment showed that loss efficiencies of extracted ⁹⁰Y after adding newly-born cattle serum 1 hour, 1 day, 3 and 7 days are 3.38%, 3.12%, 4.29% and 6.62%, respectively, and loss efficiencies of extracted ⁹⁰Y after adding human’s blood 1 hour, 1 day, 3 and 7 days are 2.55%, 5.91%, 7.88% and 5.63%, respectively. Our data also indicated that ⁹⁰Y is the most possible radioisotope for being extracted with P204 in lipiodol to treat hepatocellular carcinoma, particularly in cases of unresectable liver tumors, since ⁹⁰Y is available from several commercial sources in clinical quality. We conclude that the stability of ⁹⁰Y-P204-lipiodol tested with newly-born cattle serum and human’s blood attained great results. ⁹⁰Y-P204-lipiodol is a kind of potential and exciting pharmaceutical in inerventional therapy for liver cancer and we can carry on the further animal test and clinical trial.     

Rapid synthesis of carbon materials by microwave-assisted hydrothermal method at low temperature and its adsorption properties for uranium (VI)

The cathelicidin-derived peptide (CDP1) is a human antimicrobial peptide that preferentially targets bacterial membranes in response to infection. CDP1 was functionalised with NODAGA and DOTA for complexation with gallium-68 to evaluate its potential as an infection imaging tracer. The synthesis of [⁶⁸Ga]Ga–NODAGA–CDP1 and [⁶⁸Ga]Ga–DOTA–CDP1 were optimised for pH, molarity, incubation time and temperature, and product purification. The integrity and protein binding were investigated employing [⁶⁸Ga]GaCl₃ and [⁶⁸Ga]Ga–DOTA–TATE as internal references. [⁶⁸Ga]Ga–NODAGA–CDP1 displayed good labelling properties with higher product yield compared to [⁶⁸Ga]Ga–DOTA–CDP1. In contrast, [⁶⁸Ga]Ga–DOTA–CDP1 showed better stability and is the preferred candidate for an in vivo investigation.

     

A new technique for labeling of Lipiodol with 188Re in the treatment of hepatic tumor

A new method for the synthesis of ¹⁸⁸Re-Lipiodol without using a chelating agent and to evaluate the stability and biodistribution of the new agent in rats with hepatic tumors was attempted. Eighteen male Sprague -Dawley rats with liver tumors were sacrificed at 1, 24, and 48 hours (six rats at each time) after injection of approximately 7.4 MBq (0.2 mCi) of ¹⁸⁸Re Lipiodol via the hepatic artery. Samples of tumor, liver and other organs were collected and tissue concentration (%ID/g) of the markers were calculated. Our data showed a high level of radioactivity in the hepatic tumors at every time of the study. The ratios of tumor to normal liver tissue concentration (T/N ratio) were 7.62 at 1 hour, 8.03 at 24 hours, and 7.70 at 48 hours. Except for the liver, kidneys and lungs, concentrations in other organs were low. The new method for labeling Lipiodol with ¹⁸⁸Re is simple and has potential for the treatment of hepatic tumors This revised version was published online in July 2006 with corrections to the Cover Date.     

<Superscript>99m</Superscript>Tc-labelled biguanide derivatives: chemical speciation modelling thereof and evaluation in vervets

^99mTc-DMSA (dimercaptosuccinic acid) is known to be a safe and effective agent for static renal imaging. However, it has a long uptake time which is a limiting factor in diagnostic procedures and also leads to a relatively high radiation dose being administered to patients. There is a constant search for possible new renal imaging agents with a good resolution, kidney/liver contrast and low radiation dose to all organs. A series of biguanide derivatives (potential as non-insulin-dependent diabetes mellitus agents) labelled with ^99mTc were investigated as potential alternative kidney-imaging agents on theoretical grounds (in silico) and their biodistribution (in vivo) verified in a limited number of animal experiments. Such a dual approach has the benefit that it reduces the number of animal experiments needed to evaluate a potential radiopharmaceutical. The blood plasma model shows little or no complexation of the biguanide type ligands by the metal ions in blood plasma. It was therefore expected that these ligands will clear rapidly through the kidneys and liver (increased lipophilicity). These predictions were verified by studies on single vervets comparing them with ^99mTc-DMSA as gold standard. All the biguanide derivatives labelled with ^99mTc show liver, kidney and gallbladder uptake in vervets. It was shown that the agent ^99mTc-CBIG (carboxylbiguanide) has a very fast kidney clearance, which will reduce the dose to organs (as experienced for ^99mTc-DMSA), although it’s potential as a kidney agent is limited by its gallbladder uptake.     

新型PET核素68Ga标记D-脱氧葡萄糖的合成及生物学评价

以2-氨基-2-脱氧-D-葡萄糖为原料,通过微波加热法合成得到可用于核素标记的葡萄糖胺-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA)前体化合物.产物通过IR,1H NMR,HPLC-MS,ESI-HRMS表征.通过标记条件的优化设计,在微波作用下,与新型正电子显像核素Ga-68进行放射性标记,高产率得到68Ga-DOTA-DG.而后对该标记化合物进行了K-562,Hela,A431等肿瘤细胞摄取实验,测定了对肿瘤的特异性.68Ga-DOTA-DG的合成,拓宽了以18F-FDG为首的肿瘤代谢类显像剂的新应用,为PET(Positron emission tomography)显像肿瘤代谢情况提供了一种新的途径.     

Preparation and biological evaluation of radiogallium labeled glucagon for SPECT imaging

In this work, recently prepared ⁶⁷Ga-labeled glucagon (⁶⁷Ga-DTPA-GCG) for imaging studies (radiochemical purity >94%; HPLC, S.A. 296–370 GBq/mM) was used in biological studies. The wild-type rat biodistribution results, 2 h post injection, demonstrated high tissue:muscle ratios for target tissues (liver, kidney, heart, spleen, fat intestine stomach and pancreas), 234, 18.45, 7.12, 1.75, 128.7, 4.9, 6.3 and 1.11, respectively. The tracer binding capacity using freshly prepared rat brain homogenate demonstrated significant specific binding of the tracer to neuronal GCG receptors (⁶⁷Ga-DTPA-GCG/⁶⁷Ga:3 and ⁶⁷Ga-DTPA-GCG/⁶⁷GaDTPA:2.2 at 90 min). SPECT images also demonstrated target specific binding of the tracer at 4 h. The data suggests the tracer is accumulated in GCGR rich tissues 2–4 h post injection, suggesting potentials of the tracer for future imaging studies in glocagonoma models.     

A Tissue-Based Electrode for Peroxidase Assay: Preliminary Results in Hormone Determination by EIA

Abstract

A liver tissue based electrochemical sensor for hydrogen peroxide has been realized for determination of peroxidase activity in solution. Its behaviour is based on the oxygen electrode and an enzyme catalase largely present in liver tissue. The competition between the two enzymatic reactions based on catalase and peroxidase results in a probe for peroxidase activity determination in the range 5 · 10^?3 ? 2.5 · 10^?1 U/mL.

Digoxin and Insulin, have been determined in standard solution by the living-tissue probe, by using immunoreactions with peroxidase labelled hormones and antibodies fixed on vial wall of a commercial test kit.     

The determination of total mercury in biological tissues by a modified potassium permanganate procedure.

A simple cold-tube atomic absorption method with a silver-mercury amalgam trap and potassium permanganate as oxidizing agent is described for the determination of total mercury in tissue homogenates. Results are presented for animals fed inorganic (HgCl₂) and organic (CH₃HgOH) mercury orally at a level of 1 mg Hg kg^?1. Data are presented which compare potassium permanganate oxidation of tissue homogenates with whole tissue analysed by cold-tube atomic absorption after digestion with acid, or by neutron activation. For kidney tissue there is good agreement between all three methods for animals fed inorganic and organic mercury. For liver, however, homogenization produced an average loss of about 50 % of the mercury in rats fed mercury(II) chloride. Factors such as adsorption of mercury on sample container walls, bacterial action on the tissue and inadvertent introduction of reducing agents which could reduce the mercury to its elemental state, are not significant. Despite the loss of mercury in the liver by homogenization, rank ordering of mercury values for potassium permanganate—homogenate versus direct neutron activation analyses was essentially the same.     

Complex of tris(2-hydroxyethyl)amine with zinc bis(2-methylphenoxyacetate) as an inhibitor of the acid lipase of the aortic intima

The previously unknown ability of Zitrimin, the complex of tris(2-hydroxyethyl)amine with zinc(ii) bis(2-methylphenoxyacetate), to affect the activity of acid lipase of the aortic intima was studied. Daily administration of an aqueous solution of the tris(2-hydroxyethyl)amine complex with zinc bis(2-methylphenoxyacetate) in a dose of 10 mg kg^?1 for 3 months to rabbilts with experimental atherosclerosis was found to decrease the cholesterol and total lipid levels in the aortic tissue and to decrease the degree of aortic damage with atherosclerotic plaques. According to the results of enzymatic analysis, development of atherosclerosis is accompanied by 68% increase in the activity of acid lipase in the intima of the aorta compared to the control.

     

Indium-111 labeled bleomycin for targeting diagnosis and therapy of liver tumor: optimized preparation,biodistribution and SPECT imaging with xenograft models

In this work, the preparation of ¹¹¹In radiolabeled bleomycin (¹¹¹In-BLM) was optimized systematically and used for SPECT imaging of liver cancer xenograft models. ¹¹¹In-BLM with a high radiochemical yield (>?99%) could be obtained at pH 0.5–1 at a mixture ratio of 9:1 (¹¹¹In/BLM, mCi/mg). The radiochemical purity of ¹¹¹In-BLM retained?>?99% in either buffers or serum for 3 days. Biodistribution of ¹¹¹In-BLM revealed its excellent stability in vivo. The SPECT imaging studies showed ¹¹¹In-BLM has great specificity to liver tumor xenograft. All the results implied ¹¹¹In-BLM is a potential radiopharmaceutical for targeting diagnosis and therapy of liver tumor.

     

A New Synthesis of 3-Deoxy-D-Manno-Octulosonic Acid (KDO) From D-Mannose Via Condensation of Dioxene

Abstract

The title compound has long been known as an essential component of lipopolysaccharides (LPS) and capsular polysaccharides which exist in the outer membrane of Gram-negative bacteria. KDO has attracted additional attention as a consequence of some remarkable discoveries; i) mutants unable to produce KDO are non-viable.¹ ii) KDO is not present in mammalian cells,² iii) the 2-deoxy analog of β-KDO represents a new class of synthetic antimicrobial agent.^3,4 Although syntheses of KDO have been reported by many groups,^5–8 a more efficient synthetic method endowed with the potential for synthesis of not only complex glycoconjugates but also biologically significant analogs is required.     

Evaluation of labelling conditions,quality control and biodistribution study of 99mTc-5-aminolevulinic acid (5-ALA): a potential liver imaging agent

Labelling of 5-aminolevulinic acid (5-ALA) with ^99mTc was achieved by using SnCl₂·2H₂O as reducing agent. Radiochemical purity and labelling efficiency was determined by instant thin layer chromatography/paper chromatography. Efficiency of labelling was dependent on many parameters such as amount of ligand, reducing agent, pH, and time of incubation. ^99mTc labelled 5-ALA remained stable for 24 h in human serum. Tissue biodistribution of ^99mTc-5-ALA was evaluated in Sprague–Dawley rats. Biodistribution study (% ID/g) in rats revealed that ^99mTc-5-ALA was accumulated significantly in liver, spleen, stomach and intestine after half hour, 4 and 24 h. Significant activity was noted in bladder and urine at 4 h. High liver uptake of ^99mTc-5-ALA makes it a promising liver imaging agent.     

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, ⁶⁸Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of ⁶⁸Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded ⁶⁸Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of ⁶⁸Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.     

Pharmacokinetics and Tissue Distribution Study of Tryptanthrin in Rats by RP-HPLC with DAD Detector

A simple RP-LC-UV method was established for the determination of tryptanthrin in plasma and different tissues of rats. The separation was achieved by HPLC on a C₁₈ column with a mobile phases composed of acetonitrile–water (47:53, v/v), UV detection was used at 251 nm. Good linearity was found between 0.0183–1.1712 μg mL⁻¹ (r ² = 0.999) for plasma and 0.0937–1.7568 μg mL⁻¹ for the tissue samples, respectively (r ² ≥ 0.9932). The intra- and inter-day precisions expressed as the relative standard deviation for the method were 0.92–6.01 and 1.06–9.11 %, respectively. The relative recoveries of tryptanthrin ranged from 95.26 to 97.89 % for plasma and 82.55 to 114.99 % for tissue homogenates (except heart). The developed method was successfully applied to the pharmacokinetics and tissue distribution research after orally administration of a 56-mg kg⁻¹ dose of tryptanthrin to healthy SD rats. The main pharmacokinetics distribution results showed that liver, lung, small intestine, and large intestine were the major distribution tissues of tryptanthrin in rats, and that tryptanthrin had difficulty in crossing the blood–brain barrier.

     

Preparation and Evaluation of [18F]AlF-NOTA-NOC for PET Imaging of Neuroendocrine Tumors: Comparison to [68Ga]Ga-DOTA/NOTA-NOC

Background: The somatostatin receptors 1–5 are overexpressed on neuroendocrine neoplasms and, as such, represent a favorable target for molecular imaging. This study investigates the potential of [¹⁸F]AlF-NOTA-[1-Nal³]-Octreotide and compares it in vivo to DOTA- and NOTA-[1-Nal³]-Octreotide radiolabeled with gallium-68. Methods: DOTA- and NOTA-NOC were radiolabeled with gallium-68 and NOTA-NOC with [¹⁸F]AlF. Biodistributions of the three radioligands were evaluated in AR42J xenografted mice at 1 h p.i and for [¹⁸F]AlF at 3 h p.i. Preclinical PET/CT was applied to confirm the general uptake pattern. Results: Gallium-68 was incorporated into DOTA- and NOTA-NOC in yields and radiochemical purities greater than 96.5%. NOTA-NOC was radiolabeled with [¹⁸F]AlF in yields of 38 ± 8% and radiochemical purity above 99% after purification. The biodistribution in tumor-bearing mice showed a high uptake in tumors of 26.4 ± 10.8 %ID/g for [⁶⁸Ga]Ga-DOTA-NOC and 25.7 ± 5.8 %ID/g for [⁶⁸Ga]Ga-NOTA-NOC. Additionally, [¹⁸F]AlF-NOTA-NOC exhibited a tumor uptake of 37.3 ± 10.5 %ID/g for [¹⁸F]AlF-NOTA-NOC, which further increased to 42.1 ± 5.3 %ID/g at 3 h p.i. Conclusions: The high tumor uptake of all radioligands was observed. However, [¹⁸F]AlF-NOTA-NOC surpassed the other clinically well-established radiotracers in vivo, especially at 3 h p.i. The tumor-to-blood and -liver ratios increased significantly over three hours for [¹⁸F]AlF-NOTA-NOC, making it possible to detect liver metastases. Therefore, [¹⁸F]AlF demonstrates promise as a surrogate pseudo-radiometal to gallium-68.     

A Triazacyclononane‐Based Bifunctional Phosphinate Ligand for the Preparation of Multimeric 68Ga Tracers for Positron Emission Tomography

For application in positron emission tomography (PET), PrP9 , a N,N′,N′′‐trisubstituted triazacyclononane with methyl(2‐carboxyethyl)phosphinic acid pendant arms, was developed as ⁶⁸Ga³⁺ complexing agent. The synthesis is short and inexpensive. Ga^III and Fe^III complexes of PrP9 were characterized by single‐crystal X‐ray diffraction. Stepwise protonation constants and thermodynamic stabilities of metal complexes were determined by potentiometry. The Ga^III complex possesses a high thermodynamic stability (log K_[GaL]=26.24) and a high degree of kinetic inertness. ⁶⁸Ga labeling of PrP9 is possible at ambient temperature and in a wide pH range, also at pH values as low as 1. This means that for the first time, the neat eluate of a TiO₂‐based ⁶⁸Ge/⁶⁸Ga generator (typically consisting of 0.1 M HCl) can be directly used for labeling purposes. The rate of ⁶⁸Ga activity incorporation at pH 3.3 and 20 °C is higher than for the established chelators DOTA and NOTA. Tris‐amides of PrP9 with amino acid esters were synthesized to act as models for multimeric peptide conjugates. These conjugates exhibit radiolabeling properties similar to those of unsubstituted PrP9 .     

Development of 68Ga ethyl cysteinate dimer for PET studies

In this work development of ⁶⁸Ga-ethyl cysteinate dimer (⁶⁸Ga-ECD) a ⁶⁸Ga tracer for possible cerebral blood flow based on ^99mTc ECD homolog is reported. ⁶⁸Ga-ECD was prepared using generator-based ⁶⁸GaCl₃ and ECD at optimized conditions. Quality control, stability, partition co-efficient and the biodistribution of the tracer (by tissue counting and PET/CT in rats) was studied. Significant metabolism of the lipophilic tracer into water soluble metabolite(s) led to urinary excretion of the tracer, un-comparable to that of homologous ^99mTc-compound. Cardiac uptake of the complex suggests formation of a possible lipophil cationic complex and/or metabolite.