Design, Preparation, and Evaluation of a Novel 99mTcN Complex of Ciprofloxacin Xanthate as a Potential Bacterial Infection Imaging Agent.

In order to seek novel technetium-99m bacterial infection imaging agents, a ciprofloxacin xanthate (CPF2XT) was synthesized and radiolabeled with [99mTcN]2+ core to obtain the 99mTcN-CPF2XT complex, which exhibited high radiochemical purity, hydrophilicity, and good stability in vitro. The bacteria binding assay indicated that 99mTcN-CPF2XT had specificity to bacteria. A study of biodistribution in mice showed that 99mTcN-CPF2XT had a higher uptake in bacterial infection tissues than in turpentine-induced abscesses, indicating that it could distinguish bacterial infection from sterile inflammation. Compared to 99mTcN-CPFXDTC, the abscess/blood and abscess/muscle ratios of 99mTcN-CPF2XT were higher and the uptakes of 99mTcN-CPF2XT in the liver and lung were obviously decreased. The results suggested that 99mTcN-CPF2XT would be a potential bacterial infection imaging agent.

Investigation of inhibition of human glucose 6-phosphate dehydrogenase by some 99mTc chelators by in silico and in vitro methods.

The inhibitory effects of methoxyisobutylisonitrile (MIBI), diethylene triamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and metilendifosfonat (MDP) on human erythrocyte glucose 6-phosphate dehydrogenase (hG6PD) activity were investigated. For this purpose, hG6PD was initially purified 557-fold at a yield of 51.43% using 2',5'-adenosine diphosphate (ADP) sepharose 4B affinity gel chromatography. The in vitro effects of these chelators on hG6PD enzyme were studied. IC50 values of MIBI, DTPA, DMSA and MDP were 0.056, 0.172, 0.274 and 0.175 mM, of hG6PD, respectively. It was detected in in vitro studies that the hG6PD enzyme is inhibited due to these radiopharmaceutical chelators. In addition to in vitro studies, in order to better understand the molecular mechanism of studied compounds, combined in silico approaches, including molecular docking and molecular dynamics (MD), simulations were successfully performed. MD simulations shed light on inhibition mechanisms of the individual inhibitors into the ligand-binding pocket of hG6PD. Essential amino acids for binding are also investigated using per-residue interaction analysis studies.

Studies on batch formulation of a freeze dried kit for the preparation of 99mTc-HYNIC-TATE for imaging neuroendocrine tumors.

AIM: To formulate freeze dried cold kits for preparation of 99mTc-HYNIC-TATE suitable for use at hospital radiopharmacy and establish clinical utility of 99mTc-HYNIC-TATE prepared using kits for detection of neuroendocrine tumors (NETs). METHODS: Standardization of reagent concentrations for formulation of freeze dried kits of HYNIC-TATE was carried out. Consistency in formulation was tested by six batch preparation. Quality control tests were carried out to establish compliance of specifications of purity and safety criteria for both kits and 99mTc-HYNIC-TATE formulated using kits. Clinical utility of 99mTc-HYNIC-TATE prepared using kits was demonstrated in patients with histopathologically confirmed well-differentiated NETs. RESULTS: Pharmaceutical grade HYNIC-TATE kits compliant with all the quality control criteria were formulated and successfully radiolabeled with 99mTc. Radiopharmaceutical was successfully utilized for detection of NETs in patients and comparison with uptake of 99mTc-HYNIC-TOC and 177Lu-DOTA-TATE was made. CONCLUSION: The formulated kits are robust and provide consistently high radiolabeling yields (> 95%) with 99mTc in short time periods requiring no additional purification. Initial clinical trials demonstrate the utility of 99mTc-HYNIC-TATE using formulated kits.

The lanthanum precipitation method. Part 1: a new method for technetium(IV) speciation in humic rich natural groundwater.

A new and quick method for direct speciation of Tc(IV) in humic rich solutions, based on the induced aggregation of humic substances in the presence of the trivalent cation La3+, is presented. This method (the "La-precipitation method") allows flocculating all the humic substances and also the Tc(IV) associated with humic substances. The method is tested on solutions containing Tc(IV) and Gorleben humic substances. The influence of different parameters (humic substance concentration, Tc concentration, reaction time and pH) is investigated on the observed free Tc(IV) concentration after precipitation of all humic substances. None of these parameters had a (significant) influence on the observed Tc(IV) concentration in solution after addition of La3+ to Tc(IV)-HS containing solutions. It is therefore proposed that the method can be used to separate the Tc(IV) bound to humic substances from the free inorganic Tc species in solution.

Radiolabeled methotrexate as a diagnostic agent of inflammatory target sites: A proof-of-concept study.

Methotrexate (MTX), as a pharmaceutical, is frequently used in tumor chemotherapy and is also a part of the established treatment of a number of autoimmune inflammatory disorders. Radiolabeled MTX has been studied as a tumor­diagnostic agent in a number of published studies. In the present study, the potential use of technetium­99m­labelled MTX (99mTc­MTX) as a radiotracer was investigated for the identification of inflammatory target sites. The labelling of MTX was carried out via a 99mTc­gluconate precursor. Evaluation studies included in vitro stability, plasma protein binding assessment, partition­coefficient estimation, in vivo scintigraphic imaging and ex vivo animal experiments in an animal inflammation model. MTX was successfully labelled with 99mTc, with a radiochemical purity of >95%. Stability was assessed in plasma, where it remained intact up to 85% at 4 h post­incubation, while protein binding of the radiotracer was observed to be ~50% at 4 h. These preclinical ex vivo and in vivo studies indicated that 99mTc­MTX accumulates in inflamed tissue, as well as in the spinal cord, joints and bones; all areas with relatively high remodeling activity. The results are promising, and set the stage for further work on the development and application of 99mTc­MTX as a radiotracer for inflammation associated with rheumatoid arthritis.

Isolation, characterization, and biological evaluation of syn and anti diastereomers of [(99m)Tc]technetium depreotide: a somatostatin receptor binding tumor imaging agent.

The early and later eluting [(99m)TcO]depreotide products on RP-HPLC were confirmed to be the anti and syn diastereomers, respectively, based on proton NMR and circular dichroism spectroscopy. NMR provided evidence of a folded, conformationally constrained structure for the syn diastereomer. The syn diastereomer is predominant (anti/syn approximately 10:90) in the [(99m)TcO]depreotide preparation and shows a slightly higher affinity (IC50 = 0.15 nM) for the somatostatin receptor than the anti diastereomer (IC50 = 0.89 nM). Both diastereomers showed higher binding affinities than the free peptide (IC(50) = 7.4 nM). Biodistribution studies in AR42J tumor xenograft nude mice also showed higher tumor uptake for syn [(99m)TcO]depreotide (6.58% ID/g) than for the anti [(99m)TcO]depreotide (3.38% ID/g). Despite the differences in biological efficacy, the favorable binding affinity, tumor uptake, and tumor-to-background ratio results for both diastereomeric species predict that both are effective for imaging somatostatin receptor-positive tumors in vivo.

Studies on batch formulation of a kit for the preparation of the 99mTc-Ubiquicidin (29-41): An infection imaging agent.

The aim of this study was to formulate an indigenous cold kit of Ubiquicidin, UBI (29-41), for easy preparation of (99m)Tc-UBI (29-41) to be used as an infection imaging agent. A two component kit with the peptide and SnCl2 as vial 1 and optimum amount of NaOH as vial 2 was successfully formulated as seen from the consistent radiochemical and pharmaceutical purity of the product over six consecutive batches of kits. The utility of the kit could be demonstrated through in-vitro and in vivo specificity of (99m)Tc-UBI (29-41).

Single vial freeze-dried TRODAT-1 kit: preparation and demonstration of clinical efficacy of [(99m)Tc]TRODAT-1 in Indian scenario.

A single vial freeze-dried kit formulation for preparation of three patients' dose of [(99m)Tc]TRODAT-1 has been developed for early diagnosis of Parkinson's disease (PD). Kits were evaluated to ascertain the purity, stability and batch to batch variations. Preclinical evaluation was carried out in laboratory animals and clinical imaging was performed in human patients with PD. The labeling yield and purity of [(99m)Tc]TRODAT-1 was >90%. Swiss mice showed retention of [(99m)Tc]TRODAT-1 in the mid brain region. Clinical studies showed decreased striatal uptake with increasing severity of PD.

Development of a freeze-dried kit formulation for the preparation of 99mTc-NTP 15-5, a radiotracer for scintigraphic imaging of proteoglycans.

The cartilage-targeting strategy is based on the strong affinity of quaternary ammonium (QA) functions for cartilage proteoglycans. We use a bifunctional agent containing QA moiety and a polyazamacrocycle structure able to complex technetium-99m. (99m)Tc-NTP 15-5 was selected for its high stability and its high affinity for proteoglycans in vivo. Labeling conditions of NTP 15-5 were optimized, and a lyophilized kit was developed for radiolabeling of (99m)Tc-NTP 15-5 (radiochemical yields 94.6±1.8%). (99m)Tc-NTP 15-5 was stable and resulted in favorable biological evaluations.

Evaluation of technetium-99m-L,L-EC in renal transplant recipients: a comparative study with technetium-99m-MAG3 and iodine-125-OIH.

UNLABELLED: The clinical usefulness of kit-formulated 99mTc-L,L-EC, a new renal tubular tracer agent based on a diaminodithiol ligand was evaluated in a large population of renal transplant recipients. METHODS: Fifty patients with transplants were studied. Five patients with renal insufficiency and five normal volunteers were also included to extend the range of renal function values. The labeling efficiency of 99mTc-L,L-EC in routine conditions, i.e., without HPLC purification, and the safety of the tracer were evaluated. RESULTS: The mean radiochemical purity of 99mTc-L,L-EC determined by thin-layer chromatography was 97.4%. No side effects or significant biochemical changes were observed. The clearance of 99mTc-L,L-EC and 125I-OIH ranged from 10.7 to 417.5 and from 27.6 to 602.7 ml/min/1.73 m2, respectively. The clearance of 99mTc-L,L-EC and 99mTc-MAG3 averaged respectively 71% and 52% of that of 125I-OIH. CONCLUSION: The labeling procedure of kit-formulated 99mTc-L,L-EC is easy and efficient. This tracer is safe and suitable for both imaging and quantitative measurement of the renal tubular function. Technetium-99m-L,L-EC represents an excellent alternative to 99mTc-MAG3.

Purification and stabilization of 99mTc-d,l-HMPAO: role of organic extractants.

99mTc-d,l-HMPAO, an important SPECT agent for imaging cerebral perfusion, suffers from the disadvantage of an inherent instability and its shelf life has been reported to be 30 min. The latter is a harsh constraint and not compatible with Centralized Radiopharmacy procedures. During the attempts to improve upon the stability of 99mTc-d,l-HMPAO, preservation of product as an organic extract into suitable solvents like diethylether, ethylacetate, methylethylketone, chloroform was tried out. Chloroform extraction (yield: >90%) resulted in a product having stability not less than 5 hours. Gentle drying of the chloroform extract and reconstitution in normal saline resulted in quantitative recovery of 99mTc-d,l-HMPAO with acceptable radiochemical purity (>90%). This finding is thus of much significance, especially in the context of centralized large hospital radiopharmacy setting, by rendering convenience and flexibility in scheduling patients.

The chemical nature, purity and stability of 99mTc-N-(2,6-Diethyl-3-iodo-phenylcarbamoylmethyl)-iminodiacetic acid.

The chemical purity and stability of a radio pharmaceutical 99mTc-N-(2,6-diethyl-3-iodo-phenylcarbamoylmethyl)-iminodiacetic acid (IODIDA) has been studied using HPLC. The HPLC results indicate that IODIDA decomposed into at least three species after 99mTc labelling, and the amount of these varied with time. Mass spectra obtained by negative ion FABMS of no-carrier-added (NCA) Tc-labelled IODIDA showed, in addition to the molecular ion and the sodium adduct of N-(2,6-diethyl-3-iodo-phenylcarbamoylmethyl)-iminodiacetic acid, peaks corresponding to sodium containing dimers, trimers and tetramers. Several peaks were also found that may be assigned to 99Tc(I) containing dimers, trimers and tetramers.

Technetium-99m complexes of polydentate amine-pyrrole and amine-thiophene ligands.

Novel polydentate amine-pyrrole and amine-thiophene ligands were synthesized and characterized by 1H and 13C NMR spectroscopy. Radiochemical studies with 99mTc were carried out at 0.1-100 microM of technetium. Complexation yields were estimated from thin layer chromatography (TLC), paper electrophoresis, and solvent extraction studies. The 99mTc complexes formed were found to be neutral and lipophilic. Complexes with the corresponding imine-ligands were formed in lower yields. Biodistribution studies of the 99mTc complexes of these ligands showed no significant uptake in brain or heart, and the clearance was mainly through the hepatobiliary system.

Radiochemical purity testing for 99Tcm-exametazime: a comparison study for three-paper chromatography.

The standard radiochemical purity (RCP) determination uses a three-paper chromatography strip and solvent system (TP). The involvement of multiple strips for calculation of percentage primary, lipophilic 99Tcm-exametazime complex is tedious and time-consuming. The significant streaking of radioactivity on the ITLC/SG MEK strip of the TP method indicates that ITLC/SG MEK may not be an ideal system for RCP analysis of 99Tcm-exametazime. This study was undertaken to compare the standard TP method with two other proposed single-strip chromatography systems: Whatman 17 Chr paper with ethyl acetate (WE) and Gelman Solvent Saturation Pads paper with ether (GE). Our results showed that the solvent developing times (n = 55) and Rf values for TP, WE and GE were 130.4 +/- 9.0 s/0.5-1.0, 205.9 +/- 13.0 s/0.2-1.0 and 90.2 +/- 7.5 s/0.8-1.0, respectively. For RCP values ranging from 45.0 to 94.6% (n = 61), both WE and GE closely correlated with TP (r = 0.97 and 0.96). However, in the intermediate RCP range (i.e. 75-85%, n = 25), the false RCP acceptance rate (i.e. RCP > or = 80%) was 40% (10/25) for the WE method versus 4% (1/25) for the GE method. The GE method has the most clear separation of lipophilic 99Tcm-exametazime from other radiochemical impurities and offers the quickest RCP analysis for 99Tcm-exametazime with relatively accurate results.

Radiochemical purity of routinely prepared 99Tcm radiopharmaceuticals: a retrospective study.

Radiochemical purity is an important quality parameter for radiopharmaceuticals. In this study, the radiochemical purity of 2090 samples out of 7000 routine preparations of 20 different 99Tcm radiopharmaceuticals was tested using standard methods over a period of more than 7 years. The mean radiochemical purity was 96.92% (standard deviation = 6.71%). Seventy-four preparations failed to meet radiochemical purity limits; that is, 3.54% of all preparations tested or 1.06% of all preparations in the observation period. The reasons for substandard preparations were mainly related to laboratory-specific conditions. The introduction of a dedicated quality control protocol allowed the elimination of many sources of labelling failures and could reduce the number of administered preparations with an insufficient radiochemical purity. We stress the need for quality control in the preparation of radiopharmaceuticals and provide original radiochemical purity values of routinely prepared 99Tcm radiopharmaceuticals.

A rapid and stable ITLC procedure for the determination of the radiochemical purity of 99Tcm-tetrofosmin.

99Tcm-tetrofosmin (Myoview, Amersham Healthcare) is widely used as a radiopharmaceutical for myocardial perfusion imaging. Control of its radiochemical purity after reconstitution is usually performed by means of ITLC-SG paper chromatography in a mobile phase of methylene chloride/acetone (65/35), as recommended by the manufacturer. The present study describes the application of tetrahydrofuran in phosphate buffer for the development of 99Tcm-tetrofosmin on ITLC-SG strips. The use of this mobile phase significantly improves the separation between labelled tetrofosmin and unbound pertechnetate. The time for development is about 1 min and the solvent is stable for at least 1 year. In addition, the volume spotted on the strip does not affect the migration of 99Tcm-tetrofosmin. The labelling efficiency of 99Tcm-tetrofosmin can successfully be monitored by means of this method as a daily routine procedure.

A comparative study of 99Tcm-HMPAO and 99Tcm-ECD as a leukocyte labelling agent.

An attempt was made to use 99Tcm-ethyl cysteinate dimer (99Tcm-ECD) (Neurolite, Du Pont Merck, N. Billerica, MA) to label leukocytes. The radiochemical purity of 99Tcm-ECD, labelling efficiency of leukocytes, cell viability of labelled leukocytes and stability of 99Tcm-ECD-labelled leukocytes were calculated. Compared with the commercial cell-labelling agent, 99Tcm-hexamethylpropyleneamine oxime (99Tcm-HMPAO): (1) the radiochemical purity of 99Tcm-ECD was higher than that of 99Tcm-HMPAO and at immediately, 1, 2, 4, 6, 8 and 24 h after 99Tcm labelling; (2) the labelling efficiency of 99Tcm-ECD-labelled leukocytes was lower than that of 99Tcm-HMPAO; (3) the viability of the labelled white blood cells (WBC) was high for both agents; and (4) the stability of 99Tcm-ECD-labelled leukocytes was worse than that of 99Tcm-HMPAO at 1, 2, 4, 6, 8 and 24 h. It is concluded that although 99Tcm-ECD is more stable than 99Tcm-HMPAO, because of the lower labelling efficiency and power stability of 99Tcm-ECD-labelled leukocytes, 99Tcm-ECD is not a good choice as a leukocyte-labelling agent to replace commercial 99Tcm-HMPAO.

Effect of radiochemical purity of 99Tcm-Q12 on myocardial SPET image quality.

In myocardial perfusion SPET studies with 99Tcm-Q12, we observed that some patients had high liver uptake that interfered significantly in the assessment of the inferior wall. The aim of this study was to assess the effects of the radiochemical purity of 99Tcm-Q12 on liver uptake. Thirty-one patients undergoing routine myocardial infarction perfusion studies were evaluated. The radiochemical purity of 99Tcm-Q12 was determined using HPLC. Venous blood samples taken 50 min after injection of 99Tcm-Q12 during peak exercise were also analysed. Liver uptake was expressed as the liver-to-heart ratio. In addition, the SPET images were classified by two experienced nuclear medicine specialists into three groups representing high-quality images (n = 7), images with high general background activity (n = 13) and images with high liver and/or intestinal uptake (n = 11). The liver-to-heart ratio correlated inversely with the radiochemical purity of 99Tcm-Q12 (r = -0.65, P < 0.001) and unchanged 99Tcm-Q12 in plasma (r = -0.44, P < 0.02). The radiochemical purity of 99Tcm-Q12 was significantly lower in the group with high liver uptake (60.1 +/- 4.2%) than in the group with good-quality images (81.8 +/- 5.6%, P < 0.01) or with high background activity (82.3 +/- 2.5%, P < 0.01). In conclusion, the radiochemical purity of 99Tcm-Q12 has a significant inverse correlation with the liver-to-heart ratio; thus, the high radiochemical purity of 99Tcm-Q12 should be confirmed to prevent interference by liver uptake.

A high-efficiency, rapid and multi-dose procedure for white blood cell labelling with 99Tcm-HMPAO.

99Tcm-hexamethylpropyleneamine oxime (99Tcm-HMPAO) is presently recognized as an effective radiopharmaceutical for in vitro white blood cell (WBC) labelling in addition to its widespread utility in cerebral blood flow imaging. While performing clinical studies in patients with a wide range of inflammatory diseases, the effect of the ligand and stannous ion quantity on the labelling efficiency (LE) of WBC was examined. A mean LE of 64 +/- 7% (n = 29) was achieved when the whole HMPAO kit was used for labelling. The LEs were 78 +/- 5% (n = 43), 83 +/- 3% (n = 37) and 85 +/- 5% (n = 18) when one-half, one-third and one-fifth of the lyophilized kit was used, respectively. This is in agreement with the reports of Sampson et al. and Lang et al., suggesting that the optimal formulation of the 99Tcm-HMPAO is a faction of the whole kit. Accordingly, fractionation of a freshly prepared 99Tcm-HMPAO kit into five parts for a high-efficiency WBC labelling is proposed, encouraging the more widespread use of 99Tcm-HMPAO in WBC labelling.

Preparation of 99mTc-ethylene dicysteine (99mTc-EC) by transchelation using 99mTc-glucoheptonate (99mTc-GHA) and its evaluation for renal function studies.

The complexation of ethylene dicysteine (EC) with 99mTc needs to be carried out at pH 12 to achieve a high radiochemical yield. However, the preparation of the kit at high pH poses difficulties and requires very stringent preparation conditions, as stannous tin, one of the main ingredients in the kit, is unstable at high pH. Hence, an alternative method, involving the transchelation preparation of 99mTc-EC using 99mTc-glucoheptonate (99mTc-GHA) prepared at pH 6.5, was attempted, prompted by the reported success of the preparation of 99mTc-sestamibi (99mTc-MIBI) by this method. The preparation of 99mTc-EC by this method first involved the formation of 99mTc-GHA by the addition of sodium pertechnetate-99mTc to the Sn-GHA kit vial at pH 6.5. 99mTc-EC was formed by the addition of reconstituted EC solution at pH approximately 12 to the preformed 99mTc-GHA. The reaction was allowed to proceed both at room temperature and on a boiling water bath. The pH of the final product was adjusted to pH approximately 7 with 0.5 M phosphate buffer at pH 4-5, without affecting the quality of the product. The urinary excretion of 99mTc-EC prepared by transchelation, tested in mice, was similar to that of directly prepared 99mTc-EC, indicating that the final product prepared by the two methods was the same. The clinical evaluation of the product formulated by the new procedure showed satisfactory findings, comparable with the reports in the literature.