Exchange labeling of proteins:99mTc labeled transferrin

^99mTc-labeled transferrin was prepared by reduction of^99mTcO ₄ ⁻ ; with stannous DTPA or stannous citrate followed by equilibration of the technetium chelate with human transferrin. The rate of transfer of^99mTc to transferrin in the presence of 0.015M citrate buffer was dependent on pH in the order pH 2.1> pH 7.2> pH 4.1> pH 5.9. The incorporation rate was inversely proportional to the concentration of DTPA and citrate buffer. The replacement of citrate buffer by acetate buffer or oxalate buffer reduced drastically the formation of^99mTc-labeled transferrin at pH 4.1. The formation of^99mTc-labeled transferrin prepared from the reduction of^99mTcO ₄ ⁻ with stannous citrate was faster than that from the reduction with stannous DTPA in the presence of 0.015M citrate buffer and pH 2.5. Equilibration of transferrin with^99mTc-labeled pyrophosphate did not produce^99mTc-labeled transferrin at pH 4.5. The ligand exchange labeling of^99mTc to transferrin in 0.015M citrate did not cause appreciable denaturation of the protein at all pH values. This method also enabled labeling of the protein in a low concentration (2.6·10⁻⁴ M) via tin reduction. Sequential external imaging of the^99mTc-labeled transferrin in Sprague-Dawley rats bearing Walker-256 carcinosarcoma showed optimal tumor localization occurred at 3 hr after injection. In spite of this,^99mTc-labeled transferrin does not appear to be a suitable imaging agent because of the low tumor to blood ratio of^99mTc (0.50±0.17) at 3 hr post injection. This is similar to that of^{6 7}Ga-citrate (0.43±0.15%).     

Effect of experimental conditions on the in vitro stability of99mTc (Sn)-pyrophosphate

The in vitro stability of^99mTc (Sn)-PyP as a function of experimental conditions of the preparation of the kit and time elapsed after labeling has been tested. The preparation was protected by using nitrogen-purged reactant solutions and kit vials and by ascorbic acid. The samples under nitrogen are stable for 6 h when the content of^99mTc-pertechnetate raises to 5%. The best stability was achieved by addition of 5 g of ascorbic acid per ml of the kit (content of^99mTc-pertechnetate about 0.5%). To accelerate the decomposition, exogenous hydrogen peroxide was used. In this case it was found that the presence of 10 g of ascorbic acid inhibits the effect both of oxygen and peroxide (6 g H₂O₂/ml of the kit). Radiochemical purity of^99mTc (Sn)-PyP remains practically unchanged for 6 h (content of^99mTc-pertechnetate about 0.5%).     

The study of conditions for the preparation and application of99Mo−99mTc generators starting from irradiated molybdenum metal

⁹⁹Mo−^99mTc generators were prepared starting from irradiated molybdenum metal instead of MoO₃ in order to use reactor irradiation space more economically. The adsorption of molybdenum as sodium molybdate on different kinds of alumina was investigated. The effect of the pH of the column and the aqueous phase concentration of molybdenum were studied and related to the elution yield of^99mTc. A study of the radiation damage effect indicated that generators having a high elution efficiency of 90% could be prepared in the 100–600 mCi range. The losses of⁹⁹Mo were minimized to 10⁻⁵-10⁻⁴% and those of alumina to 2–5 μg/ml eluate.     

Comparative study of different gels for quality control of99mTc-radiopharmaceutical kits

The available six types of Sephadex and one type of Sepharose have been applied in the separation of technetium fractions in^99mTc-labelled radiopharmaceuticals produced in our laboratory using the GCS technique. By this technique the chemical state and the percentage of^99mTc-fractions have been determined. The resolution efficiency of some gel types were found to be significantly influenced by the pH of the eluent. The results obtained from the experiments indicated that Sephadex G-25 Fine was the best and can be routinely used in the radiochemical analysis of the following kits:^99mTc-HSA,^99mTc-DTPA and^99mTc-HIDA and G-100 for^99mTc-PYP. With^99mTc-HSA and^99mTc-PYP kits, 0.9% NaCl eluents at pH 3.2 and pH 2 to 2.5, respectively, were found to be necessary for the separation of^99mTc-fractions. G-50 Fine was found to be the best gel between the others in the separation of^99mTc-fractions in testing of the weak radiopharmaceuticals,^99mTc-GH and^99mTc-MDP. The development of^99mTc-MDP with the eluent at the same pH as the preparation gives negligible interaction effect.     

Preparation of113mIn radiocolloid for liver scanning

Kits were developed for the preparation of a sterile^113mIn colloid as a radiopharmaceutical for liver scanning. 2.5 ml of^113mIn sterile generator eluate was added to 0.5 ml of ferric chloride dissolved in 0.04N HCl (40 g/ml). The pH was adjusted by addition of 2 ml of phosphate buffer. The optimal pH for the formation of^113mIn colloid was found to be equal to 7.5–8.5. Liver uptake in mice was determined to be 85–90%.     

Statistical analysis of the results of99mTc-MDP quality control

The methods used for control of radiochemical purity of^99mTc-MDP are presented. TLC method on silica gel, developed with methanol and acetone (11 v/v), was convenient for determination of^99mTcO ₄ ^– with the content of 2.6±1.2%. The reliable results on detection of^99mTc hydrolyzate (2.2±1.3%) and for another^99mTc-MDP complex (13.2±2.8%) were obtained by application of ITLC (SA), developed with Sn-MDP. By Sephadex G-25 column chromatography (1.5 cm×5 cm) the separation of^99mTcO ₄ ^– was not achieved. The range of normal^99mTc-MDP biodistribution values in the organs of experimental animals have been determined. The mean value of bone distribution was 8.4±1.13%/g, in muscles 0.071±0.033%/g, while uptake in liver and kidneys was below 5%. Chi-square test and P show that the results on biodistribution of^99mTc-MDP in liver, bones and muscles are arranged around their mean values, which is statistically allowed.     

Separation of99Mo from132Te using thiourea as complexing agent. Application to the separation of99Mo from fission products

A radiochemical method to isolate⁹⁹Mo from¹³²Te, both produced in the fission of²³⁵U, has been developed. The method is based on the formation of a cationic complex of tellurium with thiourea in acid medium which is retained (98.7±0.5)% on a cation exchange resin (Dowex 50W-X8, 100–200 mesh), while (99.8±0.05)%⁹⁹Mo passes through it, due to the non-formation of such complex in the same experimental conditions. The radionuclidic purity of⁹⁹Mo was found to be suitable for the preparation of⁹⁹Mo–^99mTc generators. The retention of⁹⁹Mo on an alumina column as a function of pH was investigated and the best pH range for this purpose was found to be 4.0–4.5.     

Quality control of a detector for the calculations of radiopharmacokinetic parameters of 99mTc-glucarate in rats

Good manufacturing practices specify that a well-type scintillation NaI(Tl) crystal detector has to be validated in order to detect radioactivity from any radiopharmaceutical used to obtain radiopharmacokinetic parameters. A 5 cm well-type NaI(Tl) scintillation detector was coupled to a multi-channel analyzer centered at the 140 keV ^99mTc peak with a 20% window. The area represents counts per minute (cpm). All the net cpm were decay corrected. The activity source was ^99mTc-glucarate developed as an imaging agent for acute myocardial infarction. Wistar rats were injected in a tail vein with 0.1 ml (3.7 MBq) of ^99mTc-glucarate solution and 13 blood samples were taken. The cpm were the input data for the WINNONLN program which calculates radiopharmacokinetic parameters. The detector's efficiency for ^99mTc was 15.03% and the sensitivity 1.12 kBq/ml in plasma. The response was linear between 0.31-14.3 kBq/ml of ^99mTc-glucarate. The maximum assay variation coefficient was 2.79 and recovery of ^99mTc-glucarate in plasma was 99.8%±0.2%. LOD was 0.31 kBq and LOQ = 1.12 kBq in plasma samples. ^99mTc-glucarate follows a two-compartment model of distribution with Vd of 21.74 ml±2.71 ml; a Vdss of 74.36 ml±12.67 ml; t _{1/2 a}0.74 h±0.19 h; t _{1/2 b} 18.98 h±4.36 h; AUC = 32.75 mCi/min^.ml ± 3.73 mCi/min^.ml; MRT = 24.35 h±5.51 h and total clearance 3.05 ml/h±0.35 ml/h. The well-type detector fulfills the quality system requirements and the radiopharmacokinetic parameters for ^99mTc-glucarate in rats are reliable. This revised version was published online in July 2006 with corrections to the Cover Date.     

Technetium-99m dextran kit: formulation and biological behaviour

A new formulation of stannous-dextran (Sn-Dx) freeze dried kit, containing 60 mg dextran (Dx-70) and 0.08 mg SnCl₂·2H₂O, to be labelled with^99mTc, has been developed. At pH 6.5–7.0. the labelling efficiency was greater than 95%. Gel chromatography column scanning technique was applied for radiochemical purity determination of^99mTc-Dx preparation and the degree of in vivo plasma protein binding. Not less than 70% of the administered activity was bound to plasma and remained constant over a 1h period. The biological behaviour after intravenous injection of^99mTc-Dx kit was characterized by high and efficient yield of the radiopharmaceutical. The preliminarly clinical results on normal subjects showed that the radiopharmaceutical could be a useful agent for scintigraphy of leg lymph vesel, pelvic and inguinal lymph nodes. The activity uptake in liver and kidney (60 min) was relatively very low, whereas the urinal bladder activity (30 min) represents the drainage of the activity entering the blood stream after interdigital injection of^99mTc-Dx.     

Synthesis of <Superscript>99m</Superscript>Tc-pefloxacin: A new targeting agent for infectious foci

Summary This investigation focused on the labeling of pefloxacin, a fluoroquinolone antibacterial agent, with ^99mTc to form ^99mTc-pefloxacin complex. The labeling process was done by direct addition of pertechnetate in isotonic solution to Sn-pefloxacin solution. The labeling technique is effective, as a high labeling yield (98%) was obtained after 30-minute reaction time. Different factors were found that influenced this labeling reaction: 0.5 mg pefloxacin or more must be used to prevent the formation of colloids in the reaction medium. Fifty micrograms of stannous chloride dihydrate were found to be sufficient to reduce all pertechnetate with activity ranging from 37 to 3700 MBq without the detection of free pertechnetate or colloids in the reaction mixture. The pH of the reaction medium was found to play an important role in the labeling process. The labeling reaction proceeds well at neutral pH (pH 6) but at acidic pH value (pH 4 or below) the yield of ^99mTc-pefloxacin complex decreased markedly to a labeling yield of 5%. The reaction mixture must be heated to 100 °C in an oil bath to enhance the formation of the ^99mTc-pefloxacin complex. The biodistribution data show that ^99mTc labeled pefloxacin was retained in infectious focus. The retention was specific since the abscess uptake of ^99mTc-pefloxacin remained high as compared to the uptake of aseptic foci at 24-hour post injection. Also, the clearance of the tracer from other tissues is rapid on the contrary to its clearance from the septic focus. This supports the hypothesis that ^99mTc-pefloxacin is retained at the infectious site because of its specific binding to the gyrase enzymes of bacterial cells.     

Technetium-99m labeling of hippuric and p-amino-hippuric acid in the presence of DTPA: Biological and pharmacokinetic evaluation and comparison with99mTc-DTPA

^99mTc-Hipp as a modified^99mTc-DTPA, and^99mTc-PAH as a new renal agent are developed. Each veal of lyophilised kit contain hippuric (Hipp) or p-aminohippuric acid (PAH), diethyltriaminepentaacetic acid as calciumtrisodium salt (CaNa₃DTPA) and stannouschloride (SnCl₂·xH₂O), in molar ratio Hipp/PAH:DTPA=4∶1. They are high radiochemical purity radiopharmaceuticals, with hydrophilic character and low percentage of protein binding. The ITL chromatography and HPLC analyses of these labeled compounds have shown almost identical results as^99mTc-DTPA, but their biological behavior in rats confirm certain differences.^99mTc-Hipp is a renal agent clearing by the glomerular system, with better pharmacokinetical parameters than^99mTc-DTPA:t _1/2(α)=4.1 min,t _1/2(β)=198.6 min,K _cl=1.2·10⁻²min⁻¹ and a twofold value for blood clearance (Cl=2.07 ml/min).^99mTc-PAH is a quite different renal agent, rapidly secreted by kidney as a tubular secretion agent. Its pharmacokinetical parameters:t _1/2(α)=2.5 min,t _1/2(β)=41.7 min andK _cl=5.1·10⁻⁴ min⁻¹ are almost equal to those of^99mTc-MAG₃, but the blood clearance of Cl=5.22 ml/min is even higher than that of IOH clearance.     

Preparation of 186Re-MIBI complex for myocardial perfusion imaging as potential replacement of analogues 99mTc-MIBI complex

The optimum conditions to label 2-methoxyisobutyl-isonitrile (MIBI) compound with pure ¹⁸⁶Re as a stable contrast agent for myocardial perfusion imaging were investigated. Complexation of MIBI with ¹⁸⁶Re was carried out using anhydrous stannous chloride, gentisic acid and 1 ml of 37 MBq ¹⁸⁶ReO₄ ^? at pH 2 in a boiling water bath for 30 min. The corresponding radiochemical yield was ≈95.5 %. The biodistribution studies in mice indicated that, the complex was cleared from the body by kidneys to urinary bladder and finally into urine. ¹⁸⁶Re-MIBI demonstrated satisfactory heart uptake like to ^99mTc- MIBI (8.94 % dose/organ at 5 min). The obtained data showed that ¹⁸⁶Re-MIBI is a potential replacement of ^99mTc-MIBI for myocardial perfusion imaging.     

Development of electrochemical labelling procedures using MEK extracted99mTc

Electrolytic labelling procedures have been reported for various^99mTc radiopharmaceuticals which differ widely in the choice of the electrodes, working pH, applied voltage and the quantity of current passed. The authors have studied the electrolytic labelling of^99mTc EHDP, gluconate and glucoheptonate with MEK extracted^99mTc using tin electrodes under different experimental conditions. The results have, shown that these compounds can be efficiently labelled with^99mTc in a single step procedure avoiding multiple pH adjustments. Labelling of human serum albumin microspheres suitable for lung imaging with^99mTc by the electrolytic method is also reported.     

Radiolabeling of anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb BW 431/26) for clinical applications

A method for labeling of anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) BW 431/26 is described. For preparations of^99mTc-anti-CEA complex, a solution (1 ml) of tetrasodium-1,1,3,3-propanetetetraphosphate dihydrate (2.7 mg), stannous chloride dihydrate (0.12 mg) and sodium chloride (0.2 mg) in 5 ml of 0.9% saline was added to a vial containing monoclonal antibody (2 mg) from mouse (MAb BW 431/26), D-glucitol (2 mg) and sodium sulfate (2 mg) to obtain a clear solution. A quantitative labeling yield of the MAb BW 431/26 was achieved by addition of 5 ml (40.5 mCi, 1.5 Gbq) technetium-99m-pertechnetate (^99mTcO₄) generator eluate at room temperature within 10 minutes. The radiochemical purity determined by ITLC (Gelman, SG) plates was >95%.     

Reaction of [TcNCl4]− with ethylenediamine-N,N'-diacetic acid ligand

The reaction of [^99mTcNCl₄]^– with ethylenediamine-N,N'-diacetic acid (ENDA) has been studied. Starting from [^99mTcNCl₄]^– prepared according to the method of Apparu et al.6, the exchange reaction with ENDA in the presence of stannous tartrate led to the formation of an anionic^99mTcN-ENDA complex. The labeling yield was about 90% at pH values of 10.5 and 6.5. In the absence of the reducing agent the formation of^99mTcO₄ ^– is reflected in the decrease of the complex yield. The exchange reaction of [⁹⁹TcNCl₄]^– with ENDA at pH 6.5 and in the absence of a reducing agent resulted in the formation of the anionic⁹⁹TcN-ENDA complex and⁹⁹TcO₄ ^–. The spectrophotometric characteristics (UV-vis. and IR) of the pure complex are similar to those of some tcN²⁺-complexes with amine ligands. The electrophoretic and chromatographic behaviors of^99mTcN- and⁹⁹TcN-ENDA anions are identical; this proves the formation of the same complex with both radionuclides.     

Separation of99mTc from parent99Mo by solid-phase column extraction as a simple option for a new99mTc generator concept

Evidence is obtained to show that the liquidliquid extraction separation of^99mTc from⁹⁹Mo with methyl ethyl ketone, methyl propyl ketone and methyl isobutyl ketone can be transformed into a solid-phase column extraction procedure. The aqueous alkaline⁹⁹molybdate solution is immobilized on a column of a granular large-pore diatomaceous earch support, which is the neluted with the abovementioned extractants. Rapid and clean separation of^99mTc can be with all three solvents. The^99mTc can be back-extracted from the organic phase on a column filled with distilled water /or saline/ loaded granular diatomaceous earth /Extrelut^®/. The possibility of using the abovementioned procedure as a basis for a new^99mTc/⁹⁹Mo generator concept is envisaged.     

Effect of chemical condition of technetium-99m sodium pertechnetate on the active kit components and radiolabeling yield of Tru-Scint® ADTM monoclonal antibody kit

The chemical condition of^99mTc eluate obtained from a⁹⁹Mo-^99mTc generator is a function of the source, time elapsed after elution and age of the eluate. The radiochemical purity and stability of^99mTc labeled MAb-170 (Tru-Scint^®AD^TM, photoactivated monoclonal antibody kit) preparations was evaluated comparing pertechnetate source of known age and elution history. The effect of H₂O₂, a radiolytic impurity in^99mTc eluates, on the active kit components stannous ion and photoactivated MAb and radiolabeling, yield has been investigated. The lyophilized Tru-Scint^® AD^TM kit has been labeled with 20 to 80 mCi in 0.5 to 4.0 ml of Sodium Pertechnetate^99mTc Injection, USP. The eluates were obtained from three brands of generators and used up to six hours after elution. The kits were reconstituted either with Sodium Pertechnetate^99mTc Injection, USP or Sodium Chloride Injection, USP, 0.9% containing known amounts of H₂O₂. The reconstituted kits were analyzed for radiolabeling yield and radiochemical impurities, stannous ion and protein sulfhydryl group. The results indicated that the radiolabeling yield is a function of both the chemical condition of^99mTc eluate, generator brand and the radiolabeling parameters like reconstitution volume and activity. The observed radiolabeling yield differences did not depend on the amount of chemical technetium in the eluate. The major radiochemical impurities at 15-minute post labeling have been identified as the^99mTc-buffer complex and column adsorbed reduced^99mTc (^99mTc-Ad) species and not the unreduced^99mTcO ₄ ^– .     

Solvent extraction of99mTc/VII/ with methylene blue into nitrobenzene

The extraction of^99mtechnetium/VII/ with methylene blue into organic solvent has been investigated. Nitrobenzene, which was found to be the most effective extractant for^99mTc/VII/, was used to separate^99mTc/VII/ from other elements. The effect of various parameters on the extraction coefficient value such as effect of pH, time of equilibration, effect of anions and cations on the E value have been evaluated. The stoichiometry of metal to reagent was determined by the slope ratio method and was found to be 11.     

Standardization of quality control methods for99mTc radiopharmaceuticals

Physico-chemical characterization of^99mTc-radiopharmaceuticals is presented. Limiting pH values, iso-osmotic pressure and the apparent coefficient values between two immiscible phases are determined too. A selection of radiochromatographic methods /stationary or mobile phase/ for routine quality control of^99mTc radiopharmaceuticals for radiochemical purity was made. The methods chosen are simple, accurate, sufficiently sensitive and fast in operation. The mean values were determined for^99mTc radiopharmaceutical distribution per organs, characteristic for the tested preparates and for radiochemical purity, as well as the time interval from injection to sacrifice of the animals.     

Preparation of <Superscript>99m</Superscript>Tc-PQQ and preliminary biological evaluation for the NMDA receptor

Pyrroloquinoline quinone (PQQ), an essential nutrient, antioxidant, redox modulator and nerve growth factor found in a class of enzymes called quinoproteins, was labeled with ^99mTc by using stannous fluoride (SnF₂) method. Radiolabeling qualification, quality control and characterization of ^99mTc-PQQ and its biodistribution studies in mice were performed and discussed. Effects of pH values, temperature, time and reducing agents concentration on the radiolabeling yield were investigated. The quality control procedure of ^99mTc-PQQ was determined by thin layer chromatography (TLC), radio high-performance liquid chromatography (RHPLC) and paper electrophoresis methods. The average radiolabeling yield was 94 ± 1% under optimum conditions of 0.99 mg of PQQ, 30 μg of SnF₂, 0.5 mg of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and 18.5 MBq of Na^99mTcO₄ at pH 6 and 25 °C with a response volume of 1 ± 0.1 mL. ^99mTc-PQQ was stable and anionic. Lipid–water partition coefficient of ^99mTc-PQQ was −1.49 ± 0.16. The pharmacokinetics parameters of ^99mTc-PQQ were t _1/2α = 18.16 min, t _1/2β = 100.45 min, K ₁₂ = 0.013 min⁻¹, K ₂₁ = 0.017 min⁻¹, K _e = 0.016 min⁻¹, AUC (area under the curve) = 1040.78 ID% g⁻¹ min and CL (plasma clearance) = 0.096 mL min⁻¹. The dual-exponential equation was Y = 10.88e^−0.038t  + 5.21e^−0.0069t . The biodistribution of ^99mTc-PQQ was studied in ICR (Institute for Cancer Research 7701 Burhelme Are., Fox Chase, Philadelphia, PA 1911 USA) mice. In vitro autoradiographic studies clearly showed that the ^99mTc-PQQ radioactivity accumulated predominantly in the hippocampus and cortex, which had a high density of N-methyl-d-aspartate Receptor (NMDAR). The enrichment can be blocked by NMDAR redox modulatory site antagonists-ebselen (EB) and ^99mTc-PQQ is therefore a promising candidate for the molecular imaging of NMDAR. To date, however, there have been no studies characterizing ^99mTc-PQQ.