Direct labeling of doxorubicin with technetium-99m: its optimization, characterization and quality control

Doxorubicin (DOX) is an anthracycline antineoplastic and one of the most potent and widely used drugs in clinical oncology. It is used in the treatment of a wide variety of cancers. The aim of this study was the direct labeling of DOX with ^99mTc; its optimization, characterization and quality control of the radiolabeled DOX. Labeling efficiency was determined by paper chromatography. More than 92% labeling was obtained at pH 6?C7, 10?C12???g stannous chloride and 200???g of DOX. The stability of ^99mTc?CDOX was studied up to 5?h. All the experiments were performed at room temperature (25±?2?°C). The characterization of the labeling compound was performed by HPLC and electrophoresis. Electrophoresis indicates that labeled DOX has no charge and HPLC shows single specie of labeled compound.     

Labeling of scorpion venom with 99mTc and its biodistribution

Labeling of scorpion venom (SV) was successfully achieved with ^99mTc using direct chelating method. Venom was labeled with ^99mTc using stannous chloride as reducing agent. Preliminary studies were done to establish the optimum conditions for obtaining the highest yield of the labeled venom. The labeling technique is effective, as a maximum labeling yield (97 %) was obtained after 30-min reaction time by using 80 μg SV in phosphate buffer of pH 7 and 25 μg Sncl₂·2H₂O at room temperature. Venom was injected into normal mice to determine the excretion pathway. Biodistribution studies in normal mice with SV shows rapid clearance of the venom from blood and tissue except for kidneys. The improvement of the immunotherapeutic treatment of envenomation requires a better knowledge of the biological actions of the SV since tissue distribution studies are very important for clinical purpose.     

Synthesis of 99mTc-oxybutynin for M3-receptor-mediated imaging of urinary bladder

Radiolabeling of oxybutynin, a muscarinic acetylcholine (mACh) receptor antagonist agent with ^99mTc is of considerable interest for imaging of urinary bladder. This study is aimed to optimize radiolabeling yield of oxybutynin with ^99mTc using SnCl₂·2H₂O as a reducing agent with respect to factors that affect the reaction conditions such as oxybutynin amount, stannous chloride amount, reaction time and pH of the reaction mixture. In vitro stability of the radiolabeled complex was checked and it was found to be stable for up to 8 h. ^99mTc-oxybutynin was injected via subcutaneous and intravenous administration routes into normal Sprague?CDawley rats. Biodistribution studies have revealed that ^99mTc-oxybutynin exhibits high affinity and specificity for the muscarinic M₃ subtype located on the smooth muscle of urinary bladder relative to the M₁ and M₂ subtypes of the G protein coupled receptor (GPCR) superfamily. In vivo uptake of subcutaneous ^99mTc-oxybutynin in urinary bladder was 19.6 ± 0.42% ID at 0.5 h, whereas in intravenous administration route the accumulation in the urinary bladder was found to be 9.4 ± 0.31% ID at 0.5 h post injection. Administration of cold oxybutynin effectively blocked urinary bladder uptake and further confirms the high specificity of this complex for the M₃ receptor.     

Synthesis of 99mTc-cationic steroid antimicrobial-107 and in vitro evaluation

Ceragenins/cationic steroid antimicrobials (CSAs) are a group of cholic acid derivatives with many properties that make them favourable for application as anti-infective agents. CSA-107 is also a member of this group that was labelled with ^99mTc by using SnCl₂·2H₂O as reducing agent and Na–K tartrate as transchelating agent. Labelling efficiency was optimized by varying the amount of reducing agent, pH, and time of incubation. Labelling efficiency and the stability of ^99mTc-CSA-107 in human serum was determined by paper and thin layer chromatography, which were >95 and >90 % respectively. In vitro binding of ^99mTc-CSA-107 was >95 % determined by using Staphylococcus aureus bacteria.     

Formulation and preclinical evaluation of 99mTc–gemcitabine as a novel radiopharmaceutical for solid tumor imaging

The aim of this study is the formulation of a new radiopharmaceutical for imaging solid tumor bearing. Gemcitabine is a nucleoside analogue used as chemotherapeutic agent. Gemcitabine was formulated and radiolabeled with one of the most important diagnostic radioactive isotopes (technetium-99m) to be investigated in solid tumor imaging. The labeling parameters such as gemcitabine amount, stannous chloride amount, pH of the reaction mixture, and reaction time were optimized. ^99mTc–gemcitabine was prepared at pH 9 with a maximum labeling yield of 96 ± 0.3 % without any notable decomposition at room temperature over a period of 8 h. The preclinical evaluation and biodistribution in solid tumor bearing mice showed that ^99mTc–gemcitabine had solid tumor selectivity, preclinical high biological accumulation in tumor cells and high retention. Tumor/normal muscle (T/NT) ratios increased with time showing high T/NT ratio (T/NT = 4.9 ± 0.27 at 120 min post injection) and high Tumor/Blood ratio (3.4 ± 0.06), suggesting ^99mTc–gemcitabine as a novel solid tumor imaging agent.     

Synthesis,characterization and biodistribution of 99mTc-Bioquin-HMPAO (99mTc-BH) as a novel brain imaging agent

Recently, the development of novel brain imaging agents has aroused much interest thanks to limited number of brain cancer or diseases diagnosis agents. It is aimed to synthesize a novel brain imaging agent including a promise for further studies on AD diagnosis potential and investigate its bioaffinity with biodistribution studies on healthy Balb/c mice. A novel radiolabeled agent was synthesized and characterized. Quality control of ^99mTc-BH was performed utilizing solvent extraction and chromatographic (Radio TLC and Radio HPLC) methods. Bioaffinity of the ^99mTc-BH was investigated on male Balb/c mice at various time points (5, 30, 60, 120 min post-injection). Paper electrophoresis showed that ^99mTc-BH has a neutral structure. Radiochemical purity of ^99mTc-BH was over 95 % with appropriate stability for imaging period. Selected brain regions have uptakes over 4 % ID/g following intravenous injection. Hippocampus has uptake approximately 10 % ID/g. ^99mTc-BH has shown brain uptake, so it may prove to be valuable for brain imaging as a novel technetium-labeled agent. Further investigations with AD animal model are our on going effort to show that this agent has AD diagnosis potential.     

Synthesis and evaluation of a series of 99mTc-labelled zoledronic acid derivatives as potential bone seeking agents

Developing novel superior bone-seeking radiopharmaceuticals for the detection of malignant bone lesions could further improve the diagnostic value of routine bone scanning. A series of radiolabeled diphosphonates (^99mTc-EIPrDP, ^99mTc-EIBDP and ^99mTc-EIPeDP) have been designed and synthesized successfully in high chemical yields and radiochemical purity. The in vitro and in vivo biological properties were systematically investigated and compared. The biodistribution in mice shows that ^99mTc-EIPrDP has higher bone uptake (13.3 ± 1.23) than those of ^99mTc-EIBDP and ^99mTc-EIPeDP (11.7 ± 0.28 and 8.69 ± 0.04 %ID/g) at 1–2 h post injection. It also has the highest uptake ratio of bone to muscle, spleen and heart, respectively, and faster blood clearance in early times. The present study indicates that ^99mTc-EIPrDP holds great promise as a bone imaging agent.     

A perspective on <Superscript>99m</Superscript>Tc and <Superscript>125/131</Superscript>I labeled receptor targeted compounds and their in vitro/in vivo affinities

A novel quinoline derivative, 2,2′-[(5-chloro-8-hydroxyquinoline-7-yl) methylazanediyl] diacetic acid (CHQMADA) was labeled with ^99mTc using SnCl₂·2H₂O as a reducing agent to give a complex with a labeling yield 94 %. Also [^99mTc(H₂O)₃(CO)₃]⁺ was prepared by heating at 100 °C for 30 min using 2 mg CHQMADA at pH 8 to give a labeling yield >99 %. ^99mTc-(CO)₃ CHQMADA and ^99mTc-Sn(II)-CHQMADA showed tissue uptake (target to non target T/NT = 6.80 ± 0.22) and (T/NT = 5.65 ± 0.34) respectively in Escherichia coli induced infection, which is higher than the commercially available ^99mTc-ciprofloxacin (T/NT = 3.80 ± 0.80). In conclusion, both complexes were able to differentiate between septic and aseptic inflammation with superiority of [^99mTc-(CO)₃ CHQMADA].     

Synthesis and preliminary evaluation of 99mTc-spermine as a tumor imaging agent

Polyamines are essential for the growth and survival of all cells with biosynthesis and transportation of polyamines being very active in tumors. With the aim of developing a new tumor imaging agent, the endogenous polyamine, spermine was labeled with ^99mTc, and its characters were also evaluated via in vitro and in vivo studies. ^99mTc-labeled spermine probe (^99mTc-spermine) was synthesized by the direct pretinning procedure and the labeling procedure was optimized with regard to the pH, reaction time, amounts of spermine and SnCl₂. The stability of the ^99mTc-spermine and its capacity to accumulate into 4T1 tumor cells were also evaluated. Biodistribution of ^99mTc-spermine was studied in 4T1 tumor-bearing mice. In the optimal conditions, the whole radiosynthesis was accomplished within 10 min with a decay-corrected yield of 96.5 ± 1.3 % and radiochemical purity of >95 %.^99mTc-spermine was stable at both 37 and 4 °C for at least 6 h. In vitro tests revealed that the ability of ^99mTc-spermine to penetrate in 4T1 tumour cells and an excess of spermine blocked the accumulation of the compound in the models. Biodistribution studies showed a high tumor uptake peaked at 30 min post-injection with 1.82 ± 0.19 % ID%/g. The tumor to muscle uptake ratios of the probe were 3.60 ± 0.51, 4.48 ± 0.29, 4.82 ± 0.18, 5.64 ± 0.10, respectively at 30 min, 1, 2 and 4 h postinjection. Block studies indicated that ^99mTc-spermine had specific binding of tumor via polyamine transport systems. ^99mTc-spermine is a promising radiopharmaceutical in tumor imaging. Further studies are required to determine the usability of ^99mTc–spermine for diagnosis purposes.     

Preparation and biological distribution of 99mTc-cefazolin complex, a novel agent for detecting sites of infection

The optimization of the radiolabeling yield of cefazolin with ^99mTc was described. Dependence of the labeling yield of ^99mTc-cefazolin complex on the amounts of cefazolin and SnCl₂·2H₂O, pH and reaction time was studied. Cefazolin was labeled with ^99mTc with a labeling yield of 89.5 % by using 1 mg cefazolin, 5 μg SnCl₂·2H₂O at pH 4 and 30 min reaction time. The radiochemical purity of ^99mTc-cefazolin was evaluated with ITLC. The formed ^99mTc-cefazolin complex was stable for a time up to 3 h, after that the labeling yield decreased 64.0 % at 8 h. Biological distribution of ^99mTc-cefazolin complex was investigated in experimentally induced inflammation mice, in the left thigh, using Staphylococcus aureus (bacterial infection model) and turpentine oil (sterile inflammation model). Both thighs of the mice were dissected and counted and the ratio of bacterial infected thigh/contralateral thigh was then evaluated. In case of bacterial infection, T/NT for ^99mTc-cefazolin complex was 8.57 ± 0.4 after 0.5 h, which was higher than that of the commercially available ^99mTc-ciprofloxacin under the same experimental conditions. The ability of ^99mTc-cefazolin to differentiate between septic and aseptic inflammation indicates that ^99mTc-cefazolin could undergo further clinical trials to be used for imaging sites of infection.     

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.     

99mTc-labeling of a dithiocarbamate-DWAY fragment using [99mTcN]2+ core for the preparation of potential 5HT1A receptor imaging agents

1-(2-methoxy phenyl) piperazine fragment of WAY100635 or its phenolic analogue, derived from DWAY is used to design the desired structure of 5HT_1A receptor imaging agents. In this study a DWAY analogue was labeled with ^99mTc-nitrido ([^99mTcN]²⁺) core via dithiocarbamate. 2-(piperazin-1-yl) phenol dithiocarbamate was synthesized by the reaction of 2-(piperazin-1-yl) phenol with an equivalent amount of carbon disulfide in KOH solution then radiolabeled with ^99mTc-nitrido core. The final complex was characterized by HPLC and its radiochemical purity was more than 90 %. In vitro stability studies have shown the complex was stable at least 4 h after labeling at room temperature. The n-octanol/water partition coefficient experiment demonstrated log p = 1.34 for ^99mTcN–OHPP–DTC. Biodistribution results showed that radio tracer had moderate brain uptake (0.39 ± 0.03 %ID/g at 15 min and 0.29 ± 0.02 %ID/g at 120 min) and good retention, suggesting that this complex may lead to a further development of a radiotracer with specific binding to 5-HT_1A receptor.     

Preliminary studies of 99mTc-PQQ-NMDAR binding and effect of specificity binding by mannitol

Pyrroloquinoline quinone (PQQ) is a powerful neuroprotectant that specifically binds to brain NMDA receptors and inhibits excitotoxicity. Imaging this binding reaction in the brain remains a long sought goal in this field of study, and one of the primary challenges remaining is enabling soluble labeled PQQ to pass the blood–brain barrier (BBB). Previously, our group successfully labeled PQQ with Technetium-99m (^99mTc), a metastable nuclear isomer used in radioactive isotope medical tests. In this work, we determined the specific binding of ^99mTc-PQQ and NMDAR by radioligand receptor assay. Ebselen (EB) and MK-801 both effectively inhibited ^99mTc-PQQ binding. We then investigated methods of opening the BBB using mannitol to enable entry to the brain by ^99mTc-PQQ. Our results showed that 7.5 mL/kg of 20 % mannitol effectively opened the BBB and 20 min was the optimum treatment time. Competition studies showed that mannitol did not affect the specific binding between ^99mTc-PQQ and NMDA receptors. Using this method, the amount of ^99mTc-PQQ uptake and retention was increased most significantly in the hippocampus and cortex, and re-opening the BBB did not affect binding. Together, our results demonstrate that the use of mannitol to open the BBB may contribute significantly to improving image quality by increasing the uptake amount of a water-soluble agent in brain.     

Preparation of <Superscript>99m</Superscript>Tc-metronidazole as a model for tumor imaging

Metronidazole (MTNZ) is an antiprotozoa drug, could be labeled with the ^99mTc. MTZL could be used as an ideal vehicle to deliver radioactive decay energy of ^99mTc to the sites of tumor, thus facilitate tumor imaging. The process of labeling was done using tin chloride as reducing agent. The optimum conditions required to label 25 μg MTZL were 100 μg stannous chloride, 30 min reaction time, room temperature at pH 7–9 using 0.5 M phosphate buffer. The radiochemical purity of the labeled compound, at the above conditions, was determined using paper chromatography. The yield was about 93%. About 2.5 × l0⁶ of Ehrlich Ascites Carcinoma (EAC) was injected intrapritoneally (i.p) to produce ascites and intramuscularly (i.m) in the right thigh to produce solid tumor in female mice. Biodistribution studies were carried out by injecting solution of ^99mTc-MTZL in normal and tumor bearing mice. The uptake in ascites was over 5% of the injected dose per gram tissue body weight, at 4 h post injection and above 4% in solid tumor. These data revealed localization of the tracer in the tumor tissues with high percentage sufficient to use ^99 mTc MTZL as promising tool for diagnosis of tumor.     

Evaluation of highly loaded low specific activity 99Mo on alumina column as 99mTc generator

Adsorption behavior of molybdate on acidic alumina was studied at boiling water bath temperature (~100 °C). Various parameters affecting the adsorption of molybdenum, such as pH, amount of molybdenum, incubation period, etc., were determined. A ^99mTc generator was prepared by adsorbing low specific activity ⁹⁹Mo (150 mg) on 1 g alumina. Elutions were carried out with saline. Performance of the generator such as ⁹⁹Mo breakthrough, aluminum contents, pH, elution profile, radiochemical purity, and labeling efficiency of kits were checked.     

Synthesis, quality control and biodistribution of 99mTc-Kanamycin

Summary Kanamycin is an antibiotic used for treatment of infections when penicillin or other less toxic drugs cannot be used. Kanamycin was labeled with technetium-99m pertechnetate using SnCl₂^. 2H₂O as reducing agent. The labeling efficiency depends on the ligand/reductant ratio, pH, and volume of reaction mixture. Radiochemical purity and stability of ^99mTc-Kanamycin was determined by thin layer chromatography. Biodistribution studies of ^99mTc-Kanamycin were performed in rats and rabbits. A significantly higher accumulation of ^99mTc-Kanamycin was seen at sites of S. aureusinfected animals (rat/rabbit).     

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.     

Preparation,molecular modeling and biodistribution of 99mTc-phytochlorin complex

Phytochlorin [21H, 23H-Porphine-7-propanoicacid, 3-carboxy-5-(carboxymethyl)13-ethenyl-18-ethyl-7,8-dihydro-2,8,12,17-tetramethyl-,(7S,8S)] was labeled with ^99mTc and the factors affecting the labeling yield of ^99mTc-phytochlorin complex were studied in details. At pH 10, ^99mTc-phytochlorin complex was obtained with a high radiochemical yield of 98.4 ± 0.6 % by adding ^99mTc to 100 mg phytochlorin in the presence of 75 μg SnCl₂·2H₂O after 30 min reaction time. The molecular modeling study showed that the structure of ^99mTc-phytochlorin complex presents nearly linear HO–Tc–OH unit with an angle of 179.27° and a coplanar Tc(N1N2N3N4) unit. Biodistribution of ^99mTc-phytochlorin complex in tumor bearing mice showed high T/NT ratio (T/NT = 3.65 at 90 min post injection). This preclinical study showed that ^99mTc-phytochlorin complex is a potential selective radiotracer for solid tumor imaging and afford it as a new radiopharmaceutical suitable to proceed through the clinical trials for tumor imaging.     

Synthesis of 99mTc-gemifloxacin freeze dried kits and their biodistribution in Salmonella typhi,Pseudomonas aeruginosa and Klebsiella pneumoniae

Gemifloxacin, a novel, 4th generation fluoroquinolone derivative, was labeled with^99mTc; its freeze dried kits were prepared and used for infection imaging. Kits showed great stability with higher labeling efficiency. Kits were synthesized through a simple method; developed at room temperature without HCl and heating with low colloidal content. Reaction conditions were optimized in order to get maximum radiochemical purity. Highest labeling efficiency (99 ± 0.05)% was achieved when 1.0 mg gemifloxacin was labeled with 10 mCi sodium pertechnetate in the presence of 50 μg SnCl₂ and 300 μg D-penicillamine at room temperature. Radiolabeled antibiotic kits were preclinically assessed such as in-vitro stability, lipophilicity, protein binding, in-vitro binding with bacterial strains and pharmacokinetic investigations in animals. Kits were found highly stable for 6 h both at room temperature and at 37 °C in serum. Biodistribution showed excellent uptake of activity at infection site (in Pseudomonas aeruginosa, Salmonella typhi and Klebsiella pneumoniae). Biodistribution data showed that ^99mTc-gemifloxacin has the potential and may be used for infection imaging.     

Radiosynthesis and Biodistribution of <Superscript>99m</Superscript>Tc-Metronidazole as an <Emphasis Type="Italic">Escherichia coli</Emphasis> Infection Imaging Radiopharmaceutical

Bacterial infection poses life-threatening challenge to humanity and stimulates to the researchers for developing better diagnostic and therapeutic agents complying with existing theranostic techniques. Nuclear medicine technique helps to visualize hard-to-diagnose deep-seated bacterial infections using radionuclide-labeled tracer agents. Metronidazole is an antiprotozoal antibiotic that serves as a preeminent anaerobic chemotherapeutic agent. The aim of this study was to develop technetium-99m-labeled metronidazole radiotracer for the detection of deep-seated bacterial infections. Radiosynthesis of ^99mTc-metronidazole was carried by reacting reduced technetium-99m and metronidazole at neutral pH for 30 min. The stannous chloride dihydrate was used as the reducing agent. At optimum radiolabeling conditions, ~ 94% radiochemical was obtained. Quality control analysis was carried out with a chromatographic paper and instant thin-layer chromatographic analysis. The biodistribution study of radiochemical was performed using Escherichia coli bacterial infection-induced rat model. The scintigraphic study was performed using E. coli bacterial infection-induced rabbit model. The results showed promising accumulation at the site of infection and its rapid clearance from the body. The tracer showed target-to-non-target ratio 5.57 ± 0.04 at 1 h post-injection. The results showed that ^99mTc-MNZ has promising potential to accumulate at E. coli bacterial infection that can be used for E. coli infection imaging.