177Lu-DOTATATE Peptide Receptor Radionuclide Therapy: Indigenously Developed Freeze Dried Cold Kit and Biological Response in In-Vitro and In-Vivo Models.

Somatostatin receptors (SStR) based 177Lu-DOTATATE therapy is known as one of the highly effective neuroendocrine tumors (NETs) treatment strategy. Development of DOTATATE freeze-dried kit for imaging and therapy of SStR positive NETs is a prime goal in neuroendocrine cancer research. The present work describes the development of 177Lu-DOTATATE freeze dried cold kit for indigenous needs, through technology development fund (TDF) program offered by Higher Education Commission (HEC) Pakistan. The parameters for freeze dried kit production was optimized and tested the stored lyophilized cold kits for different time intervals after labeling with 177Lu radioisotope. The effect of ligand to radionuclide ratio, pH and reaction time at 90°C was recorded. Five times greater molar concentration of ligand, pH 5 and 30 min reaction time were the effective reaction conditions for maximum radiochemical yield. The radiolabeling yield at 1 day, 1-week and 4-week post storing period showed ∼100% radiochemical yield. The biodistribution study using rat model depicted the absence of non-targeted accumulation while glomerular filtration rate also explains the rapid renal washout. Cytotoxicity study showed quite favorable results for subjecting the radiopharmaceutical to clinical practice in Pakistan.

Dose-Dependent Internalization and Externalization Integrity Study of Newly Synthesized 99mTc-Thymoquinone Radiopharmaceutical as Cancer Theranostic Agent.

Thymoquinone (TQ) is a bioactive phytochemical isolated from Nigella sativa and has been investigated for biochemical and biological activities in both in vitro and in vivo models. It is best known for its anticancer activities. Thymoquinone accomplishes anticancer activities through targeting multiple cancer markers including PPAR-γ, PTEN, P53, P73, STAT3, and generation of reactive oxygen species at the cancer cell surface. The radiolabeling of TQ with γ- and ß-emitter radionuclide could be used as cancer diagnostic or therapeutic radiopharmaceutical, respectively. In this study, we are reporting the radiolabeling of TQ with technetium-99m (99mTc), stability in saline and blood serum, internalization and externalization of 99mTc-TQ using rhabdomyosarcoma cancer cells line. The quality control study revealed more than 95% labeling yield and stable in blood serum up to 4 hours. In vitro internalization rate was recorded 27.08% ± 0.95% at 1 hour post 2 hours internalization period and comparatively slow externalization. The results of this study are quite encourging and could be investigated for further key preclinical parameters to enter phase I clinical trials.

Development of 99mTc-SDP-choline SPECT radiopharmaceutical for imaging of cerebrovascular diseases.

Cerebrovascular diseases are known as serious public health problem worldwide, which can be addressed more precisely through molecular imaging of non-functional brain cells. CDP-choline is an active cerebrovascular chemotherapeutic agent that can be used for diagnosis of cerebrovascular diseases post radiolabeling with γ-emitter radioisotopes. In this study we developed 99mTc labeled CDP-choline for imaging of cerebrovascular diseases particularly alzheimer, stroke, and parkinson's diseases. The radiosynthesis reaction resulted 97.47±2.34% radiochemical with promising stability, that is, >95% up to 6 h in blood serum. The biodistribution study in healthy mice revealed non-accumulated uptake of radiochemical in key body organs; in brain it was 8.59±1.11% ID/g at 1h post-injection which washed-out leaving behind 0.87±0.61% ID/g at 24 h post-injection. The over-all data revealed the 99mTc-CDP-choline could be a good candidate for further imaging investigations in diseased animal model.

Technetium-99m labeled Ibuprofen: Development and biological evaluation using sterile inflammation induced animal models.

In this study we are presenting the development of technetium-99m (99mTc) labeled ibuprofen for the imaging of aseptic inflammation. 99mTc-Ibuprofen complex was developed by optimizing the radiolabeling conditions such as reaction time, ligand and reducing agent concentration, pH, reaction time and temperature. Following the addition of 600 µg of ibuprofen, 4 µg of stannous chloride as reducing agent and 300 MBq 99mTc radioactivity; the pH of reaction mixture was adjusted to 11 and allowed to react for 15 min at room temperature. Chromatography analysis revealed > 94% 99mTc-ibuprofen complex formation with promising stability in saline and blood serum up to 6 h. Biodistribution study using normal and sterile inflammation induced mice indicated low accumulation of labeled compound in key body organs; however, kidneys (14.76 ± 0.87% ID/g organ) and bladder (31.6 ± 3.0% ID/g organ) showed comparatively higher radioactivity due to main excretory path. Inflamed to normal tissues ratio (T/NT), at 1 h post-injection, showed promising value (4.57 ± 0.56). The SPECT imaging of artificially inflammation induced rabbit model also verified the biodistribution results. In conclusion, radiochemical purity and biological evaluation of 99mTc-ibuprofen complex indicates the agent can be utilized for imaging of deep seated aseptic inflammation.

Radiosynthesis, radiochemical, and biological characterization of 177 Lu-labeled diethylenetriamine penta-acetic acid.

Diethylenetriamine penta-acetic acid (DTPA), when complexed with a gamma (γ)-emitter radioisotope like 99m Tc, is used for renal function diagnosis and many other diagnostic applications. The main aim of this study was to develop a novel and versatile single-step methodology for the synthesis of a new 177 Lu-labeled radiopharmaceutical with high radiochemical yield, which can be used for diagnostic purposes and therapeutic purposes also. The single and well-defined 177 Lu-DTPA complex was radiochemically characterized by paper chromatography, thin-layer chromatography, high-performance liquid chromatography, and electrophoresis techniques. Dependence of the labeling yield of 177 Lu-DTPA complex on different factors was studied in detail. Biological evaluation was also performed in a normal rabbit by developing images under a γ camera at various time intervals. More than 99% labeling yield was obtained by reacting DTPA with 177 Lu at specific conditions (pH 7.0, 15 minutes reaction time at 100 °C). 177 Lu-DTPA complex showed high stability both at room temperature and in vitro. Biodistribution studies in normal mice indicated the fractional renal uptake of intravenously administered 177 Lu-DTPA complex, which reached in the kidneys within 2-3 minutes. Scintigraphy showed rapid clearance from the body. Based on these results, we propose that 177 Lu-DTPA complex might be used as an ideal candidate for functional evaluation of kidneys and the urinary tract, especially when needed to be transported to long-range consumer sites, because of its suitable half-life.

Susceptibility of 99mTc-Ciprofloxacin for Common Infection Causing Bacterial Strains Isolated from Clinical Samples: an In Vitro and In Vivo Study.

99mTc-ciprofloxacin scintigraphy is useful in the detection of gram-positive and gram-negative bacterial infections and also for differentiating the infection from aseptic inflammation. However, due to growing bacterial resistance to antibiotics, the 99mTc-ciprofloxacin no longer can be effective in broad-spectrum infection imaging as it is gradually losing specificity. In this study, we are presenting our findings regarding the in vitro and in vivo susceptibility of 99mTc-ciprofloxacin for multi-drug-resistant Staphylococcus aurous, Escherichia coli, and Pseudomonas aeruginosa bacterial strains which were isolated from clinical samples. The results of radiosynthesis of 99mTc-ciprofloxacin showed more the 95% radiochemical purity and less than 5% radioactive impurities. In vitro 99mTc-ciprofloxacin susceptibility test showed that E. coli offered more resistant to 99mTc-ciprofloxacin as compared to S. aurous and P. aeruginosa. In vivo study using bacterial infection induced rabbit model also revealed lowest uptake by E. coli lesion. The T/NT values were obtained 1.96 ± 0.15 in the case of E. coli; 2.81 ± 0.51 in the case of S. aurous; and 2.32 ± 0.66 in the case of P. aeruginosa at 4 h post-injection. The SPECT infection imaging of S. aurous, E. coli, and P. aeruginosa bacterial infection induced rabbit models also indicated the clear accumulation in S. aurous and P. aeruginosa lesions while negligible uptake by E. coli lesion further verify the in vitro and in vivo susceptibility profile. On the bases of the results obtained, the 99mTc-ciprofloxacin showed selective and poor broad spectrum SPECT infection imaging.

Technetium-99m radiolabeling and biological study of epirubicin for in vivo imaging of multi-drug-resistant Staphylococcus aureus infections via single photon emission computed tomography.

The development of functional imaging is a promising strategy for diagnosis and treatment of infectious and cancerous diseases. In this study, epirubicin was developed as a [99m Tc]-labeled radiopharmaceutical for the imaging of multi-drug-resistant Staphylococcus aureus infections. The labeling was carried out using sodium pertechnetate (Na99m TcO4 ; ~370 MBq). The other parameters such as amount of ligand, reducing agent (SnCl2 .2H2 O), and pH were optimized. The highest labeling yield ≥96.98% was achieved when 0.3 mg epirubicin, 13 µg SnCl2 .2H2 O, and ~370 MBq Na99m TcO4 were incubated at pH 7 for 15 min in the presence of ascorbic acid at room temperature. Radiochemical purity, stability, charge, and glomerular filtration rate were studied to evaluate the biological compatibility for in vivo administration. Biodistribution investigations showed radiotracer uptake (13.89 ± 1.56% ID/gm organ) by liver and 7.79 ± 0.38% ID/gm organ by kidneys at 30 min post-injection which promisingly wash out at 24 hr post-injection. Scintigraphy study showed selective uptake in S. aureus-infected tissues in contrast to turpentine oil-induced inflamed tissues. Target-to-non-target ratio (6.7 ± 0.05) was calculated at 1 hr post-injection using SPECT gamma camera. The results of this study reveal that the [99m Tc]-epirubicin can be a choice of imaging and monitoring the treatment process of multi-drug resistant S. aureus bacterial infections.

Correction to: Preparation, optimization and pharmacological evaluation of 99mTc-4-hydroxy-3-(2-hydroxy-3-methoxybenzylideneamino) benzoic acid complex: as a novel potential radiopharmaceutical agent with hepatobiliary excretion.

In the published original version, the complete list of authors was omitted. The complete list of contributing authors is updated with this Correction.

177Lu-DOTA-coupled minigastrin peptides: promising theranostic agents in neuroendocrine cancers.

Treatment with radionuclide labeled regulatory peptides is a promising tool in the management of patients with inoperable receptor positive neuroendocrine tumors. Peptide receptor lutetium-177 radionuclide therapy currently has gained ample attention due to high specific accumulation of regulatory peptides at tumor cell surface and promising characteristics of ß- and γ-energy photons of lutetium-177 radionuclide. In this study gastrin peptides analogues were labeled with lutetium-177 by subsequent mixing of 177LuCl3 (~ 185 MBq), ammonium acetate buffer of 5 pH, gentistic acid, aqueous solution of gastrin peptide analogues (1 mg/mL) and heating the reaction mixture at 98 °C which resulted in high radiochemical yield (> 96%). Chromatographic analysis was carried out to analyze the radiochemical purity. The shelf life and serum stability results showed the labeled peptides are sufficiently stable up to 4-h. Glomerular filtration rate study results showed moderate filtration through kidneys. The GFR values of 177Lu-MGCL2 and 177Lu-MGCL4 was noted 48 mL/min and 45 mL/min, respectively. Biodistribution and scintigraphy study using rat and rabbit models showed minimal non-target accumulation, moderate uptake by liver and kidneys. The promising radiochemical yield, stability, GFR values and biodistribution results of 177Lu-MGCL2 & 4 indicate, the agents can be tested clinically for PRRT procedures.

Ciprofloxacin: from infection therapy to molecular imaging.

Diagnosis of deep-seated bacterial infection remains a serious medical challenge. The situation is becoming more severe with the increasing prevalence of bacteria that are resistant to multiple antibiotic classes. Early efforts to develop imaging agents for infection, such as technetium-99m (99mTc) labeled leukocytes, were encouraging, but they failed to differentiate between bacterial infection and sterile inflammation. Other diagnostic techniques, such as ultrasonography, magnetic resonance imaging, and computed tomography, also fail to distinguish between bacterial infection and sterile inflammation. In an attempt to bypass these problems, the potent, broad-spectrum antibiotic ciprofloxacin was labeled with 99mTc to image bacterial infection. Initial results were encouraging, but excitement declined when controversial results were reported. Subsequent radiolabeling of ciprofloxacin with 99mTc using tricarbonyl and nitrido core, fluorine and rhenium couldn't produce robust infection imaging agent and remained in discussion. The issue of developing a robust probe can be approached by reviewing the broad-spectrum activity of ciprofloxacin, labeling strategies, potential for imaging infection, and structure-activity (specificity) relationships. In this review we discuss ways to accelerate efforts to improve the specificity of ciprofloxacin-based imaging.

Labeling of epirubicin with technetium-99m: Optimization, biodistribution and scintigraphic imaging in tumor bearing mice.

Epirubicin is an antineoplastic agent of anthracycline antibiotic, used for treating a variety of tumor types such as lymphoma, cancer of the breast, lung, ovary and stomach. The objective of this work was to demonstrate direct radiolabeling of epirubicin with 99mTc, quality control, biological characterization and scientigraphic evaluation in tumor bearing mice. The 99mTc-epirubicin labeling was optimized by varying the amounts of ligand 100-350µg, stannous chloride dihydride 20-50µg and pH range 2-10 by using NaOH or HCl. The radiochemical purity of 99mTc-epirubicin was evaluated by chromatographic techniques (Whatman No. 3 paper and ITLC-SG). HPLC analyses were performed to check purity of epirubicin and radiochemical purity of labeled 99mTc- epirubicin. Biodistribution and scintigraphic imaging of 99mTc-epirubicin was performed in normal and tumor bearing mice at various time intervals. The optimum conditions ensuring 99mTc-epirubicin labeling yield as high as 99% by adding 35µg SnCl2.2H2O, 200µg of ligand at pH 6 for 30 min at room temperature (25°C±2°C). HPLC of 99mTc-epirubicin shows about 99% binding of the compound with technetium-99m. Electrophoresis study indicated the neutral nature of 99mTc-epirubicin. Biodistribution data and scintigraphic results showed that 99mTc-epirubicin accumulated in the liver as well as in tumor with significant uptake and excellent retention. 99mTc-epirubicin shows good stability in human serum. In vitro and in vivo studies revealed the significantly uptake of 99mTc-epirubicin in the tumor, and also indicating the efficiency of 99mTc-epirubicin as a tumor diagnostic agent.

Synthesis, optimization and biological evaluation of 99mTc-digoxin as possible cardiac imaging agent.

Heart imaging radiopharmaceuticals could improve the diagnostic value of routine heart scanning for detecting cardiac disorders. The aim of the study was to prepare high radiochemical purity 99mTc-Digoxin in a yield of about 98%. The optimal conditions for labelling were as follows: 100µg of Digoxin, 2µg of SnCl2•2H2O, room temperature (25±1°C), reaction retention time of 30 min at pH 7. Under these conditions, the radiochemical yield of 99mTc-Digoxin reaches 98%. In vivo bio distribution was performed in normal Swiss Albino mice at different time intervals after administration of 99mTc-Digoxin.Scintigraphic study of 99mTc-Digoxin was performed in rabbits. The heart uptake of 99mTc-Digoxin was sufficiently high and thus may be a potential myocardial imaging radiopharmaceutical applicable in cardiology.

Synthesis, quality control, and bio-evaluation of 99m Tc-cyclophosphamide.

Cancer is found to be the leading cause of death worldwide characterized by uncontrolled cell division. Nuclear medicines imaging using radiopharmaceuticals have pronounced potential for the diagnosis and treatment of cancers. Cyclophosphamide (CPH) is an antineoplastic drug which targets selectively cancer cells. In the present work, labeling of CPH with Tc-99m is performed for diagnostic purpose, which gave labeling yield as high as 99% using 20 µg SnCl2 ·2H2 O, 200 µg of ligand at pH 7 for 10 min reaction time at room temperature. The characterization of the prepared complex was performed using ITLC, electrophoresis, and HPLC. In vitro stability was analyzed in the presence of human serum at 37°C which has maximum value of 94 ± 0.5. The biodistribution studies of 99m Tc-CPH were performed in normal and tumor bearing Swiss Webster mice. The high accumulation of 99m Tc-CPH was observed in liver and tumours respectively at 4 hr after injection. Biodistribution results revealed that 99m Tc-CPH may be a potential tumour diagnostic agent simultaneously with chemotherapy.

Radiolabeling, quality control, and biological characterization of 177 Lu-labeled kanamycin.

Kanamycin is an antibiotic, isolated from Streptomyces kanamyceticus, which is used to treat serious bacterial infections. The fact that the present radioligand 99m Tc-kanamycin used for diagnosis is short-lived, raised a need to label and study kanamycin with one of the most important beta (ß) radiation emitting isotope 177 Lu. Labeling yield of 177 Lu-kanamycin was confirmed by different chromatography techniques such as paper chromatography, TLC, HPLC. Several experiments were performed to optimize labeling with changing reaction conditions such as pH, temperature, amount of ligand, and reaction time. In vitro stability analysis was performed incubation with human serum. Electrophoresis analysis was also conducted to determine the charge on 177 Lu-kanamycin. The biodistribution and scintigraphy were performed in normal mice and rabbit, respectively, at different time intervals of postinjection. 177 Lu-kanamycin was prepared with very high yield (~100%), with excellent stability in vivo and in vitro (>99% 6 hr postprep.), at pH 7. Maximum labeling was achieved at less reaction time (15 min), with maximum conjugation of the ligand (12.5 mg) with 177 Lu. Electrophoresis analysis showed net neutral charge. The radioligand showed rapid clearance from body in biodistribution and scintigraphy studies. The preparation 177 Lu-kanamycin could be used as a radio-pharmaceutical for infection imaging purpose, especially when transporting the radioligand to long-range distances.

Preparation, biodistribution and scintigraphic evaluation of (99m)Tc-lincomycin.

A complex of lincomycin was synthesized with technetium-99m. The synthesis was carried out by using SnCl2.2H2O as reducing agent and ascorbic acid as stabilizer. The effect of various parameters such as amount of ligand/reducing agent, pH value and reaction time on radio labeling process was studied. The characterization of the (99m)Tc-Lincomycin was performed by HPLC and electrophoresis Biodistribution studies were carried out by analyzing the model of bacterial infectious rats (Sprague-Dawley). The uptake of infectious lesions at different time interval was also studied by using scintigraphic technique. The complex showed effective target to non-target ratio for various inflammatory or infectious lesions. The (99m)Tc-Lincomycin effective binding to living bacteria and could be used successfully as an infection imaging agent.

Labelling of ceftriaxone with (99m) Tc and its bio-evaluation as an infection imaging agent.

Differentiation of bacterial and sterile inflammation will have a significant impact on the current clinical practice. Ceftriaxone (CTRX) was labelled with (99m) Tc and assessed for its ability to depict infection on scintigraphy. Stoichiometry was performed to optimize labelling parameters. Stability and bacterial binding was verified and biodistribution pattern was seen in normal, infected/inflamed animal models. (99m) Tc-CTRX prepared at pH 7 with stannous chloride of 50 µg, ligand of 30 mg, and boiling for 10 min gave labelling yield of 96.2 ± 0.2% with good stability. In vitro binding was higher for Escherichia coli than Staphylococcus aureus. Biodistribution in normal rats showed high uptake in hepatobiliary system, gut and urinary system. In animal models induced with infection or inflammation, lesion to normal ratios at 4 h were 2.36 ± 0.21, 12.66 ± 1.44 and 1.40 ± 0.01 with S. aureus infection, E. coli infection and turpentine oil inflammation, respectively. Infection specificity especially for E. coli was also confirmed on scintigraphic findings. Ceftriaxone can be labelled with (99m) Tc with high labelling yield at pH compatible with that of blood. Our preparation has shown stability in vitro and in human serum, and binds preferentially with bacteria. (99m) Tc-CTRX scintigraphy can be used to delineate sites of active infection and to differentiate infection and inflammation.

Preparation, biodistribution, and scintigraphic evaluation of (99m)Tc-clindamycin: an infection imaging agent.

Bacterial infection is found to be the cause of death throughout the world. Nuclear medicine imaging with the help of radiopharmaceuticals has great potential for treating infections. In the present work, clindamycin, a lincosamide antibiotic, was labeled with technetium-99 m (~380 MBq). Clindamycin has been proven to be efficient for treating serious infections caused by bacteria such as Staphylococcus aureus. Quality control, characterization, biodistribution, and scintigraphy of radiolabeled clindamycin were done, and labeling efficiency was determined by ascending paper chromatography. More than 95 % labeling efficiency with technetium-99 m ((99m)Tc) was achieved at pH 6-7 while using 2.5-3 µg SnCl2 · H2O as a reducing agent and 100 µg of ligand at room temperature. The characterization of the compound was performed by using electrophoresis, HPLC and shake flask assay. Electrophoresis indicates the neutral behavior of (99m)Tc-clindamycin. HPLC analysis confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-clindamycin were performed Sprague Dawley rats bearing bacterial infection. Scintigraphy and biodistribution studies showed a high uptake of (99m)Tc-clindamycin in the liver, heart, lung, and stomach as well as at S. aureus-infected sites in rabbits.

Iron fortification of wheat flour: bioavailability studies.

Bioavailability refers to that fraction of nutrients which is utilized by the body out of the total indigested amount. Various direct and indirect methods for the determination of bioavailability are available. We determined the bioavailability of iron from fortified wheat flour using both in vitro and in vivo methods. The bioavailability data will be used to make the recommendation for a fortification strategy in Pakistan. The in vitro bioavailability of iron from fortified wheat flour was determined using in vitro enzymatic digestion and fermentation by simulating the condition of the small intestine and colon in the laboratory. Different products were prepared from the fortified ferrous sulfate (FeSO4) and unfortified wheat flour. The total iron of the samples was measured by the wet-digestion method and analyzed on an atomic absorption spectrometer (AAS). To obtain the percentage of iron released, the samples were subjected to pepsin digestion and dialysis. The dialysate was collected at 3, 6, 9, and 12 hours and read on an AAS. The retentates from the above were subjected to the fermentation condition of the colon by inoculating it with human fecal inoculum and incubating it for 24 hours at 37 degrees C under anerobic conditions. The dialysate was collected at 3- and 6-hour intervals and read on an AAS. More iron was released from fortified wheat flour (4.6%), leavened chapati (6.8%), and Nan (15.1%) than from the unfortified control samples. Fermentation and leavening resulted in a better release, which was evident from in vitro digestion results.