Synthesis and preliminary evaluation of a new[99m]Tc labeled substance P analogue as a potential tumor imaging agent

Neurokinin 1 receptors [NK[1]R] are overexpressed on several types of important human cancer cells. Substance P [SP] is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK[1]R positive tumors.[6-hydrazinopyridine-3-carboxylic acid [HYNIC]-Tyr[8]-Met[O][11]-SP] was synthesized and radiolabeled with 99mTc using ethylenediamine-N,N-diacetic acid [EDDA]and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity [>99%] and radiolabeled with [99m]Tc at high specific activities [84-112GBq/ micro mol] with an acceptable labeling yield [>95%]. The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 +/- 0.22% with the ratio of 60.21 +/- 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 +/- 0.43 nM and Bmax of 128925 +/- 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram [%ID/g] for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated. Therefore, this new radiolabeled peptide could be a promising radiotracer for detection of NK[1]R positive primary or secondary tumors

lyophilized peptide radiopharmaceutical kit to target neurotensin receptor for tumor imaging

Neurotensin [NT] is a tridecapeptide that binds specifically to neurotensin receptors. Several forms of cancer, including small cell lung cancer, colon, pancreatic and prostate carcinomas especially exocrine pancreatic carcinomas express receptors for neurotensin peptide. Radiolabeled neurotensin derivatives with a high affinity for these receptors might be used for scintigraphy. The aim of this work was to prepare a freeze-dried kit formulation for routine preparation of [99m]Tc labeled neurotensin in nuclear medicine center. A freeze dried kit containing reducing agent, coligands and HYNIC-Neurotensin derivative for labeling with[99m]Tc was prepared. Labeling was performed at 95°C for 15 min and radiochemical analysis involved ITLC and HPLC methods. The stability of radiopeptide was checked in the presence of human serum at 37°C up to 24 h. The receptor bound internalization rate was studied in neurotensin receptor expressing HT-29 cells. Biodistribution of radiopeptide was studied in mice. Labeling yield of >95% was obtained corresponding to a specific activity of 80 MBq/nmol. Prepared radioconjugate was stable in human serum and more than 12% of activity was specifically internalized into HT-29 cells up to 4 h. Biodistribution study showed a rapid blood clearance, with renal excretion and specific binding towards NT receptor-positive tissues such as intestines [0.99 +/- 0.16% ID/g at 1 h]. The favorable characteristics of our new designed labeled peptide formulation make it as a promising candidate for diagnosis of malignant tumors

[131]I-Chlorotoxin dosimetry in liver using MCNP simulation code

Chlorotoxin is a 36 amino acids peptide, which is able to block chloride channels isolated from mouse brain. A derivative of chlorotoxin is synthesized and it is labeled by iodine 131; then animal experiments carry out on rats. Multiple organ doses may be calculated with biological distribution results in rats with labeled compounds using simulated MCNP4C code. Human dose can be calculated using the dose distribution in rats with a conversion ratio for dose distribution. Chloramine T is our method for marking, and electrophilic substitution reactions are methods for iodize of peptides. Simulation of a human phantom to evaluate dose distribution was done using simulation code MCNP4C. To evaluate the dose distribution in the human body, using this code and the accumulated activity in each organ tissue dose is calculated. To study the biological distribution of the radiotracer 131I, 0.37 MBq radiotracer was injected into rat via the tail vein. The accumulated activity in each organ with the agent "ID / g" is determined. Biological distribution of 131I-chlorotoxine in the normal rats is obtained. Its Decay constant in the liver is 0.07h and the effective half-life of the radiotracer is 10h in rat liver. The total number of particles found in the leak from liver tissue was reported 67600. Liver tissue dosimetries originating from other sources [thyroid tissue, stomach, kidney, right and left lung, spleen, and pancreas] were examined. Then, the overall dose to the target tissue will be calculated. Leaked beta particles in liver itself [self-dose] are the most delivered dose to the liver [98%]; it is for gamma rays 1.1%, while its source is adjacent tissues in addition to liver [cross-dose]; Because of low atomic number of the tissue, delivered dose originated from Bremsstrahlung [braking radiation] is low [0.9%]. Radiation dose to the liver in intravenous injection of 0.37 MBq [131]I-chlorotoxine radiotracer is 3.44 [asterisk] 10-6

Optimization condition in labeling of ofloxacin with [99m]Tc and its biological evaluation in Staphylococcus aureus and Escherichia coli for infection imaging

The use of radiopharmaceuticals is a powerful tool in the management of patients with infectious or inflammatory diseases in nuclear medicine. In this study ofloxacin as a second-generation fluoroquinolone is used to design a desired infection imaging agent after labeling with [99m]Tc via direct labeling. Ofloxacin was radiolabeled with [99m]Tc using different concentrations of ligand, stannous chloride, sodium pertechnetate and at different pH. Then labeling yield, stability in saline and serum, lipophilicity, binding with Staphylococcus aureus and Escherichia coli and biodistribution in infected mice for labeled compound were studied. The final complex was characterized by TLC and HPLC and radiochemical purity of >90% was obtained when 1.5 mg ofloxacin in presence of 75 micro g SnCl2 was labeled with 370 MBq sodium pertechnetate. The complex showed specific binding to Staphylococcus aureus and Escherichia coli. Biodistribution results showed that radioligand had high affinity in the infected site in mice. The uptake for Staphylococcus aureus induced infections [T/NT = 2.33 +/- 0.17 at 1 h post injection] was higher than that was for Escherichia coli [T/NT = 1.96 +/- 0.13 at 1 h post injection]. This complex may lead to further development of a radiotracer for imaging of infections induced by grampositive or gram-negative bacteria

[99m]tc-ubiquicidin [29-41], a promising radiopharmaceutical to differentiate orthopedic implant infections from sterile inflammation

Ubiquicidin [UBI] [29-41] is a synthetic cationic antimicrobial peptide that preferentially binds to bacterial cell membrane at the site of infection. We aimed to assess diagnostic value of [99m]Tc-UBI [29-41] as a radiopharmaceutical in differentiation of bacterial infection from sterile inflammation in suspected orthopedic implants. Nine patients suspected for orthopedic implant infection, all males with the mean age of 41.6 +/- 20.9 years, were studied. A dose of 10 MBq/Kg [range: 555-740 MBq] [99m]Tc-UBI [29-41] was injected intravenously. A dynamic study followed by static whole body imaging at 30, 60 and 120 min post-radiotracer injection was acquired. Periprosthetic tissue culture was considered the closest test to a gold standard for diagnosing infections and scintigraphic scans were categorized as true- or false-positive and true- or false-negative, considering the bacterial culture as the gold standard. No adverse reaction was observed during or after the radiotracer injection days. There were five true positive, four true negative and no false positive and false negative scans. Sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] were all calculated as 100%. We found a high diagnostic accuracy for [99m]Tc-UBI [29-41] scintigraphy in differentiation of bacterial infection from sterile inflammation in suspected orthopedic implants. Therefore, [99m]Tc-UBI [29-41] scintigraphy might be potentially recommended as a safe and promising imaging modality in these settings. However, further studies on a larger number of patients and different pathologies are still needed

Synthesis and biological evaluation of [99m]Tc[CO][3]-OH-PP-CS[2] for brain receptor imaging

5-HT[1A] receptor is related with a variety of neuropsychiatric disorders. In this study a phenolic analogue derived from DWAY [Desmethyl WAY-100635 [N-[2-[1-[4-[2-methoxyphenyl]piperazinyl]-ethyl]]-N-[2-pyridinyl] cyclohexanecarboxamide]] is used to design the desired structure of 5-HT1A receptor imaging agents after labeling with [[99m]Tc [CO] [3][H[2]O] [3]] [+] core via dithiocarbamate moiety. 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 [[99m]Tc[CO][3][H2O][3][+] core. Radioligand chemical analysis involved high-performance liquid chromatography methods. Radioconjugate stability and lipophilicity were determined. Biodistribution of labeled compound was studied in rats. 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 6-hrs after labeling at room temperature. The n-octanol/water partition coefficient experiment demonstrated Log P = 0.74 for [99m]Tc[CO][3]-OH-PP-CS[2]. Biodistribution results showed that radio tracer had moderate brain uptake [0.32 +/- 0.03%ID/g at 30 min post injection]. This complex may lead to a further development of a radiotracer with specific binding to 5-HT[1A] receptor

Preparation and evaluation of 67Ga-DOTA-Bombesin [7-14] as a tumor scintigraphic agent

Bombesin is a 14-aminoacid peptide isolated from frog skin. The mammalian counterparts of the frog peptide are neuromedin B [NMB] and gastrin-releasing peptide [GRP]. Bombesin [BBN] is a peptide showing high affinity for the gastrin releasing peptide receptor [GRPr]. Prostate, small cell lung cancer, breast, gastric, and colon cancers are known to over express receptors to bombesin [BBN] and gastrin releasing peptide [GRP]. In this study a new 67Ga radiolabeled BBN analogue evaluated based upon the bifunctional chelating ligand DOTA [1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid] that can be used as a tool for diagnosis of GRP receptor-positive tumors. DOTA-BBN [7-14] NH2 was synthesized using a standard Fmoc strategy. Labeling with 67Ga was performed at 95°C for 30 minutes in ammonium acetate buffer [pH=4.8]. Radiochemical analysis involved ITLC and HPLC methods. The stability of radiopeptide was examined in the presence of human serum at 37°C up to 24 hours. The receptor-bound internalization and externalization rates were studied in GRP receptor expressing PC-3 cells. Biodistribution of radiopeptide was studied in nude mice bearing PC-3 tumor. Labeling yield of>90% was obtained corresponding to a specific activity of=2.48 MBq/nmol. Peptide conjugate showed good stability in the presence of human serum. The radioligand showed a good and specific internalization into PC-3 cells [14.13 +/- 0.61% at 4 h]. In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in GRP-receptor-positive organs. After 4 h, uptake in mouse pancreas was 1.08 +/- 0.29% ID/g [percentage of injected dose per gram of tissue]. These data show that [67Ga]-DOTA-Bombesin [7-14] NH2 is a specific radioligand for gastrin-releasing peptide receptor positive tumors

New freeze-dried kit for diagnosis of bombesin receptor expressing tumors

It has been shown that some primary human tumors and their metastases, including prostate and breast tumors, over-express gastrin-releasing peptide [GRP] receptors. Bombesin is a neuropeptide with a high affinity for these GRP receptors. The purpose of this study was to prepare and evaluate the characteristics of a new Freeze-dried kit, [6-hydrazinopyridine-3-carboxylic acid [HYNIC]]-GABA-Bombesin [7-14] NH[2] designed for the labeling with 99mTc using tricine and EDDA as coligand. Synthesis was performed on a solid phase using a standard Fmoc strategy and HYNIC precursor coupled at the N-terminus. Purified peptide conjugate was labeled with [99m]Tc at 100°C for 10 min. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate stability and affinity to human serum was challenged for 24 hours. The internalization rate was studied in GRP receptor expressing PC-3 cells. Biodistribution of radiopeptide was studied in rats. Radiolabeling was performed at high specific activities, and radiochemical purity was >98%. The stability of radiolabeled peptide in human serum was excellent. In vitro studies showed >14% of activity was specific internalized into PC-3 cells up to 4 h. After injection into rat biodistribution data showed a rapid blood clearance, with renal excretion and specific binding towards GRP receptor-positive tissues such as pancreas [1.15 +/- 0.19% ID/g after 4 h]. [[99m]Tc-HYNIC]-GABA-Bombesin [7-14] NH[2] showed favorable radiochemical and biological characteristics which make our new designed labeled peptide conjugate as a very suitable agent for diagnostic purposes in malignant tumors

Freeze-dried cold kit for Preparation of [99M]Tc-ciprofloxacin as an infection imaging agent

Radiolabeled antibiotics are being used for the specific diagnosis of infection by exploiting their specific binding properties to the bacterial components, thereby making it possible to differentiate infection from sterile lesions. The aim of this work was to prepare and evaluate a freeze-dried kit of ciprofloxacin designed for the labeling with [99m]Tc. Factors like amount of reducing agent and optimum pH were investigated to make the ciprofloxacin kit. The kit was reconstituted with [99m]Tc at room temperature and the radiochemical purity was evaluated by ITLC method. Stability and protein binding in human serum followed by in vitro binding to bacteria were assessed. Biodistribution of labeled kit in staphylococcus aureus infected rats muscles were studied using ex vivo counting and scintigraphy. Labeling yield of >90% was obtained corresponding to a specific activity of 178 GBq/mmol. The stability of radiolabeled kit in human serum was 84.2% after 1 hour post incubation. In-vitro studies showed 75% of radioactivity was bound to bacteria. After injection into mice clearance from the circulation occurred mainly by biliary-renal clearance and site of infection was rapidly detected within 30 min. Target to non-target muscle ratio was 3.23 +/- 0.05% at 30 min post injection. [99m]Tc-ciprofloxacin showed favorable radiochemical and biological characteristics which permitted detection of the infection with optimal visualization