Thin-layer Radiochromatography Research Articles

Synthesis of a Novel Antiestrogen Radioligand (99mTc-TOR-DTPA)

This study was aimed at developing a hydrophilic radioligand as an antiestrogen drug derivative to be used for imaging breast tumors. Toremifene [TOR; 4-chloro-1,2-diphenyl-1-(4-(2-(N,N-di-methylamino)ethoxy)phenyl)-1-butene, as citrate salt] was selected as the starting material to be derived, since it has been used extensively as an antiestrogen drug for treatment and prevention of human breast cancer. An antiestrogen drug derivative, TOR attached to diethylenetriamine pentaacetic acid (DTPA), was synthesized by two experimental treatments, including a purification and a reaction step. We described the synthesis of this TOR derivative, (3Z)-4-{4-[2-(dimethylamino) ethoxy] phenyl}-3,4-diphenylbut-3-en-1-ylN,N-bis[2-(2,6-dioxomorpholin-4-yl)ethyl]glycinate (TOR-DTPA), in detail. Mass spectroscopy confirmed the expected structures. TOR-DTPA was labeled with technetium-99m ((99m)Tc), using stannous chloride (SnCl(2)) as the reducing agent. Biodistribution studies were performed on female Albino Wistar rats. Quality controls, radiochemical yield, and stability studies were done utilizing high-performance liquid chromatography, radioelectrophoresis, thin-layer chromatography, and thin-layer radiochromatography methods. The synthesized compound was found to be hydrophilic and anionic, with high stability for the duration of the testing period in vitro. The results indicated that the radiolabeled compound has estrogen-receptor specificity, especially for the breast tissue. It is highly possible that this compound could be used for imaging breast tumors as a novel technetium-labeled hydrophilic estrogen derivative radioligand.

In vitro evaluation, biodistribution in rats of radiolabeled raloxifene

Raloxifene is a selective estrogen receptor modulator that produces both estrogen agonistic effects on bone and lipid metabolism and estrogen-antagonistic effects on uterine endometrium and breast tissue. In the present study, raloxifene was labeled with I-131 by iodogen method and investigated for its radiopharmaceutical potential. Radiolabeling yield is 91±0.7%, as determined by radio thin layer chromatography (RTLC). Results of in vitro study indicated 131I-raloxifene has high stability (4 h) in serum. Biodistribution study was carried out with Albino wistar female rats. The result has shown that the radioiodinated raloxifene has higher uptake in uterus than breast and ovarian.

The influence of stereoisomerism on the biological behavior of 99mTc labeled penicillamine

The aim of this study is to investigate stereoisomeric behavior of penicillamine and the effect of temperature on labeling. In addition, it was explored how stereoisomerism affected biological behavior of them. In the present work, D- and L-enantiomers of penicillamine(D-PA, L-PA) were labeled with 99mTc using SnCl2 as reducing agent and their radiopharmaceutical potentials were investigated. Quality control procedures were carried out using thin layer radiochromatography (TLRC), electrophoresis and high performance liquid radiochromatography (HPLRC). HPRLC chromatograms showed two peaks for 99mTc-D-PA, while a single peak was observed for 99mTc-L-PA at room temperature. However, the single peak was observed at 90 °C for both isomers. Labeling yields of each isomer were found to be over 98%. Biological activity of these complexes was determined on male Albino Wistar rats by biodistribution studies. While the biodistribution result of 99mTc-D-PA showed high uptake in the liver, maximum uptake of 99mTc-L-PA was observed in the kidneys. Both two complexes were cleared rapidly from the blood, mainly by the renal system. Since the activity concentration of 99mTc-D-PA at the 30th minute in the kidneys and the liver reached a maximum value and at the 120th minute, it was removed by renal and hepatobiliary excretion. As a result, it can be concluded that stereoisomerism affect not only the chemical behavior, but also differs their biological behavior of these compounds.

Labeling of apigenin with 131I and bioactivity of 131I-apigenin in male and female rats

Apigenin (4′,5,7-trihydroxyflavone), one of the most common flavonoids, has been shown to possess a variety of biological activities including tumor growth inhibition and chemopreventation. In the present study, apigenin was labeled with 131I using iodogen method and investigated of its bioactivity. Radiolabeling yield is 98±0.2%, as determined by radio thin layer chromatography (RTLC), electrophoresis and radio high performance liquid chromatography (RHPLC). Besides, structure analysis of synthesized cold iodoapigenin complex were assessed with LCMS/MS and 1H-NMR. Results of in vitro study indicated a high stability (3 hours) in human serum. Biodistrubition studies are performed in male and female albino Wistar rats. Biodistribution data related to the male rats showed significant uptake in the small intestine. The female rats biodistribution results indicated that the uptake of 131I-apigenin was high in the intestine and uterus.

Development of 111In labeled insulin for receptor imaging/therapy

In order to target insulin receptors in various diabetic and insulinoma conditions, human recombinant insulin was successively labeled with [111In]-indium chloride after conjugation with freshly prepared cyclic DTPA-dianhydride (ccDTPA). The best results of the conjugation were obtained by addition of 0.5 mL of an insulin pharmaceutical solution (5 mg/mL, in phosphate buffer, pH 8) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 minutes. Radio-thin layer chromatography (RTLC), instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) have shown an overall radiochemical purity of higher than 93% at optimized conditions (specific activity = 550–750 MBq/mg, radiochemical yield =81%). The white blood cell labeling capacity of the tracer was determined up to 4 hours at 37 °C. Preliminary in vivo studies in normal rat model was performed to determine the biodistribution of the radiotracer up to 48 hours. It showed a high liver and spleen uptake of the tracer which is consistent with other reported radiolabeled insulins. SPECT images have also shown high liver accumulation of the tracer.

Preparation and evaluation of [67Ga]-DTPA-β-1–24-corticotrophin in normal rats

β-1–24-corticotrophin was successively labeled with [67Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 ml of a β-1–24-corticotrophin pharmaceutical solution (1 mg/mL, pH=6.5) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 min. Radio-thin layer chromatography showed an overall radiochemical purity of >90% at optimized conditions after labeling. HPLC showed a radiochemical purity more than 95% (specific activity =300–500 MBq/mg, labeling efficiency 77%). The stability of the radioconjugate was tested in presence of human serum at 37 °C. Preliminary in vivo studies in normal rats were performed to determine the biodistribution of the radioimmunoconjugate up to 30 min. Lungs showed to have the major tracer uptake which is consistent with the natural distribution of ACTH receptors in human body. Pretreatment of animals with cold peptide following labeled compound administration reduced lung uptake at least 10 and 3 times after 15 and 30 min, respectively, as well as significant decrease in adrenal uptake after 15 min. These data support the specific receptor binding of the radiolabeled compound. This tracer can be used in detection of ACTH receptor in lung and adrenal malignancies.

Preparation and biodistribution of [111In]-rHuEpo for erythropoietin receptor imaging

Human recombinant erythropoietin (EPO) was successively labeled with [111In]-indium chloride after conjugation with freshly prepared cyclic DTPA-dianhydride (ccDTPA). The best results of the conjugation were obtained by the addition of 100 i.u. of an EPO pharmaceutical solution (in phosphate buffer, pH 7.5) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 minutes. Radio thin layer chromatography (RTLC), instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) showed overall radiochemical purity of higher than 95% at optimized conditions (specific activity = 1.2–1.5 GBq/mg, labeling efficiency 80%). Preliminary in vivo studies in normal rat model was performed to determine the biodistribution of the radiotracer up to 1 hour using scarification. The high kidney uptake of the tracer was consistent with the reported EPO receptor distribution.

Preparation and biodistribution of [67Ga]-DTPA-gonadorelin in normal rats

Gonadorelin was successively labeled with [67Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 mg of a gonadorelin to a glass tube pre-coated with DTPA-dianhydride (0.33 mg) at 25 °C with continuous mild stirring for 1 hour. Radio thin layer chromatography showed an overall radiochemical purity of >90% at optimized conditions after labeling. HPLC showed a radiochemical purity more than 95% (specific activity = 400–450 GBq/M). The stability of the radioconjugate was tested in presence of human serum at 37 °C. Preliminary in vivo studies in normal rats were performed to determine the biodistribution of the conjugate up to 48 hours. The breast and ovaries uptakes were significantly high in first 15-minute post injection which is in agreement with the other reports regarding the presence of specific GnRH receptors. This tracer can be used in detection of GnRH receptor biodistribution in various diseases and malignancies.

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

3′-Sulfonylesters of 2,5′-anhydro-1-(2-deoxy-β-d-threo-pentofuranosyl)thymine as precursors for the synthesis of [18F]FLT: syntheses and radiofluorination trials

Title compounds [3'-sulfonylesters of 2,5'-anhydro-1-(2-deoxy-beta-d-threo-pentofuranosyl)thymine: 2,5'-anhydro-1-(2-deoxy-3-methanesulfonyl-beta-d-threo-pentofuranosyl)thymine (1a); 2,5'-anhydro-1-(2-deoxy-3-(4-nitrobenzenesulfonyl)-beta-d-threo-pentofuranosyl)thymine (1b); 2,5'-anhydro-1(-2-deoxy-3-(toluenesulfonyl)-beta-d-threo-pentofuranosyl)thymine (1c); and 2,5'-anhydro-1(-2-deoxy-3-(2,2,2-trifluoroethanesulfonyl)-beta-d-threo-pentofuranosyl)thymine 1d] were synthesized, and their use as starting materials for the synthesis of 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) was investigated. Radiofluorination of Compound 1b and subsequent hydrolysis with NaOH, in solution or on solid support, yielded a product with the same retention on radio thin-layer chromatography as [(18)F]FLT. However, careful analysis with high-performance liquid chromatography could not identify the product to be [(18)F]FLT. From several options that were investigated to identify the obtained product, it was shown that fluorination had occurred at the nitro group of the nosylate, and not at the 3'-position. Other sulfonate esters (Compounds 1a, 1c and 1d) did not give any fluorination under any of the investigated reaction conditions. It had to be concluded that title compounds are not suitable as starting materials for the synthesis of [(18)F]FLT under the described conditions.

Two Distinct Pathways for Cyclooxygenase-2 Protein Degradation

Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.

Preparation, quality control and biodistribution studies of [67Ga]-DOTA-anti-CD20

Rituximab was successively labeled with [ 67 Ga]-gallium chloride. The macrocyclic bifunctional chelating agent, N -succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 °C using DOTA, N -hydroxy succinimide (NHS) in CH 2 Cl 2 . DOTA-Rituximab was obtained by the addition of 1 mL of a Rituximab pharmaceutical solution (5 mg/mL, in phosphate buffer, pH=7.8) to a glass tube pre-coated with DOTA-NHS (0.01–0.1 mg) at 25 °C with continuous mild stirring for 15 h. Radiolabeling was performed at 37 °C in 3 h. Radio-thin layer chromatography showed an overall radiochemical purity of 90%–95% at optimized conditions (specific activity = 30 GBq/mg, labeling efficacy; 82%). The final isotonic 67 Ga-DOTA-rituximab complex was checked by gel electrophoresis for radiolysis. Radio-TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary biodistribution studies in normal rat model performed to determine complex distribution of the radioimmunoconjugate up to 28 h.

Preparation and biodistribution of [67Ga]-DTPA-rituximab in normal rats

Rituximab was successively labeled with [67Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 ml of a rituximab pharmaceutical solution (5 mg/ml, in phosphate buffer, pH 8) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 minutes. Radio thin-layer chromatography showed an overall radiochemical purity of 96–99% at optimized conditions (specific activity = 300–500 MBq/mg, labeling efficiency 77%). The final isotonic 67Ga-DTPA-rituximab complex was checked by gel electrophoresis for radiolysis control. Radio-TLC was performed to ensure the formation of only one species, followed by filtration through a 0.22 μm filter. Preliminary in vivo studies in normal rat model was performed to determine the biodistribution of the radioimmunoconjugate up to 6 hours.

99mTc-glucoheptonate-guanine: Synthesis, biodistribution and imaging in animals

The aim of the current study was to design a nucleotide-based radiopharmaceutical which could be labeled with 99m Tc and to investigate its radiopharmaceutical efficiency and stability. GHA (glucoheptonate) was used as bifunctional chelate. GHA was labeled with 99m Tc by SnCl2 reduction method first, and then G (guanine) was conjugated with 99m Tc-GHA at 90 °C. In order to determine its radiopharmaceutical stability, thin layer radio chromatography (TLRC) and electrophoresis were employed. In addition, the results were confirmed using high performance liquid radio chromatography (HPLRC). Scintigraphic imaging was performed on rats with mammary tumors, while tissue distribution was determined on Albino Wistar rats. Labeling yield was found to be over 95% and the labeled complex maintained its stability during the study period. The lipophilicity of the 99m Tc-GHG was measured and the partition coefficient (logP) of the labeled compound calculated. The results demonstrated that the uptake of 99m Tc-GHG ( 99m Tc-glucoheptonate-guanine) reached its maximum at 3 hours p.i. in stomach and intestines. Main way of excretion was renal. Hepatobiliary excretion was also observed. In conclusion, 99m Tc-GHG may be useful as a nucleotide-based radiopharmaceutical for in vivo applications.

Preparation, quality control and stability of 99mTc-cefuroxime axetil

Cefuroxime axetil, a cephalosporin antibiotic used to treat bacterial infections, was investigated to label with 99mTc. Radiolabeling of cefuroxime axetil was carried out by using stannous chloride method. Effects of pH and stannous chloride amount on the radiolabeling yield were investigated. The radiochemical purity of 99mTc-cefuroxime axetil was determined by thin layer radio chromatography (TLRC), electrophoresis and high performance liquid chromatography. The maximum radiolabeling yield was 98±1%.

Radiolabeling of Bombesin-Like Peptide with 99mTc: 99mTc-Litorin and Biodistribution in Rats

Bombesin-like peptides are related to several human cancer receptors, including small cell lung, prostate, breast, colon, and pancreatic cancers. Litorin, an amphibian bombesin peptide derivative, is found to stimulate the contraction of smooth muscle, to stimulate gastrin, gastric acid, and pancreatic secretion, and to suppress the nutriment in in vivo experiments. In the present study, litorin was labeled with 99mTc by the stannous chloride procedure. Labeling yield is 95 +/- 1.4%, as determined by radio thin layer chromatography (RTLC) and radio high performance chromatography (RHPLC). Results of in vitro studies demonstrated a high stability in serum and cysteine solutions. In vivo biodistribution was investigated with normal male Albino Wistar rats. Biodistribution data showed fast clearance, low intestinal accumulation, and significant uptake in bombesin/gastrin releasing peptide (BN/GRP) receptor rich tissues such as the pancreas (23.56 +/- 0.01 %ID/g 30 min pi). It can be blocked partially by previous administration of 'cold' litorin. The results showed specificity of the uptake. As 99mTc-litorin displays good radiolabeling and biodistribution, it is a potentially useful radiopharmaceutical for detection of bombesin receptor-expressing cancers.

Development of [64Cu]-DOTA-anti-CD20 for targeted therapy

Copper-64 was produced as a by-product of 55Co via 64Ni(p,n)64Cu by 15 MeV proton bombardment of natNi resulting in a thick target yield of 5.31 MBq/μAh (143.5 μCi/μAh) and a radiochemical separation yield of 95% (radionuclide purity >97% after 25 hours of bombardment). Rituximab was successively labeled with [64Cu]-CuCl2. N-succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 °C using DOTA and N-hydroxy succinimide (NHS) in CH2Cl2 followed by the addition of 1 ml of a Rituximab pharmaceutical solution. Radiolabeling was performed at 37 °C in 3 hours. Radio thin-layer chromatography showed an overall radiochemical purity of 90–95% at optimized conditions (specific activity=30 GBq/mg, labeling efficacy; 82%) using various chromatography systems. The final isotonic 64Cu-DOTA-Rituximab complex was passed through a 0.22 μm filter and checked by gel electrophoresis for radiolysis control. Stability of the final product was checked in the formulation and in presence of human serum at 37 °C.

Investigation of new bifunctional agents: D-Penicillamine

Somatostatin inhibits the release of growth hormone (somatotropin) from the Anterior Pituitary. The main use of derivatives of somatostatin is to diagnose growth hormone problems and to use against some forms of cancer which involve growth hormone. Also somatostatin suppresses gastric acid secretion, gallbladder contractions, and pancreatic enzyme secretion. The aim of the current study is to investigate new bifunctional agents for labeling with 99mTc. Therefore D-Penicillamine was used as a bifunctional agent and compared to DTPA in the labeling with 99mTc. Quality controls were established using thin layer radio chromatography (TLRC) and electrophoresis techniques. In addition, high performance liquid radio chromatography (HPLRC) was also performed for elimination of possible uncertainties. The radiolabeled complexes maintained their stabilities throughout the study. The results obtained showed that 99mTc-D-Penicillamine-somatostatin is a promising potential radiopharm aceutical and an alternative of 99mTc-DTPA-somatostatin for in vivo and in vitro applications.

Review of advances in the thin layer chromatography of pesticides: 2004–2006

Applications of thin layer chromatography and high performance thin layer chromatography for the separation, detection, and qualitative and quantitative determination of pesticides, other agrochemicals, and related compounds are reviewed for the period from July 1, 2004 to November 1, 2006. Analyses are covered for a variety of samples, such as food, crops, biological, environmental, pharmaceuticals, and formulations, and for residues of pesticides of various types, including insecticides, herbicides, and fungicides, belonging to different chemical classes. In addition to references on residue analysis, studies such as pesticide-structure relationships, metabolism, degradation, and lipophilicity are covered, many of which make use of thin layer radiochromatography.

Biodistribution of 99m technetium- labeled creatinine in healthy rats

The distribution of creatinine, one of the toxic guanidine compounds, in various tissues has not been studied in detail by using radiolabeled creatinine. Our objective was to investigate the biodistribution of creatinine labeled with 99m technetium (99mTc) by the stannous (II) chloride method in healthy male Wistar rats. Quality controls were carried out by radio thin layer chromatography, high-performance liquid chromatography, and paper electrophoresis. The labeling yield was 85 +/- 2% under optimum conditions (pH 7 and 100 microg stannous chloride). Rats (N = 12) were injected intravenously with 99mTc-creatinine and their blood and visceral organs were evaluated for 99mTc-creatinine uptake as percent of the injected dose per gram wet weight of each tissue (%ID/g). The lowest amount of uptake was detected in the brain and testis. When the rate of uptake was evaluated, only the kidney showed increasing rates of uptake of 99mTc-creatinine throughout the study. Kidneys showed the highest amount of uptake throughout the study (P < 0.001 compared to all other organs), followed by liver, spleen and lung tissue.