Radiolabeling Reaction Research Articles

Microfluidic reactor for the radiosynthesis of PET radiotracers

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term “microfluidic PET”. The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate “proof of principle” we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[ 18F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radiofluorination of 2-[ 18F]FDG (60% radiochemical yield within 4 s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Purification of carbon-11 PET radiotracers from unlabeled precursors by preparative HPLC and SPE

A general methodology for the rapid purification of carbon-11 positron emission tomography (PET) radiotracers from radiolabeling reaction mixtures has been developed. Preparative HPLC and solid-phase extraction (SPE) techniques are described which can separate some commonly used radiopharmaceuticals such as [(11)C]raclopride, [(11)C]beta-CFT and [(11)C]choline from their unlabeled precursors.

Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb

Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T 1/2 = 52.02 h) has application potential in RII, with a γ-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T 1/2 = 10 h) is a source of highly cytotoxic α-particles via its decay to its 212Bi (T 1/2 = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC] 2+ complex was less labile to metal ion release than Pb[DOTA] 2− at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb 2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb.

A facile, water-soluble method for modification of proteins with DOTA. Use of elevated temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled immunoconjugates.

We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA) to proteins by activation of a single carboxyl group with N-hydroxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA was prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-carcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepared by adding the DOTA active ester reaction mixture to the proteins at pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reaction mixture was increased from 10:1 to 100:1, the average number of chelators attached to the protein molecule increased from 2.64 to 8.79. When DOTA active ester reacted with the antibody at a molar ratio of 100:1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with 111In(III) and 90Y(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stability of the radioconjugates increased as the molar ratio of carbodiimide, relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with 111In(III) and 90Y(III) proved to be sensitive to pH, buffer, and temperature effects. The optimum pH for the labeling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high specific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the temperature of the radiolabeling reaction from 25 to 43 degrees C greatly increased both the efficiency of radiometal incorporation and the kinetic stability of the radioconjugates. Stability studies of the conjugates in human serum and in the presence of a 5000- to 250,000-fold excess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that the radiolabeled proteins are kinetically inert under physiological conditions. In serum, the 111In(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost 111In(III) at a rate of about 0.3% per day. No loss of 90Y(III) from the conjugates was observed in serum, but in excess DTPA, both 90Y(III) labeled proteins showed a rate of radiometal loss of approximately 0.2% per day. Therefore, kinetic analysis of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.

A novel method for the non-chromatographic purification of technetium-99m-labeled monoclonal antibodies: a study with B72.3 monoclonal antibody

A cleaning resin has been developed for the non-chromatographic purification of 99mTc-labeled monoclonal antibodies (MAbs). The resin used is a modified form of thiopropylsepharose 6B resin, in which its sulfhydryl groups have been tinylated with stannous chloride. The method requires only simple stirring of the radiolabeling reaction mixture with this tinylated resin and subsequent separation of it from the resin by filtration to obtain a 99mTc-labeled MAb of radiopharmaceutical purity. The method provides an alternative to Chromatographic purification of the radiolabeled MAb (i.e. gel filtration or anion exchange chromatography) which has been used in other 99mTc-MAb preparations. For comparison studies, we labeled the B72.3 MAb with NeoRx's diamide dimercaptide chelate radiolabeling kit, split the reaction mixture into two equal portions and then purified one portion with anion exchange chromatography (NeoRx's chosen method) while the other portion was purified with our cleaning resin. Comparison of HPLC chromatograms, percent 99mTc-bound to MAb, biodistribution and scintigraphic results show that our cleaning resin methodology provides a 99mTc-labeled MAb of essentially equal purity and utility as does the established, Chromatographic one.

Radiosynthesis of [F-18]fluoxetine as a potential radiotracer for serotonin reuptake sites

Synthesis of 4-nitro-α-bromo-α,α-difluorotoluene was accomplished in two steps starting from 4-nitrobenzaldehyde, with a 30% overall yield. Radiolabeling of 4-nitro-α-bromo-α,α-difluorotoluene with no-carrier-added [ 18F]fluoride provided 4-nitro-α,α-difluoro-α-[ 18F]fluorotoluene in 2–4% yields with a specific activity of 2590 GBq/mmol (70 Ci/mmol). The effect of the reaction temperature on the radiochemical yield and specific activity of the radiolabeling reaction was studied. Radiochemical yields increased, whereas specific activity decreased, with increasing temperature. Radiosynthesis of [ 18F]fluoxetine involved coupling of 4-nitro-α,α-difluoro-α-[ 18F]fluorotoluene with the sodium alkoxide of ( S)-3-(methylamino)-1-phenyl-1-propanol. The overall yield of HPLC purified [ 18F]fluoxetine was 1–2% (decay-corrected; total radiosynthesis time, 150–180 min). The specific activity of the product was 1480 GBq/mmol (40 Ci/mmol).

Use of a microwave cavity to reduce reaction times in radiolabelling with [11C]cyanide

AbstractAdvantages of using a microwave cavity over thermal treatment are demonstrated for radiolabelling reactions with [11C]cyanide. For comparison purposes, two literature syntheses involving typical cyanide chemistry at rather vigorous conditions were investigated: cyano‐de‐halogenation with subsequent hydrolysis of the nitrile and the Bücher‐Strecker synthesis of an aromatic amino acid. Comparable yields were obtained with intensities < 100 W in reaction times that were 1/15 to 1/20th those used in thermal methods. Even rates of slower nucleophilic substitutions could be increased by manipulating the polarity of the medium. For the short‐lived radionuclide carbon‐11, such time gains result in radioactivity gains at the end‐of‐synthesis on the order of 70‐100%.

Synthesis of (R, R)123I‐QNB, A spect imaging agent for cerebral muscarinic acetylcholine receptors in vivo

AbstractThe high‐affinity muscarinic receptor antagonist (R,R) I‐QNB(1) [(R)‐(‐)‐1‐Azabicyclo [2.2.2]oct‐3‐yl‐ (R) ‐ (+) ‐ α‐hydroxy‐α‐(4‐[127I]iodophenyl)‐α‐phenyl Acetate (6a)] has been labeled with iodine‐123 to give a suitable ligand for SPECT (Single photon emission computed tomography) imaging of the human brain. The radiolabeling is achieved using a copper(I) assisted nucleophilic exchange mechanism(2) to give high specific activity (R,R)123I‐QNB in reasonable overall yield (36%). The radiolabeling reaction is carried out in the presence of excess reducing agent and the product (R,R)123I‐QNB purified on a Sep‐Pak cartridge eliminating the need for h.p.l.c. purification the synthesis of the precursor (127I‐QNB) for the radiolabeling step is a modification of the procedure reported by Rzeszotarski et. al(3) (R)‐α‐Hydroxy‐α‐(4‐nitrophenyl)‐α‐phenylacetic acid was produced from 4‐nitrobenzophenone and resolved as reported. The methyl ester of the acid was synthesised then the nitro group converted to an amino group to give Methyl, (R) ‐α‐hydroxy‐α‐ (4‐aminophenyl) ‐a‐ phenyl acetate. The iodo compound Methyl, (R) ‐α‐hydroxy‐α‐ (4‐iodophenyl)‐α‐phenyl acetate was produced from the amino compound via the diazonium salt. Transesterification of the iodo compound with (R) ‐3‐quinuclidinof gave (R, R) ‐f271‐QNB.The (R,R) 123I‐QNB produced has been successfully used to image muscarinic receptors in patients suffering from Alzheimers disease.

Microwave cavities: Some parameters affecting their use in radiolabelling reactions

The experimental conditions influencing the strength of the electromagnetic field delivered to the sample by a coaxial microwave resonance cavity were investigated. Radiofluorinations using this equipment indicate that this technique is useful for shortening reaction times and affecting the product distribution in radiolabelling reactions.

Polymer‐supported nucleophilic radiolabeling reactions with [18F]fluoride and [11C]cyanide ion on quaternary ammonium resins

Polymeric reagents are particularly valuable aids in radiopharmaceutical synthesis where fast, simple and easily automated chemical operations are necessary. Nucleophilic radiofluorination is an operation which could be simplified through use of polymeri reagents. Existing procedures require several steps to convert aqueous [ Flfluoride form the target to an organic soluble vlactivatedvv form suitable for nucleophilic displacement reactions. The steps include addition of base and solubilizing agent and azeotropic removal of water. The procedure is time c nsuming and can result in significant contamination or loss of expensive p80 target water. Furthermore, after the labeling reaction is complete an additional step is usually necessary for removal of base and solubilizing agent prior to HPLC purification of the reaction mixture.

Technetium-99 m-labeled mouse-spleen leukocytes: Radiolabeling parameters

Bouse-spleen leukocytes were radiolabeled with 99m Tc under neutral pH conditions. A low concentration of stannous chloride was used to reduce the sodium pertechnetate to a cell-binding lower-valence state. Cell radiolabeling was measured by radioactivity retention on glass-fiber filters. This procedure is quick and allows the assay of small aliquots of the radiolabeling reaction mixture. A stable cell radiolabel of 24.4% ± 5.2% was obtained which remained attached to the cell up to 27.5 h as measured by in vitro incubation in tissue culture medium. The viability of the cells after 99m Tc-cell-labeling remains as high as the control. The radiolabeling efficiency of the cells with 99m Tc was dependent on the concentration of cells and stannous chloride, but was independent of Na 99m TcO 4 concentration.