Methylated tetra-amide derivatives of paramagnetic complexes for magnetic resonance biosensing with both BIRDS and CEST.

Paramagnetic agents that utilize two mechanisms to provide physiological information by magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) are described. MRI with chemical exchange saturation transfer (CEST) takes advantage of the agent's exchangeable protons (e.g., -OH or -NHx , where 2 ≥ x ≥ 1) to create pH contrast. The agent's incorporation of non-exchangeable protons (e.g., -CHy , where 3 ≥ y ≥ 1) makes it possible to map tissue temperature and/or pH using an MRSI method called biosensor imaging of redundant deviation in shifts (BIRDS). Hybrid probes based upon 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelate (DOTA4- ) and its methylated analog (1,4,7,10-tetraazacyclododecane-α, α', α″, α‴-tetramethyl-1,4,7,10-tetraacetate, DOTMA4- ) were synthesized, and modified to create new tetra-amide chelates. Addition of several methyl groups per pendent arm of the symmetrical chelates, positioned proximally and distally to thulium ions (Tm3+ ), gave rise to favorable BIRDS properties (i.e., high signal-to-noise ratio (SNR) from non-exchangeable methyl proton peaks) and CEST responsiveness (i.e., from amide exchangeable protons). Structures of the Tm3+ probes elucidate the influence of methyl group placement on sensor performance. An eight-coordinate geometry with high symmetry was observed for the complexes: Tm-L1 was based on DOTA4- , whereas Tm-L2 and Tm-L3 were based on DOTMA4- , where the latter contained an additional carboxylate at the distal end of each arm. The distance of Tm3+ from terminal methyl carbons is a key determinant for sustaining BIRDS temperature sensitivity without compromising CEST pH contrast; however, water solubility was influenced by introduction of hydrophobic methyl groups and hydrophilic carboxylate. Combined BIRDS and CEST detection of Tm-L2, which features two high-SNR methyl peaks and a strong amide CEST peak, should enable simultaneous temperature and pH measurements for high-resolution molecular imaging in vivo.

Crystal structures of two polymorphic forms of DOTAM-mono-acid dihydrate.

The structural chemistry of 2-[4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid dihydrate, C16H31N7O5·2H2O, is described. The macrocyclic compound, also known by the abbreviation DOTAM-mono-acid, crystallized at room temperature and was isolated concomitantly as two polymorphic forms. The structures of both polymorphs were determined at 90 K. The first polymorph crystallized as a zwitterionic dihydrate [systematic name: 4,7,10-tris(carbamoylmethyl)-1-(carboxylatomethyl)-1,4,7,10-tetraazacyclododecan-1-ium dihydrate] in the space group P21/n, with Z' = 1. The second polymorph crystallized as a zwitterionic dihydrate in the space group P21 at 90 K, with Z' = 2. The two independent molecules are related by a local center. In each polymorph, the zwitterion is formed between the negatively-charged carboxylate group and the ring N atom that bears the acetate pendant arm. Extensive inter- and intramolecular hydrogen bonding exists in both polymorphic structures. In polymorph 1, an intermolecular hydrogen-bonding network propagating parallel to the a direction creates an infinite chain. A second hydrogen-bonding network is observed through a water molecule of hydration in the b direction. Polymorph 2 also has two intermolecular hydrogen-bonding networks. One propagates parallel to the a direction, while the other propagates in the [-110] direction. Increasing the temperature of polymorph 2 yields the same structure at T = 180 K, but the pseudocenter becomes exact at 299 K. The higher-temperature structure has Z' = 1 in the space group P21/c.

Unexpected Changes in the Population of Coordination Isomers for the Lanthanide Ion Complexes of DOTMA-Tetraglycinate.

Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismatic (SAP) coordination isomer in aqueous solution, a structural feature that has made them useful as water-based paraCEST agents. In an effort to create amide-based paraCEST agents with rapid water exchange rates, we prepared the analogous tetraglycinate complexes with DOTMA, a ligand known to favor the twisted square antiprismatic (TSAP) coordination structures. Unexpectedly, NMR investigations show that the LnDOTMA-(gly)4 complexes, like the LnDOTA-(gly)4 complexes, also favor the SAP isomers in solution. This observation led to density functional theory (DFT) calculations in order to identify the energy terms that favor the SAP structures in lanthanide complexes formed with macrocyclic DOTA- and DOTMA-tetraamide ligands. The DFT calculations revealed that, regardless the nature of the ligand, the TSAP isomers present more negative hydration energies than the SAP counterparts. The extent to which the TSAP isomer is stabilized varies, however, depending on the ligand structure, resulting in different isomeric populations in solution.

Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents.

EuDOTA-tetraamide complexes as paraCEST agents offer an attractive platform for designing biological sensors and responsive agents. The early versions of these agents showed low sensitivity at temperature and power levels suitable for in vivo applications partly due to non-optimal water exchange rates. Here we report two new EuDOTA derivatives having glutamyl-phosphonate side arms that display the slowest water exchange rates of any other paraCEST agent reported so far. The advantages of such systems are demonstrated experimentally both in vitro and in vivo and DFT calculations were performed to help understand the physical-chemical reasons for this interesting behavior.

CB-TE2A(+)·Cl(-)·3H2O: a short intermolecular hydrogen bond between zwitterionic bicyclo[6.6.2]tetraamine macrocycles.

1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) is of much interest in nuclear medicine for its ability to form copper complexes that are kinetically inert, which is beneficial in vivo to minimize the loss of radioactive copper. The structural chemistry of the hydrated HCl salt of CB-TE2A, namely 11-carboxymethyl-1,8-tetraaza-4,11-diazoniabicyclo[6.6.2]hexadecane-4-acetate chloride trihydrate, C16H31N4O4(+)·Cl(-)·3H2O, is described. The compound crystallized as a positively charged zwitterion with a chloride counter-ion. Two of the amine groups in the macrocyclic ring are protonated. Formally, a single negative charge is shared between two of the carboxylic acid groups, while one chloride ion balances the charge. Two intramolecular hydrogen bonds are observed between adjacent pairs of N atoms of the macrocycle. Two intramolecular hydrogen bonds are also observed between the protonated amine groups and the pendant carboxylate groups. A short intermolecular hydrogen bond is observed between two partially negatively charged O atoms on adjacent macrocycles. The result is a one-dimensional polymeric zigzag chain that propagates parallel to the crystallographic a direction. A second intermolecular interaction is a hydrogen-bonding network in the crystallographic b direction. The carbonyl group of one macrocycle is connected through the three water molecules of hydration to the carbonyl group of another macrocycle.

Amplifying the sensitivity of zinc(II) responsive MRI contrast agents by altering water exchange rates.

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r1). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM(-1) s(-1) upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r1 compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

A pH-Responsive MRI Agent that Can Be Activated Beyond the Tissue Magnetization Transfer Window.

A terbium-based complex that displays a water exchange CEST resonance well outside the normal magnetization transfer (MT) frequency range of tissues provides a direct readout of pH values by MRI. Deprotonation of the phenolic proton in this complex results in a frequency shift of 56 ppm in a bound water molecule exchange peak between pH 5 and 8. This allows direct imaging of pH without prior knowledge of the agent concentration and with essentially no interference from the tissue MT signal.

Imaging tumor vasculature noninvasively with positron emission tomography and RGD peptides labeled with copper 64 using the bifunctonal chelates DOTA, oxo-DO3a. and PCTA.

Two novel bifunctional chelates, 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (Oxo-DO3A), were found to radiolabel antibodies with copper 64 (64Cu) well for positron emission tomography (PET). In this study, the same chelators were used to radiolabel peptides with 64Cu for PET imaging of angiogenesis. PCTA, Oxo-DO3A, and 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) were conjugated to cyclic-(RGDyK), and their binding affinities were confirmed. Conditions for 64Cu radiolabeling were optimized for maximum yield and specific activity. The in vitro stability of the radiolabeled compounds was challenged with serum incubation. PET studies were carried out in a non-αvß3-expressing tumor model to evaluate the compounds' specificity for proliferating tumor vasculature and their in vivo pharmacokinetics. The PCTA and Oxo-DO3A bioconjugates were labeled with 64Cu at higher effective specific activity and radiochemical yield than the DOTA bioconjugate. In the imaging studies, all the 64Cu bioconjugates could be used to visualize the tumor and the radiotracer uptake was blocked with cyclic-(RGDyK). Target uptake of each bioconjugate was similar, but differences in other tissues were observed. 64Cu-PCTA-RGD showed the best clearance from nontarget tissue and the highest tumor to nontarget ratios. PCTA was the most promising bifunctional chelate for 64Cu peptide imaging and warrants further investigation.

A europium(III)-based PARACEST agent for sensing singlet oxygen by MRI.

A europium(III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen ((1)O2). The water soluble, thermodynamically stable complex reacts rapidly with (1)O2 to form an endoperoxide derivative that results in an ∼3 ppm shift in the position of the Eu(III)-bound water chemical exchange saturation transfer (CEST) peak. The potential of using this probe to detect accumulation of the endoperoxide derivative in biological media by ratiometric CEST imaging was demonstrated.

(68)Ga small peptide imaging: comparison of NOTA and PCTA.

In this study, a bifunctional version of the chelate PCTA was compared to the analogous NOTA derivative for peptide conjugation, (68)Ga radiolabeling, and small peptide imaging. Both p-SCN-Bn-PCTA and p-SCN-Bn-NOTA were conjugated to cyclo-RGDyK. The resulting conjugates, PCTA-RGD and NOTA-RGD, retained their affinity for the peptide target, the α(v)ß(3) receptor. Both PCTA-RGD and NOTA-RGD could be radiolabeled with (68)Ga in >95% radiochemical yield (RCY) at room temperature within 5 min. For PCTA-RGD, higher effective specific activities, up to 55 MBq/nmol, could be achieved in 95% RCY with gentle heating at 40 °C. The (68)Ga-radiolabeled conjugates were >90% stable in serum and in the presence of excess apo-transferrin over 4 h; (68)Ga-PCTA-RGD did have slightly lower stability than (68)Ga-NOTA-RGD, 93 ± 2% compared to 98 ± 1%, at the 4 h time point. Finally, the tumor and nontarget organ uptake and clearance of (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD was compared in mice bearing HT-29 colorectal tumor xenografts. Activity cleared quickly from the blood and muscle tissue with >90% and >70% of the initial activity cleared within the first 40 min, respectively. The majority of activity was observed in the kidney, liver, and tumor tissue. The observed tumor uptake was specific with up to 75% of the tumor uptake blocked when the mice were preinjected with 160 nmol (100 µg) of unlabeled peptide. Uptake observed in the blocked tumors was not significantly different than the background activity observed in muscle tissue. The only significant difference between the two (68)Ga-radiolabeled bioconjugates in vivo was the kidney uptake. (68)Ga-radiolabeled PCTA-RGD had significantly lower (p < 0.05) kidney uptake (1.1 ± 0.5%) at 2 h postinjection compared to (68)Ga-radiolabeled NOTA-RGD (2.7 ± 1.3%). Overall, (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD performed similarly, but the lower kidney uptake for (68)Ga-radiolabeled PCTA-RGD may be advantageous in some imaging applications.

Pharmacokinetic modulation of radiolabeled chelates facilitated by phosphonate ester coordinating groups.

Chelates are an important part of metal based radiopharmaceuticals. This study examines the possible application of phosphonate ester moieties incorporated into chelates, where the ester group can be changed to modulate the pharmacokinetics of the radiopharmaceutical, while the phosphonate stably binds the metal radioisotope. Two phosphonate ester containing chelates, PCTMB and PCTM(F)E, were compared to the carboxylate containing analogue, p-Bn-PCTA, with respect to radiochemistry with several radionuclides (In-111, Ga-68, Ga-67, Cu-64). The phosphonate ester derivatives were similar to p-Bn-PCTA with respect to efficient radiolabeling with each of the radiometals under mild, aqueous conditions. Each of the radiolabeled phosphonate esters was also shown to be stable under physiological conditions in vitro. The phosphonate ester moieties did exhibit a propensity to degrade under more acidic conditions. Biodistribution studies in mice with the In-111 radiolabeled versions of PCTMB, PCTM(F)E and p-Bn-PCTA demonstrated the ability of the phosphonate ester functionalities to change the pharmacokinetics of the BFCs. With increasing lipophilicity, the phosphonate ester derivatives showed increasing hepatic clearance; but no significant increase in background tissue uptake (bone, muscle) was observed, and all the In-111 radiolabeled BFCs were substantially cleared within 24 h. The substitution of phosphonate ester for carboxylate functional groups in chelates may be an effective strategy to assist in optimizing the pharmacokinetics of radiopharmaceuticals through varying of the ester group.

Nanoparticle-based PARACEST agents: the quenching effect of silica nanoparticles on the CEST signal from surface-conjugated chelates.

Silica nanoparticles of average diameter 53 ± 3 nm were prepared using standard water-in-oil microemulsion methods. After conversion of the surface Si-OH groups to amino groups for further conjugation, the PARACEST agent, EuDOTA-(gly)4 (-) was coupled to the amines via one or more side-chain carboxyl groups in an attempt to trap water molecules in the inner-sphere of the complex. Fluorescence and ICP analyses showed that approximately 1200 Eu(3+) complexes were attached to each silica nanoparticle, leaving behind excess protonated amino groups. CEST spectra of the modified silica nanoparticles showed that attachment of the EuDOTA-(gly)4 (-) to the surface of the nanoparticles did not result in a decrease in water exchange kinetics as anticipated, but rather resulted in a complete elimination of the normal Eu(3+) -bound water exchange peak and broadening of the bulk water signal. This observation was traced to catalysis of proton exchange from the Eu(3+) -bound water molecule by excess positively charged amino groups on the surface of the nanoparticles.

Quantification of water exchange kinetics for targeted PARACEST perfluorocarbon nanoparticles.

PARACEST (PARAmagnetic Chemical Exchange Saturation Transfer) agents offer the ability to generate "contrast on demand", negating the need to image before contrast agent injection. Perfluorocarbon (PFC) nanoparticles can deliver very large payloads of PARACEST agents, lowering the effective detection limit for molecular imaging of sparse biomarkers. Also, the PFC core provides a quantitative (19)F signal for measuring particle binding with high signal intensity and no background signal. (19)F quantization coupled with mathematical modeling of the PARACEST signal showed that incorporating PARACEST chelates onto the nanoparticle surface reduces the bound water lifetime and diminishes the available contrast to noise ratio compared to the parent small molecule PARACEST chelate. PARACEST nanoparticles were targeted to fibrin, an early biomarker for atherosclerotic plaque rupture, and bound to the surface of in vitro clots, yielding a detection limit of 2.30 nM at 11.7T. When the particles bind to a target surface, the image contrast is higher than predicted from phantom experiments, perhaps due to improved water exchange kinetics. We demonstrated that PARACEST PFC nanoparticles can provide two unique signatures, (19)F and PARACEST, for quantitative targeted molecular imaging of fibrin.

A lanthanide complex with dual biosensing properties: CEST (chemical exchange saturation transfer) and BIRDS (biosensor imaging of redundant deviation in shifts) with europium DOTA-tetraglycinate.

Responsive contrast agents (RCAs) composed of lanthanide(III) ion (Ln3R) complexes with a variety of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA4S) derivatives have shown great potential as molecular imaging agents for MR. A variety of LnDOTA­tetraamide complexes have been demonstrated as RCAs for molecular imaging using chemical exchange saturation transfer (CEST). The CEST method detects proton exchange between bulk water and any exchangeable sites on the ligand itself or an inner sphere of bound water that is shifted by a paramagnetic Ln3R ion bound in the core of the macrocycle. It has also been shown that molecular imaging is possible when the RCA itself is observed (i.e. not its effect on bulk water) using a method called biosensor imaging of redundant deviation in shifts (BIRDS). The BIRDS method utilizes redundant information stored in the nonexchangeable proton resonances emanating from the paramagnetic RCA for ambient factors such as temperature and/or pH.Thus, CEST and BIRDS rely on exchangeable and nonexchangeable protons, respectively, for biosensing. We posited that it would be feasible to combine these two biosensing features into the same RCA (i.e. dual CEST and BIRDS properties). A complex between europium(III) ion (Eu3R) and DOTA­tetraglycinate [DOTA­(gly)S4] was used to demonstrate that its CEST characteristics are preserved, while its BIRDS properties are also detectable. The in vitro temperature sensitivity of EuDOTA­(gly)S4 was used to show that qualitative MR contrast with CEST can be calibrated using quantitative MR mapping with BIRDS, thereby enabling quantitative molecular imaging at high spatial resolution.

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates.

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'''-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),ßAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.

Strategies for labeling proteins with PARACEST agents.

Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per 3G4 molecule and between 5.6 and 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53µs for the non-conjugated chelate to 65-77µs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73-75µs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.

Multifunctional Polymeric Scaffolds for Enhancement of PARACEST Contrast Sensitivity and Performance: The Effects of Random Copolymer Variations.

A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a single acrylamide side-chain (M1) was copolymerized with either 2-methylacrylic acid (MAA), 2-(acryloylamino)-2-methyl-1-propanesulfonic acid (AMPS) or N-isopropylacrylamide (NIPAM) to create a series of linear random copolymers using classical free radical chain polymerization chemistry. The metal ion binding properties of hydrolyzed M1 were investigated by pH potentiometry and the europium (III) complexes of the resulting heteropolymers were evaluated as PARACEST imaging agents. All polymeric agents were found to possess similar intermediate-to-slow water exchange and CEST characteristics as the parent EuDOTA-tetraamide monomer. Consistent with basic multiplexing principles, the highest molecular weight polymer, Eu-DMAA 3.1, also showed the highest CEST sensitivity with a detection limit of 20 ± 2 µM. The second arylamide component gave polymers with widely different chemical characteristics and CEST properties. In particular, the Eu-DNIPAM 4.0 and Eu-DMAA 4.1 polymers displayed different solubility characteristics as a function of pH or temperature which, in turn, affected the water exchange and CEST properties of the corresponding agents. It was concluded that introduction of hydrophobic groups into the polymer backbone reduces solvent accessibility to the Eu(3+) component, effectively slowing water exchange between the inner-sphere water coordination position at each Eu(3+) center with bulk water. The CEST properties of the heteropolymers when dissolved in plasma suggest that the more hydrophobic characteristics of these polymers could be advantageous for in vivo applications.

A responsive europium(III) chelate that provides a direct readout of pH by MRI.

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric chemical exchange saturation transfer (CEST) principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ∼5 ppm shift in the water exchange CEST peak that is easily detected by MRI. Collection of two CEST images at two slightly different activation frequencies provides a direct readout of solution pH without the need of a concentration marker.

Comparison of bifunctional chelates for (64)Cu antibody imaging.

PURPOSE: Improved bifunctional chelates (BFCs) are needed to facilitate efficient (64)Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with (64)Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). METHODS: The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. (64)Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. RESULTS: (64)Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of (64)Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that (64)Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the (64)Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the (64)Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. CONCLUSION: The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior (64)Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, (64)Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than the analogous (64)Cu-DOTA antibody conjugates.

Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine.

The positron emitter zirconium-89 ((89)Zr) has very attractive properties for positron emission tomography (PET) imaging of intact monoclonal antibodies (mAbs) using immuno-PET. This protocol describes the step-by-step procedure for the facile radiolabeling of mAbs or other proteins with (89)Zr using p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). First, Df-Bz-NCS is coupled to the lysine-NH(2) groups of a mAb at pH 9.0 (pre-modification), followed by purification using gel filtration. Next, the pre-modified mAb is labeled at room temperature by the addition of [(89)Zr]Zr-oxalic acid solution followed by purification using gel filtration. The entire process of pre-modification, radiolabeling and purification steps will take about 2.5 h.