The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

Excitation functions of (nat)Zn(p,x) nuclear reactions with proton beam energy below 18 MeV.

We measured the excitation functions of (nat)Zn (p,x) reactions up to 17.6MeV, using the stacked-foils activation technique. High-purity natural zinc (and copper) foils were irradiated with proton beams generated by an 18MeV isochronous cyclotron. Activated foils were measured using high-purity Ge gamma spectroscopy to quantify the radionuclides (61)Cu, (66)Ga, (67)Ga, and (65)Zn produced from the reactions. Thick-target integral yields were also deduced from the measured excitation functions of the produced radioisotopes. These results were compared with the published literature and were found to be in good agreement with most reports, particularly those most recently compiled.

Production of high specific activity (195m) Pt-cisplatinum at South African Nuclear Energy Corporation for Phase 0 clinical trials in healthy individual subjects.

Platinum agents continue to be the main chemotherapeutic agents used in the first-line and second-line treatments of cancer patients. It is important to fully understand the biological profile of these compounds in order to optimize the dose given to each patient. In a joint project with the Australian Nuclear Science and Technology Organisation and the Nuclear Medicine Department at Steve Biko Academic Hospital, South African Nuclear Energy Corporation synthesized and supplied (195m) Pt-cisplatinum (commonly referred to as cisplatin) for a clinical pilot study on healthy volunteers. Enriched (194) PtCl2 was prepared by digestion of enriched (194) Pt metal (>95%) followed by thermal decomposition over a 3 h period. The (194) PtCl2 was then placed in a quartz ampoule, was irradiated in SAFARI-1 up to 200 h, then decay cooled for a minimum of 34 h prior to synthesis of final product. (195m) Pt(NH3 )2 I2 , formed with the addition of KI and NH4 OH, was converted to the diaqua species [(195m) Pt(NH3 )2 (H2 O)2 ](2+) by reaction with AgNO3 . The conversion to (195m) Pt-cisplatinum was completed by the addition of concentrated HCl. The final product yield was 51.7% ± 5.2% (n = 5). The chemical and radionuclidic purity in each case was >95%. The use of a high flux reactor position affords a higher specific activity product (15.9 ± 2.5 MBq/mg at end of synthesis) than previously found (5 MBq/mg). Volunteers received between 108 and 126 MBq of radioactivity, which is equivalent to 6.8-10.0 mg of carrier cisplatinum. Such high specific activities afforded a significant reduction (~50%) in the chemical dose of a carrier cisplatinum, which represents less than 10% of a typical chemotherapeutic dose given to patients. A good manufacturing practice GMP compliant product was produced and was administered to 10 healthy volunteers as part of an ethically approved Phase 0 clinical trial. The majority of the injected activity 27.5% ± 5.8% was excreted in the urine within 5 h post injection (p.i.). Only 8.5% ± 3.1% of cisplatinum remained in blood pools at 5 h, which gradually cleared over the 6-day monitoring period p.i. At the end of the study (6 days p.i.), a total of 37.4% ± 5.3% of the product had cleared from the blood into urine, and approximately 63% remained in the body. The significantly lower concentration of carrier cisplatinum used for imaging resulted in a well-tolerated product.

Synthesis of hexa aza cages, SarAr-NCS and AmBaSar and a study of their metal complexation, conjugation to nanomaterials and proteins for application in radioimaging and therapy.

A novel hexa aza cage, N(1)-(4-isothiocyanatobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (SarAr-NCS) was synthesized in good yield and characterized by (1)H NMR and electrospray mass spectrometry. A new method for the synthesis of the related N(1)-(4-carboxybenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (AmBaSar) using the p-carboxybenzaldehyde is reported. The complexation of Cu(2+), Co(2+) and Zn(2+) by the two ligands over a range of pHs was found to be similar to the parent derivative SarAr. SarAr-NCS was conjugated to both silica particles (≈90 nm diam.) and the model B72.3 murine antibody. The SarAr-NCSN-silica particles were radiolabeled with Cu(2+) doped (64)Cu and the number of ligands conjugated was calculated to be an average of 7020 ligands per particle. Conjugation of SarAr-NCS to the B72.3 antibody was optimized over a range of conditions. The SarAr-NCSN-B72.3 conjugate was stored in buffer and as a lyophilized powder at 4 °C over 38 days. Its radiolabeling efficiency, stability and immunoreactivity were maintained. The development of a high yielding synthesis of SarAr-NCS should provide an entry point for a wide range of Cu and Zn radiometal PET imaging agents and potentially radiotherapeutic agents with (67)Cu.

Optimizing radiolabeling amine-functionalized silica nanoparticles using SarAr-NCS for applications in imaging and radiotherapy.

Silica nanoparticles functionalized with amine groups and in the size range of approximately 60-94 nm were produced by combining sol-gel processing and emulsion technology. Hexa-aza cage ligand SarAr-NCS was conjugated to the silica nanoparticles and subsequently radiolabeled with a solution of (57)Co(2+)-doped carrier Co(2+). The number of Co(2+) ions bound to the silica particles at pH 7 was used to determine the average number of available SarAr-NCS ligands conjugated to a silica particle. For organically modified silica particles of 94.0 and 59.5 nm diameter, the maximum number of metal binding sites was determined to be 11700 and 3270 sites per particle, respectively. For silica particles (63.5 nm peak diameter) produced using an water-in-oil emulsion, the calculated average was 4480 on the particle surface. The number of SarAr-NCS conjugated on the particles was easily controlled, potentially providing for a range of products for applications in the risk assessment of particles and theranostic imaging or radiotherapy when radiolabeled with a suitable radioisotope such as (64)Cu or (67)Cu.

Investigating the binding properties of porous drug delivery systems using nuclear sensors (radiotracers) and positron annihilation lifetime spectroscopy--predicting conditions for optimum performance.

Understanding how the size, charge and number of available pores in porous material influences the uptake and release properties is important for optimising their design and ultimately their application. Unfortunately there are no standard methods for screening porous materials in solution and therefore formulations must be developed for each encapsulated agent. This study investigates the potential of a library of radiotracers (nuclear sensors) for assessing the binding properties of hollow silica shell materials. Uptake and release of Cu(2+) and Co(2+) and their respective complexes with polyazacarboxylate macrocycles (dota and teta) and a series of hexa aza cages (diamsar, sarar and bis-(p-aminobenzyl)-diamsar) from the hollow silica shells was monitored using their radioisotopic analogues. Coordination chemistry of the metal (M) species, subtle alterations in the molecular architecture of ligands (Ligand) and their resultant complexes (M-Ligand) were found to significantly influence their uptake over pH 3 to 9 at room temperature. Positively charged species were selectively and rapidly (within 10 min) absorbed at pH 7 to 9. Negatively charged species were preferentially absorbed at low pH (3 to 5). Rates of release varied for each nuclear sensor, and time to establish equilibrium varied from minutes to days. The subtle changes in design of the nuclear sensors proved to be a valuable tool for determining the binding properties of porous materials. The data support the development of a library of nuclear sensors for screening porous materials for use in optimising the design of porous materials and the potential of nuclear sensors for high through-put screening of materials.

Imaging cancer using PET--the effect of the bifunctional chelator on the biodistribution of a (64)Cu-labeled antibody.

INTRODUCTION: Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the (64)Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with (64)Cu using these chelators in tumor-bearing mice. METHODS: The chelators [S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH(2)-Bn-NOTA): 6-[p-(bromoacetamido)benzyl]-1, 4, 8, 11-tetraazacyclotetradecane-N, N', N'', N'''-tetraacetic acid (BAT-6): S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododocane tetraacetic acid (p-NH(2)-Bn-DOTA): 1,4,7,10-tetraazacyclododocane-N, N', N", N"'-tetraacetic acid (DOTA): and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr)] were conjugated to the anti-GD2 antibody ch14.18, and the modified antibody was labeled with (64)Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from positron emission tomography images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. RESULTS: The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p < 0.05) were between blood, liver, spleen and kidney. For example, liver uptake of [(64)Cu]ch14.18-p-NH(2)-Bn-NOTA was 4.74 ± 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [(64)Cu]ch14.18-SarAr was 8.06 ± 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. CONCLUSIONS: The results of this study indicate that differences in the thermodynamic stability of these chelator-Cu(II) complexes were not associated with significant differences in uptake of the tracer by the tumor. However, there were significant differences in tracer concentration in other tissues, including those involved in clearance of the radioimmunoconjugate (e.g., liver and spleen).

Investigation of novel bis- and tris-tetraazamacrocycles for use in the copper-64 ((64)Cu) radiolabeling of antibodies with potential to increase the therapeutic index for drug targeting.

The (64)Cu complexes of a series of mono-, bis-, and tris-tetraazamacrocycles have been prepared, and their stability in human sera has been assessed. The ligands forming the most stable Cu(2+) complexes were then conjugated to the B72.3 antibody (mAb). Conditions for conjugation of the ligands to the mAb were optimized for the concentration of protein, ligand, pH, temperature, and time. The optimum moles of Cu(2+) attached to the mAb were as high as 3.5 for L2 and 5.5 or 2.7 for L5, and the immunoreactivity was > or =80%. Biodistribution of the radioimmunoconjugates showed good tumor localization and target-to-background ratios that were significantly enhanced compared to those achieved with monotetraazamacrocyclic derivatives.

Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates.

The advancement of positron emission tomography (PET) depends on the development of new radiotracers that will complement (18)F-FDG. Copper-64 ((64)Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging, but the high in vivo lability of conventional chelates has limited its clinical application. The objective of this work was to evaluate the novel chelating agent SarAr (1-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane-1,8-diamine) for use in developing a new class of tumor-specific (64)Cu radiopharmaceuticals for imaging neuroblastoma and melanoma. The anti-GD2 monoclonal antibody (mAb) 14.G2a, and its chimeric derivative, ch14.18, target disialogangliosides that are overexpressed on neuroblastoma and melanoma. Both mAbs were conjugated to SarAr using carbodiimide coupling. Radiolabeling with (64)Cu resulted in >95% of the (64)Cu being chelated by the immunoconjugate. Specific activities of at least 10 microCi/microg (1 Ci = 37 GBq) were routinely achieved, and no additional purification was required after (64)Cu labeling. Solid-phase radioimmunoassays and intact cell-binding assays confirmed retention of bioactivity. Biodistribution studies in athymic nude mice bearing s.c. neuroblastoma (IMR-6, NMB-7) and melanoma (M21) xenografts showed that 15-20% of the injected dose per gram accumulated in the tumor at 24 hours after injection, and only 5-10% of the injected dose accumulated in the liver, a lower value than typically seen with other chelators. Uptake by a GD2-negative tumor xenograft was significantly lower (<5% injected dose per gram). MicroPET imaging confirmed significant uptake of the tracer in GD-2-positive tumors, with minimal uptake in GD-2-negative tumors and nontarget tissues such as liver. The (64)Cu-SarAr-mAb system described here is potentially applicable to (64)Cu-PET imaging with a broad range of antibody or peptide-based imaging agents.

Molecular imaging with copper-64 in the drug discovery and development arena.

The contribution of positron emission tomography (PET) to the drug discovery and development (D3) pipeline has been inhibited by the short half-lifes of PET radioisotopes, (11)C and (18)F, poor availability and the high cost of infrastructure. Copper-64 ((64)Cu) has a 12.7 h half-life, simple yet flexible radiochemistry and imaging characteristics that make it ideal for a wider application across the D3 arena. Recent scientific breakthroughs in the production of (64)Cu show that it's, commercial production can be made more widely available. More importantly, for pharmaceutical research and development programmes wishing to incorporate the high sensitivity and spatial resolution of PET, but no desire to implement and maintain expensive radiochemistry infrastructure, (64)Cu is an exciting option.

New 64Cu PET imaging agents for personalised medicine and drug development using the hexa-aza cage, SarAr.

The success of positron emission tomography (PET) in personalised medicine and drug development requires radioisotopes that provide high quality images and flexible chemistry for a broad application. 64Cu is arguably one of the most suitable PET isotopes for imaging with the evolving target agents, but there are not many appropriate chelating agents for 64Cu and this has limited its wider application. The bi-functional chelator, SarAr is known to bind 64Cu2+ quantitatively (i.e. one metal per ligand present) and rapidly (<2 min) at 10(-6) M over a range of pH (4-9). In this paper the conjugation of SarAr to the whole and fragmented antibody is described. Conjugation of the SarAr to the protein does not impair its coordination of the 64Cu. It complexes the 64Cu2+ rapidly, quantitatively and essentially irreversibly at pH 5. Animal studies show that the 64Cu-SarAr-immunoconjugates maintain their specificity for the target and are stable in vivo. Also, SarAr is a platform technology, is easy to use in a kit formulation and is readily adaptable for the wider application in 64Cu PET imaging.

Challenges and opportunities for positron-emission tomography in personalized medicine.

Of the non-invasive functional imaging tools available, positron-emission tomography (PET) is generally expected to have the greatest potential for delivering the vision of personalized medicine. This can be achieved by the production of designer PET probes with exquisite sensitivity that can profile key biological processes that are specific to a disease. However, the challenge for the PET field will be its capability to produce and supply cost-effective PET probes to the wider community. This will most likely be achieved through the provision of long-lived PET radioisotopes with imaging qualities that match the performance of the evolving PET camera technology and chemistry that is amenable to kit formulations. Additionally, of the emerging PET radiotracers, (64)Cu has the optimum chemistry and emission characteristics for the wider application of PET in personalized medicine.

Technology evaluation: huN901-DM1, ImmunoGen.

ImmunoGen is developing huN901-DMI, a compound comprised of a CD56-targeted humanized N901 antibody conjugated to the company's proprietary cytotoxic agent. DM1, using its tumor-activated prodrug technology, for the potential treatment of cancers that express CD56, in particular, small-cell lung cancer.

Molecular imaging with copper-64.

Molecular imaging is expected to change the face of drug discovery and development. The ability to link imaging to biology for guiding therapy should improve the rate at which novel imaging technologies, probes, contrast agents, drugs and drug delivery systems can be transferred into clinical practice. Nuclear medicine imaging, in particular, positron emission tomography (PET) allows the detection and monitoring of a variety of biological and pathophysiological processes, at tracer quantities of the radiolabelled target agents, and at doses free from pharmacological effects. In the field of drug discovery and development, the use of radiotracers for radiolabelling target agents has now become one of the essential tools in identifying, screening and development of new target agents. In this regard, (64)Cu (t(1/2)=12.7 h) has been identified as an emerging PET isotope. Its half-life is sufficiently long for radiolabelling a range of target agents and its ease of production and adaptable chemistry make it an excellent radioisotope for use in molecular imaging. This review describes recent advances, in the routes of (64)Cu production, design and application of bi-functional ligands for use in radiolabelling with (64/67)Cu(2+), and their significance and anticipated impact on the field of molecular imaging and drug development.

Technology evaluation: cantuzumab mertansine, ImmunoGen.

ImmunoGen is developing cantuzumab mertansine, in which the CanAg antigen-targeted humanized antibody C242 is conjugated to the company's proprietary cytotoxic agent, DM1, using ImmunoGen's tumor-activated prodrug technology. Cantuzumab mertansine is undergoing phase II trials for the potential treatment of CanAg-expressing cancers, including pancreatic and colorectal cancers and non-small-cell lung cancer.

Insights into the mechanisms of copper tolerance of a population of black-banded rainbowfish (Melanotaenia nigrans) (Richardson) exposed to mine leachate, using 64/67Cu.

This study investigates the mechanisms of copper tolerance of a population of black-banded rainbowfish (Melanotaenia nigrans) (Richardson). The population has been exposed to elevated copper concentrations for over 40 years, due to leachate from the Rum Jungle uranium/copper mine. At the time of collection the 96 h EC(50) of exposed [E] fish was 8.3 times higher than that of reference [R] fish. The bioconcentration of 64/67Cu in fish was used to investigate the mechanism of copper tolerance in E fish. Both E and R fish were exposed to low (L(Cu), 30 microg Cu l(-1)) and elevated (E(Cu), 300 microg Cu l(-1)) copper concentrations for 24 and 48 h, respectively. Radioactivity was measured at seven or eight time points in four tissue sections: head (including gills, heart and brain), internal organs (including gastrointestinal tract, liver, kidneys and gonads), muscle and whole body. One-compartment bioconcentration models were fit to data and compared using an F-test. Copper concentrations in all tissue sections were significantly (P<0.05) less (up to 50%) in E fish compared with the respective tissue sections of R fish when exposed to both L(Cu) and E(Cu). The exception was copper accumulation in the internal organs, which was not significantly different between E and R fish exposed to E(Cu). The mechanism of copper tolerance was concluded to be reduced copper uptake in the gills, rather than increased binding or elimination. Allozyme electrophoresis was performed to determine if genetic selection had occurred in the E fish population. Allozyme frequencies at the AAT-1 and GPI-1 loci were significantly (P<0.05) different between E and R fish. Heterozygosity was reduced in E fish compared with that of R fish. Collectively these results suggest that genetic selection may have occurred in the E fish population. Consequently, the selection of allozymes less sensitive to copper may be another mechanism of copper tolerance of E fish. This is the first study on the mechanisms of copper tolerance in a wild fish population that has been exposed to elevated copper concentrations. These findings aid the understanding of metal tolerance in fish and emphasise the importance of sample selection and its implication for toxicity testing.