Vojtěch Kubíček

Gallium(III) complexes of DOTA and DOTA-monoamide: kinetic and thermodynamic studies.

Given the practical advantages of the (68)Ga isotope in positron emission tomography applications, gallium complexes are gaining increasing importance in biomedical imaging. However, the strong tendency of Ga(3+) to hydrolyze and the slow formation and very high stability of macrocyclic complexes altogether render Ga(3+) coordination chemistry difficult and explain why stability and kinetic data on Ga(3+) complexes are rather scarce. Here we report solution and solid-state studies of Ga(3+) complexes formed with the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA)(4-), and its mono(n-butylamide) derivative, (DO3AM(Bu))(3-). Thermodynamic stability constants, log K(GaDOTA) = 26.05 and log K(GaDO3AM(Bu)) = 24.64, were determined by out-of-cell pH-potentiometric titrations. Due to the very slow formation and dissociation of the complexes, equilibration times of up to ∼4 weeks were necessary. The kinetics of complex dissociation were followed by (71)Ga NMR under both acidic and alkaline conditions. The GaDOTA complex is significantly more inert (τ(1/2) ∼12.2 d at pH = 0 and τ(1/2) ∼6.2 h at pH = 10) than the GaDO3AM(Bu) analogue (τ(1/2) ∼2.7 d at pH = 0 and τ(1/2) ∼0.7 h at pH = 10). Nevertheless, the kinetic inertness of both chelates is extremely high and approves the application of Ga(3+) complexes of such DOTA-like ligands in molecular imaging. The solid-state structure of the GaDOTA complex, crystallized from a strongly acidic solution (pH < 1), evidenced a diprotonated form with protons localized on the free carboxylate pendants.

PET/CT imaging of osteoblastic bone metastases with 68Ga-bisphosphonates: first human study

Bisphosphonates are well established ligands for Tc for planar and SPECT/CT imaging of osteoblastic metastases. A novelDOTA-based bisphosphonate, (4-{[bis-(phosphonomethyl)) carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid (BPAMD) [1, 2], was labelled using the Ge/Ga generator-derived positron emitter Ga [3], resulting in the PET tracer [Ga]BPAMD. [Ga]BPAMD was injected i.v. into a patient with known extensive bone metastases of prostate cancer. [Ga]BPAMD [maximum intensity projection (MIP) 50 min post-injection (p.i.), 462 MBq] revealed intense accumulation in multiple osteoblastic lesions in the central skeleton, ribs and proximal extremities: a = coronal PET, b = sagittal PET/CT. For comparison, c shows F-fluoride PET (sagittal, MIP 90 min p.i., 270 MBq). Metastases were detected in the whole skeleton with a maximal standardized uptake value (SUVmax) of 77.1 and 62.1 in the 10th thoracic and L2 vertebra vs 39.1 and 39.2 for F-fluoride, respectively. The advantages of this new bone imaging PET tracer are the very high target to soft tissue ratios and fast clearance, its ease of use and the generator-based availability of Ga which becomes especially important in these days of Tc shortage. While F-fluoride is adsorbed on bone surface and is related to blood flow, the bisphosphonate [Ga]BPAMD is taken up also by osteoclasts reflecting the farnesyl diphosphate synthase enzyme dynamics in the HMG-CoA reductase pathway. Since this pathway is mainly responsible for the osteoclastic bone destruction, [Ga]BPAMD may be superior in osteoclastic lesions. Finally, [Ga]BPAMD seems to be an ideal PET/CT tracer to plan and monitor bisphosphonate therapy in several bone disorders like osteoporosis, osteitis deformans, bone metastases, multiple myeloma, osteogenesis imperfecta, etc. and also to monitor radionuclide therapy for palliation of bone pain.

Lanthanide(III) Complexes of Bis(phosphonate) Monoamide Analogues of DOTA : Bone-Seeking Agents for Imaging and Therapy

Lanthanide complexes of DOTA derivatives 2a (BPAMD) and 2b (BPAPD), having a monoamide pendant arm with a bis(phosphonate) moiety, were comparatively tested for application in MRI, radiotherapy, and bone pain palliation. (1)H, (31)P, and (17)O NMR spectroscopy show that they are nine-coordinated, with one water molecule in the first coordination sphere of the Ln(III) ion. The bis(phosphonate) moieties are not coordinated to the lanthanide and predominantly mono- and diprotonated at physiological pH. The parameters governing the longitudinal relaxivities of the Gd complexes are similar to those of other monoamides of DOTA reported in the literature. Upon adsorption on hydroxyapatite, the relaxivities at 20 MHz and 25 degrees C of Gd-2a and Gd-2b were 22.1 and 11 s(-1) mM(-1), respectively. An in vivo gamma-ray imaging study showed that the (177)Lu complexes of 2a and 2b have a high affinity for bones, particularly for growth plates and teeth with a prolonged retention.

Dissociation kinetics of Mn2+ complexes of NOTA and DOTA

The kinetics of transmetallation of [Mn(nota)](-) and [Mn(dota)](2-) was investigated in the presence of Zn(2+) (5-50-fold excess) at variable pH (3.5-5.6) by (1)H relaxometry. The dissociation is much faster for [Mn(nota)](-) than for [Mn(dota)](2-) under both experimental and physiologically relevant conditions (t(½) = 74 h and 1037 h for [Mn(nota)](-) and [Mn(dota)](2-), respectively, at pH 7.4, c(Zn(2+)) = 10(-5) M, 25 °C). The dissociation of the complexes proceeds mainly via spontaneous ([Mn(nota)](-)k(0) = (2.6 ± 0.5) × 10(-6) s(-1); [Mn(dota)](2-)k(0) = (1.8 ± 0.6) × 10(-7) s(-1)) and proton-assisted pathways ([Mn(nota)](-)k(1) = (7.8 ± 0.1) × 10(-1) M(-1) s(-1); [Mn(dota)](2-)k(1) = (4.0 ± 0.6) × 10(-2) M(-1) s(-1), k(2) = (1.6 ± 0.1) × 10(3) M(-2) s(-1)). The observed suppression of the reaction rates with increasing Zn(2+) concentration is explained by the formation of a dinuclear Mn(2+)-L-Zn(2+) complex which is about 20-times more stable for [Mn(dota)](2-) than for [Mn(nota)](-) (K(MnLZn) = 68 and 3.6, respectively), and which dissociates very slowly (k(3)∼10(-5) M(-1) s(-1)). These data provide the first experimental proof that not all Mn(2+) complexes are kinetically labile. The absence of coordinated water makes both [Mn(nota)](-) and [Mn(dota)](2-) complexes inefficient for MRI applications. Nevertheless, the higher kinetic inertness of [Mn(dota)](2-) indicates a promising direction in designing ligands for Mn(2+) complexation.

Design and function of metal complexes as contrast agents in MRI

Publisher Summary This chapter discusses mechanisms that relate the relaxivity of the free gadolinium ion (GdIII) complexes to the microscopic parameters of the chelate and the main streams of efforts to increase longitudinal relaxivity for GdIII complexes involving probes optimized for imaging at high magnetic fields. In addition to high relaxivity, a potential contrast agent has to fulfill several additional requirements related to safety, non-toxicity, osmolality, biodistribution, and elimination. The physiological toxicity of a LnIII poly(aminocarboxylate) complex depends mainly on the release of free LnIII, controlled by its thermodynamic stability and kinetic inertness. The chapter discusses these aspects and the new-generation MRI probes, discussing representative examples of responsive agents. It also describes paramagnetic chemical exchange saturation (PARACEST) agents and reviews the recent results on MnII complexes in the context of MRI. The chapter concludes with an overview on non-classical potential contrast agents such as gadofullerenes and carbon nanotubes.

68Ga-BPAMD: PET-imaging of bone metastases with a generator based positron emitter

PURPOSE Bone metastases are a serious aggravation for patients suffering from cancer. Therefore, early recognition of bone metastases is of great interest for further treatment of patients. Bisphosphonates are widely used for scintigraphy of bone lesions with (99m)Tc. Using the (68)Ge/(68)Ga generator together with a macroyclic bisphosphonate a comparable PET-tracer comes into focus. PROCEDURES The bisphosphonate DOTA-conjugated ligand BPAMD was labelled with (68)Ga. [(68)Ga]BPAMD was evaluated in vitro concerning binding to hydroxyapatite and stability. The tracers in vivo accumulation was determined on healthy rats and bone metastases bearing animals by μ-PET. RESULTS BPAMD was labelled efficiently with (68)Ga after 10 min at 100°C. [(68)Ga]BPAMD showed high in vitro stability within 3h and high binding to hydroxyapatite. Consequently, μ-PET experiments revealed high accumulation of [(68)Ga]BPAMD in regions of pronounced remodelling activity like bone metastases. CONCLUSIONS (68)Ga BPAMD reveals great potential for diagnosis of bone metastases via PET/CT. The straight forward (68)Ga-labelling could be transferred to a kit-preparation of a cyclotron-independent PET tracer instantaneously available in many clinical sites using the (68)Ge/(68)Ga generator.

Lanthanide(III) Complexes of 4,10‐Bis(phosphonomethyl)‐1,4,7,10‐tetraazacyclododecane‐1,7‐diacetic acid (trans‐H6do2a2p) in Solution and in the Solid State: Structural Studies Along the Series

Complexes of 4,10-bis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (trans-H(6)do2a2p, H(6)L) with transition metal and lanthanide(III) ions were investigated. The stability constant values of the divalent and trivalent metal-ion complexes are between the corresponding values of H(4)dota and H(8)dotp complexes, as a consequence of the ligand basicity. The solid-state structures of the ligand and of nine lanthanide(III) complexes were determined by X-ray diffraction. All the complexes are present as twisted-square-antiprismatic isomers and their structures can be divided into two series. The first one involves nona-coordinated complexes of the large lanthanide(III) ions (Ce, Nd, Sm) with a coordinated water molecule. In the series of Sm, Eu, Tb, Dy, Er, Yb, the complexes are octa-coordinated only by the ligand donor atoms and their coordination cages are more irregular. The formation kinetics and the acid-assisted dissociation of several Ln(III)-H(6)L complexes were investigated at different temperatures and compared with analogous data for complexes of other dota-like ligands. The [Ce(L)(H(2)O)](3-) complex is the most kinetically inert among complexes of the investigated lanthanide(III) ions (Ce, Eu, Gd, Yb). Among mixed phosphonate-acetate dota analogues, kinetic inertness of the cerium(III) complexes is increased with a higher number of phosphonate arms in the ligand, whereas the opposite is true for europium(III) complexes. According to the (1)H NMR spectroscopic pseudo-contact shifts for the Ce-Eu and Tb-Yb series, the solution structures of the complexes reflect the structures of the [Ce(HL)(H(2)O)](2-) and [Yb(HL)](2-) anions, respectively, found in the solid state. However, these solution NMR spectroscopic studies showed that there is no unambiguous relation between (31)P/(1)H lanthanide-induced shift (LIS) values and coordination of water in the complexes; the values rather express a relative position of the central ions between the N(4) and O(4) planes.

PiB-Conjugated, Metal-Based Imaging Probes: Multimodal Approaches for the Visualization of β-Amyloid Plaques.

In an effort toward the visualization of β-amyloid plaques by in vivo imaging techniques, we have conjugated an optimized derivative of the Pittsburgh compound B (PiB), a well-established marker of Aβ plaques, to DO3A-monoamide that is capable of forming stable, noncharged complexes with different trivalent metal ions including Gd(3+) for MRI and (111)In(3+) for SPECT applications. Proton relaxivity measurements evidenced binding of Gd(DO3A-PiB) to the amyloid peptide Aβ1-40 and to human serum albumin, resulting in a two- and four-fold relaxivity increase, respectively. Ex vivo immunohistochemical studies showed that the DO3A-PiB complexes selectively target Aβ plaques on Alzheimers disease human brain tissue. Ex vivo biodistribution data obtained for the (111)In-analogue pointed to a moderate blood-brain barrier (BBB) penetration in adult male Swiss mice (without amyloid deposits) with 0.36% ID/g in the cortex at 2 min postinjection.

Gd(III) complex of a monophosphinate-bis(phosphonate) DOTA analogue with a high relaxivity; Lanthanide(III) complexes for imaging and radiotherapy of calcified tissues

A new phosphinic-acid DOTA-like ligand, DO3AP(BP), containing a geminal bis(phosphonic acid) moiety as a highly effective bone-seeking group, was synthesized in high yield. Its crystal structure was determined by X-ray analysis. Complexation with lanthanide(iii) ions occurs under mild conditions (pH = 8-9, 25 degrees C, 2-3 h). (1)H, (31)P, and (17)O NMR spectroscopy show that DO3AP(BP) forms nine-coordinated lanthanide(iii) complexes with one water molecule in the first coordination sphere except for Ln = Er-Lu, which have in addition a species without lanthanide(iii)-bound water. Selective formation of only two diastereomers (out of four possible) suggests that the coordinated phosphinate phosphorus atom occurs exclusively in one of the enantiomeric forms. The ratio of the twisted square antiprism (TSA) and square antiprism (SA) diastereomers changes along the lanthanide series; the gadolinium(iii) complex has about 35% of the TSA species. The bis(phosphonate) moiety remains free for anchoring to osseous tissue. The (1)H longitudinal relaxivity of the Gd-DO3AP(BP) complex (r(1) = 7.4 s(-1) mM(-1), 20 MHz, 25 degrees C, pH = 7.5) is unexpectedly high compared to that of other monohydrated chelates of similar size thanks to a significant contribution from the second hydration sphere. The water residence time tau(M)(298) is 198 ns. Further increase in the relaxivity was observed in the presence of Zn(ii), Mg(ii) or Ca(ii) ions, due to formation of coordination polymers. Slowing down of the tumbling rate of the Gd-DO3AP(BP) complex upon adsorption on hydroxyapatite also leads to an increase of the relaxivity (r(1) = 17 s(-1) mM(-1), 20 MHz, 25 degrees C, pH = 7.5).

Phosphonate-titanium dioxide assemblies: platform for multimodal diagnostic-therapeutic nanoprobes.

Multimodal imaging-therapeutic nanoprobe TiO(2)@RhdGd was prepared and successfully used for in vitro and in vivo cell tracking as well as for killing of cancer cells in vitro. TiO(2) nanoparticles were used as a core for phosphonic acid modified functionalities, responsible for contrast in MRI and optical imaging. The probe shows high (1)H relaxivity and relaxivity density values. Presence of fluorescent dye allows for visualization by means of fluorescence microscopy. The applicability of the probe was studied, using mesenchymal stem cells, cancer HeLa cells, and T-lymphocytes. The probe did not exhibit toxicity in any of these systems. Labeled cells were successfully visualized in vitro by means of fluorescence microscopy and MRI. Furthermore, it was shown that the probe TiO(2)@RhdGd can be changed into a cancer cell killer upon UV light irradiation. The above stated results represent a valuable proof of a principle showing applicability of the probe design for diagnosis and therapy.