I Mamedov

Smart Magnetic Resonance Imaging Agents that Sense Extracellular Calcium Fluctuations

Image brain function: Gd3+ chelates linked to a modified EGTA moiety were prepared in order to respond to extracellular Ca2+ fluctuations in the brain. Upon interaction with Ca2+, they exhibit high and reversible relaxivity changes in buffered solution or in a model of the brain extracellular medium. These efficient Ca2+ magnetic resonance imaging sensors might open new perspectives in functional molecular imaging.

Synthesis and characterization of dinuclear heterometallic lanthanide complexes exhibiting MRI and luminescence response

A molecule bearing a macrocyclic DOTA-type chelator and an acyclic chelator based on the 5-aminoisophthalamide diethylenediaminetetraacid (5A-PADDTA) was synthesized by linking these two moieties via an amide bond. The ligand has the possibility to complex two identical or different lanthanide ions, depending on the desire for its potential application. Luminescence studies involving titrations of the Eu(3+) or Gd(3+) complex with Tb(3+) confirm the formation of heterometallic complexes, as well as the presence of different species in the solution. Comparative (1)H NMR spectra of the ligand, its Eu(3+) complex, and that containing both Eu(3+) and Tb(3+) proves the existence of respective monometallic or bimetallic species. NMR diffusion measurements on 5A-PADDTA as a model compound indicate the formation of aggregates upon the addition of Y(3+) (chosen as a diamagnetic analogue of lanthanide ions). Hydration values were calculated from the respective luminescence lifetime values. They show the dominance of a q = 1 species for both ions in monometallic complexes, or q = 1 and q = 2 species of ions in aggregated complexes, for DOTA and 5A-PADDTA chelators, respectively.

A smart 19F and 1H MRI probe with self-immolative linker as a versatile tool for detection of enzymes

Here we report on a dual-modal (19) F and (1) H MRI paramagnetic probe with a self-immolative linker, Gd-DOMF-Gal. The enzymatic conversion of this probe by β-galactosidase resulted in a simultaneous turning on of the fluorine signal and changed ability of the Gd(3+) complex to modulate the (1) H MR signal intensity of the surrounding water molecules. A versatile imaging platform for monitoring a variety of enzymes by (19) F and (1) H MRI using this molecular design is proposed.

In vivo characterization of a smart MRI agent that displays an inverse response to calcium concentration

Contrast agents for magnetic resonance imaging (MRI) that exhibit sensitivity toward specific ions or molecules represent a challenging but attractive direction of research. Here a Gd(3+) complex linked to an aminobis(methylenephosphonate) group for chelating Ca(2+) was synthesized and investigated. The longitudinal relaxivity (r(1)) of this complex decreases during the relaxometric titration with Ca(2+) from 5.76 to 3.57 mM(-1) s(-1) upon saturation. The r(1) is modulated by changes in the hydration number, which was confirmed by determination of the luminescence emission lifetimes of the analogous Eu(3+) complex. The initial in vivo characterization of this responsive contrast agent was performed by means of electrophysiology and MRI experiments. The investigated complex is fully biocompatible, having no observable effect on neuronal function after administration into the brain ventricles or parenchyma. Distribution studies demonstrated that the diffusivity of this agent is significantly lower compared with that of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).

Influence of Calcium-Induced Aggregation on the Sensitivity of Aminobis(methylenephosphonate)-Containing Potential MRI Contrast Agents

A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca(2+) was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca(2+)-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L(1), L(2), L(3), and L(4), respectively). Upon addition of Ca(2+), the r(1) relaxivity of their Gd(3+) complexes decreased up to 61% of the initial value for the best compounds GdL(3) and GdL(4). The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y(3+) analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca(2+). (31)P NMR experiments on EuL(1) and EuL(4) indicated the coordination of a phosphonate to the Ln(3+) for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd(3+) complexes. log K(H1) values for all complexes lie between 6.12 and 7.11 whereas log K(H2) values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu(3+) complexes confirmed the coordination of a phosphonate group to the Ln(3+) center in EuL(1). Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r(1). Variable temperature (17)O NMR experiments evidenced high water exchange rates on GdL(1), GdL(2), and GdL(3) comparable to that of the aqua ion.

Diffusion properties of conventional and calcium-sensitive MRI contrast agents in the rat cerebral cortex

Calcium-sensitive MRI contrast agents can only yield quantitative results if the agent concentration in the tissue is known. The agent concentration could be determined by diffusion modeling, if relevant parameters were available. We have established an MRI-based method capable of determining diffusion properties of conventional and calcium-sensitive agents. Simulations and experiments demonstrate that the method is applicable both for conventional contrast agents with a fixed relaxivity value and for calcium-sensitive contrast agents. The full pharmacokinetic time-course of gadolinium concentration estimates was observed by MRI before, during and after intracerebral administration of the agent, and the effective diffusion coefficient D* was determined by voxel-wise fitting of the solution to the diffusion equation. The method yielded whole brain coverage with a high spatial and temporal sampling. The use of two types of MRI sequences for sampling of the diffusion time courses was investigated: Look-Locker-based quantitative T(1) mapping, and T(1) -weighted MRI. The observation times of the proposed MRI method is long (up to 20 h) and consequently the diffusion distances covered are also long (2-4 mm). Despite this difference, the D* values in vivo were in agreement with previous findings using optical measurement techniques, based on observation times of a few minutes. The effective diffusion coefficient determined for the calcium-sensitive contrast agents may be used to determine local tissue concentrations and to design infusion protocols that maintain the agent concentration at a steady state, thereby enabling quantitative sensing of the local calcium concentration.

Multimodal contrast agents for in vivo neuroanatomical analysis of monosynaptic connections

We developed and examined the applicability of two multimodal paramagnetic contrast agents for the longitudinal in vivo investigations of the brain projections. The classical dextran based neuroanatomical tracer was conjugated with mono- and bimetal Gd(3+) complexes and an optical reporter. Relaxometric studies of both tracer molecules were performed in vitro followed by in cellulo MR and microscopy investigations. Finally, tracers were injected into the motor cortex of the rat brain; uptake and transporting properties were compared by MRI. The advantage of the multimodal approach was taken and histological studies were performed on the same animals. The histology results confirm the MRI studies demonstrating that the applied tracers labelled anterogradely the regions known for their connections with the motor cortex of the rat brain.

Development of multimodal imaging probes for neuroanatomical connectivity studies in vivo

The Phosphatidylinositide 3-kinase (PI3-K) pathway is deregulated in a range of cancers, and several inhibitors of this enzyme are entering the clinic. A current problem for such targeted therapeutics in drug discovery pipelines is the lack of biomarkers for use in clinical trials, to allow the assessment of early therapeutic response and thus better patient management. Here, we assessed uptake of the PET tracer [18F]-Fluorodeoxythymidine (FLT) in response to reduction of PI3-K signalling mediated by the novel PI-3K inhibitor GDC-0941 in both ectopic and orthotopic xenograft models. Methods Nude mice bearing U87 and HCT116 xenografts were imaged for 105 minutes dynamically with [18F]-FLT at baseline (when tumours were~200mm3) and after acute (18h) or chronic (186h) treatment with 50mg/kg GDC-0941 or vehicle twice daily by gavage. An orthotopic model consisting of HCT116 liver metastases obtained after primary inoculation of cells into the spleen was also imaged at baseline and after acute/chronic GDC-0941 therapy. Regions of Interest (ROIs) were drawn manually over tumours/metastases and maximum standard uptake value (SUV)s for tumour uptake were calculated, as was uptake normalised to mean activity in blood pool as estimated by an ROI drawn on the heart contents (NUV). Tumour tissue was analysed at sacrifice for expression of TK1, cleaved PARP and phospho-AKT. Results Growth of U87 xenografts was significantly inhibited by GDC-0941 treatment throughout the course of the study (average growth rate of treated = -1.0±9.3 mm3, control = 69.2±22.1mm3 per day, p<0.05). Growth of HCT116 xenografts was not significantly different between groups (average growth rate of treated = 37.3±8.4 mm3, control =46.2±11.9 mm3 per day); however growth of HCT116 liver metastases was significantly reduced (Liver:bodyweight ratio of 0.11±0.01 vs 0.06±0.01, p<0.01). Uptake of [18F]-FLT was reduced in GDC-0941 treated animals bearing U87 xenografts or HCT116 liver metastases at the acute timepoint compared to baseline (NUVmax of 2.17±0.38 vs 1.59±0.29, p<0.01; 2.42±0.19,1.8±0.12 p<0.05 respectively), but was unchanged in unresponsive HCT116 xenografts. Conclusion [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. In addition to FLT, we are assessing the performance of a range of other imaging agents, including proprietary tracers, that can potentially bring value in monitoring of PI3-kinase targeting therapies. Disclosure of author financial interest or relationships: C. Cawthorne, GE Healthcare, Grant/research support; I. Wilson, GE Healthcare Medical Diagnostics, Employment; N. Burrows, None; R. Gieling, None; J. Gregory, None; A.M. Smigova, None; M. Babur, None; K. Williams, GE, Grant/research support . S1286

Dual 1H/19F MR imaging approach for detection of enzyme activity

The Phosphatidylinositide 3-kinase (PI3-K) pathway is deregulated in a range of cancers, and several inhibitors of this enzyme are entering the clinic. A current problem for such targeted therapeutics in drug discovery pipelines is the lack of biomarkers for use in clinical trials, to allow the assessment of early therapeutic response and thus better patient management. Here, we assessed uptake of the PET tracer [18F]-Fluorodeoxythymidine (FLT) in response to reduction of PI3-K signalling mediated by the novel PI-3K inhibitor GDC-0941 in both ectopic and orthotopic xenograft models. Methods Nude mice bearing U87 and HCT116 xenografts were imaged for 105 minutes dynamically with [18F]-FLT at baseline (when tumours were~200mm3) and after acute (18h) or chronic (186h) treatment with 50mg/kg GDC-0941 or vehicle twice daily by gavage. An orthotopic model consisting of HCT116 liver metastases obtained after primary inoculation of cells into the spleen was also imaged at baseline and after acute/chronic GDC-0941 therapy. Regions of Interest (ROIs) were drawn manually over tumours/metastases and maximum standard uptake value (SUV)s for tumour uptake were calculated, as was uptake normalised to mean activity in blood pool as estimated by an ROI drawn on the heart contents (NUV). Tumour tissue was analysed at sacrifice for expression of TK1, cleaved PARP and phospho-AKT. Results Growth of U87 xenografts was significantly inhibited by GDC-0941 treatment throughout the course of the study (average growth rate of treated = -1.0±9.3 mm3, control = 69.2±22.1mm3 per day, p<0.05). Growth of HCT116 xenografts was not significantly different between groups (average growth rate of treated = 37.3±8.4 mm3, control =46.2±11.9 mm3 per day); however growth of HCT116 liver metastases was significantly reduced (Liver:bodyweight ratio of 0.11±0.01 vs 0.06±0.01, p<0.01). Uptake of [18F]-FLT was reduced in GDC-0941 treated animals bearing U87 xenografts or HCT116 liver metastases at the acute timepoint compared to baseline (NUVmax of 2.17±0.38 vs 1.59±0.29, p<0.01; 2.42±0.19,1.8±0.12 p<0.05 respectively), but was unchanged in unresponsive HCT116 xenografts. Conclusion [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. In addition to FLT, we are assessing the performance of a range of other imaging agents, including proprietary tracers, that can potentially bring value in monitoring of PI3-kinase targeting therapies. Disclosure of author financial interest or relationships: C. Cawthorne, GE Healthcare, Grant/research support; I. Wilson, GE Healthcare Medical Diagnostics, Employment; N. Burrows, None; R. Gieling, None; J. Gregory, None; A.M. Smigova, None; M. Babur, None; K. Williams, GE, Grant/research support . S1286