High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.     

High-throughput magnetic resonance imaging in mice for phenotyping and therapeutic evaluation

High-throughput mouse magnetic resonance imaging (MRI) is seeing rapidly increasing demand in development of therapeutics. Recent advances including higher-field systems, new gradient and radio frequency coils and new pulse sequences, coupled with efficient animal preparation and data handling, allow high-throughput MRI under certain protocols. However, with current shifts from anatomic to functional and molecular imaging, innovative technology is required to meet new throughput demands. The first multiple mouse imaging strategies have provided a glimpse of the future state-of-the-art. However, the successful translation of standard clinical MRI technology to preclinical MRI is required to facilitate next-generation high-throughput MRI.     

New vanadium-based magnetic resonance imaging probes: clinical potential for early detection of cancer

We have developed a magnetic resonance imaging (MRI) method for improved detection of cancer with a new class of cancer-specific contrast agents, containing vanadyl (VO²⁺)-chelated organic ligands, specifically bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂]. Vanadyl compounds have been found to accumulate within cells, where they interact with intracellular glycolytic enzymes. Aggressive cancers are metabolically active and highly glycolytic; an MRI contrast agent that enters cells with high glycolytic activity could provide high-resolution functional images of tumor boundaries and internal structure, which cannot be achieved by conventional contrast agents. The present work demonstrates properties of VO(acac)₂ that may give it excellent specificity for cancer detection. A high dose of VO(acac)₂ did not cause any acute or short-term adverse reactions in murine subjects. Calorimetry and spectrofluorometric methods demonstrate that VO(acac)₂ is a blood pool agent that binds to serum albumin with a dissociation constant K _d ~ 2.5 ± 0.7 × 10⁻⁷ M and a binding stoichiometry n = 1.03 ± 0.04. Owing to its prolonged blood half-life and selective leakage from hyperpermeable tumor vasculature, a low dose of VO(acac)₂ (0.15 mmol/kg) selectively enhanced in vivo magnetic resonance images of tumors, providing high-resolution images of their interior structure. The kinetics of uptake and washout are consistent with the hypothesis that VO(acac)₂ preferentially accumulates in cancer cells. Although VO(acac)₂ has a lower relaxivity than gadolinium-based MRI contrast agents, its specificity for highly glycolytic cells may lead to an innovative approach to cancer detection since it has the potential to produce MRI contrast agents that are nontoxic and highly sensitive to cancer metabolism.     

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([¹²⁵I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [¹²⁵I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [¹²⁵I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [¹²⁵I]I-NCP (11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [¹²⁵I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [¹²⁵I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [¹²⁵I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [¹²⁵I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.     

Synthesis and biological evaluation of aromatic enones related to curcumin

Curcumin, a natural product isolated from the spice turmeric, has been shown to exhibit a wide range of pharmacological activities including certain anti-cancer properties. It has been specifically shown to be an effective inhibitor of angiogenesis both in vitro and in vivo. Using curcumin as a lead compound for anti-angiogenic analog design, a series of structurally related compounds utilizing a substituted chalcone backbone have been synthesized and tested via an established SVR cell proliferation assay. The results have yielded a wide range of compounds that equal or exceed curcumin's ability to inhibit endothelial cell growth in vitro. Due to both their commercial availability and their fairly straightforward synthetic preparation, these low molecular weight compounds are attractive leads for developing future angiogenic inhibitors.     

Static magnetic field effects on human subjects related to magnetic resonance imaging systems

Goal: This paper reviews recent studies evaluating human subjects for physiologic or neuro-cognitive function adverse effects resulting from exposure to static magnetic fields of magnetic resonance imaging systems.

Materials and Methods: The results of three studies are summarized. Two studies evaluated exposure to a maximum of 8 Tesla (T). The first series studied 25 normal human subjects’ sequential vital signs (heart rate, blood pressure, blood oxygenation, core temperature, ECG, respiratory rate) measured at different magnetic field strengths to a maximum of 8 T. A second series of 25 subjects were studied at 0.05 and 8 T (out and in the bore of the magnet), performing 12 different standardized neuro-psychological tests and auditory–motor reaction times. The subjects’ comments were recorded immediately following the study and after a three-month interval. The third study contained 17 subjects, placed near the bore of a 1.5 T magnet, and it used six different cognitive, cognitive–motor, or sensory tests.

Results: There were no clinically significant changes in the subjects’ physiologic measurements at 8 T. There was a slight increase in the systolic blood pressure with increasing magnetic field strength. There did not appear to be any adverse effect on the cognitive performance of the subjects at 8 T. A few subjects commented at the time of initial exposure on dizziness, metallic taste in the mouth, or discomfort related to the measurement instruments or the head coil. There were no adverse comments at 3 months. The 1.5 T study had two of the four neuro-behavioral domains exhibiting adverse effects (sensory and cognitive–motor).

Conclusions: These studies did not demonstrate any clinically relevant adverse effects on neuro-cognitive testing or vital sign changes. One short-term memory, one sensory, and one cognitive–motor test demonstrated adverse effects, but the significance is not clear.     

Synthesis, crystal structures and biological evaluation of water-soluble zinc complexes of zwitterionic carboxylates

Three water-soluble zinc complexes, [Zn(Cbp)₂Br₂] (1) (Cbp = N-(4-carboxybenzyl)pyridinium), {[Zn(BCbpy)₂(H₂O)₄]₃Br₆·2(BCbpy)·2(4,4′-bipy)} (2) (BCbpy = 1-(4-carboxybenzyl)-4,4′-bipyridinium) and {[Zn₄(Bpybc)₆(H₂O)₁₂](OH)₈·9H₂O}_2n (3) (Bpybc = 1,1′-bis(4-carboxybenzyl)-4,4′-bipyridinium), were synthesized and characterized by IR, elemental analysis and single-crystal X-ray crystallography. In complex 1, the central Zn atom adopts a distorted tetrahedral coordination geometry that is formed from two unidentate Cbp ligands and two Br atoms. For complex 2, the Zn atom in [Zn(BCbpy)₂(H₂O)₄]²⁺ is strongly coordinated by four water molecules and two N atoms from two BCbpy ligands, hence forming an octahedral geometry. In complex 3, each Bpybc ligand bridges two [Zn(H₂O)₃]²⁺ units through two terminal carboxylate groups in a monodentate coordination mode, thus forming a flowerlike two-dimensional network. Agarose gel electrophoresis (GE) and ethidium bromide (EB) displacement experiments indicated that complex 3 was capable of converting pBR322 DNA into open circular (OC) and linear forms, and exhibited high binding affinity toward calf-thymus DNA. MTT assay showed that complex 3 displayed inhibitory activities toward the proliferation of lung adenocarcinoma A549 and mouse sarcoma S-180 cells, with the IC₅₀ values being 27.3 and 48.8 μM, respectively.     

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivatives as probes for imaging vesicular monoamine transporter-2

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4–6, targeting the vesicular monoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates prepared from rat striatal tissue homogenates with both [¹²⁵I]-iodovinyl-TBZ and [³H]DTBZ. There was a good correlation (r² = 0.925) between the affinities of the different compounds for [¹²⁵I]-iodovinyl-TBZ and [³H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter (K_i = 8.68 ± 1.26 nM and 7.01 ± 0.07 nM, respectively), which may be a good lead compound for further structural modification to develop useful probes for VMAT2.     

Paramagnetic pharmaceuticals for magnetic resonance imaging

Complexes of paramagnetic ions that are tissue-, organ- or tumor-specific will supplement routine magnetic resonance imaging, help assess organ perfusion, and in some cases assess specific organ function. Studies are described in animals and man and the results suggest that dilute iron solutions may be useful for contrast-enhancement of the gastrointestinal tract; that ferrioxamine B, a stable ferric iron complex, appears to permit identification of focal blood-brain-barrier defects and to assess renal excretory function; and that gadolinium-DTPA can produce contrast-enhancement of a variety of lesions. In addition, gadolinium-DTPA can detect a breakdown in the blood-brain-barrier and can delineate functioning myocardium in the setting of acute ischemia.     

Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.     

Arterial spin labeling blood flow magnetic resonance imaging for evaluation of renal injury

A multitude of evidence suggests that iodinated contrast material causes nephrotoxicity; however, there have been no previous studies that use arterial spin labeling (ASL) blood flow functional magnetic resonance imaging (fMRI) to investigate the alterations in effective renal plasma flow between normointensive and hypertensive rats following injection of contrast media. We hypothesized that FAIR-SSFSE arterial spin labeling MRI may enable noninvasive and quantitative assessment of regional renal blood flow abnormalities and correlate with disease severity as assessed by histological methods. Renal blood flow (RBF) values of the cortex and medulla of rat kidneys were obtained from ASL images postprocessed at ADW4.3 workstation 0.3, 24, 48, and 72 h before and after injection of iodinated contrast media (6 ml/kg). The H&E method for morphometric measurements was used to confirm the MRI findings. The RBF values of the outer medulla were lower than those of the cortex and the inner medulla as reported previously. Iodinated contrast media treatment resulted in decreases in RBF in the outer medulla and cortex in spontaneously hypertensive rats (SHR), but only in the outer medulla in normotensive rats. The iodinated contrast agent significantly decreased the RBF value in the outer medulla and the cortex in SHR compared with normotensive rats after injection of the iodinated contrast media. Histological observations of kidney morphology were also consistent with ASL perfusion changes. These results demonstrate that the RBF value can reflect changes of renal perfusion in the cortex and medulla. ASL-MRI is a feasible and accurate method for evaluating nephrotoxic drugs-induced kidney damage.     

Synthesis and biological evaluation of molecular probes based on the 9-methylstreptimidone derivative DTCM-glutarimide

Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were synthesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-glutarimide.     

Synthesis and evaluation of water-soluble paclitaxel prodrugs

A series of water-soluble 2'-paclitaxel prodrugs were synthesized by attaching paclitaxel to polyethylene glycol (PEG) through amino acid spacers. The prodrugs showed highly improved water solubility, enhanced in vitro cytotoxicity and in vivo antitumor activity compared with the native drug, paclitaxel.     

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

Synthesis and biological evaluation of cepharadiones A and B and related dioxoaporphines

Described herein is the first total synthesis and structural confirmation of cepharadione A, a naturally occurring DNA damaging agent. Also reported is the synthesis of cepharadione B, a closely related natural product, as well as the biological evaluation of both natural products. Finally, the preparation and biological evaluation of novel dioxoaporphine analogues is described.