Matthew T. Greenfield

High-relaxivity magnetic resonance imaging contrast agents. Part 2. Optimization of inner- and second-sphere relaxivity.

Rationale and Objectives:The observed relaxivity of gadolinium-based contrast agents has contributions from the water molecule(s) that bind directly to the gadolinium ion (inner-sphere water), long-lived water molecules and exchangeable protons that make up the second-sphere of coordination, and water molecules that diffuse near the contrast agent (outer-sphere). Inner- and second-sphere relaxivity can both be increased by optimization of the lifetimes of the water molecules and protons in these coordination spheres, the rotational motion of the complex, and the electronic relaxation of the gadolinium ion. We sought to identify new high-relaxivity contrast agents by systematically varying the donor atoms that bind directly to gadolinium to increase inner-sphere relaxivity and concurrently including substituents that influence the second-sphere relaxivity. Methods:Twenty gadolinium-1,4,7,10-tetraazacyclo-dodecane-N,N′,N″,N′″-tetraacetato derivatives were prepared and their relaxivity determined in presence and absence of human serum albumin as a function of temperature and magnetic field. Data was analyzed to extract the underlying molecular parameters influencing relaxivity. Each compound had a common albumin-binding group and an inner-sphere donor set comprising the 4 tertiary amine N atoms from cyclen, an &agr;-substituted acetate oxygen atom, 2 amide oxygen atoms, an inner-sphere water oxygen atom, and a variable donor group. Each amide nitrogen was substituted with different groups to promote hydrogen bonding with second-sphere water molecules. Results:Relativities at 0.47 and 1.4 T, 37°C, in serum albumin ranged from 16.0 to 58.1 mM−1s−1 and from 12.3 to 34.8 mM−1s−1, respectively. The reduction of inner-sphere water exchange typical of amide donor groups could be offset by incorporating a phosphonate or phenolate oxygen atom donor in the first coordination sphere, resulting in higher relaxivity. Amide nitrogen substitution with pendant phosphonate or carboxylate groups increased relaxivity by as much as 88% compared with the N-methyl amide analog. Second-sphere relaxivity contributed as much as 24 and 14 mM−1s−1 at 0.47 and 1.4 T, respectively. Conclusions:Water/proton exchange dynamics in the inner- and second-coordination sphere can be predictably tuned by choice of donor atoms and second-sphere substituents, resulting in high-relaxivity agents.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Molecular Magnetic Resonance Imaging of Myocardial Perfusion With EP-3600, a Collagen-Specific Contrast Agent Initial Feasibility Study in a Swine Model

Background— Cardiac magnetic resonance (MR) perfusion imaging during the first pass after intravenous administration of extracellular contrast agents is hampered by the spatial and temporal resolution achievable and by the artifacts seen in ultrafast MR imaging. Furthermore, time-consuming quantitative data analysis is often added. The use of molecular MR imaging with a target-specific contrast agent with perfusion-dependent binding to myocardium may enable prolonged visualization of perfusion defects and thus may help to overcome limitations of currently used first-pass extracellular MR imaging. EP-3600 is a new gadolinium-containing molecular contrast agent that binds reversibly to myocardial collagen. Methods and Results— A significant but nonocclusive coronary artery stenosis was modeled in 7 domestic swine with an undersized MR-compatible balloon positioned in the left anterior descending artery as verified by x-ray angiography. Two animals died before contrast injection as a result of arrhythmias. In 5 swine, high-spatial-resolution gradient echo imaging (≈1×1 mm2 in-plane resolution) was performed before and 5, 20, 40, and 60 minutes after intravenous administration of 12.3 &mgr;mol/kg EP-3600. Contrast was administered during stress induced by an infusion of 250 &mgr;mol · kg−1 · min−1 adenosine. Yb-DTPA was administered simultaneously for comparison of myocardium-to-plasma ratios. Images were assessed subjectively by 2 investigators, and signal-to-noise and contrast-to-noise ratios over time were calculated. Normal myocardium showed a significant signal-to-noise ratio increase during the entire examination time. In all animals (n=5), the perfusion defect in the left anterior descending artery territory could be visualized with a high contrast-to-noise ratio for at least 20 minutes after contrast injection. A significantly higher myocardium-to-plasma ratio was found for EP-3600 compared with the control agent Yb-DTPA (0.85±0.26 versus 0.22±0.08, respectively; P<0.01). Conclusion— EP-3600 is a new molecular MR imaging contrast agent that binds to the myocardium and enables prolonged, high-contrast, high-spatial-resolution visualization of myocardial perfusion defects.

Molecular factors that determine Curie spin relaxation in dysprosium complexes

Dysprosium complexes can serve as transverse relaxation (T2) agents for water protons through chemical exchange and the Curie spin relaxation mechanism. Using a pair of matched dysprosium(III) complexes, Dy‐L1 (contains one inner‐sphere water) and Dy‐L2 (no inner‐sphere water), it is shown that the transverse relaxation of bulk water is predominantly an inner‐sphere effect. The kinetics of water exchange at Dy‐L1 were determined by 17O NMR. Proton transverse relaxation by Dy‐L1 at high fields is governed primarily through a large chemical shift difference between free and bound water. Dy‐L1 forms a noncovalent adduct with human serum albumin which dramatically lengthens the rotational correlation time, τR, causing the dipole–dipole component of the Curie spin mechanism to become significant and transverse relaxivity to increase by 3–8 times that of the unbound chelate. These findings aid in the design of new molecular species as efficient r2 agents. Magn Reson Med 46:917–922, 2001. Published 2001 Wiley‐Liss, Inc.

Heteroditopic Binding of Magnetic Resonance Contrast Agents for Increased Relaxivity

Contrast agents for Magnetic Resonance Imaging (MRI) provide anatomical and functional detail and increasingly can convey information at the molecular level.[1] The field of molecular MRI has advanced to the point that clinical studies with molecularly targeted agents are now appearing.[2] Despite the tremendous strengths of molecular MRI (molecular specificity superimposed on a high spatial resolution anatomical image, deep tissue penetration, three dimensional imaging, and lack of ionizing radiation), the field remains limited by the relatively low sensitivity for contrast agent detection.[1b, 3] Sensitivity of contrast agents is typically described by the extent to which they can induce relaxation of tissue water, and this is termed relaxivity (r1). Molecular relaxivity can be increased either by increasing the number of paramagnetic ions in the molecule, or optimizing the molecular factors that influence relaxation, or some combination of both.

The effect of the amide substituent on the biodistribution and tolerance of lanthanide(iii) dota-tetraamide derivatives

Objectives:Recent advances in the design of MRI contrast agents have rendered the lanthanide complexes of DOTA-tetraamide ligands of considerable interest, both as responsive MR agents and paramagnetic chemical exchange saturation transfer agents. The potential utility of these complexes for in vivo applications is contingent upon them being well tolerated by the body. The purpose of this study was to examine how the nature of the amide substituent, and in particular its charge, affected the fate of these chelates postinjection. Materials and Methods:Complexes of 6 DOTA-tetraamide ligands were prepared in which the nature of the amide substituent was systematically altered. The 6 ligands formed 3 series: a phosphonate series that included tri-cationic, mono-anionic, and poly-anionic complexes; a carboxylate series made up of a tri-cationic complex and a mono-anionic complex; and lastly, a tri-cationic complex with an aromatic amide substituent. These complexes were labeled with an appropriate radioisotope, either 153Gd or 177Lu, and the biodistribution profiles in rats recorded 2 hours postinjection. Results:Biodistribution profiles were initially acquired at low doses to minimize adverse effects. All the complexes studied were found to be excreted primarily through the renal system, with the majority of the dose being found in the urine. None of the complexes exhibited substantial uptake by bone, liver, and spleen, except for a complex with 4 phosphonate groups that exhibited significant bone targeting capabilities. Increasing the dose of each complex to that of a typical MR contrast agent was found to render all 3 tri-cationic complexes studied here acutely toxic. In contrast, no ill effects were observed after administration of similar doses of the corresponding anionic complexes. Conclusions:The absence of uptake by the liver and spleen indicate that irrespective of the ligand structure and charge, these complexes are not prone to dissociation in vivo. This is in agreement with previously published work that indicates high kinetic inertness for this class of compounds. At low doses, all complexes were well tolerated; however, for applications that require higher doses, the structure and charge of the ligand becomes a fundamentally important parameter. The results reported herein demonstrate the importance of incorporating negatively charged groups on amide substituents if a DOTA-tetraamide complex is to be employed at high doses in vivo.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

Abstract 2644: AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models

In non-small cell lung cancer (NSCLC), multiple classes of activating mutations have been identified in EGFR and HER2 that vary widely in their sensitivity to available tyrosine kinase inhibitors (TKIs). Erlotinib, gefitinib, and afatinib are approved for use in patients with the most common forms of EGFR activating mutations (ie, exon 19 deletions or L858R substitutions). However, no TKIs are approved for patients with EGFR activated by any other mutation, including exon 20 insertions or other uncommon substitutions, or for patients with any class of HER2 activating mutation (including exon 20 insertions). As inhibition of wild-type (WT) EGFR is associated with dose-limiting toxicities, a TKI that inhibits oncogenic EGFR and HER2 variants more potently than WT EGFR is more likely to be able to be dosed to efficacious levels. AP32788 is a potent inhibitor of all oncogenic forms of EGFR and HER2, including exon 20 insertions, with selectivity over WT EGFR. Activity of AP32788 and other TKIs was assessed by measuring viability of Ba/F3 cell lines engineered to express 20 mutant variants of EGFR (n = 14) or HER2 (n = 6): 4 EGFR variants containing a common activating mutation with or without a T790M resistance mutation, 8 EGFR/HER2 variants containing an exon 20 activating insertion (eg, EGFR ASV, HER2 YVMA), and 8 EGFR/HER2 variants containing other uncommon activating mutations (eg, EGFR G719A, HER2 G776V). Inhibition of WT EGFR was assessed by measuring effects on EGFR phosphorylation in cells (A431) that over-express WT EGFR. Consistent with their clinical activity, erlotinib and gefitinib generally only inhibited the 2 EGFR variants with common activating mutations more potently than WT EGFR (IC50s 71 and 56 nM, respectively), and afatinib generally only inhibited EGFR with common activating mutations or uncommon substitutions more potently than WT EGFR (IC50 4 nM). In contrast, AP32788 inhibited all 14 mutant variants of EGFR (IC50s 2.4-22 nM), and all 6 mutant variants of HER2 (IC50s 2.4-26 nM), more potently than it inhibited WT EGFR (IC50 35 nM), including all 8 variants with exon 20 activating insertions. In mice implanted with a patient-derived tumor containing an EGFR exon 20 activating insertion, or with engineered Ba/F3 cells containing a HER2 exon 20 activating insertion, once daily oral dosing of AP32788 induced regression of tumors at doses that were well tolerated (30-100 mg/kg). In vivo efficacy was associated with inhibition of EGFR signaling in the tumor. AP32788 potently inhibited all activated forms of EGFR and HER2 tested, including exon 20 insertions, more potently than WT EGFR, suggesting it may have the selectivity necessary to achieve efficacious levels of exposure in patients. A phase 1/2 clinical trial of AP32788 in NSCLC patients is planned. Citation Format: Francois Gonzalvez, Xiaotian Zhu, Wei-Sheng Huang, Theresa E. Baker, Yaoyu Ning, Scott D. Wardwell, Sara Nadworny, Sen Zhang, Biplab Das, Yongjin Gong, Matthew T. Greenfield, Hyun G. Jang, Anna Kohlmann, Feng Li, Paul M. Taslimi, Meera Tugnait, Yongjin Xu, Emily Y. Ye, Willmen W. Youngsaye, Stephan G. Zech, Yun Zhang, Tianjun Zhou, Narayana I. Narasimhan, David C. Dalgarno, William C. Shakespeare, Victor M. Rivera. AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2644.

Abstract 3236: Small molecule inhibitors of choline kinase lead to reduced phosphocholine levels and induction of apoptosis in cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Choline Kinase (ChoK) catalyzes the synthesis of phosphocholine (pCho) as the first step in the Kennedy pathway towards synthesis of the major membrane phospholipid, phosphatidylcholine. Increased phosphorylation of choline is a hallmark of the malignant phenotype and ChoK over-expression (primarily ChoKβ) has been reported in a variety of human cancers including breast, lung, colorectal and prostate. These observations have recently motivated efforts to develop anti-cancer agents targeting ChoK. Here we summarize our efforts to further validate ChoKα as an oncogenic target by characterizing its tumorigenic potential and exploring the activity of novel small molecule inhibitors. We transduced the ChoKα gene into HEK293 cells to examine the effects of ChoK expression in vitro and in vivo. Under reduced serum conditions, over-expression of ChoK promoted cell growth, increased phospho-ERK and phospho-AKT levels, and reduced p21 levels. ChoKα, but not vector-expressing cells, formed tumors in immune-compromised mice and ChoKα expression levels were positively associated with tumor growth rates. Together, these data suggest that ChoK maintains proliferative pathways in the absence of growth factors, and itself provides an oncogenic driver capable of inducing tumor growth in the absence of other transforming mutations. Recently we identified a novel chemical series that inhibits ChoKα in both enzymatic and cellular assays. The binding of these inhibitors to ChoK protein was confirmed in surface plasmon resonance experiments. A representative member of this lead series, compound A, was characterized in more detail and demonstrated potent enzyme inhibition against ChoKα with an IC50 of 70 nM. Compound A also inhibited the growth of ChoKα -expressing breast cancer lines, MDA-MB-468 and MDA-MB-415, with GI50s of 7 and 2 uM respectively. In contrast, compound A exhibited much lower activity against the non-transformed breast epithelial cell line MCF-12A, with a GI50 >40 uM. Consistent with its effects on cell growth, pCho levels in MDA-MB-415 cells, as measured by NMR, were dose-dependently inhibited up to ∼80% by 24 hours with an IC50 of ∼750 nM. In MDA-MB-415 cells, but not MCF-12A cells, levels of apoptotic markers were increased at 24 hours with compound concentrations β5 uM. In summary, we demonstrated that small molecule inhibition of ChoK results in a dose-dependent decrease of pCho levels, inhibition of proliferation and induction of apoptosis in ChoKα expressing breast cancer cells. We established that exogenous expression of ChoKα in HEK293 cells drives both oncogenic transformation and constitutive activation of proliferative signaling pathways. Taken together, these data further validate ChoKα as a potential therapeutic target in cancer and support the continued investigation into the utility of ChoKα inhibitors as anti-oncogenic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3236. doi:1538-7445.AM2012-3236

207 Molecular MR-Imaging of myocardium using EP-3600, a collagen specific contrast agent: assessment of myocardial perfusion defects in a swine model

Background The non-invasive assessment of the haemodynamic significance of coronary artery disease is still challenging. Cardiac MR perfusion imaging during the first pass after i.v. administration of an extracellular contrast agents is limited by the spatial and temporal resolution achievable as well as by the artifacts seen in ultrafast MR-imaging. Moreover the patient must be stressed while being inside the magnet. Two injections of contrast are required, and a time consuming data analysis is necessary.