First-in-human imaging using [11C]MDTC: a radiotracer targeting the cannabinoid receptor type 2.

PURPOSE: We report findings from the first-in-human study of [11C]MDTC, a radiotracer developed to image the cannabinoid receptor type 2 (CB2R) with positron emission tomography (PET). METHODS: Ten healthy adults were imaged according to a 90-min dynamic PET protocol after bolus intravenous injection of [11C]MDTC. Five participants also completed a second [11C]MDTC PET scan to assess test-retest reproducibility of receptor-binding outcomes. The kinetic behavior of [11C]MDTC in human brain was evaluated using tissue compartmental modeling. Four additional healthy adults completed whole-body [11C]MDTC PET/CT to calculate organ doses and the whole-body effective dose. RESULTS: [11C]MDTC brain PET and [11C]MDTC whole-body PET/CT was well-tolerated. A murine study found evidence of brain-penetrant radiometabolites. The model of choice for fitting the time activity curves (TACs) across brain regions of interest was a three-tissue compartment model that includes a separate input function and compartment for the brain-penetrant metabolites. Regional distribution volume (VT) values were low, indicating low CB2R expression in the brain. Test-retest reliability of VT demonstrated a mean absolute variability of 9.91%. The measured effective dose of [11C]MDTC was 5.29 µSv/MBq. CONCLUSION: These data demonstrate the safety and pharmacokinetic behavior of [11C]MDTC with PET in healthy human brain. Future studies identifying radiometabolites of [11C]MDTC are recommended before applying [11C]MDTC PET to assess the high expression of the CB2R by activated microglia in human brain.

Exploring the potential of the novel imidazole-4,5-dicarboxyamide chemical exchange saturation transfer scaffold for pH and perfusion imaging.

Here, we describe and assess the potential of 14 newly synthesized imidazole-4,5-dicarboxyamides (I45DCs) for pH and perfusion imaging. A number of these aromatic compounds possess large labile proton chemical shifts (up to 7.7 ppm from water) because of their intramolecular hydrogen bonds and a second labile proton to allow for chemical exchange saturation transfer (CEST) signal ratio-based pH measurements. We have found that the contrast produced is strong for a wide range of substitutions and that the inflection points in the CEST signal ratio versus pH plots used to generate concentration-independent pH maps can be adjusted based on these subsitutions to tune the pH range that can be measured. These I45DC CEST agents have advantages over the triiodobenzenes currently employed for tumor and kidney pH mapping, both preclinically and in initial human studies. Finally, as CEST MRI combined with exogenous contrast has the potential to detect functional changes in the kidneys, we evaluated our highest performing anionic compound (I45DC-diGlu) on a unilateral urinary obstruction mouse model and observed lower contrast uptake in the obstructed kidney compared with the unobstructed kidney and that the unobstructed kidney displayed a pH of ~ 6.5 while the obstructed kidney had elevated pH and an increased range in pH values. Based on this, we conclude that the I45DCs have excellent imaging properties and hold promise for a variety of medical imaging applications, particularly renal imaging.

First-in-human use of 11C-CPPC with positron emission tomography for imaging the macrophage colony-stimulating factor 1 receptor.

PURPOSE: Study of the contribution of microglia to onset and course of several neuropsychiatric conditions is challenged by the fact that these resident immune cells often take on different phenotypes and functions outside the living brain. Imaging microglia with radiotracers developed for use with positron emission tomography (PET) allows researchers to study these cells in their native tissue microenvironment. However, many relevant microglial imaging targets such as the 18 kDa translocator protein are also expressed on non-microglial cells, which can complicate the interpretation of PET findings. 11C-CPPC was developed to image the macrophage colony-stimulating factor 1 receptor, a target that is expressed largely by microglia relative to other cell types in the brain. Our prior work with 11C-CPPC demonstrated its high, specific uptake in brains of rodents and nonhuman primates with neuroinflammation, which supports the current first-in-human evaluation of its pharmacokinetic behavior in the brains of healthy individuals. METHODS: Eight healthy nonsmoker adults completed a 90-min dynamic PET scan that began with bolus injection of 11C-CPPC. Arterial blood sampling was collected in order to generate a metabolite-corrected arterial input function. Tissue time-activity curves (TACs) were generated using regions of interest identified from co-registered magnetic resonance imaging data. One- and two-tissue compartmental models (1TCM and 2TCM) as well as Logan graphical analysis were compared. RESULTS: Cortical and subcortical tissue TACs peaked by 37.5 min post-injection of 11C-CPPC and then declined. The 1TCM was preferred. Total distribution volume (VT) values computed from 1TCM aligned well with those from Logan graphical analysis (t* = 30), with VT values relatively high in thalamus, striatum, and most cortical regions, and with relatively lower VT in hippocampus, total white matter, and cerebellar cortex. CONCLUSION: Our results extend support for the use of 11C-CPPC with PET to study microglia in the human brain.

PET/CT imaging of CSF1R in a mouse model of tuberculosis.

PURPOSE: Macrophages represent an essential means of sequestration and immune evasion for Mycobacterium tuberculosis. Pulmonary tuberculosis (TB) is characterized by dense collections of tissue-specific and recruited macrophages, both of which abundantly express CSF1R on their outer surface. 4-Cyano-N-(5-(1-(dimethylglycyl)piperidin-4-yl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole-2-carboxamide (JNJ-28312141) is a reported high affinity, CSF1R-selective antagonist. We report the radiosynthesis of 4-cyano-N-(5-(1-(N-methyl-N-([11C]methyl)glycyl)piperidin-4-yl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-2-yl)-1H-imidazole-2-carboxamide ([11C]JNJ-28312141) and non-invasive detection of granulomatous and diffuse lesions in a mouse model of TB using positron emission tomography (PET). METHODS: Nor-methyl-JNJ-28312141 precursor was radiolabeled with [11C]iodomethane to produce [11C]JNJ-28312141. PET/CT imaging was performed in the C3HeB/FeJ murine model of chronic pulmonary TB to co-localize radiotracer uptake with granulomatous lesions observed on CT. Additionally, CSF1R, Iba1 fluorescence immunohistochemistry was performed to co-localize CSF1R target with reactive macrophages in infected and healthy mice. RESULTS: Radiosynthesis of [11C]JNJ-28312141 averaged a non-decay-corrected yield of 18.7 ± 2.1%, radiochemical purity of 99%, and specific activity averaging 658 ± 141 GBq/µmol at the end-of-synthesis. PET/CT imaging in healthy mice showed hepatobiliary [13.39-25.34% ID/g, percentage of injected dose per gram of tissue (ID/g)] and kidney uptake (12.35% ID/g) at 40-50 min post-injection. Infected mice showed focal pulmonary lesion uptake (5.58-12.49% ID/g), hepatobiliary uptake (15.30-40.50% ID/g), cervical node uptake, and renal uptake (11.66-29.33% ID/g). The ratio of infected lesioned lung/healthy lung uptake is 5.91:1, while the ratio of lesion uptake to adjacent infected radiolucent lung is 2.8:1. Pre-administration of 1 mg/kg of unlabeled JNJ-28312141 with [11C]JNJ-28312141 in infected animals resulted in substantial blockade. Fluorescence microscopy of infected and uninfected whole lung sections exclusively co-localized CSF1R staining with abundant Iba1 + macrophages. Healthy lung exhibited no CSF1R staining and very few Iba1 + macrophages. CONCLUSION: [11C]JNJ-28312141 binds specifically to CSF1R + macrophages and delineates granulomatous foci of disease in a murine model of pulmonary TB.

Radiosynthesis and validation of [5-cyano-N-(4-(4-[11 C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl) furan-2-carboxamide] ([11 C]CPPC), a PET radiotracer for imaging CSF1R, a microglia-specific marker.

In this practitioner protocol, the radiochemical synthesis of [11 C]CPPC is described in detail, and a quality control summary of three validation productions is presented. The results indicate that the radiotracer product can be produced in good radiochemical yield (> 60 mCi (2.22 GBq) at end-of-synthesis (EOS)), at high specific activity (molar activity > 11,435 mCi/µmole (423 GBq/µmole) at EOS) and high chemical and radiochemical purity. The entire production conforms to current Good Manufacturing Practice (cGMP) requirements. The final product is formulated as a sterile, pyrogen-free solution suitable for human injection.

Amino Acid-Derived Sensors for Specific Zn2+ Detection Using Hyperpolarized 13 C Magnetic Resonance Spectroscopy.

Alterations in Zn2+ concentration are seen in normal tissues and in disease states, and for this reason imaging of Zn2+ is an area of active investigation. Herein, enriched [1-13 C]cysteine and [1-13 C2 ]iminodiacetic acid were developed as Zn2+ -specific imaging probes using hyperpolarized 13 C magnetic resonance spectroscopy. [1-13 C]cysteine was used to accurately quantify Zn2+ in complex biological mixtures. These sensors can be employed to detect Zn2+ via imaging mechanisms including changes in 13 C chemical shift, resonance linewidth, or T1 .

PET imaging of microglia by targeting macrophage colony-stimulating factor 1 receptor (CSF1R).

While neuroinflammation is an evolving concept and the cells involved and their functions are being defined, microglia are understood to be a key cellular mediator of brain injury and repair. The ability to measure microglial activity specifically and noninvasively would be a boon to the study of neuroinflammation, which is involved in a wide variety of neuropsychiatric disorders including traumatic brain injury, demyelinating disease, Alzheimer's disease (AD), and Parkinson's disease, among others. We have developed [11C]CPPC [5-cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide], a positron-emitting, high-affinity ligand that is specific for the macrophage colony-stimulating factor 1 receptor (CSF1R), the expression of which is essentially restricted to microglia within brain. [11C]CPPC demonstrates high and specific brain uptake in a murine and nonhuman primate lipopolysaccharide model of neuroinflammation. It also shows specific and elevated uptake in a murine model of AD, experimental allergic encephalomyelitis murine model of demyelination and in postmortem brain tissue of patients with AD. Radiation dosimetry in mice indicated [11C]CPPC to be safe for future human studies. [11C]CPPC can be synthesized in sufficient radiochemical yield, purity, and specific radioactivity and possesses binding specificity in relevant models that indicate potential for human PET imaging of CSF1R and the microglial component of neuroinflammation.

Lanthanide-Based T2ex and CEST Complexes Provide Insights into the Design of pH Sensitive MRI Agents.

The CEST and T1 /T2 relaxation properties of a series of Eu3+ and Dy3+ DOTA-tetraamide complexes with four appended primary amine groups are measured as a function of pH. The CEST signals in the Eu3+ complexes show a strong CEST signal after the pH was reduced from 8 to 5. The opposite trend was observed for the Dy3+ complexes where the r2ex of bulk water protons increased dramatically from ca. 1.5 mm-1 s-1 to 13 mm-1 s-1 between pH 5 and 9 while r1 remained unchanged. A fit of the CEST data (Eu3+ complexes) to Bloch theory and the T2ex data (Dy3+ complexes) to Swift-Connick theory provided the proton-exchange rates as a function of pH. These data showed that the four amine groups contribute significantly to proton-catalyzed exchange of the Ln3+ -bound water protons even though their pKa 's are much higher than the observed CEST or T2ex effects. This demonstrated the utility of using appended acidic/basic groups to catalyze prototropic exchange for imaging tissue pH by MRI.

Imaging Extracellular Lactate In Vitro and In Vivo Using CEST MRI and a Paramagnetic Shift Reagent.

Overproduction of lactate is a hallmark of cancer, yet a method to quantitatively measure lactate production by cancer cells is not straight-forward. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can potentially be used to image lactate but the small difference in chemical shift of the lactate -OH proton and water proton resonances make it challenging. Like other spectroscopic methods, CEST MRI cannot discriminate intracellular lactate from extracellular lactate. Herein, we demonstrate a relatively simple way to shift the lactate -OH proton resonance far away from water by addition of the paramagnetic shift reagent, EuDO3A, while retaining the CEST properties of lactate itself. The potential of the method was demonstrated by imaging extracellular lactate excreted from lung cancer cells in tissue culture without interference from other components in the culture media and by imaging excess lactate excreted into the bladder of a mouse.

Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents.

EuDOTA-tetraamide complexes as paraCEST agents offer an attractive platform for designing biological sensors and responsive agents. The early versions of these agents showed low sensitivity at temperature and power levels suitable for in vivo applications partly due to non-optimal water exchange rates. Here we report two new EuDOTA derivatives having glutamyl-phosphonate side arms that display the slowest water exchange rates of any other paraCEST agent reported so far. The advantages of such systems are demonstrated experimentally both in vitro and in vivo and DFT calculations were performed to help understand the physical-chemical reasons for this interesting behavior.

pH imaging of mouse kidneys in vivo using a frequency-dependent paraCEST agent.

PURPOSE: This study explored the feasibility of using a pH responsive paramagnetic chemical exchange saturation transfer (paraCEST) agent to image the pH gradient in kidneys of healthy mice. METHODS: CEST signals were acquired on an Agilent 9.4 Tesla small animal MRI system using a steady-state gradient echo pulse sequence after a bolus injection of agent. The magnetic field inhomogeneity across each kidney was corrected using the WASSR method and pH maps were calculated by measuring the frequency of water exchange signal arising from the agent. RESULTS: Dynamic CEST studies demonstrated that the agent was readily detectable in kidneys only between 4 to 12 min postinjection. The CEST images showed a higher signal intensity in the pelvis and calyx regions and lower signal intensity in the medulla and cortex regions. The pH maps reflected tissue pH values spanning from 6.0 to 7.5 in kidneys of healthy mice. CONCLUSION: This study demonstrated that pH maps of the kidney can be imaged in vivo by measuring the pH-dependent chemical shift of a single water exchange CEST peak without prior knowledge of the agent concentration in vivo. The results demonstrate the potential of using a simple frequency-dependent paraCEST agent for mapping tissue pH in vivo. Magn Reson Med 75:2432-2441, 2016. © 2015 Wiley Periodicals, Inc.

Amplifying the sensitivity of zinc(II) responsive MRI contrast agents by altering water exchange rates.

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r1). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM(-1) s(-1) upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r1 compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

[(68) Ga]-HP-DO3A-nitroimidazole: a promising agent for PET detection of tumor hypoxia.

The goal of this study is to evaluate a new (68) Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [(68) Ga]-HP-DO3A-nitroimidazole ([(68) Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [(68) Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2 . The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [(68) Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [(68) Ga]-HP-DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor-bearing mice breathing air versus 100% O2 . Given the commercial availability of cGMP (68) Ge/(68) Ga generators and the ease of (68) Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.

A pH-Responsive MRI Agent that Can Be Activated Beyond the Tissue Magnetization Transfer Window.

A terbium-based complex that displays a water exchange CEST resonance well outside the normal magnetization transfer (MT) frequency range of tissues provides a direct readout of pH values by MRI. Deprotonation of the phenolic proton in this complex results in a frequency shift of 56 ppm in a bound water molecule exchange peak between pH 5 and 8. This allows direct imaging of pH without prior knowledge of the agent concentration and with essentially no interference from the tissue MT signal.

A europium(III)-based PARACEST agent for sensing singlet oxygen by MRI.

A europium(III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen ((1)O2). The water soluble, thermodynamically stable complex reacts rapidly with (1)O2 to form an endoperoxide derivative that results in an ∼3 ppm shift in the position of the Eu(III)-bound water chemical exchange saturation transfer (CEST) peak. The potential of using this probe to detect accumulation of the endoperoxide derivative in biological media by ratiometric CEST imaging was demonstrated.

The importance of water exchange rates in the design of responsive agents for MRI.

The rate of water exchange in lanthanide complexes is often overlooked as an important parameter in the design of responsive MR imaging agents. Most often, the number of inner-sphere water coordination sites or the rotational mobility of the complex is considered as the central theme while water exchange is either assumed to be "fast enough" or entirely ignored. On the contrary, relaxation and shift theories predict that water exchange rates may indeed be the key parameter one should consider in any new molecular design. In this short review, the impact of water exchange rates on three classes of lanthanide-based MRI contrast agents, T1-based relaxation agents, T2 exchange line-broadening agents and chemical exchange saturation transfer (CEST) agents, is illustrated and discussed.

Advantages of paramagnetic chemical exchange saturation transfer (CEST) complexes having slow to intermediate water exchange properties as responsive MRI agents.

Paramagnetic chemical exchange saturation transfer (PARACEST) complexes are exogenous contrast agents that have great potential to further extend the functional and molecular imaging capabilities of magnetic resonance. As a result of the presence of a central paramagnetic lanthanide ion (Ln(3+) ≠ La(3+) , Gd(3+) , Lu(3+) ) within the chelate, the resonance frequencies of exchangeable protons bound to the PARACEST agent are shifted far away from the bulk water frequency. This large chemical shift, combined with an extreme sensitivity to the chemical exchange rate, make PARACEST agents ideally suited for the reporting of significant biological metrics, such as temperature, pH and the presence of metabolites. In addition, the ability to turn PARACEST agents 'off' and 'on' using a frequency-selective saturation pulse gives them a distinct advantage over Gd(3+) -based contrast agents. A current challenge for PARACEST research is the translation of the promising in vitro results into in vivo systems. This short review article first describes the basic theory behind PARACEST contrast agents, their benefits over other contrast agents and their applications to MRI. It then describes some of the recent PARACEST research results: specifically, pH measurements using water molecule exchange rate modulation, T2 exchange contrast caused by water molecule exchange, the use of ultrashort TEs (TE < 10 µs) to overcome T2 exchange line broadening and the potential application of T2 exchange as a new contrast mechanism for MRI.

The pH sensitivity of -NH exchange in LnDOTA-tetraamide complexes varies with amide substituent.

The amide proton exchange rates in various lanthanide(III) DOTA-tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA-(gly)(4)(-) (Yb-1), YbDOTA-(NHCH(2)PO(3))(4 (5-) (Yb-2) and YbDOTA-(NHCH(2)PO(3)Et(2))(4)(3+) (Yb-3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb-1, Yb-2 and Yb-3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the -NH protons in Yb-1 and Yb-2 were similar (-17 ppm) while the -NH proton in Yb-3 was at -13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb-3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted -NH proton resonances. The ratio of CEST from -NH exchange in Tm-1 compared with CEST from -NH exchange in Tm-3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA-tetraamides for mapping tissue pH by use of ratiometric CEST imaging.

Polymeric PARACEST MRI contrast agents as potential reporters for gene therapy.

Gene therapy is a potentially powerful treatment approach that targets molecular remedies for disease. Among other challenges it remains difficult to monitor gene delivery and its downstream metabolic consequences. Approaches to MRI gene reporters have been reported but few have the potential for translation beyond isolated cell systems. Herein, we report a polycationic polymer MRI contrast agent that binds to DNA in a ratio of one monomer unit per phosphate group of DNA. Significantly, this binding event diminishes the MR contrast signal from the agent itself potentially providing a platform for imaging delivery and release of a gene into cells and tissues. Importantly, we demonstrate here the proof of concept that a positively charged polymeric contrast agent can also act as a transfection agent, delivering the gene for encoding green fluorescent protein into cells. These observations provide support for the radical, new idea of creating a combined transfection/imaging agent for monitoring gene delivery in real time by MRI.

A responsive europium(III) chelate that provides a direct readout of pH by MRI.

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric chemical exchange saturation transfer (CEST) principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ∼5 ppm shift in the water exchange CEST peak that is easily detected by MRI. Collection of two CEST images at two slightly different activation frequencies provides a direct readout of solution pH without the need of a concentration marker.