Deuteration of a molecular probe for DNP hyperpolarization – a new approach and validation for choline chloride

The promising dynamic nuclear polarization (DNP) for hyperpolarized ¹³C‐MRI/MRS of real‐time metabolism in vivo is challenged by the limited number of agents with the required physical and biological properties. The physical requirement of a liquid‐state T₁ of tens of seconds is mostly found for ¹³C‐carbons in small molecules that have no direct protons attached, i.e. carbonyl, carboxyl and certain quaternary carbons. Unfortunately, such carbon positions do not exist in a large number of metabolic agents, and chemical shift dispersion often limits detection of their chemical evolution. We have previously shown that direct deuteration of protonated carbon positions significantly prolongs the ¹³C T₁ in the liquid state and provides potential ¹³C‐labeled agents with differential chemical shift with respect to metabolism. The Choline Molecular Probe [1,1,2,2‐D₄, 2‐¹³C]choline chloride (CMP2) has recently been introduced as a means of studying choline metabolism in a hyperpolarized state. Here, the biophysical properties of CMP2 were characterized and compared with those of [1‐¹³C]pyruvate to evaluate the impact of molecular probe deuteration. The CMP2 solid‐state polarization build‐up time constant (30 min) and polarization level (24%) were comparable to those of [1‐¹³C]pyruvate. Both compounds' liquid state T₁ increased with temperature. The high‐field T₁ of CMP2 compared favorably with [1‐¹³C]pyruvate. Thus, a deuterated agent demonstrated physical properties comparable to a hyperpolarized compound of already proven value, whereas both showed chemical shift dispersion that allowed monitoring of their metabolism. It is expected that the use of deuterated carbon‐13 positions as reporting hyperpolarized nuclei will substantially expand the library of agents for DNP‐MR. Copyright © 2011 John Wiley & Sons, Ltd.     

Assessment of real‐time myocardial uptake and enzymatic conversion of hyperpolarized [1‐13C]pyruvate in pigs using slice selective magnetic resonance spectroscopy

Hyperpolarization of ¹³C‐labeled energy substrates enables the noninvasive detection and mapping of metabolic activity, in vivo, with magnetic resonance spectroscopy (MRS). Therefore, hyperpolarization and ¹³C MRS can potentially become a powerful tool to study the physiology of organs such as the heart, through the quantification of kinetic patterns under both normal and pathological conditions. In this study we assessed myocardial uptake and metabolism of hyperpolarized [1‐¹³ C]pyruvate in anesthetized pigs. Pyruvate metabolism was studied at baseline and during dobutamine‐induced stimulation. We applied a numerical approach for spectral analysis and kinetic fitting (LSFIT/KIMOfit), making a comparison with a well‐known jMRUI/AMARES analysis and γ‐variate function, and we estimated the apparent conversion rate of hyperpolarized [1‐¹³ C]pyruvate into its downstream metabolites [1‐¹³C]lactate, [1‐¹³C]alanine and [¹³C]bicarbonate in a 3 T MR scanner. We detected an increase in the apparent kinetic constants (k_PX) for bicarbonate and lactate of two‐fold during dobutamine infusion. These data correlate with the double product (rate‐pressure product), an indirect parameter of cardiac oxygen consumption: we observed an increase in value by 46 ± 11% during inotropic stress. The proposed approach might be applied to future studies in models of cardiac disease and/or for the assessment of the pharmacokinetic properties of suitable ¹³C‐enriched tracers for MRS. Copyright © 2012 John Wiley & Sons, Ltd.     

Real‐time cardiac metabolism assessed with hyperpolarized [1‐13C]acetate in a large‐animal model

Dissolution‐dynamic nuclear polarization (dissolution‐DNP) for magnetic resonance (MR) spectroscopic imaging has recently emerged as a novel technique for noninvasive studies of the metabolic fate of biomolecules in vivo. Since acetate is the most abundant extra‐ and intracellular short‐chain fatty acid, we focused on [1‐¹³C]acetate as a promising candidate for a chemical probe to study the myocardial metabolism of a beating heart. The dissolution‐DNP procedure of Na[1‐¹³C]acetate for in vivo cardiac applications with a 3 T MR scanner was optimized in pigs during bolus injection of doses of up to 3 mmol. The Na[1‐¹³C]acetate formulation was characterized by a liquid‐state polarization of 14.2% and a T_1Eff in vivo of 17.6 ± 1.7 s. In vivo Na[1‐¹³C]acetate kinetics displayed a bimodal shape: [1‐¹³C]acetyl carnitine (AcC) was detected in a slice covering the cardiac volume, and the signal of ¹³C‐acetate and ¹³C‐AcC was modeled using the total area under the curve (AUC) for kinetic analysis. A good correlation was found between the ratio AUC(AcC)/AUC(acetate) and the apparent kinetic constant of metabolic conversion, from [1‐¹³C]acetate to [1‐¹³C]AcC (k_AcC), divided by the AcC longitudinal relaxation rate (r₁). Our study proved the feasibility and the limitations of administration of large doses of hyperpolarized [1‐¹³C]acetate to study the myocardial conversion of [1‐¹³C]acetate in [1‐¹³C]acetyl‐carnitine generated by acetyltransferase in healthy pigs. Copyright © 2014 John Wiley & Sons, Ltd.     

Field dependence of T1 for hyperpolarized [1‐13C]pyruvate

In vivo metabolism of hyperpolarized pyruvate has been demonstrated to be an important probe of cellular glycolysis in diseases such as cancer. The usefulness of hyperpolarized ¹³C imaging is dependent on the relaxation rates of the ¹³C‐enriched substrates, which in turn depend on chemical conformation and properties of the dissolution media such as buffer composition, solution pH, temperature and magnetic field. We have measured the magnetic field dependence of the spin–lattice relaxation time of hyperpolarized [1‐¹³C]pyruvate using field‐cycled relaxometry. [1‐¹³C]pyruvate was hyperpolarized using dynamic nuclear polarization and then rapidly thawed and dissolved in a buffered solution to a concentration of 80 mmol l^?1 and a pH of ~7.8. The hyperpolarized liquid was transferred within 8 s to a fast field‐cycling relaxometer with a probe tuned for detection of ¹³C at a field strength of ~0.75 T. The magnetic field of the relaxometer was rapidly varied between relaxation and acquisition fields where the sample magnetization was periodically measured using a small flip angle. Data were recorded for relaxation fields varying between 0.237 mT and 0.705 T to map the T₁ dispersion of the C‐1 of pyruvate. Using similar methods, we also determined the relaxivity of the triarylmethyl radical (OX063; used for dynamic nuclear polarization) on the C‐1 of pyruvate at field strengths of 0.001, 0.01, 0.1 and 0.5 T using 0.075, 1.0 and 2.0 mmol l^?1 concentrations of OX063 in the hyperpolarized pyruvate solution. Copyright © 2012 John Wiley & Sons, Ltd.     

Sub‐second proton imaging of 13C hyperpolarized contrast agents in water

Indirect proton detection of ¹³C hyperpolarized contrast agents potentially enables greater sensitivity. Presented here is a study of sub‐second projection imaging of hyperpolarized ¹³C contrast agent addressing the obstacle posed by water suppression for indirect detection in vivo. Sodium acetate phantoms were used to develop and test water suppression and sub‐second imaging with frequency‐selective RF pulses using spectroscopic and imaging indirect proton detection. A 9.8 mm aqueous solution of ¹³C PHIP hyperpolarized 2‐hydroxyethyl‐¹³C‐propionate‐d_2,3,3 (HEP), <P > ~25% was used for demonstration of indirect proton sub‐second imaging detection. Balanced 2D FSSFP (fast steady‐state free precession) allowed the recording of proton images with a field of view of 64 × 64 mm² and spatial resolution 2 × 2 mm² with total acquisition time of less than 0.2 s. In thermally polarized sodium 1‐¹³C‐acetate, ¹³C to ¹H polarization transfer efficiency of 45.1% of the theoretically predicted values was observed in imaging detection corresponding to an 11‐fold overall sensitivity improvement compared with direct ¹³C FSSFP imaging. ¹³C to ¹H polarization transfer efficiency of 27% was observed in imaging detection, corresponding to a 3.25‐fold sensitivity improvement compared with direct ¹³C FSSFP imaging with hyperpolarized HEP. The range of potential applications and limitations of this sub‐second and ultra‐sensitive imaging approach are discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">In Vivo</Emphasis> DA D<Subscript>1</Subscript> Receptor Selectivity of NNC 112 and SCH 23390

Purpose [¹¹C]NNC 112 and [¹¹C]SCH 23390 are selective positron emission tomography (PET) tracers for visualizing dopamine D₁ receptors. It is known that both have some affinity for serotonin 2A receptors, but previous studies have suggested this is negligible compared to D₁ affinity. We sought to verify this property in vivo. Procedures Two baboons were scanned to measure the selectivity of both tracers with a displacement paradigm. Four baboons were scanned to directly assess [¹¹C] NNC 112 affinity for both receptors. Results In vivo, D₁ to 5-HT_2A selectivity is six to fourteenfold, not 100-fold as previously reported by other investigators. Conclusion We conclude that about 1/4 of the cortical signal of both [¹¹C]NNC 112 and [¹¹C]SCH 23390 is due to binding to 5-HT_2A receptors. If confirmed in humans, this suggests caution should be exercised when drawing conclusions from studies using either tracer. These results also indicate the need for more selective tracers for the D₁ receptor.     

Quantitation of a spin polarization‐induced nuclear Overhauser effect (SPINOE) between a hyperpolarized 13C‐labeled cell metabolite and water protons

The spin polarization‐induced nuclear Overhauser effect (SPINOE) describes the enhancement of spin polarization of solvent nuclei by the hyperpolarized spins of a solute. In this communication we demonstrate that SPINOEs can be observed between [1,4‐¹³C₂]fumarate, hyperpolarized using the dissolution dynamic nuclear polarization technique, and solvent water protons. We derive a theoretical expression for the expected enhancement and demonstrate that this fits well with experimental measurements. Although the magnitude of the effect is relatively small (around 2% measured here), the SPINOE increases at lower field strengths, so that at clinically relevant magnetic fields (1.5–3 T) it may be possible to track the passage through the circulation of a bolus containing a hyperpolarized ¹³C‐labeled substrate through the increase in solvent water ¹H signal. © 2014 The Authors. Contrast Media & Molecular Imaging published by John Wiley and Sons, Ltd.     

超极化13C MRI研究进展

“超极化”指通过动态核极化方法使原子核自旋极化达热平衡之上，以增强分子MRI信号。近年来，超极化MRI日益发展，可克服传统MR信号低、无法实时监测体内代谢过程的缺点，在检测肿瘤及其他疾病的代谢中显示出一定应用价值。本文就常用超极化¹³C生物探针、超极化MR设备及超极化¹³C MRI的研究进展进行综述，并对其发展趋势进行展望。     

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-13C]glycine and its deuteration toward longer retention of the hyperpolarized state

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-¹³C]glycine (γ-Glu-[1-¹³C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu–Gly scaffold and develop a deuterated probe, γ-Glu-[1-¹³C]Gly-d₂, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu–Gly and the magnetic properties of ¹³C-enriched γ-Glu–Gly (γ-Glu-[1-¹³C]Gly and γ-[5-¹³C]Glu–Gly) to support the validity of γ-Glu-[1-¹³C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T₁) of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ under various conditions (D₂O, PBS, and serum) and confirmed that the T₁ of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-¹³C]Gly revealed a significant contribution of the dipole–dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T₁. In fact, by using γ-Glu-[1-¹³C]Gly-d₂ as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

By examining enzymatic and magnetic properties, γ-Glu-[1-¹³C]Gly-d₂ was developed as a long-lived DNP molecular probe for detecting γ-glutamyl transpeptidase.     

In vivo magnetic resonance imaging of glucose – initial experience

A new noninvasive, nonradioactive approach for glucose imaging using spin hyperpolarization technology and stable isotope labeling is presented. A glucose analog labeled with ¹³C at all six positions increased the overall hyperpolarized imaging signal; deuteration at all seven directly bonded proton positions prolonged the spin–lattice relaxation time. High‐bandwidth ¹³C imaging overcame the large glucose carbon chemical shift dispersion. Hyperpolarized glucose images in the live rat showed time‐dependent organ distribution patterns. At 8 s after the start of bolus injection, the inferior vena cava was demonstrated at angiographic quality. Distribution of hyperpolarized glucose in the kidneys, vasculature, and heart was demonstrated at 12 and 20 s. The heart‐to‐vasculature intensity ratio at 20 s suggests myocardial uptake. Cancer imaging, currently performed with ¹⁸ F‐deoxyglucose positron emission tomography (FDG‐PET), warrants further investigation, and glucose imaging could be useful in a vast range of clinical conditions and research fields where the radiation associated with the FDG‐PET examination limits its use. Copyright © 2012 John Wiley & Sons, Ltd.     

Modulating the relaxivity of hyperpolarized substrates with gadolinium contrast agents

Gadolinium contrast agents are known to affect the outer‐sphere relaxivities of nuclei other than protons. We have investigated the heteronuclear relaxivities of four commercial gadolinium contrast agents (Magnevist, Omniscan, Dotarem and Gadovist) on the relaxation of ¹³C in glycine, ¹³C in pyruvate and ¹⁵N in choline with long relaxation times. Marked differences in the relaxivities were found between different contrast agents and are attributed to electrostatic effects. This methodology may find applications in the field of hyperpolarized magnetic resonance and by way of example we show that injection of a bolus of contrast agent into an aqueous solution containing hyperpolarized ¹⁵N labeled tetramethylammonium or ¹³C labeled pyruvate leads to a predictable shortening of the lifetime of the hyperpolarized signal. Copyright © 2009 John Wiley & Sons, Ltd.     

In Vivo Carbon-13 Dynamic MRS and MRSI of Normal and Fasted Rat Liver with Hyperpolarized 13C-Pyruvate

Background  

The use of in vivo ¹³C nuclear magnetic resonance spectroscopy in probing metabolic pathways to study normal metabolism and characterize disease physiology has been limited by its low sensitivity. However, recent technological advances have enabled greater than 50,000-fold enhancement of liquid-state polarization of metabolically active ¹³C substrates, allowing for rapid assessment of ¹³C metabolism in vivo. The present study applied hyperpolarized ¹³C magnetic resonance spectroscopy to the investigation of liver metabolism, demonstrating for the first time the feasibility of applying this technology to detect differences in liver metabolic states.     

Clinical Implications of Cardiac Hyperpolarized Magnetic Resonance Imaging

Alterations in cardiac metabolism are now considered a cause, rather than a result, of cardiac disease. Although magnetic resonance spectroscopy has allowed investigation of myocardial energetics, the inherently low sensitivity of the technique has limited its clinical application in the study of cardiac metabolism. The development of a novel hyperpolarization technique, based on the process of dynamic nuclear polarization, when combined with the metabolic tracers [1-¹³C] and [2-¹³C] pyruvate, has resulted in significant advances in the understanding of real time myocardial metabolism in the normal and diseased heart in vivo. This review focuses on the changes in myocardial substrate selection and downstream metabolism of hyperpolarized ¹³C labelled pyruvate that have been shown in diabetes, ischaemic heart disease, cardiac hypertrophy and heart failure in animal models of disease and how these could translate into clinical practice with the advent of clinical grade hyperpolarizer systems.     

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet‐decorated VWF strings in vivo

Summary. Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C‐terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13^?/? mouse. Results: Murine MDTCS (devoid of T2‐8 and CUB domains) retained full enzyme activity in vitro towards FRETS‐VWF73 and the C‐terminal T6‐8 (del(T6‐CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet‐decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6‐CUB)) or complete (delCUB) addition of the T2‐8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2‐8 domains influence proteolysis of platelet‐decorated VWF strings in vivo.     

A novel switched frequency 3He/1H high‐pass birdcage coil for imaging at 1.5 tesla

The ability to produce hyperpolarized noble gases ³He and ¹²⁹Xe has opened up exciting possibilities for pulmonary magnetic resonance imaging (MRI). We have recently built a hyperpolarizer with the goal of using hyperpolarized ³He gas for MRI in neonatal lungs in a dedicated small foot‐print 1.5 T MR scanner developed at our institution and sited in our Neonatal Intensive Care Unit. Although hyperpolarized gas imaging can provide unique insights into lung ventilation, acinar microstructure, and gas‐exchange dynamics, there is an undiminished need for ¹H MRI of the lung to provide anatomic references, B₁ and B₀ maps, and ¹H images of lung parenchyma. To address this need, we designed, built and tested a novel radiofrequency body coil that provides a high‐pass birdcage coil that can be used for both ³He and ¹H frequencies (48.65 and 63.86 MHz, respectively, at 1.5 T). To switch between frequencies, the birdcage coil has a large mechanical actuator that simultaneously changes the capacitance between every rung of the birdcage. Advantages of this coil design include: 1) quadrature excitation and reception at the ³He and ¹H frequencies, 2) identical B₁ field maps for ³He and ¹H imaging, 3) excellent signal‐to‐noise ratio and B₁ homogeneity at both frequencies, and 4) rapid (10–20 s) switching times between ³He and ¹H operation. This report provides details of the coil's design and fabrication. Images of hyperpolarized ³He and ¹H in phantoms and ex vivo rabbit lungs demonstrate the image quality obtained with the coil. © 2015 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 45B: 174–182, 2015     

A curve fitting approach to estimate the extent of fermentation of indigestible carbohydrates

Background Information about the extent of carbohydrate digestion and fermentation is critical to our ability to explore the metabolic effects of carbohydrate fermentation in vivo. We used cooked ¹³C‐labelled barley kernels, which are rich in indigestible carbohydrates, to develop a method which makes it possible to distinguish between and to assess carbohydrate digestion and fermentation. Materials and methods Seventeen volunteers ingested 86 g (dry weight) of cooked naturally ¹³C enriched barley kernels after an overnight fast. ¹³CO₂ and H₂ in breath samples were measured every half hour for 12 h. The data of ¹³CO₂ in breath before the start of the fermentation were used to fit the curve representing the digestion phase. The difference between the area under curve (AUC) of the fitted digestion curve and the AUC of the observed curve was regarded to represent the fermentation part. Different approaches were applied to determine the proportion of the ¹³C‐dose available for digestion and fermentation. Results Four hours after intake of barley, H₂‐excretion in breath started to rise. Within 12 h, 24–48% of the ¹³C‐dose was recovered as ¹³CO₂, of which 18–19% was derived from colonic fermentation and the rest from digestion. By extrapolating the curve to baseline, it was estimated that eventually 24–25% of the total available ¹³C in barley would be derived from colon fermentation. Conclusion Curve fitting, using ¹³CO₂‐ and H₂‐breath data, is a feasible and non‐invasive method to assess carbohydrate digestion and fermentation after consumption of ¹³C enriched starchy food.     

Design of a quadrature surface coil for hyperpolarized 13C MRS cardiac metabolism studies in pigs

This work describes the design of a quadrature surface coil constituted by a circular loop and a butterfly coil, employed in transmit/receive (TX/RX) mode for hyperpolarized ¹³C studies of pig heart with a clinical 3T scanner. The coil characterization is performed by developing an SNR model for coil performance evaluation in terms of coil resistance, sample‐induced resistance and magnetic field pattern. Experimental SNR‐vs.‐depth profiles, extracted from the [1–13C]acetate phantom chemical shift image (CSI), showed good agreement with the theoretical SNR‐vs.‐depth profiles. Moreover, the performance of the quadrature coil was compared with the single TX/RX circular and TX/RX butterfly coil, in order to verify the advantage of the proposed configuration over the single coils throughout the volume of interest for cardiac imaging in pig. Finally, the quadrature surface coil was tested by acquiring metabolic maps with hyperpolarized [1–13C]pyruvate injected i.v. in a pig. © 2013 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 43B: 69–77, 2013     

A new family of insect muscarinic acetylcholine receptors

Most currently used insecticides are neurotoxic chemicals that target a limited number of sites and insect cholinergic neurotransmission is the major target. A potential target for insecticide development is the muscarinic acetylcholine receptor (mAChR), which is a metabotropic G‐protein‐coupled receptor. Insects have A‐ and B‐type mAChRs and the five mammalian mAChRs are close to the A‐type. We isolated a cDNA (CG12796) from the fruit fly, Drosophila melanogaster. After heterologous expression in Chinese hamster ovary K1 cells, CG12796 could be activated by acetylcholine [EC₅₀ (half maximal effective concentration), 73 nM] and the mAChR agonist oxotremorine M (EC₅₀, 48.2 nM) to increase intracellular Ca²⁺ levels. Thus, the new mAChR is coupled to G_q/11 but not G_s and G_i/o. The classical mAChR antagonists atropine and scopolamine N‐butylbromide at 100 μM completely blocked the acetylcholine‐induced responses. The orthologues of CG12796 can also be found in the genomes of other insects, but not in the genomes of the honeybee or parasitoid wasps. Knockdown of CG12796 in the central nervous system had no effect on male courtship behaviours. We suggest that CG12796 represents the first recognized member of a novel mAChR class.     

Non‐competitive interaction between raclopride and spiperone on human D2L‐receptors in intact Chinese hamster ovary cells

We recently investigated the binding properties of the antagonists [³H]‐raclopride and [³H]‐spiperone to intact Chinese hamster ovary cells expressing recombinant human D_2long‐dopamine receptors (CHO‐D_2L cells). Compared with saturation binding with [³H]‐raclopride, raclopride reduced [³H]‐spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [³H]‐raclopride binding non‐competitively. While raclopride only decreases the apparent K_D of [³H]‐raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [³H]‐raclopride binds with high affinity. Also, while the IC₅₀ of raclopride depends on the concentration of [³H]‐raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [³H]‐spiperone nor the rebinding of freshly dissociated [³H]‐spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [³H]‐raclopride and raclopride does not affect the (genuine) dissociation rate of [³H]‐spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D_2L‐receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [³H]‐raclopride and the other one with high affinity/accessibility for [³H]‐spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non‐reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D₂‐receptor antagonists.     

Preclinical evaluation of [111In]MICA‐401, an activity‐based probe for SPECT imaging of in vivo uPA activity

Urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐1 are key players in cancer invasion and metastasis. Both uPA and PAI‐1 have been described as prognostic biomarkers; however, non‐invasive methods measuring uPA activity are lacking. We developed an indium‐111 (¹¹¹In)‐labelled activity‐based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA‐conjugated uPA inhibitor was synthesized and radiolabelled with ¹¹¹In ([¹¹¹In]MICA‐401), together with its inactive, hydrolysed form ([¹¹¹In]MICA‐402). A biodistribution study was performed in mice (healthy and tumour‐bearing), and tumour‐targeting properties were evaluated in two different cancer xenografts (MDA‐MB‐231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [¹¹¹In]MICA‐402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [¹¹¹In]MICA‐401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA‐MB‐231 and HT‐29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA‐MB‐231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non‐invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.