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Opportunistic fungal infections caused by Cryptococcus neoformans are a significant source of mortality in immunocompromised patients. They are challenging to treat because of a limited number of antifungal drugs, and novel and more effective anticryptococcal therapies are needed. Ciclopirox olamine, a N-hydroxypyridone, has been in use as an approved therapeutic agent for the treatment of topical fungal infections for more than two decades. It is a fungicide, with broad activity across multiple fungal species. We synthesized 10 N-hydroxypyridone derivatives to develop an initial structure-activity understanding relative to efficacy as a starting point for the development of systemic antifungals. We screened the derivatives for antifungal activity against C. neoformans and Cryptococcus gattii and counter-screened for specificity in Candida albicans and two Malassezia species. Eight of the ten show inhibition at 1-3 μM concentration (0.17-0.42 μg per mL) in both Cryptococcus species and in C. albicans, but poor activity in the Malassezia species. In C. neoformans, the N-hydroxypyridones are fungicides, are not antagonistic with either fluconazole or amphotericin B, and are synergistic with multiple inhibitors of the mitochondrial electron transport chain. They appear to function primarily by chelating iron within the active site of iron-dependent enzymes. This preliminary structure-activity relationship points to the need for a lipophilic functional group at position six of the N-hydroxypyridone ring and identifies positions four and six as sites where further substitution may be tolerated. These molecules provide a clear starting point for future optimization for efficacy and target identification.

The α-hydroxytropolones (αHTs) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target HBV RNase H with submicromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays that assess mitochondrial function. Previous studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial/nuclear genome ratios, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass interfered with these processes. The troponoids had no impact on the mitochondrial DNA/nuclear DNA ratio after 3 days of compound exposure. The patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon the addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines, leading to cytotoxicity, but are not cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by coadministering ROS antagonists to ameliorate increased ROS levels.

GLS4, a potent antiviral drug candidate, has been widely studied and entered into phase II clinical trials. Nevertheless, the therapeutic application of GLS4 is limited due to poor water solubility, short half-life, and low bioavailability. In order to improve the hydrophilicity and pharmacokinetic (PK) properties of GLS4, herein, we retained the dominant fragments, and used a scaffold hopping strategy to replace the easily metabolized morpholine ring of GLS4 with diverse sizes of spiro rings consisting of hydrogen bond donor and acceptor substituents. Potent in vitro anti-HBV activity and low cytotoxicity were observed for compound 4r (EC50 = 0.20 ± 0.00 μM, CC50 > 87.03 μM), which was more potent than the positive control lamivudine (EC50 = 0.37 ± 0.04 μM, CC50 > 100.00 μM) in this assay and was about a quarter as effective as GLS4 (EC50 = 0.045 ± 0.01 μM, CC50 > 99.20 μM). Preliminary structure-activity relationship (SAR) analysis and molecular docking studies were carried out to explore potential interactions and binding mode between compounds and target protein. In terms of the physicochemical properties, 4r was predicted to be consistent with the rule-of-five, which means 4r may have favourable absorption and permeation. Finally, ADMET and PK characteristics of 4r and GLS4 were predicted to be comparable in most aspects, implying that the two compounds may have similar profiles in vivo.

Magnetic resonance imaging (MRI) can measure the effects of therapies targeting the tumor vasculature and has demonstrated that vascular-damaging agents (VDA) induce acute vascular shutdown in tumors in human and animal models. However, at subtherapeutic doses, blood flow may recover before the induction of significant levels of necrosis. We present the relationship between changes in MRI biomarkers and tumor necrosis. Multiple MRI measurements were taken at 4.7 T in athymic rats (n = 24) bearing 1.94 +/- 0.2-cm3 subcutaneous Hras5 tumors (ATCC 41000) before and 24 hours after clinically relevant doses of the VDA, ZD6126 (0-10 mg/kg, i.v.). We measured effective transverse relaxation rate (R2*), initial area under the gadolinium concentration-time curve (IAUGC(60/150)), equivalent enhancing fractions (EHF(60/150)), time constant (K(trans)), proportion of hypoperfused voxels as estimated from fit failures in K(trans) analysis, and signal intensity (SI) in T2-weighted MRI (T(2)W). ZD6126 treatment induced > 90% dose-dependent tumor necrosis at 10 mg/kg; correspondingly, SI changes were evident from T2W MRI. Although R2* did not correlate, other MRI biomarkers significantly correlated with necrosis at doses of > or = 5 mg/kg ZD6126. These data on Hras5 tumors suggest that the quantification of hypoperfused voxels might provide a useful biomarker of tumor necrosis.

The effect of two novel therapeutic agents on tumour haemodynamics was investigated using a fast dynamic contrast-enhanced (DCE)-MRI protocol (0.5 s/image) sensitive to signal changes in both the vascular input function and tumour during the administration of the macromolecular rapid clearance blood pool agent (MM-RCBPA), gadomelitol (P792, Vistarem). This enabled simultaneous measurement of the tumour blood flow per unit volume of tissue (F/V(T), mL/s/mL), the fractional plasma volume (V(p), %), and the permeability surface area product per unit volume of tissue (PSrho, s(-1)) in subcutaneous SW620 human colorectal tumour xenografts grown in nude rats before and after (at 0 and 22 h; imaging at 24 h) acute treatment with AZD2171 (3 mg/kg) and vandetanib (ZD6474, Zactima; 50 mg/kg), which have inhibitory activity against vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase. MRI was performed at 4.7 T using a single-slice, modified, T(1)-weighted, spoiled gradient-echo technique. Both compounds reduced gadomelitol uptake into the tumour. AZD2171 and vandetanib, respectively, (a) greatly reduced PSrho to 19.7 +/- 9.5% and 28.9 +/- 14.1% of baseline (P = 0.007 and P = 0.02), (b) markedly reduced V(p) to 31.2 +/- 19.1% and 54.8 +/- 21.2% of baseline (P = 0.015 and P = 0.09), and (c) had no significant effect on F/V(T). There was no significant difference between groups treated with AZD2171 and vandetanib when each variable was compared. The reductions in PSrho and V(p) are consistent with inhibition of VEGF signalling. AZD2171 (3 mg/kg) and vandetanib (50 mg/kg) were also found to produce a comparable chronic inhibition of SW620 tumour growth (89% for both). This study shows that DCE-MRI using an MM-RCPBA can be used to distinguish tumour vascular flow, volume, and permeability surface area product in a tumour model, and enables the acute effects of VEGF signalling inhibition to be examined in detail.

PURPOSE: To characterize misregistration artifact in arterial input function (AIF) pixels in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a two-dimensional non-echo-planar imaging (EPI)-based gradient-recalled echo (GRE) sequence. MATERIALS AND METHODS: Dynamic gadopentetate-enhanced MRI was acquired in the rat using a semikeyhole acquisition scheme. The AIF was obtained from abdominal aorta pixels. Different sliding-window reconstruction techniques were applied to determine which lines in a series of the semikeyhole acquisition were associated with the misregistration artifacts. RESULTS: The misregistration along the phase-encoding direction arose when k-space lines were acquired during the rise-time of the aortic gadolinium concentration. The maximum blood concentration of gadolinium estimated from the phase shift calculation agreed with that estimated from dosage. CONCLUSION: AIF misregistration results from a phase shift due to increasing gadolinium concentration in the aorta, and may need to be considered in small animal DCE-MRI studies with a high rate of rise in the AIF in high-field MR applications. (c) 2007 Wiley-Liss, Inc.

An application of independent component analysis to blood oxygenation level- dependent MRI (BOLD-MRI) results was used to detect cerebrovascular changes that followed the initiation of cortical spreading depression (CSD) in feline brain. The cortical images were obtained from a horizontal plane at 28 s intervals before, and for 1.4-1.75 h after, KCl dissolved in agar (KCl/agar) had been directly applied to the left suprasylvian gyrus of 13 anesthetized cats for 10 min. It successfully resolved, for the first time, a novel class of prolonged, and delayed, biphasic BOLD waveforms. These were larger in amplitude ( approximately 20%), longer lasting and more delayed in onset (13-33 min) than the brief propagating (90 s) BOLD increase ( approximately 4%) already known to be associated with CSD on earlier occasions. Furthermore, such changes occurred in localized regions on the hemisphere ipsilateral to the site of stimulus application in 4 out of 5 control subjects rather than themselves generating propagating waves. Finally, the biphasic waveforms were consistently abolished in the 4 experimental animals studied following the i.v. administration of sumatriptan (0.3 mg kg(-1)), an antimigraine 5-HT(1B/1D) agonist, 15 min before the application of the transient stimulus. They were abolished in 2 out of 4 animals following the intraperitoneal (i.p.) administration of SB-220453 (tonabersat: 10 mg kg(-1), 90 min before stimulus application), a novel anticonvulsant that has recently been reported to inhibit CSD. ICA has thus been successful in detecting a novel localized, as opposed to propagating, signal of potential physiological significance hidden in complex BOLD- MRI data, whose sensitivity to sumatriptan may relate it to the cerebrovascular changes reported in the headache phase of migraine.

Cortical spreading depression (CSD) in the gyrencephalic cat brain was detected with diffusion-weighted echoplanar (DWEP) magnetic resonance imaging (4-8/min for 1-2 hours) using a horizontal imaging plane through the suprasylvian (SG) and marginal gyri. A t-statistic mapping technique allowed a quantitative characterization of the passage of events through single-image pixels (0.15 mm(2)), thus providing a resolution unavailable to previous studies in which time-dependent changes instead were derived from averaging data over relatively large ROIs. Using the enhanced analysis, CSD events initiated by KCl could be quantified for the first time as primary or secondary according to their spatial and temporal features. Primary events covered 26.2 +/- 9.9 mm(2)of cortical surface (mean +/- SD, n = 7 experiments) and propagated rapidly (3.5 +/- 0.65 mm * min(-1)) with a hemispherical geometry. In contrast, the subsequent secondary events were multiple, spatially restricted (covering 7.6 +/- 4.6 mm(2), P < 0.005), slower in propagation (2.6 +/- 0.41 mm * min(-1), P < 0.012), and often confined to the originating gyrus (26 out of 59 events). However, both event types were associated with significantly reduced apparent diffusion coefficients (ADCs; from 800 to approximately 660 x 10(-6) mm(2)* s(-1), P < 0.05) that were similar for both primary (21 +/- 5.1%) and secondary waves (18 +/- 7. 7%) and that had similar durations (full width at half-maximal height: 86 +/- 17 vs. 79 +/- 20 seconds, respectively). These findings associate CSD for the first time with two categories of ADC disturbance that are similar in amplitude and duration but that differ in spatial extent, velocity, and extensiveness of spread.

Magic-angle spinning (MAS) has recently been shown to enhance spectral resolution in NMR examinations of intact biological tissue ex vivo. This work demonstrates that freezing certain tissue samples before examination by 1H MAS NMR can have a marked effect on their spectra. Spectra of rat kidney after freezing in liquid nitrogen, compared with spectra before freezing, showed a significant increase in signal intensities from alanine (>100%), glutamine (>40%), and glycine (>100%), and a decrease in signals assigned to lipids and other macromolecules. Some resonances--such as from leucine, valine, isoleucine, and aspartate--only became visible after freezing the tissue. These observations suggest that low temperature storage of tissue necropsies or biopsies might affect the results of a MAS NMR analysis, possibly resulting in the misinterpretation of metabolite changes to pathogen or disease effects.

Antibody-drug conjugates (ADCs) are optimized extensively in in vitro studies. Including drug-conjugate selection, linker stability and antibody affinity, most ADC characteristics have been studied to improve affinity, stability, efficacy and the bystander effect. Most nuclear medicine molecular imaging modalities’ resolution is too low to enable accurate in vivo intratumoral tracer distribution analysis. Thus, they have focused on understanding in vivo PK profiles and antigen-dependent tumor accumulation. We set out to develop a novel method for studying the intratumoral distribution of both antibody and drug conjugate simultaneously ex vivo. MLN0264, an ADC targeting guanylyl cyclase C (GCC) currently in phase 2 clinical trials (NCT02391038), was labeled with both 3H (MMAE drug conjugate) and 111In (DTPA-mAb) and injected into GCC-positive (GCC-293) and GCC-negative (HEK-293) subcutaneous tumor bearing female SCID mice. The tumors were excised 1, 8, 24, and 96 hours post injection (n = 2 per tumor line per time point), blocked and sectioned (30 μm) for analysis. High resolution optical images were acquired for all sections and every 10th section was evaluated for radioactivity content via autoradiography, first to evaluate the distribution of 111In immediately after tumor excision and again following 111In decay to evaluate the 3H-specific signal. Analysis of accumulation, distribution and overlap of the two signals enables the estimation of antigen-mediated metabolism of the ADC, the tumoral distribution of the drug metabolites and the time course of the bystander effect (Fig 1A). The distribution of 3H and 111In signals at 1 h was very similar for the two cell lines. At 24 and 96 hours, substantial differences in the co-localization of signals were observed between the antigen-positive and antigen-negative tumors. Quantitatively, of the top 3% of pixel values in the 3H and 111In images at 96 hours, only 0.8% of GCC-293 tumor voxels shows an overlap of signals while over 15% of voxels in the HEK-293 tumors express both signals suggesting increased ADC metabolism and bystander effect in antigen positive tumors (Fig 1B). Cryo-Imaging Quantitative Autoradiography (CIQA) is a novel technique to extend conventional autoradiography by combining it with digital imaging and advanced 3D image analysis. For the first time, we demonstrate here the use of CIQA to quantify and visualize the two major components of an ADC, the mAb and small molecule drug simultaneously in three dimensions over time (4D). We believe this powerful and unique tool will allow for increased insight into the influence of ADC properties on tumor spatial distribution, in vivo bystander effect, off-target ADC metabolism and correlation of 3D distribution and heterogeneity with immunohistochemical markers to enable more accurate pharmacodynamic profiles. Citation Format: Ohad Ilovich, Mohammed Qutaish, Jacob Hesterman, Kelly Orcutt, Jack Hoppin, Ildiko Polyak, Marc Seaman, Paige Czarnecki, Vijay Gottumukkala, Mihaela Plesescu, Ozlem Yardibi, Daniel Bradley. A dual-isotope 3D cryo-imaging quantitative autoradiography (CIQA) method for simultaneous and quantitative assessment of both antibody and drug conjugate tumor distribution and kinetics. [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 LB-185.

Cediranib (RECENTIN, AZD2171) is a highly potent inhibitor of the tyrosine kinase activity associated with all three vascular endothelial growth factor (VEGF) receptors and is currently in Phase II/III clinical trials. Preclinically, cediranib inhibits VEGF signaling and angiogenesis in vivo and impedes solid tumor growth significantly. Clinically, changes observed using dynamic contrast-enhanced MRI (DCE-MRI) with gadopentate suggest that acute cediranib treatment compromises tumor hemodynamics. In this study, a DCE-MRI baseline scan using gadopentate was performed in nude rats bearing Lovo (human colorectal carcinoma) or C6 (rat glioma) tumors. Cediranib (3 mg/kg per day) or vehicle was then dosed orally (2, 26 and 50 h after the baseline scan; 12 rats per group) and a second scan acquired 2 h after the final dosing event. Mean values for K(trans) (Tofts and Kermode-derived) [Magn Reson Med 17 (1991) 357-67] and the initial area under the gadolinium concentration curve over the first 60 s (iAUC) were reduced significantly following cediranib treatment: K(trans) by 33% (P.05) in both tumor models and iAUC by 23% (P.05) and 33% (P.005) in Lovo and C6, respectively. This is the first preclinical investigation to examine the effect of cediranib treatment on tumors by DCE-MRI with gadopentate.

Cortical spreading depression (CSD) produces propagating waves of transient neuronal hyperexcitability followed by depression. CSD is initiated by K+ release following neuronal firing or electrical, mechanical or chemical stimuli. A triphasic (30-50 s) cortical potential transient accompanies localized transmembrane redistributions of K+, glutamate, Ca2+, Na+, Cl- and H+. Accumulated K+ in the restricted interstitial space can cause both further neuronal depolarisation and inward movement of K+ into astrocytes that buffers this increased extracellular K+ concentration ([K+])o. However, astrocyte interconnections may then propagate the CSD wave by K+ liberation through an opening of remote K+ channels by volume, Ca2+ or N-methyl-D-aspartate receptor activation. Changes in cerebral blood volume and in apparent water diffusion co-efficient (ADC) accompanying CSD were first studied using magnetic resonance imaging (MRI) in whole lissencephalic brains. Diffusion-weighted echoplanar imaging in gyrencephalic brains went on to demonstrate CSD features that paralleled classical migraine aura. The ADC activity persisted minutes/hours post KCl stimulus. Pixelwise analyses distinguished single primary events and multiple, spatially restricted, slower propagating, secondary events whose detailed features varied with the nature of the originating stimulus. These ADC changes varied reciprocally with T2*-weighted (i.e. referring to spin-spin relaxation times) waveforms reflecting local blood flow. There followed prolonged decreases in cerebral blood flow culminating in late cerebrovascular changes blocked by the antimigraine agent sumatriptan. CSD phenomena have possible translational significance for human migraine aura and other cerebral pathologies such as the peri-infarct depolarisation events that follow ischaemia and brain injury.

Cortical spreading depression (CSD) was induced by transient (10 min) applications of KCl in agar upon the cortical surface of alpha-chloralose anaesthetised cats. Its features were compared with CSD resulting from sustained applications of crystalline KCl through a mapping of the apparent diffusion coefficient (ADC) using diffusion-weighted echo planar imaging (DWI) over a poststimulus period of 60-100 min. Individual CSD events were computationally detected with the aid of Savitzky-Golay smoothing applied to critically sampled data derived from regions of interest (ROIs) made up of 2 x 2 pixel matrices. The latter were consistently placed at three selected sites on the suprasylvian gyrus (SG) and six sites on the marginal gyrus (MG). The CSD events thus detected were then quantitatively characterised for each ROI using the original time series. Both stimuli consistently elicited similar spreading patterns of initial, primary CSD events that propagated over the SG and marginal MG and were restricted to the hemispheres on which the stimuli were applied. There followed secondary events over smaller extents of cortical surface. Sustained stimuli elicited primary and secondary CSD events with similar amplitudes of ADC deflection that were distributed around a single mean. The ADC deflections were also conserved in peak amplitude throughout the course of their propagation. The initial primary event showed a poststimulus latency of 1.1 +/- 0.1 min. Successive secondary events followed at longer, but uniform, time intervals of around 10 min. Primary and secondary CSDs showed significantly different velocities of conduction (3.32 +/- 0.43 mm min(-1) vs. 2.11 +/- 0.21 mm min(-1), respectively; n = 5) across the cerebral hemisphere. In contrast, transient stimuli produced significantly fewer numbers of CSD events (3.8 +/- 0.5 events per animal, n = 5) than did sustained stimuli (7.4 +/- 0.5 events per animal, mean +/- S.E.M., n = 5, P = 0.002). The peak ADC deflection of their primary CSD events declined by approximately 30 % as they propagated from their initiation site to the interhemispheric boundary. The primary CSD event following a transient stimulus showed a latency of 1.4 +/- 0.1 min. It was followed by successive and smaller secondary ADC deflections that were separated by progressively longer time intervals. Conduction velocities of secondary events were similar to those of primary events. Conduction velocities of both primary and secondary events were slower than their counterparts following a sustained stimulus. ADC changes associated with CSD thus persist at times well after stimulus withdrawal and vary markedly with the nature of the initiating stimulus even in brain regions remote from the stimulus site.

Angiogenesis, the development of new blood vessels, is essential for tumour growth; this process is stimulated by the secretion of numerous growth factors including platelet derived growth factor (PDGF). PDGF signalling, through its receptor platelet derived growth factor receptor (PDGFR), is involved in vessel maturation, stimulation of angiogenesis and upregulation of other angiogenic factors, including vascular endothelial growth factor (VEGF). PDGFR is a promising target for anti-cancer therapy because it is expressed on both tumour cells and stromal cells associated with the vasculature. MLN0518 (tandutinib) is a potent inhibitor of type III receptor tyrosine kinases that demonstrates activity against PDGFRα/β, FLT3 and c-KIT. In this study a multi-parametric MRI and histopathological approach was used to interrogate changes in vascular haemodynamics, structural response and hypoxia in C6 glioma xenografts in response to treatment with MLN0518. The doubling time of tumours in mice treated with MLN0518 was significantly longer than tumours in vehicle treated mice. The perfused vessel area, number of alpha smooth muscle actin positive vessels and hypoxic area in MLN0518 treated tumours were also significantly lower after 10 days treatment. These changes were not accompanied by alterations in vessel calibre or fractional blood volume as assessed using susceptibility contrast MRI. Histological assessment of vessel size and total perfused area did not demonstrate any change with treatment. Intrinsic susceptibility MRI did not reveal any difference in baseline R2* or carbogen-induced change in R2*. Dynamic contrast-enhanced MRI revealed anti-vascular effects of MLN0518 following 3 days treatment. Hypoxia confers chemo- and radio-resistance, and alongside PDGF, is implicated in evasive resistance to agents targeted against VEGF signalling. PDGFR antagonists may improve potency and efficacy of other therapeutics in combination. This study highlights the challenges of identifying appropriate quantitative imaging response biomarkers in heterogeneous models, particularly considering the multifaceted roles of angiogenic growth factors.