Antiviral Evaluation of Dispirotripiperazines against Hepatitis B Virus.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >296 million people worldwide, including ∼850,000 in the USA, and kills 820,000 annually worldwide. Current nucleos(t)ide analogue (NA) or pegylated interferon α therapies do not eradicate the virus and would benefit from a complementary antiviral drug. We performed a preliminary screen of 28 dispirotripiperazines against HBV, identifying 9 hits with EC50 of 0.7-25 µM. Compound 11826096 displays the most potent activity and represents a promising lead for future optimization. While the mechanism of action is unknown, preliminary assays limit possible targets to activities involved in RNA accumulation, translation, capsid assembly, and/or capsid stability. In addition, we built machine learning models to determine if they were able to predict the activity of this series of compounds. The novelty of these molecules indicated they were outside of the applicability domain of these models.

Lesion-level correspondence and longitudinal properties of paramagnetic rim and slowly expanding lesions in multiple sclerosis.

BACKGROUND: Paramagnetic rim lesions (PRLs) and slowly expanding lesions (SELs) have been posited as markers of chronic active lesions (CALs). OBJECTIVE: To assess the lesion-level concordance of PRLs and SELs in MS and to characterize changes in brain tissue integrity in CALs over time. METHODS: MRIs were analyzed from a substudy of AFFINITY [NCT03222973], a phase 2 trial of opicinumab in relapsing MS. Assessments included (1) identification of SELs based on longitudinal MRIs over 72 weeks, and identification of PRLs on susceptibility-weighted imaging (SWI) filtered phase images at week 72; (2) evaluation of subject-level correlation of SEL and PRL counts, volumes, and degree of lesion-level overlap between SELs and PRLs; and (3) characterization of tissue integrity over time in overlapping and non-overlapping SELs and PRLs. RESULTS: In 41 subjects, 119 chronic PRLs and 267 SELs were detected. Of 119 (39.5%) chronic PRLs, 47 co-localized with a SEL; 46/267 (17.2%) SELs co-localized with a PRL. PRLs co-localized with SELs showed expansion and worsening microstructural damage over time. SELs with and without co-localization with PRLs showed ongoing tissue damage. CONCLUSIONS: Chronic MS lesions identified as both PRL and SEL were associated with the most severe accumulation of tissue damage. TRIAL REGISTRATION: AFFINITY [NCT03222973].

Design, synthesis, and biological evaluation of novel sulfamoylbenzamide derivatives as HBV capsid assembly modulators.

Capsid assembly modulators (CAMs) represent a novel class of antiviral agents targeting hepatitis B virus (HBV) capsid to disrupt the assembly process. NVR 3-778 is the first CAM to demonstrate antiviral activity in patients infected with HBV. However, the relatively low aqueous solubility and moderate activity in the human body halted further development of NVR 3-778. To improve the anti-HBV activity and the drug-like properties of NVR 3-778, we designed and synthesized a series of NVR 3-778 derivatives. Notably, phenylboronic acid-bearing compound 7b (EC50 = 0.83 ± 0.33 µM, CC50 = 19.4 ± 5.0 µM) displayed comparable anti-HBV activity to NVR 3-778 (EC50 = 0.73 ± 0.20 µM, CC50 = 23.4 ± 7.0 µM). Besides, 7b showed improved water solubility (328.8 µg/mL, pH 7) compared to NVR 3-778 (35.8 µg/mL, pH 7). Size exclusion chromatography (SEC) and quantification of encapsidated viral RNA were used to demonstrate that 7b behaves as a class II CAM similar to NVR 3-778. Moreover, molecular dynamics (MD) simulations were conducted to rationalize the structure-activity relationships (SARs) of these novel derivatives and to understand their key interactions with the binding pocket, which provide useful indications for guiding the further rational design of more effective anti-HBV drugs.

Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold.

Hepatitis B virus (HBV) chronically infects >250 million people. It replicates by a unique protein-primed reverse transcription mechanism, and the primary anti-HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains compared to only two in most reverse transcriptases: the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviruses, HBV P has resisted structural analysis for decades. Here, we exploited computational advances to model P. The TP wrapped around the RT domain rather than forming the anticipated globular domain, with the priming tyrosine poised over the RT active site. The orientation of the RT and RNase H domains resembled that of the retroviral enzymes despite the lack of sequences analogous to the retroviral linker region. The model was validated by mapping residues with known surface exposures, docking nucleic acids, mechanistically interpreting mutations with strong phenotypes, and docking inhibitors into the RT and RNase H active sites. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses infecting rodent to fish hosts and a nackednavirus, but not in other non-retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will advance mechanistic analyses into the poorly understood enzymology of HBV reverse transcription and will enable drug development against non-active site targets for the first time.

Chronic lesion activity and disability progression in secondary progressive multiple sclerosis.

Objective: Slowly expanding lesions (SELs), a subgroup of chronic white matter lesions that gradually expand over time, have been shown to predict disability accumulation in primary progressive multiple sclerosis (MS) disease. However, the relationships between SELs, acute lesion activity (ALA), overall chronic lesion activity (CLA) and disability progression are not well understood. In this study, we examined the ASCEND phase III clinical trial, which compared natalizumab with placebo in secondary progressive MS (SPMS). Methods: Patients with complete imaging datasets between baseline and week 108 (N=600) were analysed for SEL prevalence (the number and volume of SELs), disability progression, ALA (assessed by gadolinium-enhancing lesions and new T2-hyperintense lesions) and CLA (assessed by T1-hypointense lesion volume increase within baseline T2-non-enhancing lesions identified as SELs and non-SELs). Results: CLA in both SELs and non-SELs was greater in patients with SPMS with confirmed disability progression than in those with no progression. In the complete absence of ALA at baseline and on study, SEL prevalence was significantly lower, while CLA within non-SELs remained associated with disability progression. Natalizumab decreased SEL prevalence and CLA in SELs and non-SELs compared with placebo. Conclusions: This study shows that CLA in patients with SPMS is decreased but persists in the absence of ALA and is associated with disability progression, highlighting the need for therapeutics targeting all mechanisms of CLA, including smouldering inflammation and neurodegeneration. Trial registration number: NCT01416181.

Effects of Troponoids on Mitochondrial Function and Cytotoxicity.

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.

Abnormalities in normal-appearing white matter from which multiple sclerosis lesions arise.

Normal-appearing white matter is far from normal in multiple sclerosis; little is known about the precise pathology or spatial pattern of this alteration and its relation to subsequent lesion formation. This study was undertaken to evaluate normal-appearing white matter abnormalities in brain areas where multiple sclerosis lesions subsequently form, and to investigate the spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis. Brain MRIs of pre-lesion normal-appearing white matter were analysed in participants with new T2 lesions, pooled from three clinical trials: SYNERGY (NCT01864148; n = 85 with relapsing multiple sclerosis) was the test data set; ASCEND (NCT01416181; n = 154 with secondary progressive multiple sclerosis) and ADVANCE (NCT00906399; n = 261 with relapsing-remitting multiple sclerosis) were used as validation data sets. Focal normal-appearing white matter tissue state was analysed prior to lesion formation in areas where new T2 lesions later formed (pre-lesion normal-appearing white matter) using normalized magnetization transfer ratio and T2-weighted (nT2) intensities, and compared with overall normal-appearing white matter and spatially matched contralateral normal-appearing white matter. Each outcome was analysed using linear mixed-effects models. Follow-up time (as a categorical variable), patient-level characteristics (including treatment group) and other baseline variables were treated as fixed effects. In SYNERGY, nT2 intensity was significantly higher, and normalized magnetization transfer ratio was lower in pre-lesion normal-appearing white matter versus overall and contralateral normal-appearing white matter at all time points up to 24 weeks before new T2 lesion onset. In ASCEND and ADVANCE (for which normalized magnetization transfer ratio was not available), nT2 intensity in pre-lesion normal-appearing white matter was significantly higher compared to both overall and contralateral normal-appearing white matter at all pre-lesion time points extending up to 2 years prior to lesion formation. In all trials, nT2 intensity in the contralateral normal-appearing white matter was also significantly higher at all pre-lesion time points compared to overall normal-appearing white matter. Brain atlases of normal-appearing white matter abnormalities were generated using measures of voxel-wise differences in normalized magnetization transfer ratio of normal-appearing white matter in persons with multiple sclerosis compared to scanner-matched healthy controls. We observed that overall spatial distribution of normal-appearing white matter abnormalities in persons with multiple sclerosis largely recapitulated the anatomical distribution of probabilities of T2 hyperintense lesions. Overall, these findings suggest that intrinsic spatial properties and/or longstanding precursory abnormalities of normal-appearing white matter tissue may contribute to the risk of autoimmune acute demyelination in multiple sclerosis.

Design, synthesis and evaluation of heteroaryldihydropyrimidine analogues bearing spiro ring as hepatitis B virus capsid protein inhibitors.

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 vitroanti-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.

Synthetic Derivatives of Ciclopirox are Effective Inhibitors of Cryptococcus neoformans.

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.

Dual-Isotope Cryoimaging Quantitative Autoradiography: Investigating Antibody-Drug Conjugate Distribution and Payload Delivery Through Imaging.

In vitro properties of antibody-drug conjugates (ADCs) such as binding, internalization, and cytotoxicity are often well characterized before in vivo studies. Interpretation of in vivo studies might be significantly enhanced by molecular imaging tools. We present here a dual-isotope cryoimaging quantitative autoradiography (CIQA) methodology combined with advanced 3-dimensional imaging and analysis allowing for the simultaneous study of both antibody and payload distribution in tissues of interest in a preclinical setting. Methods: TAK-264, an investigational ADC targeting anti-guanylyl cyclase C (GCC), was synthesized using tritiated monomethyl auristatin E. The tritiated ADC was then conjugated to diethylenetriaminepentaacetic acid, labeled with 111In, and evaluated in vivo in animals bearing GCC-positive and GCC-negative tumors. Results: CIQA revealed the time course of drug release from ADC and its distribution into various tumor regions that are less accessible to the antibody. For GCC-positive tumors, a representative section obtained 96 h after tracer injection showed only 0.8% of the voxels to have colocalized signal, versus over 15% of the voxels for a GCC-negative tumor section, suggesting successful and specific cleaving of the toxin in the GCC-positive lesions. Conclusion: The combination of a veteran established autoradiography technology with advanced image analysis methodologies affords an experimental tool that can support detailed characterization of ADC tumor penetration and pharmacokinetics.

A monomethyl auristatin E-conjugated antibody to guanylyl cyclase C is cytotoxic to target-expressing cells in vitro and in vivo.

Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.

Molecular imaging of aurora kinase A (AURKA) expression: Synthesis and preclinical evaluation of radiolabeled alisertib (MLN8237).

INTRODUCTION: Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is 36%-58%. Positron emission tomography (PET) tracers, imaging the expression of prognostic biomarkers, may contribute to assign appropriate management to individual patients. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection. METHODS: We synthesized [(3)H]alisertib and [(11)C]alisertib, starting from [(3)H]methyl nosylate and [(11)C]methyl iodide, respectively. We measured in vitro uptake of [(3)H]alisertib in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression, before and after siRNA-mediated AURKA downmodulation, as well as after inhibition of P-glycoprotein (P-gp) activity. We measured in vivo uptake and biodistribution of [(11)C]alisertib in nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice. RESULTS: [(3)H]Alisertib was synthesized with an overall yield of 42% and [(11)C]alisertib with an overall yield of 23%±9% (radiochemical purity ≥99%). Uptake of [(3)H]alisertib in Caco2 cells was higher than in A431 cells (P=.02) and higher than in SW480, HCT116 and MKN45 cells (P<.01). Uptake in A431 cells was higher than in SW480, HCT116 and MKN45 cells (P<.01). Downmodulation of AURKA expression reduced [(3)H]alisertib uptake in Caco2 cells (P<.01). P-gp inhibition increased [(3)H]alisertib uptake in Caco2 (P<.01) and MKN45 (P<.01) cells. In vivo stability of [(11)C]alisertib 90min post-injection was 94.7%±1.3% and tumor-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45). In brains of P-gp knockout mice [(11)C]alisertib uptake was increased compared to uptake in wild-type mice (P<.01) CONCLUSIONS: Radiolabeled alisertib can be synthesized and may have potential for the imaging of AURKA, particularly when AURKA expression is high. However, the exact mechanisms underlying alisertib accumulation need further investigation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Radiolabeled alisertib may be used for non-invasively measuring AURKA protein expression and to stratify patients for treatment accordingly.

Preclinical FLT-PET and FDG-PET imaging of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901.

INTRODUCTION: The Aurora kinases play a key role in mitosis and have recently been identified as attractive targets for therapeutic intervention in cancer. The aim of this study was therefore to investigate the utility of 3'-[(18)F]fluoro-3'-deoxythymidine (FLT) and 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) for assessment of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901. METHODS: Balb/c nude mice bearing HCT116 colorectal xenografts were treated with up to 30mg/kg TAK 901 or vehicle intravenously twice daily for two days on a weekly cycle. Tumor growth was monitored by calliper measurements and PET imaging was performed at baseline, day 4, 8, 11 and 15. Tumors were harvested at time points corresponding to days of PET imaging for analysis of ex vivo markers of cell proliferation and metabolism together with markers of Aurora B kinase inhibition including phospho-histone H3 (pHH3) and senescence associated ß-galactosidase. RESULTS: Tumor growth was inhibited by 60% on day 12 of 30mg/kg TAK-901 therapy. FLT uptake was significantly reduced by day 4 of treatment and this corresponded with reduction in bromodeoxyuridine and pHH3 staining by immunohistochemistry. All biomarkers rebounded towards baseline levels by the commencement of the next treatment cycle, consistent with release of Aurora B kinase suppression. TAK-901 therapy had no impact on glucose metabolism as assessed by FDG uptake and GLUT1 staining by immunohistochemistry. CONCLUSIONS: FLT-PET, but not FDG-PET, is a robust non-invasive imaging biomarker of early HCT116 tumor response to the on-target effects of the multi-targeted Aurora B kinase inhibitor, TAK-901. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This is the first report to demonstrate the impact of the multi-targeted Aurora B kinase inhibitor, TAK-901 on tumor FLT uptake. The findings provide a strong rationale for the evaluation of FLT-PET as an early biomarker of tumor response in the early phase clinical development of this compound.

A multi-parametric imaging investigation of the response of C6 glioma xenografts to MLN0518 (tandutinib) treatment.

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/ß, 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.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

PURPOSE: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). EXPERIMENTAL DESIGN: Both cell line-derived OCI-Ly10 and primary human lymphoma-derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) was used to determine antitumor activity and effects on osteolytic bone disease. RESULTS: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMyc(Cα)/Bcl-X(L) GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. CONCLUSIONS: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials.

Comparison of model-based arterial input functions for dynamic contrast-enhanced MRI in tumor bearing rats.

When using tracer kinetic modeling to analyze dynamic contrast-enhanced MRI (DCE-MRI) it is necessary to identify an appropriate arterial input function (AIF). The measured AIF is often poorly sampled in both clinical and preclinical MR systems due to the initial rapid increase in contrast agent concentration and the subsequent large-scale signal change that occurs in the arteries. However, little work has been carried out to quantify the sensitivity of tracer kinetic modeling parameters to the form of AIF. Using a preclinical experimental data set, we sought to measure the effect of varying model forms of AIF on the extended Kety compartmental model parameters (K(trans), v(e), and v(p)) through comparison with the results of experimentally acquired high temporal resolution AIFs. The AIF models examined have the potential to be parameterized on lower temporal resolution data to predict the form of the true, higher temporal resolution AIF. The models were also evaluated through application to the population average AIF. It was concluded that, in the instance of low temporal resolution or noisy data, it may be preferable to use a bi-exponential model applied to the raw data AIF, or when individual measurements are not available a bi-exponential model of the average AIF.

Examining the acute effects of cediranib (RECENTIN, AZD2171) treatment in tumor models: a dynamic contrast-enhanced MRI study using gadopentate.

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.

Correlation of MRI biomarkers with tumor necrosis in Hras5 tumor xenograft in athymic rats.

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.

Misregistration artifacts in image-derived arterial input function in non-echo-planar imaging-based dynamic contrast-enhanced MRI.

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.

Physiological studies of cortical spreading depression.

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.