Preclinical Systems Research Articles

Risk Management in Preclinical Trials as Part of an Overall Integrated Quality Management System

The implementation of a complex risk-oriented approach at all stages of drug development, including the preclinical research stage, is essential for ensuring drug safety, quality and effi cacy. The aim of the study was to justify the benefits of introducing risk management into the integrated quality management system at the preclinical trial stage. The optimal approach to implementing risk management system at the preclinical stage is an overall process approach based on the international standard ISO 31000:2009 Risk management—Principles and guidelines. This approach as applied to a preclinical research centre includes a three-tier risk management system at the level of operational process subsystems, the level of the testing centre’s departments, and the level of an individual preclinical study. The common components of the subsystems may include concepts, organisational structure, information exchange system, documentation structure, etc. The process of risk management as applied to the subsystems includes risk identification, analysis, assessment, and response to the risk. The response to the risk is an iterative process that includes selection of response options, response planning, and implementation of response activities from control procedures to corrective and preventive actions. The integration of the preclinical risk management system into the organisation’s overall management system, which includes the occupational health and safety management system and the quality management system, will enhance the capabilities of the individual systems to achieve a high quality level of research, preserve the staff ’s professional health, make informed management decisions within the organisation, increase preparedness for external audits, and gain competitive advantages.

Ensemble modeling highlights importance of understanding parasite-host behavior in preclinical antimalarial drug development

Emerging drug resistance and high-attrition rates in early and late stage drug development necessitate accelerated development of antimalarial compounds. However, systematic and meaningful translation of drug efficacy and host-parasite dynamics between preclinical testing stages is missing. We developed an ensemble of mathematical within-host parasite growth and antimalarial action models, fitted to extensive data from four antimalarials with different modes of action, to assess host-parasite interactions in two preclinical drug testing systems of murine parasite P. berghei in mice, and human parasite P. falciparum in immune-deficient mice. We find properties of the host-parasite system, namely resource availability, parasite maturation and virulence, drive P. berghei dynamics and drug efficacy, whereas experimental constraints primarily influence P. falciparum infection and drug efficacy. Furthermore, uninvestigated parasite behavior such as dormancy influences parasite recrudescence following non-curative treatment and requires further investigation. Taken together, host-parasite interactions should be considered for meaningful translation of pharmacodynamic properties between murine systems and for predicting human efficacious treatment.

Acceleration of three-dimensional diffusion magnetic resonance imaging using a kernel low-rank compressed sensing method

Diffusion Magnetic Resonance Imaging (dMRI) has shown great potential in probing tissue microstructure and structural connectivity in the brain but is often limited by the lengthy scan time needed to sample the diffusion profile by acquiring multiple diffusion weighted images (DWIs). Although parallel imaging technique has improved the speed of dMRI acquisition, attaining high resolution three dimensional (3D) dMRI on preclinical MRI systems remained still time consuming. In this paper, kernel principal component analysis, a machine learning approach, was employed to estimate the correlation among DWIs. We demonstrated the feasibility of such correlation estimation from low-resolution training DWIs and used the correlation as a constraint to reconstruct high-resolution DWIs from highly under-sampled k-space data, which significantly reduced the scan time. Using full k-space 3D dMRI data of post-mortem mouse brains, we retrospectively compared the performance of the so-called kernel low rank (KLR) method with a conventional compressed sensing (CS) method in terms of image quality and ability to resolve complex fiber orientations and connectivity. The results demonstrated that the KLR-CS method outperformed the conventional CS method for acceleration factors up to 8 and was likely to enhance our ability to investigate brain microstructure and connectivity using high-resolution 3D dMRI.

Validation of the analytical irradiator model and Monte Carlo dose engine in the small animal irradiation treatment planning system µ-RayStation 8B

Dose calculation in preclinical context with a clinical level of accuracy is a challenge due to the small animal scale and the medium photon energy range. In this work, we evaluate the effectiveness and accuracy of an analytical irradiator model combined with Monte Carlo (MC) calculations in the irradiated volume to calculate the dose delivered by a modern small animal irradiator.A model of the XRAD225Cx was created in µ-RayStation 8B, a preclinical treatment planning system, allowing arc and static beams for seven cylindrical collimators. Calculations with the µ-RayStation MC dose engine were compared with EBT3 measurements in water for all static beams and with a validated GATE model in water, heterogeneous media and a mouse CT. The GATE model is a complete MC representation of the XRAD225Cx.In water, µ-RayStation calculations, compared to GATE calculations and EBT3 measurements, agreed within a maximal error of 3.2% (mean absolute error of 0.6% and 0.8% respectively) and maximal distance-to-agreement (DTA) was 0.2 mm at 50% of the central dose. For a 5 mm static beam in heterogeneous media, the maximal absolute error between µ-RayStation and GATE calculations was below 1.3% in each medium and DTA was 0.1 mm at interfaces. For calculations on a mouse CT, µ-RayStation and GATE calculations agreed well for both static and arc beams. The 2D local gamma passing rate was >98.9% for 1%/0.3 mm criteria and >92.9% for 1%/0.2 mm criteria. Moreover, µ-RayStation reduces calculation time significantly comparing with GATE (speed-up factor between 120 and 680).These findings show that the analytical irradiator model presented in this work combined with the µ-RayStation MC dose engine accurately computes dose for the XRAD225Cx irradiator. The improvements in calculation time and availability of functionality and tools for managing, planning and evaluating the irradiation makes this platform very useful for pre-clinical irradiation research.

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT)

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a “Use of 3D Tissues in Genotoxicity Testing” working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Effects of 0.02% netarsudil ophthalmic solution on intraocular pressure of normotensive dogs.

To evaluate the effect of QD or BID 0.02% netarsudil ophthalmic solution (Aerie Pharmaceuticals) on intraocular pressure (IOP) in normotensive dogs and to describe any adverse effects. Normotensive Labrador retriever dogs were included in this study: 10 received netarsudil in one eye and artificial tears in the contralateral eye QD, and 10 received netarsudil in one eye and artificial tears in the contralateral eye BID. Intraocular pressure curves were acquired over a 3-day acclimation period, 5-day dosing period (QD or BID-10 dogs/group), and 3-day recovery period. Toxicity was assessed daily using slit-lamp biomicroscopy and the semiquantitative preclinical ocular toxicology scoring system. Once-daily dosing did not lower IOP over the entire 5-day dosing period (95% CI 0.1 to -0.9mmHg, P=.20) or on the last day of dosing (95% CI 0.4 to -0.9mmHg, P=.65). Twice-daily dosing resulted in a statistically significant, but clinically unimportant, IOP reduction over the entire 5-day dosing period (-0.6mmHg; 95% CI 0.05 to -1.1mmHg, P=.02) and on the last day of dosing (-0.9mmHg; 95% CI 0.2 to -1.5mmHg, P=.003). Adverse events were limited to transient mild-to-moderate conjunctival hyperemia during the dosing phase in eyes receiving netarsudil vs control (P<.0001). Netarsudil 0.02% ophthalmic solution twice daily resulted in a small, statistically significant, but clinically unimportant, IOP reduction in normotensive dogs. Future studies should investigate efficacy in glaucomatous dogs.

Strong demands for the new preclinical assessment system of the CNS adverse effects

Human induced pluripotent stem cell-derived neurons (hiPSC-neurons) have potentials to improve the predictability of the adverse effects (AEs) in the human central nervous system (CNS). We have succeeded in reproducing the human neural networks stably on dish. This is important because most of the CNS AEs were caused not by the neuronal death but by the abnormal neural network activities. Our group have been attempting to establish the in vitro assay system to detect abnormal neural network activities by microelectrode array system (MEA). Furthermore, the algorithm that can determine the synchronized burst firing (SBF) objectively was developed in the collaboration of our iPS Non-clinical Experiments for Nervous System (iNCENS) project and Consortium for Safety Assessment using Human iPS Cells (CSAHi). iNCENS and CSAHi are also involved in the international Life Sciences Institute (ILSI): Health and Environmental Sciences Institute (HESI) HESI NeuTox MEA sub team, and are participating in the pilot study using 12 compounds. In the big flow to develop physiological CNS safety assessment system, we have to show Japan initiative based on our innovative iPS technology we have developed so far.

Flow cytometric quantification of neutral lipids in a human skin stem cell-derived model of NASH

Non-alcoholic steatohepatitis (NASH) is a severe chronic liver disease that affects 3 to 5 percent of the world population. It is characterized by hepatic lipid accumulation and inflammation and can progress towards fibrosis, cirrhosis and hepatocellular carcinoma. Until today, no drug has been approved for the treatment of NASH. This delay relates to the complex pathogenesis of NASH and also to a lack of appropriate predictive preclinical testing systems. Furthermore, the human specificity of the NASH pathology hampers a fortiori clinical translation of animal studies.Therefore, we recently employed human skin-derived precursors (hSKP) differentiated to hepatocyte-like cells (hSKP-HPC) as a human-relevant cell source for modelling NASH in vitro. Using this in vitro NASH model, it was possible to test novel drugs being developed for anti-NASH therapy, such as elafibranor. Since steatosis is an important aspect of NASH and multiple drugs are being developed to decelerate and reduce lipid accumulation in the liver, we optimized a flow cytometric method for quantifying neutral lipids in ‘NASH’-triggered hSKP-HPC. This methodology enables efficient identification of anti-steatotic properties of new medicines.• NASH-triggered hSKP-HPC robustly accumulate lipids intracellularly.• Flow cytometric quantification of neutral lipids in NASH-triggered hSKP-HPC allows for accurate determination of the steatotic response.• This method enables efficient identification of potential anti-steatotic drugs in a human-specific model

Rapid transition of a preclinical health systems science and social justice course to remote learning in the time of coronavirus

ABSTRACT As the coronavirus pandemic started, we rapidly transitioned a preclinical social justice and health systems sciences course at our medical school to asynchronous, remote learning. We describe processes, curricular innovations, and lessons learned. Small groups were converted into independent learning modules and lectures were given live via videoconferencing technology. We started with a simplified approach and then built technological capabilities over time. Current events were incorporated into curriculum and assessment. Our course ran from 16 March–3 April 2020 for the 155-person first-year class. Student attendance for optional, synchronous remote sessions was higher than in-person attendance in previous years. Completion rates for assignments were high but with minimal student collaboration. Faculty office hours were underutilized. Focus group and formal evaluations were largely positive, with numerical ratings for quality of the course and faculty teaching higher than the 2 years prior. Student engagement with social justice topics in aremote format was successful through modifications to small groups and lecture structure. Students, faculty, and administrative staff appreciated the consistency of session format throughout the course. Students exam performance was similar to prior years. Attention should be paid to what can be learned via self-study as opposed to small group learning. Better methods of soliciting real-time student feedback, and encouraging engagement with each other and with faculty in aremote environment are needed.

Abstract C02: Establishment of a human PDAC explant culture model for treatment prediction and characterization of the tumor microenvironment

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer, with a 5-year survival of less than 8%. To date, the therapeutic approaches are chemotherapy regimens. Despite promising data from various preclinical models, new immunotherapeutic agents like checkpoint inhibitors (CPI), vaccines, oncolytic viruses, or TGFβ inhibitors do not show a benefit in the overall survival of patients. Thus, especially in PDAC and in respect to the immune system, there is an urgent need for sufficient preclinical systems that can mirror a human patient scenario. Therefore, we developed a human-based pipeline of whole explant tissue culture using PDAC to predict responses of the tumor to different types of treatments in a personalized manner. Material and Methods: Human cancer tissue is taken into culture after surgery for several days. We tested different timings and technical variations (e.g., thickness of explants, medium changing protocols) to find superior conditions for tissue viability. In culture, the explants can be treated with (pre-) clinical-grade drugs and subsequently processed for quality controls via evaluation of the tissue integrity (on H&amp;E slides) and cytokine stability. Also, histologic analyzes (e.g., cancer cells, T cells, B cells, macrophages) as well as multiplex cytokine analyzes are carried out. This combined approach enables us to study the impact of various drugs on the tissue in terms of tumor cell death, changes in the infiltration and activation of lymphocytes and macrophage polarization, etc. Results In vivo features of the tissue and the matrix architecture as well as the cytokine signatures are well maintained in the explants. Our model is reproducible among different explants of the same tissue in terms of infiltrating cells and cytokine expression. In addition, we were able to achieve an overview of the cytokine signature of PDAC in patients and to distinguish between clusters of patients by identifying different immune landscapes, for example, immunologically hot and cold subtypes. Discussion: Our model is usable as a predictive system for the response of PDAC to different types of treatments and seems to be a reliable and sufficient preclinical extension alternative to mouse models, single-cell experiments, or organoid models. The whole context of the tissue is from human origin and we aim to expand our platform for multiscreening of drug responses for personalized decision-making. Furthermore, we described the tumor microenvironment in detail, unraveling the complex cytokine signature of PDAC, and subsequently identified different clusters of immune landscapes, which may be of prognostic value. Citation Format: Azaz Ahmed, Dyke Ferber, Meggy Suarez-Carmona, Bénédicte Lenoir, Rosa Klotz, Thilo Hackert, Nathalia Giese, Inka Zörnig, Dirk Jäger, Niels Halama. Establishment of a human PDAC explant culture model for treatment prediction and characterization of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C02.

Development of a Mouse Glioblastoma Orthotopic Model Using the GLi-261 Cell Line

Glioblastoma is the most common and most aggressive type of brain tumor, with an almost 100 % mortality rate over 5 years. The search for new effective approaches to the treatment of this disease requires the development of adequate experimental models.Objective: to develop and put into practice an orthotopic model of mouse glioblastoma.Materials and methods: GLi-261 mouse glioma cells were orthotopically inoculated into the putamen of C57Bl/6 mice brain. Tumor dynamics was investigated by Preclinical MRI System 7.0T/17cm (Flexiscan) highfield magnetic resonance imager (MR Solutions, UK). Temcital® (temozolomide) was used as a positive control in the treatment of experimental glioblastoma. The neurological status of animals in the course of tumour development was assessed by specific tests.Results: a GLi-261 cell-based mouse glioblastoma orthotopic model was developed using stereotactic equipment for accurate inoculation of tumour cells, magnetic resonance imaging for non-invasive determination of tumour volume and dynamics, and special tests for determination of the neurological status of the biological test systems. This model was used to demonstrate the effectiveness of temozolomide (the «gold standard» for glioblastoma treatment).Conclusions: this model has been introduced into practice at the IBC Generium, LLC, and can be used as an in vivo test system for preclinical evaluation of efficacy of new antitumour drugs being developed, as well as brain cancer treatment regimens using combination therapy.

Regional right ventricular function in rats: a novel magnetic resonance imaging method for measurement of right ventricular strain.

The function of the right ventricle (RV) is linked to clinical outcome in many cardiovascular diseases, but its role in experimental heart failure remains largely unexplored due to difficulties in measuring RV function in vivo. We aimed to advance RV imaging by establishing phase-contrast MRI (PC-MRI) as a robust method for measuring RV function in rodents. A total of 46 Wistar-Hannover rats with left ventricular (LV) myocardial infarction and 10 control rats (sham) were examined 6 wk after surgery. Using a 9.4-T preclinical MRI system, we utilized PC-MRI to measure strain/strain rate in the RV free wall under isoflurane anesthesia. Cine MRI was used to measure RV volumes. LV end-diastolic pressure (LVEDP) was measured and used to identify pulmonary congestion. The infarct rats were divided into two groups: those with signs of pulmonary congestion (PC), with LVEDP ≥ 15 mmHg (n = 26) and those without signs of pulmonary congestion (NPC), with LVEDP < 15 mmHg (n = 20). The NPC rats exhibited preserved RV strains/strain rates, whereas the PC rats exhibited reduced strains/strain rates (26-48% lower than sham). Of the strain parameters, longitudinal strain and strain rate exhibited the highest correlations to LVEDP and lung weight (rho = 0.65-0.72, P < 0.001). Basal longitudinal strain was most closely associated with signs of pulmonary congestion and indexes of RV remodeling. Longitudinal RV strain had higher area under the curve than ejection fraction for detecting subtle RV dysfunction (area under the curve = 0.85 vs. 0.67). In conclusion, we show for the first time that global and regional RV myocardial strain can be measured robustly in rodents. Reduced RV strain was closely associated with indexes of pulmonary congestion and molecular markers of RV remodeling.NEW & NOTEWORTHY Global and regional right ventricular myocardial strain can be measured with high reproducibility and low interobserver variability in rodents using tissue phase mapping MRI. Reduced right ventricular strain was associated with indexes of pulmonary congestion and molecular markers of right ventricular remodeling. Regional strain in the basal myocardium was considerably higher than in the apical myocardium.

In vivo surface dosimetry with a scintillating fiber dosimeter in preclinical image-guided radiotherapy.

New preclinical image-guided irradiators and treatment planning systems represent a huge progress in radiobiology. Nevertheless, quality control of preclinical treatments is not as advanced as in clinical radiotherapy and in vivo dosimetry is less developed. In this study, we evaluate the use of a scintillating fiber dosimeter called DosiRat to verify the agreement between the doses planned with SmART-Plan and the measured doses during small animal irradiations. In vivo dosimetry was first evaluated with DosiRat through dose measurements performed at the surface of a 3×9×3cm3 phantom. Measured and planned doses were compared for different irradiation conditions (prescription point, anterior, and posterior beams, 5mm and 10mm irradiation fields). In a second phase, measured and planned doses were compared for rat brain irradiations performed with anterior beams, with DosiRat positioned at the beam entrance. Comparisons were performed for different tube currents (1.3 and 13mA), collimations (5, 10 and 25mm diameter), and planned doses (0.1, 0.5, 2, and 10Gy). In the case of the phantom irradiations, planned and measured doses showed discrepancies smaller than the 5% accuracy of the TPS, except in cases in which the dosimeter was not centered in the irradiation field. The differences were larger for animal irradiations (from -3.3% to 8.8%) because of variations of the beam energy spectrum and the nonequivalence between materials at medium and low energy. This study highlighted the complexity to implement one-dimension in vivo dosimetry in orthovoltage millimetric beams. Nevertheless, DosiRat is well adapted to in vivo dosimetry because of its small volume and its direct reading and allowed in vivo control of planned doses for anterior beams down to 5mm diameter.

P65 USPIO-MRI FOR PRE-OPERATIVE LYMPH NODE STAGING AFTER NEOADJUVANT CHEMORADIOTHERAPY: FEASIBILITY AND VALIDATION FRAMEWORK

Abstract Background Extensive lymph node dissections during esophagectomy may be omitted in patients with esophageal cancer without lymph node metastases, reducing associated morbidity. A promising technique to detect lymph node metastases is MRI with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, ferumoxtran-10). To validate USPIO-enhanced MRI in patients with esophageal cancer, a framework is designed to compare lymph nodes of the MRI scans with histopathology data on a node-to-node level. Methods USPIO-nanoparticles are intravenously infused 24 to 36 hours before MRI. USPIO-enhanced MRI is performed before and after nCRT. After nCRT, patients are scanned under general anesthesia immediately prior to surgery in the MR system of the hybrid MITeC operation room with controlled mechanical ventilation. During controlled prolonged apneu, a four-minute iron-sensitive MRI acquisition is used to visualize suspicious esophageal lymph nodes without motion artefacts. Resected specimens, still containing USPIO, are measured ex-vivo in a preclinical 7T MR system before histopathological examination. A radiological assessment of the presence of suspicious lymph nodes in-vivo is matched to the ex-vivo nodes on preclinical MRI, providing the ground truth for the presence of metastases. Results Currently, five patients have completed the full study protocol. Two patients underwent the first UPSIO-MRI but did not undergo surgery due to progressive disease. First results show that MRI under anesthesia prior to surgery with controlled mechanical ventilation was possible resulting in a clinically relevant spatial resolution to visualize possibly malignant lymph nodes. In these five patients, the radiologist found 58 lymph nodes with a mean diameter of 8.4 mm before nCRT and 40 lymph nodes with a mean diameter of 7.4 mm after nCRT. In total, 126 lymph nodes were analyzed by histopathology. Suspicious nodes were identified and could be matched using corresponding anatomical landmarks to the ex-vivo MRI, which showed good visual agreement with esophageal specimen after resection. Conclusions We performed USPIO-enhanced MRI in patients with esophageal cancer and proposed a method to validate USPIO-enhanced MRI to detect suspicious lymph nodes in these patients. Results on the feasibility of USPIO-enhanced MRI to detect metastatic lymph nodes after nCRT are still awaited.

CBMT-02. UP-REGULATION OF Γ-GLUTAMYL-TRANSFERASE CAN BE USED TO IMAGE GLIOBLASTOMA USING HYPERPOLARIZED Γ-GLUTAMYL-[1-13C]GLYCINE MRS

Abstract γ-glutamyl-transferase (GGT) is a key enzyme in the γ-glutamyl cycle, which regulates glutathione homeostasis. The enzyme is localized on the outer cell membrane and cleaves glutathione to glutamate and cysteinylglycine. As such, it facilitates uptake of the amino acids essential for intracellular synthesis of glutathione (GSH), which is the major thiol anti-oxidant. GGT is upregulated in glioblastoma, but remains low in normal brain. It is therefore an attractive molecular imaging target for specific detection of glioblastoma. The goal of our study was therefore to assess for the first time the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) for imaging glioblastoma in orthotopic tumor-bearing rats. Athymic nude rats with U87 glioblastoma tumors or tumor-free controls were investigated. First, we confirmed that GGT expression was significantly higher in U87 tumors compared to normal rat brain tissue. GSH levels were also higher. Imaging studies were then performed by injecting HP γ-Glu-[1-13C]Gly and acquiring dynamic 13C MR spectra using a flyback spectral-spatial slab sequence on a preclinical Bruker 3T MR system. The dynamic data were analyzed by measuring the signal-to-noise (SNR) ratios of the substrate (γ-Glu-[1-13C]Gly) and the product ([1-13C]Glycine). Comparison of control and tumor-bearing rats showed no statistically significant difference in the SNR of the substrate. In contrast, the SNR of the product demonstrated a significantly higher level of HP [1-13C]Glycine in the tumor-bearing rats compared to controls. Consistent with the higher levels of HP [1-13C]Glycine, the [1-13C]Gly-to-γ-Glu-[1-13C]Glycine ratio was also significantly higher in tumor-bearing animals relative to controls (0.046±0.004 vs 0.021±0.008 respectively; p=0.03). Further studies are needed to assess the generality of our findings. Nonetheless, this study demonstrates for the first time the feasibility of using γ-Glu-[1-13C]Gly to monitor GGT activity and thus could serve as a new approach for monitoring the presence of tumor.

Stimulated echo double diffusion encoded imaging of closed pores: Influence and removal of unbalanced terms.

Nuclear magnetic resonance (NMR) diffusion pore imaging has been proposed to study the shape of arbitrary closed pores filled with an NMR-detectable medium by use of nonclassical diffusion encoding schemes. Potential applications can be found in biomedical imaging and porous media research. When studying non-point-symmetric pores, NMR signals with nonvanishing imaginary parts arise containing the pore shape information, which is lost for classical diffusion encoding schemes. Key limitations are the required high magnetic field gradient amplitudes and T2 relaxation while approaching the diffusion long-time limit. To benefit from the slower T1 decay, we demonstrate the feasibility of diffusion pore imaging with stimulated echoes using Monte Carlo simulations and experiments with hyperpolarized xenon-129 gas in well-defined geometries and show that the necessary complex-valued signals can be acquired. Analytical derivation of the stimulated echo double diffusion encoded signal was performed to investigate the effect of the additionally arising undesired terms on the complex phase information. These terms correspond to signals arising for spin-echo sequences with unbalanced gradients. For most possible applications, the unbalanced terms can be neglected. If non-negligible, selection of the appropriate signal component using a phase cycling scheme was demonstrated experimentally. Using stimulated echoes may be a step towards application of diffusion pore imaging to larger pores with gradient amplitudes available today in preclinical systems.

Integrating Pharmacokinetics and Pharmacodynamics in Operational Research to End Tuberculosis.

Tuberculosis (TB) elimination requires innovative approaches. The new Global Tuberculosis Network (GTN) aims to conduct research on key unmet therapeutic and diagnostic needs in the field of TB elimination using multidisciplinary, multisectorial approaches. The TB Pharmacology section within the new GTN aims to detect and study the current knowledge gaps, test potential solutions using human pharmacokinetics informed through preclinical infection systems, and return those findings to the bedside. Moreover, this approach would allow prospective identification and validation of optimal shorter therapeutic durations with new regimens. Optimized treatment using available and repurposed drugs may have an increased impact when prioritizing a person-centered approach and acknowledge the importance of age, gender, comorbidities, and both social and programmatic environments. In this viewpoint article, we present an in-depth discussion on how TB pharmacology and the related strategies will contribute to TB elimination.

In Vivo Imaging of Translocator Protein in Long-term Cannabis Users

Cannabis is the most commonly used illicit drug in the world. Cannabinoids have been shown to modulate immune responses; however, the association of cannabis with neuroimmune function has never been investigated in vivo in the human brain. To investigate neuroimmune activation or 18-kDa translocator protein (TSPO) levels in long-term cannabis users, and to evaluate the association of brain TSPO levels with behavioral measures and inflammatory blood biomarkers. This cross-sectional study based in Toronto, Ontario, recruited individuals from January 1, 2015, to October 30, 2018. Participants included long-term cannabis users (n = 24) and non-cannabis-using controls (n = 27). Cannabis users were included if they had a positive urine drug screen for only cannabis and if they used cannabis at least 4 times per week for the past 12 months and/or met the criteria for cannabis use disorder. All participants underwent a positron emission tomography scan with [18F]FEPPA, or fluorine F 18-labeled N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide. Total distribution volume was quantified across regions of interest. Stress and anxiety as well as peripheral measures of inflammatory cytokines and C-reactive protein levels were also measured. In total, 24 long-term cannabis users (mean [SD] age, 23.1 [3.8] years; 15 men [63%]) and 27 non-cannabis-using controls (mean [SD] age, 23.6 [4.2] years; 18 women [67%]) were included and completed all study procedures. Compared with the controls, cannabis users had higher [18F]FEPPA total distribution volume (main group effect: F1,48 = 6.5 [P = .01]; ROI effect: F1,200 = 28.4 [P < .001]; Cohen d = 0.6; 23.3% higher), with a more prominent implication for the cannabis use disorder subgroup (n = 15; main group effect: F1,39 = 8.5 [P = .006]; ROI effect: F1,164 = 19.3 [P < .001]; Cohen d = 0.8; 31.5% higher). Greater TSPO levels in the brain were associated with stress and anxiety and with higher circulating C-reactive protein levels in cannabis users. The results of this study suggest that TSPO levels in cannabis users, particularly in those with cannabis use disorder, are higher than those in non-cannabis-using controls. The findings emphasize the need for more complementary preclinical systems for a better understanding of the role of cannabinoids and TSPO in neuroimmune signaling.

Performance Evaluation of a Novel Preclinical Micro-CT System In Vitro and In Vivo

With the advent of innovation in the preclinical studies, micro-CT provides optimized resolution by high-power X-ray output tube and flat-panel detector with ultra-short scanning time. The aim of this study was to evaluate the performance of a novel preclinical micro-CT with different parameters and protocols. Imaging quality was evaluated by micro-CT HA and wire phantom. The high power output X-ray tube provides tube voltage from 40 to 90 kV with tube current, 10 to 800 μA. The detector with 1536 × 1944 pixels was implemented by three different binning modes (1 × 1, 2 × 2 and 4 × 4). The GPU-based reconstruction was applied with Feldkamp’s algorithm. The maximum reconstruction volume was 1944 × 1944 × 4536 per scan. The phantom images were acquired by four modes with pixel sizes of 44.9, 22.5,15 and 9 μm. In the animal study, ovariectomy mice were imaged followed by analysis of bone mineral density (BMD) and BV/TV ratio (bone volume/total bone volume). Linearity (R2) measured in all imaging settings were > 0.9 and was 0.99962 at setting of 0.2 mm Cu filter, 90 kV tube voltage and 556 μA tube current. The spatial resolution as measured by full width half-maximum amplitude (FWHM) was 24 µm in the mode ultra-high resolution. The bone analysis results show the operated cohort has high significant (p < 0.05), whereas the control cohort revealed insignificant either in the BMD. In summary, this novel micro-CT system demonstrated the flexible capability and high spatial resolution with ultra-short acquisition time, and a feasible quantitative BMD software.

EGFR-Mutated Lung Cancers Resistant to Osimertinib through EGFR C797S Respond to First-Generation Reversible EGFR Inhibitors but Eventually Acquire EGFR T790M/C797S in Preclinical Models and Clinical Samples

IntroductionOsimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile. MethodsWe used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S. ResultsUse of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis. ConclusionEGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance—in clinical specimens and preclinical systems—indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.