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  • Publication
    Constructing bilayer and volumetric atrial models at scale
    (The Royal Society, 2023-12-15) Roney, Caroline H.; Solis Lemus, Jose Alonso; López Barrera, Carlos; Zolotarev, Alexander; Ulgen, Onur; Kerfoot, Eric; Bevis, Laura; Misghina, Semhar; Vidal Horrach, Caterina; Jaffery, Ovais A.; Ehnesh, Mahmoud; Rodero, Cristobal; Dharmaprani, Dhani; Ríos Muñoz, Gonzalo Ricardo; Ganesan, Anand; Good, Wilson W; Neic, Aurel; Planck, Gernot; Hopman, Luuk H G A; Götte, Marco J. W.; Honarbakhsh, Shohreh; Narayan, Sanjiv M; Vigmond, Edward; Niederer, Steven
    To enable large in silico trials and personalized model predictions on clinical timescales, it is imperative that models can be constructed quickly and reproducibly. First, we aimed to overcome the challenges of constructing cardiac models at scale through developing a robust, open-source pipeline for bilayer and volumetric atrial models. Second, we aimed to investigate the effects of fibres, fibrosis and model representation on fibrillatory dynamics. To construct bilayer and volumetric models, we extended our previously developed coordinate system to incorporate transmurality, atrial regions and fibres (rule-based or data driven diffusion tensor magnetic resonance imaging (MRI)). We created a cohort of 1000 biatrial bilayer and volumetric models derived from computed tomography (CT) data, as well as models from MRI, and electroanatomical mapping. Fibrillatory dynamics diverged between bilayer and volumetric simulations across the CT cohort (correlation coefficient for phase singularity maps: left atrial (LA) 0.27 ± 0.19, right atrial (RA) 0.41 ± 0.14). Adding fibrotic remodelling stabilized re-entries and reduced the impact of model type (LA: 0.52 ± 0.20, RA: 0.36 ± 0.18). The choice of fibre field has a small effect on paced activation data (less than 12 ms), but a larger effect on fibrillatory dynamics. Overall, we developed an open-source user-friendly pipeline for generating atrial models from imaging or electroanatomical mapping data enabling in silico clinical trials at scale (https://github.com/pcmlab/atrialmtk).
  • Publication
    Isolation of goat milk small extracellular vesicles by novel combined bio-physical methodology
    (Frontiers, 2023-09-27) González Vasco, María Isabel; Gallardo Alguacil, Begoña; Ceron, Carlos; Aguilera-Jimenez, Elena; Cortes-Canteli, Marta; Peinado, Hector; Desco Menéndez, Manuel; Salinas Rodríguez, Beatriz; Comunidad de Madrid; Ministerio de Educación, Cultura y Deporte (España)
    Introduction: Goat milk is notable as a cost-effective source of exosomes, also known as small extracellular vesicles (sEVs). These nanoparticle-like structures are naturally secreted by cells and have emerged as potential diagnostic agents and drug delivery systems, also supported by their proven therapeutic effects. However, the complexity of goat milk and the lack of standardized protocols make it difficult to isolate pure sEVs. This work presents an optimized approach that combines well-established physical isolation methods with the biological treatment of milk with rennet.Methods: sEVs derived from goat milk were purified using a methodology that combines differential ultracentrifugation, rennet, and size-exclusion chromatography. This novel strategy was compared with two of the main methodologies developed for isolating extracellular vesicles from bovine and human milk by means of physico-chemical characterization of collected vesicles using Transmission Electron Microscopy, Western blot, Bradford Coomassie assay, Dynamic Light Scattering, Nanoparticle Tracking Analysis and Zeta Potential.Results: Vesicles isolated with the optimized protocol had sEV-like characteristics and high homogeneity, while samples obtained with the previous methods were highly aggregated, with significant residual protein content.Discussion: This work provides a novel biophysical methodology for isolating highly enriched goat milk sEVs samples with high stability and homogeneity, for their further evaluation in biomedical applications as diagnostic tools or drug delivery systems.
  • Publication
    Functional screening of selective mitochondrial inhibitors of Plasmodium
    (Elsevier BV, 2018-05-15) Gómez Lorenzo, María G.; Rodríguez Alejandre, Ane; Moliner Cubel, Sonia; Martínez Hoyos, María; Bahamontes Rosa, Noemí; González Del Río, Rubén; Ródenas, Carolina; De La Fuente, Jesús; Lavandera, José Luis; García Bustos, José F.; Mendoza Losana, Alfonso
    Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.
  • Publication
    Selective sparing of bladder and rectum sub-regions in radiotherapy of prostate cancer combining knowledge-based automatic planning and multicriteria optimization
    (Elsevier, 2023-10-01) Alborghetti, Lisa; Castriconi, Roberta; Sosa Marrero, Carlos; Tudda, Alessia; Ubeira-Gabellini, Maria Guiulia; Broggi, Sara; Pascau González-Garzón, Javier; Cubero Gutierrez, Lucia; Cozzarini, Cesare; Crevoisier, Renaud De; Rancati, Tiziana; Acosta, Oscar; Fiorino, Claudio
    Background and Purpose: The association between dose to selected bladder and rectum symptom-related sub- regions (SRS) and late toxicity after prostate cancer radiotherapy has been evidenced by voxel-wise analyses. The aim of the current study was to explore the feasibility of combining knowledge-based (KB) and multi-criteria optimization (MCO) to spare SRSs without compromising planning target volume (PTV) dose delivery, including pelvic-node irradiation. Materials and Methods: Forty-five previously treated patients (74.2 Gy/28fr) were selected and SRSs (in the bladder, associated with late dysuria/hematuria/retention; in the rectum, associated with bleeding) were generated using deformable registration. A KB model was used to obtain clinically suitable plans (KB-plan). KB- plans were further optimized using MCO, aiming to reduce dose to the SRSs while safeguarding target dose coverage, homogeneity and avoiding worsening dose volume histograms of the whole bladder, rectum and other organs at risk. The resulting MCO-generated plans were examined to identify the best-compromise plan (KB + MCO-plan). Results: The mean SRS dose decreased in almost all patients for each SRS. D1% also decreased in the large majority, less frequently for dysuria/bleeding SRS. Mean differences were statistically significant (p < 0.05) and ranged between 1.3 and 2.2 Gy with maximum reduction of mean dose up to 3–5 Gy for the four SRSs. The better sparing of SRSs was obtained without compromising PTVs coverage. Conclusions: Selectively sparing SRSs without compromising PTV coverage is feasible and has the potential to reduce toxicities in prostate cancer radiotherapy. Further investigation to better quantify the expected risk reduction of late toxicities is warranted.
  • Publication
    Simulation and validation in brain image analysis [Editorial]
    (Hindawi, 2016-06-28) Tohka, Jussi; Bellec, Pierre; Grova, Christophe; Reilhac, Anthonin
  • Publication
    MouBeAT: A New and Open Toolbox for Guided Analysis of Behavioral Tests in Mice
    (Frontiers Media SA., 2018-09-07) Bello Arroyo, Elisabet; Lara Pezzi, Enrique; Roque, Helio; Caiolfa, Valeria R; Marcos, Alberto; Orihuel, Javier; Desco Menéndez, Manuel; Matas Higuera, Alejandro; Gómez Gaviro, María Victoria; Comunidad de Madrid
    Animal behavioral tests are essential to understand the bases of neurologic and psychological disorders, which can be evaluated by different methodological and experimental models. However, the quantification of behavioral tests results is limited by the considerable amount of time needed for manual evaluation and the high costs of automated analysis software. To overcome these limitations, we describe here a new, open source toolbox for ImageJ, called Mouse Behavioral Analysis Toolbox (MouBeAT), designed to analyze different behavioral tests in rodents semi-automatically. These tests include Open Field (OF), Elevated Plus Maze (EPM), Y-maze (YM) test and Morris Water Maze (MWM). MouBeAT showed a high correlation with manual evaluation in all the parameters analyzed for all the behavioral tests, reinforcing its value as an accurate analysis tool. This new tool is freely available online.
  • Publication
    Suppression of F-18-FDG signal in the bladder on small animal PET-CT
    (P. K. Garg, 2018-10-17) Cusso Mula, Lorena; Desco Menéndez, Manuel
    Introduction Retention of 2-deoxy-2-[F-18]fluoro-D-glucose F-18-FDG in the bladder causes more problems in small animal research than in human research owing to the smaller size of the subject. Catheterization has been proposed to reduce bladder spillover both in human studies and in small animal research. Noninvasive alternatives such as hydration plus furosemide also seem to be a promising pre-imaging strategy for decreasing bladder spillover. Our main goal was to measure the effects of the combination of furosemide and hydration for reducing bladder signal directly on mouse bowel F-18-FDG-PET images. Methods Nine mice were divided into two groups: the control group (C, n = 4) and the treatment group (n = 5). The clearance protocol combines hyperhydration and a single furosemide dose during the F-18-FDG uptake period. Two images were acquired on different days in treated mice to evaluate two different furosemide doses (low dose, LD, 3.5 mg/kg; and high dose, HD, 7 mg/kg). A region of interest was drawn on each computed tomography image (bladder, kidneys, liver, muscle, and bone marrow). To quantify bladder spillover, two different areas of the colon were selected. Results A remarkable reduction in bladder spillover was achieved on F-18-FDG-PET in both groups. Our imaging findings were quantified, and both furosemide doses induced a decrease in mean standard uptake values (SUVmean) compared with the controls (LD 1.46 +/- 0.54 and HD 1.05 +/- 0.29; controls: 8.90 +/- 3.4 [p-value < 0.05]). Conclusion We validated a non-invasive, easy, and harmless pre-imaging alternative for decreasing F-18-FDG bladder spillover. Our study shows the effect of furosemide on bladder spillover directly on F-18-FDG-PET images by measuring SUVmean in the bladder, colon, liver, muscle, and bone marrow.
  • Publication
    Radioimmune Imaging of α4β7 Integrin and TNFα for Diagnostic and Therapeutic Applications in Inflammatory Bowel Disease
    (MDPI, 2023-03-01) Signore, Alberto; Bonfiglio, Rita; Varani, Michela; Galli, Filippo; Campagna, Giuseppe; Desco Menéndez, Manuel; Cusso Mula, Lorena; Mattei, Maurizio; Wunder, Andreas; Borri, Filippo; Lupo, Maria T.; Bonanno, Elena; Comunidad de Madrid; Instituto de Salud Carlos III (España)
    Imaging using radiolabelled monoclonal antibodies can provide, non-invasively, molecular information which allows for the planning of the best treatment and for monitoring the therapeutic response in cancer, as well as in chronic inflammatory diseases. In the present study, our main goal was to evaluate if a pre-therapy scan with radiolabelled anti-α4β7 integrin or radiolabelled anti-TNFα mAb could predict therapeutic outcome with unlabelled anti-α4β7 integrin or anti-TNFα mAb. To this aim, we developed two radiopharmaceuticals to study the expression of therapeutic targets for inflammatory bowel diseases (IBD), to be used for therapy decision making. Both anti-α4β7 integrin and anti-TNFα mAbs were successfully radiolabelled with technetium-99m with high labelling efficiency and stability. Dextran sulfate sodium (DSS)-induced colitis was used as a model for murine IBD and the bowel uptake of radiolabelled mAbs was evaluated ex vivo and in vivo by planar and SPECT/CT images. These studies allowed us to define best imaging strategy and to validate the specificity of mAb binding in vivo to their targets. Bowel uptake in four different regions was compared to immunohistochemistry (IHC) score (partial and global). Then, to evaluate the biomarker expression prior to therapy administration, in initial IBD, another group of DSS-treated mice was injected with radiolabelled mAb on day 2 of DSS administration (to quantify the presence of the target in the bowel) and then injected with a single therapeutic dose of unlabelled anti-α4β7 integrin or anti-TNFα mAb. Good correlation was demonstrated between bowel uptake of radiolabelled mAb and immunohistochemistry (IHC) score, both in vivo and ex vivo. Mice treated with unlabelled α4β7 integrin and anti-TNFα showed an inverse correlation between the bowel uptake of radiolabelled mAb and the histological score after therapy, proving that only mice with high α4β7 integrin or TNFα expression will benefit of therapy with unlabelled mAb.
  • Publication
    Discovery of novel oral protein synthesis inhibitors of mycobacterium tuberculosis that target leucyl-tRNA synthetase
    (American Society for Microbiology, 2016-09-23) Palencia, Andrés; Mendoza Losana, Alfonso
    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.
  • Publication
    Repurposing clinically approved cephalosporins for tuberculosis therapy
    (Springer, 2016-09-28) Ramón García, Santiago; González Del Río, Rubén; Santos Villarejo, Ángel; D. Sweet, Gaye; Cunningham, Fraser; Barros, David; Ballell, Lluis; Mendoza Losana, Alfonso; Ferrer Bazaga, Santiago; J. Thompson, Charles; European Commission
    While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.
  • Publication
    New molecular settings to support in vivo anti-malarial assays
    (Springer, 2016-03-08) Bahamontes Rosa, Noemí; Rodríguez Alejandre, Ane; Gómez, Vanesa; Viera, Sara; Gómez Lorenzo, María G.; Sanz Alonso, Laura María; Mendoza Losana, Alfonso
    Background Quantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods. Methods FTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes. Results The optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed. Conclusions This is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.
  • Publication
    Identification of KasA as the cellular target of an anti-tubercular scaffold
    (Springer, 2016-09-01) Abrahams, Katherine A.; Mendoza Losana, Alfonso; European Commission
    Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.
  • Publication
    Maternal Supplementation with N-Acetylcysteine Modulates the Microbiota-Gut-Brain Axis in Offspring of the Poly I:C Rat Model of Schizophrenia
    (MDPI, 2023-04-20) Romero Miguel, Luis Diego; Casquero-Veiga, Marta; Fernández, Javier; Lamanna Rama, Nicolás; Gómez Rangel, Vanessa; Gálvez-Robleño, Carlos; Santa Marta, Cristina; Villar, Claudio J.; Lombo, Felipe; Abalo, Raquel; Desco Menéndez, Manuel; Soto Montenegro, Mª Luisa; Comunidad de Madrid; Ministerio de Ciencia e Innovación (España)
    The microbiota-gut-brain axis is a complex interconnected system altered in schizophrenia. The antioxidant N-acetylcysteine (NAC) has been proposed as an adjunctive therapy to antipsychotics in clinical trials, but its role in the microbiota-gut-brain axis has not been sufficiently explored. We aimed to describe the effect of NAC administration during pregnancy on the gut-brain axis in the offspring from the maternal immune stimulation (MIS) animal model of schizophrenia. Pregnant Wistar rats were treated with PolyI:C/Saline. Six groups of animals were studied according to the study factors: phenotype (Saline, MIS) and treatment (no NAC, NAC 7 days, NAC 21 days). Offspring were subjected to the novel object recognition test and were scanned using MRI. Caecum contents were used for metagenomics 16S rRNA sequencing. NAC treatment prevented hippocampal volume reduction and long-term memory deficits in MIS-offspring. In addition, MIS-animals showed lower bacterial richness, which was prevented by NAC. Moreover, NAC7/NAC21 treatments resulted in a reduction of proinflammatory taxons in MIS-animals and an increase in taxa known to produce anti-inflammatory metabolites. Early approaches, like this one, with anti-inflammatory/anti-oxidative compounds, especially in neurodevelopmental disorders with an inflammatory/oxidative basis, may be useful in modulating bacterial microbiota, hippocampal size, as well as hippocampal-based memory impairments.
  • Publication
    N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis
    (National Academy of Sciences, 2016-07-18) Warrier, Thulasi; Mendoza Losana, Alfonso
    The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-beta-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.
  • Publication
    Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor
    (Elsevier, 2016-05-08) Martínez Hoyos, María; Mendoza Losana, Alfonso
    Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.
  • Publication
    Understanding Deep Brain Stimulation: In Vivo Metabolic Consequences of the Electrode Insertional Effect
    (Hindawi, 2018-10-17) Casquero Veiga, Marta; García García, David; Desco Menéndez, Manuel; Soto Montenegro, Mª Luisa; Ministerio de Economía y Competitividad (España)
    Deep brain stimulation (DBS) is a neurosurgery technique widely used in movement disorders, although its mechanism of action remains unclear. In fact, apart from the stimulation itself, the mechanical insertion of the electrode may play a crucial role. Here we aimed to distinguish between the insertional and the DBS effects on brain glucose metabolism. To this end, electrodes were implanted targeting the medial prefrontal cortex in five adult male Wistar rats. Positron Emission Tomography (PET) studies were performed before surgery (D0) and seven (D7) and nine days (D9) after that. DBS was applied during the (18)FDG uptake of the D9 study. PET data were analysed with statistical parametric mapping. We found an electrode insertional effect in cortical areas, while DBS resulted in a more widespread metabolic pattern. The consequences of simultaneous electrode and DBS factors revealed a combination of both effects. Therefore, the insertion metabolic effects differed from the stimulation ones, which should be considered when assessing DBS protocols.
  • Publication
    Linking murine and human Plasmodium falciparum challenge models in a translational path for antimalarial drug development
    (American Society for Microbiology, 2016-06-01) Ferrer Bazaga, Santiago
    Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rgamma−/− (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC50s) were estimated at 2.0 mug/ml and 1.8 mug/ml in the NSG and IBSM models, respectively, aligning with 1.8 mug/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations.
  • Publication
    Characterization of Novel Antimalarial Compound ACT-451840: Preclinical Assessment of Activity and Dose-Efficacy Modeling
    (Public Library of Science, 2016-10-01) Ferrer Bazaga, Santiago
    Background Artemisinin resistance observed in Southeast Asia threatens the continued use of artemisinin- based combination therapy in endemic countries. Additionally, the diversity of chemical mode of action in the global portfolio of marketed antimalarials is extremely limited. Addressing the urgent need for the development of new antimalarials, a chemical class of potent antimalarial compounds with a novel mode of action was recently identified. Herein, the preclinical characterization of one of these compounds, ACT-451840, conducted in partnership with academic and industrial groups is presented Method and Findings The properties of ACT-451840 are described, including its spectrum of activities against multiple life cycle stages of the human malaria parasite Plasmodium falciparum (asexual and sexual) and Plasmodium vivax (asexual) as well as oral in vivo efficacies in two murine malaria models that permit infection with the human and the rodent parasites P. falciparum and Plasmodium berghei, respectively. In vitro, ACT-451840 showed a 50% inhibition concentration of 0.4 nM (standard deviation [SD]: ± 0.0 nM) against the drug-sensitive P. falciparum NF54 strain. The 90% effective doses in the in vivo efficacy models were 3.7 mg/kg against P. falciparum (95% confidence interval: 3.3±4.9 mg/kg) and 13 mg/kg against P. berghei (95% confidence interval: 11±16 mg/kg). ACT-451840 potently prevented male gamete formation from the gametocyte stage with a 50% inhibition concentration of 5.89 nM (SD: ± 1.80 nM) and dose-dependently blocked oocyst development in the mosquito with a 50% inhibitory concentration of 30 nM (range: 23±39). The compound's preclinical safety profile is presented and is in line with the published results of the first-in-man study in healthy male participants, in whom ACT-451840 was well tolerated. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was applied using efficacy in the murine models (defined either as antimalarial activity or as survival) in relation to area under the concentration versus time curve (AUC), maximum observed plasma concentration (Cmax), and time above a threshold concentration. The determination of the dose±efficacy relationship of ACT- 451840 under curative conditions in rodent malaria models allowed prediction of the human efficacious exposure. Conclusion The dual activity of ACT-451840 against asexual and sexual stages of P. falciparum and the activity on P. vivax have the potential to meet the specific profile of a target compound that could replace the fast-acting artemisinin component and harbor additional gametocytocidal activity and, thereby, transmission-blocking properties. The fast parasite reduction ratio (PRR) and gametocytocidal effect of ACT-451840 were recently also confirmed in a clinical proof-of-concept (POC) study.
  • Publication
    Response to deep brain stimulation in three brain targets with implications in mental disorders: a PET study in rats
    (PLOS, 2016-12-29) Casquero-Veiga, Marta; Hadar, Ravit; Pascau González-Garzón, Javier; Winter, Christine; Desco Menéndez, Manuel; Soto Montenegro, Mª Luisa; Comunidad de Madrid
    Objective: To investigate metabolic changes in brain networks by deep brain stimulation (DBS) of the medial prefrontal cortex (mPFC), nucleus accumbens (NAcc) and dorsomedial thalamus (DM) using positron emission tomography (PET) in naïve rats. Methods: 43 male Wistar rats underwent stereotactic surgery and concentric bipolar platinum-iridium electrodes were bilaterally implanted into one of the three brain sites. [18F]-fluoro-2-deoxy-glucose-PET (18FDG-PET) and computed tomography (CT) scans were performed at the 7th (without DBS) and 9th day (with DBS) after surgery. Stimulation period matched tracer uptake period. Images were acquired with a small-animal PET-CT scanner. Differences in glucose uptake between groups were assessed with Statistical Parametric Mapping. Results: DBS induced site-specific metabolic changes, although a common increased metabolic activity in the piriform cortex was found for the three brain targets. mPFC-DBS increased metabolic activity in the striatum, temporal and amygdala, and reduced it in the cerebellum, brainstem (BS) and periaqueductal gray matter (PAG). NAcc-DBS increased metabolic activity in the subiculum and olfactory bulb, and decreased it in the BS, PAG, septum and hypothalamus. DM-DBS increased metabolic activity in the striatum, NAcc and thalamus and decreased it in the temporal and cingulate cortex. Conclusions: DBS induced significant changes in 18FDG uptake in brain regions associated with the basal ganglia-thalamo-cortical circuitry. Stimulation of mPFC, NAcc and DM induced different patterns of 18FDG uptake despite interacting with the same circuitries. This may have important implications to DBS research suggesting individualized target selection according to specific neural modulatory requirements.
  • Publication
    Monitoring vascular normalization induced by antiangiogenic treatment with F-18-fluoromisonidazole-PET
    (FEBS Press, 2016-05) Hernandez Agudo, Elena; Mondejar, Tamara; Soto Montenegro, Mª Luisa; Megias, Diego; Mouron, Silvana; Sanchez, Jesus; Hidalgo, Manuel; Lopez Casas, Pedro Pablo; Mulero, Francisca; Desco Menéndez, Manuel; Quintela Fandino, Miguel
    Background: Rationalization of antiangiogenics requires biomarkers. Vascular re normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with F-18-misonidazole ([F-18]-FMISO, a probe that detects hypoxia) PET, in response to window-of-opportunity (WoO) treatment with the antiangiogenic dovitinib. Methods: Two patient-derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV-PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [F-18]-FMISO-PET, [F-18]-FDG-PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase-3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. Results: [F-18]-FMISO SUV did not change after dovitinib-WoO treatment compared to vehicle-WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P < 0.05). In parallel, 10-KDa perivascular dextran extravasation was not reduced with dovitinib or vehicle-WoO treatment in Panc215, but it was reduced in Panc286. Whereas the addition of dovitinib to gemcitabine was indifferent in Panc215, it increased TGI in Panc286 (TGI switched from -59% to +49%). [F-18]-FMISO SUV changes were accompanied by an almost 100% increase in interstitial gemcitabine delivery (665-1260 ng/mL). The results were validated in the PyMT model. Conclusions: [F-18]-FMISO accurately monitored vascular re-normalization and improved interstitial chemotherapy delivery.