RESUMEN
Self-assembly through dynamic covalent chemistry (DCC) can yield a range of multi-component organic assemblies. The reversibility and dynamic nature of DCC has made prediction of reaction outcome particularly difficult and thus slows the discovery rate of new organic materials. In addition, traditional experimental processes are time-consuming and often rely on serendipity. Here, we present a streamlined hybrid workflow that combines automated high-throughput experimentation, automated data analysis, and computational modelling, to accelerate the discovery process of one particular subclass of molecular organic materials, porous organic cages. We demonstrate how the design and implementation of this workflow aids in the identification of organic cages with desirable properties. The curation of a precursor library of 55 tri- and di-topic aldehyde and amine precursors enabled the experimental screening of 366 imine condensation reactions experimentally, and 1464 hypothetical organic cage outcomes to be computationally modelled. From the screen, 225 cages were identified experimentally using mass spectrometry, 54 of which were cleanly formed as a single topology as determined by both turbidity measurements and 1H NMR spectroscopy. Integration of these characterisation methods into a fully automated Python pipeline, named cagey, led to over a 350-fold decrease in the time required for data analysis. This work highlights the advantages of combining automated synthesis, characterisation, and analysis, for large-scale data curation towards an accessible data-driven materials discovery approach.
RESUMEN
BACKGROUND: Previous investigators have shown that high heels decrease the muscle activity of the gastrocnemius muscle during gait. However, it is not known whether commonly used in-shoe heel lifts of lower heights will demonstrate similar effects on muscle activity. The aim of this study was to determine whether heel lifts alter the muscle activity of the ankle plantarflexors and dorsiflexors during the stance phase of gait among individuals with limited gastrocnemius extensibility. MATERIALS AND METHODS: This study used a repeated measures design. Twenty-four healthy volunteers (12 males and 12 females) with less than 5 degrees of passive ankle dorsiflexion with the knee extended participated in the study. Electromyography (EMG), computerized motion analysis, and a force plate were used to measure mean muscle activity of the lateral gastrocnemius, medial gastrocnemius, soleus and tibialis anterior muscles during the stance phase of gait across three walking conditions. Muscle activity was measured as participants ambulated at a self-selected speed in athletic shoes alone and with heel lifts of 6 mm and 9 mm inserted in athletic shoes. RESULTS: Between heel-strike and heel-off, the mean EMG amplitude of the medial gastrocnemius increased with both 6 and 9 mm heel lifts and the amplitude of the tibialis anterior increased with 9 mm heel lifts compared to shoes alone. Between heel-strike and heel-off, there were no significant differences in mean EMG amplitude of the lateral gastrocnemius or soleus muscles walking in heel lifts compared to shoes alone. Between heel-off and toe-off, there were no significant differences in mean EMG amplitude of the lateral gastrocnemius, medial gastrocnemius, soleus, or tibialis anterior muscles when walking in heel lifts compared to shoes alone. CONCLUSION: Heel lifts increase muscle activity of the medial gastrocnemius and tibialis anterior muscles between heel-strike and heel-off among individuals with limited gastrocnemius extensibility. CLINICAL RELEVANCE: We were unable to confirm a decrease in muscle activity when using heel lifts.
Asunto(s)
Pie/fisiología , Marcha/fisiología , Músculo Esquelético/fisiología , Aparatos Ortopédicos , Adolescente , Adulto , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Glioblastoma multiforme (GBM) is an aggressive and difficult to treat form of brain cancer. In this work, we report on a novel chronotherapeutic polymeric drug, PEAMOtecan, for GBM therapy. PEAMOtecan was synthesized by conjugating camptothecin, a topoisomerase I inhibitor, to our proprietary, 'clickable' and modular polyoxetane polymer platform consisting of acetylene-functionalized 3-ethyl-3-(hydroxymethyl)oxetane (EAMO) repeat units (Patent No.: US 9,421,276) via the linker 3,3'-dithiodipropionic acid (DDPA) with a disulfide bond (SS) extended by short-chain polyethylene glycol (PEG). We show that PEAMOtecan is a highly modular polymer nanoformulation that protects covalently bound CPT until slowly being released over extended periods of time dependent on the cleavage of the disulfide and ester linkages. PEAMOtecan kills glioma cells by mitotic catastrophe with p53 mutant/knockdown cells being more sensitive than matched wild type cells potentially providing cancer-specific targeting. To establish proof-of-principle therapeutic effects, we tested PEAMOtecan as monotherapy for efficacy in a mouse orthotopic glioma model. PEAMOtecan was administered by one-time, convection-enhanced delivery (CED) intra-tumorally to achieve superior distribution and extended drug release over time. In addition, the near-infrared (NIR) dye Cy5.5 was coupled to the polymer providing live-animal imaging capability to track tissue distribution and clearance of the injected polymer over time. We show that PEAMOtecan significantly improves the survival of mice harboring intra-cranial tumors (p = .0074 compared to untreated group). Altogether, these results support further development and testing of our nanoconjugate platform.
Asunto(s)
Neoplasias Encefálicas , Glioma , Preparaciones Farmacéuticas , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Cronoterapia de Medicamentos , Sistemas de Liberación de Medicamentos , Glioma/tratamiento farmacológico , Ratones , Polímeros/uso terapéuticoRESUMEN
Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro AZ32, with enhanced blood-brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637-47. ©2018 AACR.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Glioma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Administración Oral , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Inhibidores de Proteínas Quinasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/química , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/química , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Deer mice (Peromyscus) are the most common native North American mammals, and exhibit great natural genetic variation. Wild-derived stocks from a number of populations are available from the Peromyscus Genetic Stock Center (PGSC). The PGSC also houses a number of natural variants and mutants (many of which appear to differ from Mus). These include metabolic, coat-color/pattern, neurological, and other morphological variants/mutants. Nearly all these mutants are on a common genetic background, the Peromyscus maniculatus BW stock. Peromyscus are also superior behavior models in areas such as repetitive behavior and pair-bonding effects, as multiple species are monogamous. While Peromyscus development generally resembles that of Mus and Rattus, prenatal stages have not been as thoroughly studied, and there appear to be intriguing differences (e.g., longer time spent at the two-cell stage). Development is greatly perturbed in crosses between P. maniculatus (BW) and Peromyscus polionotus (PO). BW females crossed to PO males produce growth-restricted, but otherwise healthy, fertile offspring which allows for genetic analyses of the many traits that differ between these two species. PO females crossed to BW males produce overgrown but severely dysmorphic conceptuses that rarely survive to late gestation. There are likely many more uses for these animals as developmental models than we have described here. Peromyscus models can now be more fully exploited due to the emerging genetic (full linkage map), genomic (genomes of four stocks have been sequenced) and reproductive resources.
Asunto(s)
Desarrollo Embrionario , Modelos Animales , Peromyscus/embriología , Animales , Variación Genética , Peromyscus/genética , Pigmentación , ReproducciónRESUMEN
This work investigates the effect of cell-collagen I interactions on the synthesis and activation of MMP-2, as well as synthesis of MT1-MMP and TIMP-1, by using an in vitro model with 3D fibrillar and 2D monomeric collagen. In order to reveal whether the metastasis-associated protein S100A4 can influence the cell's response to the two forms of collagen, osteosarcoma cell lines with high and low endogenous levels of S100A4 were used. Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric collagen resulted in opposite effects on MMP-2 activation. Attachment to 3D fibrillar collagen decreased activation of proMMP-2, with a corresponding reduction in MT1-MMP. By contrast, attachment to monomeric collagen increased the amount of fully active MMP-2. This was caused by a reduction in TIMP-1 levels when cells were attached to monomeric 2D collagen. The effect of collagen on proMMP-2 activation was independent of endogenous S100A4 levels, whereas synthesis of TIMP-1 was dependent on S100A4. When cells were attached to monomeric collagen, cells with a high level of S100A4 showed a greater reduction in the synthesis of TIMP-1 than did those with a low level of S100A4. Taken together, this study shows that synthesis and activation of MMP-2 is affected by interactions between osteosarcoma cells and collagen I in both fibrillar and monomeric form.