RESUMO
Resistance to antibiotics among bacterial pathogens is rapidly spreading, and therapeutic options against multidrug-resistant bacteria are limited. There is an urgent need for new drugs, especially those that can circumvent the broad array of resistance pathways that bacteria have evolved. In this study, we assessed the pharmacokinetic/pharmacodynamic relationship of the novel ß-lactamase inhibitor relebactam (REL; MK-7655) in a hollow-fiber infection model. REL is intended for use with the carbapenem ß-lactam antibiotic imipenem for the treatment of Gram-negative bacterial infections. In this study, we used an in vitro hollow-fiber infection model to confirm the efficacy of human exposures associated with the phase 2 doses (imipenem at 500 mg plus REL at 125 or 250 mg administered intravenously every 6 h as a 30-min infusion) against imipenem-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumoniae Dose fractionation experiments confirmed that the pharmacokinetic parameter that best correlated with REL activity is the area under the concentration-time curve, consistent with findings in a murine pharmacokinetic/pharmacodynamic model. Determination of the pharmacokinetic/pharmacodynamic relationship between ß-lactam antibiotics and ß-lactamase inhibitors is complex, as there is an interdependence between their respective exposure-response relationships. Here, we show that this interdependence could be captured by treating the MIC of imipenem as dynamic: it changes with time, and this change is directly related to REL levels. For the strains tested, the percentage of the dosing interval time that the concentration remains above the dynamic MIC for imipenem was maintained at the carbapenem target of 30 to 40%, required for maximum efficacy, for imipenem at 500 mg plus REL at 250 mg.
Assuntos
Imipenem/farmacologia , Inibidores de beta-Lactamases/farmacologia , Animais , Compostos Azabicíclicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.
Assuntos
Ensaios de Triagem em Larga Escala , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Ensaios de Triagem em Larga Escala/métodos , Descoberta de Drogas/métodos , Reprodutibilidade dos Testes , HumanosRESUMO
The discovery of peptide therapeutics represents a fast-growing segment of pharmaceutical research. During the early discovery process, a large number of peptide candidates needs to be rapidly screened for metabolic stability in relevant biological matrices. In most cases, peptide stability assays are quantified using LC-MS/MS, which may take hours to analyze 384 samples and generates liters of solvent waste. Herein, we introduce a high-throughput screening (HTS) platform for peptide stability assessment founded on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS). Full automation has been implemented for sample preparation with minimal manual intervention. The limit of detection, linearity, and reproducibility of the platform were evaluated, and metabolic stabilities have been determined for a number of peptide candidates. The MALDI-MS-based HTS workflow is able to analyze 384 samples in less than 1 h while only using 115 µL of total solvent. Although this process allows for very rapid assessment of peptide stability, given the nature of the MALDI process, it is noteworthy that spot-to-spot variations and ionization bias are observed. Therefore, LC-MS/MS may still be needed for confident, quantitative measurements and/or when the ionization efficiency of certain peptides is inadequate using MALDI.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Peptídeos/química , AutomaçãoRESUMO
Past publications have highlighted the influence of postdialysis plasma pH on the measured fraction unbound in plasma (fup). There is disparity in the industry as to which of two main methods is more suitable for controlling postdialysis plasma pH: the use of either a stronger buffer or a CO(2) atmosphere for the incubation. In the current study, it has been found that 10% CO(2) could be too high for the buffering capacities of both 100 mM sodium phosphate (pH 7.40 decreased to pH 6.90 after a 6-h incubation) and plasma (decreased below pH 7.40 after a 6-h incubation). To provide appropriate control over the postdialysis plasma pH, for a range of species, it is proposed that a standard phosphate buffer strength (100 mM) and pH (7.40) in combination with a 5% CO(2) atmosphere be used for equilibrium dialysis. Furthermore, statistically significant differences in fup values obtained with a pH difference of less than 0.32 pH unit have been demonstrated. An acceptance range for postdialysis plasma pH in routine in vitro fup screening assays of pH 7.40 ± 0.10 is recommended.
Assuntos
Proteínas Sanguíneas/metabolismo , Preparações Farmacêuticas/sangue , Animais , Soluções Tampão , Dióxido de Carbono , Fenômenos Químicos , Diálise , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , Camundongos , Concentração Osmolar , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Ligação Proteica , Ratos , Reprodutibilidade dos TestesRESUMO
We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.
Assuntos
Compostos Heterocíclicos/química , Indóis/química , Receptor CB1 de Canabinoide/agonistas , Tiazóis/química , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacocinética , Camundongos , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinéticaRESUMO
BACKGROUND: Real time clinical gait analysis (RTCGA) is often incorporated as part of a general or lower limb musculoskeletal (MSK) adult patient assessment. However, it is not known if RTCGA is clinically effective as a useful outcome measure or aids in decision making. Whether there is a clinical worth in conducting RTCGA in adult MSK consultations remains controversial. The aim of this study was to provide unique insights into MSK podiatrists use and opinions of RTCGA, using Posterior Tibial Tendon Dysfunction (PTTD) as an exemplar adult condition. METHODS: A qualitative approach was employed to explore MSK podiatrists' views and experiences of RTCGA when assessing or treating patients with PTTD. Semi-structured interviews were conducted via Skype video calls which were transcribed using an orthographic transcription method. Thematic analysis was employed to identify key meanings and report patterns within the data. RESULTS: Twenty nine MSK podiatrists who used RTCGA in the assessment and treatment of PTTD participated in the study. Five themes were identified as 1) RTCGA Method; 2) Working with RTCGA; 3) RTCGA uses; 4) What could aid RTCGA; 5) How RTCGA skills are acquired. This is the first known study to explore this topic of relevance to clinicians and researchers alike. Clinical observations were not only kinematic, but also included patient perceived experiences such as pain and orthotic comfort with normative kinematic reference values not perceived as important to that management goal. The most common barefoot RTCGA observations performed were the rearfoot to leg angle, medial bulge, forefoot abduction and arch integrity. However, a high amount of variation in many gait observations was noted between participants. Documentation methods also varied with a four-point scale system to grade motion and position most often employed and RTCGA was most often learnt through experience. The main barriers to performing RTCGA were clinical time and space restrictions. CONCLUSION: Findings from this study have provided a view of how podiatry MSK clinicians utilise RTCGA within their practice. MSK podiatrists use RTCGA as both an outcome measure and as an aid in decision making. This implies a perceived worth in conducting RTCGA, however further work is recommended that focusses on development of a national guideline to RTCGA to be adopted.
Assuntos
Análise da Marcha , Médicos/psicologia , Podiatria , Disfunção do Tendão Tibial Posterior/diagnóstico , Avaliação de Sintomas/psicologia , Adulto , Feminino , Humanos , Masculino , Padrões de Prática Médica , Avaliação de Sintomas/métodosRESUMO
BACKGROUND: Diverse theories of orthoses application have evolved with the continual development of podiatric biomechanics and orthotic management. This theoretical disparity can lead to confusion in clinical, educational, and research situations. However, although approaches are varied, the common consensus is that foot orthoses outcomes are generally positive. METHODS: Three main podiatric theories exist: the foot morphology theory, the sagittal plane facilitation theory, and tissue stress theory. By researching the available literature, the perspectives of all three theories are summarized, emphasizing areas of conflict and agreement. RESULTS: Through a unified theory, we introduce a premise by which the similar orthotic outcomes obtained from the three main podiatric theories may be explained. CONCLUSIONS: It remains up to the individual podiatric physician to decide which method to use to prescribe a foot orthosis. It may be of benefit to encompass all approaches rather than be dogmatic or exclusive.
Assuntos
Articulações do Pé/fisiopatologia , Pé/fisiopatologia , Aparelhos Ortopédicos , Fenômenos Biomecânicos , Humanos , Perna (Membro)/fisiopatologia , Articulação Metatarsofalângica/fisiopatologia , Modelos Teóricos , Rotação , Resultado do TratamentoRESUMO
Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low µM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Ácidos Pentanoicos/química , Peptídeos/análise , Peptídeos/farmacocinética , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Penetradores de Células/análise , Precipitação Química , Estabilidade de Medicamentos , Fluorocarbonos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Peptídeos/química , Ratos Wistar , Extração em Fase Sólida , Distribuição Tecidual , Ácido Tricloroacético/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/análise , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
The use of subtalar joint neutral (STJN) in the assessment and treatment of foot-related musculoskeletal symptomology is common in daily practice and still widely taught. The main pioneer of this theory was Dr Merton L. Root, and it has been labeled with a variety of names: "the foot morphology theory," "the subtalar joint neutral theory," or simply "Rootian theory" or "Root model." The theory's core concepts still underpin a common approach to musculoskeletal assessment of the foot, as well as the consequent design of foot orthoses. The available literature continues to point to Dr Root's theory as the most prevalently utilized. Concurrently, the worth of this theory has been challenged due to its poor reliability and limited external validity. This Viewpoint reviews the main clinical areas of the STJN theory, and concludes with a possible explanation and concerns for its ongoing use. To support our view, we will discuss (1) historical inaccuracies, (2) challenges with reliability, and (3) concerns with validity. J Orthop Sports Phys Ther 2018;48(3):130-132. doi:10.2519/jospt.2018.0604.
Assuntos
Medicina Baseada em Evidências , Órtoses do Pé , Articulação Talocalcânea/anatomia & histologia , Fenômenos Biomecânicos , Pesquisa Biomédica , Desenho de Equipamento , Doenças do Pé/reabilitação , Humanos , Reprodutibilidade dos Testes , Articulação Talocalcânea/fisiologia , Suporte de CargaRESUMO
BACKGROUND: The aim of this review was to evaluate and summarise the current evidence on non-computerised or non-recorded real time adult gait assessment conducted within the clinical musculoskeletal setting. It was hoped a protocol for best practice and a framework for further research could be developed from this search. RESEARCH QUESTION: Can a protocol for best practice and a framework for further research be established from previous literature relating to non-computerised or non-recorded real time adult gait analysis in a musculoskeletal clinical setting. METHODS: A literature review with no limitation on date of publication was conducted on the 18th February 2017. RESULTS: The review found no significantly informative papers relating to the search SIGNIFICANCE: The lack of research on the accuracy, reliability and therefore worth of this highly recommended area of musculoskeletal assessment raises concerns over current assessment and treatment pathways. Further work to develop a method by which gait analysis can be routinely employed in musculoskeletal clinics as a diagnostic tool is required, with any new approach undertaking robust methodological testing.
Assuntos
Marcha/fisiologia , Extremidade Inferior/fisiopatologia , Doenças Musculoesqueléticas/reabilitação , Modalidades de Fisioterapia , Adulto , Humanos , Doenças Musculoesqueléticas/fisiopatologiaRESUMO
Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.
Assuntos
Proteínas Sanguíneas/metabolismo , Interações Medicamentosas/fisiologia , Preparações Farmacêuticas/química , Preparações Farmacêuticas/normas , Ligação Proteica/fisiologia , Animais , Indústria Farmacêutica/normas , Humanos , Preparações Farmacêuticas/metabolismo , Plasma/metabolismoRESUMO
The experimental measurement of plasma protein binding is a useful in vitro Absorption Distribution Metabolism and Excretion(ADME) assay currently conducted in both screening and definitive early development candidate modes. The fraction unbound is utilized to calculate important pharmacokinetic (PK) parameters such as unbound clearance and unbound volume of distribution in animals that can be used to make human PK and dose predictions and estimate clinically relevant drug-drug interaction potential. Although these types of assays have been executed for decades, a rigorous statistical analysis of sources of variability has not been conducted because of the tedious nature of the manual experiment. Automated conduct of the incubations using a 96-well equilibrium dialysis device as well as high-throughput liquid chromatography-mass spectrometry quantitation has now made this level of rigor accessible and useful. Sources of variability were assessed including well position, day-to-day, and site-to-site reproducibility. Optimal pH conditions were determined using a design of experiments method interrogating buffer strength, CO2 % and device preparation conditions. Variability was minimized by implementing an in-well control that is concurrently analyzed with new chemical entity analytes. Data acceptance criteria have been set for both the in-well control and the range of analyte variability, with a sliding scale tied to analyte-binding characteristics. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3302-3309, 2014.
Assuntos
Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Ligação ProteicaRESUMO
A medially deviated axis has been cited as an etiologic factor in increasing pronatory moments across the subtalar joint axis. Orthoses are often used to reduce these pronatory moments, aiming to off-load related injured structures. By aligning the posting or incline of an orthosis shell medial to the axis and parallel to it, the amount of moments applied will be theoretically greater than if prescribed at a less-than-optimal angle. We first published the medial oblique shell inclination as a method to increase supinatory moments to a medially deviated subtalar joint axis in 2008. This paper summarizes the theoretical reasons for use and introduces original methods of construction.