A Model-Based Pharmacokinetic/Pharmacodynamic Analysis of the Combination of Amoxicillin and Monophosphoryl Lipid A Against S. pneumoniae in Mice.

Combining amoxicillin with the immunostimulatory toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) represents an innovative approach for enhancing antibacterial treatment success. Exploiting pharmacokinetic and pharmacodynamic data from an infection model of Streptococcus pneumoniae infected mice, we aimed to evaluate the preclinical exposure-response relationship of amoxicillin with MPLA coadministration and establish a link to survival. Antibiotic serum concentrations, bacterial numbers in lung and spleen and survival data of mice being untreated or treated with amoxicillin (four dose levels), MPLA, or their combination were analyzed by nonlinear mixed-effects modelling and time-to-event analysis using NONMEM® to characterize these treatment regimens. On top of a pharmacokinetic interaction, regarding the pharmacodynamic effects the combined treatment was superior to both monotherapies: The amoxicillin efficacy at highest dose was increased by a bacterial reduction of 1.74 log10 CFU/lung after 36 h and survival was increased 1.35-fold to 90.3% after 14 days both compared to amoxicillin alone. The developed pharmacometric pharmacokinetic/pharmacodynamic disease-treatment-survival models provided quantitative insights into a novel treatment option against pneumonia revealing a pharmacokinetic interaction and enhanced activity of amoxicillin and the immune system stimulator MPLA in combination. Further development of this drug combination flanked with pharmacometrics towards the clinical setting seems promising.

The Biosynthetic Monophosphoryl Lipid A Enhances the Therapeutic Outcome of Antibiotic Therapy in Pneumococcal Pneumonia.

Alternative treatment strategies against bacterial infections are required to decrease the use of antibiotics. This study tested the hypothesis that stimulation of the innate immune receptor Toll-like receptor 4 can be combined with antibiotics to improve the treatment of invasive pneumonia. The efficacy of the biosynthetic monophosphoryl lipid A (MPLA), a clinically approved Toll-like receptor 4 activator, was tested in a mouse model of Streptococcus pneumoniae respiratory infection. Interestingly, administration of amoxicillin or MPLA decreased 400- to 11 000-fold the bacterial load in the lungs and spleen but did not enhance survival compared to mock treatment. The single administration of a combination of MPLA and amoxicillin further reduced 10- to 18-fold the bacterial colonization and invasion and significantly improved protection against lethal disease. The combined administration of MPLA and amoxicillin in a context of infection was associated with transient increase of the serum concentrations of amoxicillin and pro-inflammatory cytokines and chemokines as well as the expression of immune genes in lung tissue. Remarkably, the systemic and lung immune activation extended beyond amoxicillin elimination, suggesting a two-step and cooperative anti-infective effect, i.e., rapid antibiotic-mediated alteration of bacteria and a long-lasting impact through mucosal and systemic immunity. Our proof-of-concept study demonstrated for the first time that boosting Toll-like receptor 4 signaling can synergize with antibiotics in order to increase the efficacy of therapy of bacterial pneumonia, thereby in fine reducing the dose or regimen of antibiotics.

The Use of Translational Modelling and Simulation to Develop Immunomodulatory Therapy as an Adjunct to Antibiotic Treatment in the Context of Pneumonia.

The treatment of respiratory tract infections is threatened by the emergence of bacterial resistance. Immunomodulatory drugs, which enhance airway innate immune defenses, may improve therapeutic outcome. In this concept paper, we aim to highlight the utility of pharmacometrics and Bayesian inference in the development of immunomodulatory therapeutic agents as an adjunct to antibiotics in the context of pneumonia. For this, two case studies of translational modelling and simulation frameworks are introduced for these types of drugs up to clinical use. First, we evaluate the pharmacokinetic/pharmacodynamic relationship of an experimental combination of amoxicillin and a TLR4 agonist, monophosphoryl lipid A, by developing a pharmacometric model accounting for interaction and potential translation to humans. Capitalizing on this knowledge and associating clinical trial extrapolation and statistical modelling approaches, we then investigate the TLR5 agonist flagellin. The resulting workflow combines expert and prior knowledge on the compound with the in vitro and in vivo data generated during exploratory studies in order to construct high-dimensional models considering the pharmacokinetics and pharmacodynamics of the compound. This workflow can be used to refine preclinical experiments, estimate the best doses for human studies, and create an adaptive knowledge-based design for the next phases of clinical development.

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry assay to determine amoxicillin concentrations in biological matrix of little volume.

To assess pharmacokinetics of amoxicillin (AMX) in mice, limitations such as a small sampling volume and low drug concentrations have to be addressed. Similar challenges are faced in a clinical framework, e.g. for therapeutic drug monitoring in neonates or small-scale in vitro investigations. An assay enabling quantification of small sample volumes but still at very low concentrations covering a broad concentration range is thus needed. A simple, rapid and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and successfully validated for quantification of AMX in mouse serum according to European Medicines Agency guidelines. Sample preparation enabled the use of only 10 µL of serum, which is 5-fold less than comparable assays and allows to reduce the number of mice used in pharmacokinetic studies. After protein precipitation with 40 µL chilled methanol and dilution of the supernatant with water, the sample was injected into the LC system on a Poroshell 120 Phenyl Hexyl column (2.1 × 100 mm, 2.7 µm). Chromatographic separation was achieved using a gradient method consisting of acetonitrile and ultra-pure water, both with 0.1% (V/V) formic acid. Positive electrospray ionisation in multiple reaction monitoring mode was used for detection and quantification of AMX. Application to murine study samples demonstrated the reliability of the developed method being accurate and precise with a quantification range from 0.01 to 10 µg/mL. The assay is easily transferable due to a simple sample preparation and confirmed stability of AMX under various applied conditions.