In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection.

Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

Physiologically Based Pharmacokinetic/Pharmacodynamic Modeling Accurately Predicts the Better Bronchodilatory Effect of Inhaled Versus Oral Salbutamol Dosage Forms.

BACKGROUND: Predicting local lung tissue pharmacodynamic (PD) responses of inhaled drugs is a longstanding challenge related to the lack of experimental techniques to determine local free drug concentrations. This has prompted the use of physiologically based pharmacokinetic (PBPK) modeling to potentially predict local concentration and response. A unique opportunity for PBPK model evaluation is provided by the clinical PD data for salbutamol, which in its inhaled dosage form (400 µg), produces a higher bronchodilatory effect than in its oral dosage form (2 mg) despite lower drug concentrations in blood. The present study aimed at evaluating whether inhalation PBPK model predictions of free drug in tissue would be predictive of these observations. METHODS: A PBPK model, including 24 airway generations, was parameterized to describe lung, plasma, and epithelial lining fluid concentrations of salbutamol administered intratracheally and intravenously to rats (100 nmol/kg). Plasma and lung tissue concentrations of unbound (R)-salbutamol, the active enantiomer, were predicted with a humanized version of the model and related to effect in terms of forced expiratory volume in 1 second (FEV1). RESULTS: In contrast to oral dosing, the model predicted inhalation to result in spatial heterogeneity in the target site concentrations (subepithelium) with higher free drug concentrations in the lung as compared with the plasma. FEV1 of inhaled salbutamol was accurately predicted from the PK/PD relationship derived from oral salbutamol and PBPK predictions of free concentration in airway tissue of high resistance (e.g., 6th generation). CONCLUSION: An inhalation PBPK-PD model was developed and shown predictive of local pharmacology of inhaled salbutamol, thus conceptually demonstrating the validity of PBPK model predictions of free drug concentrations in lung tissue. This achievement unlocks the power of inhalation PBPK modeling to interrogate local pharmacology and guide optimization and development of inhaled drugs and their formulations.

A Partial Differential Equation Approach to Inhalation Physiologically Based Pharmacokinetic Modeling.

The heterogeneous nature of the lungs and the range of processes affecting pulmonary drug disposition make prediction of inhaled drugs challenging. These predictions are critical, as the local exposure cannot be measured and current inhalation physiologically based pharmacokinetic (PBPK) models do not capture all necessary features. Utilizing partial differential equations, we present an inhalation PBPK model to describe the heterogeneity in both lung physiology and particle size. The model mechanistically describes important processes, such as deposition, mucociliary clearance, and dissolution. In addition, simplifications are introduced to reduce computational cost without loss of accuracy. Three case studies exemplify how the model can enhance our understanding of pulmonary drug disposition. Specific findings include that most small airways can be targeted by inhalation, and overdosing may eradicate the advantage of inhalation. The presented model can guide the design of inhaled molecules, formulations, as well as clinical trials, providing opportunities to explore regional targeting.

Lung Retention by Lysosomal Trapping of Inhaled Drugs Can Be Predicted In Vitro With Lung Slices.

Modulating and optimizing the local pharmacokinetics of inhaled drugs by chemical design or formulation is challenged by the lack of predictive in vitro systems and in vivo techniques providing a detailed description of drug location in the lung. The present study investigated whether a new experimental setup of freshly prepared agarose-filled lung slices can be used to estimate lung retention in vitro, by comparing with in vivo lung retention after intratracheal instillation. Slices preloaded with inhaled ß-adrenergic compounds (salbutamol, formoterol, salmeterol, indacaterol or AZD3199) were incubated in a large volume of buffer (w/wo monensin to assess the role of lysosomal trapping), and the amount remaining in slices at different time points was determined with liquid chromatography-tandem mass spectrometry. The in vitro lung retention closely matched the in vivo lung retention (half-lives within 3-fold for 4/5 compounds), and monensin shortened the half-lives for all compounds. The results suggest that freshly prepared rat lungs slices can be used to predict lung retention and that slow kinetics of lysosomal trapping is a key mechanism by which retention in the lung and the effect duration of inhaled ß-adrenergic bronchodilators are prolonged.

Development of a Novel Lung Slice Methodology for Profiling of Inhaled Compounds.

The challenge of defining the concentration of unbound drug at the lung target site after inhalation limits the possibility to optimize target exposure by compound design. In this study, a novel rat lung slice methodology has been developed and applied to study drug uptake in lung tissue, and the mechanisms by which this occurs. Freshly prepared lung slices (500 µm) from drug-naive rats were incubated with drugs followed by determination of the unbound drug volume of distribution in lung (Vu,lung), as the total concentration of drug in slices divided by the buffer (unbound) concentration. Vu,lung determined for a set of inhaled drug compounds ranged from 2.21 mL/g for salbutamol to 2970 mL/g for dibasic compound A. Co-incubation with monensin, a modulator of lysosomal pH, resulted in inhibition of tissue uptake of basic propranolol to 13%, indicating extensive lysosomal trapping. Partitioning into cells was particularly high for the cation MPP+ and the dibasic compound A, likely because of the carrier-mediated transport and lysosomal trapping. The results show that different factors are important for tissue uptake and the presented method can be used for profiling of inhaled compounds, leading to a greater understanding of distribution and exposure of drug in the lung.

A novel in vivo receptor occupancy methodology for the glucocorticoid receptor: toward an improved understanding of lung pharmacokinetic/pharmacodynamic relationships.

Investigation of pharmacokinetic/pharmacodynamic (PK/PD) relationships for inhaled drugs is challenging because of the limited possibilities of measuring tissue exposure and target engagement in the lung. The aim of this study was to develop a methodology for measuring receptor occupancy in vivo in the rat for the glucocorticoid receptor (GR) to allow more informative inhalation PK/PD studies. From AstraZeneca's chemical library of GR binders, compound 1 [N-(2-amino-2-oxo-ethyl)-3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-methyl-benzamide] was identified to have properties that are useful as a tracer for GR in vitro. When given at an appropriate dose (30 nmol/kg) to rats, compound 1 functioned as a tracer in the lung and spleen in vivo using liquid chromatography-tandem mass spectrometry bioanalysis. The methodology was successfully used to show the dose-receptor occupancy relationship measured at 1.5 hours after intravenous administration of fluticasone propionate (20, 150, and 750 nmol/kg) as well as to characterize the time profile for receptor occupancy after a dose of 90 nmol/kg i.v. The dose giving 50% occupancy was estimated as 47 nmol/kg. The methodology is novel in terms of measuring occupancy strictly in vivo and by using an unlabeled tracer. This feature confers key advantages, including occupancy estimation not being influenced by drug particle dissolution or binding/dissociation taking place postmortem. In addition, the tracer may be labeled for use in positron emission tomography imaging, thus enabling occupancy estimation in humans as a translatable biomarker of target engagement.