Transfer learning predicts species-specific drug interactions in emerging pathogens.

Machine learning (ML) algorithms are necessary to efficiently identify potent drug combinations within a large candidate space to combat drug resistance. However, existing ML approaches cannot be applied to emerging and under-studied pathogens with limited training data. To address this, we developed a transfer learning and crowdsourcing framework (TACTIC) to train ML models on data from multiple bacteria. TACTIC was built using 2,965 drug interactions from 12 bacterial strains and outperformed traditional ML models in predicting drug interaction outcomes for species that lack training data. Top TACTIC model features revealed genetic and metabolic factors that influence cross-species and species-specific drug interaction outcomes. Upon analyzing ~600,000 predicted drug interactions across 9 metabolic environments and 18 bacterial strains, we identified a small set of drug interactions that are selectively synergistic against Gram-negative (e.g., A. baumannii) and non-tuberculous mycobacteria (NTM) pathogens. We experimentally validated synergistic drug combinations containing clarithromycin, ampicillin, and mecillinam against M. abscessus, an emerging pathogen with growing levels of antibiotic resistance. Lastly, we leveraged TACTIC to propose selectively synergistic drug combinations to treat bacterial eye infections (endophthalmitis).

Respirable Clofazimine Particles Produced by Air Jet Milling Technique Are Efficacious in Treatment of BALB/c Mice with Chronic Mycobacterium tuberculosis Infection.

Tuberculosis (TB) remains a major cause of morbidity and mortality, particularly in low- and middle-income countries where access to health care workers, cold-chain storage, and sterile water sources may be limited. Inhaled drug delivery is a promising alternative to systemic delivery of antimycobacterial drugs, as it enables rapid achievement of high infection-site drug concentrations. The off-patent drug clofazimine (CFZ) may be particularly suitable for this route, given its known systemic toxicities. In this study, micronized CFZ particles produced by air jet milling were assessed for shelf-stability, pharmacokinetics, and anti-TB efficacy by the oral and pulmonary routes in BALB/c mice. Intratracheal instillation of micronized CFZ particles produced several-fold higher lung concentrations after a single 30 mg/kg dose compared to delivery via oral gavage, and faster onset of bactericidal activity was observed in lungs of mice with chronic Mycobacterium tuberculosis infection compared to the oral route. Both infection status and administration route affected the multidose pharmacokinetics (PK) of micronized CFZ. Increased lung and spleen accumulation of the drug after pulmonary administration was noted in infected mice compared to naive mice, while the opposite trend was noted in the oral dosing groups. The infection-dependent PK of inhaled micronized CFZ may point to a role of macrophage trafficking in drug distribution, given the intracellular-targeting nature of the formulation. Lastly, air jet milled CFZ exhibited robustness to storage-induced chemical degradation and changes in aerosol performance, thereby indicating the suitability of the formulation for treatment of TB in regions with limited cold chain supply.

Comparison of HPMC Inhalation-Grade Capsules and Their Effect on Aerosol Performance Using Budesonide and Rifampicin DPI Formulations.

Despite the fact that capsules play an important role in many dry powder inhalation (DPI) systems, few studies have been conducted to investigate the capsules' interactions with respirable powders. The effect of four commercially available hydroxypropyl methylcellulose (HPMC)inhalation-grade capsule types on the aerosol performance of two model DPI formulations (lactose carrier and a carrier-free formulation) at two different pressure drops was investigated in this study. There were no statistically significant differences in performance between capsules by using the carrier-based formulation. However, there were some differences between the capsules used for the carrier-free rifampicin formulation. At 2-kPa pressure drop conditions, Embocaps® VG capsules had a higher mean emitted fraction (EF) (89.86%) and a lower mean mass median aerodynamic diameter (MMAD) (4.19 µm) than Vcaps® (Capsugel) (85.54%, 5.10 µm) and Quali-V® I (Qualicaps) (85.01%, 5.09 µm), but no significant performance differences between Embocaps® and ACGcaps™ HI. Moreover, Embocaps® VG capsules exhibited a higher mean respirable fraction (RF)/fine particle fraction (FPF) with a 3-µm-sized cutoff (RF/FPF< 3 µm) (33.05%/35.36%) against Quali-V® I (28.16%/31.75%) (P < 0.05), and a higher RF/FPF with a 5-µm-sized cutoff (RF/FPF< 5 µm) (49.15%/52.57%) versus ACGcaps™ HI (38.88%/41.99%) (P < 0.01) at 4-kPa pressure drop condition. Aerosol performance variability, pierced-flap detachment, as well as capsule hardness and stiffness, may all influence capsule type selection in a carrier-based formulation. The capsule type influenced EF, RF, FPF, and MMAD in the carrier-free formulation.

Identification of Stability Constraints in the Particle Engineering of an Inhaled Monoclonal Antibody Dried Powder.

Monoclonal antibody (mAb) based therapies may provide a valuable new treatment modality for acute and chronic lung diseases, including asthma, respiratory infections, and lung cancer. Currently mAbs are delivered via systemic administration routes, but direct delivery to the lungs via the inhaled route could provide higher concentrations at the site of disease and reduced off-target effects. Though lyophilized mAbs may be reconstituted and delivered to the lungs using nebulizers, dry powder inhalers provide a more patient-friendly delivery method based upon their fast administration time and portability. However, particle engineering processes required to prepare respirable dried powders for DPI delivery involve multiple potential stressors for mAbs, which have not been fully explored. In this study, a systematic examination of various aspects of the particle engineering process (atomization, freezing, drying, and storage) was performed to further understand their impact on mAb structure and aggregation. Using anti-streptavidin IgG1 as a model mAb, atomization settings were optimized using a design of experiments approach to elucidate the relationship between feed flow rate, formulation solid content, and atomization airflow rate and protein structural changes and aggregation. The optimized atomization conditions were then applied to spray drying and spray freezing drying particle engineering processes to determine the effects of freezing and drying on IgG1 stability and aerosol performance of the powders. IgG1 was found to be particularly susceptible to degradation induced by the expansive air-ice interface generated by spray freeze drying and this process also produced powders that exhibited decreased storage stability. This study further delineates the design space for manufacturing of respirable biologic therapies and is intended to serve as a roadmap for future development work.

A Quality by Design Framework for Capsule-Based Dry Powder Inhalers.

Capsule-based dry powder inhalers (cDPIs) are widely utilized in the delivery of pharmaceutical powders to the lungs. In these systems, the fundamental nature of the interactions between the drug/formulation powder, the capsules, the inhaler device, and the patient must be fully elucidated in order to develop robust manufacturing procedures and provide reproducible lung deposition of the drug payload. Though many commercially available DPIs utilize a capsule-based dose metering system, an in-depth analysis of the critical factors associated with the use of the capsule component has not yet been performed. This review is intended to provide information on critical factors to be considered for the application of a quality by design (QbD) approach for cDPI development. The quality target product profile (QTPP) defines the critical quality attributes (CQAs) which need to be understood to define the critical material attributes (CMA) and critical process parameters (CPP) for cDPI development as well as manufacturing and control.

Inhaled fixed-dose combination powders for the treatment of respiratory infections.

INTRODUCTION: Respiratory infections are a major cause of morbidity and mortality. As an alternative to systemic drug administration, inhaled drug delivery can produce high drug concentrations in the lung tissue to overcome resistant bacteria. The development of inhaled fixed-dose combination powders (I-FDCs) is promising next step in this field, as it would enable simultaneous drug-drug or drug-adjuvant delivery at the site of infection, thereby promoting synergistic activity and improving patient compliance. AREAS COVERED: This review covers the clinical and pharmaceutical rationales for the development of I-FDCs for the treatment of respiratory infections, relevant technologies for particle and powder generation, and obstacles which must be addressed to achieve regulatory approval. EXPERT OPINION: I-FDCs have been widely successful in the treatment of asthma and chronic obstructive pulmonary disease; however, application of I-FDCs towards the treatment of respiratory infections carries additional challenges related to the high dose requirements and physicochemical characteristics of anti-infective drugs. At present, co-spray drying is an especially promising approach for the development of composite fixed-dose anti-infective particles for inhalation. Though the majority of fixed-dose research has thus far focused on the combination of multiple antibiotics, future work may shift to the additional inclusion of immunomodulatory agents or repurposed non-antibiotics.

Development and evaluation of inhalable composite niclosamide-lysozyme particles: A broad-spectrum, patient-adaptable treatment for coronavirus infections and sequalae.

Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, administration of the currently available oral formulation results in systemic drug levels that are too low for the inhibition of SARS-CoV-2. We hypothesized that the co-formulation of NIC with an endogenous protein, human lysozyme (hLYS), could enable the direct aerosol delivery of the drug to the respiratory tract as an alternative to oral delivery, thereby effectively treating COVID-19 by targeting the primary site of SARS-CoV-2 acquisition and spread. To test this hypothesis, we engineered and optimized composite particles containing NIC and hLYS suitable for delivery to the upper and lower airways via dry powder inhaler, nebulizer, and nasal spray. The novel formulation demonstrates potent in vitro and in vivo activity against two coronavirus strains, MERS-CoV and SARS-CoV-2, and may offer protection against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to SARS-CoV-2 infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure rapid clinical development and wide-spread utilization.

Effect of Particle Formation Process on Characteristics and Aerosol Performance of Respirable Protein Powders.

Pulmonary delivery of biopharmaceuticals may enable targeted local therapeutic effect and noninvasive systemic administration. Dry powder inhaler (DPI) delivery is an established patient-friendly approach for delivering large molecules to the lungs; however, the complexities of balancing protein stability with aerosol performance require that the design space of biopharmaceutical DPI formulations is rigorously explored. Utilizing four rationally selected formulations obtained using identical atomization conditions, an extensive study of the effect of the particle formation process (spray drying or spray freeze-drying) on powder properties, aerosol performance, and protein stability was performed. Multiple linear regression analysis was used to understand the relationship between powder properties, device dispersion mechanism, and aerosol performance. Spray drying and spray freeze-drying, despite the same spraying conditions, produced powders with vastly different physical characteristics, though similar aerosol performance. The resulting regression model points to the significance of particle size, density, and surface properties on the resulting aerosol performance, with these factors weighing differently according to the device dispersion mechanism utilized (shear-based or impaction-based). The physical properties of the produced spray dried and spray freeze-dried powders have differing implications for long-term stability, which will be explored extensively in a future study.

Formulation techniques for high dose dry powders.

Delivery of drugs to the lungs via dry powder inhaler (DPI) is a promising approach for the treatment of both local pulmonary conditions and systemic diseases. Though DPIs are widely used for the pulmonary deposition of potent bronchodilators, anticholinergics, and corticosteroids, there is growing interest in the utilization of this delivery system for the administration of high drug doses to the lungs, as made evident by recent regulatory approvals for anti-microbial, anti-viral and osmotic agents. However, the formulation of high dose DPIs carries several challenges from both a physiological and physicochemical standpoint. This review describes the various formulation techniques utilized to overcome the barriers associated with the pulmonary delivery of high dose powders.

Influence of Formulation Factors on the Aerosol Performance and Stability of Lysozyme Powders: a Systematic Approach.

With the growing interest in developing biologics for pulmonary delivery, systematic fast screening methods are needed for rapid development of formulations. Due to the labile nature of macromolecules, the development of stable, biologically active formulations with desired aerosol performance imposes several challenges both from a formulation and processing perspective. In this study, spray-freeze-drying was used to develop respirable protein powders. In order to systematically map the selected design space, lysozyme aqueous pre-formulations were prepared based on a constrained mixture design of experiment. The physicochemical properties of the resulting powders were characterized and the effects of formulation factors on aerosol performance and protein stability were systematically screened using a logic flow chart. Our results elucidated several relevant formulation attributes (density, total solid content, protein:sugars ratio) required to achieve a stable lysozyme powder with desirable characteristics for pulmonary delivery. A similar logical fast screening strategy could be used to delineate the appropriate design space for different types of proteins and guide the development of powders with pre-determined aerodynamic properties.

Excipient-Free Pulmonary Delivery and Macrophage Targeting of Clofazimine via Air Jet Micronization.

Clofazimine (CFZ) is highly active against mycobacterium, including resistant Mycobacterium tuberculosis, but its therapeutic efficacy via the oral route is limited by severe adverse effects, poor aqueous solubility, and slow onset of action. Pulmonary delivery of CFZ is an attractive alternative to target mycobacterium-harboring alveolar macrophages. This study explores the use of air jet milling to develop a respirable, cost-effective CFZ formulation. Jet milled CFZ was readily dispersed from an off-the-shelf dry powder inhaler without the need for additional excipients or carrier particles. Additionally, milled CFZ was internalized by J774.A1 alveolar macrophages within 8 h, with evidence of intracellular biotransformation of the CFZ crystals and macrophage sequestration by 24 h. Less macrophage toxicity was noted in comparison to solubilized drug. Compared to macrophage uptake rate, dissolution of milled CFZ was limited, thereby potentially reducing systemic absorption and subsequent side effects. These results suggest that jet milling is an effective manufacturing method in the development of a CFZ formulation for pulmonary delivery and alveolar macrophage targeting.

Connexin membrane materials as potent inhibitors of breast cancer cell migration.

Gap junction (GJ) channels facilitate cell-cell communication through the exchange of chemical and mechanical signals, ensuring proper tissue development and homeostasis. The complex, disease stage-dependent role of connexins in breast cancer progression has been extensively studied over the past two decades. In the early stages of breast cancer, substantial evidence supports the role of GJ channels, formed by connexins at the interfaces between neighbouring cells, as suppressors of cell migration and proliferation. These findings suggest that materials that reintroduce connexins into the tumour cell environment have the potential to inhibit cell migration. Here, we report that exposure of highly metastatic MDA-MB-231 breast tumour cells to connexin-rich biovesicle materials potently suppresses cell migration. Specifically, these biovesicles, which can form GJ interfaces with cells, were extracted from the plasma membrane of donor cells engineered to express a high concentration of functional connexin 43 channels. These connexin-rich membrane materials dramatically reduced cell migration in both a transwell migration assay and a scratch closure assay. Collectively, these results suggest that using membrane materials to reintroduce connexins into the tumour cell environment provides a novel approach for combating cell migration and invasion.