Differential effects of antibiotics on neutrophils exposed to lipoteichoic acid derived from Staphylococcus aureus.

BACKGROUND: Persistent bacteremia occurs in at least 30% of patients with Staphylococcus aureus bloodstream infection (SAB) and may be attributable to a dysregulated host immune response. Neutrophils interact with a variety of S. aureus microbial factors, including lipoteichoic acid (LTA), to activate phagocytic function in a concentration-dependent manner. Antibiotics have been shown to exert both direct antimicrobial action as well as immunomodulatory effects. In this study, we compared the effects of different anti-staphylococcal antibiotics on LTA-mediated immune activation of neutrophils. METHODS: Neutrophils obtained from healthy volunteers were exposed to two levels of LTA (1 and 10 µg/ml) with or without addition of antibiotics from different pharmacologic classes (vancomycin, daptomycin, ceftaroline). Neutrophil function was assessed by examining phagocytic response, activation (CD11b, CD62L expression), Toll-like receptor-2 expression, cell survival and apoptosis, and CXCL8 release. RESULTS: Differential LTA-mediated antibiotic effects on neutrophil function were observed primarily at the high LTA exposure level. Ceftaroline in the presence of 10 µg/ml LTA had the most prominent effects on phagocytosis and CD11b and CD62L expression, with trends towards increased neutrophil survival and preservation of CXCL8 release when compared to daptomycin and vancomycin with the latter significantly dampening PMN CXCL8 release. CONCLUSIONS: Select antimicrobial agents, such as ceftaroline, exert immunostimulatory effects on neutrophils exposed to S. aureus LTA, which when confirmed in vivo, could be leveraged for its dual immunomodulatory and antibacterial actions for the treatment of persistent SAB mediated by a dysregulated host response.

Real-time algorithmic exchange and processing of pharmaceutical quality data and information.

Herein, a modern method is proposed for exchanging and processing real-time medicinal product information using Health Level 7 International's (HL7) Fast Healthcare Interoperability Resources (FHIR®) standard, Application Programming Interfaces (API), digitization and artificial intelligence. FHIR is presently in use largely to facilitate interactions between patient-facing healthcare institutions, such as hospitals, doctor's offices, and laboratories, for electronic health record management and exchange. There are several ongoing efforts to adapt the FHIR standard for regulatory use cases to support the needs of the global biopharmaceutical industry, including the exchange of Electronic Product Information (ePI); chemistry, manufacturing, and controls (CMC) data; and adverse event reporting. Once in place, this new method of data exchange is expected to (1) improve efficiency by reducing the time and effort needed to manage regulatory information; (2) accelerate decision making; (3) encourage innovation in pharmaceutical manufacturing; (4) improve the ability and agility of information exchange. Currently, the end-to-end timescale for the pharmaceutical regulatory workflow is measured in months and years. This new paradigm will use FHIR APIs and other supporting technologies to reduce the potential time for data exchange from months to days, hours, minutes, and eventually sub-seconds. With such drastic improvements in speed provided by digitization, automation, and interoperability, the biopharmaceutical industry can reach more patients, and more quickly than at any time in the industry's 100+ year history. The present work will focus on examining specific real-world implementation examples for using FHIR to support exchange of CMC information within and across the biopharmaceutical industry.

Patient-Centric Product Development: A Summary of Select Regulatory CMC and Device Considerations.

Patient-centric drug development describes the systematic approach to incorporating the patient's perspectives and preferences into the design, assessment, and production of a therapeutic product. While a patient centric approach can be applied at any stage of the drug development lifecycle, an integrated end-to-end strategy is often most effective to create an optimized product for the patient at the earliest possible timepoint. The importance of patient centricity is well recognized by health authorities and biopharmaceutical organizations which have established toolsets, guidances, and methodologies for incorporating patient input during the clinical stage of development. However, in addition to clinical research, there are other significant aspects of product development that profoundly impact the patient experience. Specifically, chemistry, manufacturing, and control (CMC) and device aspects must also be acknowledged and addressed as part of a cohesive patient-centric development strategy. This review explores current applications and regulatory considerations for patient-centric approaches across the product lifecycle, including R&D, early product development, clinical development, device and combination product development, and post-approval change management. Specific topics of discussion include the contributions of product modality, formulation, and devices to the patient experience; usage of the Quality Target Product Profile (QTPP) as a patient-centered design tool; and post-approval product optimization. Future advancements in regulatory data management and information exchange are also explored as potential enablers of patient engagement which support enhanced communication and interconnectivity between stakeholders. Multidisciplinary collaboration between patients, health authorities, health care providers, and the biopharmaceutical industry is ultimately necessary for ensuring that medicinal products, and their corresponding regulatory processes, take on a patient-first mindset that prioritizes patient needs, values, and preferences.

Structured content and data management-enhancing acceleration in drug development through efficiency in data exchange.

Innovation in pharmaceutical therapeutics is critical for the treatment of serious diseases with unmet medical need. To accelerate the approval of these innovative treatments, regulatory agencies throughout the world are increasingly adopting the use of expedited pathways and collaborative regulatory reviews. These pathways are primarily driven by promising clinical results but become challenging for Chemistry, Manufacturing, and Controls (CMC) information in regulatory submissions. Condensed and shifting timelines present constraints that require new approaches to the management of regulatory filings. This article emphasizes technological advances that have the potential to tackle the underlying inefficiencies in the regulatory filing eco-system. Structured content and data management (SCDM) is highlighted as a foundation for technologies that can ease the burden on both sponsors and regulators by streamlining data usage in regulatory submissions. Re-mapping of information technology infrastructure will improve the usability of data by moving away from document-based filings towards electronic data libraries. Although the inefficiencies of the current regulatory filing eco-system are more evident for products that are filed using expedited pathways, it is envisioned that the more widespread adoption of SCDM, across standard filing and review processes, will improve overall efficiency and speed in the compilation and review of regulatory submissions.

In-Human Multiyear Evolution of Carbapenem-Resistant Klebsiella pneumoniae Causing Chronic Colonization and Intermittent Urinary Tract Infections: A Case Study.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a frequent pathogen of the urinary tract, but how CRKP adapts in vivo over time is unclear. We examined 10 CRKP strains from a patient who experienced chronic colonization and recurrent urinary tract infections over a period of 4.5 years. We performed whole-genome sequencing and phenotypic assays to compare isolates that had evolved relative to the first isolate collected and to correlate genetic and phenotypic changes over time with the meropenem-containing regimen received. Phylogenetic analysis indicated that all 10 strains originated from the same sequence type 258 (ST258) clone and that three sublineages (SL) evolved over time; strains from two dominant sublineages were selected for detailed analysis. Up to 60 new mutations were acquired progressively in genes related to antibiotic resistance, cell metabolism, and biofilm production over time. Doubling of meropenem MICs, increases in biofilm production and blaKPC expression, and altered carbon metabolism occurred in the latter strains from the last sublineage compared to the initial strain. Subinhibitory meropenem exposure in vitro significantly induced or maintained high levels of biofilm production in colonizing isolates, but isolates causing infection were unaffected. Despite acquiring different mutations that affect carbon metabolism, overall carbon utilization was maintained across different strains. Together, these data showed that isolated urinary CRKP evolved through multiple adaptations affecting carbon metabolism, carbapenem resistance, and biofilm production to support chronic colonization and intermittent urinary tract infections. Our findings highlight the pliability of CRKP in adapting to repeated antibiotic exposure and should be considered when developing novel therapeutic and stewardship strategies. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) can cause a variety of infections such as recurrent urinary tract infections (rUTI) with the ability to change with the host environment over time. However, it is unclear how CRKP adapts to the urinary tract during chronic infections and colonization. Here, we studied the evolution of CRKP strains from a patient who experienced chronic colonization and recurrent UTIs over a period of 4.5 years despite multiple treatment courses with meropenem-containing regimens. Our findings show the flexibility of CRKP strains in developing changes in carbapenem resistance, biofilm production, and carbon metabolism over time, which could facilitate their persistence in the human body for long periods of time in spite of repeated antibiotic therapy.

Re-Envisioning Pharmaceutical Manufacturing: Increasing Agility for Global Patient Access.

The traditional paradigm for pharmaceutical manufacturing is focused primarily upon centralized facilities that enable mass production and distribution. While this system reliably maintains high product quality and reproducibility, its rigidity imposes limitations upon new manufacturing innovations that could improve efficiency and support supply chain resiliency. Agile manufacturing methodologies, which leverage flexibility through portability and decentralization, allow manufacturers to respond to patient needs on demand and present a potential solution to enable timely access to critical medicines. Agile approaches are particularly applicable to the production of small-batch, personalized therapies, which must be customized for each individual patient close to the point-of-care. However, despite significant progress in the advancement of agile-enabling technologies across several different industries, there are substantial global regulatory challenges that encumber the adoption of agile manufacturing techniques in the pharmaceutical industry. This review provides an overview of regulatory barriers as well as emerging opportunities to facilitate the use of agile manufacturing for the production of pharmaceutical products. Future-oriented approaches for incorporating agile methodologies within the global regulatory framework are also proposed. Collaboration between regulators and manufacturers to cohesively navigate the regulatory waters is ultimately needed to best serve patients in the rapidly-changing healthcare environment.

Meeting report: Advancing accelerated regulatory review with Real-Time Oncology Review (RTOR), Project Orbis, and the Product Quality Assessment Aid.

The American Association of Pharmaceutical Scientists (AAPS) Chemistry, Manufacturing, and Controls (CMC) Community hosted two virtual panel discussions focusing on several novel regulatory review pathways for innovative oncology products: Real-Time Oncology Review (RTOR), Project Orbis, and the Product Quality Assessment Aid (PQAAid). The panel sessions were held on August 27, 2021, for the discussion of RTOR, and January 21, 2022, for the discussion of Project Orbis and the PQAAid. Both panel sessions included representatives from the US Food and Drug Administration (FDA) and subject matter experts from the pharmaceutical and biotechnology industries, with the aim of facilitating knowledge sharing on CMC-specific advantages, challenges, eligibility criteria for participation, and operational modifications instituted through the utilization of these acceleration initiatives. Key topics included managing cross-regional regulatory CMC requirements, adapting to expedited development timelines, coordinating interactions between health authorities and industry, and potential opportunities for future improvement and expansion of these programs. As RTOR, Project Orbis, and PQAAid are relatively new initiatives, the experiences shared by the panel experts are valuable for providing deeper insight into these new regulatory pathways and processes.

Anti-Inflammatory Effects of RTD-1 in a Murine Model of Chronic Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB, Inflammasome Gene Expression, and Pro-IL-1ß Biosynthesis.

Vicious cycles of chronic airway obstruction, lung infections with Pseudomonas aeruginosa, and neutrophil-dominated inflammation contribute to morbidity and mortality in cystic fibrosis (CF) patients. Rhesus theta defensin-1 (RTD-1) is an antimicrobial macrocyclic peptide with immunomodulatory properties. Our objective was to investigate the anti-inflammatory effect of RTD-1 in a murine model of chronic P. aeruginosa lung infection. Mice received nebulized RTD-1 daily for 6 days. Bacterial burden, leukocyte counts, and cytokine concentrations were evaluated. Microarray analysis was performed on bronchoalveolar lavage fluid (BALF) cells and lung tissue homogenates. In vitro effects of RTD-1 in THP-1 cells were assessed using quantitative reverse transcription PCR, enzyme-linked immunosorbent assays, immunoblots, confocal microscopy, enzymatic activity assays, and NF-κB-reporter assays. RTD-1 significantly reduced lung white blood cell counts on days 3 (-54.95%; p = 0.0003) and 7 (-31.71%; p = 0.0097). Microarray analysis of lung tissue homogenates and BALF cells revealed that RTD-1 significantly reduced proinflammatory gene expression, particularly inflammasome-related genes (nod-like receptor protein 3, Mediterranean fever gene, interleukin (IL)-1α, and IL-1ß) relative to the control. In vitro studies demonstrated NF-κB activation was reduced two-fold (p ≤ 0.0001) by RTD-1 treatment. Immunoblots revealed that RTD-1 treatment inhibited proIL-1ß biosynthesis. Additionally, RTD-1 treatment was associated with a reduction in caspase-1 activation (FC = -1.79; p = 0.0052). RTD-1 exhibited potent anti-inflammatory activity in chronically infected mice. Importantly, RTD-1 inhibits inflammasome activity, which is possibly a downstream effect of NF-κB modulation. These findings support that this immunomodulatory peptide may be a promising therapeutic for CF-associated lung disease.

Antibiotics Differentially Modulate Lipoteichoic Acid-Mediated Host Immune Response.

In Staphylococcus aureus bacteremia, our group has shown that a dysregulated balance of pro- and anti-inflammatory cytokine response biased towards an immunoparalysis phenotype is predictive of persistence and mortality, despite receipt of antibiotics. Certain antibiotics, as well as lipoteichoic acid (LTA) released from S. aureus, can modulate immune response ex vivo. Here, we evaluated the effects of three anti-staphylococcal antibiotics (vancomycin, tedizolid, and daptomycin) on the expression of cytokines and cell surface markers of immune activation (TNFα, HLA-DR) and immunoparalysis (IL-10, PD-L1) in human peripheral blood mononuclear cells (PBMC) exposed to high (10 µg) and low (1 µg) doses of LTA. Results suggested a dose-dependent relationship between LTA and induction of anti- and pro-inflammatory immune responses. Differential antibiotic effects were prominently observed at high but not low LTA condition. Vancomycin significantly induced IL-10 and TNFα expression, whereas daptomycin had no effects on cytokine response or expression of cell surface receptors. Tedizolid increased TNFα and modestly increased HLA-DR expression, suggesting a stimulatory effect. These findings suggest that anti-staphylococcal agents differentially alter LTA-mediated immune cell activation status and cytokine response, providing support for future clinical studies to better elucidate the complexities of host-microbial-antibiotic interaction that can help direct precision therapy for S. aureus bacteremia.

Variable Release of Lipoteichoic Acid From Staphylococcus aureus Bloodstream Isolates Relates to Distinct Clinical Phenotypes, Strain Background, and Antibiotic Exposure.

BACKGROUND: Staphylococcus aureus is a leading cause of bacterial bloodstream infections. The heterogeneity in patient outcomes in S. aureus bacteremia (SAB) can be attributed in part to strain characteristics, which may influence host response to infection. We specifically examined the relationship between lipoteichoic acid (LTA) release from S. aureus and disease phenotype, strain background, and antibiotic exposure. METHODS: Seven strains of S. aureus causing different clinical phenotypes of bacteremia and two reference strains (LAC USA 300 and Mu3) were analyzed for LTA release at baseline and following exposure to antibiotics from different pharmacologic classes (vancomycin, ceftaroline, and tedizolid). LTA release was quantified by LTA-specific ELISA. Whole genome sequencing was performed on the clinical strains and analyzed using open-source bioinformatics tools. RESULTS: Lipoteichoic acid release varied by 4-fold amongst the clinical strains and appeared to be related to duration of bacteremia, independent of MLST type. Low LTA releasing strains were isolated from patients who had prolonged duration of bacteremia and died. Antibiotic-mediated differences in LTA release appeared to be associated with MLST type, as ST8 strains released maximal LTA in response to tedizolid while other non-ST8 strains demonstrated high LTA release with vancomycin. Genetic variations related to the LTA biosynthesis pathway were detected in all non-ST8 strains, though ST8 strains showed no variations despite demonstrating differential LTA release. CONCLUSION: Our findings provide the basis for future studies to evaluate the relationship between LTA release-mediated host immune response and clinical outcomes as well as the potential for antibiotic modulation of LTA release as a therapeutic strategy and deserve confirmation with larger number of strains with known clinical phenotypes.

Transitioning Chemistry, Manufacturing, and Controls Content With a Structured Data Management Solution: Streamlining Regulatory Submissions.

The process of assembling regulatory documents for submission to multiple global health agencies can present a repetitive cycle of authoring, editing, and data verification, which increases in complexity as changes are made for approved products, particularly from a chemistry, manufacturing, and controls (CMC) perspective. Currently, pharmaceutical companies rely on a workflow that involves manual CMC change management across documents. Similarly, when regulators review submissions, they provide feedback and insight into regulatory decision making in a narrative format. As accelerated review pathways are increasingly used and pressure mounts to bring products to market quickly, innovative solutions for assembling, distributing, and reviewing regulatory information are being considered. Structured content management (SCM) solutions, in which data are collated into centrally organized content blocks for use across different documents, may aid in the efficient processing of data and create opportunities for automation and machine learning in its interpretation. The US Food and Drug Administration (FDA) has recently created initiatives that encourage application of SCM for CMC data, though many challenges could impede their success and efficiency. The goal is for industry and health authorities to collaborate in the development of SCM for CMC applications, to potentially streamline compilation of quality data in regulatory submissions.