Identification of New Modulators and Inhibitors of Palmitoyl-Protein Thioesterase 1 for CLN1 Batten Disease and Cancer.

Palmitoyl-protein thioesterase 1 (PPT1) is an understudied enzyme that is gaining attention due to its role in the depalmitoylation of several proteins involved in neurodegenerative diseases and cancer. PPT1 is overexpressed in several cancers, specifically cholangiocarcinoma and esophageal cancers. Inhibitors of PPT1 lead to cell death and have been shown to enhance the killing of tumor cells alongside known chemotherapeutics. PPT1 is hence a viable target for anticancer drug development. Furthermore, mutations in PPT1 cause a lysosomal storage disorder called infantile neuronal ceroid lipofuscinosis (CLN1 disease). Molecules that can inhibit, stabilize, or modulate the activity of this target are needed to address these diseases. We used PPT1 enzymatic assays to identify molecules that were subsequently tested by using differential scanning fluorimetry and microscale thermophoresis. Selected compounds were also tested in neuroblastoma cell lines. The resulting PPT1 screening data was used for building machine learning models to help select additional compounds for testing. We discovered two of the most potent PPT1 inhibitors reported to date, orlistat (IC50 178.8 nM) and palmostatin B (IC50 11.8 nM). When tested in HepG2 cells, it was found that these molecules had decreased activity, indicating that they were likely not penetrating the cells. The combination of in vitro enzymatic and biophysical assays enabled the identification of several molecules that can bind or inhibit PPT1 and may aid in the discovery of modulators or chaperones. The molecules identified could be used as a starting point for further optimization as treatments for other potential therapeutic applications outside CLN1 disease, such as cancer and neurological diseases.

Luminal Delivery of Pectin-Modified Oxygen Microbubbles Mitigates Rodent Experimental Intestinal Ischemia.

INTRODUCTION: Ischemic gut injury is common in the intensive care unit, impairs gut barrier function, and contributes to multiorgan dysfunction. One novel intervention to mitigate ischemic gut injury is the direct luminal delivery of oxygen microbubbles (OMB). Formulations of OMB can be modified to control the rate of oxygen delivery. This project examined whether luminal delivery of pectin-modified OMB (OMBp5) can reduce ischemic gut injury in a rodent model. METHODS: The OMBp5 formulation was adapted to improve delivery of oxygen along the length of small intestine. Adult Sprague-Dawley rats (n = 24) were randomly allocated to three groups: sham-surgery (SS), intestinal ischemia (II), and intestinal ischemia plus luminal delivery of OMBp5 (II + O). Ischemia-reperfusion injury was induced by superior mesenteric artery occlusion for 45 min followed by reperfusion for 30 min. Outcome data included macroscopic score of mucosal injury, the histological score of gut injury, and plasma biomarkers of intestinal injury. RESULTS: Macroscopic, microscopic data, and intestinal injury biomarker results demonstrated minimal intestinal damage in the SS group and constant damage in the II group. II + O group had a significantly improved macroscopic score throughout the gut mucosa (P = 0.04) than the II. The mean histological score of gut injury for the II + O group was significantly improved on the II group (P ≤ 0.01) in the proximal intestine only, within 30 cm of delivery. No differences were observed in plasma biomarkers of intestinal injury following OMBp5 treatment. CONCLUSIONS: This proof-of-concept study has demonstrated that luminal OMBp5 decreases ischemic injury to the proximal small intestine. There is a need to improve oxygen delivery over the full length of the intestine. These findings support further studies with clinically relevant end points, such as systemic inflammation and vital organ dysfunction.

Inhaled "Muco-Trapping" Monoclonal Antibody Effectively Treats Established Respiratory Syncytial Virus (RSV) Infections.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable "muco-trapping" variant of motavizumab (Mota-MT), a potent neutralizing mAb against RSV F is engineered. Mota-MT traps RSV in AM via polyvalent Fc-mucin bonds, reducing the fraction of fast-moving RSV particles in both fresh pediatric and adult AM by ≈20-30-fold in a Fc-glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota-MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota-MT to RSV-infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000-fold and 100 000-fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota-MT-treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco-trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.

Practical Considerations in Dose Extrapolation from Animals to Humans.

Animal studies are an important component of drug product development and the regulatory review process since modern practices have been in place, for almost a century. A variety of experimental systems are available to generate aerosols for delivery to animals in both liquid and solid forms. The extrapolation of deposited dose in the lungs from laboratory animals to humans is challenging because of genetic, anatomical, physiological, pharmacological, and other biological differences between species. Inhaled drug delivery extrapolation requires scrutiny as the aerodynamic behavior, and its role in lung deposition is influenced not only by the properties of the drug aerosol but also by the anatomy and pulmonary function of the species in which it is being evaluated. Sources of variability between species include the formulation, delivery system, and species-specific biological factors. It is important to acknowledge the underlying variables that contribute to estimates of dose scaling between species.

iBCS: 3. A Biopharmaceutics Classification System for Orally Inhaled Drug Products.

In this article, we specify for the first time a quantitative biopharmaceutics classification system for orally inhaled drugs. To date, orally inhaled drug product developers have lacked a biopharmaceutics classification system like the one developed to navigate the development of immediate release of oral medicines. Guideposts for respiratory drug discovery chemists and inhalation product formulators have been elusive and difficult to identify due to the complexity of pulmonary physiology, the intricacies of drug deposition and disposition in the lungs, and the influence of the inhalation delivery device used to deliver the drug as a respirable aerosol. The development of an inhalation biopharmaceutics classification system (iBCS) was an initiative supported by the Product Quality Research Institute (PQRI). The goal of the PQRI iBCS working group was to generate a qualitative biopharmaceutics classification system that can be utilized by inhalation scientists as a "rule of thumb" to identify desirable molecular properties and recognize and manage CMC product development risks based on physicochemical properties of the drug and the deposited lung dose. Herein, we define the iBCS classes quantitatively according to the dose number and permeability. The proposed iBCS was evaluated for its ability to categorize marketed inhaled drugs using data from the literature. The appropriateness of the classification of each drug was assessed based on published development, clinical and nonclinical data, and mechanistic physiologically based biopharmaceutics modeling. The inhaled drug product development challenges for each iBCS classification are discussed and illustrated for different classes of marketed inhaled drugs. Finally, it is recognized that discriminatory laboratory methods to characterize regional lung deposition, dissolution, and permeability will be key to fully realizing the benefits of an iBCS to streamline and derisk inhaled drug development.

The Pharmacokinetics of CPZEN-45, a Novel Anti-Tuberculosis Drug, in Guinea Pigs.

CPZEN-45 is a novel compound with activity against drug-susceptible and drug-resistant tuberculosis (TB). The present study was undertaken to determine the best dose and dosing regimen of inhalable CPZEN-45 powders to use in efficacy studies with TB-infected guinea pigs. The disposition of CPZEN-45 after intravenous, subcutaneous (SC), and direct pulmonary administration (INS) was first determined to obtain their basal pharmacokinetic (PK) parameters. Then, the disposition of CPZEN-45 powders after passive inhalation using consecutive and sequential doses was evaluated. Plasma concentration versus time curves and PK parameters indicated that the absorption of CPZEN-45 after INS was faster than after SC administration (Ka = 12.94 ± 5.66 h-1 and 1.23 ± 0.55 h-1, respectively), had a longer half-life (2.06 ± 1.01 h versus 0.76 ± 0.22 h) and had higher bioavailability (67.78% and 47.73%, respectively). The plasma concentration versus time profiles and the lung tissue concentration at the end of the study period were not proportional to the dose size after one, two, and three consecutive passive inhalation doses. Three sequential passive inhalation doses maintained therapeutic concentration levels in plasma and lung tissue for a longer time than three consecutive doses (10 h vs. 3 h, respectively). Future studies to evaluate the efficacy of inhaled CPZEN-45 powders should employ sequential doses of the powder, with one nominal dose administered to animals three times per day.

Tyvaso DPI: Drug-device characteristics and patient clinical considerations.

Tyvaso DPI is a drug-device combination therapy comprised of a small, portable, reusable, breath-powered, dry powder inhaler (DPI) for the delivery of treprostinil. It is approved for the treatment of pulmonary arterial hypertension and pulmonary hypertension associated with interstitial lung disease. Tyvaso DPI utilizes single-use prefilled cartridges to ensure proper dosing. Unlike nebulizer devices, administration of Tyvaso DPI is passive and does not require coordination with the device. The low-flow rate design results in targeted delivery to the peripheral lungs due to minimal drug loss from impaction in the oropharynx. The inert fumaryl diketopiperazine (FDKP) excipient forms microparticles that carry treprostinil into the airways, with a high fraction of the particles in the respirable range. In a clinical study in patients with pulmonary arterial hypertension, Tyvaso DPI had similar exposure and pharmacokinetics, low incidence of adverse events, and high patient satisfaction compared with nebulized treprostinil solution. Tyvaso DPI may be considered as a first prostacyclin agent or for those that do not tolerate other prostacyclin formulations, patients with pulmonary comorbidities, patients with mixed Group 1 and Group 3 pulmonary hypertension, or those that prefer an active lifestyle and need a portable, non-invasive treatment. Tyvaso DPI is a patient-preferred, maintenance-free, safe delivery option that may improve patient compliance and adherence.

A Modified BPaL Regimen for Tuberculosis Treatment replaces Linezolid with Inhaled Spectinamides.

The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse effects (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here we hypothesize that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the Balb/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCE: Tuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure ∼90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse effects (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.

Dry Powder Inhalers: An Overview.

Dry powder inhaler products have played an important role in the treatment and prevention of asthma and more recently chronic obstructive pulmonary disease. The considerations that go into formulation development to support these products cover a unique range of disciplines including analytical and physical chemistry, aerosol physics, device technology, process engineering and industrial design. An enormous research effort has been expended in the last half century to provide understanding of this complex dosage form. The guiding principles in considering the development of dry powder inhaler products encompass requirements for disease therapy, advantages and limitations of adopting certain technological approaches, and desirable features to facilitate patient use, which are all embodied in the target product profile.

Succinate-mediated reactive oxygen species production in the anoxia-tolerant epaulette (Hemiscyllium ocellatum) and grey carpet (Chiloscyllium punctatum) sharks.

Anoxia/re-oxygenation (AR) results in elevated unchecked oxidative stress and mediates irreversible damage within the brain for most vertebrates. Succinate accumulation within mitochondria of the ischaemic brain appears to increase the production of reactive oxygen species (ROS) upon re-oxygenation. Two closely related elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) repeatedly experience near anoxia and re-oxygenation in their habitats and have adapted to survive AR at tropical temperatures without significant brain injuries. However, these anoxia-tolerant species display contrasting strategies to survive AR, with only H. ocellatum having the capacity to supress metabolism and H. ocellatum mitochondria the capacity to depress succinate oxidation post-AR. We measured oxygen consumption alongside ROS production mediated by elevated succinate in mitochondria of permeabilized cerebellum from both shark species. Although mitochondrial respiration remained similar for both species, the ROS production in H. ocellatum was half that of C. punctatum in phosphorylating and non-phosphorylating mitochondria. Maximum ROS production in H. ocellatum was mediated by succinate loads 10-fold higher than in C. punctatum mitochondria. The contrasting survival strategies of anoxia-tolerant sharks reveal the significance of mitigating ROS production under elevated succinate load during AR, shedding light on potential mechanisms to mitigate brain injury.

Metabolic resilience of the Australasian snapper (Chrysophrys auratus) to marine heatwaves and hypoxia.

Marine organisms are under threat from a simultaneous combination of climate change stressors, including warming sea surface temperatures (SST), marine heatwave (MHW) episodes, and hypoxic events. This study sought to investigate the impacts of these stressors on the Australasian snapper (C. auratus) - a finfish species of high commercial and recreational importance, from the largest snapper fishery in Aotearoa New Zealand (SNA1). A MHW scenario was simulated from 21°C (current February SST average for north-eastern New Zealand) to a future predicted level of 25°C, with the whole-animal and mitochondrial metabolic performance of snapper in response to hypoxia and elevated temperature tested after 1-, 10-, and 30-days of thermal challenge. It was hypothesised that key indicators of snapper metabolic performance would decline after 1-day of MHW stress, but that partial recovery might arise as result of thermal plasticity after chronic (e.g., 30-day) exposures. In contrast to this hypothesis, snapper performance remained high throughout the MHW: 1) Aerobic metabolic scope increased after 1-day of 25°C exposure and remained high. 2) Hypoxia tolerance, measured as the critical O2 pressure and O2 pressure where loss of equilibrium occurred, declined after 1-day of warm-acclimation, but recovered quickly with no observable difference from the 21°C control following 30-days at 25°C. 3) The performance of snapper mitochondria was also maintained, with oxidative phosphorylation respiration and proton leak flux across the inner mitochondrial membrane of the heart remaining mostly unaffected. Collectively, the results suggest that heart mitochondria displayed resilience, or plasticity, in snapper chronically exposed to 25°C. Therefore, contrary to the notion of climate change having adverse metabolic effects, future temperatures approaching 25°C may be tolerated by C. auratus in Northern New Zealand. Even in conjunction with supplementary hypoxia, 25°C appears to represent a metabolically optimal temperature for this species.

Pharmacokinetic Considerations for Optimizing Inhaled Spray-Dried Pyrazinoic Acid Formulations.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of anti-TB regimens, but its prolonged use has the potential to drive the development of PZA-resistant Mtb. PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA, and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung; hence, direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA-mutant PZA-resistant Mtb. The objectives of the current study were to (i) develop novel dry powder POA formulations, (ii) assess their feasibility for pulmonary delivery using physicochemical characterization, (iii) evaluate their pharmacokinetics (PK) in the guinea pig model, and (iv) develop a mechanism-based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous, and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF, and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3) and (ii) the highest concentration in ELF (CmaxELF: 171 nM) within 15.5 min, correlating with a fast transfer into ELF after pulmonary administration (KPM: 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product.

Disposable Dosators Intended for Dry Powder Delivery to Mice.

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models' utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 µg. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Dynamic defence? Intertidal triplefin species show better maintenance of mitochondrial membrane potential than subtidal species at low oxygen pressures.

Oxygen is essential for most eukaryotic lifeforms, as it supports mitochondrial oxidative phosphorylation to supply ∼90% of cellular adenosine triphosphate (ATP). Fluctuations in O2 present a major stressor, with hypoxia leading to a cascade of detrimental physiological changes that alter cell operations and ultimately induce death. Nonetheless, some species episodically tolerate near-anoxic environments, and have evolved mechanisms to sustain function even during extended hypoxic periods. While mitochondria are pivotal in central metabolism, their role in hypoxia tolerance remains ill defined. Given the vulnerability of the brain to hypoxia, mitochondrial function was tested in brain homogenates of three closely related triplefin species with varying degrees of hypoxia tolerance (Bellapiscis medius, Forsterygion lapillum and Forsterygion varium). High-resolution respirometry coupled with fluorometric measurements of mitochondrial membrane potential (mtMP) permitted assessment of differences in mitochondrial function and integrity in response to intermittent hypoxia and anoxia. Traditional steady-state measures of respiratory flux and mtMP showed no differences among species. However, in the transition into anoxia, the tolerant species B. medius and F. lapillum maintained mtMP at O2 pressures 7- and 4.4-fold lower, respectively, than that of the hypoxia-sensitive F. varium and exhibited slower rates of membrane depolarisation. The results indicate that dynamic oxic-hypoxic mitochondria transitions underlie hypoxia tolerance in these intertidal fish.

Development of a Minimalistic Physiologically Based Pharmacokinetic (mPBPK) Model for the Preclinical Development of Spectinamide Antibiotics.

Spectinamides 1599 and 1810 are lead spectinamide compounds currently under preclinical development to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. These compounds have previously been tested at various combinations of dose level, dosing frequency, and route of administration in mouse models of Mycobacterium tuberculosis (Mtb) infection and in healthy animals. Physiologically based pharmacokinetic (PBPK) modeling allows the prediction of the pharmacokinetics of candidate drugs in organs/tissues of interest and extrapolation of their disposition across different species. Here, we have built, qualified, and refined a minimalistic PBPK model that can describe and predict the pharmacokinetics of spectinamides in various tissues, especially those relevant to Mtb infection. The model was expanded and qualified for multiple dose levels, dosing regimens, routes of administration, and various species. The model predictions in mice (healthy and infected) and rats were in reasonable agreement with experimental data, and all predicted AUCs in plasma and tissues met the two-fold acceptance criteria relative to observations. To further explore the distribution of spectinamide 1599 within granuloma substructures as encountered in tuberculosis, we utilized the Simcyp granuloma model combined with model predictions in our PBPK model. Simulation results suggest substantial exposure in all lesion substructures, with particularly high exposure in the rim area and macrophages. The developed model may be leveraged as an effective tool in identifying optimal dose levels and dosing regimens of spectinamides for further preclinical and clinical development.

Electron transfer and ROS production in brain mitochondria of intertidal and subtidal triplefin fish (Tripterygiidae).

While oxygen is essential for oxidative phosphorylation, O2 can form reactive species (ROS) when interacting with electrons of mitochondrial electron transport system. ROS is dependent on O2 pressure (PO2) and has traditionally been assessed in O2 saturated media, PO2 at which mitochondria do not typically function in vivo. Mitochondrial ROS can be significantly elevated by the respiratory complex II substrate succinate, which can accumulate within hypoxic tissues, and this is exacerbated further with reoxygenation. Intertidal species are repetitively exposed to extreme O2 fluctuations, and have likely evolved strategies to avoid excess ROS production. We evaluated mitochondrial electron leakage and ROS production in permeabilized brain of intertidal and subtidal triplefin fish species from hyperoxia to anoxia, and assessed the effect of anoxia reoxygenation and the influence of increasing succinate concentrations. At typical intracellular PO2, net ROS production was similar among all species; however at elevated PO2, brain tissues of the intertidal triplefin fish released less ROS than subtidal species. In addition, following in vitro anoxia reoxygenation, electron transfer mediated by succinate titration was better directed to respiration, and not to ROS production for intertidal species. Overall, these data indicate that intertidal triplefin fish species better manage electrons within the ETS, from hypoxic-hyperoxic transitions.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Preparation Strategies of the Anti-Mycobacterial Drug Bedaquiline for Intrapulmonary Routes of Administration.

Mycobacterium tuberculosis (M.tb) has infected one-quarter of the world's population and led to the deaths of 1.6 million individuals in 2021 according to estimates from the World Health Organization. The rise in prevalence of multidrug-resistant and extensively drug-resistant M.tb strains coupled with insufficient therapies to treat such strains has motivated the development of more effective treatments and/or delivery modalities. Bedaquiline, a diarylquinoline antimycobacterial agent, effectively targets mycobacterial ATP synthase but may lead to systemic complications upon oral delivery. Targeted delivery of bedaquiline to the lungs represents an alternative strategy to harness the sterilizing benefits of the drug against M.tb while mitigating off-target side effects. Two pulmonary delivery modalities were developed herein, including dry powder inhalation and liquid instillation. Despite bedaquiline's poor water solubility, spray drying was performed in predominantly aqueous conditions (≥80%) to avoid a closed-loop, inert system. Aerosols of spray-dried bedaquiline with L-leucine excipient outperformed spray-dried bedaquiline alone, demonstrating superior fine particle fraction metrics (~89% of the emitted dose below <5 µm), suitable for inhalation therapies. Furthermore, the use of a 2-hydroxypropyl-ß-cyclodextrin excipient allowed a molecular dispersion of bedaquiline in an aqueous solution for liquid instillation. Both delivery modalities were successfully administered to Hartley guinea pigs for pharmacokinetic analysis and were well-tolerated by the animals. Intrapulmonary liquid delivery of bedaquiline led to adequate serum absorption and appropriate peak serum concentrations of the drug. The liquid formulation was superior in systemic uptake compared to the powder formulation. The predominant route via which M.tb bacilli enter the body is aerosol droplets that are deposited onto airway surfaces. For this reason, we believe that further studies should focus on inhalation or intrapulmonary therapies that target the site of entry and primary site of infection for M.tb.

Pharmacokinetic considerations for optimizing inhaled spray-dried pyrazinoic acid formulations.

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of anti-TB regimens, but its prolonged use has the potential to drive development of PZA resistant Mtb . PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA , and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung, hence direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA- mutant PZA-resistant Mtb . The objectives of the current study were to i) develop novel dry powder POA formulations ii) assess their feasibility for pulmonary delivery using physicochemical characterization, iii) evaluate their pharmacokinetics (PK) in the guinea pig model and iv) develop a mechanism based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3); (ii) the highest concentration in ELF ( Cmac ELF : 171 nM) within 15.5 minutes, correlating with a fast transfer into ELF after pulmonary administration ( k PM : 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product.

Characterization of spectinamide 1599 efficacy against different mycobacterial phenotypes.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.