Optimization and Scale Up of Spray Dried CPZEN-45 Aerosol Powders for Inhaled Tuberculosis Treatment.

PURPOSE: Tuberculosis (TB) remains one of the most serious diseases caused by a single organism. Multiple (MDR) and extensively (XDR) drug resistant disease poses a threat to global health and requires new drugs and/or innovative approaches to treatment. A number of drugs have been proposed as inhaled therapy for TB, frequently prepared by spray drying. CPZEN-45 is a novel anti-tubercular drug that has poor oral bioavailability but has shown promise when administered via inhalation. METHODS: Excipient-free CPZEN-45 HCl has been spray dried into a powder with physicochemical characteristics, aerodynamic particle size distribution, and delivered dose suitable for consideration as an inhaled product. RESULTS: The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the powder delivered using a RS01 inhaler were 2.62 ± 0.04 µm and 1.76 ± 0.09, respectively. Additionally, the powder was physically and chemically stable after storage at ambient conditions for >1.5 years with particle size similar to freshly manufactured product. Overages in spray dried powder were recycled the powder and resprayed into drug product likewise resulting in negligible change in quality thus allowing for further preclinical characterization as necessary. CPZEN-45 was scaled up using pilot-scale manufacturing equipment where the density of the powder was increased to facilitate larger delivered doses without affecting the aerodynamic performance properties. CONCLUSION: The spray dried powders were suitable for pharmacokinetics, efficacy and preclinical toxicology studies. The final method of manufacture may be used directly for CGMP particle manufacture to support IND and Phase I clinical trials and beyond.

Nitric Oxide-Releasing Hyaluronic Acid as an Antibacterial Agent for Wound Therapy.

Taking advantage of their respective wound-healing roles in physiology, the dual activity of hyaluronic acid (HA) and nitric oxide (NO) was combined to create a single-agent wound therapeutic. Carboxylic acid groups of HA (6 and 90 kDa) were chemically modified with a series of alkylamines via carbodiimide chemistry to provide secondary amines for subsequent N-diazeniumdiolate NO donor formation. The resulting NO-releasing HA derivatives stored 0.3-0.6 µmol NO mg-1 and displayed diverse release kinetics (5-75 min NO-release half-lives) under physiological conditions. The 6 kDa HA with terminal primary amines and intermediate release kinetics exhibited broad-spectrum bactericidal activity against common wound pathogens, including planktonic methicillin-resistant Staphylococcus aureus as well as planktonic and biofilm-based multidrug-resistant Pseudomonas aeruginosa. The treatment of infected murine wounds with NO-releasing HA facilitated more rapid wound closure and decreased the quantity of the P. aeruginosa genetic material in the remaining wound tissue. Hyaluronidase readily degraded the HA derivatives, indicating that NO donor modification did not prohibit endogenous biodegradation pathways.

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.

Spray dried tigecycline dry powder aerosols for the treatment of Nontuberculous mycobacterial pulmonary infections.

Nontuberculous mycobacterial (NTM) pulmonary infections are a global health concern and a significant contributor to lung disease. Systemic therapies of a cocktail of antibiotics administered over a long period often lead to adverse reactions and/or treatment failure. NTM pathogens, such as Mycobacterium abscessus (Mabs), are notoriously difficult to treat due to resistance to many traditional antibiotics. However, the antibiotic tigecycline has demonstrated efficacy in vitro and in vivo against Mabs strains varying in drug susceptibility. Tigecycline exhibits instability in aqueous medium, posing delivery challenges, and has caused severe adverse gastrointestinal effects following intravenous administration, requiring treatment discontinuation. To mitigate both of these concerns, inhalation therapies using dry powder aerosols are proposed as an alternative administration route and means of delivery. Tigecycline dry powder formulations were prepared, characterized, and optimized to develop a therapeutic aerosol with low moisture, high dispersibility, and a large fraction of particles in the respirable size range (1-5 µm). The addition of lactose, leucine, and phosphate buffer salts was investigated to achieve additional stability, dispersibility, and tolerability. Preliminary delivery of the dry powders to Mabs-infected mice for 30 min per day over 7 d demonstrated a 0.91-log (87.7%) decrease in lung bacterial burden.

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.

Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions.

In an attempt to address the significant morbidity, mortality, and economic cost associated with tunneled dialysis catheter (TDC) dysfunction, we report the development of nitric oxide-releasing dialysis catheter lock solutions. Catheter lock solutions with a range of NO payloads and release kinetics were prepared using low-molecular-weight N-diazeniumdiolate nitric oxide donors. Nitric oxide released through the catheter surface as a dissolved gas was maintained at therapeutically relevant levels for at least 72 h, supporting clinical translatability (interdialytic period). Slow, sustained NO release from the catheter surface prevented bacterial adhesion in vitro by 88.9 and 99.7% for Pseudomonas aeruginosa and Staphylococcus epidermidis, respectively, outperforming a burst NO-release profile. Furthermore, bacteria adhered to the catheter surface in vitro prior to lock solution use was reduced by 98.7 and 99.2% for P. aeruginosa and S. epidermidis, respectively, when using a slow releasing NO donor, demonstrating both preventative and treatment potential. The adhesion of proteins to the catheter surface, a process often preceding biofilm formation and thrombosis, was also lessened by 60-65% by sustained NO release. In vitro cytotoxicity of catheter extract solutions to mammalian cells was minimal, supporting the non-toxic nature of the NO-releasing lock solutions. The use of the NO-releasing lock solution in an in vivo TDC porcine model demonstrated decreased infection and thrombosis, enhanced catheter functionality, and improved outcome (i.e., likelihood of survival) as a result of catheter use.

Injectable polysaccharide hydrogels as localized nitric oxide delivery formulations.

A series of injectable polysaccharide hydrogels were prepared with oxidized dextran and diethylenetriamine-modified carboxymethylcellulose or hyaluronic acid. Rheological evaluation revealed that carboxymethylcellulose-based hydrogels achieved the largest storage moduli (>1 kPa) when prepared from 5 wt. % solutions. However, carboxymethylcellulose-based hydrogels with storage moduli >100 Pa were prepared from solutions with concentrations as low as 2 wt. %. Hyaluronic acid-based hydrogels demonstrated smaller storage moduli but had swelling ratios more than four times that of the carboxymethylcellulose systems at the same polymer concentrations. The incorporation of N-diazeniumdiolate NO donors into the hydrogels resulted in reduced hydrogel storage moduli as a function of NO donor concentration. The impact of the hydrogel architecture on NO-release kinetics proved dependent on the identity of the NO donor. Hydrogel degradation over 14 d was measured at pH 5.4 and 7.4 and indicated that hyaluronic acid-based hydrogels degraded more rapidly than carboxymethylcellulose hydrogels and that the addition of NO to the hydrogels increased the rate at which they degraded. In vitro cytotoxicity of hydrogel extracts was evaluated against five cell lines, with no observed toxicity except for that of hyaluronic acid-based hydrogel extracts against human gingival fibroblasts. The diverse properties, versatility, and non-toxic characteristics of these injectable hydrogels should facilitate local delivery of nitric oxide for a range of biomedical applications.

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.

Role of Nitric Oxide-Releasing Glycosaminoglycans in Wound Healing.

Two glycosaminoglycan (GAG) biopolymers, hyaluronic acid (HA) and chondroitin sulfate (CS), were chemically modified via carbodiimide chemistry to facilitate the loading and release of nitric oxide (NO) to develop a multi-action wound healing agent. The resulting NO-releasing GAGs released 0.2-0.9 µmol NO mg-1 GAG into simulated wound fluid with NO-release half-lives ranging from 20 to 110 min. GAGs containing alkylamines with terminal primary amines and displaying intermediate NO-release kinetics exhibited potent, broad spectrum bactericidal action against three strains each of Pseudomonas aeruginosa and Staphylococcus aureus ranging in antibiotic resistance profile. NO loading of the GAGs was also found to decrease murine TLR4 activation, suggesting that the therapeutic exhibits anti-inflammatory mechanisms. In vitro adhesion and proliferation assays utilizing human dermal fibroblasts and human epidermal keratinocytes displayed differences as a function of the GAG backbone, alkylamine identity, and NO-release properties. In combination with antibacterial properties, the adhesion and proliferation profiles of the GAG derivatives enabled the selection of the most promising wound healing candidates for subsequent in vivo studies. A P. aeruginosa-infected murine wound model revealed the benefits of CS over HA as a pro-wound healing NO donor scaffold, with benefits of accelerated wound closure and decreased bacterial burden attributable to both active NO release and the biopolymer backbone.

Nitric Oxide Therapy for Diabetic Wound Healing.

Nitric oxide (NO) represents a potential wound therapeutic agent due to its ability to regulate inflammation and eradicate bacterial infections. Two broad strategies exist to utilize NO for wound healing; liberating NO from endogenous reservoirs, and supplementing NO from exogenous sources. This progress report examines the efficacy of a variety of NO-based methods to improve wound outcomes, with particular attention given to diabetes-associated chronic wounds.

Nitric oxide diffusion through cystic fibrosis-relevant media and lung tissue.

A simplified diffusion cell methodology was employed to measure the diffusion coefficient of nitric oxide (NO) through phosphate buffered saline (PBS) and artificial sputum medium (ASM)-an in vitro analog for airway mucus. Diffusion through the proteinaceous ASM yielded a significantly lower diffusion coefficient compared to PBS, which is attributed to both the physical obstruction by the mucin mesh and reactive nature of NO radicals towards the biological compounds in ASM. To further confirm that ASM was restricting NO from diffusing freely, a macromolecular propylamine-modified cyclodextrin donor (CD-PA) was employed to release the NO more slowly. The NO diffusion characteristics in ASM via the NO donor were also slower relative to PBS. As NO is likely to interact with lung cells after passing through the mucus barrier, the diffusion of both NO and the CD-PA macromolecular NO donor through differentiated lung tissue was investigated with and without an ASM layer. Comparison of NO diffusion through the three diffusion barriers indicated that the lung tissue significantly impeded NO penetration over the course of the experiment compared to PBS and ASM. In fact, the diffusion of CD-PA through the lung tissue was hindered until after the release of its NO payload, potentially due to the increased net charge of the NO donor structure. Of importance, the viability of the tissue was not influenced by the NO-releasing CD-PA at bactericidal concentrations.