Effect of the amount of cationic lipid used to complex siRNA on the cytotoxicity and proinflammatory activity of siRNA-solid lipid nanoparticles.

When preparing siRNA-encapsulated solid lipid nanoparticles (siRNA-SLNs), cationic lipids are commonly included to condense and lipophilize the siRNA and thus increase its encapsulation in the SLNs. Unfortunately, cationic lipids also contribute significantly to the cytotoxicity and proinflammatory activity of the SLNs. Previously, our group developed a TNF-α siRNA-SLN formulation that showed strong activity against rheumatoid arthritis unresponsive to methotrexate in a mouse model. The siRNA-SLNs were composed of lecithin, cholesterol, an acid-sensitive stearoyl polyethylene glycol (2000) conjugate, and siRNA complexes with 1,2-dioleoyl-3trimethylammonium-propane (DOTAP), a cationic lipid. The present study was designed to study the effect of the amount of DOTAP used to complex the siRNA on the cytotoxicity and proinflammatory activity of the resultant siRNA-SLNs. A small library of siRNA-SLNs prepared at various ratios of DOTAP to siRNA (i.e., nitrogen to phosphate (N/P) ratios ranging from 34:1 to 1:1) were prepared and characterized, and the cytotoxicity and proinflammatory activity of selected formulations were evaluated in cell culture. As expected, the siRNA-SLNs prepared at the highest N/P ratio showed the highest cytotoxicity to J774A.1 macrophage cells and reducing the N/P ratio lowered the cytotoxicity of the siRNA-SLNs. Unexpectedly, the cytotoxicity of the siRNA-SLNs reached the lowest at the N/P ratios of 16:1 and 12:1, and further reducing the N/P ratio resulted in siRNA-SLNs with increased cytotoxicity. For example, siRNA-SLNs prepared at the N/P ratio of 1:1 was more cytotoxic than the ones prepared at the N/P ratio 12:1. This finding was confirmed using neutrophils differentiated from mouse MPRO cell line. The DOTAP release from the siRNA-SLNs prepared at the N/P ratio of 1:1 was faster than from the ones prepared at the N/P ratio of 12:1. The siRNA-SLNs prepared at N/P ratios of 12:1 and 1:1 showed comparable proinflammatory activities in both macrophages and neutrophils. Additionally, the TNF-α siRNA-SLNs prepared at the N/P ratios of 12:1 and 1:1 were equally effective in downregulating TNF-α expression in J774A.1 macrophages. In conclusion, it was demonstrated that at least in vitro in cell culture, reducing the amount of cationic lipids used when preparing siRNA-SLNs can generally help reduce the cytotoxicity of the resultant SLNs, but siRNA-SLNs prepared with the lowest N/P ratio are not necessarily the least cytotoxic and proinflammatory.

Development of low-cost, weight-adjustable clofazimine mini-tablets for treatment of tuberculosis in pediatrics.

Clofazimine (CFZ) is an important component of the World Health Organization's (WHO) recommended all-oral drug regimen for treatment of multi-drug resistant tuberculosis (MDR-TB). However, the lack of a dividable oral dosage form has limited the use of the drug in pediatric populations, who may require lowering of the dose to reduce the likelihood of adverse drug events. In this study, pediatric-friendly CFZ mini-tablets were prepared from micronized powder via direct compression. Rapid disintegration and maximized dissolution in GI fluids was achieved using an iterative formulation design process. Pharmacokinetic (PK) parameters of the optimized mini-tablets were obtained in Sprague-Dawley rats and compared against an oral suspension of micronized CFZ particles to examine the effect of processing and formulation on the oral absorption of the drug. Differences in maximum concentration and area under the curve between the two formulations were non-significant at the highest dosing level tested. Variability between rats prevented bioequivalence from being determined according to guidelines outlined by the Food and Drug Administration (FDA). These studies provide an important proof-of-concept for an alternative, low-cost formulation and processing approach for the oral delivery of CFZ in manner that is suitable for children as young as 6 months of age.

Twin-Screw Continuous Mixing Can Produce Dry Powder Inhalation Mixtures for Pulmonary Delivery.

The feasibility of twin-screw corotating extruder as a continuous process mixer to prepare dry powder inhalation (DPI) powders was investigated. Interactive mixtures of 1% micronized budesonide, 0.3% magnesium stearate and 98.7% alpha-lactose monohydrate were manufactured using a Leistritz Nano-16 extruder at various processing conditions. One set of GFM (grooved mixing) elements were included in the screw profile to provide distributive mixing of conveyed powders with the goal of resulting in a homogeneous mixture. Residence time in the twin-screw mixer was modelled to quantify mixing efficiency. Comparative powders were also prepared using either low or high-shear batch mixing to compare the effect of mixing methods on the properties of the budesonide dry powder inhalation formulation. Twin screw mixing results in homogeneous mixtures with aerosol performance comparable to that of high-shear batch mixing. Scanning electron microscopy confirmed that twin screw mixing produces particles with morphology like that of low and high-shear batch mixing. X-ray diffraction (XRD) analysis verified that there was no form change of the drug due to twin-screw processing. Statistical regression was used to probe the relationship between twin screw mixing process parameters such as screw speed and feed rate and aerosol performance. The twin screw mixing process was found to be robust, as no significant differences in aerosol performance were found for various processing parameters.

Oral Delivery of Niclosamide as an Amorphous Solid Dispersion That Generates Amorphous Nanoparticles during Dissolution.

Niclosamide is an FDA-approved anthelmintic that is being studied in clinical trials as a chemotherapeutic and broad-spectrum antiviral. Additionally, several other applications are currently in the preclinical stage. Unfortunately, niclosamide is a poorly water soluble molecule, with reduced oral bioavailability, which hinders its use for new indications. Moreover, niclosamide is a poor glass former; in other words, the molecule has a high tendency to recrystallize, and it is virtually impossible to generate a stable amorphous solid employing the neat molecule. Previously, our group reported the development of an amorphous solid dispersion (ASD) of niclosamide (niclosamide ASD) that generates nanoparticles during its dissolution, not only increasing niclosamide's apparent solubility from 6.6 ± 0.4 to 481.7 ± 22.2 µg/mL in fasted state simulated intestinal fluid (FaSSIF) but also its oral bioavailability 2.6-fold in Sprague-Dawley rats after being administered as a suspension. Nevertheless, niclosamide ASD undergoes recrystallization in acidic media, and an enteric oral dosage form is needed for its translation into the clinic. In this work, we further characterized the nanoparticles that generated during the dissolution of the niclosamide ASD. Cryogenic transmission electron microscopy (Cryo-TEM) and wide-angle X-ray scattering (WAXS) revealed that the nanoparticles were amorphous and had a particle size of ~150 nm. The oral dosage forms of niclosamide ASD were formulated using commercial enteric capsules (Capsuline® and EudracapTM) and as enteric-coated tablets. The enteric dosage forms were tested using pH-shift dissolution and acid-uptake tests, using the USP type II dissolution apparatus and the disintegration apparatus, respectively. The capsules exhibited a higher percentage of weight gain, and visual rupture of the Capsuline capsules was observed. Eudracap capsules protected the formulation from the acidic media, but polymer gelling and the formation of a nondispersible plug were noted during dissolution testing. In contrast, enteric-coated tablets protected the formulation from acid ingress and maintained the performance of niclosamide ASD granules during dissolution in FaSSIF media. These enteric-coated tablets were administered to beagle dogs at a niclosamide dose of 75 mg/kg, resulting in plasma concentrations of niclosamide higher than those reported in the literature using solubilized niclosamide at a higher dose (i.e., 100 mg/kg). In summary, an enteric oral dosage form of niclosamide ASD was formulated without hindering the generation of nanoparticles while maintaining the increase in the niclosamide's apparent solubility. The enteric-coated tablets successfully increased the niclosamide plasma levels in dogs when compared to a niclosamide solution prepared using organic solvents.

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.

Niclosamide inhalation powder made by thin-film freezing: Multi-dose tolerability and exposure in rats and pharmacokinetics in hamsters.

In this work, we have developed and tested a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters to gather data about its toxicology and pharmacokinetics. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with pharmacological effect against SARS-CoV-2 infection. TFF was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 µm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose tolerability and exposure study in rats as evidenced by histopathology analysis, and also was able to achieve lung concentrations above the required IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation that overcomes niclosamide's limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

PD-1 siRNA-Encapsulated Solid Lipid Nanoparticles Downregulate PD-1 Expression by Macrophages and Inhibit Tumor Growth : PD-1 siRNA-Encapsulated Solid Lipid Nanoparticles.

The present study was designed to test the hypothesis that programmed cell death-1 (PD-1) siRNA can downregulate PD-1 expression in macrophages in culture and in tumor tissues in mice and inhibit tumor growth in a mouse model. PD-1 siRNA was encapsulated in solid lipid nanoparticles (SLNs), and the physical properties of the resultant SLNs were characterized. The ability of the PD-1 siRNA-SLNs to downregulate PD-1 expression was confirmed in J774A.1 macrophages in culture and in tumor tissues in mice. Moreover, the antitumor activity of the PD-1 siRNA-SLNs was evaluated in a mouse model. The PD-1 siRNA-SLNs were roughly spherical, and their particle size, polydispersity index, and zeta potential were 141 ± 5 nm, 0.17 ± 0.02, and 20.7 ± 4.7 mV, respectively, with an siRNA entrapment efficiency of 98.9%. The burst release of the PD-1 siRNA from the SLNs was minimal. The PD-1 siRNA-SLNs downregulated PD-1 expression on J774A.1 macrophage cell surface as well as in macrophages in B16-F10 tumors pre-established in mice. In mice with pre-established B16-F10 tumors, the PD-1 siRNA-SLNs significantly inhibited the tumor growth, as compared with siRNA-SLNs prepared with non-functional, negative control siRNA. In conclusion, the PD-1 siRNA-SLNs inhibited tumor growth, likely related to their ability to downregulate PD-1 expression by tumor-associated macrophage (TAMs).

In vivo pharmacokinetic study of remdesivir dry powder for inhalation in hamsters.

Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-h pharmacokinetic study in hamsters demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 h. The half-life of GS-4412524 following dry powder insufflation was about 7 h, suggesting the dosing regimen would be twice-daily administration. Although the remdesivir-Captisol® (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease.

How Does the Dissimilarity of Screw Geometry Impact Twin-screw Melt Granulation?

Using a model formulation of 80% gabapentin and 20% hydroxypropyl cellulose (KlucelTM), we investigate how differences in the geometry of mixing elements in the Leistritz Nano-16 and Micro-18 extruders affect granulation mechanisms and the properties of the resulting granules. Two extruders, Leistritz Nano-16 and Micro-18, commonly used in development and manufacturing, respectively, were used. The kneading blocks of the Nano-16 extruder are less efficient in dispersive mixing than the kneading blocks of the Micro-18 due to the thinner discs (2.5 mm wide) of the Nano-16. Therefore, our model formulation could be granulated only under a higher degree of fill (DF) by enhancing the axial compaction and heating of the barrel. In contrast, the thicker (5 mm wide) kneading blocks of the Micro-18 extruder provide efficient dispersive mixing that enables granulation without axial compaction and barrel heating. The higher specific mechanical energy (SME) achieved at higher screw speeds and lower feed rates led to more granulation. Because of the difference in granulation mechanisms between the two extruders, critical processing parameters also differed. Tabletability and degradant content of granules correlated positively with DF for the Nano-16 but with SME for the Micro-18 extruder.

Development of Remdesivir as a Dry Powder for Inhalation by Thin Film Freezing.

Remdesivir exhibits in vitro activity against SARS-CoV-2 and was granted approval for emergency use. To maximize delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (TFF). TFF produces brittle matrix nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. In vitro aerodynamic testing demonstrated that drug loading and excipient type affected the aerosol performance of remdesivir. Remdesivir combined with optimal excipients exhibited desirable aerosol performance (up to 93.0% FPF< 5 µm; 0.82 µm mass median aerodynamic diameter). Remdesivir was amorphous after the TFF process, which benefitted drug dissolution in simulated lung fluid. TFF remdesivir formulations are stable after one month of storage at 25 °C/60% relative humidity. An in vivo pharmacokinetic evaluation showed that TFF remdesivir-leucine was poorly absorbed into systemic circulation while TFF remdesivir-Captisol® demonstrated increased systemic uptake compared to leucine. Remdesivir was hydrolyzed to the nucleoside analog GS-441524 in the lung, and levels of GS-441524 were greater in the lung with leucine formulation compared to Captisol®. In conclusion, TFF technology produces high-potency remdesivir dry powder formulations for inhalation that are suitable to treat patients with COVID-19 on an outpatient basis and earlier in the disease course where effective antiviral therapy can reduce related morbidity and mortality.

Using thin film freezing to minimize excipients in inhalable tacrolimus dry powder formulations.

We investigated the feasibility of preparing high-potency tacrolimus dry powder for inhalation using thin film freezing (TFF). We found that using ultra-rapid freezing can increase drug loading up to 95% while maintaining good aerosol performance. Drug loading affected the specific surface area and moisture sorption of TFF formulations, but it did not affect the chemical stability, physical stability, and dissolution of tacrolimus. Tacrolimus remained amorphous after storage at 40 °C/75% RH, and 25 °C/60% RH for up to 6 months. Lactose functioned as a bulking agent, and it had little to no effect as a stabilizer for amorphous tacrolimus due to a lack of interaction between the drug and excipient. Additionally, the aerosol performance of TFF tacrolimus/lactose (95/5) did not significantly change after six months of storage at 25 °C/60% RH. For processing parameters, the solids content and the processing temperature did not affect the aerosol performance of tacrolimus. Furthermore, both low- and high-resistance RS01 showed optimal and consistent aerosol performance over the 1-4 kPa pressure drop range. In conclusion, TFF is a suitable technology for producing inhalable powder that contain high drug loading and have less flow rate dependence.

Development of an Excipient-Free Peptide Dry Powder Inhalation for the Treatment of Pulmonary Fibrosis.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 µm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Formulation Composition and Process Affect Counterion for CSP7 Peptide.

Counterions commonly remain with peptides in salt form after peptide purification. In animal and human studies, acetate counterions are a safer and more acceptable choice for peptides than others (e.g., trifluoroacetate counterions). Various salt forms of caveolin-1 scaffolding domain (CSP7) affect counterion volatilization. The development of lyophilized formulations containing volatile compounds is a challenge because these compounds sublimate away during the process. This work aims to investigate the effect of excipients and lyophilization parameters on the preservation of volatile compounds after lyophilization. The peak areas obtained from 1H and 19F NMR spectra were used to calculate the molar ratio of counterions to CSP7. We found that the pH modifier excipient had the greatest impact on the loss of counterions. By optimizing the molar ratio of bulking agent to CSP7, volatile compounds can be preserved after lyophilization. Higher chamber pressure during lyophilization can lower the sublimation rate of volatile compounds. Moreover, the loss of volatile compounds affects the stability of CSP7 due to the pH shift of reconstituted solutions, thereby causing peptide aggregation. The optimization of the formulation and processing helps preserve volatile compounds, thus minimizing the pH change of reconstituted solutions and maintaining the stability of peptide.

Effect of the Ratio of Betamethasone to TNF-α siRNA Coencapsulated in Solid Lipid Nanoparticles on the Acute Proinflammatory Activity of the Nanoparticles.

There is evidence that encapsulating glucocorticoids into nucleic acid-containing nanoparticles reduces the inflammatory toxicities of the nanoparticles. Herein, using betamethasone acetate (BA), a glucocorticoid, and a solid lipid nanoparticle formulation of siRNA, we confirmed that coencapsulating BA into the siRNA solid lipid nanoparticles significantly reduced the proinflammatory activity of the siRNA nanoparticles in a mouse model. Using TNF-α siRNA, we then showed that the BA and TNF-α siRNA coencapsulated into the solid lipid nanoparticles acted as a dual anti-inflammatory and synergistically reduced TNF-α release by mouse macrophages in culture following stimulation with lipopolysaccharide, as compared to solid lipid nanoparticles encapsulated with TNF-α siRNA or BA alone. Importantly, upon studying the effect of the ratio of BA and TNF-α siRNA on the proinflammatory activity of the resultant nanoparticles, we identified that BA and TNF-α siRNA coencapsulated solid lipid nanoparticles prepared with a BA to TNF-α siRNA weight ratio of 2:1 induced the lowest proinflammatory cytokine production by macrophages in culture. This result was in comparison to nanoparticles prepared with BA to TNF-α siRNA ratios both higher and lower than 2:1 (i.e., 4:1, 1:1, and 0.5:1) and is likely due to differences in molecular interactions among the various components in the BA and TNF-α-siRNA coencapsulated solid lipid nanoparticles at these ratios. Encapsulating glucocorticoids into siRNA-nanoparticles represents a viable strategy to reduce the proinflammatory activity of the nanoparticles; however, the ratio of the glucocorticoid to siRNA in the nanoparticles requires optimization.

The influence of polymeric subcoats and pellet formulation on the release of chlorpheniramine maleate from enteric coated pellets.

The influences of aqueous polymeric subcoats and pellet composition on the release properties of a highly water-soluble drug, chlorpheniramine maleate (CPM), from enteric coated pellets were investigated. Three different aqueous polymeric subcoats, Eudragit RD 100, Eudragit RS 30D, and Opadry AMB, were applied to 10% w/w CPM-loaded pellets that were then enteric coated with Eudragit L 30D-55. Observed drug release from the coated pellets in acidic media correlated with water vapor transmission rates derived for the subcoat films. The influence of pellet composition on retarding the release of CPM from enteric coated pellets in 0.1 N HCl was investigated. The rate of drug release was greatest for pellets prepared with lactose, microcrystalline cellulose, or dibasic calcium phosphate compared with pellets formulated with citric acid and microcrystalline cellulose. Citric acid reduced the pellet micro-environmental pH, decreasing the amount of drug leakage in 0.1 N HCL during the first 2 hr of dissolution. Polymer flocculation was observed when CPM was added to the Eudragit L 30D-55 dispersion. An adsorption isotherm was generated for mixtures of CPM and the polymer and the data were found to fit the Freundlich model for adsorption. Adsorption of CPM to the polymer decreased with the addition of citric acid to the drug-polymer mixtures.

Stability of polyethylene oxide in matrix tablets prepared by hot-melt extrusion.

The thermal stability of polyethylene oxide (PEO) in sustained release tablets prepared by hot-melt extrusion was investigated. The weight average molecular weight of the polymer was studied using gel permeation chromatography. The chemical stability of PEO was found to be dependent on both the storage and processing temperature, and the molecular weight of the polymer. Storage of the polymer above its melting point significantly increased polymer degradation, and the degradation process was accelerated as the molecular weight was reduced. The thermal stability of PEO MW = 1,000,000 (PEO 1 M) in sustained release chlropheniramine maleate (CPM) tablets prepared by hot-melt extrusion was found to depend on the processing temperature and screw speed. Lower molecular weight PEO MW = 100,000 (PEO 100 K) was demonstrated to be a suitable processing aid for PEO 1 M. Incorporation of PEO 100 K reduced degradation of PEO 1 M and did not alter the release rate of CPM. Vitamin E, Vitamin E Succinate and Vitamin E TPGS were found to be suitable stabilizers for PEO, however, ascorbic acid was shown to degrade the polymer in solution. Thermal analysis demonstrated that Vitamin E Succinate and Vitamin E TPGS were dispersed at the molecular level in hot-melt extruded tablets. Solubilized Vitamin E Succinate and Vitamin E TPGS suppressed the melting point of the polyethylene oxide. Drug release rates from hot-melt extruded tablets stabilized with antioxidants were found to be dependent on the hydrophilic nature of the antioxidant.

Production of spherical pellets by a hot-melt extrusion and spheronization process.

Controlled-release theophylline containing spherical pellets were successfully produced by a hot-melt extrusion (HME) and spheronization process. A powder blend of anhydrous theophylline, Eudragit Preparation 4135 F, microcrystalline cellulose and polyethylene glycol 8000 powder was sieved, blended and then melt-extruded in a Randcastle Microtruder. The hot-melt extruded pellets were prepared by first cutting a thin, extruded composite rod into symmetrical pellets. The pellets were then spheronized in a traditional spheronizer at an elevated temperature. Thermal properties of the pellet formulation components and the hot-melt extrudate were studied to determine suitability of the formulation for HME. Pellets were examined using scanning electron microscopy to determine the effect of spheronization time on surface morphology. The rate of release of theophylline from the hot-melt extruded spherical pellets was characterized using USP 24 Apparatus 2 dissolution testing after initial pellet production and after 1 year storage in sealed HDPE containers at 25 degrees C/60% RH.