John A. Denman

A novel dry powder inhalable formulation incorporating three first-line anti-tubercular antibiotics

Treatment for tuberculosis (TB) using the standard oral antibiotic regimen is effective but inefficient, requiring high drug dosing and lengthy treatment times. Three concurrent first-line antibiotics recommended by the World Health Organization (WHO) guidelines are pyrazinamide, rifampicin and isoniazid. Combining these antibiotics in a novel formulation for dry powder inhalation (DPI) may facilitate rapid and efficient resolution of local and systemic infection. However, spray-dried individually, these antibiotics were found to be physically unstable. A solution of the three antibiotics, at the WHO-recommended ratio, was spray-dried. The collected powder was assessed by a series of in vitro methods to investigate aerosol performance, particle physico-chemical characteristics and dissolution profile. Particles obtained were spherical with a surface composed primarily of rifampicin, as identified by TOF-SIMS. A mass median aerodynamic diameter of 3.5 ± 0.1 μm and fine particle fraction (<5 μm) of 45 ± 3% indicated excellent aerosol performance. The combination powder was differentiated by the presence of rifampicin dihydrate and the delta polymorph of pyrazinamide. Quantitative analysis indicated individual particles contained the three antibiotics at the expected proportions (400:150:75 w/w). This excipient-free triple antibiotic DPI formulation could be used as a significant enhanced treatment for TB.

Characterization of the surface properties of a model pharmaceutical fine powder modified with a pharmaceutical lubricant to improve flow via a mechanical dry coating approach

The aim of this study is to investigate the changes in physical and chemical surface properties of a fine lactose powder, which has been processed by a mechanical dry coating approach. A commercially available milled lactose monohydrate powder (median diameter around 20 μm) was dry coated with a pharmaceutical lubricant, magnesium stearate (MgSt). Substantial changes in bulk behavior have been shown previously and the purpose of the current work was to understand the relationship between these bulk changes and physico-chemical changes in the surface. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry results demonstrated both qualitatively and quantitatively how the chemical properties of the lactose particle surfaces had been altered. The characterization results indicated that a high-level coverage of a thin coating layer of MgSt has been created through the coating. Inverse gas chromatography was used to probe the surface energetic changes, and at conditions of finite dilution, provided a new insight into surface energy changes. This work demonstrated that the modifications of the surface physical and chemical properties correlated with the reduction in powder cohesion and improvement in powder flow.

Investigation of the extent of surface coating via mechanofusion with varying additive levels and the influences on bulk powder flow properties.

The objective of this study was to investigate if the coating extent created by a mechanofusion process corresponded with observed changes in bulk powder properties. A fine lactose powder (approximate median diameter 20 μm) was dry coated with magnesium stearate using from 0.1 to 5% (w/w) content. An ultra-thin coating layer of magnesium stearate was anticipated, but previous attempts to determine such thin layers on these fine particles have had limited success, with poor resolution. In this study, the surface coating was examined using the state-of-the-art XPS and ToF-SIMS systems. The powder flow was characterized by Carr index and shear cell testing. XPS was successfully applied to demonstrate variations in surface coverage, as a function of additive levels, and indicated near complete coating coverage at additive levels of 1% (w/w) and above. ToF-SIMS results supported such coating coverage assessment, and indicated coating uniformly across the fine particle surfaces. The flow metrics employed could then be related to the coating coverage metrics. The mechanofusion process also modified the apparent surface roughness observed by SEM and BET. It was suggested that the changes in the surface chemical composition exerted a more evident and direct impact on the powder cohesion and flow characteristics than the changes in the surface morphological properties after the mechanofusion in this study.

Synergistic Antibiotic Combination Powders of Colistin and Rifampicin Provide High Aerosolization Efficiency and Moisture Protection

For many respiratory infections caused by multidrug-resistant Gram-negative bacteria, colistin is the only effective antibiotic despite its nephrotoxicity. A novel inhaled combination formulation of colistin with a synergistic antimicrobial component of rifampicin was prepared via co-spray drying, aiming to deliver the drug directly to the respiratory tract and minimize drug resistance and adverse effects. Synergistic antibacterial activity against Acinetobacter baumannii was demonstrated for the combination formulation with high emitted doses (96%) and fine particle fraction total (FPFtotal; 92%). Storage of the spray-dried colistin alone formulation in the elevated relative humidity (RH) of 75% resulted in a substantial deterioration in the aerosolization performance because the amorphous colistin powders absorbed significant amount of water up to 30% by weight. In contrast, the FPFtotal values of the combination formulation stored at various RH were unchanged, which was similar to the aerosolization behavior of the spray-dried rifampicin-alone formulation. Advanced surface chemistry measurements by XPS and ToF-SIMS demonstrated a dominance of rifampicin on the combination particle surfaces, which contributed to the moisture protection at the elevated RH. This study shows a novel inhalable powder formulation of antibiotic combination with the combined beneficial properties of synergistic antibacterial activity, high aerosolization efficiency, and moisture protection.

Detecting the Presence of Denatured Human Serum Albumin in an Adsorbed Protein Monolayer Using TOF−SIMS

We demonstrate the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in conjunction with multivariate statistics to differentiate trace levels of denatured proteins in adsorbed monolayers; specifically, human serum albumin (HSA) on oxidized silicon substrates. Subtle differences in protein conformation due to thermal denaturation of HSA, unable to be determined by dynamic light scattering nor circular dichroism, were differentiated by TOF-SIMS. The fragmentation pattern is highly sensitive to protein conformation, allowing assessment of relative amounts of proteins in mixtures and quantifying amounts of denatured protein in a sample. Discussion is presented on ascribing orientation and conformational differences between samples based upon TOF-SIMS spectra. This has implications for detecting denatured protein in biotechnology and medical applications.

Relationship between surface concentration of L-leucine and bulk powder properties in spray dried formulations.

The amino acid L-leucine has been demonstrated to act as a lubricant and improve the dispersibility of otherwise cohesive fine particles. It was hypothesized that optimum surface L-leucine concentration is necessary to achieve optimal surface and bulk powder properties. Polyvinylpyrrolidone was spray dried with different concentration of L-leucine and the change in surface composition of the formulations was determined using X-ray photoelectron spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS). The formulations were also subjected to powder X-ray diffraction analysis in order to understand the relationship between surface concentration and solid-state properties of L-leucine. In addition, the morphology, surface energy and bulk cohesion of spray dried formulations were also assessed to understand the relation between surface L-leucine concentration and surface and bulk properties. The surface concentration of L-leucine increased with higher feed concentrations and plateaued at about 10% L-leucine. Higher surface L-leucine concentration also resulted in the formation of larger L-leucine crystals and not much change in crystal size was noted above 10% L-leucine. A change in surface morphology of particles from spherical to increasingly corrugated was also observed with increasing surface l-leucine concentration. Specific collapsed/folded over particles were only seen in formulations with 10% or higher l-leucine feed concentration suggesting a change in particle surface formation process. In addition, bulk cohesion also reduced and approached a minimum with 10% L-leucine concentration. Thus, the surface concentration of L-leucine governs particle formation and optimum surface L-leucine concentration results in optimum surface and bulk powder properties.

Characterization of chemoselective surface attachment of the cationic peptide melimine and its effects on antimicrobial activity.

Antimicrobial peptides (AMPs) are promising alternatives to current treatments for bacterial infections. However, our understanding of the structural-functional relationship of tethered AMPs still requires further investigation to establish a general approach for obtaining consistent antimicrobial surfaces. In this study, we have systematically examined the effects of surface orientation of a broad-spectrum synthetic cationic peptide, melimine, on its antibacterial activity against Gram-positive and Gram-negative bacteria. The attachment of melimine to maleimide-functionalized glass was facilitated by addition of a single cysteine amino acid into the peptide sequence at the N-terminus (CysN) or C-terminus (CysC), or at position 13 (Cys13, approximately central). The successful attachment of the modified melimine was monitored using X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principle component analysis. The ToF-SIMS analysis clearly demonstrated structural difference between the three orientations. The peptide density for the modified surfaces was found to be between 3.5-4.0×10(-9)molcm(-2) using a modified Bradford assay. The ability of the surfaces to resist Pseudomonas aeruginosa and Staphylococcus aureus colonization was compared using fluorescence confocal microscopy. Reductions in total P. aeruginosa and S. aureus adhesion of 70% (p<0.001) and 83% (p<0.001), respectively, after 48h were observed for the melimine samples when compared to the blank control. We found that melimine attached via the N-terminus was the most effective in reducing total bacterial adhesion and bacterial viability with two- and four times (p<0.001) more activity than melimine attached via the C-terminus for P. aeruginosa and S. aureus, respectively. Furthermore, for Cys13, despite having the highest measured peptide density of the three surfaces, the higher concentration did not confer the greatest antibacterial effect. This highlights the importance of orientation of the peptides on the surface to efficacy. Our results suggest that the optimal orientation of the cationic residues is essential for maximum surface activity, whereby the optimal activity is obtained when the cationic portion is more available to interact with colonizing bacteria.

l-Leucine as an excipient against moisture on in vitro aerosolization performances of highly hygroscopic spray-dried powders

L-Leucine (LL) has been widely used to enhance the dispersion performance of powders for inhalation. LL can also protect powders against moisture, but this effect is much less studied. The aim of this study was to investigate whether LL could prevent moisture-induced deterioration in in vitro aerosolization performances of highly hygroscopic spray-dried powders. Disodium cromoglycate (DSCG) was chosen as a model drug and different amounts of LL (2-40% w/w) were added to the formulation, with the aim to explore the relationship between powder dispersion, moisture protection and physicochemical properties of the powders. The powder formulations were prepared by spray drying of aqueous solutions containing known concentrations of DSCG and LL. The particle sizes were measured by laser diffraction. The physicochemical properties of fine particles were characterized by X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vapor sorption (DVS). The surface morphology and chemistry of fine particles were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In vitro aerosolization performances were evaluated by a next generation impactor (NGI) after the powders were stored at 60% or 75% relative humidity (RH), and 25°C for 24h. Spray-dried (SD) DSCG powders were amorphous and absorbed 30-45% (w/w) water at 70-80% RH, resulting in deterioration in the aerosolization performance of the powders. LL did not decrease the water uptake of DSCG powders, but it could significantly reduce the effect of moisture on aerosolization performances. This is due to enrichment of crystalline LL on the surface of the composite particles. The effect was directly related to the percentage of LL coverage on the surface of particles. Formulations having 61-73% (molar percent) of LL on the particle surface (which correspond to 10-20% (w/w) of LL in the bulk powders) could minimize moisture-induced deterioration in the aerosol performance. In conclusion, particle surface coverage of LL can offer short-term protection against moisture on dispersion of hygroscopic powders.

Investigation of the potential for direct compaction of a fine ibuprofen powder dry-coated with magnesium stearate

Abstract Intensive dry powder coating (mechanofusion) with tablet lubricants has previously been shown to give substantial powder flow improvement. This study explores whether the mechanofusion of magnesium stearate (MgSt), on a fine drug powder can substantially improve flow, without preventing the powder from being directly compacted into tablets. A fine ibuprofen powder, which is both cohesive and possesses a low-melting point, was dry coated via mechanofusion with between 0.1% and 5% (w/w) MgSt. Traditional low-shear blending was also employed as a comparison. No significant difference in particle size or shape was measured following mechanofusion. For the low-shear blended powders, only marginal improvement in flowability was obtained. However, after mechanofusion, substantial improvements in the flow properties were demonstrated. Both XPS and ToF-SIMS demonstrated high degrees of a nano-scale coating coverage of MgSt on the particle surfaces from optimized mechanofusion. The study showed that robust tablets were produced from the selected mechanofused powders, at high-dose concentration and tablet tensile strength was further optimized via addition of a Polyvinylpyrrolidone (PVP) binder (10% w/w). The tablets with the mechanofused powder (with or without PVP) also exhibited significantly lower ejection stress than those made of the raw powder, demonstrating good lubrication. Surprisingly, the release rate of drug from the tablets made with the mechanofused powder was not retarded. This is the first study to demonstrate such a single-step dry coating of model drug with MgSt, with promising flow improvement, flow-aid and lubrication effects, tabletability and also non-inhibited dissolution rate.

How Much Surface Coating of Hydrophobic Azithromycin Is Sufficient to Prevent Moisture-Induced Decrease in Aerosolisation of Hygroscopic Amorphous Colistin Powder?

ABSTRACTAerosolisation performance of hygroscopic particles of colistin could be compromised at elevated humidity due to increased capillary forces. Co-spray drying colistin with a hydrophobic drug is known to provide a protective coating on the composite particle surfaces against moisture-induced reduction in aerosolisation performance; however, the effects of component ratio on surface coating quality and powder aerosolisation at elevated relative humidities are unknown. In this study, we have systematically examined the effects of mass ratio of hydrophobic azithromycin on surface coating quality and aerosolisation performance of the co-spray dried composite particles. Four combination formulations with varying drug ratios were prepared by co-spray drying drug solutions. Both of the drugs in each combination formulation had similar in vitro deposition profiles, suggesting that each composite particle comprises two drugs in the designed mass ratio, which is supported by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) data. XPS and ToF-SIMS measurements also revealed that 50% by weight (or 35% by molecular fraction) of azithromycin in the formulation provided a near complete coating of 96.5% (molar fraction) on the composite particle surface, which is sufficient to prevent moisture-induced reduction in fine particle fraction (FPF)recovered and FPFemitted. Higher azithromycin content did not increase coating coverage, while contents of azithromycin lower than 20% w/w did not totally prevent the negative effects of humidity on aerosolisation performance. This study has highlighted that a critical amount of azithromycin is required to sufficiently coat the colistin particles for short-term protection against moisture.