Understanding the dehydration of levofloxacin hemihydrate.

Levofloxacin is a broad-spectrum antibiotic that exists as a hemihydrate under ambient conditions. In addition to the hemihydrate, there are three known crystalline anhydrate forms, denoted as α, ß, and γ. In this study, differential scanning calorimetry (DSC), thermogravimetric analysis, Raman spectroscopy, single-crystal and powder X-ray diffraction, and solid-state NMR spectroscopy were used to investigate the transitions that occurred upon dehydration to the anhydrate as well as additional transitions that occurred to the anhydrous material upon heating/cooling. An enantiotropic conversion was observed in the DSC around 54°C corresponding to the conversion of the γ form to a new form, denoted as the δ form. Raman spectroscopy, powder X-ray diffraction, and solid-state NMR spectroscopy confirmed that a new crystalline form was being produced.

Photolysis of recombinant human insulin in the solid state: formation of a dithiohemiacetal product at the C-terminal disulfide bond.

PURPOSE: Exposure of protein pharmaceuticals to light can result in chemical and physical modifications, potentially leading to loss of potency, aggregation, and/or immunogenicity. To correlate these potential consequences with molecular changes, the nature of photoproducts and their mechanisms of formation must be characterized. The present study focuses on the photochemical degradation of insulin in the solid state. METHODS: Solid insulin was characterized by solid-state NMR, polarized optical microscopy and scanning electron microscopy; various insulin preparations were exposed to UV light prior to product analysis by mass spectrometry. RESULTS: UV-exposure of solid human insulin results in photodissociation of the C-terminal intrachain disulfide bond, leading to formation of a CysS(•) thiyl radical pair which ultimately disproportionates into thiol and thioaldehyde species. The high reactivity of the thioaldehyde and proximity to the thiol allow the formation of a dithiohemiacetal structure. Dithiohemiacetal is formed during the UV-exposure of both crystalline and amorphous insulin. CONCLUSIONS: Dithiohemiacetals represent novel structures generated through the photochemical modification of disulfide bonds. This is the first time that such structure is identified during the photolysis of a protein in the solid state.

Carbon-deuterium rotational-echo double-resonance NMR spectroscopy of lyophilized aspartame formulations.

In this study, changes in the local conformation of aspartame were observed in annealed lyophilized glasses by monitoring changes in the distance between two labeled sites using C-(2)H rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR) spectroscopy. Confirmation that the REDOR experiments were producing accurate distance measurement was ensured by measuring the (13)C-(15)N distance in glycine. The experiment was further verified by measuring the REDOR dephasing curve on (13)C-(2)H methionine. (13)C-(2)H REDOR dephasing curves were then measured on lyophilized aspartame-disaccharide formulations. In aspartame-sucrose formulation, the internuclear distances increased upon annealing, which correlated with decreased chemical reactivity. By contrast, annealing had only a minimal effect on the dephasing curve in aspartame-trehalose formulation. The results show that stability is a function of both mobility and local structure (conformation), even in a small molecule system such as lyophilized aspartame-sucrose.

Budesonide nanoparticle agglomerates as dry powder aerosols with rapid dissolution.

Nanoparticle technology represents an attractive approach for formulating poorly water-soluble pulmonary medicines. Unfortunately, nanoparticle suspensions used in nebulizers or metered dose inhalers often suffer from physical instability in the form of uncontrolled agglomeration or Ostwald ripening. In addition, processing such suspensions into dry powders can yield broad particle size distributions. To address these encumbrances, a controlled nanoparticle flocculation process has been developed. Nanosuspensions of the poorly water-soluble drug budesonide were prepared by dissolving the drug in organic solvent containing surfactants followed by rapid solvent extraction in water. Different surfactants were employed to control the size and surface charge of the precipitated nanoparticles. Nanosuspensions were flocculated using leucine and lyophilized. Selected budesonide nanoparticle suspensions exhibited an average particle size ranging from approximately 160 to 230 nm, high yield and high drug content. Flocculated nanosuspensions produced micron-sized agglomerates. Freeze-drying the nanoparticle agglomerates yielded dry powders with desirable aerodynamic properties for inhalation therapy. In addition, the dissolution rates of dried nanoparticle agglomerate formulations were significantly faster than that of stock budesonide. The results of this study suggest that nanoparticle agglomerates possess the microstructure desired for lung deposition and the nanostructure to facilitate rapid dissolution of poorly water-soluble drugs.

Nifedipine nanoparticle agglomeration as a dry powder aerosol formulation strategy.

Efficient administration of drugs represents a leading challenge in pulmonary medicine. Dry powder aerosols are of great interest compared to traditional aerosolized liquid formulations in that they may offer improved stability, ease of administration, and simple device design. Particles 1-5microm in size typically facilitate lung deposition. Nanoparticles may be exhaled as a result of their small size; however, they are desired to enhance the dissolution rate of poorly soluble drugs. Nanoparticles of the hypertension drug nifedipine were co-precipitated with stearic acid to form a colloid exhibiting negative surface charge. Nifedipine nanoparticle colloids were destabilized by using sodium chloride to disrupt the electrostatic repulsion between particles as a means to achieve the agglomerated nanoparticles of a controlled size. The aerodynamic performance of agglomerated nanoparticles was determined by cascade impaction. The powders were found to be well suited for pulmonary delivery. In addition, nanoparticle agglomerates revealed enhanced dissolution of the drug species suggesting the value of this formulation approach for poorly water-soluble pulmonary medicines. Ultimately, nifedipine powders are envisioned as an approach to treat pulmonary hypertension.

Pure insulin nanoparticle agglomerates for pulmonary delivery.

Diabetes is a set of diseases characterized by defects in insulin utilization, either through autoimmune destruction of insulin-producing cells (Type I) or insulin resistance (Type II). Treatment options can include regular injections of insulin, which can be painful and inconvenient, often leading to low patient compliance. To overcome this problem, novel formulations of insulin are being investigated, such as inhaled aerosols. Sufficient deposition of powder in the peripheral lung to maximize systemic absorption requires precise control over particle size and density, with particles between 1 and 5 microm in aerodynamic diameter being within the respirable range. Insulin nanoparticles were produced by titrating insulin dissolved at low pH up to the pI of the native protein, and were then further processed into microparticles using solvent displacement. Particle size, crystallinity, dissolution properties, structural stability, and bulk powder density were characterized. We have demonstrated that pure drug insulin microparticles can be produced from nanosuspensions with minimal processing steps without excipients, and with suitable properties for deposition in the peripheral lung.

NanoCipro encapsulation in monodisperse large porous PLGA microparticles.

Pulmonary drug delivery of controlled release formulations may provide an effective adjunct approach to orally delivered antibiotics for clearing persistent lung infections. Dry powder formulations for this indication should possess characteristics including; effective deposition to infected lung compartments, persistence at the infection site, and steady release of antibiotic. Large porous particles ( approximately 10-15 microm) have demonstrated effective lung deposition and enhanced lung residence as a result of their large diameter and reduced clearance by macrophages in comparison to small microparticles ( approximately 1-5 microm). In this report, Precision Particle Fabrication technology was used to create monodisperse large porous particles of poly(d,l-lactic-co-glycolic acid) (PLGA) utilizing oils as extractable porogens. After extraction, the resulting large porous PLGA particles exhibited a low density and a web-like or hollow interior depending on porogen concentration and type, respectively. Ciprofloxacin nanoparticles (nanoCipro) created by homogenization in dichloromethane, possessed a polymorph with a decreased melting temperature. Encapsulating nanoCipro in large porous PLGA particles resulted in a steady release of ciprofloxacin that was extended for larger particle diameters and for the solid particle morphology in comparison to large porous particles. The encapsulation efficiency of nanoCipro was quite low and factors impacting the entrapment of nanoparticles during particle formation were elucidated. A dry powder formulation with the potential to control particle deposition and sustain release to the lung was developed and insight to improve nanoparticle encapsulation is discussed.

3-Methylglutaric acid as a 13C solid-state NMR standard.

The calibration of a solid-state NMR spectrometer requires setting the magic angle, setting the reference and decoupler frequencies, ensuring that the magnetic field is homogeneous across the sample volume, optimizing the signal-to-noise ratio, determining the pi/2 pulse durations, and optimizing the Hartman-Hahn matching condition. Each task has one or more widely accepted standards, such as potassium bromide for setting the magic angle, adamantane for optimizing magnet homogeneity, and hexamethylbenzene or glycine for measuring the signal-to-noise ratio. We show that all of these tasks can be performed using 3-methylglutaric acid (MGA). In the case of high-powered decoupling, the CH(2) and CH carbon peaks of MGA provide an opportunity to evaluate the decoupling in a manner that is superior to any of the commonly used standard compounds. Thus, MGA can be used as a single solid-state NMR standard compound to perform all calibration steps except for magnet shimming.

Quantitation of crystalline and amorphous forms of anhydrous neotame using 13C CPMAS NMR spectroscopy.

Although most drugs are formulated in the crystalline state, amorphous or other crystalline forms are often generated during the formulation process. The presence of other forms can dramatically affect the physical and chemical stability of the drug. The identification and quantitation of different forms of a drug is a significant analytical challenge, especially in a formulated product. The ability of solid-state 13C NMR spectroscopy with cross polarization (CP) and magic-angle spinning (MAS) to quantify the amounts of three of the multiple crystalline and amorphous forms of the artificial sweetener neotame is described. It was possible to quantify, in a mixture of two anhydrous polymorphic forms of neotame, the amount of each polymorph within 1-2%. In mixtures of amorphous and crystalline forms of neotame, the amorphous content could be determined within 5%. It was found that the crystalline standards that were used to prepare the mixtures were not pure crystalline forms, but rather a mixture of crystalline and amorphous forms. The effect of amorphous content in the crystalline standards on the overall quantitation of the two crystalline polymorphic forms is discussed. The importance of differences in relaxation parameters and CP efficiencies on quantifying mixtures of different forms using solid-state NMR spectroscopy is also addressed.

Effects of acidic N + 1 residues on asparagine deamidation rates in solution and in the solid state.

The deamidation kinetics of four model peptides (AcGQNGG, AcGQNDG, AcGQNEG, and AcGQNQG) were studied in solution (70 degrees C, pH 5-10) and in lyophilized solids [70 degrees C, 50% relative humidity, "effective pH" ('pH') 5-10] containing polyvinyl pyrrolidone. AcGQNGG, AcGQNEG, and AcGQNQG degraded exclusively through Asn deamidation, whereas AcGQNDG also displayed Asp isomerization, and Asp-Gly peptide bond cleavage. The pH/'pH'-rate profiles were consistent with a shift in the rate-determining step of Asn deamidation from carbonyl addition to expulsion of ammonia with increasing pH. In solution, AcGQNGG deamidated up to 38-fold faster than the other peptides, indicating the importance of steric effects of the N + 1 residue. AcGQNGG and AcGQNQG had up to 60 times slower rates of deamidation in the solid state than in solution. In contrast, the deamidation rates of AcGQNEG and AcGQNDG in the solid state were similar to those in solution. N + 1 Glu or Asp residue may enhance local hydration, so that the deamidation of Asn in the solid formulations actually proceeds in a solution-like environment.