Karen C. Thompson

The structure of human acidic fibroblast growth factor and its interaction with heparin

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.

Characterization of Crystalline Drug Nanoparticles Using Atomic Force Microscopy and Complementary Techniques

AbstractPurpose. The purpose of this work was to image crystalline drug nanoparticles from a liquid dispersion and in a solid dosage form for the determination of size, shape, and distribution. Methods. Crystalline drug nanoparticles were adsorbed from a colloidal dispersion on glass for atomic force microscopy (AFM) imaging. Nanoparticles that were spray coated onto a host bead were exposed by ultramicrotomy for scanning electron microscopy and AFM examination. Results. The adsorbed drug nanoparticles were measured by AFM to have a mean diameter of 95 nm and an average aspect ratio of 1.3. Nanoparticles observed in the solid dosage form had a size and shape similar to drug nanoparticles in the dispersion. Particle size distribution from AFM measurement agreed well with data from field emission scanning electron microscopy, static light scattering, and X-ray powder diffraction. Conclusions. AFM is demonstrated to be a valuable tool in visualization and quantification of drug nanoparticle crystals in formulations. In addition to accurate size measurement, AFM readily provides shape and structural information of nanoparticles, which cannot be obtained by light scattering. Ultramicrotomy is a good sample preparation method to expose the interior of solid dosage forms with minimal structural alteration for microscopic examination.

Discovery of a stable molecular complex of an API with HCl: A long journey to a conventional salt

We report formation and characterization of the first pharmaceutically acceptable and stable molecular complex of a mono-HCl salt of Compound 1 with HCl. The novelty of this discovery is due to the fact that there is only one major basic site in the molecule. Thus this complex is reminiscent of other noncovalent crystalline forms including solvates, hydrates, cocrystals and others. To the best of our knowledge, the observed bis-HCl salt appears to be the first example of an active pharmaceutical ingredient in a form of a stable HCl complex. The paucity of stable complexes of APIs with HCl is likely due to the fact that HCl is a gas at ambient conditions and can easily evaporate compromising physical (and chemical) stability of a drug. The bis-HCl salt was chemically/physically stable at low humidity and the molecular HCl stays in the lattice until heated above 140 degrees C under nitrogen flow. Structure solution from powder diffraction using the Monte Carlo simulated annealing method as well as variable temperature ATR-FTIR suggest the possibility of weak hydrogen bonding between the molecular HCl and the nitrogen atom of the amide group. Two years later after the search for a suitable pharmaceutical salt began, the elusive conventional mono-HCl salt was obtained serendipitously concluding the lengthy quest for a regular salt. This work emphasizes the necessity to be open-minded during the salt selection process. It also highlights the difficult, lengthy and often serendipitous path of finding the most appropriate form of an API for pharmaceutical development.

High-performance liquid chromatographic separation of an HIV-1 reverse transcriptase inhibitor and its enantiomer.

This article describes the direct separation of an HIV-1 reverse transcriptase inhibitor and its enantiomer by HPLC on a silica-bonded polyacrylamide (ChiraSpher) column. The column selection was based on specific interactions between the individual enantiomers and the chiral stationary phase. The influence of some chromatographic conditions, such as concentration of the polar modifier in the mobile phase, column flow-rate and column temperature, on column performance was investigated. The separation was applied to the determination of the minor enantiomer in the bulk drug and as low as 0.3% of minor enantiomer was detectable.

Leveraging Solid State Form and Physiochemical Properties for Early Clinical Formulation Efforts: Opportunities and Challenges During Telcagepant Liquid Capsule Development

Lipid-based formulations (LBF) have emerged as an attractive formulation strategy to deliver poorly water-soluble drug molecules. The final dosage form, which can be either a liquid or a semisolid, is typically filled into hard-shell or soft gelatin capsules. Due to the relatively straightforward formulation development steps required, LBFs are especially suitable to support early phase clinical studies for a new chemical entity. However, line-of-sight to late stage clinical studies and commercialization requires a thorough understanding of the physical and chemical properties of the API, as well as potential interactions with excipients and the capsule shell materials used. This report describes the formulation development efforts of telcagepant, a novel candidate for the treatment of acute migraine. The key challenges associated with the formulation are discussed, including low API solubility in the excipient matrix, capsule cross-linking, as well as physical and chemical instability. Initially, telcagepant was formulated as a liquid LBF using the crystalline neutral form of the molecule, aided by in situ salt formation, and filled into hard-shell gelatin capsules. Following an extensive search and evaluation of potential options, a liquid formulation using the potassium ethanolate form of the molecule, filled into soft gelatin capsules, was selected for late stage clinical studies and commercialization. However, disproportionation of the salt form, which was further exacerbated by hydrolysis of the excipients, eventually led to the loss of API solubilization within the vehicle matrix. The development challenges encountered with stability requirements, pharmacokinetic targets, and commercial market demands are discussed in details, as well as the risk assessment and mitigation strategies employed in delivering this molecule to patients.