Richard A. Kenley

Biotechnology and bone graft substitutes

Trauma, disease, developmental deformities, and tumor resection frequently cause bone defects that seriously challenge the skills of orthopedic and maxillofacial surgeons. Currently, repairing osseous deficiencies involves various medical surgical techniques, including autogenous grafts, allografts, internal and external fixation devices, electrical stimulation, and alloplastic implants. The existing technology, though effective in many cases, still is beset with numerous difficulties and disadvantages. A critical need for improved treatment methods exists today. Biotechnology now provides access to new bone repair concepts via administration of protein growth and morphogenic factors. Implantable device and drug delivery system technologies also have advanced. The converging biopharmaceutical, device, and delivery technologies represent an opportunity to improve the quality of health care for individuals with orthopedic and maxillofacial deficiencies. This report reviews current concepts in fracture healing and bone repair and examines existing treatment modalities. It also addresses novel protein drugs that stimulate osseous regeneration and delivery systems for these drugs.

Osseous regeneration in preclinical models using bioabsorbable delivery technology for recombinant human bone morphogenetic protein 2 (rhBMP-2)

Abstract Novel delivery systems for the bone morphogenetic protein rhBMP-2, consisting of poly( d,l -lactide-co-glycolide) porous microspheres as bioabsorbable filling material with either autologous blood clot or carboxymethylcellulose as binding agent, were tested in several preclinical models including a rat calvarial defect model, a rabbit radius segmental defect model, and a rabbit ulna segmental defect model. In the rat calvarial defect model, these novel delivery systems were shown to be comparable to inactivated collagenous bone matrix as a matrix for rhBMP-2 delivery at the appropriate dose. The incidence of union in the rabbit long bone studies exhibited a rhBMP-2 dose-response, achieving an incidence of union of nearly 100% at the higher doses tested. Histological evaluation showed remodelling of newly generated bone, and biomechanical testing found that the healed limbs were as strong as untreated control limbs. These studies demonstrate that it is possible to obtain osseous regeneration of critical-size defects by combining rhBMP-2 with synthetic delivery systems.

Degradation Pathways for Recombinant Human Macrophage Colony-Stimulating Factor in Aqueous Solution

Recombinant human macrophage colony-stimulating factor (rhM-CSF) promotes macrophage proliferation and activity. rhM-CSF clinical trials are currently in progress and require a stable, pharmaceutically acceptable dosage form. This report documents pH effects on rhM-CSF degradation profiles in aqueous solution, with an emphasis on identifying degradation products. Thus, highly purified rhM-CSF was maintained at 30 to 50°C in solutions adjusted to pH 2 to 10. Stressed samples were analyzed by SDS-PAGE, reverse-phase HPLC, size exclusion HPLC, scanning microcalorimetry, and murine bone marrow activity. The results show maximal protein stability in the region pH 7 to 8. Degradation product chromatographic and electrophoretic analyses show distinctly different degradation product profiles in acidic versus alkaline solution. For samples stressed in acidic solution, degradation products were isolated chromatographically and electrophoretically. These degradation products were characterized by N-terminal amino acid sequencing, fast-atom bombardment mass spectrometry, and peptide mapping. The results show that the major degradation pathway in acidic solution involves peptide cleavage at two sites: aspartate169-proline170 and aspartate213-proline214. A third potential cleavage site (aspartate45-proline46) remains intact under conditions that cleave Asp169-Pro170 and Asp213-Pro214. In alkaline solution, degradation proceeds via parallel cleavage and intramolecular cross-linking reactions. A β-elimination mechanism is proposed to account for the degradation in alkaline solution. Consistent with literature observations, the rhM-CSF N-terminal cleavage products retain biological activity.

Acid-Catalyzed Peptide Bond Hydrolysis of Recombinant Human Interleukin 11

Recombinant human interleukin 11 (rhIL-11) is a multispectrum cytokine that plays an important role in megakaryocytopoiesis and platelet production. Probing rhIL-11 chemical reactivity in aqueous solution is an important initial step in developing a dosage form for rhIL-11 clinical trials. This report documents rhIL-11 degradation kinetics at 50°C in solutions adjusted to pH 3.0 to 9.5. Stressed samples were analyzed by reverse-phase HPLC and degradation product peaks were isolated for structural characterization. The results show maximal stability in the region pH 6.5 to 7.0. Degradation product identification shows that the major reaction pathway in acidic solution involves peptide cleavage at aspartate133–proline134. In alkaline solution, protein disappearance proceeds via nonspecific loss to container surfaces. Degradation products at alkaline pH have not been identified.

HPLC Analysis of Thimerosal in Ophthalmic Solution. Comparing Electrochemical with Spectrophotometric Detectors

Electrochemical and spectrophotometric detectors for the reversed phase HPLC analysis of thimerosal in an ophthalmic formulation were evaluated with respect to response linearity, recovery, and lower limit of detection. The electrochemical oxidation of thimerosal at a glassy carbon electrode was characterized by both cyclic voltammetry and thin layer amperometry. The hydrodynamic half-wave potential for thimerosal (+0.6 V) was determined using a coulometric detector. The relatively low oxidation potential for thimerosal forms the basis for this highly sensitive and selective analytical technique. The amperometric lower limit of detection for thimerosal (at signal/noise = 2) was less than 400 pg. The detection limit with spectrophotometric detection at 254 nm was approximately 20 ng, a 50 fold increase.

Temperature and pH dependence of fluocinolone acetonide degradation in a topical cream formulation

AbstractWe investigated the degradation of fluocinolone acetonide (FA) incorporated into an oil-in-water cream base. The study examined the influence of temperature (23 to 80°C) and cream pH (pH 2.3 to 6) on FA degradation rates. FA degradation followed pseudo-first-order kinetics and adhered to the Arrhenius expression over the entire temperature range investigated. At all temperatures, the pH strongly influenced the observed degradation rate constant (kobs) values, with rate minima observed near pH 4. The FA log(degradation rate)–pH profiles were consistent with a reaction mechanism requiring drug hydrolysis catalyzed by hydroxide and hydrogen ions. Taking into account both the temperature and the pH dependence of FA degradation permits calculating kobs values from the following equation:

Effects of PAM, proPAM, and DFP on behavior, thermoregulation, and brain AChE in rats

\begin{gathered} k_{{\text{obs}}} = \exp \left\{ {22.5 - \left( {17,200/RT} \right)} \right\} + \exp \left\{ {38.7 - \left( {22,200/RT} \right)} \right\} \times \left[ {H^ + } \right] \hfill \\ {\text{ + exp}}\left\{ {{\text{49}}{\text{.5}} - \left( {21,100/RT} \right)} \right\} \times \left[ {OH^ - } \right] \hfill \\ \end{gathered}

Sulconazole reactions with peracetic acid and hydrogen peroxide

where the three bracketed terms represent Arrhenius expressions for neutral, acid-catalyzed, and base-catalyzed hydrolysis reactions. FA degradation in the cream base parallels the degradation of a related steroid (triamcinolone acetonide) in an aqueous alcohol solution. The equivalence between FA and triamcinolone acetonide kinetics in the different reaction media suggests that in the cream base, FA degradation is limited to an aqueous phase largely unperturbed by the presence of nonaqueous constituents that comprise the cream formulation.