Cynthia L. Stevenson

An in vivo/in vitro comparison with a leuprolide osmotic implant for the treatment of prostate cancer

An osmotically driven implantable system was designed and characterized for the delivery of leuprolide over a year-long duration. Leuprolide has been used in the treatment of prostate cancer since the 1980s. The DUROS implant consists of a titanium alloy cylinder, measures 4 mm in diameter by 45 mm in length and holds approximately 150 microl of formulation. Stability studies indicated that leuprolide was stable, as a solution formulation in DMSO, for several years at 37 degrees C. In vitro release rate testing, at weekly intervals, showed zero-order delivery for 1 year. DUROS implants demonstrated release rates that were reproducible and similar to one another after storage at 25 degrees C for 18 months prior to testing. In vivo studies, with implants placed subcutaneously, revealed delivery rates comparable to those observed under in vitro conditions. Leuprolide stability was also comparable between in vivo and in vitro conditions. Steady leuprolide serum levels produced by the implant resulted in the desired pharmacodynamic efficacy endpoint of testosterone suppression, both in canines and in humans. The good agreement between in vivo/in vitro delivery rates was as expected for a delivery system based on the principles of osmosis.

Estimation of recombinant bovine somatotropin solubility by excluded-volume interaction with polyethylene glycols.

AbstractPurpose. The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt). Methods. Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed. Results. An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG. Conclusions. Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.

Solid-State Stability of Spray-Dried Insulin Powder for Inhalation: Chemical Kinetics and Structural Relaxation Modeling of Exubera Above and Below the Glass Transition Temperature

The effect of temperature on the chemical stability of an amorphous spray-dried insulin powder formulation (Exubera) was evaluated in the solid state at constant moisture content. The chemical stability of the powder was assessed using reversed-phase high-performance liquid chromatography (RP-HPLC) and high-performance-size exclusion chromatography (HP-SEC). The major degradants in spray-dried insulin produced during heat stressing were identified as A21-desamidoinsulin (A21) and high molecular weight protein (HMWP). As expected, the rates of formation of A21 and HMWP were observed to increase with temperature. A stretched-time kinetic model (degradation rate is proportional to the square root of time) was applied to the degradant profiles above and below the glass transition temperature (T(g)) and apparent reaction rate constants were determined. Below T(g), isothermal enthalpy of relaxation measurements were used to assess the effect of temperature on molecular mobility. The formation of A21 and HMWP was found to follow an Arrhenius temperature dependence above and below the T(g). Comparison of reaction rate constants to those estimated from structural relaxation experiments suggests that the reaction pathways to form A21 and HMWP below the T(g) may be coupled with the molecular motions involved in structural relaxation.

Effect of gelation on the chemical stability and conformation of Leuprolide

AbstractPurpose. The purpose of this study was to characterize the conformation, aggregation, and stability of leuprolide on gelation. Methods. Infrared spectra (FTIR) of leuprolide solutions and gels were collected in water, propylene glycol (PG), dimethyl sulfoxide (DMSO), and trifluoroethanol (TFE). Leuprolide solution and gel stability data were obtained by SEC and RP-HPLC. Results. Leuprolide was induced to gel with increasing peptide concentration, introduction of salts, and gentle agitation. Leuprolide dissolved in water (400 mg/ml) demonstrated FTIR spectra consisting of two major bands of equal intensity at 1615 cm−1 and 1630 cm−1, similar to inter- and intra-molecular β-sheet structure in proteins. When samples were gently agitated for 24 hours at 25°C, the formulation was observed to change from a viscous liquid to an opaque gel with a concomitant shift in infrared spectra from the equal intensity bands to mostly 1630 cm−1, indicating a shift to a preferred β-sheet structure. Incubation of leuprolide with 20−200 mM salts at 25°C and 37°C also produced gels ranging from clear to cloudy and stringy white precipitates. The gel and precipitate were marked by a shift of the predominant p-sheet band to 1630 cm−1 and 1615 cm−1, respectively. Leuprolide was also observed to gel and/or precipitate in mixtures of water, PG or TFE, but not in DMSO. Conclusions. Birefringence was noted in many of the firmer gels. Both solutions and gels demonstrated minimal dimer or trimer formation, with no larger order aggregates detected. The chemical stability profile of gelled leuprolide was similar to that of the non-gelled water formulation by RP-HPLC.

Delivery of Peptides and Proteins via Long Acting Injections and Implants

Biomolecules are rarely orally bioavailable and parenteral injections are the industry standard. Development of long acting delivery systems will continue to help biopharmaceuticals reach their full therapeutic potential. Long acting injections have manifested into a range of products designed to target optimal therapeutic dosing requirements (prefilled syringes, long acting injections, sustained release depots, controlled release implants, targeted delivery, and larger payloads). Sustained release implants usually provide a longer term delivery duration than long acting injections and can be categorized into bioerodible systems (rods and cylinders) and implantable devices. This chapter reviews biomolecules currently marketed or in clinical trials, utilizing long acting parenteral technology or sustained/controlled release implants.

Development of the Exubera® Insulin Pulmonary Delivery System

Development of Exubera® (insulin powder for inhalation) presented numerous challenges as a first-in-class product. These challenges included developing (a) the first room temperature stable insulin formulation, (b) a spray-drying process to produce a fine respirable powder with the physical attributes needed for efficient aerosolization, (c) a filling and packaging process capable of accurately and reproducibly dispensing fine powders, (d) a durable inhaler that enables reliable dosing to the patient under a variety of dosing scenarios and environmental conditions, and (e) a clinical program for Type 1 and Type 2 diabetics involving parallel measurements of pulmonary function. Insulin was formulated with sodium citrate, mannitol, and glycine in a homogenous aqueous solution prior to spray drying. Spray-drying equipment was designed and scaled up, to produce powder with tight control over particle size (mean ≈ 2 µm) and moisture content (< 2 %). The resulting amorphous powder was purposely designed to provide a high glass transition temperature (T g) that minimized insulin mobility (thus reactivity). Fine powder handling and packaging technology was developed to reproducibly fill blister packages containing 1.7 and 5.1 mg of powder (1 and 3 mg insulin per blister, respectively). The heat-sealed perimeter of the blisters prevented moisture ingress and provided room temperature storage for 18 and 24 months in the USA and EU, respectively. The Exubera® inhaler was designed for reproducible delivery and simple 5-step operation: (1) extension of the device chamber, (2) insertion of blister, (3) manual pump of handle to compress a defined volume of air (as the energy source for aerosolization), (4) button actuation to release the compressed air and thereby extract the insulin powder from the blister and disperse it as a fine aerosol into the chamber, and (5) rotation of the chamber mouthpiece to enable dose administration via inhalation of the standing aerosol cloud. Exubera® delivered insulin to the deep lung where it was absorbed into the blood stream with similar reproducibly and effectiveness as subcutaneous injections. Exubera® gained FDA and EMA approval in 2006, validating the technological and clinical efforts of Pfizer, Nektar, and Sanofi-Aventis. However, Exubera® was not a commercial success and was removed from the market in 2007. Lessons learned from the technology integration, the partnership between technology provider and sponsor, and the brief market experience are discussed.