Deborah M. Lidgate

Sterile Filtration of a Parenteral Emulsion

The Syntex adjuvant formulation (SAP) containing [thr1]-muramyldipeptide in an oil-in-water emulsion has proven to be an effective adjuvant eliciting both cell-mediated and humoral immune response. As a parenteral emulsion, sterility of the final product was a concern, and various methods of achieving sterility were considered. For emulsions, most conventional sterilization methods are not viable, requiring the more cumbersome technique of sterilizing individual components and assembling/manufacturing under sterile conditions. Emulsion vehicles were manufactured with various models in the Microfluidizer M110 series. All equipment examined was capable of reducing the average dispersed oil droplet size to approximately 160 nm, with varying size ranges. Operating at an internal equipment pressure of greater than 16,000 psi, with at least five cycles through the interaction chamber, the resulting emulsion had a narrow droplet size range distribution, with the largest droplets being small enough to enable sterile filtration. Under specific-manufacturing conditions, the adjuvant emulsion becomes easily filtered through a 0.22-µm cartridge filter, thus yielding a sterile end product. This is the first published example of emulsion sterilization being achieved by terminal filtration.

Formulation of vaccine adjuvant muramyldipeptides. 3. Processing optimization, characterization, and bioactivity of an emulsion vehicle.

An efficacious vaccine adjuvant which elicits both cell-mediated immunity (CMI) and humoral immune response was developed using [thr1-Muramyldipeptide (MDP) in an oil-in-water emulsion vehicle containing poloxamer 401, polysorbate 80, and squalane. Processing optimization was performed to increase the physical stability of this adjuvant emulsion which, when prepared by conventional mixing methods, demonstrated good bioactivity but poor physical stability. Various manufacturing methods were compared with a microfluidization process, which produced the most stable and elegant emulsion vehicle. The microfluidized emulsion also elicited equivalent biological response in the animal model tested.

Influence of Ferrous Sulfate on the Solubility, Partition Coefficient, and Stability of Mycophenolic Acid and the Ester Mycophenolate Mofetil

ABSTRACT Studies were performed to (1) evaluate whether the presence of iron affected the physicochemical properties of mycophenolate mofetil (MMF) and mycophenolic acid (MPA), and (2) determine whether alteration of these properties was indicative of formation of an MMF–iron complex. The solubility, stability (chemical reactivity), and partitioning properties of MMF and MPA were evaluated over a pH range of 2–7 in the presence and absence of ferrous sulfate. In addition, the solubility and partitioning properties of MMF were assessed after the MMF drug product, CellCept® capsules, was combined with an iron tablet (Fero-Gradumet®, ferrous sulfate, tablets). The results of studies showed that: The solubility of MMF in the presence of ferrous sulfate was generally unaffected over a pH range of 2–7; a small increase in solubility was observed in pH 5.2 buffer solution. The solubility of MPA decreased in pH 5.2 and 7.0 buffer solutions. Both MMF and MPA were more stable in the presence of ferrous sulfate at pH 2.0; ferrous sulfate had no effect on the stability of MMF and MPA at pH 7.0. Overall, the partitioning of MMF and MPA was unaffected by the addition of ferrous sulfate. The solubility and partitioning of MMF from CellCept® capsules combined with Fero-Gradumet® (ferrous sulfate) tablets showed a twofold increase in aqueous solubility of MMF as well as increased concentration of MMF in both the n-octanol and aqueous phases, leading to a decrease in the octanol/water partition coefficient due to a reduction in pH of the aqueous phase. Based on these results, it was concluded that the physicochemical properties of MMF and MPA were generally not affected by the presence of ferrous sulfate. Further, the presence of ferrous sulfate did not suggest the formation of an MMF–iron complex.

Development of an emulsion-based muramyl dipeptide adjuvant formulation for vaccines.

This chapter contains a summary of the development of a very effective adjuvant that contains a muramyl dipeptide (MDP) analogue (threonyl-MDP, temurtide) in an oil-inwater emulsion vehicle. The oil-in-water emulsion system contains squalane, Pluronic® LI21, and polysorbate 80 in an isotonic, pH 7.4, phosphate-buffered saline solution. This adjuvant elicits both cell-mediated and humoral immune responses. While threonyl-MDP serves to increase antibody production and cell-mediated responses, the emulsion vehicle enhances immunogenicity by facilitating presentation of antigens to responding lymphocytes. Because threonyl-MDP does not exhibit toxicity usually associated with alanyl-MDP (pyrogenicity, uveitis, adjuvant-induced arthritis), no safety concerns are anticipated at therapeutic doses. In several animal species, this vehicle proved safe and efficacious, having been used successfully with a variety of antigens.

In Vitro Rabbit Corneal Permeability Study of Ketorolac, Tromethamine, a non-Steroidal Anti-Inflammatory Agent

AbstractIn vitro rabbit corneal studies utilizing ketorolac tromethamine were designed to 1) determine its permeability at different pH levels; 2) study the effects of two commonly used ophthalmic preservatives on corneal permeability; and 3) study the corneal penetration effects through ion pairing of the anionic ketorolac with positively charged counter ions. The results indicate that ketorolac shows a pH dependent penetration; it also exhibits penetration in both the unionized as well as ionized form. Results of the preservative evaluation shows that benzalkonium chloride enhances penetration while Thimerosal has no effect. Evaluation of the counter ions paired with ketorolac gave penetration rates in the following descending, relative, order: quaternary ammonium compounds > guanidino compounds > aliphatic amines > alkali metal.

Conductivity measurement as a convenient technique for determination of liposome capture volume

Abstract The reduction in conductivity seen between a buffer solution and a liposome preparation in that buffer was evaluated as a means of measuring liposome capture volume. Using DOPC and DOPG lipid to form negatively charged liposomes, conductivity measurements showed that conductivity of the liposome dispersion decreased as lipid concentrations of liposome preparations increased. Independent measurement of capture volumes by gel filtration chromatography showed that conductivity changes correlated with a liposome concentration dependent increase in capture volume. It is proposed that ions from the hydrating/suspending buffer normally contributing to conductivity were trapped within liposomes upon vesicle formation. These internalized and therefore shielded ions were not able to effectively contribute to conductivity of the liposome dispersion. For multilamellar vesicles (MLVs), capture volume was determined by reduction in conductivity over a large lipid concentration range and a broad buffer ionic strength range. Capture volume could also be determined for small unilamellar vesicles (SUVs). However, the greater number of exposed phospholipid head groups in high surface area SUVs contributed to conductivity of the liposome dispersion thereby limiting range of utility. A much higher ionic strength buffer (relative to MLVs) was required before conductivity of phospholipid no longer influenced conductivity of the dispersion. To expand this study, multilamellar vesicles having either neutral (DOPC) or positive (DOPC/stearylamine) charge were evaluated. Similar correlations were found between reduction in conductivity and mannitol entrapment (capture volume). These studies have confirmed that measurement of reduction in conductivity provides an easy and convenient method for determining liposome capture volume.

Corneal permeability of ketorolac tromethamine when formulated with tobramycin

AbstractIn vitro rabbit corneal penetration studies were designed to determine the effect tobramycin (an antibiotic) has on the diffusion of ketorolac tromethamine (a nonsteroidal anti-inflammatory compound). Evaluation was performed in two vehicle solutions: (1) a simple sodium chloride vehicle and (11) a suitable ophthalmic formulation. Quantitation of both ketorolac tromethamine and tobramycin were performed to determine the corneal penetration of each drug. Tobramycin was found to penetrate rabbit cornea to a limited extent. Also, tobramycin proved neither to impede nor enhance ketorolacs corneal diffusion. Both compounds showed greater penetration in an ophthalmic formulation, presumably due to the effects of the preservative, benzalkonium chloride (a quaternary ammonium compound)—known for disrupting corneal integrity.