Michael F. Powell

A compendium of vaccine adjuvants and excipients.

In the early 20th century, researchers experimented with diverse compounds such as alum (aluminum salts), mineral oil, and killed mycobacteria to improve the immunogenicity of vaccines. These first empirical studies demonstrated the adjuvant activity of many substances, but several products also elicited significant local and systemic adverse reactions that precluded their use in human vaccine formulations. Alum adjuvant, first described in 1926, remains the only immunologic adjuvant used in human vaccines licensed in the United States.

QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immmunization in humans

Three separate studies were undertaken in HIV-1 uninfected persons to determine if the adjuvant QS-21 improves the magnitude or kinetics of immune responses induced by recombinant soluble gp120 HIV-1(MN) protein (rsgp120) immunization. The QS-21 was administered at two doses (50 and 100 microg), either alone or in combination with aluminum hydroxide (600 microg). At the highest doses of rsgp120 (100, 300, and 600 microg), QS-21 exerted no significant effect on either binding or neutralizing antibody titers. Antibody binding and neutralizing responses fell dramatically when rsgp120, formulated with alum alone, was given at low doses (3 and 30 microg). In contrast, antibody responses similar in titer to those in the high dose antigen groups were induced with the low dose rsgp120 formulated with QS-21. In addition, the lymphocyte proliferation and delayed type hypersensitivity skin testing were superior in the QS-21 recipients compared with the alum recipients at the low antigen doses. Moderate to severe pain was observed in majority of the volunteers receiving QS-21 formulations, and vasovagal episodes and hypertension were not infrequent. Thus, the use of QS-21 may provide a means to reduce the dose of a soluble protein immunogen.

Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy

The saponin QS-21, derived from the bark of the Quillaja saponaria Molina tree, has shown great potential as an adjuvant with a number of vaccines. Kinetic studies carried out to establish the stability of vaccine formulations show that commercially supplied QS-21 (primarily QS-21A) is converted slowly at pH 5.5, and rapidly at higher pH, to an equilibrium mixture of two regioisomers, QS-21A and QS-21B, in a ratio of 20:1. NMR studies show that QS-21A and QS-21B differ only in the point of attachment of the fatty acyl moiety to the fucose sugar ring. The major isomer, QS-21A, has the fatty acyl portion attached at the 4-hydroxyl group whereas the minor isomer, QS-21B, has the fatty acyl portion attached at the 3-hydroxyl group. The isomerization most likely involves ionization of the 3-hydroxy group and intramolecular acyl transfer from the 4-hydroxy group. The relative stereochemistry of the triterpene and the sugar anomeric centers is also established by NMR methods.

Development of a single-shot subunit vaccine for HIV-1: Part 4. Optimizing microencapsulation and pulsatile release of MN rgp120 from biodegradable microspheres

The successful development of an AIDS vaccine will require formulations that not only invoke the desired immunological response, but also are stable and easy to administer. A single shot MN rgp120 vaccine formulation comprised of MN rgp120 encapsulated in poly (lactic-coglycolic) acid (PLGA) microspheres was developed to provide an in vivo autoboost of antigen. These formulations were designed to yield an in vivo autoboost at 1, 2, 3 or 4-6 months. In addition, PLGA microspheres containing the adjuvant, QS21, were also prepared to provide an in vivo autoboost concomitant with antigen. In guinea pigs, these formulations yielded higher anti-MN rgp120 and anti-V3 loop antibody titers than alum formulations that were administered at higher antigen doses. Different doses of encapsulated MN rgp120 provided a clear and well-defined dose response curve for both anti-MN rgp120 and anti-V3 loop antibody titers. When soluble QS21 was mixed with the encapsulated MN rgp120, the antibody titers were increased by a factor of 5 over the titers with encapsulated MN rgp120 alone. An additional fivefold increase in antibody titers was observed for guinea pigs immunized with encapsulated MN rgp120 and QS21 on the same microspheres. These results suggest that the adjuvant properties of QS21 can be increased by microencapsulation in PLGA. Furthermore, antibodies induced by these preparations neutralized the MN strain of HIV-1. The neutralization titers for sera from animals immunized with MN rgp120-PLGA and soluble QS21 were greater than the titers obtained from guinea pigs that were treated with MN rgp120 and soluble QS21 at the same dose. Overall, these studies validate the in vivo autoboost concept, reveal a method for improving the adjuvant properties of QS21, and indicate the potential of future single shot vaccine formulations.

Stability of Lidocaine in Aqueous Solution: Effect of Temperature, pH, Buffer, and Metal Ions on Amide Hydrolysis

The degradation of lidocaine in aqueous solution obeys the expression kobs = (kH+[H +] + ko ) [H+]/([H + ] + Ka + k′oKa([H + ] + Ka) where kH+ is the rate constant for hydronium ion catalysis, and ko and k′o are the rate constants for the spontaneous (or water-catalyzed) reactions of protonated and free-base lidocaine. At 80°C, the rate constants for these processes are 1.31 × 10−7M−l sec−1, 1.37 × 10−9 sec−1, and 7.02 × 10−9sec−1; the corresponding activation energies are 30.5, 33.8, and 26.3 kcal mol−1, respectively. It was found that the room temperature pH of maximum stability is ∼3–6 and that lidocaine is more reactive in the presence of metal ions such as Fe2+ and Cu2+. The dissociation constant, Ka, for lidocaine at 25–80°C was also measured at 0.1 M ionic strength and a plot of pKa versus 1/T gave a slope of (1.88 ± 0.05) × 103 K−1 and intercept 1.56 ± 0.16.

Parenteral Peptide Formulations: Chemical and Physical Properties of Native Luteinizing Hormone-Releasing Hormone (LHRH) and Hydrophobic Analogues in Aqueous Solution

The degradation of native LHRH in aqueous buffers of pH ∼1–10 obeyed the rate equation, kobs = kH+aH+ + ko + kHO-(aHO-)x, where x at 60–100°C was ∼0.64 and temperature independent. Extrapolation to 25°C using the Arrhenius equation and secondary rate constants showed that native LHRH is reasonably stable at pH 5.4, giving a shelf life (t90) of approximately 5 years. Regarding physical properties, hydrophobic LHRH analogues nafarelin and detirelix were found to be surface active as demonstrated by a decrease in apparent surface tension with increased peptide concentration. The CMC for detirelix at pH 7.4 was determined to be 5.3 × 10−4M (0.88 mg/ml), and that for nafarelin, >2 mg/ml. At higher concentrations (∼4–8 mg/ml), nafarelin and detirelix formed nematic liquid crystals of undulose extinction (birefringence, <0.001). The thermo-dynamic stability of these peptide liquid crystals was probed by determining their melting points (Tcm) in the presence of propylene glycol, a solvent which proved to be efficacious at suppressing gelation and at destabilizing liquid crystals as measured by a reduction in Tcm.

Immunological and Formulation Design Considerations for Subunit Vaccines

Vaccination represents the most successful attempt yet to protect humans and domestic animals from infectious diseases. Most vaccines operate by limiting infections, not necessarily by preventing them; it is the host immune system that mediates control and ultimate clearance of the infectious agent. Many of the most widely used vaccines developed for use in humans are based on live replicating organisms that have been attenuated. Vaccination with these agents results in a limited infection, but measurable disease pathogenesis is usually avoided and recovery is complete. The resultant immune responses are protective against the fully virulent pathogens and long-term immunological memory is induced. Today, attenuated vaccines are still used and efforts to develop the next generation are active.

Characterization of the MN gp120 HIV-1 Vaccine: Antigen Binding to Alum

AbstractPurpose. The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. Methods. Several observations suggest that the gpl20-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. Results. Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. Conclusions. These observations indicate that both the antigen and the adjuvant require optimization together.

Peptide Liquid Crystals: Inverse Correlation of Kinetic Formation and Thermodynamic Stability in Aqueous Solution

Luteinizing Hormone Releasing Hormone (LHRH) is a highly potent decapeptide that stimulates the release of luteinizing hormone and follicle stimulating hormone. Although it was initially thought that the LHRH agonists would be useful for the treatment of infertility, continuous administration of agonist resulted in a parodoxical desensitization of pituitary gonadotropes by receptor down regulation, causing suppression of fertility. 1 These suppressive effects, however, may be very useful for the treatment of other diseases, including endometriosis, breast cancer, precocious puberty and prostatic cancer

Chapter 30. Peptide Stability in Drug Development: in vitro Peptide Degradation in Plasma and Serum

Publisher Summary This chapter discusses chemical and physical stability of pharmaceuticals, the effect of formulation stabilizers on peptide stability and protein/peptide degradation pathways. Analytical methods to study polypeptide degradation and peptide adsorption are reviewed. Probably the greatest problem in using peptides as drugs is the nature of peptides themselves since there exist a whole class of molecules bent on peptide destruction––the peptidases. Even if efficient peptide delivery is achieved, many peptides do not circulate in blood for more than a few minutes due to enzymatic degradation, thus preventing their usefulness as therapeutic agents. It is critical in peptide drug development to determine which peptide candidates are enzymatically stable. The methodology for measuring peptide biostability––the in vitro degradation of peptides in serum and plasma is presented. A stability-specific assay method (one that is able to differentiate between the substrate and its degradation products) is also required to determine peptide stability.