David R. Cassatt

Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen

The development of peptide-based vaccines that elicit antibody (Ab) and cellular immune responses has been hampered by the lack of highly immunogenic formulations. In this study, we compared the induction of Ab and cytotoxic T-lymphocyte (CTL) responses to a peptide derived from the V3 loop of HIV-1 gp120 (P18 and its cysteine-glycine derivative (CG-P18)) when incorporated into liposomes with lipid A (LA) or mixed with aluminum hydroxide. P18-specific CTL were only observed with liposomes with LA. P18-specific Ab responses were found with liposomes containing CG-P18 but not P18. Increased surface expression of the former, resulted in enhancement of the Ab response without loss of CTL induction. Thus, the manner in which a peptide is localized can influence the outcome of the response induced by highly immunogenic liposome formulations.

Subcutaneous administration of amifostine (ethyol) is equivalent to intravenous administration in a rat mucositis model

PURPOSE Amifostine (Ethyol) is currently approved for intravenous (IV) administration to prevent xerostomia in patients receiving radiotherapy for head-and-neck cancer. Recently, subcutaneous (SC) administration has been explored as an alternative route. To determine whether SC administration was equivalent to IV administration, we used models to follow pharmacokinetics and oral mucosal protection in rats. METHODS Amifostine was administered to rats at doses of 200, 100, or 50 mg/kg (1300, 650, or 325 mg/m(2)) IV or SC at various times before radiation at 15.3 Gy (protection studies) or harvest of blood and tissues for analysis by HPLC (pharmacokinetic studies). RESULTS Amifostine administered IV or SC 1 h before radiation protected rats from mucositis, but the protective effect was more prolonged when amifostine was administered SC. Tissue levels of the active metabolite (WR-1065) were equivalent after SC administration. The correlation between tissue levels of WR-1065 and protection was strong, but that between blood levels of WR-1065 and protection was only weak. CONCLUSION These data demonstrate that, in a rat model, SC administration of amifostine was at least as effective as that by IV.

Evidence for Vaccine Synergy between Borrelia burgdorferi Decorin Binding Protein A and Outer Surface Protein A in the Mouse Model of Lyme Borreliosis

ABSTRACT Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected againstBorrelia burgdorferi challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous B. burgdorferi, B. garinii, andB. afzelii isolates, suggesting that there is synergy between these two immunogens.

Preclinical studies on the radioprotective efficacy and pharmacokinetics of subcutaneously administered amifostine

The radioprotective effects and pharmacokinetics of subcutaneously (SC) administered amifostine have been investigated in animal studies. Studies in rats using a single dose of amifostine showed that SC administration gave protection from radiation-induced mucositis that is at least equivalent to that achieved by intravenous administration of the drug. These studies also indicate that tissue levels of the active metabolite WR-1065 correlated better with the radioprotective effects of amifostine than do plasma WR-1065 levels. Multiple-dose studies in rats show radioprotective effects equal to or greater than those obtained with intravenous dosing in the setting of fractionated irradiation. In addition, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. Preliminary data from studies of SC amifostine in monkeys indicate a plasma pharmacokinetic profile similar to that reported earlier in humans. Tissue WR-1065 levels were higher at 30 minutes after SC dosing than they were after intravenous dosing and were comparable for the two routes at 60 minutes.

Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

ABSTRACT Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.

Tissue Levels of WR-1065, the Active Metabolite of Amifostine (Ethyol®), Are Equivalent following Intravenous or Subcutaneous Administration in Cynomolgus Monkeys

Amifostine (Ethyol®) is a cytoprotective drug approved for the reduction of xerostomia in head and neck cancer when administered to patients receiving postoperative radiation therapy. Although amifostine is approved for intravenous infusion, the off-label subcutaneous route of administration has become more prevalent. Although human patient data indicate higher plasma bioavailability of the active metabolite (WR-1065) following intravenous compared to subcutaneous administration, there are no corresponding data showing human tissue levels of WR-1065 following either route of administration due to the difficulty in obtaining human specimens. In our study we compared plasma and tissue pharmacokinetics of WR-1065 in primates following both routes of administration. Monkeys received amifostine at a dose of 260 mg/m2 either intravenously or subcutaneously. Plasma samples were analyzed for total WR-1065 by reverse-phase high-pressure liquid chromatography (HPLC) and fluorescence detection up to 4 h after amifostine administration. Tissues were analyzed for free WR-1065 by reverse-phase HPLC and electrochemical detection 30 and 60 min after administration. Following intravenous administration, plasma WR-1065 levels peaked rapidly and showed a bi-exponential decline, while following subcutaneous administration WR-1065 levels rose slowly and declined exponentially. The relative plasma bioavailability of WR-1065 given subcutaneously was lower at 30 and 60 min. Interestingly, after 30 min, tissues showed equal or slightly greater concentrations of WR-1065 following subcutaneous administration. Levels following 60 min were comparable following both routes. The plasma bioavailability studies performed in primates confirm human plasma data. Expanding the study to evaluate primate tissue levels of WR-1065 revealed that despite lower plasma bioavailability following subcutaneous administration, tissue levels of the active metabolite were surprisingly greater than or equal to those measured in animals that received the drug intravenously. These studies strengthen the argument for subcutaneous administration of amifostine in radiation oncology.