Todd Talarico

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock.

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.

A Novel Alphavirus Vaccine Encoding Prostate-Specific Membrane Antigen Elicits Potent Cellular and Humoral Immune Responses

Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). Experimental Design: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-γ enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice–compliant toxicology study was conducted in rabbits. Results: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 × 105 infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 102 infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. Conclusions: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was developed in the 1980s as an oxygen carrier and is now under development for treatment of nitric oxide-dependent, volume refractory shock. PHP is made by derivatizing human stroma-free hemoglobin with pyridoxal-5-phosphate and polyoxyethylene (POE). A unique aspect of using POE for modification is that unlike its mono-methoxy polyethylene glycol (PEG) relatives, POE is bifunctional. The result of derivatization of stroma-free hemoglobin is a complex mixture of modified hemoglobin and other red cell proteins. The molecular weight profile, based on size exclusion chromatography, is bimodal and has a number average molecular weight of approximately 105¿ omitted¿000 and a weight average molecular weight of approximately 187¿ omitted¿000. The mixture of hemoglobin molecules has on average 3.3 pyridoxal and 5.0 polyoxyethylene units per tetramer. A portion of the tetramers are linked by POE crosslinks. The hemoglobin tetramers retain their ability to dissociate into dimer pairs and only a small percentage of the dimer pairs are not modified with POE. The SDS-PAGE profile exhibits the ladder-like appearance commonly associated with polyethylene glycol-modified proteins. The isoelectric focusing profile is broad, demonstrating a pI range of 5.0-6.5. The hydrodynamic size of PHP was determined to be approximately 7.2 nm by dynamic light scattering. Soluble red blood cell proteins, such as catalase, superoxide dismutase, and carbonic anhydrase, are present in PHP and are also modified by POE.

Inter-molecular degradation of signal peptidase I in vitro.

Highly purified preparations of signal peptidase I (36 kDa) were found to undergo an apparent inter-autocatalytic degradation at 4 degrees C and 37 degrees C. The disappearance of the 36 kDa protein coincided with the stable appearance of a 31 kDa and a 5 kDa species. Amino-terminal sequencing of the 31 kDa product indicated a site specific cleavage following Ala38-Gln-Ala of signal peptidase I. The 31 kDa fragment was purified and shown to have 100-fold less activity than the native enzyme, with pre-maltose binding protein as a substrate.

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

Background The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. Methodology/Principal Findings The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. Conclusions/Significance We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

Detection of residual polyethylene glycol derivatives in pyridoxylated-hemoglobin-polyoxyethylene conjugate

Purified hemoglobin solutions have been shown to cause renal toxicity in animals. Safe use of hemoglobin based therapeutics in humans requires modification of the hemoglobin molecule to prevent this toxicity. Hemoglobin modification may be accomplished by crosslinking the dimers within the hemoglobin tetramer or by derivatization of the alpha and/or beta subunits such that their size and/or charge prevents filtration by the glomeruli. Pyridoxylated hemoglobin polyoxyethylene conjugate (PHP) consists of hemoglobin molecules modified with alpha-carboxymethyl, omega-carboxymethoxy polyoxyethylene (POE). We have developed a high performance liquid chromatography-based (HPLC) method which can quantitate residual POE at levels of 0.1 mg/ml or greater. The detection of POE at this level of sensitivity requires the use of an evaporative light scattering detector (ELSD). A differential refractometer may also be used for POE detection, however the limit of quantitation for this detector is approximately 10 fold greater than that observed for the evaporative light scattering detector, resulting in a reduction in sensitivity. The successful use of this method requires sample deproteination using trichloroacetic acid. The reliability of the method has been demonstrated by spike recovery, precision, and reproducibility studies in PHP and buffer solutions.