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Theodore W Randolph

Publications and source records attributed to Theodore W Randolph.

16 recordsLinked to original sources
PubMed2003

Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis.

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.

Amino Acid Substitution↗
PubMed2003

Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation.

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.

Amyloid↗
PubMed2003

Effects of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-gamma.

The purpose of this study was to examine the effects of adsorption of recombinant human interferon-gamma (rhIFN-gamma) on ice surfaces and subsequent drying during processing by spray-lyophilization and lyophilization. Ice/liquid interfacial areas were manipulated by the freezing method as well as by the addition of an annealing step during lyophilization; that is, rhIFN-gamma adsorption was modified by the addition of nonionic surfactants. rhIFN-gamma was lyophilized or spray-lyophilized at a concentration of 1 mg/mL in 5% sucrose, 5% hydroxyethyl starch (HES) +/- 0.03% polysorbate 20 in 140 mM KCl, and 10 mM potassium phosphate, pH 7.5. After the samples were frozen, half were annealed on the lyophilizer shelf. Recovery of soluble protein was measured at intermediate points during processing. On drying, the secondary structure of rhIFN-gamma was determined by second-derivative infrared (IR) spectroscopy, specific surface areas (SSAs) were measured, scanning electron micrographs (SEM) were taken, and dissolution times were recorded. Adsorption of rhIFN-gamma to ice/liquid interfaces alone was not responsible for aggregation. Rather, drying was necessary to cause aggregation in lyophilized sucrose formulations. Addition of an annealing step to the lyophilization cycle resulted in more native-like secondary protein structure in the dried solid, eliminated cracking of the dried cakes, and suppressed both the formation of air/liquid interfaces and rhIFN-gamma aggregation on reconstitution.

Adsorption↗
PubMed2003

Computer simulation of convective and diffusive transport of controlled-release drugs in the vitreous humor.

PURPOSE: Biodistribution of drugs in the eye is central to the efficacy of pharmaceutical ocular therapies. Of particular interest to us is the effect of intravitreal transport on distribution of controlled-released drugs within the vitreous. METHODS: A computer model was developed to describe the three-dimensional convective-diffusive transport of drug released from an intravitreal controlled release source. Unlike previous studies, this work includes flow of aqueous from the anterior to the posterior of the vitreous. The release profile was based on in vitro release of gentamicin from poly(L-lactic acid) microspheres into vitreous. RESULTS: For small drugs, convection plays a small role, but for large (slower diffusing) drugs, convection becomes more important. For the cases studied, the predicted ratio of drug reaching the retina to drug cleared by the aqueous humor was 2.4 for a small molecule but 13 for a large molecule. Transport in neonatal mouse eye, in contrast, was dominated by diffusion, and the ratio decreased to 0.39. CONCLUSIONS: The interaction among convection, diffusion, and geometry causes significant differences in biodistribution between large and small molecules or across species. These differences should be considered in the design of delivery strategies or animal studies.

Animals↗
PubMed2003

Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor.

We studied the non-native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (DeltaG(unf)) and osmotic second virial coefficient (B(22)), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large DeltaG(unf) and negative B(22)), the first step is rate-limiting, and increasing DeltaG(unf) (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B(22) values) the second step is rate-limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate-limiting.

Colloids↗
PubMed2003

Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins.

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native-state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native-state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far- and near-UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native-state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl-induced unfolding, increased in proportion to sucrose concentration. Native-state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange-resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large-scale than on small-scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native-state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.

Animals↗
PubMed2002

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.

Chemical Precipitation↗
PubMed2002

Kinetics and energetics of assembly, nucleation, and growth of aggregates and fibrils for an amyloidogenic protein. Insights into transition states from pressure, temperature, and co-solute studies.

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.

Amyloid↗
PubMed2002

High hydrostatic pressure as a tool to study protein aggregation and amyloidosis.

Aggregation of proteins is a serious problem, affecting both industrial production of proteins and human health. Despite recent advances in the theories and experimental techniques available to address understanding of protein aggregation processes, mechanisms of aggregate formation have proved challenging to study. This is in part because the typical irreversibility of protein aggregation processes at atmospheric conditions complicates analysis of their kinetics and thermodynamics. Because high hydrostatic pressures act to disfavor the hydrophobic and electrostatic interactions that cause protein aggregation, studies conducted under high hydrostatic pressures may allow protein aggregates to be formed reversibly, enabling thermodynamic and kinetic parameters to be measured in greater detail. Although application of high hydrostatic pressures to protein aggregation problems is rather recent, a growing literature, reviewed herein, suggests that high pressure may be a useful tool for both understanding protein aggregation and reversing it in industrial applications.

Amyloidosis↗
PubMed2002

A new mechanism for decreasing aggregation of recombinant human interferon-gamma by a surfactant: slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20.

To study the mechanisms by which Tween 20 (polysorbate 20) used in a reconstitution solution affects the aggregation of lyophilized recombinant human interferon-gamma (rhIFN-gamma), we used four types of buffered formulations containing 0.4-5 mg/mL rhIFN-gamma in either 10 mM potassium phosphate or phosphate buffered saline: (1) without excipients, (2) with 5% sucrose, (3) with 0.03% polysorbate 20, or (4) with the combination of 5% sucrose and 0.03% polysorbate 20. After lyophilization, infrared spectroscopy was used to analyze the secondary structure of the protein in the freeze-dried solid. Each solid showed structural perturbation of the protein. Each formulation was reconstituted with water or a 0.03% polysorbate 20 solution. Aggregation of rhIFN-gamma after reconstitution was measured by optical density at A(350), and recovery of soluble protein was determined by high-performance liquid chromatography and ultraviolet spectroscopy. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. These results revealed the potential for recovery of native protein using the appropriate reconstitution conditions, even though the protein is non-native in the lyophilized state. Urea-induced unfolding with and without polysorbate 20 as measured by second-derivative ultraviolet spectroscopy indicated that a concentration of 0.03% polysorbate 20 lowered the free energy of unfolding for rhIFN-gamma (destabilizing). Polysorbate 20 also retarded refolding from urea solutions and increased aggregation. At a level of 0.03%, polysorbate 20 did not protect the protein against surface-induced aggregation during agitation. Dissolution times in water versus a 0.03% polysorbate 20 solution were measured using a rotating disk electrode for lyophilized formulations containing an electrochemically reactive species. The presence of 0.03% polysorbate 20 in the reconstitution solution nearly doubled the time required for dissolution of the phosphate buffered saline formulation, and the sucrose formulations dissolved 33-57% more slowly. Slowing the dissolution rates of lyophilized powders allows more time for the protein to refold while it decreases the maximum concentration of the protein at the dissolution interface, thus reducing the total amount of aggregation.

Buffers↗
PubMed2002

Hydrophobic ion pairing of isoniazid using a prodrug approach.

Inhalation therapy for infectious lung diseases, such as tuberculosis, is currently being explored, with microspheres being used to target alveolar macrophages. One method of drug encapsulation into polymeric microspheres to form hydrophobic ion-paired (HIP) complexes, and then coprecipitate the complex and polymer using supercritical fluid methodology. For the potent antituberculosis drug, isoniazid (isonicotinic acid hydrazide, INH), to be used in this fashion, it was modified into an ionizable form suitable for HIP. The charged prodrug, sodium isoniazid methanesulfonate (Na-INHMS), was then ion paired with hydrophobic cations, such as alkyltrimethylammonium or tetraalkylammonium. The logarithms of the apparent partition coefficients (log P') of various HIP complexes of INHMS display a roughly linear relationship with the numbers of carbon atoms in the organic counterions. The water solubility of the tetraheptylammonium-INHMS complex is about 220-fold lower than that of Na-INHMS, while the solubility in dichloromethane exceeds 10 mg/mL, which is sufficient for microencapsulation of the drug into poly(lactide) microspheres. The actual logarithm of the dichloromethane/water partition coefficient (log P) for tetraheptylammonium-INHMS is 1.55, compared to a value of - 1.8 for the sodium salt of INHMS. The dissolution kinetics of the tetraheptylammonium-INHMS complex in 0.9% aqueous solutions of NaCl was also investigated. Dissolution of tetraheptylammonium-INHMS exhibited a first-order time constant of about 0.28 min(-1), followed by a slower reverse ion exchange process to form Na-INHMS. The half-life of this HIP complex is on the order of 30 min, making the enhanced transport of the drug across biological barriers possible. This work represents the first use of a prodrug approach to introduce functionality that would allow HIP complex formation for a neutral molecule.

Administration, Inhalation↗
PubMed2002

Surface adsorption of recombinant human interferon-gamma in lyophilized and spray-lyophilized formulations.

Recombinant human interferon-gamma (rhIFN-gamma) was lyophilized or spray-lyophilized in 9.5% trehalose, +/- 0.12% polysorbate 20 in 10 mM potassium phosphate, pH 7.5. We measured recovery of soluble protein after spraying, freeze-thawing, and drying and reconstitution. Infrared spectroscopy showed rhIFN-gamma secondary structure to be native-like in all dried powders. Powders were characterized using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and gas adsorption isotherms. rhIFN-gamma adsorbed at air/liquid interfaces during spraying, and to ice/liquid interfaces during lyophilization. The concentration of rhIFN-gamma at ice/liquid interfaces was approximately one-fourth that adsorbed at air/liquid interfaces. Addition of 0.12% polysorbate 20 reduced the concentration of rhIFN-gamma at both interfaces. Time-of-flight secondary ion mass spectroscopy detected polysorbate 20 on surfaces of lyophilized powders. Lyophilized samples dried more slowly but reconstituted more quickly than spray-lyophilized samples. rhIFN-gamma aggregated after nebulization, but aggregation decreased in 0.12% polysorbate 20. Addition of 0.12% polysorbate 20 reduced protein surface adsorption and decreased but did not completely prevent aggregation. Insignificant aggregation occurred after exposure to ice/liquid interfaces, but subsequent drying and reconstitution caused aggregation. The majority of the aggregation is due to adsorption at air-liquid and solid-air interfaces formed during spray-lyophilization or lyophilization.

Chemistry, Pharmaceutical↗
PubMed2002

Encapsulating DNA within biodegradable polymeric microparticles.

In order for genetic medicines to become viable commercial products, the active form of the drug (e.g., DNA) must be able to reach the site of action and remain there long enough to accomplish its intended function. Encapsulation of plasmid DNA into biodegradable microspheres is one approach towards solving this challenge. This review describes the primary methods for satisfactorily entrapping intact DNA into biodegradable polymeric matrices. In particular, the materials, processes, and equipment required for each encapsulation method are described in detail. The resulting microspheres could be used for parenteral, oral, and inhalation therapy.

Biodegradation, Environmental↗
PubMedDate not supplied

High-pressure refolding of disulfide-cross-linked lysozyme aggregates: thermodynamics and optimization.

Previous exploratory work revealed that high pressure (200 MPa), in combination with oxido-shuffling agents such as glutathione, effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules, at concentrations up to 2 mg/mL (1). To understand further and optimize this process, in the current study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH). Yields decreased dramatically at more oxidizing conditions ([GSSG] > [GSH]). Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions. At GSSG:GSH ratios of 4:1, 1:1, and 1:16, lysozyme dissolved and refolded with time constants of 62, 20, and 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.

Disulfides↗