Douglas Decker

Use of a Biophysical-Kinetic Model to Understand the Roles of Protein Binding and Membrane Partitioning on Passive Diffusion of Highly Lipophilic Molecules Across Cellular Barriers

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

Localization of damage induced by reactive oxygen species in cultured cells

N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single-hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process.

Efficacy of lipid soluble, membrane-protective agents against hydrogen peroxide cytotoxicity in cardiac myocytes

We examined the efficacy of a group of drugs that stabilize the cell membrane and can potentially prevent cytotoxicity in cultured fetal chick cardiac myocytes exposed to hydrogen peroxide (H2O2). The effects of various membrane-protective agents were determined by analysis of the kinetics of lactic dehydrogenase (LDH) release. The kinetic parameters calculated from the data include a rate constant for release of LDH (kb) and the fraction of total LDH that is released from the cells (CIIMax). The CIIMaxs derived from a range of H2O2 concentrations reveal that the mean toxic concentration of H2O2 is 1.1 mM and that the pattern of toxicity is consistent with the damage being directly proportional to the concentration of the free radicals generated from the H2O2. Maximum nontoxic concentrations of three amphiphilic membrane protective agents had no effect upon cytotoxicity from H2O2. The slightly polar lipophilic agent, Trolox C, a vitamin E derivative, was also without protective effect at a maximum nontoxic concentration. The highly lipophilic agent, probucol, had a small protective effect at 50 microM, the maximum concentration we succeeded in solubilizing in the culture medium. However, the lipophilic 21-aminosteroid U74500, delivered to the cells in an emulsion, markedly reduced cytotoxicity from H2O2. The CII Max was significantly reduced and the protection was concentration dependent over a range of concentrations from 50-400 nmol/ml. Furthermore, the inhibition by U74500 was fully consistent with a mechanism of scavenging of free radicals formed during lipid peroxidation. In support of this hypothesis, a dose of 400 nmoles/ml completely prevented an increase in lipid peroxides due to H2O2 exposure, whereas there was a sixfold increase during exposure to H2O2 in untreated myocytes. Thus, a lipid soluble 21-aminosteroid prevented lipid peroxidation and reduced cardiac myocyte injury during exposure to H2O2, probably by scavenging of free radicals formed during lipid peroxidation in the cell membrane, whereas amphiphilic agents, which probably altered the physicochemical structure of the cell membrane but did not scavenge free radicals, were not protective.

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus‐infected Spodoptera frugiperda (SF‐9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single‐step affinity chromatography procedure using a high‐affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF‐9 cells coded for pro‐NGF, the NGF recovered after purification was > 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3‐day primary response and a 1‐day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub‐maxillary glands.

Kinetics and thermodynamics of emulsion delivery of lipophilic antioxidants to cells in culture

Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

THE AMPHIPHILIC PROPERTIES OF NOVENAMINES DETERMINE THEIR ACTIVITY AS INHIBITORS OF HIV-1 RNASE H

Few inhibitors of the RNase H function associatesd with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U-34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules from stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (∼pH5) the three componds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than theKd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd=10−9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.

Inhibition of oxidative insult in cultured cells by a novel 6-chromanol-containing antioxidant

N18-RE-105 neuronal hybridoma cells were used in a cell culture system to evaluate the protective effects of a novel 6-chromanol-containing antioxidant, U78517F. First, the incorporation of the compound into the cells was evaluated, using a serum albumin carrier. Then the cells were exposed to peroxide-generating compounds, and the cell injury was estimated from the loss of alpha-aminoisobutyric acid (AIB) transport. We found that U78517F only protected the cells significantly when the degree of oxidative insult was below a certain limit; the measurable protection of cells by U78517F against either cumene hydroperoxide or H2O2 was limited to a narrow range of concentrations of the reactive oxygen species generator. Additionally, the protection provided by U78517F was largely localized to the cell membrane and did not extend to protection of mitochondrial function. The action of U78517 was fully consistent with a direct radical scavenging in the cells. The results indicate that the following factors must be taken into account for evaluation of antioxidants in cell culture: (a) the delivery of a compound to cells, especially when the compound is lipophilic; (b) the nature and extent of the oxidative insult used to evaluate protection; and (c) the location of the protective agent in the cells.