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In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

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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.

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Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2',7'-dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for the these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U-87,663, and the bulk of the ROS prevented detection of any protection of U-87,663 may offer.

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Few inhibitors of the RNase H function associated 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 form 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 (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.

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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.

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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.

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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.

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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.

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The design and fabrication of large-area, high-efficiency metallic gratings for use in high-power laser systems is described. The gratings exhibit a diffraction efficiency in excess of 95% in the m = -1 order (Littrow mount) and have a high threshold for laser damage. Computations and experimental measurements are presented that illustrate the effect of grating shape and polarization on efficiency. A simple theory for optical damage to metallic diffraction gratings is developed and compared with experimental measurements of the laser-damage threshold over the pulse range from 400 fs to >1 ns.

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We examined the effects of 6-hydroxydopamine (6-OHDA) treatment on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the rat pheochromocytoma cell line, PC12. Structural and metabolic integrity was tested by measuring the ability of cells to transport the non-metabolizable amino acid analogue [3H]-alpha-aminoisobutyric acid (AIB). We determined that treatment with 6-OHDA at concentrations of 49 microM and 62 microM inhibited 50% of the AIB uptake in SY5Y and PC12 cells, respectively. Inhibition of AIB uptake was prevented by the addition of catalase, but was not influenced by the addition of 1 mM dopamine. This indicated that cell damage resulted from the generation of H2O2 and was independent of the catecholamine uptake system. Effects directly on the catecholamine uptake system were observed by measuring the uptake of 3H-dopamine. In contrast to the effects on amino acid uptake, dopamine uptake was significantly inhibited by 6-OHDA treatment, and this inhibition was not prevented by the addition of catalase. The results indicate a Ki of 430 microM for inhibition of dopamine uptake by 6-OHDA treatment of PC12 cells. The results are consistent with a competitive irreversible inhibition of the dopamine uptake sites by 6-OHDA or one of its metabolites. Thus, the lack of a catecholamine uptake-dependent cellular toxicity appears to result from the direct inactivation of catecholamine uptake sites. Similarly, the inhibition of dopamine uptake in vivo by 6-OHDA may be explained, at least in part, by direct inactivation of dopamine uptake sites rather than exclusively by intracellular transport and action of 6-OHDA.

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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.

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In an effort to determine if PC12 cells have functional dopamine autoreceptors we found that carbachol-stimulated release of dopamine from undifferentiated PC12 cells was inhibited by the dopamine autoreceptor agonists apomorphine and U-68553B. Studies were conducted to determine the mechanism of this effect. The inhibition of dopamine release by apomorphine or U-68553B did not appear to result from effects on dopamine metabolism. When cells were exposed to the dopamine agonists for 2 minutes no changes in dopa, DOPAC or dopamine were observed. Over this same time interval, apomorphine and U68553B at 10 microM inhibited carbachol-stimulated dopamine release by 45.6% and 57.4% respectively. However, these drugs failed to inhibit the potassium dependent release of dopamine from cells suggesting no direct involvement with ion fluxes. Furthermore, haloperidol did not block the inhibitory effects of U-68553B on PC12 cells. This would appear to preclude activation of a dopamine autoreceptor as a possible mechanism. Kinetic analysis revealed that U-68553B is most likely a non-competitive inhibitor (Ki = 2 microM) of the nicotinic acetylcholine receptor. These data do not provide evidence for functional dopamine autoreceptors on undifferentiated PC12 cells but rather indicate that dopamine agonists may alter dopamine release by alternative mechanisms.

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Studies were performed to elucidate the ontogeny of a single class of androgen binding protein in male rat liver cytosol which exhibits characteristics of a ligand specific, high affinity (Kd = 2.3 nM), 8S-receptor capable of nuclear translocation. Detectable levels of receptor first appear at 45 days of age in the male and reach maximum concentration at 65 days. Barely detectable levels are seen in females throughout the duration of study (80 days). Gonadectomy in both sexes (65 days) and androgen treatment of oophorectomized females do not alter the normal development of sexual differentiation of the high affinity androgen receptor. After neonatal castration (2 days) and DES replacement however, receptor sites do not undergo differentiation and adult males exhibit female levels. Conversely, neonatal androgen replacement in 2-day castrates partially restores the level of binding sites to control males values (TP, 71%; DHT, 51%). Neonatal castration without replacement retards but does not fully eliminate sexual differentiation of levels of receptor sites in adult males. Likewise, neonatal androgen treatment in females results in a partial masculinization of binding sites. Following hypophysectomy, levels of receptor sites in females are similar to intact or hypophysectomized males; sexual differences in the adult are abolished. These studies suggest that sexual differentiation of specific liver cytosol androgen binding sites in the adult may be partially programmed at birth by testicular androgen and furthermore, adult sexual dimorphism is maintained through an inhibitory influence of the pituitary in the female.

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The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.

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To further delineate the mechanism responsible for the differences in xanthine oxidase activity in male and female Sprague-Dawley rats, a sensitive and specific radioimmunoassay (RIA) was developed for the measurement of hepatic xanthine oxidase. The RIA could detect as little as 5 mg of liver enzyme. Specificity of the RIA was confirmed by 1) Ouchterlony double immuno-diffusion in which a single precipitin band exhibited xanthine oxidase activity, when crude liver homogenate and an enzyme-specific stain were used; 2) parallelism between purified 125I-labeled xanthine oxidase and serial dilutions of crude liver homogenate; 3) a linear correlation between xanthine oxidase activity and the level of enzyme protein; and 4) a single protein band coincident with purified xanthine oxidase, when an immunoprecipitate prepared from antisera and crude liver homogenate was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Whether xanthine oxidase activity was assayed in the absence of nicotinamide adenine dinucleotide (NAD+) (oxidase form) or in the presence of NAD+ (dehydrogenase), male values were consistently higher, and both forms of the enzyme correlated significantly with each other. When purified to homogeneity, neither form of the enzyme was appreciably affected by 17 beta-estradiol or testosterone propionate. When the RIA was employed, levels of hepatic xanthine oxidase were significantly greater in male than in female rats. We concluded from these data that increased xanthine oxidase activity in the male corresponds to a greater quantitative complement of xanthine oxidase protein. Furthermore, lower xanthine oxidase activity in the female cannot be explained by immunologically cross-reactive material without enzyme activity nor by a direct sex-steroid enzyme interaction.

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The uricosuric response to 80 mg of micronized Benzbromarone was employed to assess the renal tubular secretory site for uric acid in patients with primary gout. Since Benzbromarone selectively inhibits tubular reabsorption of secreted urate, the maximum uricosuria induced by this drug can be equated with the minimal secretory rate. Furthermore, a significant relationship was noted in normal controls between urate secretion and the plasma urate concentration (r = 0.956, p less than 0.005). Using the Benzbromarone response as a measure of tubular secretion, gouty patients with normal production hyperuricemia had a significantly lower secretory rate by comparison to patients with overproduction of uric acid. These data indicate that in patients with primary normal production hyperuricemia, the renal tubular defect is related to a decreased secretory response for a given plasma concentration of uric acid.

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The cardiovascular responses to cold were studied in a group of 28 subjects who enjoyed swimming in ice-cold water in winter ("ice-bears"). History and clinical examination had revealed no abnormalities except hypertension (180/105 mm Hg) in 1, while 3 other subjects had a diastolic value of 95 mm Hg. Systolic blood-pressure increased significantly while the subjects were waiting undressed in cold air in the cabin by the pond. Neither immersion nor swimming in the ice-cold water caused further increase in systolic blood-pressure, and diastolic blood-pressure showed only a modest rise. 4 min later, blood-pressure had returned to control values. Electro and vector cardiographic signs remained unchanged. Although very high pressures were recorded in several subjects, no signs of left ventricular hypertrophy or of cardiovascular of cerebrovascular damage could be detected.

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