Potentiometric determination of the thermodynamic ionization constants of deferoxamine.
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Deferoxamine↗
Biomedical subjects
D H Robinson
Publications and source records attributed to D H Robinson.
Pharmaceutical properties, biodistribution, and imaging characteristics of manganese-mesoporphyrin. A potential hepatobiliary contrast agent for magnetic resonance imaging.
OBJECTIVES: Manganese (III) mesoporphyrin (Mn-mesoporphyrin) was investigated for its pharmaceutical properties and magnetic resonance imaging characteristics as a potential hepatobiliary contrast agent. METHODS: Solubility, partition coefficient, plasma binding, proton relaxation enhancement, biodistribution, biliary excretion, liver extraction ratio, and liver enhancement were measured in various in-vitro and in-vivo systems. RESULTS: Mn-mesoporphyrin was soluble and stable at moderate alkaline pH in phosphate buffer. The octanol/water coefficient was 25.98, and the compound was highly protein bound. R1 for water and plasma were 1.94 and 2.35 L/mmol sec, respectively. R1 in liver was calculated to be 15.72 L/mmol sec. Biodistribution studies in rats and mice confirmed hepatotrophic properties and biliary excretion was 65% over 24 hours. First pass liver uptake was 15%. Magnetic resonance imaging studies showed persistent liver enhancement at 0.05 mmol/kg. CONCLUSION: Mn-mesoporphyrin is a lipophilic compound that shows potential as a hepatobiliary magnetic resonance contrast agent.
Animals↗
Complement activation during saturation diving.
In this study, the levels of activated complement fragments C3a and C5a were measured on 11 U.S. Navy divers as they performed a 28-day saturation dive to a pressure equivalent of 1,000 feet of seawater (fsw, 31.3 atm abs). Two subjects developed symptoms consistent with the high pressure nervous syndrome (HPNS) and three were treated for type I DCS (joint pain only). These events allowed us to test two hypotheses: a) alterations in C3a or C5a levels during compression are related to the occurrence of HPNS and b) increases in complement fragments are an indicator of decompression stress associated with type I DCS. There was no correlation between changes in C3a and C5a levels during compression and the diagnosis of HPNS. Our results suggest that an increase in C3a and C5a levels during saturation diving correlates with decompression stress and the clinical diagnosis of type I DCS.
Analysis of Variance↗
The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy.
(R)-(2-dodecanamidoisohexyl)phosphocholine (DAHPC), labelled with 13C at the amide carbonyl group, has been synthesized and its binding to bovine pancreatic phospholipase A2 (PLA2) studied by n.m.r. and i.r. spectroscopy. Two-dimensional 1H-n.m.r. spectra show that, in the presence of Ca2+, DAHPC binds to the active site of the enzyme in a similar manner to other phospholipid amide substrate analogues. The environment of the labelled carbonyl group has been investigated by a combination of 13C n.m.r. and difference-Fourier-transform i.r. spectroscopy. The carbonyl resonance shifts 3 p.p.m. downfield on the binding of DAHPC to PLA2. The carbonyl absorption frequency decreases by 14-18 cm-1, accompanied by a marked sharpening of the absorption band. These results indicate that the carbonyl bond undergoes significant polarization in the enzyme-ligand complex, facilitated by the enzyme-bound Ca2+ ion. This suggests that ground-state strain is likely to promote catalysis in the case of substrate binding. Simple calculations, based on the i.r. data, indicate that the carbonyl bond is weakened by 5-9 kJ.mol-1. This is the first report of observation of the amide vibration of a bound ligand against the strong background of protein amide vibrations.
Animals↗
Design and synthesis of some substrate analogue inhibitors of phospholipase A2 and investigations by NMR and molecular modeling into the binding interactions in the enzyme-inhibitor complex.
A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.
Animals↗
Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties.
We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony-stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage-like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the "reticuloendothelial" phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.
Animals↗
Epithelial sodium conductance in rabbit preimplantation trophectodermal cells.
We examined the development of epithelial Na+ conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na+ channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na+ current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na+ with the impermeant cation N-methyl-D-glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na(+)-dependent. Treatment of 7-day p.c. cells with 100 microM amiloride, benzamil, or ethylisopropyl amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by amiloride. The amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an amiloride-free solution. The apparent dissociation constant (Ki) for amiloride, benzamil, and EIPA was 12, 50, and 16 microM, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high amiloride affinity Na+ channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c.(ABSTRACT TRUNCATED AT 250 WORDS)
Amiloride↗
Design, synthesis, and physicochemical properties of a novel, conformationally restricted 2,3-dihydro-1,3,4-thiadiazole-containing angiotensin converting enzyme inhibitor which is preferentially eliminated by the biliary route in rats.
Two novel series of dihydrothiadiazole ring containing inhibitors of angiotensin converting enzyme have been designed and synthesized. The compounds are highly potent enzyme inhibitors and, as a consequence of conformational restriction, chemically stable with respect to undesirable cyclization reactions. The most interesting compound from this series, 5a (FPL 63547), is the monoethyl ester prodrug of the highly potent "aminocarboxy" inhibitor 5b (FPL 63674). It produces an antihypertensive effect of long duration in animal models after oral dosing. Unlike other ACE inhibitors, 5b is eliminated almost entirely by biliary clearance in the rat. The favorable pharmacological properties of 5a and 5b are rationalized in terms of their unique physicochemical profiles. The clear preference for biliary clearance seen with 5b is consistent with its lipophilicity and its high degree of net ionization at physiological pH, which results from the very low pKa of the C-terminus carboxylic acid function. FPL 63547 is presently undergoing clinical investigation in man.
Angiotensin-Converting Enzyme Inhibitors↗
Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo.
The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.
Animals↗
Drug delivery systems.
New and emerging drug delivery systems for traditional drugs and the products of biotechnology are discussed, and the role of the pharmacist in ensuring the appropriate use of these systems is outlined. Advantages of advanced drug delivery systems over traditional systems are the ability to deliver a drug more selectively to a specific site; easier, more accurate, less frequent dosing; decreased variability in systemic drug concentrations; absorption that is more consistent with the site and mechanism of action; and reductions in toxic metabolites. Four basic strategies govern the mechanisms of advanced drug delivery: physical, chemical, biological, and mechanical. Oral drug delivery systems use natural and synthetic polymers to deliver the product to a specific region in the gastrointestinal tract in a timely manner that minimizes adverse effects and increases drug efficacy. Innovations in injectable and implantable delivery systems include emulsions, particulate delivery systems, micromolecular products and macromolecular drug adducts, and enzymatic-controlled delivery. Options for noninvasive drug delivery include the transdermal, respiratory, intranasal, ophthalmic, lymphatic, rectal, intravaginal, and intrauterine routes as well as topical application. Rapid growth is projected in the drug delivery systems market worldwide in the next five years. Genetic engineering has mandated the development of new strategies to deliver biotechnologically derived protein and peptide drugs and chemoimmunoconjugates. The role of the pharmacist in the era of advanced drug delivery systems will be broad based, including administering drugs, compounding, calculating dosages based on pharmacokinetic and pharmacodynamic monitoring, counseling, and research. The advent of advanced drug delivery systems offers pharmacists a new opportunity to assume an active role in patient care.
Administration, Oral↗
Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides.
New indirect spectrophotometric methods of analysis of tobramycin using the derivatizing agents o-phthaldialdehyde, fluorescamine, and dansyl chloride were developed and compared with existing methods. Optimum reaction conditions were established for the complex formation between tobramycin and each reagent. The sensitivity and reproducibility of these new procedures were compared with results obtained using published methods. Of the new methods, the o-phthaldialdehyde and fluorescamine procedures were more sensitive and reproducible than the dansyl chloride procedure. Further, these procedures were subsequently applied to the analysis of aqueous solutions of other aminoglycosides including amikacin, neomycin, gentamicin, and kanamycin.
Anti-Bacterial Agents↗
Morphologic plasticity and periodicity: porcine cerebral microvascular cells in culture.
Porcine cerebral microvascular (PCMV) endothelial cell cultures and pericyte-endothelial cell cocultures were established and the self-organizational properties of the cells were examined in various culture conditions. Cultured PCMV endothelial cells were characterized by the capacity to produce prostacyclin in response to bradykinin. Cultured PCMV pericytes were identified with a smooth muscle actin-specific stain. PCMV endothelial cells organized into cord structures when left in culture for several weeks without passage. Lumina were observed in cross sections of these cords and appeared to form through a process of cell-selective autolysis. PCMV endothelial cells required three dimensions for self-organization, forming suspended cords in planes that either intersected or paralleled the culture vessel floor. After formation, suspended cords continued to exhibit a morphologic plasticity punctuated by the coordinated migrations of PCMV endothelial cells en masse. Sequential propagation of PCMV endothelial cell monolayers and development of suspended capillarylike cords recurred cyclically when cells were left in culture without passage for several weeks. Cord development was also observed in PCMV pericyte-endothelial cell cocultures with large proportions of pericytes. However, pericytes were not located in cross sections of suspended cords formed in coculture. Apparently, in some conditions of PCMV coculture, populations of PCMV endothelial cells and pericytes segregate. Retina-derived growth factor (RDGF) promoted this cell-type segregation and the subsequent formation of suspended cords in PCMV cocultures, although its exact mode of action is unclear. These results indicate that cultured cerebral microvascular endothelial cells and pericytes have capacities for complex, temporal self-organization that varies according to culture conditions.
Animals↗
Preferential biliary elimination of FPL 63547, a novel inhibitor of angiotensin-converting enzyme, in the rat.
1. The route of elimination of FPL 63547, a novel inhibitor of angiotensin-converting enzyme (ACE), has been investigated in the anaesthetized rat. Comparisons have been made with other ACE inhibitors. 2. Bile and urine samples were collected over a 5 hour period following a single i.v. dose of ACE inhibitor (2 mumol kg-1). Samples were bioassayed for ACE inhibitory activity using affinity-purified rabbit lung ACE and the amounts of the active form of inhibitor present in each sample were calculated by comparison with a standard curve. 3. FPL 63547 was rapidly and extensively excreted as the diacid in the bile but appeared in the urine in negligible amounts. The bile:urine ratio was 21.4:1 indicating a marked preference for the biliary route. A similar elimination profile was observed when the compound was dosed in its active form (FPL 63547 diacid), 87.9% of which was found in the bile over the 5 h collection period, with a bile: urine ratio of 14.6:1. 4. The marked preference of FPL 63547 for biliary elimination was not shared by the other ACE inhibitors tested in this study. Lisinopril demonstrated the opposite pattern, being excreted almost exclusively by the kidney (bile:urine ratio 0.06:1). Enalapril was eliminated in approximately equal amounts in bile and urine (ratio 0.7:1) while spirapril diacid showed a slight preference for the bile (ratio 2.6:1). 5. The physical chemical properties of FPL 63547 diacid may be responsible for its unusual preference for biliary elimination. In particular, the amphipathic character and strong acid functionality of the compound are thought to favour transport into the bile. 6. Elimination by the biliary route will be preferred in patients whose renal function is impaired as a result of disease or age. In such patients the elimination of renally-excreted ACE inhibitors is known to be compromised, resulting in compound accumulation and the need for closer monitoring. Therefore, the elimination profile of FPL 63547, if confirmed in man, may prove to be clinically advantageous.
Anesthesia↗
Hexose transport in preimplantation rabbit blastocysts.
Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.
3-O-Methylglucose↗
Stability of esmolol hydrochloride in the presence of aminophylline, bretylium tosylate, heparin sodium, and procainamide hydrochloride.
The visual and chemical compatibility of esmolol hydrochloride mixed with aminophylline, heparin sodium, bretylium tosylate, or procainamide hydrochloride in 5% dextrose injection was studied. Esmolol hydrochloride 600 mg was injected into polyvinyl chloride bags containing 100 mL of 5% dextrose injection with aminophylline 100 mg, heparin sodium 5000 units, bretylium tosylate 100 mg, or procainamide hydrochloride 400 mg. All admixtures were prepared in triplicate and stored at room temperature under fluorescent light. Esmolol concentrations were measured with high-performance liquid chromatography at 0, 2, 4, 8, and 24 hours. Samples were also examined for precipitate formation and pH and color changes by using visual, microscopic, and spectrophotometric methods. No detectable changes in color or pH and no particulate formation were observed in any of the sample bags. Esmolol concentrations varied by less than 5% throughout the 24-hour study period. Esmolol hydrochloride was visually compatible and chemically stable for at least 24 hours when mixed with aminophylline, heparin sodium, bretylium tosylate, or procainamide hydrochloride in polyvinyl chloride bags containing 5% dextrose injection.
Aminophylline↗
In vitro differentiation of rabbit blastocyst cells.
Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for trophectodermal cells from 6-d p.c. embryos to attach to the collagen substrate. Culture of 7-d p.c. embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was -59 +/- 1.2 mV and the threshold potential was -53 +/- 2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect.
Animals↗
Macromolecular transport in rabbit blastocysts: evidence for a specific uteroglobin transport system.
Though uterine proteins are found within the blastocoel of the rabbit blastocyst, the mechanisms involved in protein entry into the blastocoel have not been studied. To investigate potential avenues of protein entry into the blastocoel, we have monitored uptake of a fluorescent, fluid-phase marker (lucifer yellow) into the rabbit blastocyst trophectodermal cell. In addition, we have measured the transtrophectodermal permeabilities of several uterine proteins (uteroglobin (UTG), rabbit serum albumin, rabbit IgG), and radiolabeled fluid-phase markers (sucrose, polyethylene glycol, dextran) in the 6- and 7-day post-coitus rabbit blastocyst. Extrablastocoelic lucifer yellow (LY) was rapidly endocytosed by the trophectodermal cell and was subsequently trapped in a perinuclear compartment for at least 30 min after removal of the extracellular dye. The slow rate of LY turnover within the endocytic compartments implies that the rate of fluid-phase transcytosis was negligible. Permeability coefficients of the fluid-phase markers and proteins, with the exception of UTG, decreased with increasing molecular weight of each compound tested. These data are consistent with the conclusion that these compounds traverse the trophectoderm via a 'leak' pathway, as opposed to a transcytotic pathway. In contrast, permeability of 125I-UTG when compared to the other compounds, was 10-fold greater than would be predicted based on its molecular weight. Furthermore, the flux of radioiodinated UTG displayed saturation kinetics with a Km of 19.5 micrograms/ml and a Vmax of 132 ng.cm-2.h-1. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the radioiodinated protein recovered from the blastocoel was of a lower molecular weight than native UTG and was not immunoreactive with goat anti-UTG antibody. Our data are consistent with the idea that UTG is transported across the trophectoderm by a receptor-mediated system, and UTG is modified intracellularly during transport to a protein of a lower molecular weight.
Animals↗
Morphology of ventral epidermis of Rana catesbeiana during metamorphosis.
A detailed morphological examination of the bullfrog tadpole ventral epidermis and changes in structure that occur during metamorphosis has not been done. Knowledge of this is crucial to interpretation of physiological studies such as those dealing with development of transepithelial Na+ transport. Examination of tadpole epidermis with light microscopy reveals the presence of three different cell types: apical, basal, and skein. This epidermal morphology is constant until Taylor and Kollros (Anat. Rec. 94:7-23, 1946) stage 19 when degeneration of apical cells is noted. Stages 20 and 21 are characterized by rapid proliferation of basal cells and development of a true stratum germinativum together with the disappearance of other tadpole cell types. By stage 22, epidermal morphology is similar to that of the adult frog. Studies with the electron microscope reveal that as the proliferation proceeds during metamorphosis, the skein cells, at stage 20, differentiate to form the apical border of the skin. The development of the adult frog cell phenotype appears to mimic the cellular differentiation that occurs in the adult epidermis with the cells first developing into progranular cells in the intermediate stratum of the skin and then progressing to granular cells in the outermost living cell layer. The granular cells then undergo cornification to form the stratum corneum. Mitochondria rich cells are not seen in the developing epidermis until stage 21. These observations, when considered with previous results from Na+ transport studies (Hillyard et al.: Biochim. Biophys. Acta 692:455-461, 1982), suggest that both the physiological differentiation and morphological differentiation are simultaneous events.
Animals↗