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Biomedical subjects

D S Fletcher

Publications and source records attributed to D S Fletcher.

16 recordsLinked to original sources
PubMed1995

Orally active beta-lactam inhibitors of human leukocyte elastase. 3. Stereospecific synthesis and structure-activity relationships for 3,3-dialkylazetidin-2-ones.

The stereospecific synthesis of several 4-[(4-carboxyphenyl)oxy]- 3,3-dialkyl-1-[[(1-phenylalkyl)-amino]carbonyl]azetidin-2-on es 3 is described in which the C-3 alkyl groups were varied from methyl to butyl as well as allyl, benzyl and methoxymethyl. The structure-activity relations for these compounds are discussed in terms of the hydrolytic stability of the beta-lactam ring, their in vitro inhibitory potency for human leukocyte elastase (HLE), and their in vivo oral efficacy in an HLE-mediated hamster lung hemorrhage assay. Further alkyl substitution on the benzylic urea moiety, especially in the R configuration, afforded enhanced HLE inhibition and in vivo efficacy. The stereochemical assignments for (3R,4S)-4-[(4-carboxyphenyl)oxy]-3-ethyl-3-methyl-1-[[((R)-1- phenylpropyl)amino]carbonyl]azetidin-2-one (42a) (kobs/[I] = 91,000 M-1 s-1) were confirmed with an X-ray structure determination, which was also utilized to develop an HLE inhibition model.

Administration, Oral↗
PubMed1995

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

Amino Acid Oxidoreductases↗
PubMed1995

A synthetic inhibitor of interleukin-1 beta converting enzyme prevents endotoxin-induced interleukin-1 beta production in vitro and in vivo.

A potent, reversible, tetrapeptide inhibitor of interleukin-1 beta converting enzyme (ICE), L-709,049, has been shown to suppress the in vitro production of mature IL-1 beta. We now report that this inhibitor also effectively suppresses the production of mature IL-1 beta in a murine model of endotoxic shock. Intraperitoneal administration of L-709,049 reduced the elevations of IL-1 beta in the plasma and peritoneal fluid of mice treated with LPS in a dose-related manner (ED50 = 2 +/- 0.9 mg/kg). LPS-induced elevations in IL-1 alpha and IL-6 in these mice were unaffected, indicating that the inhibitor specifically affected IL-1 beta production. Immunoblot analysis of plasma and peritoneal fluid indicated that L-709,049 suppressed the formation of mature IL-1 beta production in vivo. When mouse blood was incubated in vitro with LPS, IL-1 beta was released into the plasma. This assay was used to determine ex vivo the activity of an ICE inhibitor in the blood following its administration to mice. Blood obtained 15 minutes after ip administration of 10 mg/kg of L-709,049 to mice produced 80% less IL-1 beta than control blood, and IL-1 beta production returned to control levels in blood obtained 30 minutes after injection of this inhibitor. In addition, the capacity of the blood plasma obtained from these animals to prevent the cleavage of a synthetic substrate by ICE disappeared within 1 h of ip administration of 50 mg/kg of inhibitor.

Animals↗
PubMed1995

An inhibitor of leukocyte elastase prevents immune complex-mediated hemorrhage in the rat lung.

The typical reverse passive Arthus reaction (RPA) was attained in rats by the instillation of a rabbit antiovalbumin serum into the lungs and intravenous injection of ovalbumin. Instillation of antiserum alone caused accumulation of polymorphonuclear leukocytes (PMN) and increased vascular permeability, but did not cause hemorrhage. However, when an intravenous injection of ovalbumin was also given, the vascular permeability of the lungs increased dramatically and PMN, as well as hemoglobin, were measurable in the lung lavage fluids by 4 hr after initiation of the reaction. Various proteinase inhibitors were instilled into the lungs after the initial stages of the RPA had developed, specifically to investigate their effect on the development of the hemorrhage, which we chose to monitor as an indicator of severe vascular damage. A cephalosporin-based beta-lactam, L-658,758, which is a time-dependent inhibitor of human and rat PMN elastase, effectively prevented the lung hemorrhage associated with the RPA reaction (ED50 = 2 x 55 micrograms doses/animal when instilled at 1.5 and 2.5 hr after initiating the RPA). The PMN elastase inhibitor, methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone, also inhibited hemorrhage in this model. Compounds of the same chemical class as these elastase inhibitors, but having no activity against PMN elastase in vitro, did not affect the hemorrhage associated with the RPA. Several specific inhibitors of proteinases other than PMN elastase (e.g., pepstatin and methoxysuccinyl-prolyl-glycyl-alanyl-lysine-chloromethylketone) were found to have little effect on the hemorrhage associated with the RPA reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1990

A comparison of alpha 1-proteinase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone and specific beta-lactam inhibitors in an acute model of human polymorphonuclear leukocyte elastase-induced lung hemorrhage in the hamster.

A pharmacokinetic model is described for testing of polymorphonuclear leukocyte (PMN) elastase inhibitors administered by intratracheal or aerosol dosing of hamsters. Acute lung injury, measured as hemorrhage occurring within hours after intratracheal instillation of human PMN elastase, correlated directly with the amount of active enzyme instilled. Hemorrhage began within minutes of elastase instillation, was maximal within 1 h, and remained constant for up to 5 h subsequently. Therefore, inhibition of hemorrhage was used as an assay of the effectiveness of various PMN elastase inhibitors given by the intratracheal route. Lung hemorrhage could also be induced by intratracheal instillation of other elastolytic enzymes, such as thermolysin, and inhibition of hemorrhage was seen only with inhibitors active against the type of elastase used. Methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone (MeOSuc-AAPV-CMK), as well as alpha 1-proteinase inhibitor (alpha 1PI) but not tosyl-lysine-chloromethylketone (tosyl-lysine-CMK), inhibited the hemorrhage caused by human PMN elastase, but the specific inhibitors of this enzyme had no effect on thermolysin-induced lung hemorrhage. The duration of activity of these compounds as elastase inhibitors in this model correlated directly with the extent of their persistence in lung lavage fluid as determined by HPLC analysis of compound recovered by bronchoalveolar lavage. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1987

A model for the separation of large DNA molecules by crossed field gel electrophoresis.

The idea that large DNA molecules adopt a stretched conformation as they pass through gels suggests a simple mechanism for the separation of DNA by crossed field electrophoresis: at each change in field direction a DNA molecule takes off in the new direction of the field by a movement which is led by what was formerly its back end. The effect of this ratcheting motion is to subtract from the DNA molecule's forward movement, at each step, an amount which is proportional to its length. We find that this model explains most of the features of the separation, and we describe experiments, using a novel electrophoresis apparatus, which support the model. The apparatus turns the gel between two preset orientations in a uniform electric field at preset time intervals. This separation method has the practical advantage over some others that the DNA molecules follow straight tracks. A further advantage is that the parameters which determine the separation are readily predicted from the simple theory describing their motion.

DNA↗
PubMed1986

Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse passive Arthus reaction.

The reverse passive Arthus (RPA) reaction performed in the skin of rats was modified to allow for the determination of polymorphonuclear leukocyte (PMN) infiltration and hemorrhage, as well as changes in vascular permeability. After initiation of the RPA reaction, PMN infiltration, monitored by measurement of tissue myeloperoxidase (MPO, EC 1.11.1.7) content, increased dramatically with time. Depending on the experimental conditions used, PMN accumulation reached a maximum 2-10 hr after increased vascular permeability (125I-labeled albumin content) had peaked. Hemorrhage (59Fe-labeled erythrocyte accumulation) began to occur only after significant levels of PMN were reached and continued to increase proportionately to the level of PMN infiltration attained. Indomethacin administered 30 min prior to initiating the RPA reaction had no effect on vascular permeability increase but suppressed both PMN accumulation and hemorrhage development about 50%. When indomethacin was given 2 hr after the RPA reaction was begun, no effect on any of the RPA variables was noted. Dexamethasone suppressed the increase in vascular permeability (53%), PMN accumulation (78%), and hemorrhage (90%) when given 30 min prior to initiation of the reaction. Dexamethasone given 2 hr after initiating the RPA suppressed the entire reaction, but to a lesser extent. Catalase, as well as trasylol, alpha-1-antiproteinase and soybean trypsin inhibitor, inhibited PMN accumulation as well as hemorrhage when given intravenously at plus 2 hr. These results indicate that the damage to blood vessels during a severe RPA reaction is a direct consequence of PMN activity.

Animals↗
PubMed1983

Inhibition of immune aggregate-induced activation of the first complement component by cationic polypeptides.

A cationic amino acid copolymer (CP530) with a molar ratio of lysine, leucine, tryptophan and phenylalanine of 11:2:1:1 and a Mr of about 2300 was prepared and its inhibitory effects on the complement cascade was compared with those of polylysine with a Mr of about 3000. The effects by these two cationic peptides appeared to be at the early stage of complement activation. CP530 and polylysine inhibited the binding of C1q to insoluble IgG aggregates with a concentration required for 50% inhibition of 0.7 and 0.9 mM, respectively. Both compounds were also potent inhibitors of immune hemolysis (a concentration causing 50% inhibition, 0.5 and 3.5 microM respectively) as well as assembly of EAC cell intermediates required for formation of C3 and C5 convertases (a concentration of 50% inhibition of 1.0 microM for CP530 and 3.8 microM for polylysine). However, CP530 was shown to be distinctly more effective against the activation of C1r . Cls complex induced by insoluble IgG aggregate-bound C1q, requiring 0.15 mM for 50% inhibition compared to greater than 10 mM for polylysine. The 50% inhibition value for soluble IgG aggregate-induced activation of C1 in whole serum was 0.7 mM for CP530 and 5.0 mM for polylysine. The greater inhibition of C1 activation by CP530 than that exerted by polylysine could be attributable to the presence of non-lysyl residues which provide the structural basis for specificity and potency.

Animals↗
PubMed1980

Inhibition of immune complex-mediated activation of complement. Effects of agents modulating activation of, and the activated C1 complex.

Several known chemical compounds were shown to selectively inhibit the interaction between immune aggregates and C1q, the activation of C1r-C1s complex by immune aggregate-bound C1q, and the esterolytic activity of the activated C1s, C1s. These reactions are relevant to the functions of the first complement component, C1, and its activation induced by immune complexes. The effects of these inhibitors on tissue injury mediated by immune complex-induced complement activation, such as immune hemolysis, passive cutaneous anaphylaxis, and experimental glomerulonephritis were examined. The results suggest an approximate correlation between the activity shown on the molecular level and that obtained in vivo. One such compound, suramin, was shown to be an effective inhibitor of PCA and the proteinuria manifestation of EGN while not affecting antibody fixation to tissue or histamine-mediated skin reaction. These results suggest that effective suppression of the initial steps of complement activation may be of value of controlling immune complex-mediated tissue injuries in disease.

Animals↗
PubMed1977

Quantitation of immune complexes by competitive inhibition of binding of Clq to insoluble IgG aggregates.

An assay system for the binding of Clq to insoluble IgG aggregates was found useful for the quantitation of immune complexes in biological fluids. The assay, both easily and rapidly performed, is based on the competition of Clq binding substances with IgG aggregates. Serum from rheumatoid arthritis patients showed an increase in Clq binding substances over normal serum and this increase could be abolished by pretreatment of the serum with D-penicillamine.

Antigen-Antibody Complex↗