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A Janoff

Publications and source records attributed to A Janoff.

At least 19 recordsLinked to original sources
PubMed2001

Hydrolyzable hydrophobic taxanes: synthesis and anti-cancer activities.

A series of taxane prodrugs with 2-bromoacyl chains attached at the 2'-position of the paclitaxel side chain, varying from six, eight, 12, 14 to 16 carbons in length, were synthesized, characterized and evaluated against human breast MCF-7 cancer cell line for their growth inhibitory (GI50) activities. The GI50 is the drug concentration required to inhibit cell growth by 50%. For comparison, hydrophobic taxanes varying in acyl chain lengths from six to 16 carbons were also synthesized and compared for their G050s with taxanes having equivalent bromoacyl chain lengths. The bromoacyl taxanes bearing six, eight and 12 carbon acyl chain lengths had GI50 values very similar to parent paclitaxel. The GI50 was 3 nM for three taxanes versus 1 nM for paclitaxel on the MCF-7 cell line. Increasing the acyl chain length to 14 or 16 carbons resulted in a significant decrease in cytotoxicity and an increase in the GI50 to 20 or 70 nM, respectively. In general, the GI50 values were directly related to the bromoacyl chain lengths in cultured tumor cells. Unlike bromoacyl taxanes, the taxanes lacking bromine in their acyl chain composition were 50- to 250-fold less active, suggesting that the heteroatom facilitated the hydrolysis of acyl chains to yield free paclitaxel. These differences in growth inhibitory activities may indirectly reflect differences in the susceptibility of the acyl chain to bromine-induced hydrolysis after association of the derivative with cell membranes. Liposome formulations of 2-bromoacyl taxanes bearing six, eight, 12 and 16 carbons were prepared and tested in SCID mice against a xenografted human ovcar-3 ovarian tumor. In vivo results showed that bromoacyl taxanes with a longer chain were therapeutically more efficacious than those with a short chain, presumably due to slow hydrolysis of the prodrug followed by sustained delivery of paclitaxel to the tumor.

Animals↗
PubMed2000

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes.

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC >> DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

A novel N-acyl phosphatidylethanolamine-containing delivery vehicle for spermine-condensed plasmid DNA.

A unique method for formulation of plasmid DNA with phospholipids has been devised for the purpose of producing vehicles that can mediate gene delivery and transfection of living cells. The polycation, spermine, was used to condense plasmid DNA within a water-in-chloroform emulsion stabilized by phospholipids. After organic solvent removal, the particles formed could be extruded to a number average size of about 200 nm and retained DNA that was protected from nuclease digestion. This resulted in a relatively high protected DNA-to-lipid ratio of approximately 1 microg DNA/micromol lipid. The size distribution of the preparation was relatively homogeneous as judged by light microscopy and quasi-elastic light scattering. Electron microscopic studies showed structural heterogeneity, but suggested that at least some of the plasmid DNA in this preparation was in the form of the previously observed spermine-condensed bent rods and toroids and was encapsulated within liposomal membranes. Preparations with the fusogenic phospholipid composition, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3-phosphocholine, showed transfection activity for several cells lines, particularly OVCAR-3 cells. The transfection activity sedimented with the lipid during centrifugation, confirming the association of active plasmid DNA with phospholipids. Transfection efficiency in culture was found to be of the same order of magnitude as cationic lipoplexes but much less toxic to the cells. Significant transfection of OVCAR-3 cells in tissue culture could also be observed, even in the presence of the intraperitoneal fluid from a mouse with an OVCAR-3 ascites tumor. These data indicate a new type of liposomal gene delivery system devoid of cationic lipids, phosphatidylethanolamine, cationic polymers and viral components.

Animals↗
PubMed1997

The release and detection of endotoxin from liposomes.

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulate Limulus amebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxin in vitro. This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C12E10) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C12E10 varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC >> DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.

Animals↗
PubMed1997

Activity of paclitaxel liposome formulations against human ovarian tumor xenografts.

Although the current clinical formulation of paclitaxel (Taxol) is an important new anti-cancer agent, it has significant side effects, some of which are related to its formulation in Cremophor/ethanol. Paclitaxel is difficult to formulate for i.v. administration because of its poor aqueous solubility. Here, we report the therapeutic effects of 2 liposome formulations of paclitaxel against human ovarian A121 tumor growing as an s.c. xenograft in athymic nude mice. The liposome formulations used were ETL and TTL, which have I or 3 lipid components, respectively. TTL was used as a reconstituted lyophilate or as a stable aqueous suspension. ETL was used as a reconstituted lyophilate only. Both paclitaxel-liposome formulations were much better tolerated than Taxol after i.v. or i.p. administration. The acute reactions seen after Taxol administration did not occur when paclitaxel-liposome formulations were administered. All ETL and TTL preparations significantly delayed A121 tumor growth similarly to Taxol at equivalent doses and schedules. Based on pharmacokinetic data, it is possible that paclitaxel rapidly dissociates from ETL or TTL after i.v. administration and distributes in a manner similarly to Taxol. ETL and TTL formulations may be useful clinically not only for eliminating toxic effects of the Cremophor/ethanol vehicle but also for allowing alterations in route and schedule of drug administration.

Animals↗
PubMed1995

Infarct salvage with liposomal prostaglandin E1 administered by intravenous bolus immediately before reperfusion in a canine infarction-reperfusion model.

BACKGROUND: Prostaglandin E1 (PGE1) inhibits leukocyte and platelet function and reduces infarct size during left atrial infusion. Intravenous liposomal PGE1 (TLC C-53) accelerates thrombolysis and prevents reocclusion in canine coronary thrombosis. We tested the hypothesis that intravenous TLC C-53 would attenuate reperfusion injury in a canine infarction-reperfusion model. METHODS AND RESULTS: Twenty-one open-chest dogs were randomized to receive a 10-minute intravenous infusion of either liposome diluent (placebo), free PGE1 (2 micrograms/kg), or TLC C-53 (2 micrograms/kg PGE1) after 2 hours of left anterior descending (LAD) occlusion just before reperfusion. Hemodynamic assessment, regional myocardial blood flow determination with radioactive microspheres, myocardial leukocyte infiltration by myeloperoxidase assay, and estimation of infarct size using triphenyl tetrazolium chloride staining were performed. Regional fractional shortening was measured with sonomicrometer crystals implanted in the midmyocardium. Infarct size as a percentage of the risk region was significantly reduced (P < .05) with TLC C-53 (37.9 +/- 17.4%) compared with PGE1 (56.7 +/- 13.9%) or placebo (58.0 +/- 9.9%) infusion. Infarct salvage with TLC C-53 was independent of collateral blood flow by ANCOVA. There was a dramatic reduction in myeloperoxidase activity in the infarct, risk, and border regions of dogs treated with TLC C-53 compared with placebo. Enzyme activity was also significantly reduced (P < .05) in the infarct zone with TLC C-53 (0.11 +/- 0.1 U/100 mg) treatment compared with PGE1 (0.38 +/- 0.3 U/100 mg). No significant differences in regional myocardial blood flow or myocardial function among treatment groups were identified, although there was a trend toward improved function in the TLC C-53 dogs. CONCLUSIONS: Bolus intravenous administration of TLC C-53 immediately before reperfusion results in reduced leukocyte infiltration and substantial infarct salvage. TLC C-53 mah be useful in limiting reperfusion injury during treatment of acute myocardial infarction.

Alprostadil↗
PubMed1990

Interactions of human mast cell tryptase with biological protease inhibitors.

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.

Antithrombin III↗
PubMed1988

Degradation of plasmodial antigens by human neutrophil elastase.

Human neutrophil elastase (HNE) has been well-studied with respect to its role in pathologic states, but less is known about the physiologic functions of this important granulocyte enzyme. In the present study, we show that HNE can degrade the major circumsporozoite protein of the infective (sporozoite) stage of Plasmodium vivax malaria, and that this enzyme can also interfere with the cytoadherence of human E infected with Plasmodium falciparum (strain K+ FMG-FCR3) (IE). Cytoadherence reactions are not only blocked by treatment of IE with as little as 10 fg HNE/IE, but already adherent IE are also removed by the enzyme. Normal E surface Ag are not extensively destroyed by these doses of HNE. This suggests that the effect of HNE on cytoadherence is selective and probably due to degradation of the malarial Ag exported to the IE surface and responsible for the formation of "recognition knobs" upon which the cytoadherence reaction depends. This conclusion, in turn, was supported by the results of Western blot analysis showing that HNE degrades a high m.w. Ag found exclusively in membrane extracts of IE. Our results suggest that one physiologic role of HNE may be degradation of parasitic antigens during host defense against malaria.

Amino Acid Sequence↗
PubMed1988

Characterization of sheep alpha-1-proteinase inhibitor. Important differences from the human protein.

The plasma proteinase inhibitor corresponding to alpha-1-proteinase inhibitor (alpha 1Pl) in humans was isolated from sheep plasma. Ovine alpha 1Pl is of higher molecular weight (62,000 daltons) than is human alpha 1Pl, is resistant to chemical oxidation by N-chlorosuccinimide, and has poor elastase-inactivating power compared with the corresponding inhibitor in humans. However, ovine alpha 1Pl is a potent trypsin inhibitor. Despite the differences indicated above, a partial homology (22 to 35%) exists between human and sheep alpha 1Pl, at least as analyzed through the first 20 residues of the sheep inhibitor. The weak elastase-inhibitory capacity of sheep alpha 1Pl is paralleled by the low content of elastase in the sheep neutrophil granule. These important differences between sheep and human neutrophils and plasma proteinase inhibitors should be borne in mind in designing experiments related to proteolytically mediated lung injury in the former species.

Amino Acid Sequence↗
PubMed1988

Elevated histamine and tryptase levels in smokers' bronchoalveolar lavage fluid. Do lung mast cells contribute to smokers' emphysema?

Human lung mast cells have been reported recently to contain small amounts of the elastolytic protease present in the neutrophil and implicated in the pathogenesis of alveolar wall destruction in emphysema. Since mast cells are numerous within alveolar walls, release of inflammatory mediators (and possibly elastase) by cigarette smoking could contribute to alveolar injury in this disease. We therefore examined bronchoalveolar lavage (BAL) fluid for the mast cell granule constituents histamine and tryptase. The results, while not conclusive, supported the possibility that cigarette smoking increases secretion of histamine releasing activity by alveolar macrophages with subsequent degranulation of local mast cells. Mast cell discharge of inflammatory mediators (including neutrophil chemotactic factors and perhaps the elastolytic protease) could then participate in the destruction of alveolar walls.

Adult↗
PubMed1988

Inhibition of human neutrophil elastase by bacterial polyanions.

We previously demonstrated that pneumococcal extracts contain a highly specific inhibitor of human neutrophil elastase (HNE). We now show that the active inhibitor in these extracts is a high-molecular-weight, heat-stable substance that appears to be RNA, since inhibitory activity of pneumococcal extracts is decreased by incubation with ribonuclease but not by incubation with deoxyribonuclease or proteinase K. Moreover, metabolically labeled ([3H]uridine) pneumococcal RNA, isolated by phenol extraction followed by ethanol precipitation, strongly inhibits HNE. Pneumococcal capsular polysaccharide, although polyanionic, is only weakly inhibitory toward HNE and is not a major source of elastase-inhibitory activity in pneumococcal extracts. On the other hand, the capsule of Haemophilus influenzae type b contains polyribosylribitol phosphate. This highly charged polyanion possesses HNE-inhibitory activity, but only under special circumstances to be discussed below. Pneumococci (type I, type II smooth, type II rough) and H. influenzae (type b) all release HNE-inhibitory activity into their culture medium during growth. By contrast, Klebsiella pneumoniae and Staphylococcus aureus release little (if any) stable HNE-inhibitory activity during growth. We propose that some bacterial pneumonias may spare host tissue because polyanions released by the invading microorganisms (e.g. RNA from autolysing pneumococci) inhibit elastase released from inflammatory neutrophils and thereby modulate accompanying tissue proteolysis. Pneumonias caused by microorganisms that do not release stable polyanionic inhibitors of HNE (e.g., Staphylococcus and Klebsiella) may be correspondingly more injurious to the lung.

Haemophilus influenzae↗
PubMed1988

Inhibition of human neutrophil elastase by polyguanylic acid and other synthetic RNA homopolymers.

Human neutrophils contain large amounts of a neutral serine protease, human neutrophil elastase (HNE), which has been implicated as a mediator of acute and chronic lung injury. We found that this enzyme is effectively inhibited, at physiological ionic strength, by several synthetic non-base-paired polyribonucleotides. Among the most active of these is polyguanylic acid (poly G). Inhibitory activity is greatest with high-molecular-weight poly G fractions, but poly G fractions even as low as 60K Mr (app) are effective. Both amidolysis of synthetic elastase substrates, such as succinyl-ala-ala-ala-p-nitroanilide, and proteolysis of elastin are blocked. Poly G inhibits elastin proteolysis even when subsequently added to mixtures of elastin and HNE that have first been preincubated together for 10 min. Under these conditions, polyribosylribitol phosphate, a polyanion derived from Haemophilus influenzae capsular polysaccharide, is not inhibitory. Complex formation between HNE and poly G is dependent on ionic rather than covalent interactions, since it is blocked by 0.6 M NaCl but not by inactivation of the enzyme's catalytic-site serine residue with diisopropylfluorophosphate. However, nonspecific ionic interactions alone cannot explain complex formation, since pancreatic elastase and cathepsin G, an even more basic serine protease from human neutrophils, do not form complexes with poly G, even at low ionic strength. Moreover, in the presence of the amphiphiles taurocholic acid and glycocholic acid, HNE is much less effectively blocked by poly G. Peptide chloromethyl ketone-inactivate HNE (which has its extended substrate-binding pocket occupied by the peptidyl inactivator) also fails to form complexes with poly G. These results indicate that HNE may utilize both hydrophobic and ionic binding sites to couple with poly G, and suggest that these sites may be close to or within the extended substrate-binding pocket of the enzyme.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Characterization of two distinct transglutaminases of murine bone marrow-derived macrophages: effects of exposure of viable cells to cigarette smoke on enzyme activity.

The present study examines the effects of water soluble extracts of gas-phase cigarette smoke on intracellular transglutaminase activities of intact, murine, bone marrow-derived macrophages maintained in culture. Western blotting of cell lysates utilizing noncross-reactive antisera indicate that mouse bone marrow-derived macrophages contain both tissue-type transglutaminase and factor XIII-associated transglutaminase. This finding is also supported by data indicating that the intracellular transglutaminase activity of these cells contains thrombin-dependent and -independent components. Macrophages incubated with cigarette smoke solutions for 15 minutes at 37 degrees C display a dose-dependent decrease (maximum inhibition = 55%, p less than .001) in tissue-type (thrombin-independent) transglutaminase activity, as compared to control cells incubated with phosphate-buffered saline. Factor XIII (zymogen) is not inactivated following incubation of macrophages with smoke extracts. Smoke exposure under the conditions employed has no effect on either cell viability or adherence. Incubation with 2 microM retinoic acid for 24 hours leads to a modest (2-fold) induction of tissue transglutaminase, but does not induce factor XIII; in contrast, incubation with 10% homologous serum for 24 hours results in a decrease in factor XIII, but does not affect tissue transglutaminase. These data indicate that: bone marrow-derived macrophages contain factor XIII as well as tissue-type transglutaminase; and gas-phase cigarette smoke can inactivate tissue transglutaminase within viable murine bone marrow-derived macrophages, but cannot inactivate zymogenic factor XIII.

Animals↗
PubMed1987

Characterization of the third component of complement (C3) after activation by cigarette smoke.

Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of [14C]methylamine (as does native C3), and relatively little [14C]methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers.

Chemical Phenomena↗
PubMed1987

The fifth component of complement (C5) is necessary for maximal pulmonary leukocytosis in mice chronically exposed to cigarette smoke.

Recruitment of elastase-containing leukocytes to the alveoli and small airways of cigarette smokers is thought to be a major contributing factor in the pathogenesis of pulmonary emphysema. The fifth component of complement (C5), when activated, acts as a potent chemoattractant for these cells. We therefore examined the effect of chronic cigarette smoke exposure on numbers of pulmonary leukocytes in congenic strains of C5-deficient (C5-) and C5-sufficient (C5+) mice. Animals were exposed daily to 15 puffs (1.5 cigarettes) or unfiltered smoke from 2A1 Kentucky Reference cigarettes. After 8 weeks of exposure, the total number of bronchoalveolar leukocytes, recovered by lavage, was significantly increased in cigarette smoke-exposed animals (both C5- and C5+) vs their sham-exposed counterparts. In addition, cigarette smoke exposure also significantly increased the total number of recovered alveolar polymorphonuclear leukocytes (PMNs) and the PMN chemotactic activity of cell-free bronchoalveolar lavage supernatants in both strains vs those of sham controls. However, total numbers of recovered bronchoalveolar leukocytes and PMNs in smoke-exposed animals were significantly greater (P less than 0.001) in C5+ mice than in their congenic C5- counterparts. In C5+ mice, acute smoke exposure caused an immediate but transient increase in chemotactic activity of lung fluids, which was not observed in C5- mice following acute smoke exposure. These results suggest that cigarette smoking induces an increase in leukocytes in the lungs of mice by mechanisms which are partly dependent on C5.

Animals↗