New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Novel sulfonate derivatives: potent antimitotic agents.

The synthesis and biological evaluation of novel sulfonate analogues of E-7010 are reported. Several of the compounds are potent inhibitors of cell proliferation and tubulin polymerization. Importantly, these compounds are also active against P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.

Novel sulfonate analogues of combretastatin A-4: potent antimitotic agents.

Sulfonate analogues of combretastatin A-4 have been prepared. These compounds compete with colchicine and combretastatin A-4 for the colchicine binding site on tubulin and are potent inhibitors of tubulin polymerization and cell proliferation. Importantly, these compounds also inhibit the proliferation of P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.

Modulation of drug resistance by alpha-tubulin in paclitaxel-resistant human lung cancer cell lines.

Beta(beta)-tubulin isotype variation has recently been implicated in the modulation of resistance to paclitaxel in human lung cancer cells and in primary human ovarian tumour samples. Whether alpha-tubulin is involved in drug resistance has not been reported. We have generated a paclitaxel-resistant cell line (H460/T800) from the sensitive human lung carcinoma parental cell line NCI-H460. The resistant cells are more than 1000-fold resistant to taxol and overexpress P-glycoprotein. Interestingly, H460/T800 cells also overexpress alpha- and beta-tubulin as detected by Western blot analysis. From Northern blot analysis, the mechanism of tubulin overexpression appears to be post-transcriptional. To understand whether alpha-tubulin plays a role in drug resistance, we transfected antisense human kalpha1 cDNA construct into the H460/T800 paclitaxel-resistant cells. The antisense clones displayed a reduced alpha-tubulin expression, and the cells were 45-51% more sensitive to paclitaxel and other known antimitotic drugs, compared with vector transfected controls. Complementary experiments of transfecting the sense kalpha1 cDNA into H460 cells conferred a 1.8- to 3.3-fold increase in the IC(50) of several antimitotic agents. Our study suggests that alpha-tubulin is one of the factors that contributes to drug resistance.

Fibrinolytic mechanism, biochemistry, and preclinical pharmacology of recombinant prourokinase.

The purpose of this review is to provide a biochemical characterization of recombinant prourokinase (r-ProUK [ABT-187]), including a description of its clot-specific fibrinolytic mechanism. In addition, the preclinical data will be briefly reviewed, demonstrating the efficacy of r-ProUK as a potent therapeutic plasminogen activator. r-ProUK was purified to homogeneity from the culture medium of SP2/0 mouse hybridoma cells. The fibrinolytic potency of r-ProUK was characterized by both in vitro clot lysis experiments in human plasma and a canine femoral artery thrombosis model. In the in vitro clot lysis system, with use of clots prepared from fresh frozen human plasma, r-ProUK exhibits a lag phase to the onset of lysis and a concentration-dependent threshold effect due to the presence of the inhibitors alpha 2-antiplasmin and plasminogen activator inhibitor (PAI-1). Effective clot lysis can be achieved without degradation of the fibrinogen in the surrounding plasma. Over a dose range of 50,000-220,000 IU, the canine femoral artery thrombosis model shows a dose-dependent relationship for r-ProUK and effective clot lysis. The lytic activity of r-ProUK is significantly enhanced in this model by the concomitant administration of heparin as an adjunctive agent for thrombolytic treatment. Fibrinogen, plasminogen, and alpha 2-antiplasmin levels in the systemic circulation were unaltered during the 30-minute infusion period and a 4-hour observation period, in which 85% lysis was achieved with r-ProUK (100,000 IU) and heparin. Moreover, restoration of blood flow in the previously fully occluded femoral artery was achieved within minutes of the start of the r-ProUK infusion. The experimental findings presented here are consistent with the clot-specific fibrinolytic mechanism of r-ProUK. Effective clot lysis can be achieved without alteration of the systemic coagulation and fibrinolytic parameters.

Alpha-chain domain of fibrinogen controls generation of fibrinoligase (coagulation factor XIIIa). Calcium ion regulatory aspects.

Fibrinogen (approximately 10(-5) M) labilizes heterologous interactions within the thrombin-modified factor XIII zymogen (i.e., XIII' = a2'b2) so that, in the time frame (ca. 10 min) of normal clotting in plasma (37 degrees C, mu = 0.15, pH 7.5), 1.5 mM Ca2+ is sufficient to cause the release of the noncatalytic b subunits and also the unmasking of 1 equiv of iodo[1-14C]acetamide-titratable group per catalytic a subunit. Under similar conditions, but in the absence of fibrinogen, approximately 10 mM Ca2+ would be needed to achieve the same effect. Thus, by promoting the conversion of XIII' to XIIIa (i.e., a2'b2 leads to a2* + b2), fibrinogen functions as a physiologically important Ca2+-modulator protein. Total plasmin digests of fibrinogen display the regulatory phenomenon nearly as well as the parent protein. In an attempt to identify the structural domain on the fibrinogen which is responsible for this novel function of the molecule, it was found that two overlapping fragments derived from the midsections of the alpha chains, either by CNBr cleavage (residues 243-476) or by plasmin digestion (residues 242-424), are active.

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages.

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg-dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g-Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.

Ca2+-related regulatory function of fibrinogen.

Fibrinogen displays a regulation of considerable physiological significance by lowering the Ca2+ requirement for the conversion of the fibrin-stabilizing factor (Factor XIII) zymogen to the range of concentrations of this ion found in plasma. Fibrinogen modulates both Ca2+-dependent steps in the complex process of zymogen activation, involving the heterologous dissociation of subunits of the thrombin-modified zymogen (Factor XIII') species : formula: (see text) and the unmasking of iodoacetamide titratable sites during generation of transamidating activity : formula: (see text). It is interesting that a thrombin-independent pathway of zymogen activation : formula: (see text), which we found to operate at Ca2+ concentrations above 50 mM, is not affected by the presence of fibrinogen. Regulation by fibrinogen thus appears to be specific for controlling only the physiological pathway of zymogen conversion.

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.