Tetramethylpiperidine-substituted phenazines inhibit the proliferation of intrinsically multidrug resistant carcinoma cell lines.

The effects of nine new tetramethylpiperidine (TMP)-substituted phenazines on the growth of a human esophageal cancer cell line (WHCO3), two human hepatocellular carcinoma cell lines (PLC and HepG2) and three human colon cancer cell lines (CaCo2, COLO 320DM and HT29) were compared to those of clofazimine, B669 and five standard chemotherapeutic agents. The three most active TMP-substituted phenazines against these cell lines were B3962, B4126 and B4125 with mean IC50 values for all the cancer cell lines tested of 0.36, 0.47 and 0.48 microg/ml respectively. B3962 and B4126, but not B4125 were also the most active against a semi-continuous human fibroblast culture (MRC5). The compound with the highest tumor specificity relative to the fibroblast culture, was B4125. Importantly, there was minimal variation in sensitivity of the different cell lines, including a multidrug resistant cell line (COLO 320DM) expressing high levels of P-glycoprotein, to the TMP-substituted phenazines. This was not the case with the standard chemotherapeutic agents. The efficacy of compounds such as B4125 against a broad spectrum of multidrug resistant cancer cell lines, together with their relatively high tumor specificity, suggests that these agents may be useful in the treatment of intrinsically resistant cancers such as colon and liver cancer.

Tetramethylpiperidine-substitution increases the antitumor activity of the riminophenazines for an acquired multidrug-resistant cell line.

The multidrug resistance (MDR)-neutralizing and cytotoxic properties of five tetramethylpiperidine (TMP)-substituted phenazines were compared with those of their corresponding isopropyl-substituted analogues using a P-glycoprotein (P-gp)-expressing small cell lung cancer cell line (H69/LX4). All of the TMP-substituted phenazines tested outperformed their isopropyl analogues with respect to both cytotoxic and chemosensitizing properties, indicating the importance of TMP-substitution when designing novel riminophenazines with increased activity against MDR cancer cell lines. Of the TMP-substituted phenazines tested, B4112, chlorinated at position 3 of the phenyl- and anilino-rings, had the most potent anti-cancer activity in vitro, making this agent a potential candidate for evaluation in experimental and clinical oncology.

Clofazimine and B4121 sensitize an intrinsically resistant human colon cancer cell line to P-glycoprotein substrates.

The potential of B4121 to sensitize three intrinsically resistant human colon cancer cell lines (CaCo2, ATCC HTB 37; COLO 32 DM, ATCC CCL 220; HT-29, ATCC HTB 38) to vinblastine, doxorubicin, daunorubicin, paclitaxel, taxotere and cisplatin at a non-toxic, therapeutically relevant concentration of 0.25 microg/ml was compared with that of clofazimine at a similar concentration. The cell line expressing high levels of P-glycoprotein (P-gp), COLO 320 DM, was susceptible to chemosensitization by the experimental agents for the P-gp substrates (paclitaxel, taxotere, daunorubicin, vinblastine and doxorubicin) but not for cisplatin. CaCo2 cells expressed lower levels of P-gp and were only marginally susceptible to sensitization by any one of these drugs, except in the case of sensitization by B4121 for doxorubicin and taxotere, whereas the HT-29, a P-gp negative cell line, was unaffected. The riminophenazines, especially B4121, might prove useful as combination treatment in circumventing P-gp mediated resistance of colon cancers.

In vitro investigation of the antimicrobial activities of novel tetramethylpiperidine-substituted phenazines against Mycobacterium tuberculosis.

The intra- and extracellular activities of 5 novel tetramethylpiperidine (TMP)-substituted phenazines against Mycobacterium tuberculosis H37Rv (ATCC 27294) were determined and compared with those of clofazimine and rifampicin. Two of these agents, together with clofazimine, were also tested for their activities against drug-resistant strains of M. tuberculosis. Three of the TMP-substituted phenazine compounds were significantly more active than clofazimine against M. tuberculosis, including multidrug-resistant clinical strains of this microbial pathogen, demonstrating a lack of cross-resistance between the riminophenazines and standard anti-tuberculous drugs. Using M. tuberculosis-infected monocyte-derived macrophages, all of the TMP-substituted phenazines were found to possess intracellular activity which was superior to that of both clofazimine and rifampicin. In this model of intracellular bioactivity, the experimental compounds inhibited bacterial growth at concentrations which were approximately 10-fold lower than the corresponding minimal inhibitory concentration values obtained using conventional in vitro sensitivity testing procedures. These results demonstrate that the novel TMP phenazines are active against multidrug-resistant M. tuberculosis strains, and particularly effective intracellularly.

The chemosensitizing potential of GF120918 is independent of the magnitude of P-glycoprotein-mediated resistance to conventional chemotherapeutic agents in a small cell lung cancer line.

GF120918, at 250 ng/ml, increased the sensitivity of a P-glycoprotein (P-gp)-mediated multidrug resistant (MDR) small cell lung cancer cell line (H69/LX4) to the P-gp substrates, paclitaxel, taxotere, vinblastine, vinorelbine, daunorubicin and etoposide to levels which were either greater (in the case of etoposide) or close to that of the parent cell line (H69/P). This was achieved in spite of the great variation in the levels of resistance of the MDR cell line for the various anti-cancer drugs tested. These data suggest that GF120918 is a potent antagonist of P-gp mediated multidrug resistance, even in the case of high levels of resistance, as was the case with paclitaxel and taxotere (2560 and 2215 fold more than the sensitive parent cell line respectively).

Alpha-tocopherol antagonizes the multidrug-resistance-reversal activity of cyclosporin A, verapamil, GF120918, clofazimine and B669.

The effects of the membrane-stabilizing agent, alpha-tocopherol (25 microg/ml), on the chemosensitizing interactions of cyclosporin A (5 microg/ml), verapamil (2 microg/ml), clofazimine (1 microg/ml), B669 (0.5 microg/ml) and GF120918 (0.015 microg/ml) with a P-glycoprotein-expressing human lung cancer cell line (H69/LX4) have been investigated in vitro. In an assay of cell proliferation, all the chemosensitizing agents restored the sensitivity of H69/LX4 cells to doxorubicin and vinblastine. The inclusion of alpha-tocopherol (25 microg/ml) antagonized the multidrug-resistance (MDR)-modifying activity of all five chemosensitizing agents, effectively preventing restoration of sensitivity to both doxorubicin and vinblastine in H69/LX4 cells.

Novel tetramethylpiperidine-substituted phenazines are potent inhibitors of P-glycoprotein activity in a multidrug resistant cancer cell line.

The multidrug resistance (MDR)-neutralizing and cytotoxic properties of 16 novel tetramethylpiperidine (TMP)-substituted phenazines were compared with those of clofazimine and B669 using a P-glycoprotein (P-gp)-expressing undifferentiated, human leukemia cell line (K562/MMB). Unchlorinated TMP-substituted phenazine molecules were more cytotoxic than their chlorinated counterparts, while the halogenated molecules, especially those with chlorine atoms at position 3 on the aniline and phenyl rings, were less cytotoxic but more effective as chemosensitizing, P-gp-neutralizing agents. One of the TMP-substituted phenazines, B4121, increased the sensitivity of K562/MMB cells to vinblastine by 100-fold. TMP-substituted phenazines are a novel class of pharmacologic anti-cancer agents with both direct cytotoxic, as well as MDR-neutralizing anti-tumor properties.

An in vitro investigation of the bioactivities of ciprofloxacin and the new fluoroquinolone agents clinafloxacin (CI-960) and PD 131628 against Mycobacterium tuberculosis in human macrophages.

In this study the intracellular bioactivity of ciprofloxacin and the new fluoroquinolone agents clinafloxacin (CI-960) and PD 131628 against Mycobacterium tuberculosis (H37Rv) was compared with rifampicin using human macrophages. Monocyte-derived macrophages were infected with M. tuberculosis in the presence of 10% autologous serum and treated with the antibiotics for 2 days, either immediately after infection or 3 days post-infection. The survival of the intracellular microorganisms was determined using the BACTEC tuberculosis system. Clinafloxacin, although not as active, compared favourably with rifampicin at concentrations ranging from 0.1 to 5 micrograms/ml in both systems, whereas PD 131628 performed reasonably well only when added directly after infection. However, ciprofloxacin was relatively unimpressive with intracellular bioactivity detected only with the highest concentration used (5 micrograms/ml). The ability of clinafloxacin, but not PD 131628, to inhibit mycobacteria after most of the organisms have escaped from the fused phagosomes emphasizes the importance of using a prolonged incubation time after infection when screening new antituberculosis drugs for intracellular bioactivity.

Comparison of the composition and opsonic activities of imported and South African-manufactured intravenous and intramuscular immunoglobulin preparations.

We compared the composition and opsonic activities for two common microbial pathogens (Staphylococcus aureus and Streptococcus pyogenes) of various imported intravenous (IV) (Sandoglobulin, Octagam and Gammagard) and intramuscular (IM) (Beriglobin and Globuman Berna) immunoglobulin (Ig) preparations with those of the corresponding locally manufactured products, Polygam (IV) and Intragam (IM). When tested at equivalent concentrations (1 g/100 ml) the total IgG and IgG subclass concentrations of the various IV and IM preparations were similar. All the test preparations (IV and IM) possessed similar opsonic activity for S. aureus and S. pyogenes. These findings demonstrate that, in respect of IgG content and protective biological activity, Intragam and Polygam, the locally manufactured IM and IV Ig preparations, respectively, compared extremely favourably with the corresponding imported products.

The riminophenazine agents clofazimine and B669 reverse acquired multidrug resistance in a human lung cancer cell line.

The potential of the riminophenazine agents clofazimine and B669, at therapeutically relevant concentrations, to reverse P-glycoprotein-mediated multidrug-resistance (MDR) in a human lung cancer cell line (H69/LX4) has been investigated in vitro. Cyclosporin A, a well-documented MDR-modifying agent, was included for comparison. Clofazimine, B669 and cyclosporin A at minimally cytotoxic concentrations of 1, 0.5 and 5 micrograms/ml, respectively, were equally effective in restoring sensitivity to vinblastine, doxorubicin, daunorubicin and mitomycin C in the H69/LX4 cell line. All three chemosensitizing agents also increased the accumulation of [14C]vinblastine by H69/LX4 cells. Riminophenazines, which are relatively non-toxic, non-carcinogenic and non-myelosuppressive agents, are promising contenders for evaluation in experimental and clinical oncology as modulators of acquired MDR.

In vitro investigation of the intraphagocytic bioactivities of ciprofloxacin and the new fluoroquinolone agents, clinafloxacin (CI-960) and PD 131628.

In this study the intraphagocytic bioactivities of the new fluoroquinolone antimicrobial agents clinafloxacin (CI-960) and PD 131628 (the active metabolite of CI-990) were investigated in vitro at final concentrations of 0.0005-0.5 microgram/ml using human neutrophils and the combination of a radiometric and a colony-counting method, which enabled us to distinguish between intracellular bacteriostatic and bactericidal mechanisms. Ciprofloxacin was included for comparison. Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used as the test intraphagocytic microbial pathogens. Clinafloxacin (> or = 0.05 microgram/ml) displayed potent intraphagocytic bactericidal activity against S. aureus, while PD 131628 was merely bacteriostatic. Ciprofloxacin displayed relatively unimpressive bacteriostatic rather than bactericidal activity for S. aureus. Against E. coli, the intraphagocytic activity of clinafloxacin (0.001-0.005 microgram/ml and above) was superior to that of PD 131628 or ciprofloxacin, which were approximately equipotent. Clinafloxacin is a potent intraphagocytic bactericidal agent for both gram-positive and gram-negative bacteria.

Antimicrobial activities of clofazimine and B669 are mediated by lysophospholipids.

The susceptibilities of a range of gram-positive and gram-negative microbial pathogens to clofazimine and its analog B669 (0.1 to 32 micrograms/ml), as well as the effects of these agents on membrane phospholipid metabolism in Staphylococcus aureus and Escherichia coli, have been investigated in vitro. Gram-positive bacteria were found to be generally susceptible to these agents, whereas gram-negative organisms were uniformly resistant. Exposure of S. aureus to both agents (1 to 5 micrograms/ml), especially B669, caused dose-related enhancement of the activity of phospholipase A2, according to an increase in the release of 3H-radiolabeled arachidonate and lysophosphatidylethanolamine ([3H]LPE) from bacterial-membrane phospholipids. Treatment of E. coli with the riminophenazines also increased the release of [3H]arachidonate and [3H]LPE. Growth of gram-positive but not gram-negative bacteria was inhibited by LPE and lysophosphatidylcholine. Moreover, coincubation with alpha-tocopherol (vitamin E), a lysophospholipid complex-forming agent, or with lysophospholipase protected gram-positive bacteria against the riminophenazines as well as against lysophospholipids. The results from this study are consistent with a mechanism whereby lysophospholipids mediate the activities of the two drugs.

Investigation of the in-vitro uptake, intraphagocytic biological activity and effects on neutrophil superoxide generation of dirithromycin compared with erythromycin.

The cellular uptake by human neutrophils and the intraphagocytic biological activity of the new macrolide antimicrobial agent dirithromycin (0.01-2 mg/L) compared with erythromycin was investigated in vitro. Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila were used as the test intracellular microbial pathogens. After coincubation (45 min at 37 degrees C) of neutrophils with a fixed concentration of 2 mg/L of each antibiotic the respective intracellular/extracellular ratios for erythromycin and dirithromycin were 6.1 +/- 2.5 and 10.6 +/- 2 respectively (P < 0.005). Using a combination of techniques (colony counting, radiometry and fluorescence microscopy) both erythromycin and dirithromycin at concentrations of 0.01 and 0.5 mg/L and higher, respectively, were found to possess dose-related intraphagocytic bacteristatic activity for each of the test microbial pathogens. The effects of dirithromycin and erythromycin (1-20 mg/L) on neutrophil chemotaxis and generation of reactive oxidants by these cells were also investigated in vitro. Both antimicrobial agents caused a dose-related stimulation of neutrophil migration which was associated with inhibition of leucoattractant-activated generation of superoxide and activity of the myeloperoxidase/H2O2/halide system. However, superoxide generation by neutrophils activited with opsonized zymosan or phorbol myristate acetate was unaffected by the macrolides. These findings demonstrate that dirithromycin accumulates in human neutrophils, is biologically active intracellularly and modulates leucoattractant-activated superoxide generation and chemotaxis.

Clindamycin, erythromycin, and roxithromycin inhibit the proinflammatory interactions of Pseudomonas aeruginosa pigments with human neutrophils in vitro.

The Pseudomonas aeruginosa-derived phenazine pigments pyocyanin and 1-hydroxyphenazine (1-hp) prime human neutrophils for enhanced, stimulus-activated release of superoxide and myeloperoxidase (MPO), respectively. In the present study, the modulatory potentials of the antimicrobial agents clindamycin, erythromycin, and roxithromycin (10 and 20 micrograms/ml) on the prooxidative interactions of pyocyanin and 1-hp (12.5 microM) with human neutrophils have been investigated. Clindamycin, erythromycin, and especially roxithromycin caused dose-related inhibition of the generation of superoxide by both untreated and pyocyanin-treated neutrophils during activation with either the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or the calcium ionophore A23187. The antimicrobial agents also inhibited the generation of reactive oxidants by the MPO-H2O2-halide system during activation of both untreated and 1-hp-treated neutrophils by FMLP. These effects appeared to be due to drug-related interference with membrane-associated oxidative metabolism, since none of the antimicrobial agents inhibited the release of MPO by activated neutrophils, nor did they possess oxidant-scavenging properties. These data demonstrate that clindamycin, erythromycin, and especially roxithromycin antagonize the proinflammatory interactions of pyocyanin and 1-hp with neutrophils and indicate a possible therapeutic role for these antimicrobial agents in the prevention of tissue damage in diseases characterized by P. aeruginosa infection.

Auranofin inactivates phosphofructokinase in human neutrophils, leading to depletion of intracellular ATP and inhibition of superoxide generation and locomotion.

The effects of the oral gold compound auranofin (AF), at concentrations well within the therapeutic range (0.04-1.5 microM), on human neutrophil functions and energy metabolism were investigated in vitro. At the concentrations tested, this agent had minimal effects on neutrophil degranulation and phagocytosis. However, AF caused dose-related inhibition of neutrophil chemotaxis and stimulus-activated generation of superoxide, which was evident at concentrations as low as 0.04 microM. Inhibition of superoxide generation by activated neutrophils increased with the time of preincubation of the cells with AF at 37 degrees. At low concentrations of AF (less than 0.75 microM), early events (within 5 min) involved in the transduction, assembly, and activity of the neutrophil superoxide-generating enzyme NADPH oxidase appeared to be normal, but the cells were unable to sustain the level of oxygen consumption, superoxide production, and NADPH oxidase activity of the corresponding drug-free control cells. On a mechanistic level, coincubation of neutrophils with AF was associated with decreased glycolytic activity and depletion of intracellular ATP, apparently due to drug-mediated, dose-related inactivation of the glycolytic enzyme phosphofructokinase (PFK). Using purified PFK, the triethylphosphine gold (TEPG) moiety of AF, but not AF per se, caused dose-related inactivation of enzyme activity. These data indicate that the potent inhibition of neutrophil migration and reactive oxidant generation observed during treatment of neutrophils with low, therapeutically attainable concentrations of AF is related to TEPG-mediated inactivation of PFK and consequent interference with cellular energy metabolism and functions.

Interactions of the oxygen-dependent antimicrobial system of the human neutrophil with difloxacin, ciprofloxacin, pefloxacin and fleroxacin in the intraphagocytic eradication of Staphylococcus aureus.

The effect of the O2-dependent antimicrobial systems of the human neutrophil on the intraphagocytic activity of difloxacin, ciprofloxacin, pefloxacin and fleroxacin was determined by use of a radioassay with Staphylococcus aureus as the test organism. The fluoroquinolones exhibited good intraphagocytic activity with normal neutrophils. However the intracellular bioactivities of the four antimicrobial agents were substantially less in tests with neutrophils from two patients with chronic granulomatous disease. These observations suggest a synergic interaction between fluoroquinolones and the O2-dependent antimicrobial systems of phagocytes in the eradication of intracellular microbial pathogens.

An in vitro investigation of the intraphagocytic bioactivity of difloxacin, ciprofloxacin, pefloxacin and fleroxacin.

In this study the intraphagocytic bioactivity of difloxacin, ciprofloxacin, pefloxacin and fleroxacin was investigated using human neutrophils and a combination of a radioassay, a colony-counting method and a fluorescence microassay which enables us to differentiate between intracellular bacteriostatic and bactericidal mechanisms. Staphylococcus aureus and Listeria monocytogenes were used as the test intraphagocytic microbial pathogens. It was found that difloxacin, ciprofloxacin and to a lesser extent pefloxacin and fleroxacin possess intracellular bacteriostatic activity for S. aureus and L. monocytogenes.

An in-vitro evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A-56268, TE-031), a new macrolide antimicrobial agent.

Erythromycin base and its 6-0-methyl derivative clarithromycin were actively accumulated 7.3 +/- 1.2-fold and 9.2 +/- 2-fold respectively by human neutrophils in vitro. The intraphagocytic bioactivities of the antimicrobial agents were investigated using the combination of a radioassay, colony counting method and a fluorescence microassay which facilitates the distinction between intracellular bacteriostatic and bactericidal mechanisms. Staphylococcus aureus, Listeria monocytogenes and Legionella micdadei were used as the test intraphagocytic microbial pathogens. Both agents were found to possess intracellular bioactivity for all three species of bacteria with clarithromycin being consistently more active than erythromycin. Under the assay conditions used both agents were bacteriostatic (intracellularly) for S. aureus and Leg. micdadei and bactericidal for List. monocytogenes. Clarithromycin is clearly a potent intraphagocytic antibiotic and potentially superior in this respect to erythromycin.