The influence of structural modifications of dihydrophenazines on arachidonic acid mobilization and superoxide generation by human neutrophils.

In this study the effects of nine dihydrophenazine derivatives, relative to clofazimine (B663), on the N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) stimulated release of superoxide anion and on the spontaneous generation of arachidonic acid by human neutrophils were investigated. Previous findings that the pro-oxidative activity of the agents depended largely on the substitution in position 2 of the phenazine molecule and on chlorination in the paraposition of the phenyl and anilino rings were confirmed. Only riminophenazines, but not aposafranone derivatives or the imidazophenazine B621, could enhance superoxide release from activated neutrophils. The lack of chlorination of the phenyl and anilino rings could be compensated for by chlorine substitution in position 7 of the phenazine core. The priming effect of the agents on FMLP stimulated superoxide generation was completely prevented by the phospholipase A2 inhibitor 4-p-bromophenacyl bromide. Furthermore pro-oxidative activities correlated closely with a stimulatory effect of the agents on arachidonic acid release. It was therefore concluded that dihydrophenazine derivatives with pro-oxidative properties can prime neutrophils for FMLP-stimulated superoxide release by modulation of phospholipase A2 activity.

Investigation of the structural properties of dihydrophenazines which contribute to their pro-oxidative interactions with human phagocytes.

Twenty-six dihydrophenazine compounds, including clofazimine, were investigated, at a fixed concentration of 1 mg/l, for their effects on spontaneous and stimulus-activated generation of superoxide by human neutrophils in vitro. The synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L phenyl-alanine (FMLP) (0.1 microM) was used as a stimulant of membrane-associated oxidative metabolism. None of the agents tested influenced basal levels of superoxide generation by neutrophils. However sixteen of these compounds, all rimino phenazines, significantly increased the production of superoxide by FMLP-activated neutrophils. These pro-oxidative, priming interactions of the rimino phenazines with neutrophils were largely dependent on the nature of the alkylimino group at position 2 on the phenazine nucleus, and to a lesser extent on halogenation. Cycloalkylimino groups were generally less potent stimulants of superoxide generation by FMLP-activated neutrophils than clofazimine, and their pro-oxidative properties were independent of mono- or dichlorination. However the halogen-free cycloalkylimino compound, B669, was an exceptionally potent pro-oxidative agent. Chlorination was promotive to pro-oxidative activity in the case of dihydrophenazines with linear or branched alkylimino substituents. The pharmaco-therapeutic mechanisms of dihydrophenazines may be related to their pro-oxidative interactions with phagocytes.

Mononuclear leucocyte function in patients with lichen planus and cutaneous lupus erythematosus during chemotherapy with clofazimine.

Mitogen-induced transformation and the production of reactive oxidants by mononuclear leucocytes (MNLs) from patients with chronic dermatological disorders were investigated in vitro before and during the administration of the antimycobacterial/immunosuppressive agent clofazimine (200 mg 3 times weekly). Seven patients, 4 with lichen planus and 3 with cutaneous lupus erythematosus, were included in the study. Clofazimine administration did not influence the mitogen-induced proliferative responses of patients' MNLs. However, chemotherapy with this drug stimulated the production of reactive oxidants by MNLs. Since reactive oxidants are immunosuppressive it is possible that these effects may be involved in the pharmacotherapeutic activity of clofazimine.

Clofazimine-mediated enhancement of reactive oxidant production by human phagocytes as a possible therapeutic mechanism.

Clofazimine, at concentrations within the therapeutic range (0.01-5 micrograms/ml), stimulated human polymorphonuclear leucocytes (PMNL) to generate increased amounts of reactive oxidants (RO) when activated with the tripeptide leucoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), calcium ionophore, phorbol myristate acetate and opsonised zymosan. Clofazimine per se did not activate the membrane-associated oxidative metabolism of PMNL, but rather primed these cells to hyperreact to the various stimuli. To investigate the therapeutic significance of these observations clofazimine was administered to patients with various chronic inflammatory diseases (lichen planus, discoid lupus erythematosus and rheumatoid arthritis) and generation of RO by FMLP-activated phagocytes was measured before and during clofazimine administration. A statistically significant potentiation of RO generation by FMLP-activated phagocytes was observed during clofazimine administration. Since RO are immunosuppressive and antimicrobial the therapeutic mechanisms of clofazimine may be related to pro-oxidative interactions of this agent with phagocytes.

Antimycobacterial drugs and the production of reactive oxidants by polymorphonuclear leucocytes in vitro.

The effect of five antimycobacterial drugs isoniazid, rifampicin, streptomycin, pyrazinamide and ethambutol on the generation of reactive oxidants by polymorphonuclear leucocytes was investigated in vitro, using N-formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) activated and spontaneous luminol-enhanced chemiluminescence and myeloperoxidase-mediated iodination. Streptomycin, pyrazinamide and ethambutol had no effect on the assays at the concentrations investigated. Isoniazid as concentrations of 1.25 and 5 micrograms/ml and rifampicin at 100 micrograms/ml significantly inhibited iodination. Rifampicin also caused dose-dependent inhibition of chemiluminescence which was partly due to its light-absorbing activities. It is concluded that isoniazid, and to a lesser extent, rifampicin at therapeutic concentrations possess anti-oxidative properties.

Prooxidative activities of 10 phenazine derivatives relative to that of clofazimine.

The objective of this study was to investigate the relationship between the antimycobacterial properties of the antileprosy drug clofazimine and its stimulatory effect on the release of reactive oxidants by polymorphonuclear leukocytes by using a variety of phenazine derivatives. The effects of these compounds on myeloperoxidase-mediated iodination, luminol-enhanced chemiluminescence, and the release of superoxide anion by polymorphonuclear leukocytes were investigated. Dissociation of the antimycobacterial and prooxidative effects of clofazimine was possible by manipulation of the chemical group in position 2 of the phenazine molecule. When nitrogen-containing substituents in this position were replaced by oxygen, the mode of the prooxidative action of the compounds was altered.

Effects of anti-tuberculosis drugs on the production of prostaglandin E2 and on mononuclear leucocyte transformation.

The effects of the anti-tuberculosis (TB) drugs isoniazid, rifampicin, streptomycin, pyrazinamide and ethambutol on the synthesis of prostaglandin E2 (PGE2) by polymorphonuclear leucocytes were investigated. Only rifampicin at a concentration of 100 micrograms/ml caused statistically significant stimulation of PGE2 production whereas the other compounds had no effect. Immunomodulatory properties of the compounds individually and in combination were investigated using phytohaemagglutinin-stimulated mononuclear leucocyte transformations. Rifampicin caused dose-dependent inhibition of blastogenesis and combinations containing this agent also had an inhibitory effect. The prostaglandin synthesis inhibitor indomethacin caused stimulation of phytohaemagglutinin responsiveness and this effect was not influenced by the presence of the anti-TB drugs.

Clofazimine-mediated stimulation of prostaglandin synthesis and free radical production as novel mechanisms of drug-induced immunosuppression.

The effects of clofazamine (3-(p-chloroanilino)-10-(p-chlorophenyl)-2, 10-dihydro-2-(isopropylimino)-phenazine) at concentrations of 0.625-20 micrograms/ml on the mitogen-induced transformation, luminol-enhanced chemiluminescence, arachidonic acid metabolism and sulphydryl content of human mononuclear leucocytes (MNL) were investigated in vitro. The drug at all concentrations tested decreased MNL sulphydryl content and inhibited mitogen-induced transformation. Clofazimine increased the spontaneous luminol-enhanced chemiluminescence and activated the arachidonic acid cascade in MNL. The anti-oxidants ascorbic acid and cysteine and the prostaglandin (PG) synthesis inhibitor indomethacin were used individually and in combination to identify the primary mediators of the anti-proliferative effects of clofazimine on MNL. Combinations of an anti-oxidant with a PG synthesis inhibitor completely protected MNL from clofazimine mediated inhibition of mitogen-induced transformation. These results show that the anti-proliferative activity of clofazimine is related to both the pro-oxidative and PG synthesis enhancing effects of the drug on MNL.