Role of oxidative stress in clofazimine-induced cardiac dysfunction in a zebrafish model.

BACKGROUND: Clofazimine (CFZ), a riminophenazine, is now commonly used in the treatment of multidrug-resistant tuberculosis. However, its use may be potentially associated with cardiac dysfunction in some individuals. In this study, the zebrafish heart, by merit of its developmental and genetic characteristics being in homology with that of human, was chosen as an animal model for evaluation of such dysfunction. METHODS: Morphological and physiological parameters were used to assess cardiac dysfunction. Transcriptome analysis was performed, followed by validation with real-time quantitative PCR, for delineation of the relevant genomics. RESULTS: Exposure of 2 dpf zebrafish to 4 mg/L CFZ for 2 days, adversely affected cardiac functions including significant decreases in HR, SV, CO, and FS, with observable pathophysiological developments of pericardial effusion and blood accumulation in the heart, in comparison with the control group. In addition, genes which respond to xenobiotic stimulus, related to oxygen transport, glutathione metabolism and extracellular matrix -receptor interactions, were significantly enriched among the differentially up-regulated genes. Antioxidant response element motif was enriched in the 5000 base pair upstream regions of the differentially expressed genes. Co-administration of N-acetylcysteine was shown to protect zebrafish against the development of CFZ-induced cardiac dysfunction. CONCLUSIONS: This study suggests an important role of oxidative stress as a major pathogenetic mechanism of riminophenazine-induced cardiac dysfunction.

The Physicochemical Basis of Clofazimine-Induced Skin Pigmentation.

Clofazimine is a weakly basic, Food and Drug Administration-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Due to the drug's red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine-treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.

Tissue concentration, systemic distribution and toxicity of clofazimine--an autopsy study.

There are very few autopsy studies available on systemic distribution of clofazimine, a drug with anti-mycobacterial activity, used in multidrug therapy (MDT) regimen of leprosy and in erythema nodosum leprosum (ENL). An autopsy study was done on a 45 year old female of lepromatous leprosy (LL) on MDT and long term high dosage of clofazimine. Patient succumbed to intractable abdominal pain, diarrhoea, hypokalemia following clofazimine treatment. Autopsy study revealed yellowish brown discoloration of skin, viscera and body fluids. Chemical extraction of the drug revealed the highest concentration of the drug in jejunum (1.5mg/gm),followed by spleen (1.2mg/gm), pancreas (0.4mg/gm), adrenal (0.25mg/gm), liver (0.21mg/gm), and less than 0.2mg/gm in lung, fat, large intestine and stomach. It can be inferred from the present study that the drug is absorbed from the jejunum and gets deposited in fat, reticulo-endothelial cells (R-E cells) and hepatocytes. The drug is best demonstrated in cryostat sections and is lost partly during tissue processing and staining. The drug toxicity can be fatal as seen in the present case.

Efficacy of clofazimine-modified cyclodextrin against Mycobacterium avium complex in human macrophages.

Clofazimine, a water insoluble substituted iminophenazine derivative with anti-mycobacterial and anti-inflammatory activity, is recommended by the WHO for the treatment of leprosy. It is also active against disseminated Mycobacterium avium complex (MAC) disease in HIV-infected patients. Recently, we achieved a 4000-fold increase of clofazimine water solubility using a novel modified clofazimine-cyclodextrin complex synthesized and patented by our group [Wasserlösliche, Iminiophenazinderivate enthaltende pharmazeutische Zusammensetzungen, deren Herstellung und Verwendung, German Patent, DE19814814C2]. In this paper we examine the activity of this complex against MAC in human macrophages, and evaluate its cytotoxicity. MAC-infected macrophages were treated for 24h with free or complexed clofazimine. The in vitro minimum inhibitory concentrations of both free and complexed clofazimine were 0.1 microg/ml. Free and complexed clofazimine inhibited the growth of MAC inside macrophages to a similar extent, while modified cyclodextrin alone had no observable effects on MAC or macrophages. Complexed clofazimine was not toxic to cells at concentrations effective against MAC. The TD(50) of the modified cyclodextrin in THP-1 cell line was 297 microg/ml.

Efficacy of clofazimine - modified cyclodextrin against Mycobacterium avium complex in human macrophages

Clofazimine, a water insoluble substituted iminophenazine derivative with anti-mycobacterial and anti-inflammatory activity, is recommended by the WHO for the treatment of leprosy. It is also active against disseminated Mycobacterium avium complex (MAC) disease in HIV-infected patients. Recently, we achieved a 4000-fold increase of clofazimine water solubility using a novel modified clofazimine-cyclodextrin complex synthesized and patented by our group [Wasserlösliche, Iminiophenazinderivate enthaltende pharmazeutische Zusammensetzungen, deren Herstellung und Verwendung, German Patent, DE19814814C2]. In this paper we examine the activity of this complex against MAC in human macrophages, and evaluate its cytotoxicity. MAC-infected macrophages were treated for 24h with free or complexed clofazimine. The in vitro minimum inhibitory concentrations of both free and complexed clofazimine were 0.1 microg/ml. Free and complexed clofazimine inhibited the growth of MAC inside macrophages to a similar extent, while modified cyclodextrin alone had no observable effects on MAC or macrophages. Complexed clofazimine was not toxic to cells at concentrations effective against MAC. The TD(50) of the modified cyclodextrin in THP-1 cell line was 297 microg/ml.

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.

Liposome encapsulation of clofazimine reduces toxicity in vitro and in vivo and improves therapeutic efficacy in the beige mouse model of disseminated Mycobacterium avium-M. intracellulare complex infection.

Disseminated infections caused by the Mycobacterium avium-M. intracellulare complex (MAC) are the most frequent opportunistic bacterial infections in patients with AIDS. MAC isolates are resistant to many of the standard antituberculous drugs. Failure to obtain significant activities of certain drugs is due to difficulty in achieving high concentrations at the sites where the infections reside. New and improved agents for the treatment of mycobacterial infections are therefore required. Earlier, the anti-MAC activities of various agents in free or liposomal form were studied; liposomes were used as drug carriers to ultimately target the drugs to macrophages where mycobacterial infections reside. Clofazimine was chosen for further studies because it could be effectively encapsulated and its activity was well maintained in liposomal form. The present studies with both erythrocytes and macrophages as the model systems show that liposomal drug is far less toxic in vitro than the free drug. The in vivo toxicity of clofazimine was also significantly reduced after liposome encapsulation. The therapeutic efficacies of free and liposomal drugs were compared in a beige mouse model of disseminated MAC infection. An equivalent dose of liposomal drug (10 mg/kg of body weight) was more effective in eliminating the bacterial from the various organs studied, particularly from the liver. Moreover, because of the reduced toxicity of liposomal drug, higher doses could be administered, resulting in a significant reduction in the numbers of CFU in the liver, spleen, and kidneys. The data demonstrate that liposomal clofazimine is highly effective in the treatment of MAC infections, even if the treatment is initiated after a disseminated infection has been established. The present studies thus suggest the potential usefulness of liposomal clofazimine for the treatment of disseminated MAC infections.

Tissue distribution and deposition of clofazimine in rat following subchronic treatment with or without rifampicin.

Tissue distribution and deposition characteristics of clofazimine (CAS 2030-63-9), an antileprotic drug in rats have been investigated following controlled sub-chronic administration (p.o.) for a period of 1-2 months. The drug was administered alone at a dose of 20 mg/kg body weight and in combination with rifampicin (CAS 13292-46-1) (20 mg/kg p.o.). Various tissues (liver, lung, spleen, small intestine, brain, heart, kidney, skin, stomach and subcutaneous fat) were analyzed for clofazimine in all the treated groups. High levels (range 0.9-3.6 mg/g of wet tissue) were observed in tissues having reticuloendothelial components. In other tissues the levels were relatively lower (range 3-114 micrograms/g of wet tissue). Histopathological studies revealed that clofazimine is deposited in many tissues in the form of reddish-orange crystals. Concomitant treatment with rifampicin did not significantly alter tissue distribution or deposition profile of clofazimine nor did it influence the histopathology.

Reduction of clastogenic effect of clofazimine, an antileprosy drug, by vitamin A and vitamin C in bone marrow cells of mice.

Clofazimine (CLF), an antileprosy drug, has earlier been proved to be clastogenic in mice in vivo. It is an important constituent of the triple-drug regimen recommended by WHO for the treatment of leprosy. In this study the protective role of vitamins A and C (vit A and vit C) against the clastogenic effect of CLF in mouse bone marrow cells has been evaluated. Two doses (20 and 40 mg/kg) of vit C and two doses (2500 and 5000 IU/kg) of vit A were tested against a dose of 40 mg CLF/kg. The drug alone induced chromosomal aberrations of about 8 times the control value. Neither of the doses of vit C exhibited any clastogenic effect and, when administered simultaneously with CLF, both reduced the effect of CLF very significantly, the higher dose reducing chromosomal aberrations almost to the control value. Conversely, both doses of vit A, when administered alone, brought about significant increases in chromosome aberrations over the control value; the higher, but not the lower dose, given simultaneously with CLF, minimized the effect of CLF significantly but not as greatly as vit C. A scavenging effect of the vitamins, removing free radicals produced by CLF, is assumed to be responsible for modulation of the clastogenic effect of CLF.

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. III. Sister chromatid exchange analysis in bone marrow cells.

The potential genotoxicity of an antileprosy drug, clofazimine, was evaluated in mice in an in vivo model by sister chromatid exchange (SCE) analysis. Three different dose levels (4, 20 and 40 mg/kg) were tested, and the animals were treated once daily for 15 days. Sister chromatid differential staining was done by BrdU-tablet implantation and FPG technique. All the doses tested here elevated the SCE frequencies significantly and the increases showed a significant positive correlation with the doses. The results confirm our earlier findings based on metaphase analysis and micronucleus test in the same species.

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. II. Micronucleus test in bone marrow and hepatocytes.

The antileprosy drug, clofazimine, was tested for its possible genotoxicity using micronucleus (MN) tests in mice. A significantly higher incidence of MN in bone marrow erythrocytes, particularly in polychromatic erythrocytes, as well as in regenerated hepatocytes revealed a positive clastogenic effect of the drug. The drug also had a marked antimitotic effect as indicated by a negative correlation with the dose.

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. I. Chromosome analysis in bone marrow and spermatocytes.

Clofazimine, an antileprosy drug, was tested for its cytogenetic effect in mouse bone marrow and testis. Bone marrow metaphase analysis in adults treated directly for different periods (1, 2 and 4 weeks, 40 mg/kg/day) and with different doses (4, 20 and 40 mg/kg/day for 7 days) as well as in young animals exposed through lactation for different periods (2, 3, and 4 weeks) revealed significant increases in chromosomal aberrations over the controls. Analysis of diakinesis-metaphase I stages also exhibited a significantly elevated incidence of chromosome aberrations over controls after treatment for different periods. On the basis of the present result the drug may be considered a potential clastogen in mice.

Clofazimine enteropathy: possible relation to Peyer's patches.

Clofazimine administered orally to rats and mice caused pigmentation of the intestines, draining lymph nodes, fat, and other tissues and organs. Peyer's patches were always more deeply colored than the remainder of the intestine. A microscopic study revealed crystal-containing epithelioid cell granulomas in the patches and in the draining mesenteric lymph nodes but not in the remainder of the gut. During the evolution of the granulomas, some of the epithelioid cells were capable of phagocytosing an iron complex, a circumstance which made it possible to get detailed views of the clofazimine crystals in paraffin sections by negative contrast in histochemical stains for iron. The granulomas appeared after three oral treatments during 1 week, but were better developed after six or more treatments during 2 or more weeks. Similar observations were made in three strains of rats and in mice. We hypothesize that the greater pigmentation of Peyer's patches and their granulomatous response to clofazimine might indicate a special susceptibility to toxic effects of the drug. Whether or not this susceptibility is the starting point for an enteropathy can only be determined by examination of affected human tissues and by further animal experimentation.

Mutagenic activity of antileprosy drugs and their derivatives.

We tested the mutagenic activity of antileprosy drugs (clofazimine, ethionamide, prothionamide, prothionamide-S-oxide, rifampin, and dapsone and many of its derivatives) using the Ames Salmonella/microsome assay system. None of these, including N-acetylated and N-hydroxylated derivatives of dapsone, were found to be positive with or without metabolic activation of this test. However, the sulfoxide and sulfide analogs of dapsone were found to be mutagenic with metabolic activation. These two analogs could not be detected in pharmaceutical preparations of dapsone (less than 0.01%), nor could they be found (in either unconjugated or conjugated form) in urine from volunteers taking a single oral dose of 50 mg of dapsone or from patients receiving daily oral doses of 100 mg of dapsone. Also, urine concentrates from volunteers taking 50 mg of dapsone did not exhibit mutagenic activity in the Ames screen. These results indicate that patients receiving antileprosy therapy with clofazimine, dapsone, ethionamide, prothionamide, and/or rifampin are not being exposed to mutagenic (and thereby possible carcinogenic) drugs.