Simultaneous determination of the potent anti-tuberculosis regimen-Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC-MS/MS method coupled with 96-well format plate.

Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 µL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 µm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r2 > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.

Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulations.

The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.

Critical Review on the Analytical Methods for the Estimation of Clofazimine in Bulk, Biological Fluids and Pharmaceutical Formulations.

Clofazimine (CFZ), a riminophenazine derivative and a crucial drug in the treatment of lepromatous leprosy, has been reintroduced clinically to treat multidrug-resistant tuberculosis. CFZ holds both antimycobacterial and anti-inflammatory properties. But, due to its highly hydrophobic, polar and photosensitive nature, it is challenging to extract and quantify the drug from different biological fluids and its pharmaceutical formulations. This has also hampered the pharmacokinetic evaluation of the CFZ. This article accentuates various analytical methods viz. Identification methods, titrimetric methods, spectrometric methods such as colorimetric, fluorometric, mass spectroscopy and UV/Vis spectroscopy, Chromatographic methods like paper chromatography, thin-layer chromatography, high-performance thin layer chromatography, high-performance liquid chromatography, liquid chromatography tandem mass spectrometry for the estimation of CFZ in bulk, biological fluids and its pharmaceutical formulations.

Development and validation of the simultaneous determination of artemisone, clofazimine and decoquinate with HPLC.

The aim of this study was to develop and validate a novel HPLC method for the simultaneous analysis of artemisone, clofazimine and decoquinate. Detection was obtained at two wavelengths; 284 nm (clofazimine) and 210 nm (artemisone and decoquinate). Gradient elution was used with mobile phase A (A) consisting of 0.005 M sodium octanesulphonic-acid (pH 3.5) and mobile phase B (B) of HPLC grade acetonitrile. The flow rate was set to 1.0 ml/min with (A) at 35% and (B) at 65% for 2 min, followed by a gradient shift of 10/90% ((A)/(B)) over a duration of 4 min. After 10 min, the initial gradient conditions were readjusted to 35/65% ((A)/(B)). Distinctive peaks were identified for clofazimine, artemisone and decoquinate, respectively. The proposed HPLC assay method was validated and found to be reliable, reproducible and accurate for simultaneous analysis of the three compounds.

"Ghost peak" of clofazimine: A solution degradation product of clofazimine via nucleophilic substitution by nitrite leaching from certain glass HPLC vials.

Analytical solutions of clofazimine drug substance stored in glass HPLC vials were found to undergo degradation at room temperature occasionally. At the time of each sample preparation, it was unpredictable if a particular solution would undergo such solution degradation. Once the degradation peak was observed in a particular vial, typically within 24h, it would keep growing until reaching a total yield of approximately 2%. By using a strategy that combines LC-PDA/UV-MSn with mechanism-based stress studies, followed by preparative HPLC separation and subsequent structure characterization by 1D and 2D NMR, the unknown peak was identified as a clofazimine nitrite ester. It apparently results from nucleophilic substitution of clofazimine by residual nitrite leaching out of the inner surface of the glass HPLC vials used in the sample preparation. Overall, the percentage of the sample solutions that underwent solution degradation is approximately ∼10% to 15%, when the sample solutions were stored in glass HPLC vials at room temperature. Over the period of the analytical method development, it was found that the occurrence of the degradation can be suppressed when the solutions were stored under refrigerated condition (2 - 8°C) or when the samples were prepared in less acidic diluents.

A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

Bone distribution study of anti leprotic drug clofazimine in rat bone marrow cells by a sensitive reverse phase liquid chromatography method.

The aim of the present study was to investigate the distribution of clofazimine (CLF) in rat bone marrow cells by a validated reverse phase high performance liquid chromatography. CLF and chlorzoxazone (I.S) were extracted by liquid-liquid extraction from plasma and rat bone marrow cells. The chromatographic separation was performed in isocratic mode by the mobile phase consisting of 10mM ammonium formate (pH 3.0 with formic acid) and acetonitrile in a ratio of 50:50 (v/v). The method was accurate and precise in the linear range of 15.6-2000.0 ng/mL with a correlation coefficient (r(2)) of 0.996 and 0.995 in rat plasma and bone marrow cells, respectively. After single oral dose of 20mg/kg, the maximum concentration of CLF in plasma and bone marrow cells were obtained at 12h with the concentrations of 593.2 and 915.4 ng/mL, respectively. The AUC0-t and mean elimination half life (t1/2) of CLF in bone marrow cells were 54339.02 ng h/mL and 52.46 h, respectively, which signified the low body clearance and high distribution of CLF in bone marrow cells. The single oral dose pharmacokinetic investigation was confirmed the CLF endure for a long period in rat due to high distribution in various tissues. The developed method was successfully applied to the estimation of the pharmacokinetic parameters of CLF in plasma and bone marrow cells after administration of single oral dose of 20mg/kg to rats.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

Clofazimina e laritromicina são utilizadas no tratamento da hanseníase e em infecções causadas pelo complexo Mycobacterium avium. Devido à escassez de dados sobre a toxicidade de esquemas terapêuticos que associam estes fármacos, este estudo teve por objetivo avaliar os efeitos adversos desta terapia, em ratos machos Wistar, por meio da determinação da contagem global e específica de leucócitos e ensaios de fagocitose e burst oxidativo de neutrófilos por citometria de fluxo. Houve aumento do número de leucócitos (dose múltipla) e de células polimorfonucleares (doses única e múltipla) nos grupos tratados com claritromicina em monoterapia ou associada à clofazimina e redução das células mononucleares, em doses única e múltipla, nos mesmos grupos. Os fármacos parecem inverter a proporção entre células mono e polimorfonucleares. Observou-se aumento do burst oxidativo nos animais tratados com os fármacos isolados ou associados. Concluindo, clofazimina e claritromicina provocam alterações leucocitárias e os resultados podem contribuir para melhor entendimento dos mecanismos relacionados aos efeitos da administração dos fármacos em associação.

Molecular imaging of intracellular drug-membrane aggregate formation.

Clofazimine is a lipophilic antibiotic with an extremely long pharmacokinetic half-life associated with the appearance of crystal-like drug inclusions, in vivo. Here, we studied how clofazimine accumulates inside cells in the presence of supersaturating, extracellular concentrations of the drug (in the range of physiological drug concentrations). Based on a combination of molecular imaging, biochemical analysis and electron microscopy techniques, clofazimine mass increased inside cells in vitro, over a period of several days, with discrete clofazimine inclusions forming in the cytoplasm. These inclusions grew in size, number and density, as long as the drug-containing medium was replenished. With Raman confocal microscopy, clofazimine's spectral signature in these inclusions resembled that of amorphous clofazimine precipitates and was unlike that of clofazimine crystals. Additional experiments revealed that clofazimine first accumulated in mitochondria, with ensuing changes in mitochondrial structure and function. In turn, the degenerating organelles coalesced, fused with each other and condensed to form prominent drug-membrane aggregates (dubbed autophagosome-like drug inclusions or "aldis"). Like clofazimine, it is possible that intracellular drug-membrane aggregate formation is a common phenomenon underlying the reported phenotypic effects of many other small molecule drugs.

Probing cell chemistry with time-of-flight secondary ion mass spectrometry: development and exploitation of instrumentation for studies of frozen-hydrated biological material.

Imaging static secondary ion mass spectrometry (SIMS) offers a powerful method of obtaining molecular information from biological systems with good spatial resolution. However, the technique needs further development to make it suitable for routine analysis of cells. We report here the development of a new freeze-facture device to facilitate the manipulation and analysis of biological cell material, with the cell chemistry preserved intact by rapid freezing. We illustrate performance characteristics with high-contrast images of freeze-fractured, frozen-hydrated liposomes with the drug clofazamine constrained within the lipid bilayer providing a marker to determine the fracture plane across the liposome structure. By monitoring and imaging clofazamine on the surface of yeast cells in the frozen-hydrated state, and demonstrating its absence within molecular information from a cell fractured to reveal the cell ultrastructure, we demonstrate that the molecule does not penetrate the cell wall.

Determination of serum and tissue levels of phenazines including clofazimine.

A rapid and sensitive HPLC method is described for the analysis of synthetic phenazines, including clofazimine, from a variety of biological samples. Phenazines were extracted from serum, tissue and fat using a mixture of dichloromethane and sodium hydroxide. The drugs were then quantified on a reversed-phase C18 column using a mobile phase consisting of 594 ml of water, 400 ml of tetrahydrofuran, 6 ml of concentrated acetic acid and 0.471 g of hexanesulfonic acid. In this mobile phase, each phenazine tested had its own retention time. This allowed one phenazine to be used as an internal standard for the analysis of other phenazines. The method was validated for clofazimine [3-(4-chloroanilino)-10-(4-chlorophenyl)-2,10-dihydro-2-(isopro pylimino) phenazine] and B4090 [7-chloro-3-(4-chloranilino)-10-(4-chlorophenyl)-2, 10-dihydro-2-(2,2,6,6-tetramethylpiperid-4-ylimino)phenazine ] (VI) and shown to be accurate and precise across a broad concentration range from 0.01 to 50 micrograms/g (microgram/ml). Extraction was 100% for each agent across this range. This system was used to measure clofazimine and VI levels following their administration to rats. The pharmacokinetic profile of VI was different to that of clofazimine, with high tissue concentrations but lower fat levels.

Study of intracellular deposition of the anti-leprosy drug clofazimine in mouse spleen using laser microprobe mass analysis.

Laser microprobe mass analysis (LAMMA) was used to study the composition of the brick-red crystalline material which had accumulated in the spleen of mice that had received the anti-leprosy drug Clofazimine in their diet for several months. The crystalline deposits light-microscopically resembled pure Clofazimine crystals. The presence of the drug in the crystals was indicated by LAMMA by the appearance of the chloride mass peaks in the negative mass spectra. More specific information was obtained from the positive mass spectra. A mass signal for the protonated molecule was present.

Red discoloration of the sputum by clofazimine simulating haemoptysis--a case report.

A patient of lepromatous leprosy, who received a high dose of clofazimine as part of multidrug therapy, for chronic erythema nodosum leprosum (ENL) had frequent 'haemoptysis'. The haemoptysis was later found to be due to expectoration of clofazimine. This interesting, and perhaps first case of such an occurrence, is reported.

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.

[Exudative enteropathy caused by clofazimine: apropos of a case]. / Entéropathie exsudative à la clofazimine: à propos d'une observation.

The authors report a case of a forty-six year old woman suffering from generalized prurigo nodularis. This dermatosis was associated with a cellular immunodeficiency; therefore a clofazimine therapy 300 mg/a day was instituted for six months. Ten months after the cessation of the clofazimine therapy, there appeared a malabsorption syndrome that was temporarily improved by gluten--free diet the real etiology was only ascertained during laparotomy, when masses of crystals in the small intestine mucosa as well as in mesenteric lymph nodes were observed. Therefore when it is necessary to prescribe clofazimine to take advantage of become its immunoregulating properties, it must always become in mind that an intestinal complication may ensue: this is well recognized in articles appearing in journals devoted to leprosy.