Metabolism and interactions of antileprosy drugs.

Leprosy is a chronic infectious disease caused my Mycobacterium leprae that primarily affects peripheral nervous system and extremities and is prevalent in tropical countries. Treatment for leprosy with multidrug regimens is very effective compared to monotherapy especially in multibacillary cases. The three major antileprosy drugs currently in use are 4, 4'-diaminodiphenyl sulfone (DDS, dapsone), rifampicin, and clofazimine. During multidrug therapy, the potent antibiotic rifampicin induces the metabolism of dapsone, which results in decreased plasma half-life of dapsone and its metabolites. Furthermore, rifampicin induces its own metabolism and decreases its half-life during monotherapy. Rifampicin upregulates several hepatic microsomal drug-metabolizing enzymes, especially cytochrome P450 (CYP) family that in turn induce the metabolism of dapsone. Clofazimine lacks significant induction of any drug-metabolizing enzyme including CYP family and does not interact with dapsone metabolism. Rifampicin does not induce clofazimine metabolism during combination treatment. Administration of dapsone in the acetylated form (acedapsone) can release the drug slowly into circulation up to 75 days and could be useful for the effective treatment of paucibacillary cases along with rifampicin. This review summarizes the major aspects of antileprosy drug metabolism and drug interactions and the role of cytochrome P450 family of drug metabolizing enzymes, especially CYP3A4 during multidrug regimens for the treatment of leprosy.

Evaluating the safety, tolerability, pharmacokinetics and efficacy of clofazimine in cryptosporidiosis (CRYPTOFAZ): study protocol for a randomized controlled trial.

BACKGROUND: Cryptosporidium infection and diarrhea (cryptosporidiosis) is a life-threatening infection in persons with HIV and also in children of 6-18 months of age in the developing world. To date, only nitazoxanide is licensed for treatment of cryptosporidiosis, and only in persons after the first year of life and with healthy immune systems. Clofazimine (CFZ: Lamprene®), an established drug that has been used for leprosy for more than 50 years, recently has been described as effective against Cryptosporidium in vitro and in mouse infections. The efficacy and pharmacokinetics of CFZ in vivo, in HIV-infected patients with cryptosporidial diarrhea are not known. METHODS: CRYPTOFAZ includes a randomized, double-blind, placebo-controlled study of the safety, tolerability and Cryptosporidium inhibitory activity of orally administered CFZ in subjects with HIV infection and chronic diarrhea with Cryptosporidium. An additional open label aspect of the study will compare the pharmacokinetics (PK) of orally administered CFZ in HIV-infected individuals with and without Cryptosporidium-associated diarrhea. The study will recruit a total of 66 subjects. Study participants will be given either CFZ or a placebo for 5 days while in hospital and will be followed up after discharge. Cryptosporidium will be diagnosed by quantitative PCR as the definitive test and by stool ELISA, which will also be used to quantify the shedding of Cryptosporidium in stool. PK will be studied on plasma and stool samples. Primary endpoints include reduction in the number of Cryptosporidium shed in stools over a 5-day period and compared to placebo recipients and the PK of CFZ in plasma assessed by area under the curve, peak plasma concentration, and half-life (T ½) determined after the last dose. DISCUSSION: This study provides an opportunity to explore a possible treatment option for HIV-infected patients with cryptosporidial diarrhea, who, as of now in Malawi and most of sub-Saharan Africa, do not have a definitive treatment apart from supportive care. The strength of this study lies in it being a randomized, double-blind, placebo-controlled trial. If shown to be effective and safe, the findings will also lay a foundation for a future study of the use of CFZ in children 6-18 months of age. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03341767 . Registered on 14 November 2017.

Clofazimine protects against Mycobacterium tuberculosis dissemination in the central nervous system following aerosol challenge in a murine model.

Tuberculosis (TB) has been the scourge of the human race for many decades, claiming countless number of lives. This is further complicated by the ability of Mycobacterium tuberculosis to infect extrapulmonary sites, specifically the brain. These extrapulmonary forms of TB are difficult to treat owing to problems associated with drug delivery across the blood-brain barrier. Linezolid (LIN) and clofazimine (CFZ) are two of the more promising anti-TB drugs in recent times. In this study, BALB/c mice were aerosol-infected with M. tuberculosis H37Rv and were treated for 4 weeks with LIN [100 mg/kg body weight (BW)] or CFZ (100 mg/kg BW). Concurrently, it was investigated whether an aerosol TB infection would lead to dissemination of TB bacilli into the brain. Post-treatment brain and lung CFUs were determined together with serum, lung and brain drug concentrations. CFZ displayed a strong bactericidal effect in the lung, whilst LIN had a bacteriostatic effect. Mycobacterium tuberculosis appeared at 2 weeks post-infection in the untreated group (2.38 ± 0.43 log10 CFU) and more surprisingly at 3 weeks post-infection in the LIN-treated group (1.14 ± 0.99 log10 CFU). TB bacilli could not be detected in the brains of the CFZ-treated group. To the best of our knowledge, this is the first study showing the appearance of M. tuberculosis in the brain following a murine aerosol TB infection. This study may advocate the use of CFZ as prophylactic treatment to prevent the development of extrapulmonary TB of the central nervous system using a two-pronged approach.

Quantitative analysis of clofazimine (Lamprene®), an antileprosy agent, in human dried blood spots using liquid chromatography-tandem mass spectrometry.

An LC-MS/MS method was developed and validated for bioanalysis of clofazimine in human dried blood spot (DBS) samples in support of a clinical study on multidrug-resistant tuberculosis in developing countries. The validated assay dynamic range was from 10.0 to 2000 ng/mL using a 1/8 inch DBS punch. The accuracy and precision of the assay were ±11.0% (bias) and ≤13.5% (CV) for the LLOQs (10.0 ng/mL) and ±15% (bias) and ≤15% (CV) for all other QC levels. The assay was proved to be free from the possible impact owing to spot size and storage temperature (e.g. at 60°C, ≤ - 60°C). The validated assay is well suited for the intended clinical study where conventional pharmacokinetic sample collection is not feasible.

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.

Multiscale distribution and bioaccumulation analysis of clofazimine reveals a massive immune system-mediated xenobiotic sequestration response.

Chronic exposure to some well-absorbed but slowly eliminated xenobiotics can lead to their bioaccumulation in living organisms. Here, we studied the bioaccumulation and distribution of clofazimine, a riminophenazine antibiotic used to treat mycobacterial infection. Using mice as a model organism, we performed a multiscale, quantitative analysis to reveal the sites of clofazimine bioaccumulation during chronic, long-term exposure. Remarkably, between 3 and 8 weeks of dietary administration, clofazimine massively redistributed from adipose tissue to liver and spleen. During this time, clofazimine concentration in fat and serum significantly decreased, while the mass of clofazimine in spleen and liver increased by >10-fold. These changes were paralleled by the accumulation of clofazimine in the resident macrophages of the lymphatic organs, with as much as 90% of the clofazimine mass in spleen sequestered in intracellular crystal-like drug inclusions (CLDIs). The amount of clofazimine associated with CLDIs of liver and spleen macrophages disproportionately increased and ultimately accounted for a major fraction of the total clofazimine in the host. After treatment was discontinued, clofazimine was retained in spleen while its concentrations decreased in blood and other organs. Immunologically, clofazimine bioaccumulation induced a local, monocyte-specific upregulation of various chemokines and receptors. However, interleukin-1 receptor antagonist was also upregulated, and the acute-phase response pathways and oxidant capacity decreased or remained unchanged, marking a concomitant activation of an anti-inflammatory response. These experiments indicate an inducible, immune system-dependent, xenobiotic sequestration response affecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.

In vitro and in vivo activity of clofazimine against Mycobacterium tuberculosis persisters.

OBJECTIVE: To assess the activity of clofazimine (CFZ) against Mycobacterium tuberculosis persisters using an oxygen depletion model and a low-dose aerosol mouse model of chronic tuberculosis (TB). DESIGN: In in vitro experiments, CFZ showed much better activity than isoniazid under anaerobic conditions. In a low-dose aerosol mouse model of TB, we evaluated the efficacy of CFZ and moxifloxacin at different doses following treatment durations of 30, 60 and 90 days. RESULTS: CFZ showed significant bactericidal activity in the mouse model over the wide dose range of 2-200 mg/kg. CFZ activity was dose-dependent. The bacilli were eradicated in the CFZ 200 mg/kg group after treatment for 60 days, and in the CFZ 20 mg/kg group after 90 days of treatment. CONCLUSION: CFZ exhibits dose-dependent, sustained bactericidal activity against M. tuberculosis persisters, and thus warrants further study to demonstrate its potential to contribute significantly in a novel treatment-shortening regimen.

Pharmacokinetics and relative bioavailability of clofazimine in relation to food, orange juice and antacid.

BACKGROUND: Clofazimine is potentially useful for the treatment of disease due to multidrug resistant Mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases. Its pharmacokinetics have been incompletely characterized. This study was conducted to explore issues relating to bioavailability in the presence of food, orange juice, and antacid. METHODS: A 5 drug regimen consisting of clofazimine, cycloserine, ethionamide, para-aminosalicyclic acid, and pyridoxime was administered to healthy subjects four times using a four period cross-over design with two weeks washout between treatments. Subjects also received orange juice, a high fat meal, aluminum/magnesium antacid, or only water in random order with the drug regimen. The pharmacokinetics of clofazimine were assessed using individual- and population-based methods and relative bioavailability compared to fasting administration was determined. RESULTS: Clofazimine exhibited a sometimes prolonged and variable lag-time and considerable variability in plasma concentrations. From the population analysis (one-compartment model), the mean oral clearance was 76.7 l/h (CV=74.2%) and mean apparent volume of distribution was 1470 l (CV=36.3%). The first-order absorption rate constant ranged from 0.716 to 1.33 h(-1) (pooled CV=61.7%). Residual (proportional) error was 49.1%. Estimates of bioavailability compared to fasting administration were 145% (90% CI, 107-183%) for administration with high fat food, 82.0% (63.2-101%) for administration with orange juice, and 78.5% (55.1-102%) for administration with antacid. CONCLUSION: Administration of clofazimine with a high fat meal provides the greatest bioavailability, however, bioavailability is associated with high inter- and intra-subject variability. Both orange juice and aluminum-magnesium antacid produced a reduction in mean bioavailability of clofazimine.

Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture.

The activity of oral clofazimine against intracellular Mycobacterium tuberculosis was compared to that of ofloxacin in healthy volunteers by the use of whole-blood cultures. Clofazimine was inactive whether it was tested alone or combined with other drugs that are used to treat multidrug-resistant tuberculosis, despite a total dose of 2 g. Kanamycin was the most active drug tested.

HPLC determination of clofazimine in tissues and serum of mice after intravenous administration of nanocrystalline or liposomal formulations.

A simple HPLC method is described for the determination of clofazimine in mouse tissues and in serum. The main application of the method was the determination of the drug in mouse tissues after i.v. administration of nanocrystalline suspensions or liposomal encapsulated clofazimine. Tissues were extracted with a 10-fold (w/v) volume of an extraction solution consisting of methanol/glacial acetic acid 9:1 (v/v). Serum proteins were precipitated with a 2-fold volume of acetonitrile. Isocratic chromatography was performed using an anion exchange column (Nucleosil 100-5 SA, Macherey & Nagel) for separation. The mobile phase was a mixture of acetonitrile and 0.1 mol/l aqueous phosphoric acid (75:25, v/v), adjusted to pH 2.9 with sodium hydroxide solution. Absorption of the eluate was monitored at 495 nm. The assay was precise, simple to perform and fast. Recovery from tissues was > or = 98%, from nanoparticles > or = 98%, and from liposomes > or = 96%. No interference was observed in extracts from mouse liver, spleen, lungs and human serum.

Combined dapsone and clofazimine intoxication.

We report clinical findings and pharmacokinetic data regarding a combined dapsone and clofazimine intoxication in a man, who ingested 50 tablets of dapsone (100 mg) 20 capsules of clofazimine (100 mg) and two tablets of rifampicin (600 mg). Oral administration of activated charcoal (50 grams) and sodium sulphate (20 grams) after gastric lavage resulted in an elimination half-life in plasma of 11.1 and 10.8 h for dapsone and its main metabolite, monoacetyldapsone, respectively. A rapid initial decrease of the plasma concentration of clofazimine was observed after gastric lavage and administration of activated charcoal and sodium sulphate. 15 h after this treatment, clofazimine plasma levels remained relatively constant. Dapsone-induced methaemoglobinaemia (48% at admission) was treated successfully with methylene blue.

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.

Comparative bioavailability of clofazimine coevaporate in the pig.

The systemic availability of a solid dispersion (coevaporate) of clofazimine (CLF) in poly(vinyl methyl ether maleic anhydride) copolymer (PVM/MA) was tested in the pig. Single 100 mg oral doses of the coevaporate and the commercial product, Lamprene (Ciba-Geigy) were administered on separate occasions (separated by a two-week washout period) to four pigs (two males, two females) in a random cross-over study. Multiple plasma samples, obtained from an indwelling jugular-vein cannula, following drug administration, were analysed by an HPLC method for CLF. Pharmacokinetic analyses of the plasma CLF concentration-time data were performed. A paired t-test indicated significant differences (p < 0.05) between the coevaporate and Lamprene in the Cpmax, tmax, and AUC. The calculated relative systemic bioavailability (Frel) of CLF from the coevaporate, relative to that from Lamprene, was three. It is concluded that formulation of CLF, as a solid dispersion, may provide enhanced aqueous dissolution and systemic absorption and may also provide high therapeutic blood levels. These could lead to reduction in the current therapeutic doses and, consequently, minimization of drug-related side effects.

Low serum levels of oral antimycobacterial agents in patients with disseminated Mycobacterium avium complex disease.

Twenty-seven human immunodeficiency virus-infected patients with disseminated Mycobacterium avium complex disease who were treated with oral antimycobacterial agents (clofazimine, ciprofloxacin, ethambutol, and rifampin) were studied to evaluate the usefulness of monitoring serum drug concentrations and testing in vitro susceptibility of M. avium complex (MAC) isolates. Twenty patients tolerated treatment with three or four antimycobacterial agents for at least 8 weeks; mycobacteremia was eradicated in 7 (35%). The in vitro susceptibilities of MAC isolates to antimycobacterial agents were similar for these 7 and for the 13 who did not respond to antimycobacterial treatment. Serum drug levels were below the expected range in 6 of the 7 whose mycobacteremia was cleared and in 9 of the 13 nonresponders (P = .41). These low serum concentrations of antimycobacterial drugs may be due to impaired drug absorption in patients with AIDS and disseminated MAC disease.

A rapid and sensitive high performance liquid chromatographic analysis of clofazimine in plasma.

The high performance liquid chromatographic (HPLC) method of Gidoh, et al. has been modified substantially to provide a simple, rapid, and relatively inexpensive procedure for measuring clofazimine in plasma. The modification involves the use of commonly available laboratory reagents instead of custom-made ones. It also employs a solid phase system for efficient extraction instead of the conventional, less efficient and more labor intensive, liquid-liquid extraction. The inclusion of an internal standard (salicylic acid) improves the precision and reproducibility. It is demonstrated that the method can be used to monitor in vivo clofazimine levels as may be required in formal pharmacokinetic studies or therapeutic drug monitoring.

Determination of three main antileprosy drugs and their main metabolites in serum by high-performance liquid chromatography.

The simultaneous analysis of main antileprosy drugs such as 4,4'-diaminodiphenyl sulfone (DDS), clofazimine, rifampicin and their main metabolites in serum was examined by high-performance liquid chromatography using a muBondapak C18 column. When the drugs dissoluted from serum were developed by tetrahydrofuran-0.5% acetic acid (40:60), clofazimine and rifampicins could be analyzed separately. Apart from the mutual separation of water-soluble conjugates of DDS, the individual analysis of DDS, its main liposoluble metabolite and a few related sulfone compounds is possible when the drugs are first developed by acetonitrile-water (20:80). By the use of tetrahydrofuran-water (50:50) containing PIC B-5, the rapid measurement of clofazimine isolated from the other compounds is also possible.

Quantitative estimation of clofazimine in tissue.

The histological examination and the chemical estimation of clofazimine in the organs removed at autopsy of a patient receiving the drug indicated the accumulation of the drug in the organs of the reticulo-endothelial system and those having a large number of macrophages. The clofazimine levels in the skin from patients receiving the drug and those in whom the drug had been stopped for different periods of time were compared. The skin levels of clofazimine bore a direct relationship to the size of the granuloma. A slow elimination of the drug from the tissues was indicated by the presence of traces of the skin tissue even 1-2 years after stopping the drug. The clinical implication of these findings are discussed.