Safety and tolerability of clofazimine as salvage therapy for atypical mycobacterial infection in solid organ transplant recipients.

BACKGROUND: The treatment of Mycobacterium avium complex (MAC) requires prolonged, multidrug therapy, which is often not well tolerated. In solid organ transplant (SOT) recipients, drug-drug interactions complicate treatment further. Failure or intolerance requires the use of salvage regimens, and clofazimine is one of the drugs that can be used. No data are available on the safety and tolerability of clofazimine for the treatment of MAC in SOT recipients. METHODS: Retrospective review of all SOT recipients treated for MAC infection with clofazimine at a large transplant center between 2006 and 2013. RESULTS: Five SOT recipients received clofazimine as salvage therapy. Transplanted organs were lungs in 3 patients, and kidney and liver in 1 patient each. Infection was diagnosed at a median of 22 months (range 4-57) post transplant. Sites of infection were the lungs in 2 patients, and septic arthritis, mesenteric, and disseminated disease in 1 patient each. All patients received standard anti-MAC therapy for a median of 26 weeks (range 18-45) before starting clofazimine. Indications for use of clofazimine included a lack of response to previous therapy (3 patients), and poor tolerance of other regimens (3 patients). All patients received at least 2 additional drugs besides clofazimine. Median duration of clofazimine-containing regimen was 8 months (range 2-18). Clofazimine was discontinued because of gastrointestinal intolerance in 1 of the 5 patients. The most common adverse event from clofazimine was skin discoloration, in 60% of patients. No hepatotoxicity or hematologic toxicity occurred. Microbiological clearance and resolution of clinical disease was documented in 2 of 5 patients; and 2 of the 5 patients died of other causes while on therapy. CONCLUSIONS: Clofazimine appears safe and may be considered as a salvage therapeutic option in SOT recipients with MAC infection who are intolerant or unresponsive to standard therapy. The small sample size does not allow conclusions regarding efficacy.

Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011.

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993­2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.

Mechanism of thioamide drug action against tuberculosis and leprosy.

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target-drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.

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.

Natural and acquired macrolide resistance in mycobacteria.

The genus Mycobacterium contains two of the most important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, the etiologic agents of tuberculosis and leprosy, respectively. Other mycobacteria are mostly saprophytic organisms, living in soil and water, but some of them can cause opportunistic infections. The increasing incidence of tuberculosis as well as infections with non-tuberculous mycobacteria (NTM) in AIDS patients has renewed interest in molecular mechanisms of drug resistance in these pathogens. Mycobacteria show a high degree of intrinsic resistance to most common antibiotics. For instance, species from the M. tuberculosis complex (MTC) are intrinsically resistant to macrolides. Nevertheless, some semi-synthetic macrolides as the erythromycin derivatives clarithromycin, azithromycin and most recently the ketolides, are active against NTM, particularly Mycobacterium avium, and some of them are widely used for infection treatment. However, shortly after the introduction of these new drugs, resistant strains appeared due to mutations in the macrolide target, the ribosome. The mycobacterial cell wall with its specific composition and structure is considered to be a major factor in promoting the natural resistance of mycobacteria to various antibiotics. However, to explain the difference in macrolide sensitivity between the MTC and NTM, the synergistic contribution of a specific resistance mechanism might be required, in addition to possible differences in cell wall permeability. This mini-review summarizes the current knowledge on the natural and acquired macrolide resistance in mycobacteria, gives an overview of potential mechanisms implicated in the intrinsic resistance and brings recent data concerning a macrolide resistance determinant in the MTC.

Potential role of roxithromycin against the Mycobacterium avium complex.

Until the recent experience with azithromycin and clarithromycin, macrolides were not considered to be important agents against mycobacteria. Clinical evidence is now growing that the newer 14 and 15 membered macrolide compounds have therapeutic activity against Mycobacterium avium, Mycobacterium chelonae and Mycobacterium leprae. Several years ago, when evaluating the activity of roxithromycin using one of the more virulent M. avium in our collection, the authors found that roxithromycin exerted a bacteriostatic effect in cultured human macrophages. However, in combination with tumour necrosis factor, which induces macrophage activation, roxithromycin caused enhanced intracellular killing. The significance of this finding is that tumour necrosis factor can be elaborated by activated macrophages during the course of infection. The roxithromycin doses that were chosen for these studies were less than achievable blood levels. More recently, the in vitro effect of roxithromycin against a panel of isolates from AIDS patients has been assessed and it was found that some (but not all) of the inhibitory concentrations, by the T-100 method of Inderlied, are within achievable serum levels. This, however, may not be the basis for anticipating in vivo activity since macrolide compounds are known to be concentrated within cells and particularly within phagolysosomes. Demonstration of effect in an in vitro test system is encouraging, but should be considered only as a preliminary step to careful assessments in experimental animals, such as the beige mouse, and studies in humans.

Postantibiotic effect of amikacin and rifapentine against Mycobacterium avium complex.

Postantibiotic effect (PAE) has received little attention in the therapy of chronic intracellular infections, such as those caused by mycobacteria. Amikacin is active therapeutically against Mycobacterium avium complex, even though serum levels exceed the MIC for only a few hours. To determine the PAE of amikacin and rifapentine for M. avium, bacteria were exposed to concentrations of 1x, 4x, and 10x the MIC of each drug for up to 120 min. Regrowth of M. avium was compared with similarly diluted untreated cultures. No PAE was observed on an inoculum of 10(4) bacteria when rifapentine was used at 5x MIC, although a slight inhibition of growth was obtained at 10x MIC for 2 h. For amikacin, PAE was observed up to 48 h at concentrations of 4x and 8x MIC and exposure times of 30-120 min. A PAE of 22 h was seen with 10(7) cfu of M. avium during incubation for 30 min with amikacin at 4x MIC. These results show that amikacin, unlike rifapentine, has a long PAE against M. avium.

Correlation between inhibitory effect of quinolones and mycolic acid metabolism in mycobacteria.

Mycolic acids are important components having a significant role in maintaining the rigidity of mycobacterial cell wall. They could also be the barrier for penetration of certain drugs into the bacterial cell. A novel in vitro model system was established for assessing the effect of Ciproflaxacin on mycolic acid metabolism in pathogenic mycobacteria M. Kansasii (which has similar mycolic acid pattern to that from M. leprae) and the effect of norfloxacin in M. intracellulare. These test mycobacteria were exposed in their midlogarithmic phase of growth to 0.5, 1, 2, 3, 4, 5 and 6 micrograms ml of ciprofloxacin and norfloxacin respectively for 1, 2 and 24 hours. Ciprofloxacin completely inhibited the synthesis of mycolates in M. kansasii at 3, 4 and 5 micrograms/ml; whereas norfloxacin exhibited its maximum inhibitory action on mycolic acids in M. intracellulare at 6 micrograms/ml for all the durations of exposure. Inhibition of mycolates directly correlated with bacterial viability which was estimated by colony forming units. The effect of quinolones on mycolic acid metabolism appears to be direct and not secondary to DNA gyrase. The results obtained from this study and our previous findings show that mycolic acid metabolism is affected by various groups of drugs, whose primary sites of activity may be different. The findings of the present study may have significant therapeutic implications in leprosy and other mycobacterial diseases.

Activities of sparfloxacin, azithromycin, temafloxacin, and rifapentine compared with that of clarithromycin against multiplication of Mycobacterium avium complex within human macrophages.

The activities of sparfloxacin, azithromycin, temafloxacin, and rifapentine against two virulent strains of the Mycobacterium avium complex isolated from patients with AIDS were evaluated in a model of intracellular infection and were compared with that of clarithromycin. Human monocyte-derived macrophages were infected with the M. avium complex at day 6 of culture. The intracellular CFU was counted 60 min after inoculation. The intracellular and supernatant CFU was counted on days 4 and 7 after inoculation. The concentrations used, which were equal to peak levels in serum, were 10 micrograms of rifapentine per ml (MICs for the two strains, 4 and 16 micrograms/ml), 4 micrograms of clarithromycin per ml (MICs, 8 and 4 micrograms/ml), 1 microgram of azithromycin per ml (MICs, 32 and 16 micrograms/ml), 4 micrograms of temafloxacin per ml (MICs, 2 and 16 micrograms/ml), and 1 microgram of sparfloxacin per ml (MICs, 0.5 and 2 micrograms/ml). Compared with controls on day 7 after inoculation, clarithromycin (P less than 0.001), sparfloxacin (P less than 0.001), and azithromycin (P less than 0.001 for the first strain, P less than 0.02 for the second) slowed intracellular replication. Rifapentine (P less than 0.001) and temafloxacin (P less than 0.001) slowed intracellular replication of the first strain but not of the second strain. Azithromycin plus sparfloxacin was as effective as sparfloxacin alone. In this macrophage model, sparfloxacin or clarithromycin (difference not significant) exhibited a better efficacy than rifapentine, azithromycin, or temafloxacin against intracellular M. avium complex infection.

Susceptibilities of Mycobacterium leprae and M. avium complex to the H2O2-Fe-mediated halogenation system supplemented with antimicrobial agents.

The susceptibilities of Mycobacterium leprae and M. avium complex (MAC) to the H2-O2-Fe-mediated halogenation system supplemented with antimicrobial agents were evaluated by fluorescein diacetate-ethidium bromide (FDA/EB) staining. In the case of M. leprae, the number of greenstained bacteria (intact cells) was reduced in the presence of the H2O2-Fe-mediated halogenation system supplemented with agents possessing antileprosy activity, such as rifampin, 4,4'-diaminodiphenylsulfone (dapsone), clofazimine, and ofloxacin. In the case of the MAC strain, although viable units of the organisms were reduced by the halogenation system alone, the number of greenstained cells in the FDA/EB stain was not reduced, even when the halogenation system was used in combination with ofloxacin. Because stainability of the cells is related to structural and functional intactness of the membrane, differences between M. leprae and the MAC strain imply possible differences in the rigidity of the cell membrane.

Studies on the mechanisms of the synergistic effects of ethambutol and other antibacterial drugs on Mycobacterium avium complex.

Synergistic effects of combinations of anti-mycobacterial drugs on Mycobacterium avium complex (MAC) in vitro was studied by radiometric respirometry. Pronounced synergy was seen for several drug combinations where ethambutol was found to be the key drug in the synergistic potentiation. Microcalorimetric studies show that a very rapid physico-chemical interaction occurs between the cell-surface of MAC and ethambutol. When MAC cells were pretreated with ethambutol and then subjected to streptomycin the thermal response significantly differed from that seen with MAC cells which had not been pretreated. The typical thermal effects of the interaction of ethambutol with live and UV-killed MAC cells was not seen with heat-killed MAC cells. It is proposed that specific cell-surface protein(s) act as receptors in the initial interaction with ethambutol.