Lack of Activity of Orally Administered Clofazimine against IntracellularMycobacterium tuberculosisin Whole-Blood Culture

Abstract

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.Multidrug-resistant tuberculosis (MDR TB) poses a growing public health problem. Studies to assess tissue sterilization in MDR TB are particularly long and complex. Members of our laboratory recently described a method for studying the ability of administered chemotherapy to kill intracellular Mycobacterium tuberculosis cells by using whole-blood cultures (14). In patients with drug-sensitive TB, the whole-blood bactericidal activity (WBA) was correlated with two sputum markers of tissue sterilization, the 8-week sputum culture conversion and the serial sputum CFU slope (16). In the present study, the whole-blood model was used to examine the potential role of clofazimine in the treatment of MDR TB.Our subjects consisted of 10 healthy tuberculin-nonreactive volunteers who provided written informed consent according to an Institutional Review Board-approved protocol. They were randomly assigned to receive only ofloxacin (600 mg) or clofazimine (200 mg) daily for 5 days. Pyrazinamide (25 mg/kg of body weight) was added on days 6 to 10. Ethambutol (25 mg/kg) was added on day 10. This schedule permitted the intracellular accumulation of clofazimine, ofloxacin, and pyrazinamide in phagocytic cells. Kanamycin was added to wholeblood cultures at final concentrations of 20 and 10 g/ml in blood specimens at 2 and 6 h postdose, respectively, to mimic blood levels of 40 and 20 g/ml, respectively. Drug addition was delayed 30 min after infection to allow for phagocytosis.WBA was assessed as previously described (5, 14, 16). Briefly, heparinized blood was diluted 1:1 with medium and infected with M. tuberculosis H37Rv. Members of our laboratory previously documented the high efficiency of phagocytosis in whole-blood cultures (Ͼ95%) by examining CFU counts in the liquid and cellular partitions after low-speed sedimentation and by showing the lack of effect of amoxicillin-clavulanate, which does not reach adequate intracellular levels to act against M. tuberculosis (14); this eliminates the need to remove extracellular bacilli. After 72 h of incubation, the host cells were disrupted by hypotonic lysis, and bacilli were sedimented and inoculated into BACTEC medium. Bacillus killing was calculated based on days-to-positivity by using a standard curve. The total bactericidal effect was assessed as the area under the curve (AUC) (16). The results were examined by a paired or unpaired t test.At the baseline, there was a net growth of M. tuberculosis H37Rv of 0.41 log CFU over 72 h in the subjects' blood cells. A single dose of clofazimine had no effect in blood sampled 2 h afterward (mean, 0.42 log CFU; P ϭ 0.3), whereas the corresponding effect of ofloxa...

Biomarker-Assisted Dose Selection for Safety and Efficacy in Early Development of PNU-100480 for Tuberculosis

“…Interactions of infected cells with lymphocytes and other cells result in restriction of intracellular mycobacterial growth: partial restriction in the blood of healthy volunteers and full bacteriostasis in the blood of TB patients (10,26,27). Cumulative WBA during anti-TB therapy correlates with 2-month sputum culture status and serial sputum CFU counts, is greater for rifampin than isoniazid, and is greater for the standard four-drug therapy than for current treatment regimens for MDR-TB (11,25,27). It is therefore indirectly related to treatment outcome (24).…”

“…WBA was measured as previously described (11,23,25,27). Briefly, M. tuberculosis H37Rv was grown in mycobacterial growth indicator tubes (MGIT; Becton-Dickinson) and frozen in aliquots.…”

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“…Clofazimine is primarily used in the treatment of leprosy but is sometimes incorporated in the treatment regimens for MDR tuberculosis, although data on its clinical efficacy [143] and tolerance [144] are limited and conflicting. Rifabutin has very limited potential utility in treatment of MDR tuberculosis due to its high cross-resistance rate with rifampicin [16].…”

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“…In these assays, metabolic activity is measured as a correlate of viable mycobacterial and three different systems have been designed in the last 10 years using either uracil incorporation 4,5 , reporter-gene tagged mycobacteria [6][7][8][9][10][11] or a radiometric detection system via BacTec/MDGIT bottles 7,8 .…”