Single-Dose Rifapentine in Household Contacts of Patients with Leprosy.

BACKGROUND: Previous studies have suggested that a single dose of rifampin has protective effects against leprosy in close contacts of patients with the disease. Rifapentine was shown to have greater bactericidal activity against Mycobacterium leprae than rifampin in murine models of leprosy, but data regarding its effectiveness in preventing leprosy are lacking. METHODS: We conducted a cluster-randomized, controlled trial to investigate whether single-dose rifapentine is effective in preventing leprosy in household contacts of patients with leprosy. The clusters (counties or districts in Southwest China) were assigned to one of three trial groups: single-dose rifapentine, single-dose rifampin, or control (no intervention). The primary outcome was the 4-year cumulative incidence of leprosy among household contacts. RESULTS: A total of 207 clusters comprising 7450 household contacts underwent randomization; 68 clusters (2331 household contacts) were assigned to the rifapentine group, 71 (2760) to the rifampin group, and 68 (2359) to the control group. A total of 24 new cases of leprosy occurred over the 4-year follow-up, for a cumulative incidence of 0.09% (95% confidence interval [CI], 0.02 to 0.34) with rifapentine (2 cases), 0.33% (95% CI, 0.17 to 0.63) with rifampin (9 cases), and 0.55% (95% CI, 0.32 to 0.95) with no intervention (13 cases). In an intention-to-treat analysis, the cumulative incidence in the rifapentine group was 84% lower than that in the control group (cumulative incidence ratio, 0.16; multiplicity-adjusted 95% CI, 0.03 to 0.87; P = 0.02); the cumulative incidence did not differ significantly between the rifampin group and the control group (cumulative incidence ratio, 0.59; multiplicity-adjusted 95% CI, 0.22 to 1.57; P = 0.23). In a per-protocol analysis, the cumulative incidence was 0.05% with rifapentine, 0.19% with rifampin, and 0.63% with no intervention. No severe adverse events were observed. CONCLUSIONS: The incidence of leprosy among household contacts over 4 years was lower with single-dose rifapentine than with no intervention. (Funded by the Ministry of Health of China and the Chinese Academy of Medical Sciences; Chinese Clinical Trial Registry number, ChiCTR-IPR-15007075.).

Post-exposure prophylaxis (PEP) efficacy of rifampin, rifapentine, moxifloxacin, minocycline, and clarithromycin in a susceptible-subclinical model of leprosy.

BACKGROUND: Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: Athymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation. CONCLUSIONS/SIGNIFICANCE: The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3-4 months) and late (8-9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.

Evaluation of anti-bacterial activity of Rifapentine, Clarithromycin, Minocycline, Moxifloxacin, Ofloxacin and their combinations in Murine Model of Rifampicin Resistant Leprosy.

Leprosy, a debilitating disease of the skin and peripheral nerves is caused by Mycobacterium leprae (M. leprae) and is treated by multidrug therapy (MDT) comprising of Dapsone, Rifampicin and Clofazimine. Resistance to any of these drugs poses a threat to the current disease control strategies. With the emergence of Rifampicin resistance in leprosy, it is important that alternative drugs need to be tested to develop a treatment strategy to combat drug resistant leprosy. In the current study, we have investigated WHO MDT, Rifapentine, Clarithromycin, Minocycline, Moxifloxacin, Ofloxacin and their combinations in intermittent and daily dose regimens in rifampicin resistant strains of M. leprae through mouse foot pad experiments in order to determine the loss in viability of M. leprae in response to these drugs and their combinations. Our findings suggest that WHO MDT is still the best combination in Rifampicin resistance cases. Combination of Moxifloxacin with Minocycline and Clarithromycin may also be taken up for clinical trials in cases with Rifampicin resistant leprosy. Rifapentine and Moxifloxacin can be effective alternative drugs to replace Rifampicin where required either in daily dose shorter duration regimens or intermittent dose longer regimen to treat resistant strains.

Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

Rifapentine, moxifloxacin, or DNA vaccine improves treatment of latent tuberculosis in a mouse model.

RATIONALE: Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms. OBJECTIVES AND METHODS: By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin. MEASUREMENTS: Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined. MAIN RESULTS: Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6-9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin. CONCLUSIONS: Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection.

In-vitro activity of sitafloxacin (DU-6859a), either singly or in combination with rifampin analogs, against Mycobacterium leprae.

The in-vitro antibacterial activity of sitafloxacin (DU-6859a) against Mycobacterium leprae was evaluated and compared with those of ofloxacin, levofloxacin, and ciprofloxacin. Two biochemical indicators (intracellular ATP and uptake of [(3)H]-thymidine) were used to measure the in-vitro growth of M. leprae in Dhople-Hanks (DH) medium. Sitafloxacin was found to be more potent than the other three commonly used fluoroquinolones, with the minimum inhibitory concentration (MIC) against M. leprae being 0.1875 microg/ml and the action being bactericidal. The MICs of ofloxacin, levofloxacin, and ciprofloxacin were 1.5, 0.75, and 3.0 microg/ml, respectively. Similar to ofloxacin and levofloxacin, sitafloxacin also exhibited synergistic activity when combined with either rifabutin or KRM-1648, but not with rifampin. Thus, further studies on the incorporation of sitafloxacin in multidrug therapy regimens in treating leprosy patients are suggested.

In vivo susceptibility of Mycobacterium leprae to sitafloxacin (DU-6859a), either singly or in combination with rifampicin analogues.

The antimicrobial effects of sitafloxacin (DU-6859a) against Mycobacterium leprae, either singly or in combination with either rifampicin, rifabutin or KRM-1648, were studied using a mouse footpad assay technique and the results were compared with those obtained with ofloxacin. When used singly, the minimum concentrations of sitafloxacin and ofloxacin needed to inhibit completely the growth of M. leprae were 25 and 100 mg per kg body weight per day, respectively, and the effects were bactericidal. Both sitafloxacin and ofloxacin exhibited excellent synergistic effects when combined with either rifabutin or KRM-1648, but not with rifampicin. Thus, incorporation of sitafloxacin and rifabutin (or KRM-1648) in the multidrug regimen for treating leprosy patients is suggested.

Bactericidal activities of HMR 3647, moxifloxacin, and rifapentine against Mycobacterium leprae in mice.

Bactericidal activities of HMR 3647 (HMR), moxifloxacin (MXFX), and rifapentine (RPT) against Mycobacterium leprae, measured by the proportional bactericidal technique in the mouse footpad system, were compared with those of the established antileprosy drugs clarithromycin (CLARI), ofloxacin (OFLO), and rifampin (RMP). Administered in five daily doses of 100 mg/kg of body weight, HMR appeared slightly more bactericidal than CLARI. In a single dose, MXFX at 150 mg/kg was more active than the same dose of OFLO and displayed exactly the same level of activity as RMP at 10 mg/kg; the combination MXFX-minocycline (MINO) (MM) was more bactericidal than the combination OFLO-MINO (OM); RPT at 10 mg/kg was more bactericidal than the same dose of RMP and even more active than the combination RMP-OFLO-MINO (ROM); the combination RPT-MXFX-MINO (PMM) killed 99.9% of viable M. leprae and was slightly more bactericidal than RPT alone, indicating that the combination PMM showed an additive effect against M. leprae.

Search for newer antileprosy drugs.

In 1991 World Health Organization proclaimed the goal of global elimination of leprosy as a public health problem by year 2000 by implementing multidrug therapy (MDT). Since then the prevalence rate has declined by 85%. However, during the same period the incidence rate of leprosy has remained constant or even has been increasing. This suggests that it will take a long time for the eradication of leprosy and that without in-vitro cultivation of M. leprae, eradication of leprosy is not likely to be achieved. While in-vitro cultivation is a long-term goal, as an immediate measure, there is an urgent need for the development of newer drugs and newer multidrug therapy regimens. Using the in-vitro system for screening potential antileprosy drugs and also using the mouse foot-pad system we have evaluated several compounds in four classes of drugs--dihydrofolate reductase inhibitors, fluoroquinolones, rifampicin analogues and phenazines--and identified at least two compounds that appear to be more potent than dapsone, rifampicin and clofazimine. Newer combinations of rifampicin analogues and fluoroquinolones have also been identified that seem to be better than the combination of rifampicin and ofloxacin.

Combination of rifapentine-moxifloxacin-minocycline (PMM) for the treatment of leprosy.

To further the development of a multidrug regimen for treatment of leprosy that is suitable for monthly administration and fully supervisable, the bactericidal activities against Mycobacterium leprae of HMR 3647 (HMR), moxifloxacin (MXFX) and rifapentine (RPT) were measured by the proportional bactericide technique in the mouse footpad system, and compared with those of the established antileprosy drugs clarithromycin (CLARI), ofloxacin (OFLO) and rifampicin (RMP). Administered in five daily doses of 100 mg per kg body weight, HMR appeared slightly more bactericidal than CLARI, but the difference did not attain statistical significance. Administered as single doses, MXFX in a dosage of 150 mg per kg was more active than OFLO in the same dosage, and displayed the same level of activity as RMP in a dosage of 10 mg per kg; the combination MXFX-minocycline (MINO) (MM) was more bactericidal than the combination OFLO-MINO (OM); RPT in a dosage of 10 mg per kg was more bactericidal than RMP administered in the same dosage, and even more active than the combination RMP-OFLO-MINO (ROM); the combination RPT-MXFX-MINO (PMM) killed 99.9% of viable M. leprae, and was slightly more bactericidal than was RPT alone, indicating that the combination PMM showed an additive effect against M. leprae. These promising results justify a clinical trial among lepromatous patients, in which MM is being compared with OM, and PMM with ROM, in terms of efficacy and tolerance.

Comparative in vitro activities of rifamycin analogues against Mycobacterium leprae.

Comparative activities of various rifamycin analogues against leprosy were studied by evaluating their effects on in vitro growth of Mycobacterium leprae in DH medium as described earlier. Among the seven analogues studied, KRM-1648 was found to be the most potent in inhibiting the growth of rifampicin-sensitive strains of M. leprae, MIC being 0.05 microgram/ml. This was followed by KRM-2312 and T9 (MIC of each being 0.1 microgram/ml) and rifabutin (MIC, 0.2 microgram/ml). Rifampicin, along with KRM-1657 and KRM-1668, were least effective, with MIC for each being 0.4 microgram/ml. The effects of each at their respective MICs were bactericidal. The results were similar for rifampicin-resistant strains of M. leprae, but the MICs were higher than those obtained with rifampicin-sensitive strains of M. leprae. Thus, even though rifampicin has been the first-line drug in the treatment of leprosy, the results in present studies suggest that other rifamycin analogues are available that are more potent than rifampicin against both rifampicin-sensitive as well as rifampicin-restraint strains of M. leprae.

Structure determination of ribosylated rifampicin and its derivative: new inactivated metabolites of rifampicin by mycobacterial strains.

Rifampicin (I) was converted into two inactivated products RIP-Ma and RIP-Mb by Mycobacterium smegmatis DSM43756. MS, NMR and chromatographic analysis showed the compounds to be 3-formyl-23-[O-(alpha-D-ribofuranosyl)]rifamycin SV (6) and 23-[O-(alpha-D-ribofuranosyl)]rifampicin (7), respectively.

Methods for determining concentrations of antimicrobial agents in human monocytes.

Human monocytes can be derived from the leukocyte-rich by-product of donors' blood available after platelet separation. Large volumes of the monocyte samples obtained from this product provided an opportunity to conduct experiments with relatively high concentrations of the antimicrobial agents sufficient for their detection in bioassays, thus avoiding the necessity of working with the radiolabelled drugs. Washing of the cells after their exposure to the drug may lead to an extraction of the tested agent from the cell, especially if it is a substance of low molecular weight. In our experiments we excluded the washing step, and separated the monocytes from the extracellular medium by velocity gradient centrifugation. In experiments with two rifamycins, the cell pellet as well as the extracellular fluid were subjected to a bioassay using Micrococcus luteus as a target organism. The method showed good reproducibility and consistency in results obtained.

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.

High-performance liquid chromatographic determination of rifapentine in serum using column switching.

A high-performance liquid chromatographic method with column switching has been developed for the determination of rifapentine in serum. The serum samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard in a 1% ascorbic acid solution. Polar serum components were washed out using 0.05 M phosphate buffer. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by a mu Bondapak C18 column with acetonitrile-tetrahydrofuran-0.05 M phosphate buffer (pH 7.0) (42:5:53, v/v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.1 microgram/ml. The total analysis time was less than 25 min and the mean coefficients of variation for intra- and inter-assay were less than 4.8%. The method has been successfully applied to serum samples from dogs after the oral administration of rifapentine.

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.

Inductive effects of rifapentine on mice hepatic mixed function oxidase system.

Rifapentine (R773, DL473) is a long-acting antituberculous drug used in China. In our experiments we have found some manifestations of induction of hepatic mixed function oxidase system in mice following pretreatment with rifapentine or phenobarbital. Both rifapentine and phenobarbital significantly increased the rate of antipyrine and pentobarbital metabolism in vivo. They also increased liver weight, the content of liver microsomal protein and cytochrome P-450, the activity of NADPH-cytochrome C reductase and NADPH oxidase. SDS-polyacylamide gel electrophoresis showed that the relative proportions of some polypeptide bands in mice microsomal fraction were significantly changed following rifapentine or phenobarbital pretreatment. The results indicate that rifapentine, like phenobarbital, is a potent inducer of hepatic mixed function oxidase system in mice and that it should be used carefully in clinical therapy, when combined with other drugs.