Activity of capuramycin analogues against Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in vitro and in vivo

Tetsufumi Koga1,*, Takashi Fukuoka1, Norio Doi2, Tamako Harasaki1, Harumi Inoue1, Hitoshi Hotoda3, Masayo Kakuta1, Yasunori Muramatsu4, Naotoshi Yamamura5, Misa Hoshi5 and Takashi Hirota5

1 Biological Research Laboratories, Sankyo Co., Ltd, 2–58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710; 2 Research Institute of Tuberculosis; 3 Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd; 4 Lead Discovery Laboratories, Sankyo Co., Ltd; 5 Drug Metabolism & Pharmacokinetics Research Laboratories, Sankyo Co., Ltd, Tokyo, Japan

Received 13 April 2004; returned 13 July 2004; revised 27 July 2004; accepted 28 July 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The antimycobacterial activities of RS-112997, RS-124922 and RS-118641, three capuramycin analogues that inhibit phospho-N-acetylmuramyl-pentapeptide translocase, were tested against clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare.

Methods and results: MICs were determined by the broth microdilution method using a modified Middlebrook 7H9 broth. RS-118641 was the most potent compound overall. The MIC50/90 (mg/L) results for RS-118641 were: M. tuberculosis, 1/2; multidrug-resistant (MDR) M. tuberculosis, 0.5/2; M. avium, 4/8; and M. intracellulare, 0.06/0.5. No statistically significant differences in MIC distributions were observed between non-MDR and MDR M. tuberculosis for any of the capuramycin analogues tested. In order to evaluate the therapeutic efficacy of RS-112997 and RS-124922 in a murine lung model of tuberculosis, both compounds were administered intranasally at 0.1 or 1 mg/mouse/day for 12 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls. Additional experiments were performed to evaluate the therapeutic efficacy of the three compounds against the M. intracellulare infection in mice. All compounds were administered intranasally at 0.1 mg/mouse/day for 21 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls.

Conclusions: These results suggest that capuramycin analogues exhibit strong antimycobacterial potential and should be considered for further evaluation in the treatment of M. tuberculosis and M. aviumM. intracellulare complex infections in humans.

Keywords: mycobacteria , antimycobacterials , translocase I , peptidoglycan


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tuberculosis (TB) continues to be the leading cause of death worldwide due to an infectious agent. Approx. 8 million people develop active TB each year, and almost 2 million of these ultimately die from the disease.1 Moreover, there has recently been a disturbing increase in the number of TB cases caused by organisms resistant to the two most important drugs for the treatment of TB, rifampicin and isoniazid. A recent survey in 72 countries suggested that multidrug-resistant TB is more widespread than previously thought and is likely to be worsening.2 Meanwhile, the Mycobacterium aviumMycobacterium intracellulare complex (MAC) infection is a common cause of bacteraemia and is disseminated in patients in the advanced stages of AIDS.3,4 It is known that only the macrolides, ethambutol, rifabutin and a limited number of other compounds have activity against MAC in vivo.5,6 Worse still, the emergence of macrolide resistance and drug interactions between rifamycins and protease inhibitors has been reported.5,7 Without question, the development of new drugs will be essential to combat both drug-resistant Mycobacterium tuberculosis and opportunistic infections by non-tuberculous mycobacteria such as MAC.

Since peptidoglycan is an essential bacterial cell wall polymer, peptidoglycan biosynthesis provides a unique and selective target for the mechanism of action of bacteria. Phospho-MurNAc-pentapeptide translocase (translocase I) is an integral membrane protein that catalyses the first step of the intramembrane cycle of reactions involved in peptidoglycan assembly. In the course of screening for new antibiotics with translocase I inhibitory activity, we identified a series of capuramycin analogues that proved to have selective antibacterial activity against mycobacteria.8 In this study, we evaluated the in vitro and in vivo activities of these capuramycin analogues against clinically important mycobacteria, namely, M. tuberculosis, M. avium and M. intracellulare.


    Materials and methods
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 Abstract
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 Materials and methods
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 References
 
Compounds

RS-112997 (A-500359A) was isolated as described previously.9,10 RS-124922 (compound No. 65)10 and RS-118641 (compound No. 20)11 were synthesized at the Lead Discovery Research Laboratories of Sankyo Co., Ltd (Tokyo, Japan) (Figure 1). Sterile distilled water was used for RS-112997 and RS-124922, and sterile distilled water supplemented with 50% methanol was used for RS-118641 as the solvent. Clarithromycin was purchased from the National Institute of Infectious Diseases (Tokyo). Rifampicin and isoniazid stored on air-dried microplates of BrothMIC MTB were purchased from Kyokuto Pharmaceutical Industrial Company (Tokyo).12



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Figure 1. Structures of capuramycin analogues. (a) RS-112997, (b) RS-124922 and (c) RS-118641.

 
Isolates

M. tuberculosis (33 strains), M. avium (33 strains) and M. intracellulare (17 strains) isolated from patients with tuberculosis or MAC infections in Japan were grown in 7H9 broth (Becton Dickinson and Co.). M. tuberculosis and MAC were isolated in several hospitals in different districts of Japan and identified by standard biochemical tests. M. tuberculosis strains were divided into two groups on the basis of their susceptibilities to rifampicin and isoniazid, as follows. One group was rifampicin- and isoniazid-susceptible TB [non-MDR-TB (where MDR stands for multidrug-resistant); 22 strains] and the other group was rifampicin- and isoniazid-resistant TB (MDR-TB; 11 strains) by classification based on Kochi et al.13 M. avium and M. intracellulare were identified by DNA probe testing (AccuProbe test, Kyokuto Pharmaceutical Industrial Company).

Inoculum preparation

The organisms were grown in Middlebrook 7H9 broth with 10% Middlebrook albumin/dextrose/catalase (ADC; Becton Dickinson and Co.) enrichment and 0.05% Tween 80 at 37°C in a 5% CO2 incubator. This was carried on for 5–7 days until the absorbances of the pre-cultures were ~0.1 at 660 nm (OD660) measured with a spectrophotometer (Mini Photo 518; Taitec Co., Ltd, Saitama, Japan). The inoculum size was verified by plating serial dilutions of the cell suspension in triplicate onto 7H10 agar (Becton Dickinson and Co.) plates supplemented with 10% Middlebrook oleic acid/albumin/dextrose/catalase (OADC; Becton Dickinson and Co.) enrichment. This was followed by incubation at 37°C in a CO2 incubator for 4 weeks for M. tuberculosis and 7 days for M. avium and M. intracellulare.

Broth dilution method

The microdilution MICs for the mycobacteria isolates were determined by a procedure reported earlier with slight modification.12 In brief, the MICs were determined by serially diluting each test compound in Middlebrook 7H9 broth with 10% OADC and 0.5% glycerol in 96-well plates. Two-fold serial dilutions of the test compounds were prepared with dimethyl sulphoxide. The dilution series were initially prepared at 200-fold final concentrations from this stock to provide a final test range of 32–0.016 mg/L. One microlitre of each dilution and 200 µL of test organisms (~105 cfu) were then dispensed into each well. Air-dried plates were prepared for rifampicin and isoniazid ranging from 32–0.03 mg/L. Two hundred microlitres of each test organism was dispensed into each well. The MIC was defined as the lowest concentration of compound at which no visible growth could be seen.

Animals

Female BALB/c (Charles River Inc., Nagano, Japan) SPF mice were used in this study. All animal experiments were carried out according to the guidelines provided by the Institutional Animal Care and Use Committee of Sankyo Co., Ltd.

Infection

Stock cultures of M. tuberculosis H37Rv ATCC 27294 (approx. 4 x 107 cfu/mL) and a clinical isolate of M. intracellulare N-256 (approx. 2 x 108 cfu/mL) stored at –80°C were diluted four-fold with sterile distilled water and were used in this study. A murine intratracheal infection model of mycobacteria in mice was established, using a previously reported method with slight modification.14 Mice were anaesthetized by intraperitoneal injection with dimorpholamine (Theraptique; Eisai Co., Ltd, Tokyo, Japan), xylazine (Bayer Yakuhin Ltd, Tokyo, Japan) and pentobarbital sodium (Nembutal, Dainippon Pharmaceutical Co., Ltd, Osaka, Japan) (1:8:40). This was followed by inoculation intratracheally with cell suspensions of M. tuberculosis at a volume of 100 µL (1.0 x 106 cfu/mouse) or M. intracellulare at a volume of 50 µL (2.4 x 106 cfu/mouse) using a tuberculin syringe connected to a 23 G needle.

Treatment

Sterile physiological saline was used for RS-112997, RS-124922 and RS-118641 as the solvent, and the compound suspensions were sonicated for 2–3 min to ensure uniform dispersion. For the M. tuberculosis infection, RS-112997 and RS-124922 were administered at doses of 0.1 and 1 mg/mouse for 12 days, starting from the seventh day after inoculation (n=4). For the M. intracellulare infection, RS-112997, RS-124922 and RS-118641 were administered at doses of 0.1 mg/mouse for 21 days, starting from the third day after inoculation (n=4–5). To prepare for the administration of the test compounds, the mice were anaesthetized with a mixture of ether and chloroform (1:1). Compound suspension at a volume of 25 µL for M. tuberculosis or 50 µL for M. intracellulare was dropped slowly and gently into the nose using a micropipette. Saline was administered to the untreated control mice by the same method at the same volumes.

Therapeutic efficacy

The therapeutic efficacy was defined as the reduction in bacterial colony formation in the lungs of infected mice. Mice were sacrificed by CO2 asphyxiation 2 days after the last administration for M. tuberculosis or 1 day after the last administration for M. intracellulare. Each pair of lungs was removed aseptically, and homogenized in 5 mL of sterile distilled water. After diluting the homogenates 10-fold serially with sterile distilled water, 100 µL of each dilution was spread onto a Middlebrook 7H10 agar plate containing 10% Middlebrook OADC enrichment and 0.5% glycerol. This was followed by incubation at 37°C in a CO2 incubator for 4 weeks for M. tuberculosis or for 7 days for M. intracellulare. The number of viable organisms in the lungs was determined by counting the colonies on the plates. Once the number of viable organisms was taken, the values were converted into common logarithms, and the mean and standard error were calculated for each group.

Statistical analysis

For the evaluation of MICs of the compounds, the differences between non-MDR and MDR M. tuberculosis were analysed by Wilcoxon's test. To evaluate the efficacy of the compounds, the differences in the number of viable organisms in the lungs between the untreated control and treated groups were analysed by parametric Dunnett's test.15 P values <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs of RS-112997, RS-124922 and RS-118641 for M. tuberculosis, M. avium and M. intracellulare

Tables 1 and 2 summarize the MIC range and MICs at which 50% and 90% of the test strains were inhibited (MIC50 and MIC90, respectively). The MICs of the test compounds for non-MDR-TB and MDR-TB were in the order of RS-118641 < RS-124922 < RS-112997. No statistically significant differences in MIC distributions of the three test compounds used were observed between non-MDR-TB and MDR-TB. Rifampicin and isoniazid were active against non-MDR-TB, with MIC90s of ≤0.03 and 0.125 mg/L, respectively. Clarithromycin was less active, with an MIC90 of 8 mg/L. In contrast, the MICs of these three drugs for MDR-TB were much higher than those for non-MDR-TB. The MICs of RS-112997 and RS-124922 for M. tuberculosis H37Rv were 8 and 2 mg/L, respectively. The MICs of the test compounds for M. avium were in the order of RS-118641 = RS-124922 < RS-112997. The MIC50 of rifampicin for M. avium was superior to those of all the other compounds tested, and the MIC90 of rifampicin was comparable to those of all the other compounds tested. The MICs of the test compounds for M. intracellulare were in the order of RS-118641 < RS-124922 < RS-112997. The MICs of rifampicin for M. intracellulare were comparable to those of RS-118641 for the same microorganism. The MICs of RS-112997, RS-124922 and RS-118641 for M. intracellulare N-256 were 4, 2 and 0.5 mg/L, respectively.


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Table 1. MICs of RS-112997, RS-124922 and RS-118641 for M. tuberculosis

 

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Table 2. MICs of RS-112997, RS-124922 and RS-118641 for M. avium and M. intracellulare

 
Treatment of M. tuberculosis-infected mice by intranasal administrations of RS-112997 and RS-124922

The therapeutic efficacies of RS-112997 and RS-124922 are shown in Table 3. The number of viable organisms in lungs after RS-112997 administration at doses of 0.1 mg/mouse and at 1 mg/mouse was significantly lower than that of the untreated controls (P < 0.001 and P < 0.01). The number of viable organisms in lungs after RS-124922 administration at doses of 0.1 and 1 mg/mouse was significantly lower than that of the untreated controls (P < 0.001), although 4.1–5.5 log10 cfu/lungs of viable organisms persisted.


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Table 3. Therapeutic efficacy of RS-112997 and RS-124922 against M. tuberculosis in a murine model

 
Treatment of M. intracellulare-infected mice by intranasal administrations of RS-112997, RS-124922 and RS-118641

The therapeutic efficacies of RS-112997, RS-124922 and RS-118641 are shown in Table 4. The number of viable organisms in lungs after administration with all three of the respective test compounds at doses of 0.1 mg/mouse was significantly lower than that of the untreated controls (P < 0.001). The test compounds had comparable activities against the M. intracellulare infection model.


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Table 4. Therapeutic efficacy of RS-112997, RS-124922 and RS-118641 against M. intracellulare in a murine model

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study demonstrates that capuramycin analogues are active against pathogenic mycobacteria in vitro and in vivo. The capuramycin analogues were originally found during the course of screening for new antibiotics with inhibitory activity against translocase I, an enzyme required for peptidoglycan biosynthesis. Peptidoglycan biosynthesis inhibitors are excellent targets from the viewpoint of selective toxicity between humans and bacteria.16 Interestingly, mycobacteria tend to be resistant to many of the ß-lactam antibiotics, a group of antibiotics that belong to the same category as the peptidoglycan biosynthesis inhibitors. ß-Lactamase production by mycobacteria is likely to be an important factor in the expression of resistance to ß-lactam antibiotics.17,18 Capuramycin analogues could be active because of their ß-lactamase stability. Capuramycin analogues have weak activity against some Gram-positive bacteria such as Streptococcus pneumoniae, but they do not show any activity against Staphylococcus aureus or any Gram-negative bacteria other than Moraxella catarralis.8 Although the precise mechanism of cell permeability of capuramycin analogues against mycobacteria is not yet fully understood, it may involve a special uptake mechanism of capuramycin analogues in mycobacteria. No cross-resistance was found between non-MDR-TB and MDR-TB. This result was as expected, as translocase I is a new target not inhibited by clinically active antituberculosis drugs. Different mycobacterial species showed different degrees of susceptibility to capuramycin analogues. This result appears to be attributable to the cell permeability or affinity to translocase I of the compounds. However, little difference in the order of the MICs of the three capuramycin analogues was observed among the same mycobacterial species. Although there is growing evidence that the lipid-rich cell wall structures of mycobacteria form a unique asymmetric bilayer responsible for the extremely low permeability of the cell wall to both hydrophilic and hydrophobic compounds,19,20 a higher hydrophobicity of the capuramycin analogues was found to correlate with increased antimycobacterial activity. Within the range of this hydrophobicity, a higher hydrophobicity would interact with and permeate the outer lipid layers of these organisms.

We treated the infected mice with capuramycin analogues by the intranasal route of administration. Inhalation is a well-established means for targeting drugs to the lungs for the treatment of respiratory diseases such as cystic fibrosis.21 The minimal systemic absorption of drugs delivered to the lungs by aerosol administration allows the use of high antibiotic concentrations with reduced risk of systemic toxic reactions because of minimal absorption into the circulation.22 As a result, the drugs can be administered to patients over long periods. In pulmonary TB, aerosolized administration of aminoglycosides as an adjunctive salvage therapy has been used in clinical practice;23,24 however, this therapy raises some concerns that inhaled aminoglycosides would be deposited in chronic cavitary lesions in concentrations that would be sufficient to exert real killing of mycobacteria.25 As this mouse model did not progress to chronic cavitation,14 this concern is not clear. Evaluation with a rabbit model in which cavitary tuberculosis can be readily produced26 is needed to clarify this. Furthermore, the distribution of aerosol aminoglycosides in the lungs in clinical practice requires further investigation to confirm whether or not they penetrate into the pulmonary cavitation.

Although RS-118641 is the most active among the capuramycin analogues in vitro,11 we could not evaluate RS-118641 at the dose of 1 mg/mouse because of poor solubility in the M. tuberculosis infection model. RS-124922 and RS-112997 caused significant reduction in the number of viable organisms in the lungs compared with that in the untreated controls; however, neither compound displayed dose-dependency. The reason for this lack of dose-dependency is presently unknown, but the low solubility of these compounds may be one reason.

In the M. intracellulare infection model, all capuramycin analogues tested produced significantly lower numbers of viable organisms in the lungs compared with that of the untreated controls. While the antimycobacterial activities of the capuramycin analogues against M. intracellulare N-256 varied in strength (in the order of RS-118641 > RS-124922 > RS-112997) in vitro, these compounds were found to possess similar antimycobacterial activities in vivo overall. Since M. intracellulare are intracellular bacteria,27 we conjecture that this similarity might be due to the differences in the cellular uptake of the capuramycin analogues, or possibly differences in the pharmacokinetic parameters of the capuramycin analogues.

In both infection models treated with capuramycin analogues, the reduction in the number of viable organisms was in the range 0.9–3.5 log units; however, 4–5 log units persisted in the lung. Further studies are needed to confirm longer-term treatment efficacy and combination therapy such as rifampicin for M. tuberculosis and clarithromycin for M. intracellulare infection models.

Extrapolation of animal experimental data to humans is very important. The dose of capuramycin analogues at 0.1 mg/mouse (~20 g of body weight) used in our study was larger than the dose that can be administered as an inhaled powder via an inhaler device in clinical practice.28 Thus, further studies will have to be conducted to determine the minimum effective dose. Another delivery method to the lung is administration of inhaled aerosol via nebulizer. This method of delivery would enable the administration of higher doses to humans.

In conclusion, these results suggest that capuramycin analogues exhibit strong antimycobacterial potential and are excellent candidates for further evaluation in the treatment of M. tuberculosis and MAC infections in humans.


    Footnotes
 
* Corresponding author. Tel: +81-3-3492-3131, ext. 3511; Fax: +81-3-5436-8565; Email: tekoga{at}sankyo.co.jp


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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28 . Le Brun, P. P., de Boer, A. H., Mannes, G. P. et al. (2002). Dry powder inhalation of antibiotics in cystic fibrosis therapy: part 2. Inhalation of a novel colistin dry powder formulation: a feasibility study in healthy volunteers and patients. European Journal of Pharmaceutics and Biopharmaceutics 54, 25–32.[CrossRef][ISI][Medline]





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