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The activity of rifabutin against Mycobacterium leprae in armadillos
International Journal of Antimicrobial Agents 9 (1998) 169 – 173
The activity of rifabutin against Mycobacterium leprae in armadillos Arvind M. Dhople *, Sharon L. Williams Department of Biological Sciences, Infectious Diseases Laboratory, Florida Institute of Technology, 150 West Uni6ersity Boule6ard, Melbourne, FL 32901 -6975, USA Accepted 29 October 1997
Abstract The activity of rifabutin (LM 427) against Mycobacterium leprae was evaluated in armadillos inoculated earlier with human-derived M. leprae. Rifabutin was administered daily at a dose of 6 mg/kg body weight/day. The effect of rifabutin on M. leprae harvested from armadillos was determined by measuring the intracellular levels of ATP (an indicator of metabolic activity) of M. leprae and also their ability to multiply in the mouse footpads and in vitro in DH medium. Within 2 weeks of initiating the treatment, ATP levels declined to 21% of the original (pre-treatment level) and these M. leprae failed to multiply in the footpads of mice as well as in the in vitro culture system. This suggests that rifabutin was able to kill all M. leprae within 2 weeks. After 8 weeks the treatment was terminated and results showed that M. leprae from the treated armadillos remained non-viable in the mouse footpad system as well as in the in vitro system, indicating bactericidal action of rifabutin. The results suggest that rifabutin can be a substitute for rifampin in the leprosy multi-drug therapy regimen. © 1998 Elsevier Science B.V.
1. Introduction Rifampin is the most potent bactericidal drug available today in the treatment of leprosy [1] and is considered a standard drug worldwide, as part of multi-drug therapy (MDT) regimen recommended by the World Health Organization [2]. A semi-synthetic rifamycin, rifabutin (Ansamycin LM 427; spiropiperidyl rifamycin), has been reported as having substantial in vitro activity against several species of mycobacteria [3 – 7]. Its minimal inhibitory concentrations (MICs) are from 2 [5] to 20 [8] times lower than those of rifampin. Hastings and Jacobson [9] were the first to study the action of rifabutin against M. leprae in mouse footpads. Subsequently, Hastings and associates [10] have shown Abbre6iations: Mycobacterium leprae; Armadillo; Rifabutin; Multidrug therapy. * Corresponding author. Tel.: + 1 407 6747253; fax: + 1 407 9848461; e-mail:[email protected] 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 4 - 8 5 7 9 ( 9 7 ) 0 0 0 4 8 - 4
that the MIC of rifabutin against Mycobacterium leprae in mouse footpads was several times lower than that of rifampin. Using the in vitro system developed in our laboratory [11], superiority of rifabutin over rifampin was demonstrated against both rifampin-sensitive and rifampin-resistant strains of M. leprae [12–15]. Similar superiority of rifabutin has also been shown in our mouse footpad studies [16,17] for both rifampin-sensitive and rifampin-resistant strains of M. leprae. In the early 1970s, Kirchheimer and Storrs [18,19] have demonstrated that the nine-banded armadillo (Dasypus no6emcinctus) developed disseminated leprosy if inoculated intravenously with human-derived M. leprae. Subsequently, we have shown that this animal model can be used for chemotherapeutic investigations in leprosy [20]. Since armadillo is a better model than mouse for disseminated leprosy, it was decided to extend the in vivo studies on the effects of rifabutin against M. leprae using armadillo as a model of multibacillary leprosy.
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2. Materials and methods
2.1. Armadillo inoculation Rifampin-susceptible M. leprae were harvested from the livers of nine-banded armadillos which had been previously inoculated with a human-derived, rifampinsensitive strain of M. leprae. M. leprae cells were purified by first treating the liver suspensions with DNAase and then separating the bacteria from the host tissue with a Percoll liquid density gradient (Pharmacia LKB Biotechnology, Piscataway, NJ) and centrifugation [21]. The cells were counted microscopically by the pinhead method [22]. A total of 20 males were selected for the study. Average weight was 4.9 lb and all were in good health. Smears from blood buffy coats, ear clippings and nasal swabs failed to show any acid-fast bacilli. All the animals were anesthetized using Innovar-vet (0.15 ml/ kg) plus atropin sulfate (0.04 ml/kg). Each animal was inoculated subcutaneously at ten different sites on the abdomen, each site receiving 0.1 ml suspension containing 106 M. leprae. Animals were housed in ‘vari-kennel’cages and were fed daily with moistened cat chow and provided water ad libitum. Between 9 and 11 months post inoculations, 14 animals developed subcutaneous lepromas (approximately 10 mm diameter in size) at the sites of inoculations. These 14 animals were divided into two groups; control, receiving only cat chow and the experimental, receiving rifabutin along with cat chow.
2.2. Drug administration Rifabutin was a gift from Pharmacia-Adria Laboratories, Dublin, OH. All seven animals in the experimental group received rifabutin every day (7 days a week) for a total of 8 weeks. Rifabutin was administered through food at a dose of 6mg/kg per day. Required quantity of the drug was mixed in small amount of moistened cat chow and fed to the animals early in the morning, when they are most hungry. After consuming all the food, a bowl with moistened cat chow was placed in each cage to eat ad libitum.
2.3. Susceptibility testing Before initiating rifabutin treatment, all animals were anesthetized and with 5-mm punch biopsy, biopsy specimens from one leproma of each of the 14 animals were collected aseptically. Also, 5 ml of blood from each animal was drawn through the saphenous vein and collected in heparinized vacutainers. This procedure was repeated at the end of 1, 2, 4 and 8 weeks of treatment. After 8 weeks the administration of rifabutin was terminated and all animals in the experimental
group were fed moistened cat chow, similar to those in the control group. The biopsy procedure was repeated at 4 and 8 weeks after withdrawing rifabutin.
2.4. Assays for the 6iability of M. leprae Suspensions from each biopsy specimen were prepared and the suspensions were purified by the method described above. Aliquots were used for microscopic enumeration of M. leprae, for the assay of intracellular adenosine triphosphate (ATP) determinations, for in vitro culture of M. leprae and also for inoculations into footpads of mice. ATP assays were performed by the firefly bioluminescence method described earlier [23] and the amount of intracellular ATP was expressed as picogram. of ATP per 106 M. leprae cells. For the in vitro culture, DH medium was used and the culture system which was previously described, using ATP and [3H]thymidine uptake as metabolic indicators of the growth of M. leprae [23–25]. Inoculation of M. leprae into footpads of BALB/c mice (Jackson Laboratories, Bar Harbor, ME) and subsequent harvests of footpads were carried out by the standard technique developed by Shepard [26].
2.5. Rifabutin assays Blood samples were centrifuged for 10 min at 1200× g and the plasma was removed and stored at −76°C. The concentration of rifabutin and its major metabolite, 25-deacetylrifabutin, was determined by high-pressure liquid chromatography using the method of Skinner and associates [27].
3. Results The results on the effect of rifabutin on M. leprae harvested from armadillos treated with rifabutin are presented in Table 1. During the 16-week period of observation, the load of acid-fast bacteria in subcutaneous nodules from animals in the control group, as counted by the pin-head method [22], increased from 100 to 226%. In case of armadillos treated with rifabutin, the number of acid-fast bacteria decreased from 100 to 77% during the same period. However, a different picture emerged with the metabolism of these bacteria, even though the metabolism of M. leprae from untreated animals remained constant, it declined steadily with rifabutin treatment, reaching 9 and 0% at the end of 4 and 8 weeks, respectively. This suggests that the viability of these organisms was also affected with the rifabutin (discussed in Table 2). The rifabutin levels in plasma of animals from the treated group remained steady during treatment, varying from 0.38 mg/ml at the end of first week to 0.49 mg/ml at the end
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Table 1 Anti-leprosy activity of rifabutin in armadillos Weeks
Microscopic counts: M. leprae/g (×108)
ATP (pg/106 M. leprae)
Plasma levelsb (mg/ml)
Control group
Experimental groupa
Control group
Experimental group
Rifabutin
25-d.a. rifabutinc
0 1 2 4 8
72.3 9 7.95 (100)e 79.8 9 9.38 (110) 90.3 98.01 (125) 102.1 9 14.29 (141) 117.3 9 10.59 (162)
80.99 11.33 (100) 81.79 7.39 (101) 79.39 8.72 (98) 76.1 9 6.71 (94) 72.3 9 10.72 (89)
25.1 93.00 25.693.80 27.3 93.00 24.6 93.20 26.1 93.70
24.4 9 3.70 17.6 9 2.50 5.1 9 0.56 2.2 9 0.33 0
0 0.38 90.05 0.45 90.04 0.51 90.07 0.49 90.07
0 0.04290.006 0.05090.005 0.055 90.007 0.05290.007
12 16
137.3 919.22 (190) 163.4 9 17.19 (226)
67.2 9 7.92 (83) 62.5 9 5.62 (77)
26.9 92.90 (107) 27.993.60 (111)
0.09 90.01 0
0 0
(100) (102) (109) (98) (104)
0 0
(100) (72) (21) (9)
a
Rifabutin, 6 mg/kg per day. Experimental group only. c 25-Deacetylrifabutin. d Rifabutin administration terminated. e % Values. b
of 8 weeks. On withdrawl of rifabutin, levels in plasma started declining, with complete disappearance occuring between 12 and 16 weeks. A similar pattern was also observed in the case of 25-deacetylrifabutin, except that the plasma levels were approximately 10% of those of rifabutin. The viability of M. leprae harvested from these animals was established using three indicators as described earlier and the results are presented in Table 2. M. leprae harvested from armadillos in the control group retained their viability during the period of observation. Organisms harvested at 0, 2, 8 and 16 weeks and inoculated into footpads of mice multiplied in normal fashion. Also, these organisms retained their metabolic activity when inoculated in the DH medium and incubated at 34°C for 16 weeks. The zero hour values (100%) for ATP varied between 202 and 218 pg/ml per culture, indicating that the % increase at the end of 16 weeks of incubation was between 1706 and 2010, which was normal. In the case of M. leprae harvested from armadillos treated with rifabutin, the organisms lost their viability as well as metabolic activity. M. leprae obtained 2 weeks after initiating treatment failed to multiply in the footpads of mice and remained non-viable during the entire period of treatment. In the in vitro culture system, the zero hour (100%) ATP values of organisms harvested at 0 and 2 weeks were 2099 25 pg/ml culture and 48 92.5 pg/ml culture, respectively, thus giving a % increase of 2010 and 710, respectively, at the end of 16 weeks of incubation in DH medium. Similarly, the zero hour values for [3H]thymidine uptake for organisms harvested at 0 and 2 weeks were 0.72 90.10 pmol/5 ml culture and 0.1990.02 pmol/5 ml culture, giving the % increase of 2490 and 850, respectively, at the end of 16 weeks of incubation in DH medium. Organisms harvested 8 weeks after initiating rifabutin treatment did not show presence of ATP
and also failed to incorporate [3H]thymidine. These organisms also failed to multiply in DH medium, since at the end of 16 weeks of incubation, neither the presence of ATP nor the uptake of [3H]thymidine was detected. Even 8 weeks after terminating the rifabutin treatment, organisms from this group of animals failed to multiply in the footpads of mice and also failed to demonstrate any metabolic activity (ATP and [3H]thymidine uptake). Furthermore, these organisms also failed to grow in DH medium. Thus, the results suggest that the effects of rifabutin on M. leprae were bactericidal.
4. Discussion We have demonstrated earlier the bactericidal activity of rifabutin in both the in vitro system [12–15] and in the mouse footpad system [16,17]. Even though the mouse footpad system had been widely used for evaluating potential anti-leprosy compounds, the important drawbacks of this model have been limited multiplication of M. leprae in footpads and failure to develop disseminated infection resembling human lepromatous leprosy. Previously, we demonstrated the suitability of armadillos for such chemotherapeutic studies based on the pattern of dapsone metabolism by these organisms, which was similar to that observed in humans [20]. The results presented here on the anti-leprosy effects of rifabutin in armadillos confirm our earlier findings using the in vitro culture system and the mouse footpad system. Microscopic counts of acid-fast bacilli do not differentiate between viable and non-viable organisms and thus do not indicate the viability or metabolic status of the organisms. Even though the microscopic counts of M. leprae from armadillos in the control group in-
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Table 2 Growth characteristics of M. leprae harvested from rifabutin-treated aramaillos Characteristics M. leprae harvested from armadillos (–) weeks after initiating rifabutin treatment 0 Week Control Mouse foot3.51 90.39 pada In vitro 3957 9 475 growthb ATPc [3H]thymidined 16.90 92.19
2 Weeks Experimental
Control
3.24 9 0.45
3.36 9 0.44
4194 9 461
37989 425
17.92 9 2.15
15.89 9 2.27
8 Weeks Experimental
Control
16 Weeks Experimental
Control
Experimental
3.64 90.350 0
3.29 9 0.30
0
342 947
4392 9614
0
3685 9 479
0
1.61 9 0.18
19.09 92.86
0
15.74 91.73
0
0
a
Footpad harvested 9 months after inoculations (×106 per footpad). Metabolic activity after 16 weeks of incubation in DH medium (Inoculum: 1×107/ml). c Picograms of ATP in M. leprae from 1 ml of culture. d Picomols of [3H]thymidine uptake by M. leprae from 5 ml of culture. b
creased in 16 weeks, those in the experimental group decreased by about 23%. However, bacilli from the experimental group failed to multiply in the footpads of mice, suggesting loss of viability. Dhople [28] was the first to demonstrate the applicability of ATP assays for monitoring the response of leprosy patients to chemotherapy. It was demonstrated that ATP content of M. leprae progressively declined with therapy and responses correlated with loss of infectivity in mouse footpads. This was further confirmed by Seydel et al. [29], Katoch [30] and Dietz et al. [31]. Furthermore, ATP levels have been correlated with in vitro growth of several mycobacteria, including M. leprae and M. tuberculosis [23,32–34]. In the present study, ATP levels per million cells of M. leprae declined progressively with treatment, however the microscopic counts remained some 2 weeks after initiating treatment. The bacilli obtained at 2 weeks had lost their viability as indicated by their failure to multiply in the footpads of mice. During this period, the ATP levels of the organisms declined to 21% of the original. This loss of metabolic activity suggest that bacteria were not in a position to multiply even when given a favorable environment. Using ATP and [3H]thymidine as biochemical indicators, we have demonstrated that M. leprae can multiply, to a limited extent, in DH medium [23 – 25]. M. leprae obtained from armadillos in the experimental group before initiating treatment multiplied in DH medium in the same fashion as those obtained from animals in the control group. However, after 2 weeks of rifabutin treatment, levels of ATP and [3H]thymidine fell to 23–26% of those taken before treatment; this was reflected in lower growth of these organisms in the DH medium. At the end of 8 weeks of treatment, no detectable levels of ATP and [3H]thymidine uptake were found and in addition, these organisms failed to
show any growth in the DH medium. This proves the correlation between the two metabolic indicators and the viability of the organisms. Using the in vitro drug susceptibility method developed by us [11], we have shown that the mean inhibitory concentration (MIC) of rifabutin against rifampin-sensitive M. leprae was 0.2 mg/ml [16,17]. In the present studies, the rifabutin levels in plasma of armadillos treated with rifabutin was at least twice the MIC level during the treatment and thus were adequate to exert the killing action on M. leprae. While studying the pharmacokinetics of rifabutin in humans, Skinner and co-workers [27] showed that the 25-deacetylated metabolite of rifabutin reaches peak values of about 10% of the parent compound and demonstrated a similar plasma decay curve to rifabutin. Results in this present study agree well with those of Skinner and co-workers.
5. Conclusions Thus, the present studies suggest two important points: (a) the armadillo is a suitable model for the chemotherapeutic studies in leprosy; and (b) rifabutin is a potent bactericidal drug that should be evaluated in leprosy patients.
Acknowledgements Financial support from the German Leprosy Relief Association, Wurzburg, Germany and from the National Institutes of Health (NO1-AI-05052), Bethesda, MD, USA, is gratefully acknowledged. The authors wish to thank Pharmacia-Adria Laboratories for the generous supply of rifabutin and Ms Janet Stoddard for excellent care of the armadillos.
A.M. Dhople, S.L. Williams / International Journal of Antimicrobial Agents 9 (1998) 169–173
References [1] Bullock WE. Rifampicin in the treatment of leprosy. Rev Infect Dis 1983;5:S606 – 13. [2] World Health Organization. A Guide to Leprosy Control, 2nd ed. Geneva: World Health Organization, 1988. [3] Sanfillipo A, Della Bruna C, Marsilli L, et al. Biological activity of a new class of rifamycins, spiropiperidyl rifamycins. J Antibiot 1980;33:1193 –8. [4] Woodley CL, Kilburn JO. In vitro susceptibility of Mycobacterium a6ium complex and Mycobacterium tuberculosis strains to a spiropiperidyl ansamycin. Am Rev Resp Dis 1982;126:586 – 7. [5] Della Bruna C, Schioppacassi G, Ungheri D, Jabes D, Morvillo E, Sanfillipo A. LM427, a new spiropiperidyl rifamycin:in vitro and in vivo studies. J Antibiot 1983;36:1502–6. [6] Cynamon MH. Comparative in vitro activities of MDL473, rifampin and ansamycin against Mycobacterium intracellulare. Antimicrob Agent Chemother 1985;28:440–1. [7] Heifets LB, Iseman MD, Lindholm-Levy PJ, Kanes W. Determination of ansamycin MICs for Mycobacterium a6ium complex in liquid medium by radiometric and conventional methods. Antimicrob Agent Chemother 1985;28:570–5. [8] Truffot-Pernot C, Giroir AM, Grosset J. A study of the minimal inhibitory concentrations of rifabutin (ansamycin LM 427) for Mycobacterium tuberculosis, Mycobacterium a6ium-intracellulare and Mycobacterium xenopi. Rev Mal Resp 1988;5:401–6. [9] Hastings RC, Jacobson RR. Activity of ansamycin against Mycobacterium leprae in mice. Lancet 1983;2:1079–80. [10] Hastings RC, Richard VR, Jacobson RR. Ansamycin activity against rifampicin-resistant Mycobacterium leprae. Lancet 1984;1:1130. [11] Dhople AM, Ortega I. An in vitro culture method for screening new drugs against Mycobacterium leprae. Indian J Lepr 1990;62:66 – 75. [12] Dhople AM, Ibanez MA, Gardner GD. In vitro synergistic activity between ofloxacin and ansamycins against Mycobacterium leprae. Arzneim-Forsch/Drug Res 1993;43:384–6. [13] Dhople AM, Ibanez MA. In vitro activities of novel benzoxazinorifamycins against Mycobacterium leprae. J Antomicrob Chemother 1995;35:463–71. [14] Dhople AM, Ibanez MA. In vitro activity of levofloxacin, singly or in combination with rifamycin analog, against Mycobacterium leprae. Antimicrob Agent Chemother 1995;39:2116–9. [15] Dhople AM, Dimova V. In vitro activity of a new rifamycin derivative against Mycobacterium leprae. Arzneim-Forschung/ Drug Res 1996;46:210–2. [16] Dhople AM, Ibanez MA. In vivo susceptibility of Mycobacterium leprae to ofloxacin either singly or in combination with rifampicin and rifabutin. Arzneim-Forsch/Drug Res 1994;44:563 – 5.
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[17] Dhople AM, Ibanez MA. In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae. Indian J Lepr 1995;67:375 – 82. [18] Kirchheimer WF, Storrs EE. Attempts to establish the armadillo (Dasypus no6emcinctus) as a model for the study of leprosy. Int J Leprosy 1971;39:693 – 702. [19] Kirchheimer WF, Storrs EE. Leprosy in experimentally infected armadillos. Int J Lepr 1972;40:212 – 3. [20] Dhople AM. Armadillo as a model for studying chemotherapy of leprosy. Indian J Lepr 1986;58:19 – 28. [21] World Health Organization. Purification of Mycobacterium leprae. Proceedings of the Fifth meeting of the Scientific Working Group on the Immunology of Leprosy. Document no. TDR/ IMMLEP-SWG (5) 80.3, Geneva: World Health Organization, 1980. [22] Hanks JH, Chatterjee BR, Lechat MF. A guide to the counting of mycobacteria in clinical and experimental materials. Int J Lepr 1964;32:156 – 67. [23] Dhople AM, Green KJ, Osborne LJ. Limited in vitro multiplication of Mycobacterium leprae. Ann Inst Pasteur/Microbiol 1988;139:213 – 23. [24] Dhople AM, Lamoureux LC. Factors influencing the in vitro growth of Mycobacterium leprae; effect of sulfhydryl compounds. Microbiol Immunol 1991;35:209 – 13. [25] Dhople AM, Lamoureux LC. Factors influencing in vitro growth of Mycobacterium leprae; effect of oxygen. Microbiol Immunol 1991;35:507 – 14. [26] Shepard CC. The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J Exp Med 1960;112:445 – 54. [27] Skinner MH, Hsieh M, Torseth J, et al. Pharmacokinetics of rifabutin. Antimicrob Agent Chemother 1989;33:1237 –41. [28] Dhople AM. Adenosine triphosphate content of Mycobacterium lepraefrom leprosy patients. Int J Lepr 1984;52:183 – 8. [29] Seydel U, Lindner B, Dhople AM. Results from cations and mass fingerprint analysis of single cells and from ATP measurements of Mycobacterium leprae. Int J Lepr 1985;53:365–72. [30] Katoch VM. Application of bioluminescent technology to patient care and research in leprosy. Indian J Lepr 1989;61:313–22. [31] Dietz M, Hass M, Lindner B, Dhople AM, Tebebe YB, Seydel U. Intrabacterial sodium-to-potassium ratios and ATP contents of Mycobacterium leprae from ofloxacin-treated patients. Int J Lepr 1991;59:548 – 57. [32] Dhople AM, Hanks JH. In vitro growth of Mycobacterium lepraemurium, an obligate intracellulare microbe. Science 1977;197:379 – 81. [33] Dhople AM, Green KJ. An in vitro system using adenosine triphosphate and [3H]thymidine to determine drug sensitivity of Mycobacterium leprae. IRCS J Med Sci 1986;14:807 – 8. [34] Beckers B, Lang HRM, Schimke D, Lammers A. Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria. Eur J Clin Microbiol 1985;4:556–61.
The activity of rifabutin against Mycobacterium leprae in armadillos Arvind M. Dhople *, Sharon L. Williams Department of Biological Sciences, Infectious Diseases Laboratory, Florida Institute of Technology, 150 West Uni6ersity Boule6ard, Melbourne, FL 32901 -6975, USA Accepted 29 October 1997
Abstract The activity of rifabutin (LM 427) against Mycobacterium leprae was evaluated in armadillos inoculated earlier with human-derived M. leprae. Rifabutin was administered daily at a dose of 6 mg/kg body weight/day. The effect of rifabutin on M. leprae harvested from armadillos was determined by measuring the intracellular levels of ATP (an indicator of metabolic activity) of M. leprae and also their ability to multiply in the mouse footpads and in vitro in DH medium. Within 2 weeks of initiating the treatment, ATP levels declined to 21% of the original (pre-treatment level) and these M. leprae failed to multiply in the footpads of mice as well as in the in vitro culture system. This suggests that rifabutin was able to kill all M. leprae within 2 weeks. After 8 weeks the treatment was terminated and results showed that M. leprae from the treated armadillos remained non-viable in the mouse footpad system as well as in the in vitro system, indicating bactericidal action of rifabutin. The results suggest that rifabutin can be a substitute for rifampin in the leprosy multi-drug therapy regimen. © 1998 Elsevier Science B.V.
1. Introduction Rifampin is the most potent bactericidal drug available today in the treatment of leprosy [1] and is considered a standard drug worldwide, as part of multi-drug therapy (MDT) regimen recommended by the World Health Organization [2]. A semi-synthetic rifamycin, rifabutin (Ansamycin LM 427; spiropiperidyl rifamycin), has been reported as having substantial in vitro activity against several species of mycobacteria [3 – 7]. Its minimal inhibitory concentrations (MICs) are from 2 [5] to 20 [8] times lower than those of rifampin. Hastings and Jacobson [9] were the first to study the action of rifabutin against M. leprae in mouse footpads. Subsequently, Hastings and associates [10] have shown Abbre6iations: Mycobacterium leprae; Armadillo; Rifabutin; Multidrug therapy. * Corresponding author. Tel.: + 1 407 6747253; fax: + 1 407 9848461; e-mail:[email protected] 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 4 - 8 5 7 9 ( 9 7 ) 0 0 0 4 8 - 4
that the MIC of rifabutin against Mycobacterium leprae in mouse footpads was several times lower than that of rifampin. Using the in vitro system developed in our laboratory [11], superiority of rifabutin over rifampin was demonstrated against both rifampin-sensitive and rifampin-resistant strains of M. leprae [12–15]. Similar superiority of rifabutin has also been shown in our mouse footpad studies [16,17] for both rifampin-sensitive and rifampin-resistant strains of M. leprae. In the early 1970s, Kirchheimer and Storrs [18,19] have demonstrated that the nine-banded armadillo (Dasypus no6emcinctus) developed disseminated leprosy if inoculated intravenously with human-derived M. leprae. Subsequently, we have shown that this animal model can be used for chemotherapeutic investigations in leprosy [20]. Since armadillo is a better model than mouse for disseminated leprosy, it was decided to extend the in vivo studies on the effects of rifabutin against M. leprae using armadillo as a model of multibacillary leprosy.
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2. Materials and methods
2.1. Armadillo inoculation Rifampin-susceptible M. leprae were harvested from the livers of nine-banded armadillos which had been previously inoculated with a human-derived, rifampinsensitive strain of M. leprae. M. leprae cells were purified by first treating the liver suspensions with DNAase and then separating the bacteria from the host tissue with a Percoll liquid density gradient (Pharmacia LKB Biotechnology, Piscataway, NJ) and centrifugation [21]. The cells were counted microscopically by the pinhead method [22]. A total of 20 males were selected for the study. Average weight was 4.9 lb and all were in good health. Smears from blood buffy coats, ear clippings and nasal swabs failed to show any acid-fast bacilli. All the animals were anesthetized using Innovar-vet (0.15 ml/ kg) plus atropin sulfate (0.04 ml/kg). Each animal was inoculated subcutaneously at ten different sites on the abdomen, each site receiving 0.1 ml suspension containing 106 M. leprae. Animals were housed in ‘vari-kennel’cages and were fed daily with moistened cat chow and provided water ad libitum. Between 9 and 11 months post inoculations, 14 animals developed subcutaneous lepromas (approximately 10 mm diameter in size) at the sites of inoculations. These 14 animals were divided into two groups; control, receiving only cat chow and the experimental, receiving rifabutin along with cat chow.
2.2. Drug administration Rifabutin was a gift from Pharmacia-Adria Laboratories, Dublin, OH. All seven animals in the experimental group received rifabutin every day (7 days a week) for a total of 8 weeks. Rifabutin was administered through food at a dose of 6mg/kg per day. Required quantity of the drug was mixed in small amount of moistened cat chow and fed to the animals early in the morning, when they are most hungry. After consuming all the food, a bowl with moistened cat chow was placed in each cage to eat ad libitum.
2.3. Susceptibility testing Before initiating rifabutin treatment, all animals were anesthetized and with 5-mm punch biopsy, biopsy specimens from one leproma of each of the 14 animals were collected aseptically. Also, 5 ml of blood from each animal was drawn through the saphenous vein and collected in heparinized vacutainers. This procedure was repeated at the end of 1, 2, 4 and 8 weeks of treatment. After 8 weeks the administration of rifabutin was terminated and all animals in the experimental
group were fed moistened cat chow, similar to those in the control group. The biopsy procedure was repeated at 4 and 8 weeks after withdrawing rifabutin.
2.4. Assays for the 6iability of M. leprae Suspensions from each biopsy specimen were prepared and the suspensions were purified by the method described above. Aliquots were used for microscopic enumeration of M. leprae, for the assay of intracellular adenosine triphosphate (ATP) determinations, for in vitro culture of M. leprae and also for inoculations into footpads of mice. ATP assays were performed by the firefly bioluminescence method described earlier [23] and the amount of intracellular ATP was expressed as picogram. of ATP per 106 M. leprae cells. For the in vitro culture, DH medium was used and the culture system which was previously described, using ATP and [3H]thymidine uptake as metabolic indicators of the growth of M. leprae [23–25]. Inoculation of M. leprae into footpads of BALB/c mice (Jackson Laboratories, Bar Harbor, ME) and subsequent harvests of footpads were carried out by the standard technique developed by Shepard [26].
2.5. Rifabutin assays Blood samples were centrifuged for 10 min at 1200× g and the plasma was removed and stored at −76°C. The concentration of rifabutin and its major metabolite, 25-deacetylrifabutin, was determined by high-pressure liquid chromatography using the method of Skinner and associates [27].
3. Results The results on the effect of rifabutin on M. leprae harvested from armadillos treated with rifabutin are presented in Table 1. During the 16-week period of observation, the load of acid-fast bacteria in subcutaneous nodules from animals in the control group, as counted by the pin-head method [22], increased from 100 to 226%. In case of armadillos treated with rifabutin, the number of acid-fast bacteria decreased from 100 to 77% during the same period. However, a different picture emerged with the metabolism of these bacteria, even though the metabolism of M. leprae from untreated animals remained constant, it declined steadily with rifabutin treatment, reaching 9 and 0% at the end of 4 and 8 weeks, respectively. This suggests that the viability of these organisms was also affected with the rifabutin (discussed in Table 2). The rifabutin levels in plasma of animals from the treated group remained steady during treatment, varying from 0.38 mg/ml at the end of first week to 0.49 mg/ml at the end
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171
Table 1 Anti-leprosy activity of rifabutin in armadillos Weeks
Microscopic counts: M. leprae/g (×108)
ATP (pg/106 M. leprae)
Plasma levelsb (mg/ml)
Control group
Experimental groupa
Control group
Experimental group
Rifabutin
25-d.a. rifabutinc
0 1 2 4 8
72.3 9 7.95 (100)e 79.8 9 9.38 (110) 90.3 98.01 (125) 102.1 9 14.29 (141) 117.3 9 10.59 (162)
80.99 11.33 (100) 81.79 7.39 (101) 79.39 8.72 (98) 76.1 9 6.71 (94) 72.3 9 10.72 (89)
25.1 93.00 25.693.80 27.3 93.00 24.6 93.20 26.1 93.70
24.4 9 3.70 17.6 9 2.50 5.1 9 0.56 2.2 9 0.33 0
0 0.38 90.05 0.45 90.04 0.51 90.07 0.49 90.07
0 0.04290.006 0.05090.005 0.055 90.007 0.05290.007
12 16
137.3 919.22 (190) 163.4 9 17.19 (226)
67.2 9 7.92 (83) 62.5 9 5.62 (77)
26.9 92.90 (107) 27.993.60 (111)
0.09 90.01 0
0 0
(100) (102) (109) (98) (104)
0 0
(100) (72) (21) (9)
a
Rifabutin, 6 mg/kg per day. Experimental group only. c 25-Deacetylrifabutin. d Rifabutin administration terminated. e % Values. b
of 8 weeks. On withdrawl of rifabutin, levels in plasma started declining, with complete disappearance occuring between 12 and 16 weeks. A similar pattern was also observed in the case of 25-deacetylrifabutin, except that the plasma levels were approximately 10% of those of rifabutin. The viability of M. leprae harvested from these animals was established using three indicators as described earlier and the results are presented in Table 2. M. leprae harvested from armadillos in the control group retained their viability during the period of observation. Organisms harvested at 0, 2, 8 and 16 weeks and inoculated into footpads of mice multiplied in normal fashion. Also, these organisms retained their metabolic activity when inoculated in the DH medium and incubated at 34°C for 16 weeks. The zero hour values (100%) for ATP varied between 202 and 218 pg/ml per culture, indicating that the % increase at the end of 16 weeks of incubation was between 1706 and 2010, which was normal. In the case of M. leprae harvested from armadillos treated with rifabutin, the organisms lost their viability as well as metabolic activity. M. leprae obtained 2 weeks after initiating treatment failed to multiply in the footpads of mice and remained non-viable during the entire period of treatment. In the in vitro culture system, the zero hour (100%) ATP values of organisms harvested at 0 and 2 weeks were 2099 25 pg/ml culture and 48 92.5 pg/ml culture, respectively, thus giving a % increase of 2010 and 710, respectively, at the end of 16 weeks of incubation in DH medium. Similarly, the zero hour values for [3H]thymidine uptake for organisms harvested at 0 and 2 weeks were 0.72 90.10 pmol/5 ml culture and 0.1990.02 pmol/5 ml culture, giving the % increase of 2490 and 850, respectively, at the end of 16 weeks of incubation in DH medium. Organisms harvested 8 weeks after initiating rifabutin treatment did not show presence of ATP
and also failed to incorporate [3H]thymidine. These organisms also failed to multiply in DH medium, since at the end of 16 weeks of incubation, neither the presence of ATP nor the uptake of [3H]thymidine was detected. Even 8 weeks after terminating the rifabutin treatment, organisms from this group of animals failed to multiply in the footpads of mice and also failed to demonstrate any metabolic activity (ATP and [3H]thymidine uptake). Furthermore, these organisms also failed to grow in DH medium. Thus, the results suggest that the effects of rifabutin on M. leprae were bactericidal.
4. Discussion We have demonstrated earlier the bactericidal activity of rifabutin in both the in vitro system [12–15] and in the mouse footpad system [16,17]. Even though the mouse footpad system had been widely used for evaluating potential anti-leprosy compounds, the important drawbacks of this model have been limited multiplication of M. leprae in footpads and failure to develop disseminated infection resembling human lepromatous leprosy. Previously, we demonstrated the suitability of armadillos for such chemotherapeutic studies based on the pattern of dapsone metabolism by these organisms, which was similar to that observed in humans [20]. The results presented here on the anti-leprosy effects of rifabutin in armadillos confirm our earlier findings using the in vitro culture system and the mouse footpad system. Microscopic counts of acid-fast bacilli do not differentiate between viable and non-viable organisms and thus do not indicate the viability or metabolic status of the organisms. Even though the microscopic counts of M. leprae from armadillos in the control group in-
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Table 2 Growth characteristics of M. leprae harvested from rifabutin-treated aramaillos Characteristics M. leprae harvested from armadillos (–) weeks after initiating rifabutin treatment 0 Week Control Mouse foot3.51 90.39 pada In vitro 3957 9 475 growthb ATPc [3H]thymidined 16.90 92.19
2 Weeks Experimental
Control
3.24 9 0.45
3.36 9 0.44
4194 9 461
37989 425
17.92 9 2.15
15.89 9 2.27
8 Weeks Experimental
Control
16 Weeks Experimental
Control
Experimental
3.64 90.350 0
3.29 9 0.30
0
342 947
4392 9614
0
3685 9 479
0
1.61 9 0.18
19.09 92.86
0
15.74 91.73
0
0
a
Footpad harvested 9 months after inoculations (×106 per footpad). Metabolic activity after 16 weeks of incubation in DH medium (Inoculum: 1×107/ml). c Picograms of ATP in M. leprae from 1 ml of culture. d Picomols of [3H]thymidine uptake by M. leprae from 5 ml of culture. b
creased in 16 weeks, those in the experimental group decreased by about 23%. However, bacilli from the experimental group failed to multiply in the footpads of mice, suggesting loss of viability. Dhople [28] was the first to demonstrate the applicability of ATP assays for monitoring the response of leprosy patients to chemotherapy. It was demonstrated that ATP content of M. leprae progressively declined with therapy and responses correlated with loss of infectivity in mouse footpads. This was further confirmed by Seydel et al. [29], Katoch [30] and Dietz et al. [31]. Furthermore, ATP levels have been correlated with in vitro growth of several mycobacteria, including M. leprae and M. tuberculosis [23,32–34]. In the present study, ATP levels per million cells of M. leprae declined progressively with treatment, however the microscopic counts remained some 2 weeks after initiating treatment. The bacilli obtained at 2 weeks had lost their viability as indicated by their failure to multiply in the footpads of mice. During this period, the ATP levels of the organisms declined to 21% of the original. This loss of metabolic activity suggest that bacteria were not in a position to multiply even when given a favorable environment. Using ATP and [3H]thymidine as biochemical indicators, we have demonstrated that M. leprae can multiply, to a limited extent, in DH medium [23 – 25]. M. leprae obtained from armadillos in the experimental group before initiating treatment multiplied in DH medium in the same fashion as those obtained from animals in the control group. However, after 2 weeks of rifabutin treatment, levels of ATP and [3H]thymidine fell to 23–26% of those taken before treatment; this was reflected in lower growth of these organisms in the DH medium. At the end of 8 weeks of treatment, no detectable levels of ATP and [3H]thymidine uptake were found and in addition, these organisms failed to
show any growth in the DH medium. This proves the correlation between the two metabolic indicators and the viability of the organisms. Using the in vitro drug susceptibility method developed by us [11], we have shown that the mean inhibitory concentration (MIC) of rifabutin against rifampin-sensitive M. leprae was 0.2 mg/ml [16,17]. In the present studies, the rifabutin levels in plasma of armadillos treated with rifabutin was at least twice the MIC level during the treatment and thus were adequate to exert the killing action on M. leprae. While studying the pharmacokinetics of rifabutin in humans, Skinner and co-workers [27] showed that the 25-deacetylated metabolite of rifabutin reaches peak values of about 10% of the parent compound and demonstrated a similar plasma decay curve to rifabutin. Results in this present study agree well with those of Skinner and co-workers.
5. Conclusions Thus, the present studies suggest two important points: (a) the armadillo is a suitable model for the chemotherapeutic studies in leprosy; and (b) rifabutin is a potent bactericidal drug that should be evaluated in leprosy patients.
Acknowledgements Financial support from the German Leprosy Relief Association, Wurzburg, Germany and from the National Institutes of Health (NO1-AI-05052), Bethesda, MD, USA, is gratefully acknowledged. The authors wish to thank Pharmacia-Adria Laboratories for the generous supply of rifabutin and Ms Janet Stoddard for excellent care of the armadillos.
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