a Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK; b NeXstar Pharmaceuticals Inc., Boulder, CO 80301, USA; c Medical Research Council Clinical Trials Unit, 222 Euston Road, London NW1 2DA, UK
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Abstract |
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Introduction |
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Materials and methods |
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Female outbred, specific pathogen-free MF1 white mice with a starting weight of 2022 g were supplied by Bantin & Kingman (Hull, UK) or by Harlan & Olac (Bicester, UK). They resemble the Swiss Albino mice used in previous classical studies.12,13 Thawed aliquots of M. tuberculosis and suspensions of BCG were appropriately diluted in 0.1% buffered gelatin saline and were sonicated for 18 s to remove clumps before injection iv into mice in 200 µL volumes. Mice were challenged with 1.3 x 105 cfu of a recently mouse-passaged, virulent, drug-susceptible H37Rv strain of M. tuberculosis that had been stored in liquid N2 previously. Mice were immunized with 9.4 x 106 cfu Mycobacterium bovis BCG (Glaxo, Greenford, Middlesex, UK), which was prepared and freeze-dried by Evans Medical Ltd (Leatherhead, UK).
Organ culture
The method used has been described previously.14 In brief, the spleen and lungs were placed after sterile autopsy into motorized polytetrafluorethylene/glass grinders and homogenized in 5 mL of water. Homogenates were diluted in 10-fold steps and 100 µL were spread on segments of 7H11 medium made selective by the addition of 100 µg carbenicillin, 200 U polymyxin B, trimethoprim 20 µg and amphotericin B 10 µg per mL of medium; these antibiotics do not influence the growth of M. tuberculosis.15 Colonies were counted after incubation for 3 weeks at 37°C in sealed plastic bags; the counts were expressed as cfu/organ.
Liposomal amikacin
The liposomal amikacin (MiKasome), supplied by NeXstar Pharmaceuticals Inc. (San Dimas, CA, USA), is a preparation of amikacin entrapped within the aqueous core of small unilamellar liposomes <100 nm diameter. The liposomes are made of hydrogenated soy phosphatidylcholine, cholesterol and distearoylphosphatidyl glycerol in a 2:1:0.1 molar ratio.8 They were formed by rehydration of the spray-dried lipids in drug-containing buffer, sized by homogenization, and unencapsulated drug removed by dialysis against formulation buffer (9% sucrose/succinate at pH 6.5). They contained amikacin c. 10 mg/mL, with a lipid:drug ratio of c. 5:1. More than 85% of the amikacin in the final formulation was contained within the liposomes whereas the remainder was externally associated with the liposome bilayer.
Drug dosage
The maximal dose size of streptomycin is 200 mg/kg body weight when given by im injection, whereas 50 mg/kg has little effect.16 The highest dose of liposomal amikacin used was 160 mg/kg, to avoid particularly large volumes of the iv doses; lower doses were 80 and 40 mg/kg.
Experimental layout
In the first two experiments in the acute tuberculosis model, liposomal amikacin was given iv three times a week. The rationale for this frequency arose from the pharmacokinetic study in mice (see below) which indicated that a frequency of three times a week would maintain an elevated plasma concentration without the accumulation that might occur with more frequent dosage. In experiment 1, free amikacin and streptomycin were given by daily im injection five times a week to mimic the optimal frequency of dosage in humans. In experiment 2, they were given iv three times a week at the same frequency as for liposomal amikacin. The numbers of mice at each time point in the three experiments are set out in Table 1. In experiments 1 and 2, four mice were killed for organ counts 2 h after challenge with M. tuberculosis. After 4 days, further counts were made and the remaining mice were divided into treatment groups, some of which were dosed three times a week with liposomal amikacin 160, 80 or 40 mg/kg iv. In experiment 1, further groups were given free amikacin and streptomycin 160 or 80 mg/kg im five times a week. In experiment 2, groups were given free amikacin or streptomycin 80 or 40 mg/kg iv three times a week, as the 160 mg/kg doses of both free aminoglycosides were rapidly lethal. In both experiments, there was a control group that received drug-free liposomes in the same concentration as in the liposomal amikacin suspension in the 160 mg/kg dose. Mice were then killed for organ counts at 7, 14, 21 and 28 days (t7, t14, t21, t28) after t0. In experiment 3, mice were first immunized with BCG. After 5 weeks, all mice were challenged with M. tuberculosis. Organ counts for BCG were made after immunization (t-49) and just before challenge (t-14). In subsequent organ cfu counts, 2 mg/L thiophen-2-carboxylic acid (TCH) was added to the 7H11 medium to inhibit BCG. Organ counts were done at weekly intervals during a 2 week stabilization period (t-14 and t-7). At the end of stabilization (t0), the mice were then divided into treatment groups to receive liposomal amikacin 160 or 80 mg/kg, free amikacin 160 mg/kg, isoniazid 25 mg/kg or rifampicin 15 mg/kg. Liposomal amikacin was given iv three times a week for 4 weeks and then once a week for 4 weeks. Free amikacin was given by im injection, and rifampicin and isoniazid by oral gavage, on 5 days in each week. A control group was given drug-free liposomes in the same concentration as in the liposomal amikacin 160 mg/kg preparation. Mice were humanely killed at t0 and at t14, t28, t42 and t56 for organ counts. At t28, t42 and t56, organ homogenates were plated on medium containing 0.1 mg/L isoniazid to test for the emergence of resistance. Resistance to the other drugs was unlikely because of their low mutation rates.
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Tissues were homogenized in trichloroacetic acid (10 mL/g), centrifuged (1 h at 9000 r.c.f.) and the supernatants analysed for amikacin. Total amikacin in pooled mouse plasma and tissue homogenates was measured using a commercial fluorescence polarization immunoassay (TDx/FLx, Abbott Diagnostics, Abbott Park, IL, USA) as described previously.17 Triton X-100 was added in a final concentration of 0.05% to the assay dilution buffer to release liposome-associated amikacin. The lower limit of quantification was 0.3 mg/L for plasma and 6.6 mg/L for tissues.
Statistical assessments
In order to obtain a single measure of the response within each treatment group of mice, straight lines were fitted to the cfu counts from t7 to t28 in experiments 1 and 2. The estimated cfu counts, taken as the intercept of t28 with the lines, were chosen as the best single estimates of response, as some of the fitted lines diverged slightly as the experiments proceeded. The estimated cfu counts at t28, obtained from the four sets of spleen and lung log cfu counts in experiments 1 and 2 (y-axis), were then related to log dose size (x-axis) in regression models (Stata release 6.0; Stata Corp., College Station, TX, USA). In each of the four sets of data, no significant deviations from a common regression line were found using a statistical test for interaction. The log potency of liposomal amikacin relative to the other aminoglycosides was then estimated as follows:
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The variance of log potency was derived, using a standard variance formula for the ratio of two random variables,18 which can be considered as a naive version of Fieller's Theorem.
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Results |
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To determine the pharmacokinetic profile of liposomal amikacin, uninfected mice were injected iv with liposomal amikacin 160 mg/kg body weight. In groups of three mice, blood was obtained from tail veins or the heart at 0.33, 0.5, 1, 3, 5, 24, 48, 73 and 96 h and lungs and spleens were taken at 1, 6, 24 and 72 h after the dose (Figure 1). In plasma, there was an initial rapid fall from a Cmax of 2920 mg/L at 0.5 h to 855 mg/L at 5 h. Thereafter, the fall was slower to 264 mg/L at 48 h, 26 mg/L at 73 h and 3.4 mg/L at 96 h. The plasma half-life was initially 25 h, falling to 7.5 h after the 48 h time point. Tissue concentrations were more prolonged with a Cmax of 245 and 263 mg/L, half-lives of 23 and 31 h, and Kp (ratio of tissue AUC to plasma AUC) of 0.22 and 0.73 in lungs and spleens, respectively. Additional estimations were done on heart blood obtained from groups of four to eight infected mice treated with liposomal amikacin 160 mg/kg in experiment 3. During the initial three times a week dosing phase, the total plasma amikacin concentration was 19.6 mg/L 3 days after the sixth dose and 50 mg/L 3 days after the 12th dose, indicating that liposomal amikacin maintained continuously elevated plasma levels during thrice weekly dosage. During the subsequent once weekly dosing phase, no amikacin was detected (<3 mg/L) at 7 days after the second and the fourth doses, indicating that weekly dosing with liposomal amikacin did not provide continuous plasma exposure in mice.
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In addition to those in Table 1, one mouse died in experiment 1, probably as a result of iv mis-injection of streptomycin 160 mg/kg, whereas four mice were killed by the initial attempts to give streptomycin and free amikacin 160 mg/kg iv in experiment 2, and a further two mice died in experiment 3, one because of air embolism and one was humanely killed because its veins were sclerosed.
Acute murine tuberculosis
(i) Controls dosed im. In experiment 1, liposomal amikacin at 160, 80 and 40 mg/kg was given iv, three times a week, while free amikacin and streptomycin at 160 and 80 mg/kg were given im five times a week. Spleen cfu counts (Figure 2) in the control group rose rapidly during the first week and then levelled off as immunity developed. The counts in the treated groups rose less rapidly during the first 7 days but thereafter all were approximately parallel. The curves for the 160 and 80 mg/kg doses of liposomal amikacin rose to a lesser extent than the groups treated with free amikacin or streptomycin at the same dose levels. Almost all of the differences between the treatment regimens in their cfu counts occurred during the first 7 days of treatment. In the lungs (Figure 2
), immunity developed more slowly in the control mice than in spleens. Again the differences between the treated groups arose during the first 14 days while immunity was not yet effective. The curves with liposomal amikacin were again lower than those for the corresponding dose sizes of amikacin and streptomycin.
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The mean counts in spleens and lungs, estimated as intercepts of fitted straight lines at t28, are considered to be the best single estimate of response in each treatment group. These estimated t28 counts are plotted against log dose size in Figure 4 for both experiments. The lines relating count to dose for liposomal amikacin, free amikacin and streptomycin within the four sets of data from the spleen and lung tissues in the two experiments were found by regression analysis to be parallel. Using the regression models, the potency of liposomal amikacin relative to amikacin or streptomycin was then calculated (see Statistical methods). In experiment 1, liposomal amikacin was 2.414.98 times more active than amikacin (weight for weight) and was 6.606.73 times more active than streptomycin (Table 2
). In experiment 2, the corresponding liposomal amikacin potencies are 2.692.94 relative to amikacin and 3.715.60 relative to streptomycin. Thus, liposomal amikacin was the most active, and free amikacin appeared slightly more active than streptomycin. As the potency ratios were more than twice their standard errors (s.e.) in experiment 1, the differences between liposomal amikacin and free amikacin or streptomycin are both statistically significant, although they are not in experiment 2. The failure to achieve statistical significance in experiment 2 is likely to be due to the absence of the 160 mg/kg dose of amikacin and streptomycin.
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In experiment 3, cfu counts in spleens (Figure 5) showed a c. 10-fold fall in the counts of BCG between the immunizing dose and challenge with M. tuberculosis H37Rv. After the challenge, the spleen counts increased during the 14 day stabilization period but remained constant in the control group thereafter at c. 105 cfu/spleen. The counts in mice treated with amikacin 160 mg/kg five times a week were only slightly lower than in the controls. Mice treated with liposomal amikacin three times a week showed dose-dependent reductions in cfu, so that after 4 weeks treatment, counts in the liposomal amikacin 160 mg/kg group were 10-fold lower than controls, and similar to those in mice treated with five times a week isoniazid 25 mg/kg. Counts from both liposomal amikacin doses then levelled off between the fourth and eighth week of treatment so that by 8 weeks neither liposomal amikacin group was as effective in sterilization as the five times a week isoniazid. The counts on mice treated five times a week with the potent sterilizing drug rifampicin fell more rapidly than in any other treatment group over the whole period. In the lungs (not tabulated here), counts in control mice failed to stabilize, continuing to rise from t0 to t8. The counts in the treatment groups were similar to those in the spleens except that there was less difference between the isoniazid and the rifampicin groups. Resistance to isoniazid was not found. It is of interest that the neat dilutions of the spleen and lungs of the mice treated with 160 or 80 mg/kg liposomal amikacin, which were exceptionally plated out without further dilution at t28, t42 and t56, completely inhibited growth of M. tuberculosis but growth occurred freely from 1:10 dilutions. As the organs, usually weighing c. 1 g, were homogenized in 5 mL diluent, organ concentration must have been between five and 50 times the MIC of amikacin (2 mg/L).
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For those used to error bars on graphs, variation between the log cfu counts within each treatment group and sampling time were calculated. The pooled estimates of the standard deviation (s.d.) in experiment 1 were 0.23 for spleen counts and 0.21 for lung counts with 119 degrees of freedom (df) for each whereas in experiment 2, the corresponding estimates were 0.25 for spleens and 0.26 for lungs with 108 df. In experiment 3, the pooled s.d. increased slightly in the spleen counts as the experiment proceeded starting with 0.24 in groups up to t4 (27 df), then 0.29 at t6 (22 df) and 0.44 at t8 (23 df). From these estimates, 95% confidence limits were calculated and entered as single error bars in Figures 1 and 2. As they apply to a single time point only, they cannot be used to test the overall difference between the curves, which has been assessed over several time points in Table 2
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Discussion |
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Experiments 1 and 2 demonstrate that liposomal amikacin is more active weight for weight than free amikacin or streptomycin in the treatment of acute murine tuberculosis. Liposomal amikacin was estimated to be 2.45.0 times more potent than free amikacin and 3.76.7 times more potent than streptomycin (Table 2). Liposomal amikacin was also found to be more active than free amikacin in a murine Klebsiella pneumoniae model.8 The greater activity of liposomal amikacin is not surprising in view of the high levels of amikacin (50 mg/L) found 3 days after an iv dose of liposomal amikacin 160 mg/kg. To exert antimicrobial activity, amikacin must be released from the liposomes of liposomal amikacin, probably after phagocytosis by macrophages, and reach the tubercle bacilli in mouse tissues, which are usually considered to be intracellular. In the cavities of human pulmonary tuberculosis, the great majority of bacilli are, however, extracellular and may lie some distance from blood vessels in caseum near the air interface. The delivery of amikacin from the liposomes to these bacilli is more problematic and has been explored in studies of the early bactericidal activity of liposomal amikacin in pulmonary tuberculosis.10 Although streptomycin is highly bactericidal against rapidly dividing M. tuberculosis, it has little activity against bacilli that are not multiplying,20 for instance in the stationary phase of growth.21 A similar dependency on bacterial multiplication was shown by liposomal amikacin, free amikacin and streptomycin as all three drugs were actively bactericidal in the early phases of experiments 1 and 2 before the onset of immunity, but showed little further activity once immunity had developed. Again, in experiment 3, there was initial bactericidal activity, reducing counts by 10-fold, during the first fortnight of treatment with liposomal amikacin, but thereafter during the next fortnight the counts did not change. Presumably all bacilli that were actively metabolizing at the start of treatment had been killed, in the same manner as occurred during the first week in the spleen cfu counts of experiments 1 and 2. The levelling off of the counts might be thought to be due to the change from thrice weekly to once weekly administration of liposomal amikacin, which allowed a period of sub-bacteriostatic plasma concentrations at the end of each week. This is an unlikely explanation because of the longer persistence of tissue concentrations in the mice and because of a long post-antibiotic lag lasting at least 8 days22 and the absence of perceptible diminution of activity when dosage of aminoglycosides is spaced out from daily to twice weekly in either animal experiments23 or in clinical trials.24 The inactivity of aminoglycosides against dormant and semi-dormant organisms accounts for their failure to increase sterilizing activity in experimental murine tuberculosis25 and also to have no more than a minimal effect in decreasing relapse rates when added to short-course regimens in the treatment of pulmonary tuberculosis.26,27 Although liposomal amikacin appears to be more effective, weight for weight, than free amikacin or streptomycin, it still had little long-term sterilizing activity and thus appears subject to the same limitations as streptomycin. In summary, encapsulation in low-clearance liposomes increases the activity and prolongs the residence times of amikacin, permitting the widely spaced dosing regimen desirable for treatment of human tuberculosis. However, the effectiveness of liposomal amikacin is limited by the poor intrinsic activity of amikacin against persisting organisms. Nevertheless, this study does not exclude the possibility that monthly administration of a more powerful sterilizing drug, such as rifampicin, in similar low-clearance liposomes could be of benefit in the treatment of human tuberculosis.
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Acknowledgements |
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Notes |
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References |
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Received 19 February 2001; returned 21 June 2001; revised 3 August 2001; accepted 12 September 2001