The early bactericidal activity of a low-clearance liposomal amikacin in pulmonary tuberculosis

Peter R. Donalda, Frederick A. Sirgelb, Amour Venterb, Elizabeth Smita, Donald P. Parkina, Bernard W. Van de Wala and Denis A. Mitchisonc,*

a Departments of Paediatrics and Child Health, Pharmacology, Medical Biochemistry and Internal Medicine, University of Stellenbosch, Cape Town; b Medical Research Council, Tygerberg, South Africa; c St George's Hospital Medical School, London SW17 0RE, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The early bactericidal activity (EBA) of a liposomal preparation of amikacin (MiKasome) with a long plasma half-life of 120–200 h was examined in seven patients with newly diagnosed, smear-positive pulmonary tuberculosis. Liposomal amikacin was given in slow iv infusions of 30 mg total amikacin/kg body weight on three successive days. Cfu counts were set up on 16 h sputum collections preceding the first dose and following each dose and were used for calculating the EBA. Despite the high concentrations of total amikacin, >1000 mg/L, obtainable in plasma, no evidence of EBA was obtained. In view of the considerable activity of liposomal amikacin in experimental murine tuberculosis, this finding indicates that liberation of amikacin from the long-life liposomes occurs only in macrophages that are not usually present in the vicinity of the large extracellular clumps of bacilli in the cavity caseum.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The aminoglycoside amikacin is a semi-synthetic derivative of kanamycin A,1 supposedly with less auditory toxicity.2 Amikacin is, therefore, sometimes used as an injectable drug in the treatment of tuberculosis when the strain is resistant to streptomycin, as there is no cross-resistance between the two aminoglycosides.3,4 A long-life liposomal preparation of amikacin (MiKasome) given by iv injection has been found to produce high and prolonged concentrations of total amikacin in the blood of animals and humans, accompanied by little evidence of toxicity.5 Concentrations of total amikacin well above the MIC of amikacin were found to be present for at least 1 month in volunteers. These findings indicate that it might be possible to give doses at very widely spaced intervals, at least 1 month apart, so avoiding problems in maintaining compliance and reducing toxicity. The liposomal drug was two to six times more active than free amikacin or streptomycin in an acute experimental model of murine tuberculosis.6 In view of this activity in the mouse, we investigated its early bactericidal activity (EBA) in patients with pulmonary tuberculosis, as the EBA is a convenient and economical way of measuring drug activity in tuberculous cavities.7,8 The EBA of antituberculosis drugs can be measured by the fall in the number of viable Mycobacterium tuberculosis in the sputum of patients with pulmonary tuberculosis during the initial days of treatment. The largest differences in the EBA between various drugs and drug combinations occurred during the first 2 days of chemotherapy and the EBA over this period has been taken as the standard measure.7 In view of the accumulation of liposomal amikacin during daily iv administration, so that the plasma concentration would be considerably higher during the third day than in the preceding days, the period during which the drug was administered was extended to 3 days. The study was originally designed for an intake of 13 patients. However, long-term toxicological studies on rabbits indicate that continuous dosage with the liposomal amikacin over a 6 month period might result in the development of atheroma. When this information became available, the intake to the study was immediately terminated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients

All patients were of mixed race (previously called ‘Cape coloured’) living in the Western Cape district, where there is a serious epidemic of tuberculosis. They were admitted for the study into Tygerberg Hospital, Cape Town, South Africa. Permission for all studies was obtained from local ethics committees and the patients gave written informed consent. Normal renal function of all patients was confirmed as a criterion for admission. Studies were carried out before and after the test period on each patient of haematology, urinalysis and serum chemistry, including liver function tests, electrolytes, cholesterol, triglycerides and glucose. No estimates of concentrations of total amikacin in blood were made, as these had already been estimated in healthy volunteers, nor were concentrations of amikacin in sputum measured.

Estimation of the EBA

Details of the procedures have been described elsewhere.9 In all patients, a 16 h collection of sputum was made on the night of the first day (S1 collection). Soon after the completion of this collection the first dose of liposomal amikacin 30 mg/kg body weight was given by slow iv drip over a 2 h period. A further 16 h sputum collection (S2 collection) was made on the second day followed by another iv dose of liposomal amikacin 30 mg/kg. This procedure was repeated once more on the third day (S3 collection). Finally one further 16 h collection was made (S4 collection) and the patient was then given standard multidrug chemotherapy. Thus, each patient was treated for 3 days and had four sputum collections.

Microbiological methods

Sputum in the S1 and S4 collections was examined conventionally by direct smear, culture and susceptibility testing. The procedure for sputum processing was similar to that described previously.9 Briefly, sputum collections were homogenized on a magnetic stirrer by adding a teflon-coated follower bar to the entire specimen which was then stirred for 20–30 min. Ten millilitres of the homogenate was added to an equal volume of 1:10 dithiothreitol (Sputasol, Oxoid, Basingstoke, UK) in a 50 mL screw-capped tube containing three to six glass beads (10 mm) and vortex mixed for 20 s. The specimen was then mechanically shaken for c. 30 min until well digested. Sets of dilutions were prepared for each collection in sterile distilled water by making 10-fold dilutions from neat to 10-2 and thereafter by serial five-fold steps. From each of the dilutions, aliquots of 100 µL were spread in duplicate on to half selective 7H11 oleic acid albumin agar plates. These were placed into polyethylene bags together with a plate inoculated with Mycobacterium phlei to provide CO2 and were incubated for 3 weeks at 37°C before colonies were counted at that dilution which permitted counting between 20 and 200 colonies.

Statistical methods

The results of the cfu counts were entered on Microsoft Excel worksheets and the corresponding log10 counts/mL sputum, Y1, Y2, Y3 and Y4, were calculated from the cfu counts on the S1, S2, S3 and S4 collections, respectively. Three EBAs were then calculated: S1–S2 EBA = Y1–Y2; S1–S3 EBA = (Y1–3)/2; and S1–S4 EBA = (Y1–Y4)/3. Shapiro-Francia tests for normality of distribution, ANOVA and regression analyses were done on these estimates using Stata release 6 (Stata Corp., College Station, TX, USA)


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the nine patients admitted to the study two were excluded, one because of contamination of the cultures from the sputum collections and the other because of low sputum volumes. The remaining seven patients were all male, with age ranging from 19 to 36 years and weight ranging from 45 to 66 kg (mean 54.7 kg). All had extensive cavitated pulmonary disease with sputum strongly positive on direct smear examination. No toxicity from the administration of liposomal amikacin was encountered. Bacterial drug resistance did not develop during the 3 days of the study. The distributions of cfu counts (Table 1Go) on the pre-treatment sputum collection (S1) and the collections after each of the succeeding 3 days of treatment (S2, S3 and S4) were all normally distributed. Although there appears to be a slight fall in the mean during treatment, the regression of the mean on treatment days is not significant. EBAs calculated over various treatment periods are set out in Table 2Go. The lower 95% confidence limits for all of the calculated EBAs were all negative. This indicates that there is no significant EBA. There is some indication that the S3–S4 EBA (0.1320) was greater than either the S1–S2 EBA (0.0160) or the S2–S3 EBA (0.0097), but the differences are not significant. Equally, there is an indication that the S1–S4 EBA (0.0526) was greater than the S1–S3 EBA (0.0128) but this also fails to attain significance.


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Table 1. Cfu counts of M. tuberculosis in the S1, S2, S3 and S4 collections from seven patients
 

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Table 2. EBA of liposomal amikacin in seven patients over various dosage periods
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The MIC of amikacin is 1.0 mg/L in 7H11 oleic acid agar medium.10 The concentration of total amikacin in plasma in healthy volunteers was found to increase after three successive infusions of liposomal amikacin 30 mg/kg, as in the present study, from c. 700 mg/L to just over 1000 mg/L.11 After this, the concentration fell slowly and was still >100 mg/L 30 days later. At the beginning of treatment the great majority of tubercle bacilli lay in dense masses within the caseum of cavities just under the air/caseum interface.12 If sufficient amikacin was released from the liposomes and arrived at these bacillary sites, there should be killing of rapidly growing organisms within the clumps of bacilli. Despite the high total amikacin concentrations, we have been unable to detect any EBA, in agreement with an earlier report on a single patient.13 Justification for the assumption that the EBA should be tested against zero has been presented elsewhere.14 In another report, the EBA of free amikacin given to patients has been examined in a group of 59 patients treated with amikacin 5–15 mg/kg by im injection.9 The S1–S3 EBA over the standard 2 day period was 0.046, a value that just differed significantly (P = 0.03) from zero. Values of the S1–S3 EBA of 0.0533 found in patients receiving amikacin 15 mg/kg were similar to the S1–S4 EBA of 0.0526 in the present study. Furthermore, the mean peak amikacin concentrations of 39.2 mg/L at 1 h after the im dose was similar to a mean of 43.5 mg/L free amikacin at 1 h during the alpha phase of excretion in healthy volunteers given three doses of liposomal amikacin 30 mg/kg.11 The similarities of these values indicates that the S1–S4 EBA of the present study could arise solely from the free amikacin available in plasma after administration of liposomal amikacin.

The failure to detect EBA indicates a failure of liberation of the amikacin from the liposomes in liposomal amikacin. It seems probable that liberation happens only when the liposomes are phagocytosed by macrophages. This accounts for the high activity of liposomal amikacin in acute murine tuberculosis, where bacilli are mainly intracellular within macrophages. As there are few macrophages close to tubercle bacilli in cavities,12,15 this mechanism may not produce an adequate concentration in those parts of the caseum where the great majority of the bacilli exist. More amikacin might, however, be released in areas of pneumonic infiltration where macrophages abound and there is a closer resemblance to murine histology. Even so, the mouse experiments clearly demonstrate that amikacin had little bactericidal activity once immunity has developed,6 which is certain to have happened in human lesions. In HIV-seropositive patients, macrophages are less likely to cause bacillary stasis, so that it is still possible that liposomal amikacin might be of value in their treatment. The very low EBA of amikacin whether administered in the free form or in liposomes may result from an acid micro-environment of the bacilli due to the presence of acute inflammation, as argued elsewhere.9 It is necessary to postulate such an acid micro-environment to account for the sterilizing activity of pyrazinamide.16 Thus, it appears that amikacin in any form has low initial bactericidal activity, mainly because of the lesional pH. While the great majority of the bacilli at the start of treatment are extracellular, it has been postulated that the persisting bacilli in the later stages of treatment have often been phagocytosed.15 Drugs incorporated in liposomes would then be liberated close to bacilli after they had been phagocytosed by macrophages containing bacilli. This mechanism might allow liposomal amikacin to be more bactericidal in the later stages of treatment. Unfortunately, because all aminoglycosides are poor sterilizing drugs, as shown in the treatment of chronic murine tuberculosis6,17 and of pulmonary tuberculosis,18,19 they are also ineffective in killing bacilli during the later stages of treatment and do not shorten the duration of treatment. Nevertheless, whereas aminoglycosides and amikacin in particular may be less bactericidal in the treatment of human tuberculosis than other drugs, they serve a purpose in preventing the emergence of drug resistance. Although the role of liposomal amikacin appears to be strictly limited in the treatment of pulmonary tuberculosis, enclosure of drugs with higher sterilizing activity, particularly rifampicin, in liposomes or by micro-encapsulation20 could offer a mechanism for drug delivery that has advantages over conventional oral administration.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by NeXstar, San Dimas, CA, USA (now Gilead Sciences Inc., Boulder, CO, USA), who also provided the liposomal amikacin.


    Notes
 
* Corresponding author. Tel: +44-20-8725-5704; Fax: +44-20-8672-0234; E-mail: dmitchis{at}sghms.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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6 . Dhillon, J., Fielding, R., Adler-Moore, J., Goodall, R. & Mitchison, D. A. (2001). The activity of low-clearance liposomal amikacin in experimental murine tuberculosis. Journal of Antimicrobial Chemotherapy 48, 869–76.[Abstract/Free Full Text]

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8 . Mitchison, D. A. & Sturm, A. W. (1997). The measurement of early bactericidal activity. In Bailliere's Clinical Infectious Diseases, Vol. 4, No. 2, (Malin, A. & McAdam, K. P. W. J., Eds), pp. 185–206. Bailliere Tindall, London.

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11 . Data on file at NeXstar.

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17 . Grumbach, F., Canetti, G. & Le Lirzin, M. (1969). Rifampicin in daily and intermittent treatment of experimental murine tuberculosis, with emphasis on late results. Tubercle 50, 280–93.[Medline]

18 . Hong Kong Chest Service/British Medical Research Council. (1991). Controlled trial of 2, 4 & 6 months of pyrazinamide in 6-month, 3x weekly regimens for smear-positive pulmonary tuberculosis, including an assessment of a combined preparation of isoniazid, rifampicin & pyrazinamide. American Review of Respiratory Disease143, 700–6.[ISI][Medline]

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Received 19 February 2001; returned 21 June 2001; revised 3 August 2001; accepted 10 September 2001