Difficulties in the assay of liposomal amikacin (MiKasome) in serum
A. M. Lovering,
L. O. White and
A. P. MacGowan
Bristol Centre for Antimicrobial Research and Evaluation, Southmead Hospital, Westbury
on Trym, Bristol BS10 5NB, UK
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Abstract
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Antibiotic-free human serum was spiked with known concentrations of liposomal
amikacin and
assayed on the Abbott TDx System, using polarization fluoroimmuno assay (PFIA) kits from
Abbott Laboratories, Oxis and Sigma. Although all three kits gave a linear response, the Abbott
and Oxis kits showed very low recovery (>21%) with only the Sigma kit giving near 100%
recovery. Heating samples at 56°C for 30 min improved recovery with the Abbott and Oxis
kits (75- 80% of target value), but decreased recovery with the Sigma kit (85% of target value).
The loss of amikacin from liposomal amikacin, as measured using the Sigma kit, was related to
both temperature and duration of heating, reaching a maximal loss of 21% after 1 h at 60°C.
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Introduction
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For the past 40 years, the aminoglycosides have been one of the most important classes of
antimicrobial used in the treatment of serious sepsis. They are characterized by a narrow
therapeutic index, most being potentially both nephrotoxic and ototoxic. Serum levels are
monitored to avoid toxicity.
1 In recent years aminoglycosides, and amikacin in
particular, have been used increasingly in the treatment of mycobacterial infection, especially
infections caused by atypical or drug-resistant mycobacteria, often in HIV-infected patients.
2, 3 In such cases
patients normally receive treatment for many months and the risk of toxicity presents a
significant
clinical problem.
The incorporation of aminoglycosides into liposomal carriers appears to offer a number of
advantages over the use of conventional dosing. Early studies suggest that liposomal
preparations are significantly less toxic than conventionally administered drug, that they have
pharmacokinetics that favour less frequent dosing, and that they exhibit better penetration into
sites of infection.
4, 5, 6, 7, 8, 9, 10 Despite
this lower potential for toxicity, there is a need
to assay liposomal preparations in order to establish pharmacokinetic parameters and to develop
optimum treatment regimens. In this paper the suitabilities of three commercially available
polarization fluoroimmuno assay (PFIA) methods for the assay of liposomal amikacin
(MiKasome, NeXstar Pharmaceuticals, San Dimas, CA, USA) are investigated.
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Materials and methods
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Antibiotic-free human serum was spiked with known concentrations (2.3- 45.0 mg/L) of
liposomal amikacin and assayed on the Abbott TDx System using PFIA kits from Abbott
(Abbott
Diagnostics, Chicago, IL, USA), Oxis (Oxis, Portland, OR, USA) and Sigma (Sigma Chemical
Company, St Louis, MO, USA). The three kits, and calibrators, controls and buffers, were
purchased from their UK suppliers and calibrated and used according to the
manufacturers' recommendations. Additional serum samples were spiked with
liposomal
amikacin and either frozen at - 70°C, thawed and then assayed or heated at 56°C for 30
min and then assayed. A further set of samples were spiked with known concentrations of
amikacin (Bristol-Myers Squibb, Hounslow, UK) and treated as described for the liposomal
preparations. To establish whether the loss of amikacin seen on heating liposomal preparations at
56°C was specific to amikacin, samples containing known concentrations of either
gentamicin or vancomycin were spiked with 10 mg/L liposomal amikacin. After they had been
heated at 56°C for 30 min these samples were assayed using the Abbott TDx System and
kits from either Abbott (gentamicin) or Sigma (vancomycin).
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Results and discussion
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The apparent concentrations of amikacin detected in the samples spiked with liposomal amikacin
are shown in the.Figure Although all three kits gave a linear response
over the concentration
range (R2= 0.994- 0.997), two kits showed very low recovery
(Abbott, 18.0 ± 1.3% of target value; Oxis, 18.2 ± 0.6% of target value) with
only the Sigma kit giving near 100% recovery (92.3 ± 2.2% of target value). One freeze-
thaw cycle of the samples did not affect these recoveries (data not shown). Since information on
kit composition is not usually available, it is difficult to postulate reasons for the differences in
results between the kits. It is likely that these relate to differences in the degree of lysis of
liposomes obtained. This may be influenced by surfactants used in the kits. Although this could
be addressed by pre-treatment of samples with surfactant, or possibly phospholipase, such
procedures would constitute a major deviation from normal operating procedures, and might
result in damage to either the kits or TDx analyser.
Liposomal amikacin may be used in patients who are HIV antibody positive. As specimens from
these patients can be subjected to heat inactivation, the effect of heating serum on the
performance of the kits was investigated. Heating samples at 56°C for 30 min improved
recovery with the Abbott (80% of target value) and Oxis (76% of target value) kits but decreased
recovery with the Sigma kit (87% of target value). Heating had no effect on the recovery of
non-encapsulated amikacin from serum, or of liposomal amikacin in the absence of serum
(Table).
The loss of amikacin from liposomal amikacin, as measured using the Sigma kit, was related to
both temperature and duration of heating, reaching a maximal loss of 21% after 1 h at
60°C.
Heating samples containing either gentamicin or vancomycin, in the presence of liposomal
amikacin, did not affect the concentrations of either analyte (data not shown); this suggests that
the loss of amikacin, seen on heating serum samples containing liposomal amikacin, is specific
to
the encapsulated drug.
View this table:
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Table. The concentrations of amikacin (mg/L) in samples spiked with liposomal amikacin or
conventional amikacin and assayed using Abbott, Oxis, or Sigma kits after 30 min at either (a)
room temperature or (b) 56°C
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We conclude that not all the kits currently available for the PFIA assay of amikacin are suitable
for the assay of liposomal preparations and also that there is a loss of drug from liposomal
amikacin on heating serum to inactivate HIV. We advise caution in the application of existing
assay methodologies for the measurement of liposomal aminoglycosides.
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Received 28 July 1998;
returned 19 October 1998; revised 13 November 1998;
accepted 6 January 1999