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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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.



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Figure. The concentration of amikacin in samples spiked with liposomal amikacin and assayed using Abbott ({triangleup}), Oxis ({diamond}), or Sigma ({square}) kits.

 
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.


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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
 
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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Lovering, A. M. (1999). Aminoglycosides. In Clinical Antimicrobial Assays, (Reeves, D. S., Wise, R., Andrews, J. & White, L. O., Eds), pp. 123- 35. Oxford University Press, Oxford.

2 . Peloquin, C. A. (1997). Using therapeutic drug monitoring to dose the antimycobacterial drugs.Clinics in Chest Medicine 18, 79–87.[ISI][Medline]

3 . Jenner, P. A. (1999). Antimycobacterial drugs. In Clinical Antimicrobial Assays, (Reeves, D. S., Wise, R., Andrews, J. & White, L. O., Eds), pp. 205- 19. Oxford University Press, Oxford.

4 . Tomioka, H., Saito, H., Sato, K. & Yoneyama, T. (1991). Therapeutic efficacy of liposome-encapsulated kanamycin against Mycobacterium intracellulare infection induced in mice. American Review of Respiratory Disease 144, 575–9.[ISI][Medline]

5 . Swenson, C. E., Stewart, K. A., Hammett, J. L., Fitzsimmons, W. E. & Ginsberg, R. S. (1990). Pharmacokinetics and in vivo activity of liposome-encapsulated gentamicin. Antimicrobial Agents and Chemotherapy 34,235 – 40.[ISI][Medline]

6 . Sanderson, N. M. & Jones, M. N. (1996). Encapsulation of vancomycin and gentamicin within cationic liposomes for inhibition of growth of Staphylococcus epidermidis. Journal of Drug Targeting 4, 181– 9.[ISI][Medline]

7 . Khalil, R. M., Murad, F. E., Yehia, S. A., El-Ridy, M. S. & Salama, H. A. (1996). Free versus liposome-entrapped streptomycin sulfate in treatment of infections caused by Salmonella enteritidis. Pharmazie 51, 182– 4.[ISI][Medline]

8 . Omri, A. & Ravaoarinoro, M. (1996). Comparison of the bactericidal action of amikacin, netilmicin and tobramycin in free and liposomal formulation against Pseudomonas aeruginosa. Chemotherapy 42,170 – 6.[ISI][Medline]

9 . Ladigina, G. A. & Vladimirsky, M. A. (1986). The comparative pharmacokinetics of 3H-dihydrostreptomycin in solution and liposomal form in normal and Mycobacterium tuberculosis infected mice. Biomedicine and Pharmacotherapy 40, 416– 20.

10 . Nightingale, S. D., Saletan, S. L., Swenson, C. E., Lawrence, A. J., Watson, D. A., Pilkiewicz, F. G. et al. (1993). Liposome- encapsulated gentamicin treatment of Mycobacterium avium- Mycobacterium intracellulare complex bacteremia in AIDS patients.Antimicrobial Agents and Chemotherapy 37, 1869– 72.[Abstract]

Received 28 July 1998; returned 19 October 1998; revised 13 November 1998; accepted 6 January 1999





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