dc.description.abstract
Microdialysis represents a minimally invasive sampling technique to collect samples and also to
administer drugs at target site, which was first introduced in 1974 by Ungerstedt and Pycock to
sample endogenous dopamine from rat brain [1]. Until then, the only methods available to obtain in
vivo concentrations of analytes were tissue destroying or only possible with a limited number of
repetitions of sample taking. In contrast, since the method of microdialysis was introduced, the
advantages over more invasive methods due to its minimally invasive character were repeatedly
shown in clinical studies. In addition, microdialysis enables the continuous sampling of analytes over
a predefined time interval, allowing the determination of concentration-time profiles of analytes
over hours and even days without loss of body fluids, associated with other sampling methods.
Moreover, samples can be taken directly from the target site (for example of an infection) located in
virtually every tissue. Especially in the research field of infectious diseases the method is often used,
particularly when investigating the sources of antimicrobial resistance. At present, research tackling
antimicrobial resistance is not primarily focussing on bacterial or viral pathogens any more, but
gradually shifting its focus also towards fungal infections. These infections are on the rise but still
remain underestimated in comparison to the threads caused by bacteria.
Since microdialysis is an important technique for sampling pharmacologically active analytes, the
number of in vivo clinical studies using microdialyis increased. However, thorough in vitro
investigations to characterise the analyte and ensure optimal study conditions prior to in vivo studies
are often missing/not performed. Due to the significance of these in vitro investigations and rising
importance of antifungal research, the two first-line antifungals anidulafungin and voriconazole were
investigated with the static and dynamic in vitro microdialysis system in the present thesis. Thus, in a
first step a bioanalytical assay for the quantification of anidulafungin samples from in vitro
microdialysis was developed and validated. A previous bioanalytical assay [2] for quantitative analysis
of voriconazole in vitro samples from microdialysis, was further revised and validated. Both assays,
for anidulafungin and voriconazole, were validated according to the Guideline on bioanalytical
method validation [3].
After development of the bioanalytical assay, in vitro microdialysis of anidulafungin was performed.
As described for in vitro microdialysis investigations, the aim was to investigate and proof the
feasibility of anidulafungin for microdialysis as a prerequisite for potential in vivo clinical studies.
Anidulafungin was found to adsorb on catheter material in the static in vitro microdialysis system and
therefore to bias the forthcoming results. In order to enable in vivo investigations despite adsorption,
various parameters were investigated, for example catheter design (20 kDa or 100 kDa cut-off), perfusate composition (different combinations of Ringer’s solution and human serum albumin,
dextran) and pre-coating of catheters with caspofungin or anidulafungin. The investigations
ultimately resulted in the recommendation to perform in vivo microdialysis after an equilibration
time of at least 3 h in steady state at the target site to allow for equilibration of the adoption process
with a perfusate of Ringer’s solution and human serum albumin (0.5%) in catheters with 100 kDa cutoff.
Previous investigations on voriconazole using static in vitro microdialysis by Simmel et al. resulted in
a relative recovery of approximately 100% [4]. To verify the results, a shortened in vitro microdialysis
investigation was performed with the static in vitro microdialysis system. The dependence of relative
recovery and relative delivery on flow rate and independence of relative recovery and relative
delivery on concentration was investigated with the static microdialysis system. Since voriconazole is
an easy to handle drug and the results for relative recovery were also close to 100%, it is an ideal
drug for the first investigations with the dynamic in vitro microdialysis system. Thus, the developed
bioanalytical methods and the static in vitro microdialysis system were used to develop a dynamic in
vitro microdialysis system to mimic concentration-time profiles of analytes, to gain further
knowledge of the microdialysis specific characteristics of the analyte in vivo. The validation of the
system demonstrated that it is possible to perform in vitro microdialysis with this model. First,
concentration-time profiles of anidulafungin and voriconazole were predicted in silico, based on in
vivo data from clinical studies. Then, the concentration-time profiles were mimicked in vitro with the
dynamic microdialysis system. Finally, microdialysis with subsequent calibration by retrodialysis of
the single drugs was performed. The resulting microdialysis concentration-time profiles for
voriconazole had a high accuracy compared to the concentration in the medium mimicking the
tissue, whereas experiments with anidulafungin resulted in a misleading prediction of concentrations
due to adsorption of the analyte on catheter material.
During this work, it was emphasised that it is indispensable to perform adequate in vitro
microdialysis before starting in vivo studies. Apart from experiments with the static in vitro
microdialysis system on flow rate, concentration and perfusate also investigations based on
concentration-time profiles of the analyte (i.e. decision about calibration technique) should be
conducted for a more detailed characterisation of the analyte of interest and thus reliable clinical
results.
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