Anidulafungin and Micafungin Concentrations in Cerebrospinal Fluid and in Cerebral Cortex

Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01 to 0.66 μg/ml) and micafungin levels (<0.01 to 0.16 μg/ml) were lower than those in plasma concentrations (0.77 to 5.07 and 1.21 to 8.70 μg/ml, respectively) drawn simultaneously. In cerebral cortex, anidulafungin and micafungin levels were 0.21 to 2.34 and 0.18 to 2.88 μg/g, respectively.

The cerebral cortex of deceased patients who had received anidulafungin or micafungin within their last 30 days of life was sampled during autopsy, which is routinely performed for quality assurance in Austrian hospitals. Anidulafungin and micafungin were extracted from 0.2 g of tissue by addition of 250 l of acetonitrile and 250 l of methanol (Sigma-Aldrich, Vienna, Austria), homogenization with a Precellys homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) at 4,500 rpm (twice for 25 s, 2-s break), and centrifugation for 5 min at 8°C and 4,665 ϫ g. For calibration, we used porcine brain samples spiked with anidulafungin or micafungin and homogenized and proceeded in the same manner. Both echinocandins were quantified by HPLC-UV at 306 nm (5). The lower limit of quantification was 0.05 g/g for anidulafungin and 0.10 g/g for micafungin. The extraction recovery from brain was ϳ40% and ϳ60% for anidulafungin and micafungin, respectively.
The significance of the difference between CSF and plasma concentrations was assessed by Wilcoxon matched-pairs test, and the difference between the penetration ratio (PR) of anidulafungin and PR of micafungin was calculated by Mann-Whitney U test using IBM SPSS statistics 24.0. PR was the ratio between the AUC 0 -24 in CSF and that in plasma over 24 h (AUC 0 -24 CSF /AUC 0 -24 plasma ), when multiple samples were drawn via EVD. For single samples drawn by LP, PR was the ratio between the concentration in CSF and that in plasma (C CSF /C plasma ).
CSF samples were obtained from three patients on anidulafungin and three patients on micafungin. One patient on anidulafungin (patient 2) and one patient on micafungin (patient 5) had undergone EVD (Table 1; Fig. 1). CSF concentrations of anidulafungin (Ͻ0.7 g/ml) and micafungin (Ͻ0.2 g/ml) were lower than the corresponding plasma concentrations (P Ͻ 0.05 and P Ͻ 0.01, respectively) ( Table 1; Fig. 1). PRs of anidulafungin and micafungin were similar (P ϭ 0.40). In the cerebral cortex of four deceased patients, anidulafungin had reached concentrations of 0.21 to 2.34 g/g. Brain concentrations of micafungin were Ͻ0.10 to 2.88 g/g (n ϭ 6). Anidulafungin and micafungin were measurable even 13 and 10 days, respectively, after treatment (Table 2).
In some of the CSF and brain specimens, measured anidulafungin and micafungin concentrations were below the MICs reported for relevant Candida species (i.e., 0.008 to 4.0 g/ml) (6). In the CSF of patients 2 and 5, the target AUC 0 -24 /MIC ratios of 2,782 and 5,299 suggested for anidulafungin and micafungin, respectively, have not been achieved, not even for highly susceptible Candida strains (6,7). However, the relevance of in vitro MIC values for antifungal activity of echinocandins in CSF and in CNS remains to be clarified. In CSF, protein binding of anidulafungin and micafungin is unknown. In vitro, protein binding obviously affects MICs (8,9). We did not separate protein-bound from free echinocandins. In our small and heterogeneous study population, only two patients (patients 3 and 6) reached steady state, when CSF was taken by LP, and only two patients (patients 2 and 4) suffered from CNS infections that might have enhanced permeability of the blood-brain barrier (10). However, CSF concentrations and PRs of anidulafungin and micafungin in patients 2 and 4 were similar to those in our other patients.
During cerebral aspergillosis, the 2-fold maximum dose of micafungin (i.e., 300 mg/ day) achieved CSF concentrations of Ͻ0.02 g/ml (11). After intracranial hemorrhage, micafungin CSF levels amounted to 0.019 to 4.66 g/ml (12). Caspofungin was undetectable in CSF during meningeal coccidioidomycosis and in 9 of 11 CSF samples from children with hematological malignancies (13,14). In infants with meningitis, daily high doses of 8 to 10 mg/kg of micafungin resulted in CSF concentrations of 0.80 to 1.80 g/ml (15). In neonatal rats, anidulafungin brain concentrations of 1.60 and 4.40 g/g were measured (16). Along with the higher dosage, the immaturity of the blood-brain barrier might explain the higher penetration into CSF and brain.
In brain specimens, we cannot rule out minor agonal or postmortem changes of anidulafungin and micafungin concentrations, although anidulafungin and micafungin were stable in the brains of deceased patients for at least 96 h. Brain specimens consist of various compartments, e.g., different cells, extracellular matrix, and blood vessels. Anidulafungin and micafungin were not quantified on a cellular level or in different brain areas. Echinocandin extraction from human brain might yield slightly lower recovery than extraction from external standards. In rats, [ 3 H]caspofungin brain concentrations were Յ0.16 g eq/g (17). In rabbits, micafungin brain concentrations were Ͻ0.19 g/g, and those of anidulafungin were Ͻ4.0 g/g (18,19). In a biopsy sample of a brain abscess, a micafungin concentration of 0.26 g/g was measured (20).
In conclusion, anidulafungin and micafungin concentrations in CSF and in brain specimens were below the in vitro MIC values of several pathogenic Candida strains. Studies on target-site pharmacodynamics are required for assessment of antifungal efficacy.
(Parts of the data were presented at the 23rd and 25th Scientific Symposium of the Austrian Pharmacological Society, 2017 and 2019, Innsbruck, Austria.)

ACKNOWLEDGMENTS
This study was supported by the Austrian Science Fund (FWF) (grant KLI 565-B31). We thank Thomas Nachtigall, Obersöchering, Germany, for donation of the HPLC system and for technical support. Porcine brain was kindly provided by Landmetzgerei Piegger, Sistrans, Austria.
R.B. has received an IIR grant from Pfizer; research support from Rokitan, Vienna, Austria; and a lecture fee from Basilea Pharmaceutica, Basel, Switzerland. He is a member of an advisory board of Merck Sharp & Dohme. . AFG (Ecalta, Pfizer Limited, Sandwich, Kent, UK) and MFG (Mycamine, Astellas, Tokyo, Japan) were stable in brain tissue for at least 96 h at 4°C, which was the storage temperature of the corpses. AFG and MFG had been administered for suspected or proven invasive candidiasis at the discretion of the treating physician. The lower limit of quantification is 0.05 g/g for AFG and 0.10 g/g for MFG.